bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 648: Semi-synthetic peptibodies are a novel personalized therapeutic with activity against lymphoma in vitro and in vivo
Authors: Torchia James , Ng Patrick , Chen Homer , Weiskopf Kipp , Levy Ronald
Published: May 2015

The complementarity determining region, or idiotype, of the surface immunoglobulin receptor is a true tumor-specific marker on B-cell lymphomas unique to each patient. Antibodies against idiotype can induce complete regression of lymphoma in patients, but since this requires the generation of a custom monoclonal antibody for each patient, it has not been practical.

We have developed a method for targeting idiotype using a novel construct that we refer to as a “semi-synthetic peptibody”. It consists of a synthetic peptide ligand for idiotype that is covalently linked to the amino terminus of a recombinant IgG Fc domain. Peptide ligands for idiotype can be identified by high throughput screens of random peptide libraries and produced inexpensively by solid-phase synthesis. Linkage of these idiotype ligands to the Fc domain enhances their pharmacokinetics and augments their anti-tumor effect by activating innate immune effectors. Since each patient-specific synthetic peptide can be chemically linked to a common IgG Fc domain, this modular design yields a custom therapeutic that may be more practical to produce than a unique biologic monoclonal antibody.

We demonstrate that semi-synthetic peptibodies bind specifically to the idiotype of tumor cells and induce apoptosis by crosslinking surface immunoglobulin. Additionally, they trigger antibody dependent cellular cytotoxicity, antibody mediated phagocytosis, and complement-mediated lysis of opsonized lymphoma cells in vitro. They possess a favorable pharmacokinetic profile and are sufficient to clear tumor in SCID mice challenged intravenously with a luciferase-labeled human lymphoma cell line. Tumor clearance in vitro and in vivo mediated by peptibody was superior to that achievable with an anti-idiotype monoclonal antibody.

Thus, semi-synthetic anti-idiotype peptibodies demonstrate multimodal activity against lymphoma cells in vitro and clear human lymphoma in a disseminated xenograft model. This modular, semi-synthetic design may enable a personalized and targeted therapy that is feasible to produce for patients with B-cell lymphoma.

Citation Format: James Torchia, Patrick Ng, Homer Chen, Kipp Weiskopf, Ronald Levy. Semi-synthetic peptibodies are a novel personalized therapeutic with activity against lymphoma in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 648. doi:10.1158/1538-7445.AM2014-648
Abstract 649: Development and preclinical characterization of an Fc-optimized CD133 antibody for improved induction of NK cell reactivity against myeloid leukemia
Authors: Koerner Samuel , Leibold Julia , Grosse-Hovest Ludger , Bühring Hans-Jörg , Salih Helmut R. , Jung Gundram
Published: May 2015

NK cells are cytotoxic lymphocytes that play a major role in anti-tumor immunity and largely contribute to the efficacy of allogenic stem cell transplantation (SCT) in leukemia. Another clinically important feature of NK cells is their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of anti-tumor antibodies (mAb) such as Rituximab. Modifications of the human IgG1 Fc-part of anti-tumor antibodies may lead to markedly improved capability to recruit Fc-receptor bearing effector cells. Here we report on the development and preclinical characterization of an Fc-optimized mAb directed towards CD133, a molecule expressed on a wide variety of malignant cell types. Initially we evaluated binding of three different mouse anti-human CD133 mAbs (clones AC133, W6B3 and 293C3) to 20 AML and 6 CML samples to identify a clone with optimal binding characteristics. Binding of AC133 and W6B3 to the leukemic cells in 11/20 AML and 5/6 CML samples was comparable. In contrast, binding of 293C3 was observed in 18/20 AML cases and 5/6 CML cases. This indicates that 293C3 recognizes a particular epitope, which is expressed in a high proportion of AML cases. Therefore mAb 293C3 was selected for generation of chimeric mAbs with either a wildtype Fc part (293C3-WT) or a variant containing distinct modifications (S239D/I332E) to enhance its affinity to the Fc receptor CD16 (293C3-SDIE). The binding specificity of 293C3-WT and 293C3-SDIE was validated in analyses with CD133 transfectants and the respective mock controls. In cytotoxicity assays and analyses of NK cell degranulation, 293C3-WT clearly induced NK cell ADCC against primary leukemia cells. However, ADCC activity of the Fc-optimized mAb 293C3-SDIE markedly exceeded that of its counterpart containing the wild type Fc part. Considering the expression of CD133 on healthy hematopoietic progenitor cells, we presently evaluate potential toxicity of the antibodies towards these cells and other healthy tissue. We envisage that 293C3-SDIE might be an attractive immunotherapeutic reagent for the elimination of minimal residual disease in CD133 expressing leukemia, particularly in the context of allogenic SCT.

Citation Format: Samuel Koerner, Julia Leibold, Ludger Grosse-Hovest, Hans-Jörg Bühring, Helmut R. Salih, Gundram Jung. Development and preclinical characterization of an Fc-optimized CD133 antibody for improved induction of NK cell reactivity against myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 649. doi:10.1158/1538-7445.AM2014-649
Abstract 65: Ink4a/Arf locus drives gastric tumorigenesis through induction of parietal cell loss
Authors: Seishima Ryo , Wada Takeyuki , Hasegawa Hirotoshi , Ishii Yoshiyuki , Okabayashi Koji , Tsuruta Masashi , Kitagawa Yuko , Saya Hideyuki , Nagano Osamu
Published: May 2015

Background: Cancer development is often preceded by the appearance of preneoplastic lesions. In gastric carcinogenesis, chronic inflammation and histopathologic progression of the stomach epithelium lead to the development of metaplasia and eventually adenocarcinoma. Spasmolytic polypeptide-expressing metaplasia (SPEM) is one of the preneoplastic lesions in the stomach and is triggered by the loss of parietal cells that secrete gastric acid. The Ink4a/Arf locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p19(ARF) in mice. However, little is known regarding the role of these proteins encoded by the INK4a/ARF locus in gastric tumorigenesis.Methods: To evaluate the functional role of Ink4a/Arf locus, we crossed mouse gastric cancer modelK19-Wnt1/C2mE mice with Ink4a/Arf (-/-) mouse and generated Ink4a/Arf (-/-) K19-Wnt1/C2mE mice and evaluated the effect of deletion of Ink4a/Arf locus on the parietal cell loss and formation of SPEM and gastric tumor. Results: In K19-Wnt1/C2mE mice, accumulation of reactive oxygen species (ROS) and the increase of p19 expression were observed in gastric epithelium along with the parietal cells loss leading to SPEM development. However, the parietal cells loss followed by formation of SPEM and gastric tumor were drastically suppressed in Ink4a/Arf (-/-) K19-Wnt1/C2mE mice. Furthermore, treatment of tamoxifen, an inducer of parietal cells loss, in Ink4a/Arf (-/-) mice revealed that deletion of Ink4a/Arf locus suppresses parietal cells loss in mice. Conclusion: These results suggest that activation of p19(ARF) tumor suppressor protein by ROS accumulation paradoxically play a key role in the parietal cells loss leading to gastric tumorigenesis.

Citation Format: Ryo Seishima, Takeyuki Wada, Hirotoshi Hasegawa, Yoshiyuki Ishii, Koji Okabayashi, Masashi Tsuruta, Yuko Kitagawa, Hideyuki Saya, Osamu Nagano. Ink4a/Arf locus drives gastric tumorigenesis through induction of parietal cell loss. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 65. doi:10.1158/1538-7445.AM2014-65
Abstract 651: Ex vivo and in vivo characterization of IPH4102, a humanized anti-KIR3DL2 antibody for the treatment of cutaneous T-cell lymphomas
Authors: Marie-Cardine Anne , Viaud Nicolas , Dujardin Arnaud , Joly Rachel , Gauthier Laurent , Bonnafous Cecile , Blery Mathieu , Paturel Carine , Bensussan Armand , Bagot Martine , Sicard Helene
Published: May 2015

KIR3DL2 belongs to the killer immunoglobulin (Ig)-like receptors (KIRs) family and is composed of three extracellular Ig-like domains. KIR3DL2 is naturally expressed on some NK cells and minor subpopulations of CD8+ and CD4+ T cells. Physiologically, KIRD3L2 is an inhibitory receptor for human leukocyte antigen (HLA) class I molécules regulating NK cell activation. Interestingly, KIR3DL2 is also expressed on several subtypes of T lymphomas/leukemias, and in particular on Advanced cutaneous T cell lymphomas (CTCL), making it a unique therapeutic target in cancer. From a wide collection of anti-KIR3DL2 monoclonal antibodies (mAbs) with distinct properties related to several epitopes, we have selected IPH4102 as the lead therapeutic candidate. IPH4102 is a humanized IgG1 that selectively kills KIR3DL2-positive cells in vitro, mainly through antibody-dependent cell cytotoxicity (ADCC). IPH4102 also improves survival of mice engrafted with KIR3DL2-positive tumors. More importantly, IPH4102 exerts remarkable anti-tumor activity against primary KIR3DL2-positive tumor cells ex vivo: using blood taken from Sézary Syndrome patients as experimental model, we demonstrated the efficient killing of primary leukemic cells by autologous NK cells engaged by IPH4102. IPH4102 IND-enabling studies are ongoing. In parallel, other anti-KIR3DL2 mAbs were also developed as unique and sensitive tools for the detection by immunohistochemistry of KIR3DL2 on tumor biopsies. Owing to its efficacy profile and to the highly restricted expression pattern of its target KIR3DL2, IPH4102 stands as a very promising therapeutic option for the treatment of advanced cutaneous T cell lymphomas.

Citation Format: Anne Marie-Cardine, Nicolas Viaud, Arnaud Dujardin, Rachel Joly, Laurent Gauthier, Cecile Bonnafous, Mathieu Blery, Carine Paturel, Armand Bensussan, Martine Bagot, Helene Sicard. Ex vivo and in vivo characterization of IPH4102, a humanized anti-KIR3DL2 antibody for the treatment of cutaneous T-cell lymphomas . [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 651. doi:10.1158/1538-7445.AM2014-651
Abstract 658: A bispecific HER2 targeting FynomAb with superior anti-tumor activity and novel mode of action
Authors: Schade Babette , Brack Simon , Attinger-Toller Isabella , Klupsch Kristina , Woods Richard , Hachemi Helen , Bey Ulrike von der , Koönig-Friedrich Susann , Bertschinger Julian , Grabulovski Dragan
Published: May 2015

Upregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2 targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance.

Fynomers are small 7 kDa globular proteins derived from the SH3 domain of the human Fyn kinase (Fyn SH3) that can be engineered to bind with antibody-like affinity and specificity to virtually any target of choice. Fynomers can be fused to N-terminal and/or C-terminal ends of antibodies to generate multispecific therapeutics (FynomAbs) with tailored architectures. FynomAbs can be produced using standard antibody technology (GMP production yield of 3.3 g/L at 1000 L scale achieved), and show IgG-like biophysical properties and pharmacokinetic profiles.

Based on the approved antibody pertuzumab we have created a panel of bispecific FynomAbs which target two epitopes on HER2. The activity of the HER2 targeting FynomAbs was found to depend on the FynomAb architecture, i.e. the spatial arrangement of the binding sites of antibody and the Fynomer.

The most potent of these FynomAbs, termed COVA208, demonstrated superior tumor cell growth inhibition in vitro compared to pertuzumab and trastuzumab. COVA208 was characterized in detail and showed an increased ability to induce rapid HER2-internalization and apoptosis in vitro. Moreover, it elicited a stronger inhibition of downstream HER2 signaling which was accompanied by a reduction of HER2, HER3 and EGFR levels in vitro and in vivo. The therapeutic potential of COVA208 has been demonstrated in vivo in four different HER2 mouse models, where COVA208 exhibited excellent anti-tumor activity. Importantly, COVA208 demonstrated superior activity in vivo compared to trastuzumab and pertuzumab.

The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets.

Citation Format: Babette Schade, Simon Brack, Isabella Attinger-Toller, Kristina Klupsch, Richard Woods, Helen Hachemi, Ulrike von der Bey, Susann Koönig-Friedrich, Julian Bertschinger, Dragan Grabulovski. A bispecific HER2 targeting FynomAb with superior anti-tumor activity and novel mode of action. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 658. doi:10.1158/1538-7445.AM2014-658
Abstract 659: Integrated therapeutic antibody development at the National Research Council of Canada
Authors: Jaramillo Maria L. , Marcil Anne , Durocher Yves , Gilbert Renald , Mullick Alaka , Kelly John , O'Connor-McCourt Maureen , Massie Bernard
Published: May 2015

Advances in genomics and antibody engineering have enabled the development of an innovative class of targeted therapies, namely therapeutic antibodies, for the treatment of diseases with significant unmet medical needs such as cancer. Therapeutic antibodies represent one of the largest and fastest growing classes of medications. The National Research Council of Canada (NRC) has built a chain of cutting edge technology platforms needed to discover, engineer and produce therapeutic monoclonal antibodies with the goal of partnering with industrial and academic centers to advance research and development of this important class of therapeutics.

Target Identification- Tumor targets were identified using a combination of proteomics, transcriptomics and bioinformatic approaches. Out of these lists, approximately 40 tumor targets were selected and over 3,000 antibodies of mouse and camelid origin were generated against these targets.

Antibody generation can be done conventionally, using the recombinant target protein produced using NRC's high efficiency cell expression platforms in CHO or HEK293 cells or by direct immunization with plasmid DNA constructs. Clone selection is carried out by ELISA and typically 50 antibodies/target are identified for further characterization.

Antibody characterization and validation- The affinities of the antibodies are determined by SPR biosensor analysis. Epitope mapping can be carried out so that representative antibodies can be selected for further validation in appropriate cell-based assays (many of which are established at NRC) and animal models.

Therapeutic antibody Optimization, Bioprocessing and Biomanufacturing- Therapeutic antibodies selected for development can be further optimized using antibody engineering technologies to humanize them and/or modify their glycosylation patterns to improve their effector function, pharmacokinetics, solubility and stability as well as reduce their immunogenicity. The NRC platform for large scale protein production has the capacity to manufacture up to 500 g of commercial grade antibody in a cGMP certified CHO cell line which is ready for transfer to CMOs .

Citation Format: Maria L. Jaramillo, Anne Marcil, Yves Durocher, Renald Gilbert, Alaka Mullick, John Kelly, Maureen O'Connor-McCourt, Bernard Massie. Integrated therapeutic antibody development at the National Research Council of Canada. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 659. doi:10.1158/1538-7445.AM2014-659
Abstract 660: Engineering strategies to control stoichiometry, site of conjugation, serum stability and effector functions of antibody-drug conjugates
Authors: Dimasi Nazzareno , Fleming Ryan
Published: May 2015

A promising approach to enhance the antitumor activity of antibodies and reduce the systemic toxicity of small molecule anti-cancer drugs is to covalently attach the small molecule to the antibody thus forming antibody drug conjugates (ADCs). Conventionally, ADCs are produced by chemical conjugation of drugs to antibodies through either lysine side chain amines or native cysteine sulfhydryl groups generated by reduction of interchain disulfide bonds. Both of these methods yield heterogeneous conjugates with complex biophysical properties. To limit these liabilities, we have designed, characterized, and validated antibody variants, which allow precise control of the site of conjugation and the drug load; such as two and/or four drugs per antibody can be site-specific conjugated. These engineered antibody variants can be efficiently and site-specifically conjugated with drug payloads at milligram and gram scales to yield homogenous ADC products. Engineered antibody variants described here retain antigen-binding affinities, FcRn binding, and have biochemical, biophysical and manufacturability properties similar to their non-engineered parent antibodies. Upon conjugation to highly cytotoxic drugs with anti-mitotic and/or DNA alkylating activities, the antibody variants have potent in vitro and in vivo anti-tumor activity. Furthermore, we describe ADCs vehicles that provide efficient scaffolds for site-specific conjugation of drugs while offering the opportunity to minimize off-target toxicities often observed with ADCs as a result of their binding to cells expressing Fc-gamma receptors. Finally, we show the impact of the identified sites for drug conjugation on the in vivo pharmacokinetic and safety profile of ADCs in rats. Our strategies for engineering ADCs with defined sites for conjugation, controlled drug loads, potentially decreased off-target toxicity, and optimal serum stability are broadly applicable to any full-length IgG or Fc-containing therapeutics as they involve the Fc constant domain of the antibody.

Citation Format: Nazzareno Dimasi, Ryan Fleming. Engineering strategies to control stoichiometry, site of conjugation, serum stability and effector functions of antibody-drug conjugates. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 660. doi:10.1158/1538-7445.AM2014-660
Abstract 662: Generation and characterization of antibodies specific for IL1RAP antigen to target quiescent and proliferating AML leukemic stem cells
Authors: Jiang Ping , Roy Sharmili , Huang Jen , Dao-Pick Trang , Lu Jennifer , Mishra Madhavi , Lin Jeffrey , Hsi Eric D. , Tressler Robert J. , Karsunky Holger
Published: May 2015

We have isolated and characterized blast cells and leukemic stem cells (LSCs) enriched from AML patients and identified cell surface targets selectively expressed on AML blast cells and LSCs and not their normal hematopoietic stem cell counterparts. A series of potent, specific, monoclonal antibodies (mAbs) have been generated against one of these AML LSC targets, IL1RAP. IL1RAP mAb clones were characterized for patient binding properties and also evaluated in in vitro cell based assays and in in vivo tumor xenograft models. These monoclonal antibodies demonstrated promising binding profiles in primary AML patients’ samples and robust anti-tumor activity in in vitro cell based assays and in vivo tumor xenograft models. Specifically, select clones have a high degree of specific binding to AML primary patient samples (>90%), and specifically kill primary patient AML cells in complement-dependent cytotoxicity assays. These IL1RAP clones are being evaluated for inhibition of clonogenic potential of AML patient samples in vitro in colony forming assays and against normal clonogenic hematopoietic bone marrow derived stem cells. Select IL1RAP mAb clones evaluated in orthotopic AML tumor xenograft models have shown potent inhibition of tumor engraftment as well as significant tumor growth inhibition effects ranging from 80-90%. Because a key driver of LSC resistance is their quiescence, we also evaluated the antitumor effect of IL1RAP mAbs on quiescent AML cells in vitro. In summary, the patient binding profiles and in vitro and in vivo efficacy against AML cells indicates that targeting IL1RAP is a promising therapeutic approach for the treatment of AML and has the potential for effective eradication of AML LSCs and long term remission in the clinic.

Citation Format: Ping Jiang, Sharmili Roy, Jen Huang, Trang Dao-Pick, Jennifer Lu, Madhavi Mishra, Jeffrey Lin, Eric D. Hsi, Robert J. Tressler, Holger Karsunky. Generation and characterization of antibodies specific for IL1RAP antigen to target quiescent and proliferating AML leukemic stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 662. doi:10.1158/1538-7445.AM2014-662
Abstract 663: Development of a cancer-specific monoclonal antibody LpMab-2 specific for cancer-type podoplanin
Authors: Kato Yukinari , Kaneko Mika K.
Published: May 2015

Podoplanin is a platelet aggregation-inducing mucin-like sialoglycoprotein that is highly expressed in brain tumors, malignant mesotheliomas, testicular tumors, and lung, esophageal, or head and neck squamous cell carcinomas. Podoplanin has also been reported to be a marker to enrich tumor-initiating cells. We previously established a neutralizing monoclonal antibody (mAb), NZ-1, which inhibits the association between podoplanin and C-type lectin-like receptor-2 (CLEC-2) and blocks the podoplanin-induced platelet aggregation and tumor metastasis. A human chimeric mAb, NZ-8 possesses extremely high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and showed high anti-tumor activities in several xenograft models. However, those mAbs also react with normal cells including lung type I alveolar cells, renal podocytes, and lymphatic endothelial cells throughout the body. Recently, we established the platform to produce a cancer-specific mAb (CasMab) by combining glycobiology and cell engineering technologies. Herein, we report a novel cancer-specific anti-podoplanin mAb, LpMab-2. In Western-blot analyses, LpMab-2 did not react with sialic acid-deficient podoplanin and O-glycan deficient podoplanin, which were expressed in Lec2 and Lec8 cells, respectively. In enzyme-linked immunosorbent assay using several deletion mutants of podoplanin, the epitope of LpMab-2 was shown to be included in 55-88 amino acids of podoplanin, although almost all anti-podoplanin mAbs recognize platelet aggregation-stimulating (PLAG) domain near the N-terminus. Analyses of glycopeptides produced by Edman degradation revealed that O-glycans were attached to glycosylation sites at residues Thr65, Thr66, Thr70, Ser71, Ser74, and Thr76 around LpMab-2 epitope. LpMab-2 did not react with podoplanin-negative cell lines, indicating that not only glycans but also amino acids of podoplanin are necessary for LpMab-2 reaction. In flow cytometric and immunohistochemical analyses, LpMab-2 reacted with tumor cells, but not with lung type I alveolar cells and lymphatic endothelial cells, suggesting that LpMab-2 recognizes the cancer-type conformation of podoplanin. Furthermore, human chimeric LpMab-2 also showed cancer-specific reaction against podoplanin. Taken together, a cancer-specific mAb LpMab-2 should be useful for molecular targeting therapy against podoplanin without side effects such as respiratory or renal failures and lymphatic edema. The CasMab-producing platform could lead to establishment of the side effect-reducing mAbs.

Note: This abstract was not presented at the meeting.

Citation Format: Yukinari Kato, Mika K. Kaneko. Development of a cancer-specific monoclonal antibody LpMab-2 specific for cancer-type podoplanin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 663. doi:10.1158/1538-7445.AM2014-663
Abstract 665: Therapeutic targeting of breast cancer stem cells using PG-101 improves breast cancer survival
Authors: Wadehra Madhuri , Kiyohara Meagan
Published: May 2015

Since the original description of CSCs in solid tumors in 2003, emerging evidence suggests the presence of multiple breast CSC states (CD44+CD24-EpCAM+, ALDH+) that might be responsible for distinct processes in breast cancer progression such as metastasis, invasion, and proliferation. It has been hypothesized that CSCs contribute to the cellular heterogeneity within cancer, the metastatic spread, and eventual recurrence. As chemotherapy and radiation are relatively ineffective at targeting CSCs, therapeutic agents that target the relatively rare CSC population are highly needed.

To this end, we define another marker epithelial membrane protein-2 (EMP2) as a novel target for this population of cells. EMP2 is an oncogenic protein whose expression has been shown to correlate with tumor progression and survival in a number of human cancers including breast, ovarian, and endometrial tumors. Clinical data also revealed higher levels of EMP2 in metastatic lesions compared to primary tumors and high expression in triple negative breast cancer. In this study, we show new evidence that EMP2 is highly expressed in CSCs and regulates their proposed functions, namely mammosphere formation and invasion. Moreover, in vivo modulation of EMP2 expression in both triple negative and HER2 positive tumors show that EMP2 promotes ALDH1 expression as well as increases tumor load.

We have recently developed a novel IgG1 monoclonal antibody to EMP2 termed PG-101, and we have shown that this therapy reduces primary. We now have evidence that PG-101 treatment in the primary tumor is sufficient to reduce secondary tumor formation in triple negative breast cancer cells on the order of 1:20,000 compared to the control treated mice. To extend on the benefits of PG-101, we have combined it with docetaxel in an effort to mimic the clinical setting given that the vast majority of patients treated in the neoadjuvant setting have a taxane as part of the regimen. Data using triple negative xenografts show that PG-101 in combination with docetaxel can cure up to 80% of animals treated. This effect is lasting as these mice have shown no rebound in tumor load more than 2 months following the last treatment. Collectively, these results show that PG-101 therapy has the potential to revolutionize the outcomes for women with breast cancer.

Citation Format: Madhuri Wadehra, Meagan Kiyohara. Therapeutic targeting of breast cancer stem cells using PG-101 improves breast cancer survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 665. doi:10.1158/1538-7445.AM2014-665
Abstract 674: ODM-201 - New generation androgen receptor inhibitor targeting resistance mechanisms to androgen signalling-directed prostate cancer therapies
Authors: Moilanen Anu-Maarit , Riikonen Reetta , Kallio Pekka J.
Published: May 2015

Androgen receptor (AR) mutations have been described to emerge in response to prostate cancer treatment with first generation AR antagonists, resulting in broadened ligand specificity and AR antagonist-to-agonist switch. Second-generation AR antagonists enzalutamide and ARN-509 function as antagonists in the presence of AR overexpression. They express high clinical efficacy in castration resistant prostate cancer (CRPC) but eventually resistance will emerge.

Recently, a F876L missense mutation in the ligand-binding domain of AR has been described that confers resistance to enzalutamide and ARN-509 in preclinical models of prostate cancer. AR F876L mutation has also been identified in plasma DNA from ARN-509-treated CRPC patients with rising PSA.

ODM-201 is a new generation AR inhibitor with high anticancer activity in patients with CRPC and has unique properties compared to first and second generation antiandrogens. ODM-201 and its major metabolite bind to wt AR with excellent affinity (Ki 8 and 9 nM, respectively) and selectivity. In functional cell-based assays, ODM-201 functions as antagonist in highly AR overexpressing cells and does not induce AR T877A or W741L transcriptional activity. In contrast to enzalutamide and ARN-509, ODM-201 and its metabolite have been found to potently inhibit the activity of AR F876L while enzalutamide and ARN-509 were agonistic.

ODM-201 is a promising new generation AR inhibitor for the treatment of advanced prostate cancer with superior antiandrogenic activity in preclinical models of AR function.

Citation Format: Anu-Maarit Moilanen, Reetta Riikonen, Pekka J. Kallio. ODM-201 - New generation androgen receptor inhibitor targeting resistance mechanisms to androgen signalling-directed prostate cancer therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 674. doi:10.1158/1538-7445.AM2014-674
Abstract 68: Tumor operational signaling pathways in an Nf1 mutant mouse model system
Authors: Braunstein Steve , Mroue Rana , Huang Brian , Bandyopadhyay Sourav , Nakamura Jean
Published: May 2015

Purpose/Objectives: NF1 is recognized as a tumor suppressor, via its role as a Ras GTPase activating protein. However, the precise mechanisms critical to tumor development in NF1 patients remain unclear. NF1 heterozygosity confers susceptibility to tumorigenesis, promoted by genotoxins such as radiation. We mutagenized Nf1 heterozygous mice using radiation to generate a diverse panel of Nf1 null tumors. Using these novel reagents we interrogated signaling pathways essential for tumor development and propagation towards identification of potential targets for therapeutic approaches.

Materials/Methodology: Cell lines were derived from solid tumors developing after focal irradiation of Nf1 heterozygous mice. Radiation-induced tumors included mammary carcinomas, squamous carcinomas, and sarcomas. We employed a high-throughput toxicity screen of 94 conventional and targeted FDA-approved chemotherapeutics directed across diverse core cellular signaling pathways. Drug sensitivity profiles were determined by quantifying cell numbers after a 72 hour drug exposure, and tumor cell lines, non-transformed wild-type and Nf1 null MEFs, and mutant Ras-transformed cell lines (added as controls) were compared. Validation of identified drug sensitivities was performed by enzyme activity assays, phosphoprotein analysis, and soft-agar colony formation.

Results: Nf1 mutant tumors failed to demonstrate a common drug sensitivity signature, but instead revealed a heterogeneous pattern of differential sensitivities, with subgroups clustering independent of tumor histology. However, radiation-induced Nf1 null tumor lines exhibited increased sensitivity to inhibition of the Ras-Erk and PI3K-mTOR axis as compared to non-transformed controls. Enhanced sensitivity to agents targeting cell cycle and survival pathways was also observed selectively within the Nf1 null tumor cell lines.

Conclusions: These studies indicate that Nf1 null tumors, even those of the same histology, do not intrinsically share a signature sensitivity profile to pharmacologic inhibition. This suggests that therapeutic targeting of signaling pathways in Nf1 mutant tumors is not likely to be defined strictly by Nf1 loss alone. Notably, these Nf1 null tumors evidence varied operational dependence upon PI3K-mTOR and Raf-MEK activity. Current efforts are directed towards further resolving essential mediators, with the goal of more precisely defining signaling pathways which drive progression of the malignant state.

Citation Format: Steve Braunstein, Rana Mroue, Brian Huang, Sourav Bandyopadhyay, Jean Nakamura. Tumor operational signaling pathways in an Nf1 mutant mouse model system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 68. doi:10.1158/1538-7445.AM2014-68
Abstract 683: Sensitizing tumors to pro-oxidant therapy by inhibiting gamma-glutamyl transpeptidase
Authors: Hanigan Marie H. , Wakeham Nancy , Wickham Stephanie , Terzyan Simon S.
Published: May 2015

We have reported that 73% of ovarian tumors express the cell surface enzyme gamma-glutamyl transpeptidase (GGT). Clinical studies have shown a poorer prognosis for ovarian tumors that are GGT-positive compared to GGT-negative tumors. GGT-positive tumors are able to use the glutathione in the interstitial fluid as an additional source of cysteine, the limiting component of intracellular glutathione synthesis in tumors. The concentration of cysteine/cystine in standard tissue culture media is more than 3 times the concentration in interstitial fluid. In this study we show that in studies of cultured cells, the high concentration of cysteine in tissue culture media obscured the role of GGT expression in drug resistance. However, culturing human ovarian tumor cells in tissue culture media containing physiological concentrations of cystine and glutathione demonstrated that the supply of cysteine/cystine regulates intracellular glutathione concentrations. In these studies, inhibition of GGT blocked the use of extracellular glutathione as a source of cysteine and sensitized the tumors to cisplatin, carboplatin and melphalan. Inhibiting GGT in patients would also inhibit GGT in the kidney resulting in the excretion of glutathione in the urine and depletion of cysteine from serum and interstitial fluid. As a result, inhibition of GGT in patients would sensitize both GGT-positive and GGT-negative tumors to pro-oxidant chemotherapy. Clinical application of this therapeutic strategy is limited by the fact that most inhibitors of GGT are glutamate analogs and too toxic for use in humans. We discovered a non-glutamate analog that is an uncompetitive inhibitor of GGT, and are developing more potent, non-toxic GGT inhibitors. We recently published the crystal structure of human GGT. We have now also solved the structure of the substrate-bound enzyme, the form of the enzyme to which our uncompetitive inhibitors bind. Using structure-based drug design and our physiologically relevant tissue culture system we are designing new GGT inhibitors and evaluating them for their ability to sensitize tumors to chemotherapy regimens used in the clinic. Supported by an Institutional Development Award (IDeA) from the National Institutes of Health under grant P20GM103640.

Citation Format: Marie H. Hanigan, Nancy Wakeham, Stephanie Wickham, Simon S. Terzyan. Sensitizing tumors to pro-oxidant therapy by inhibiting gamma-glutamyl transpeptidase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 683. doi:10.1158/1538-7445.AM2014-683
Abstract 685: Antagonism of sorafenib and regorafenib-mediated HCC growth inhibition by platelet factors
Authors: Carr Brian I. , D'Alessandro Rosalba , Refolo Maria Grazie , Lippolis Catia , Messa Caterina , Cavallini Aldo
Published: May 2015

Background. Circulating blood platelet numbers are frequently altered in hepatocellular carcinoma (HCC) patients. Thrombocytopenia is associated with smaller tumors and thrombocytosis with larger tumors (Oncology 2013;85:153-9). Recently, platelet extracts have been shown to enhance HCC cell growth and have anti-apoptotic effects.

Methods. Human HCC cell lines were used to evaluate the actions of time-expired mixed human platelet lysates on the inhibition of HCC growth by MTT assay, as well as on migration and Matrigel invasion mediated by Sorafenib or Regorafenib in vitro and Western blots were performed for cell signaling and apoptosis.

Results. Sorafenib or Regorafenib-mediated inhibition of cell growth, migration and invasion was antagonized by platelet extracts. The multi-kinase inhibitor-mediated induction of apoptosis and decrease in phospho-ERK levels were both blocked by the platelet extracts, which also increased anti-apoptotic phospho-STAT, Bax and Bcl-XL levels.

Conclusions. In addition to their effects on coagulation and wound healing, platelet factors can antagonize growth inhibition and apoptosis induction by multi-kinase inhibitors Sorafenib or Regorafenib. This could be due to several growth factors or inflammatory cytokines that are produced by platelets. Targeting platelets might be a novel approach to increasing multi-kinase inhibitor actions.

Citation Format: Brian I. Carr, Rosalba D'Alessandro, Maria Grazie Refolo, Catia Lippolis, Caterina Messa, Aldo Cavallini. Antagonism of sorafenib and regorafenib-mediated HCC growth inhibition by platelet factors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 685. doi:10.1158/1538-7445.AM2014-685
Abstract 695: p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling
Authors: Axelrod Mark J. , Mendez Rolando E. , Gioeli Daniel G. , Jameson Mark J. , Weber Michael J.
Published: May 2015

Targeted cancer therapies often induce compensatory adaptive responses that blunt drug effectiveness, resulting in therapeutic resistance - intrinsic or acquired. Adaptive responses are characteristic of the complex nature of the cancer cell signaling network. Within this network, nodes of convergence between signaling modules are critical mediators of adaptive responses to targeted drugs. The critical nodes in an oncogenically-activated signaling network represent potential therapeutic vulnerabilities because their inhibition could result in collapse of the network and hence enhanced cytotoxicity. We have previously used high-throughput combinatorial drug screening to empirically identify adaptive survival responses to targeted therapies. We found that HER-family and PI3K represented compensatory signaling pathways, and combination therapy caused synergistic cytotoxicity in cases where inhibition of neither target was effective as a monotherapy. RPPA analysis identified ribosomal protein S6 as being synergistically down-regulated upon HER-family and PI3K co-inhibition. Expression of a constitutively active construct of the upstream activator of S6, p70S6K, was protective against apoptosis induced by combined HER-family and PI3K inhibition. Direct inhibition of p70S6K using small molecule inhibitors phenocopied the growth inhibition and apoptosis caused by HER-family and PI3K inhibition. These data implicate p70S6K as a critical node and druggable target in the HER-family / PI3K signaling network. The ability of direct inhibitors of p70S6K to phenocopy the co-inhibition of upstream signaling indicates that identification and targeting of critical nodes may be a way to overcome adaptive resistance to targeted therapies.

Citation Format: Mark J. Axelrod, Rolando E. Mendez, Daniel G. Gioeli, Mark J. Jameson, Michael J. Weber. p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 695. doi:10.1158/1538-7445.AM2014-695
Abstract 696: ASS1, ANXA4 and MVP underlie acquired multicellular resistance of mesothelioma 3D cell cultures
Authors: Barbone Dario , Dam Loes van , Zhang Shu-Dong , Puthen Jithesh , Fennell Dean , Broaddus V. Courtney
Published: May 2015

Cancer cells are usually studied in flat 2D monolayers however, solid tumors, such as malignant mesothelioma, grow as 3D masses. When tumor cells are grown as 3D structures, they acquire multi-cellular resistance to apoptosis, resembling the stubborn chemoresistance seen in patients. In this study, we have identified a gene expression signature that is specific for 3D spheroids, possibly underlying multicellular resistance. By comparing this signature with the data from two sets of publicly-available gene expression datasets of patients’ specimens we have identified three genes in common, genes that may play a role in multicellular resistance: ASS1, ANXA4 and MVP. Moreover, we have studied the levels of the proteins encoded by the three genes and have begun investigating how their ablation by RNAi influences the response to chemotherapy. Furthemore, by Connectivity Analysis, we have found two safe and patent-free candidate drugs that can reduce the expression levels of the three genes and undermine the acquired chemoresistance of spheroids. Such drugs would have potential as adjuncts to chemotherapy. These studies will not only clarify the role of ASS1, ANXA4 and MVP in mesothelioma and provide therapeutic insights but also advance the use of spheroids as a more realistic model for studying tumor biology.

Citation Format: Dario Barbone, Loes van Dam, Shu-Dong Zhang, Jithesh Puthen, Dean Fennell, V. Courtney Broaddus. ASS1, ANXA4 and MVP underlie acquired multicellular resistance of mesothelioma 3D cell cultures. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 696. doi:10.1158/1538-7445.AM2014-696
Abstract 697: Development of spheroid based high-throughput screening of cell-cell adhesion inhibitors to reverse acquired multicellular resistance
Authors: Arai Kazuya , Itho Manabu , Mizuno Atsushi , Miura Hiromi
Published: May 2015

Background: Solid tumors are more resistant to chemo- and radio-therapy than in vitro cancer cells under conventional monolayer culture condition. This phenomenon can be explained by “multicellular resistance” (MCR). It is considered that one of approach for keeping out cancer cells from acquired MCR is inhibition of cell-cell/cell-matrix adherence. Several therapeutic approaches increasing effect of chemo- and radio-therapy by preventing MCR are proposed. However, reports of the approaches are still few. Therefore, we tried to develop a spheroid-based high-throughput screening method of cell-cell adherence inhibitor, for discovery of drug capable of reversing the acquired MCR.

Method: For spheroid formation, A549 cells were cultured under three-dimensional condition using NanoCulture® Plate, which is compatible with imaging and HTS systems. After 3 days culture, A549 spheroids were treated with library compounds for 4 days. To evaluate a degree of cell-cell adherence of the spheroids, we used Hypoxia Probe, which is a phosphorescence substrate for visualizing of intra-spheroid hypoxicity. And the signals were detected using Celigo® imaging cytometer.

Results: In NanoCulture® Plate, A549 cells formed a number of spheroids. Intra-spheroid hypoxicity was correlated with degree of cell-cell adherence. After screening of inhibitor of cell-cell adherence without cytotoxity, several candidates were evaluated the ability as chemosensitizing agent. Combination treatment with the hit compound and anticancer drug was more effective than each drug alone.

Conclusion: We developed a novel high throughput screening method of cell-cell adherence inhibitors. And we acquired several hit compounds capable of reversing the acquired MCR.

Note: This abstract was not presented at the meeting.

Citation Format: Kazuya Arai, Manabu Itho, Atsushi Mizuno, Hiromi Miura. Development of spheroid based high-throughput screening of cell-cell adhesion inhibitors to reverse acquired multicellular resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 697. doi:10.1158/1538-7445.AM2014-697
Abstract 7: Assessment of FSH receptor expression in the major molecular subtypes of breast cancer
Authors: Planeix François , Siraj Mohammad-Ahsan , Bidard François-Clément , Pierga Jean-Yves , Sastre Xavier , Antoine Martine , Ghinea Nicolae
Published: May 2015

Recent data from our laboratory show that the follicle-stimulating hormone receptor (FSHR) is expressed on the endothelial surface of tumor peripheral blood vessels in different types of tumors including breast cancer. The main goal of the present study was to determine differences in the endothelial FSHR expression level among the breast cancer types, and within the same tumor type, among tumor stages. We have also compared the FSHR expression with the receptor status of three most important markers for breast cancer diagnosis and therapy (estrogen receptor, progesterone receptor, and HER-2/neu). By using a highly specific monoclonal antibody raised against the human FSHR, we determined the density of FSHR-positive vessels in a band with a width of 10 mm (centered on the demarcation line between the tumor and the normal breast tissue) on the tissue sections by standard peroxidase-immunohistochemistry and immunofluorescent confocal microscopy. We examined 35 patients of Luminal A, 13 patients of Luminal B, 19 patients of HER-2 positive, and 16 patients of Triple negative/basal breast cancer. All four groups expressed almost similar densities of FSHR stained blood vessels with 36.7 ± 4.0/mm2 in Luminal A, 31.4 ± 5.2/mm2 in Luminal B, 39.6 ± 6.0/mm2 in HER-2 positive tumors, and 38.4 ± 8.0/mm2 in triple negative/basal group. A positive correlation has been observed between the percentage of FSHR-positive vessels and total blood vessels in the tumor periphery (r = 0.551; p = 7e-08). We have also noticed a tendency (not reaching statistical significance) towards an overall decrease in the extent of the vascular bed in elder patients as compared to younger patients. No correlation was found between the percentage of FSHR-positive vessels and the expression of receptors for estrogen, progesterone, and HER-2/neu.

In conclusion, our data show that: (i) FSHR is robustly expressed in breast cancer, and (ii) the main types of breast cancers and stages do not differ significantly in terms of vascular FSHR expression. These facts strongly increase FSHR potential relevance as a clinical target for breast cancer imaging and for therapy, especially for triple negative/basal breast cancers, a group with only chemotherapy options.

Citation Format: François Planeix, Mohammad-Ahsan Siraj, François-Clément Bidard, Jean-Yves Pierga, Xavier Sastre, Martine Antoine, Nicolae Ghinea. Assessment of FSH receptor expression in the major molecular subtypes of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 7. doi:10.1158/1538-7445.AM2014-7