Potent Tumor Inhibition Research Articles

Characterization of a humanized PD-L1 blocking antibody with pH-dependent antigen binding in a humanized syngenic tumor model.

81 Background: Checkpoint inhibition is a promising approach for cancer treatment with durable responses and survival benefits. Currently, immune-checkpoint inhibitors are delivered at maximally tolerated doses, resulting in significant toxicities and cost burden. There is significant need to develop more efficacious and safer immune checkpoint inhibitor. Towards this goal, we have developed a next-generation humanized PD-L1 antibody with pH-dependent antigen binding and evaluated its biological activities in vitro and in vivo. Methods: Hybridoma approach was employed to generate anti-PD-L1 antibodies. A cell-based receptor-ligand blocking assay was employed to identify neutralizing antibodies. Antibodies with pH-dependent antigen binding property were identified and characterized further with those without using PD-L1/PD-1 binding assay, mixed lymphocyte reaction assay, and in vivo tumor growth inhibition assay in syngeneic MC38 tumor model expressing human PD-L1 protein. Results: A panel of neutralizing antibodies blocking PD-L1/PD-L1 binding was isolated. Among them we identified antibodies with or without pH-dependent binding property. While the in vitro activities of these antibodies are similar, their tumor growth inhibition activities differ significantly. The in vivo activity of 23F11, an antibody with ph-dependent binding, is maximized at 1mg/kg in MC38/hPD-L1 model. At 1 mg/kg, 23F11 induced complete tumor regression in 7/12 of the mice treated, more potent than antibodies without pH-dependent binding. Imaging study revealed that significantly higher drug concentration in the tumor for 23F11 and correlated with extended period of target occupancy. Furthermore 23F11 treatment is correlated with sustained up-regulation of plasma mIL-18, a potential pharmacodynamics response marker. Conclusions: We have identified antibodies with pH-dependent PD-L1 binding property. These antibodies can maintain high antibody level in PD-L1 expressing tumor and have potent tumor inhibition activity at low dose. Current work will evaluate whether humanized 23F11 can be at least as efficacious as benchmark antibodies yet with improved safety profile in patients.

AvidinOX-anchored biotinylated trastuzumab and pertuzumab induce down-modulation of ErbB2 and tumor cell death at concentrations order of magnitude lower than not-anchored antibodies.

The oxidized version of Avidin, known as AvidinOX, was previously shown to link to tissue proteins upon injection or nebulization, thus becoming a stable receptor for biotinylated therapeutics. AvidinOX is currently under clinical investigation to target radioactive biotin to inoperable tumor lesions (ClinicalTrials.gov NCT02053324). Presently, we show that the anti-ErbB2 monoclonal antibodies Trastuzumab and Pertuzumab can be chemically biotinylated while maintaining their biochemical and biological properties. By using several and diverse experimental conditions, we show that when AvidinOX is conjugated to tumor cells, low antibody concentrations of biotinylated Trastuzumab (bTrast) or Pertuzumab (bPert) prevent internalization of ErbB2, induce endoplasmic reticulum stress, cell cycle arrest and apoptosis leading to inhibition of proliferation and ErbB2 signaling. Moreover, we found that the treatment is able to induce down-modulation of ErbB2 thus bypassing the known resistance of this receptor to degradation. Interestingly, we show that AvidinOX anchorage is a way to counteract agonistic activities of Trastuzumab and Pertuzumab. Present data are in agreement with previous observations from our group indicating that the engagement of the Epidermal Growth Factor Receptor (EGFR) by AvidinOX-bound biotinylated Cetuximab or Panitumumab, leads to potent tumor inhibition both in vitro and in animal models. All results taken together encourage further investigation of AvidinOX-based treatments with biotinylated antibodies directed to the members of the EGFR family.

Dual SYK/JAK inhibition overcomes ibrutinib resistance in chronic lymphocytic leukemia: Cerdulatinib, but not ibrutinib, induces apoptosis of tumor cells protected by the microenvironment.

Ibrutinib (BTK inhibitor) has generated remarkable responses in CLL. However, the drug, to a large extent, does not cause cell death directly and does not eradicate CLL malignant clones. Inability to eradicate CLL has fostered resistance generation. Once patients become resistant, they do poorly with a median survival of 3-4 months. Novel therapeutic strategies are needed to prevent resistance, improve treatment outcome and ultimately cure the disease. Herein, we explore dual targeting of the BCR and JAK-STAT pathways with a novel single agent, cerdulatinib, which selectively inhibits both SYK (a BCR component) and JAK kinases. We demonstrated that cerdulatinib delivered potent tumor inhibition in 60 primary CLL patient samples, especially in those with poor prognostic indicators. Importantly, cerdulatinib, but not ibrutinib, is able to overcome the support of microenvironment and induces CLL cell death at clinically achievable concentrations. Notably, cerdulatinib blocked proliferation of ibrutinib-resistant primary CLL cells and of BTKC481S-transfected/ibrutinib-resistant lymphoma cells. These anti-tumor effects are well correlated with the inhibition of BCR and JAK-STAT signaling and downstream inhibition of the functions of AKT, ERK and NF?B. Collectively, our results show that simultaneous targeting of BCR and JAK-STAT pathways is a more effective strategy relative to single BTK inhibition.

Anti-Angiogenic Effects of Some (5-hydroxy-4-methyl-2-((1-methyl-1H-indol-3-yl)methyl)-Thieno[2,3-d] Pyrimidine Derivatives

Some previously reported pyrimidine candidates 1–7 were founded to have potent tumor inhibition activities on diffrrent models of human primary and secondary brain tumors and in the same time these derivatives were founded to have potent anti-angiogenic effects on Ehrlich Ascites Tumor [EAT] cells in-vivo. These pyridine derivatives were founded to be selectively inhibit the p110α isoform. These pyridine derivatives were potent inhibited the histone deactylase. Also, they are inhibited the Sphingosine kinase SK1 activities.

Dual SYK/JAK Inhibition Overcomes Ibrutinib Resistance in Chronic Lymphocytic Leukemia

Ibrutinib (a BTK inhibitor) has generated remarkable responses in CLL. However, the drug, to a large extent, does not cause cell death directly and does not eradicate CLL malignant clones. Inability to eradicate CLL has fostered resistance generation. Once patients become resistant, they do poorly with a median survival of 3-4 months. Novel therapeutic strategies are needed to prevent resistance, improve treatment outcome and ultimately cure the disease. Herein, we explore dual targeting of the BCR and JAK-STAT pathways with a novel single agent, cerdulatinib, which selectively inhibits both SYK (a BCR component) and JAK kinases. We demonstrated that cerdulatinib delivered potent tumor inhibition in 60 primary CLL patient samples, especially in those with poor prognostic indicators. Importantly, cerdulatinib, but not ibrutinib, is able to overcome the support of microenvironment and induces CLL cell death at clinically achievable concentrations. Further, cerdulatinib blocked proliferation of ibrutinib-sensitive and ibrutinib-resistant primary CLL cells and of BTKC481S-transfected cells. These anti-tumor effects are correlated with the inhibition of BCR and JAK-STAT signaling and downstream inhibition of the functions of AKT, ERK and NFκB. Collectively, our results show that simultaneous targeting of BCR and JAK-STAT pathways is a more effective strategy relative to single BTK inhibition. DisclosuresCoffey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding.

47 Gambogic acid delivery using lipid nanoparticles modified with cell-penetrating peptide

Objectives Gambogic acid (GA) is a novel tissue-specific proteasome inhibitor which can potentially be used to treat cancer with low toxicity. However, poor aqueous solubility (∼10 μg/mL) and low tumor cell-specific delivery have limited its clinical application. Clinical application of GA requires the development of delivery vehicles. Methods In this study, we developed a novel nanoparticle GA delivery system. The nanoparticles incorporate a cell-penetrating peptide conjugated to myristic acid (MA-R7W), a folate modified lipid (FA-PEG2000-DSPE), a pH-sensitive lipid (PEG1000-hyd-PE), eggPC and cholesterol. The lipids formed the nanoparticle shells, and GA was loaded into the lipid bilayer of the nanoparticles. PEG on the surface of the nanoparticles provides a long circulation time. Folate is incorporated to enable targeting of tumor cells with amplified folate receptor expression. PEG1000-hyd-PE can shield/unshield R7W on the nanoparticle surface according to the pH difference between normal tissues and cancer. Results In vitro, FA/MA-R7W nanoparticles improved cellular uptake 2.5-fold compared to GA liposomes (without FA-PEG2000-DSPE, AA-R8 and PEG1000-hyd-PE) at pH 5. In vivo, GA encapsulated in FA/MA-R7W nanoparticles induced potent tumor inhibition (62.6%), showed lengthy circulation (Figure 1) and tumor cell targeting. Conclusions In conclusion, FA/MA-R7W nanoparticles are promising vehicles for GA delivery and warrant further investigation. Acknowledgments This research was financially supported by Jilin Province Science and Technology Development Program (Grant No. 20140311072YY) and Jilin Province Science and Technology Development Program (Grant No.20150520141JH).

Delivery of siRNA Using Lipid Nanoparticles Modified with Cell Penetrating Peptide.

Clinical development of siRNA has been hindered by the lack of an effective delivery system. Here, we report the construction of a novel siRNA delivery system, sTOLP, which is based on cell penetrating peptide oleoyl-octaarginine (OA-R8) modified multifunctional lipid nanoparticles. sTOLP nanoparticles are composed of a protamine complexed siRNA core, OA-R8, cationic and PEGylated lipids, and transferrin as a targeting ligand. sTOLP formulation was optimized and characterized in vitro and showed excellent gene silencing activity. In vivo, siRNA encapsulated in sTOLP exhibited potent tumor inhibition (61.7%) and was preferentially taken up by hepatocytes and tumor cells in HepG2-bearing nude mice without inducing immunogenicity or hepatic or renal toxicity. Furthermore, sTOLP-loaded siRNA had stability in circulation greater than that of free siRNA. These data demonstrated potential utility of sTOLP-mediated siRNA delivery in cancer therapy.

Synthesis and biological evaluation of Hederacolchiside A1 derivatives as anticancer agents

Modification of Hederacolchiside A1 (HA1) on 28-COOH gave a series of novel triterpenoid saponin compounds containing ester or amide group. Comparing with natural product HA1, several derivatives showed decreased toxicity in the mice acute toxicity trial and increased the anticancer activity in vitro. Especially compound 1 exhibited the strongest antiproliferative activities against human cancer cell lines tested (IC50=1.1–4.6μM) and potent tumor inhibition rate in vivo (46.8%).

Abstract B19: Vascular normalization suppresses tumor progression and potentiates docetaxel chemotherapy in prostate cancer

Abstract Background: Tumor requires excessive oxygen and nutrients due to their rapid growth. Tumor is generally vascular but the vasculature is not physiologic but leaky, dilated, and tortuous. The inefficiency of tumor vessels induces hypoxic stress that makes the tumor more aggressive. Here, we investigated the role of normalizing tumor vasculature on tumor progression and treatment efficacy of conventional chemotherapy in prostate cancer. Methods: Human vascular endothelial cell (HUVEC) was used to evaluate the effects of SAC-1004 on endothelial cells. Immunofluorescent confocal imaging with immunocytochemistry was performed for E-cadherin expression in HUVECs. MTS assay was performed to assess cell proliferation of HUVECs and PC-3 cells after SAC-1004 treatment. Immunohistochemical staining with anti-CD31 and anti-SMA antibodies was performed to assess vasculature in vivo. PC-3 subcutaneous xenografts were developed to evaluate the effects of SAC-1004 on tumor vasculature and tumor growth in prostate cancer. Magnetic resonance images were taken to assess vascular perfusion in xenografts. SAC-1004 was administrated via tail vein once a day for 7 days. Docetaxel was administrated intravenously once a week. Results: SAC-1004 restored junction protein E-cadherin in vitro in endothelial cells and significantly reduced VEGF-induced retinal vascular leakage. SAC-1004 promoted proliferation of HUVECs in vitro in a dose-dependent manner. However, proliferation of cancer cells was not significantly affected by SAC-1004 treatment. Reduced vascular leakiness and improved hypoxia were found in PC-3 xenografts treated with SAC-1004 treatment and magnetic resonance imaging after SAC-1004 treatment for 7 days confirmed improved perfusion in the PC-3 xenografts treated with SAC-1004 compared with control. Interestingly, SAC-1004 treatment suppressed tumor growth in PC-3 xenografts but SAC-1004 followed by intravenous docetaxel treatment showed the most potent tumor suppression compared with control in PC-3 xenografts. Conclusions: SAC-1004 improves hypoxic tumor microenvironment and restores abnormal leak vasculature of tumor by restoring endothelial cell junction in prostate cancer. Vascular normalization induced tumor regression and docetaxel combination following vascular normalization sensitized prostate cancer to docetaxel treatment. Citation Format: You Hyun Kang, Hyun A Jin, Jun Hyeok Heo, Ki Hong Kim, Kyung Seok Han, Sung Joon Hong. Vascular normalization suppresses tumor progression and potentiates docetaxel chemotherapy in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B19.

Self-sufficing H2O2-responsive nanocarriers through tumor-specific H2O2 production for synergistic oxidation-chemotherapy

One of distinct features in tumor tissues is the elevated concentration of reactive oxygen species (ROS) during tumor immortality, proliferation and metastasis. However, ROS-responsive materials are rarely utilized in the field of in vivo tumoral ROS-responsive applications due to the fact that the intrinsic ROS level in the tumors could not escalate to an adequate level that the developed materials can possibly respond. Herein, palmitoyl ascorbate (PA) as a prooxidant for hydrogen peroxide (H2O2) production in tumor tissue is strategically compiled into a H2O2-responsive camptothecin (CPT) polymer prodrug micelle, which endowed the nanocarriers with self-sufficing H2O2 stimuli in tumor tissues. Molecular oncology manifests the hallmarks of tumoral physiology with deteriorating propensity in eliminating hazardous ROS. H2O2 production was demonstrated to specifically sustain in tumors, which not only induced tumor cell apoptosis by elevated oxidation stress but also served as autochthonous H2O2 resource to trigger CPT release for chemotherapy. Excess H2O2 and released CPT could penetrate into cells efficiently, which showed synergistic cytotoxicity toward cancer cells. Systemic therapeutic trial revealed potent tumor suppression of the proposed formulation via synergistic oxidation-chemotherapy. This report represents a novel nanomedicine platform combining up-regulation of tumoral H2O2 level and self-sufficing H2O2-responsive drug release to achieve novel synergistic oxidation-chemotherapy.

A novel PI3K axis selective molecule exhibits potent tumor inhibition in colorectal carcinogenesis.

Phosphatidylinositol-3-kinase (PI3K) pathway deregulation is responsible for initiation, chemo-resistance, and poor prognosis of colorectal cancer (CRC). Therefore, PI3K pathway inhibition can provide a plausible way of attaining CRC treatment. We report PI3K target specific synthesis and selection of a potent molecule, that is, 2,3-dihydro-2-(naphthalene-1-yl) quinazolin-4(1H)-one (DHNQ) from quinazolinone series based on the structural activity relationship after evaluation in diverse cancers. This molecule inhibited the PI3K enzyme activity and transcriptional as well as translational expression levels in colorectal cancer (CRC) models. This was associated with subsequent decrease in phosphorylation of its downstream effector proteins, that is, p-Akt(Ser-473) and p-mTORC1(Ser-2448) and decreased ERK signaling. Furthermore, DHNQ decreased expression of cyclins that caused G1 arrest and decreased Bcl-2/Bax ratio after mitochondrial membrane potential loss, reactive oxygen species generation, and an increase in cytosolic Ca2+ loads that is responsible for the decreased CRC cell proliferation and survival. These biochemical changes triggered apoptotic cell death with altered autophagic Beclin-1 and LC3β expression. It seemed that the PI3K-Akt signaling regulated apoptosis and autophagy through different mechanisms but mTORC1 mediated autophagy appeared not to be involved in the cell death induction by DHNQ. The molecule also showed significant anticancer efficacy in in vivo tumor models without any mortality indicating its non-toxic nature with possible clinical significance. Overall, the selective elucidation of DHNQ molecular mechanism will provide the possible strategies for the clinical development in CRC that may respond to this specific, potent and novel P13K inhibitor. © 2016 Wiley Periodicals, Inc.

Abstract B12: Endothelial stabilizer SAC-1004 normalizes tumor vasculature and potentiates docetaxel chemotherapy in prostate cancer

Abstract Background: Since tumor requires excessive oxygen and nutrients due to their rapid growth, tumor is generally vascular. However, tumor vasculature is not physiologic but leaky, dilated, and tortuous. The inefficiency of tumor vessels in terms of perfusion induces hypoxic stress that makes the tumor more aggressive. Here, we investigated the role of a novel endothelial stabilizer SAC-1004 on tumor vascular normalization and its effects on tumor growth in prostate cancer. Methods: Human vascular endothelial cell (HUVEC) was used to evaluate the effects of SAC-1004 on endothelial cells. Immunocytochemical staining and immunofluorescent confocal imaging was performed for E-cadherin expression in HUVECs. MTS assay was performed to assess cell proliferation of HUVECs and PC-3 cells after SAC-1004 treatment. Immunohistochemical staining with anti-CD31 and anti-SMA antibodies was performed to assess vasculature in vivo. PC-3 subcutaneous xenografts were developed to evaluate the effects of SAC-1004 on tumor vasculature and tumor growth in prostate cancer. Magnetic resonance images were taken to assess vascular perfusion in xenografts. SAC-1004 was administrated via tail vein once a day for 7 days. Docetaxel was administrated intravenously once a week. Results: SAC-1004 restored junction protein E-cadherin in vitro in endothelial cells and significantly reduced VEGF-induced retinal vascular leakage. SAC-1004 promoted proliferation of HUVECs in vitro in a dose-dependent manner. However, proliferation of cancer cells was not significantly affected by SAC-1004 treatment. Reduced vascular leakiness was found in PC-3 xenografts treated with SAC-1004 treatment and magnetic resonance imaging after SAC-1004 treatment for 7 days confirmed improved perfusion in the PC-3 xenografts treated with SAC-1004 compared with control. Interestingly, SAC-1004 treatment suppressed tumor growth in PC-3 xenografts but SAC-1004 followed by intravenous docetaxel treatment showed the most potent tumor suppression compared with control in PC-3 xenografts. Conclusions: SAC-1004 restored abnormal leak vasculature of tumor by restoring endothelial cell junction in prostate cancer. Vascular normalization by SAC-1004 induced tumor regression and combination with docetaxel following vascular normalization potentiated anti-tumor effects of docetaxel in prostate cancer. Citation Format: Yoo Hyun Kang, Jun Hyeok Heo, Hyun A Jin, Ki Chung Park, Sung Joon Hong, Kyung Seok Han. Endothelial stabilizer SAC-1004 normalizes tumor vasculature and potentiates docetaxel chemotherapy in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B12.

HOXB9 induction of mesenchymal-to-epithelial transition in gastric carcinoma is negatively regulated by its hexapeptide motif.

HOXB9, a transcription factor, plays an important role in development. While HOXB9 has been implicated in tumorigenesis and metastasis, its mechanisms are variable and its role in gastric carcinoma (GC) remains unclear. In the present study, we demonstrated that the expression of HOXB9 decreased in gastric carcinoma and was associated with malignancy and metastasis. Re-expression of HOXB9 in gastric cell lines resulted in the suppression of cell proliferation, migration, and invasion, which was accompanied by the induction of mesenchymal-to-epithelial transition (MET). Comparative sequence analysis and examination of a HOXB9 structural model indicated that three sites might possibly be involved in MET regulation. The in vitro study of HOXB9 mutants showed that these were unable to inhibit MET induction. However, when overexpressing a HOXB9 mutant lacking the hexapeptide motif, a more potent MET induction and tumor suppression was observed compared to that of the wild-type, indicating that the presence of the hexapeptide motif reduced HOXB9 MET induction and tumor suppression activity. Therefore, the results of the present study suggested that HOXB9 is a tumor suppressor in gastric carcinoma, and its activity was controlled by different regulatory mechanisms such as the hexapeptide motif as a "brake" in this case. The results of these regulatory effects could lead to either oncogenic or tumor suppressive roles of HOXB9, depending on the context of the particular type of cancer involved.

The MDM2 small-molecule inhibitor RG7388 leads to potent tumor inhibition in p53 wild-type neuroblastoma.

Neuroblastoma is an aggressive pediatric malignancy which is >98% p53 wild-type at diagnosis. As a primary repressor of p53 activity and part of a p53-activated negative feedback loop, targeting of mouse double minute 2 homolog (MDM2) is an attractive therapeutic approach to reactivation of p53. Since development of the first selective MDM2 inhibitor, Nutlin-3a, newer compounds have been developed for increased potency and improved bioavailability. Herein, we sought to determine the efficacy and specificity of a second-generation MDM2 inhibitor, RG7388, in neuroblastoma cell lines and xenografts and examine its effect on the p53-independent pathway of hypoxia-inducible factor-1 alpha (HIF-1α)/vascular endothelial growth factor (VEGF). Cell viability and apoptosis studies were performed on the neuroblastoma cell lines, NGP, SH-SY5Y, LAN-5, LAN-5 si-p53 (p53 silenced), and SK-N-AS (p53 null). RG7388 potently decreased cell proliferation and activated p53-dependent apoptosis. Tumor-bearing mice treated with RG7388 demonstrated significant tumor inhibition by 59% in NGP (P=0.003), 67% in SH-SY5Y (P=0.006), and 75% in LAN-5 (P=0.0019) p53 wild-type xenograft tumors, but no inhibitory effect on LAN-5 si-p53 or SK-N-AS p53-silenced/null xenograft tumors. Moreover, RG7388 was found to inhibit the p53-independent pathway of HIF-1α/VEGF with decreased gene expression and alteration of angiogenesis. Our study supports the further evaluation of RG7388 as a novel treatment option in p53 wild-type neuroblastoma at diagnosis and relapse.

MiR-127 Regulates Cell Proliferation and Senescence by Targeting BCL6

Cellular senescence occurs as a response to extracellular and intracellular stresses and contributes to aging and age-related pathologies. Emerging evidence suggests that cellular senescence also acts as a potent tumor suppression mechanism that prevents the oncogenic transformation of primary human cells. Recent reports have indicated that miRNAsact as key modulators of cellular senescence by targeting critical regulators of the senescence pathways. We previously reported that miR-127 is up-regulated in senescent fibroblasts. In this report, we identified miR-127 as a novel regulator of cellular senescence that directly targets BCL6. We further showed that miR-127 is down-regulated in breast cancer tissuesand that this down-regulation is associated with up-regulation of BCL6. Over-expression of miR-127 or depletion of BCL6 inhibits breast cancer cell proliferation. Our data suggest that miR-127 may function as a tumor suppressor that modulates the oncogene BCL6.

Inhibition of Tumor Growth in a Mouse Xenograft Model by the Humanized Anti-HGF Monoclonal Antibody YYB-101 Produced in a Large-Scale CHO Cell Culture

The humanized anti-hepatocyte growth factor (HGF) monoclonal antibody (mAb) YYB-101 is a promising therapeutic candidate for treating various cancers. In this study, we developed a bioprocess for large-scale production of YYB-101 and evaluated its therapeutic potential for tumor treatment using a xenograft mouse model. By screening diverse chemically defined basal media formulations and by assessing the effects of various feed supplements and feeding schedules on cell growth and antibody production, we established an optimal medium and feeding method to produce 757 mg/l of YYB-101 in flask cultures, representing a 7.5-fold increase in titer compared with that obtained under non-optimized conditions. The optimal dissolved oxygen concentration for antibody production was 70% pO2. A pH shift from 7.2 to 7.0, rather than controlled pH of either 7.0 or 7.2, resulted in productivity improvement in 5 L and 200 L bioreactors, yielding 737 and 830 mg/ml of YYB-101, respectively. The YYB-101 mAb highly purified by affinity chromatography using a Protein A column and two-step ion exchange chromatography effectively neutralized HGF in a cell-based assay and showed potent tumor suppression activity in a mouse xenograft model established with human glioblastoma cells.

Abstract 4751: Simultaneous inhibition of EGFR, HER2 and HER3 by an antibody mixture provides broad and potent tumor inhibition.

Abstract Deregulation of HER family members plays an important role in the development and progression of human cancer, and EGFR and HER2 are also clinically validated targets in many cancer indications. Accumulating evidence indicates, however, a high degree of HER family cross-talk and compensatory signaling leading to resistance upon therapeutic intervention with one of the receptors. Simultaneous targeting of more than one HER family receptor in vivo is frequently able to overcome resistance to the initial drug and the combined treatment is often also more efficacious than single-receptor targeting alone. Blocking of signaling from more than one HER family member may thus be necessary to effectively treat cancer and limit drug resistance. We have generated Sym013, a mixture of monoclonal antibodies specifically targeting EGFR, HER2 and HER3. Preclinical testing has shown that Sym013 displays potent growth inhibitory activity in a broad range of cell lines of diverse tissue origin and genetic background, including cell lines with acquired resistance to cetuximab, trastuzumab, or pertuzumab. Sym013 effectively inhibits all three targets simultaneously and thus prevents compensatory receptor up-regulation/activation, a commonly observed cellular response that can lead to drug escape when a single receptor is targeted. Furthermore, Sym013 is highly efficacious in the presence of EGFR and HER3 ligands, indicating that it is capable of overcoming acquired resistance due to increased ligand production. Overall, simultaneous targeting of three receptors provides broader efficacy than targeting of a single receptor or any combination of two receptors in the HER family. The ability to simultaneously inhibit EGFR, HER2 and HER3 in cancer cells in vitro also translates into broad and efficacious tumor growth suppression in vivo. All three target specificities have been demonstrated to contribute to the efficacy of Sym013 in vivo, and there is a clear benefit of combining HER family target specificities compared to single-receptor targeting. Sym013 is also highly efficacious in hard-to-treat KRAS mutated patient-derived pancreatic cancer models, and is frequently able to overcome resistance to HER family targeted therapeutics. Our data suggest that a mixture of antibodies, which simultaneously inhibits EGFR, HER2 and HER3, is superior to existing targeted therapies in dealing with both primary and acquired resistance due to intra-tumor heterogeneity and plasticity in terms of HER family dependency. Citation Format: Johan Lantto, Mikkel W. Pedersen, Helle J. Jacobsen, Thomas T. Poulsen, Ida Kjær, Klaus Koefoed, Jette W. Sen, Dietmar Weilguny, Bolette Bjerregaard, Christina R. Andersen, Ivan D. Horak, Michael Kragh. Simultaneous inhibition of EGFR, HER2 and HER3 by an antibody mixture provides broad and potent tumor inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4751. doi:10.1158/1538-7445.AM2013-4751

Abstract 790: An alternative tetramerization domain of p53 for exclusive homo-oligomerization and potent tumor suppression.

Abstract The tumor suppressor p53 is mutated in more than 50% of all cancers. Our data have shown that swapping the tetramerization domain (TD) of p53 with an alternative oligomerization domain enhances the utility of p53 for cancer gene therapy. Previously, we have swapped the tetramerization domain of p53 with the coiled-coil (CC) domain from breakpoint cluster region (Bcr). This alteration of the oligomerization motif of the tumor suppressor allowed for our construct, namely p53-CC, to evade hetero-oligomerization with endogenous mutant p53 commonly found in cancer cells. This proves to be critical since mutant p53 has a transdominant inhibitory effect over wild-type p53 upon hetero-oligomerization. A co-immunopreciptation (co-IP) experiment in human breast ductal carcinoma (T47D) cells validated our hypothesis that endogenous p53 interacts directly with exogenous wild-type p53, which is due to hetero-oligomerization via their TDs. In contrast, p53-CC, which lacks the TD, evaded binding to endogenous p53. Furthermore, overexpression of a potent mutant p53 (contains three hotspot mutations; R175H, R248W, and R273H) significantly impaired the function of exogenous wild-type p53, while the tumor suppressor activity of p53-CC was not affected. To further increase the apoptotic potential of p53-CC, rational design of mutations in the CC domain were investigated for two purposes: first to increase the dimerization affinity of CC, and second to prevent any potential interaction with endogenous Bcr. Leucine at residue 62 of the CC domain forms a critical hydrophobic pocket at the dimeric interface. Therefore, mutating this residue into the more hydrophobic isoleucine (with similar molecular weight),, should increase CC dimerization affinity. Results from a standard mammalian two-hybrid assay will be presented to validate the binding affinity of our mutant CC compared to the native CC. Furthermore, two additional mutations on the CC domain (R55E and E34K) were carried out to prevent any potential interaction of our construct with endogenous Bcr. These two mutations introduce a salt bridge that promotes homo-dimerization of mutant CC as well as preventing hetero-dimerization with native CC representative of endogenous Bcr. This will be confirmed using another co-IP experiment that shows no interaction between mutant CC and endogenous Bcr. Finally, two apoptotic assays were carried out to verify that introducing the aforementioned mutations did not cause any loss of tumor suppressor activity of our enhanced p53-CC variant. In summary, a p53 with altered TD has been designed for exclusive homo-dimerization and has potent activity as a tumor suppressor. Our ultimate goal is to use this version of p53 as a new gene therapeutic, capable of bypassing the dominant negative effect in cancers with mutated or mislocalized p53. Citation Format: Abood Okal, Mohanad Mossalam, Karina J. Matissek, Carol S. Lim. An alternative tetramerization domain of p53 for exclusive homo-oligomerization and potent tumor suppression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 790. doi:10.1158/1538-7445.AM2013-790

Tumor suppression via paclitaxel-loaded drug carriers that target inflammation marker upregulated in tumor vasculature and macrophages

Clinically approved chemotherapeutic nanoparticles may provide advantages over free drugs by achieving slower clearance and preferential accumulation in tumors. However, the lack of leaky vasculatures can create barriers to the permeation of ∼100nm-sized nanoparticles in solid tumors. We hypothesized that nanoparticles designed to target both tumor and tumor stroma would penetrate deeper into the tumors. To construct such comprehensive drug carriers, we utilized cross-linked amphiphilic polymer nanoparticles and functionalized them to target ICAM-1, a biomarker prevalent in various tumors and inflamed tumor stroma. The targeting moiety was derived from the modular domain present in αL integrin, which was engineered for high affinity and cross-reactivity with human and murine ICAM-1. ICAM-1-selective delivery of paclitaxel produced potent tumor suppression of not only ICAM-1-positive cervical cancer cells but also ICAM-1-negative tumors, presumably by causing cytotoxicity in tumor-associated endothelium (CD31+) and macrophages (CD68+) over-expressing ICAM-1. Contrary to the strategies of targeting only the tumor or specific tumor stromal constituents, we present a strategy in delivering therapeutics to the major cellular components of solid tumors. Drug carriers against inflammation-biomarkers may be effective against many different types of tumors, while being less susceptible to the highly mutable nature of tumor markers.

Development of tetravalent IgG1 dual targeting IGF-1R–EGFR antibodies with potent tumor inhibition

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine–glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R–EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R–EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.