Evaluation of the Bioactive Compounds of Apis mellifera Honey Obtained from the Açai (Euterpe oleracea) Floral Nectar.

The biodiversity of Brazil provides an excellent climate and favorable pollination conditions for Apis mellifera L., especially in the Eastern Amazon region, which boasts vast floral wealth, including an abundance of açaí (Euterpe oleracea) flowers and fruits. In the present study, seven types of honey were evaluated: three containing floral nectar from açaí (Açaí honey) collected in the Eastern Amazon region (Açaí honey from Breu Branco (AH1 and AH2) and Açaí honey from Santa Maria (AH3), both from the state of Pará, Brazil) and four honeys from different regions of Brazil (Aroeira honey from Minas Gerais, Cipó-Uva honey from Distrito Federal, Mangue honey from Pará, and Timbó honey from Rio Grande do Sul). The characteristics of these honeys were evaluated by examining their physicochemical properties, melissopalynological aspects, color, antioxidant potential, and their constituent compounds, which were confirmed through GC-MS analysis. Açaí floral nectar honeys presented physicochemical results similar to those of other honeys, aligning with Brazilian legislation norms, but differed in their high values of free acidity, apparent sugars, and lower reducing sugars, which are directly related to their botanical origin. These differences correlate with unique flavor and distinct aroma characteristics. Melissopalynological analysis confirmed the botanical origin of the honeys containing açaí floral nectar, which had a color range from amber to dark amber. The three açaí honeys demonstrated high antioxidant capacity and superior flavonoid and polyphenol content compared to other honeys, particularly the açaí honey from Breu Branco (AH1), which had four times the content to combat free radicals compared to the honey with the highest potential (Aroeira honey). GC-MS analysis confirmed the presence of antioxidant properties as well as potential anti-inflammatory, antibacterial, antimicrobial, and antitumor capabilities in açaí honeys, which have not yet been fully studied.

Healing Activity of Propolis of Stingless Bee (Scaptotrigona aff. postica), Reared in Monoculture of Açaí (Euterpe oleracea), in Induced Wounds in Rats.

Wound healing is a complex and coordinated process involving interactions between cells and various messenger systems. This study conducted in vivo tests to determine the healing effect of propolis (PR)-based cream derived from the Amazon stingless bee, Scaptotrigona aff. postica, reared in açaí (Euterpe oleracea) monoculture, on induced wounds in rats. Data were obtained by monitoring injuries on 14 Wistar rats, divided into three groups (G1, G2 and G3), each receiving specific treatments: propolis-based cream (PR), collagenase (PC) and neutral cream (NC). Over the seven days of treatment, the lesions were measured using photographic records and ImageJ software to evaluate the healing effectiveness of the test cream. ImageJ software version 1.53g was used to compare the wound diameters for each treatment. After seven days, histopathological analyses of the induced lesions were performed. It was observed that collagenase (PC) and the test cream (PR) did not differ significantly in terms of wound diameter reduction. However, the propolis-based cream directly influenced the lesion maturation process and exhibited a milder inflammatory response compared to the positive control (PC). This effect is possibly associated with antimicrobial and anti-inflammatory compounds identified by GC/MS analysis in the propolis. Notably, this is the first report describing propolis of Scaptotrigona aff. postica obtained from açaí monocultures with strong healing potential, highlighting the identification of a high concentration of phenolic compounds that aid directly in wound repair.

Clinical proof of concept for anti-FGF2 therapy in exudative age-related macular degeneration (nAMD): phase 2 trials in treatment-naïve and anti-VEGF pretreated patients.

BACKGROUND/OBJECTIVE: Intravitreal injections of anti-vascular endothelial growth factor (VEGF) agents are the first-line treatment for exudative age-related macular degeneration (nAMD). Due to the limitations of these standard therapies, targeting alternative mechanisms of action may be helpful for treatment of this very common disease. Here, we investigated an anti-fibroblast growth factor-2 (FGF2) aptamer, umedaptanib pegol, a next generation therapeutic for the treatment of nAMD. METHODS: Three phase 2 studies were designed. First, a multicentre, randomized, double-masked TOFU study assessed the efficacy of intravitreal injections of umedaptanib pegol monotherapy or in combination with aflibercept, compared to aflibercept monotherapy in 86 subjects with anti-VEGF pretreated nAMD. Second, 22 subjects who had exited the TOFU study received 4 monthly intravitreal injections of umedaptanib pegol (extension, RAMEN study). Third, as an investigator-sponsored trial (TEMPURA study), a single-center, open-label, 4-month study was designed to evaluate the safety and treatment efficacy of umedaptanib pegol in five naïve nAMD patients who had not received any prior anti-VEGF treatment. RESULTS: The TOFU study demonstrated that umedaptanib pegol alone or in combination with aflibercept did not improve best-corrected visual acuity (BCVA) and central subfield thickness (CST) over aflibercept alone. However, the change in BCVA and CST at primary endpoint was marginal in all the three treatment groups, suggesting that umedaptanib pegol is effective to prevent the disease progression. The RAMEN study confirmed the cessation of disease progression. In the TEMPURA study, naïve nAMD patients showed improvement and no further macular degeneration, with striking improvement of visual acuity and central subfield thickness in some of the patients. CONCLUSIONS: These results demonstrate, for the first time, clinical proof of concept for aptamer based anti-FGF2 therapy of nAMD.

Safety and tolerability of intravitreal umedaptanib pegol (anti-FGF2) for neovascular age-related macular degeneration (nAMD): a phase 1, open-label study.

OBJECTIVE: To evaluate the efficacy and safety of a single-dose intravitreal umedaptanib pegol (anti-FGF2, investigational new drug) for the treatment of neovascular age-related macular degeneration (nAMD). METHODS: Nine participants who had a diagnosis of refractory nAMD were enrolled and received a single intravitreal injection of umedaptanib pegol at increasing doses of 0.2, 1.0 or 2.0 mg in the study eye. RESULTS: All three doses of umedaptanib pegol evaluated in the study were safe and well tolerated. No severe adverse event (AE) was observed in the study. There was an improvement in retinal fluid measured by central subfield thickness (CST) in most subjects. Remarkably, all three subjects who received 2.0 mg/eye showed improvement of more than 150 µm. CONCLUSIONS: Intravitreal umedaptanib pegol was safe, well tolerated, and demonstrated an indication of bioactivity in participants that have persistent subretinal fluid refractory to the treatment with anti-VEGFs.

Novel SIRPα Antibodies That Induce Single-Agent Phagocytosis of Tumor Cells while Preserving T Cells.

The signal regulatory protein α (SIRPα)/CD47 axis has emerged as an important innate immune checkpoint that enables cancer cell escape from macrophage phagocytosis. SIRPα expression is limited to macrophages, dendritic cells, and neutrophils-cells enriched in the tumor microenvironment. In this study, we present novel anti-SIRP Abs, SIRP-1 and SIRP-2, as an approach to targeting the SIRPα/CD47 axis. Both SIRP-1 and SIRP-2 bind human macrophage SIRPα variants 1 and 2, the most common variants in the human population. SIRP-1 and SIRP-2 are differentiated among reported anti-SIRP Abs in that they induce phagocytosis of solid and hematologic tumor cell lines by human monocyte-derived macrophages as single agents. We demonstrate that SIRP-1 and SIRP-2 disrupt SIRPα/CD47 interaction by two distinct mechanisms: SIRP-1 directly blocks SIRPα/CD47 and induces internalization of SIRPα/Ab complexes that reduce macrophage SIRPα surface levels and SIRP-2 acts via disruption of higher-order SIRPα structures on macrophages. Both SIRP-1 and SIRP-2 engage FcγRII, which is required for single-agent phagocytic activity. Although SIRP-1 and SIRP-2 bind SIRPγ with varying affinity, they show no adverse effects on T cell proliferation. Finally, both Abs also enhance phagocytosis when combined with tumor-opsonizing Abs, including a highly differentiated anti-CD47 Ab, AO-176, currently being evaluated in phase 1 clinical trials, NCT03834948 and NCT04445701 SIRP-1 and SIRP-2 are novel, differentiated SIRP Abs that induce in vitro single-agent and combination phagocytosis and show no adverse effects on T cell functionality. These data support their future development, both as single agents and in combination with other anticancer drugs.

Development of AO-176, a Next-Generation Humanized Anti-CD47 Antibody with Novel Anticancer Properties and Negligible Red Blood Cell Binding.

Inhibitors of adaptive immune checkpoints have shown promise as cancer treatments. CD47 is an innate immune checkpoint receptor broadly expressed on normal tissues and overexpressed on many tumors. Binding of tumor CD47 to signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggers a "don't eat me" signal that inhibits phagocytosis enabling escape of innate immune surveillance. Blocking CD47/SIRPα interaction promotes phagocytosis reducing tumor burden in numerous xenograft and syngeneic animal models. We have developed a next-generation humanized anti-CD47 antibody, AO-176, that not only blocks the CD47/SIRPα interaction to induce tumor cell phagocytosis, but also induces tumor cytotoxicity in hematologic and solid human tumor cell lines, but not normal noncancerous cells, by a cell autonomous mechanism (not ADCC). AO-176 also binds preferentially to tumor versus many normal cell types. In particular, AO-176 binds negligibly to RBCs in contrast to tumor cells, even at high concentrations up to 200 µg/mL and does not agglutinate RBCs up to 1 mg/mL in vitro These properties are expected not only to decrease the antigen sink, but also to minimize on-target clinical adverse effects observed following treatment with other reported RBC-binding anti-CD47 antibodies. When tested in cynomolgus monkeys, AO-176 was well tolerated with no adverse effects. Finally, we show that AO-176 demonstrates dose-dependent antitumor activity in tumor xenograft models. Taken together, the unique properties and antitumor activity of our next-generation anti-CD47 antibody, AO-176, distinguishes it from other CD47/SIRPα axis targeting agents in clinical development.

The Discovery and Preclinical Development of ASG-5ME, an Antibody-Drug Conjugate Targeting SLC44A4-Positive Epithelial Tumors Including Pancreatic and Prostate Cancer.

Here, we report the development of an antibody-drug conjugate, ASG-5ME, which targets the solute carrier receptor SLC44A4. SLC44A4 is a member of a family of putative choline transporters that we show to be markedly upregulated in a variety of epithelial tumors, most notably prostate and pancreatic cancer. SLC44A4 is normally expressed on the apical surface of secretory epithelial cells, but in cancer we show expression is not restricted to the luminal surface in advanced and undifferentiated tumors. ASG-5ME consists of a human IgG2 anti-SLC44A4 antibody conjugated through a cleavable linker to the microtubule-disrupting agent monomethylauristatin E. It has potent antitumor activity in both cell line - and patient-derived xenograft models of pancreatic and prostate cancers. Combination studies with ASG-5ME and nab-paclitaxel demonstrated combination effect in both pancreatic and prostate tumor models. Altogether, the data presented here suggest that ASG-5ME may have the potential to offer a new therapeutic option for the treatment of pancreatic and prostate cancers. Mol Cancer Ther; 15(11); 2679-87. ©2016 AACR.

Development of ASG-15ME, a Novel Antibody-Drug Conjugate Targeting SLITRK6, a New Urothelial Cancer Biomarker.

SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody-drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301-10. ©2016 AACR.

Enfortumab Vedotin Antibody-Drug Conjugate Targeting Nectin-4 Is a Highly Potent Therapeutic Agent in Multiple Preclinical Cancer Models.

The identification of optimal target antigens on tumor cells is central to the advancement of new antibody-based cancer therapies. We performed suppression subtractive hybridization and identified nectin-4 (PVRL4), a type I transmembrane protein and member of a family of related immunoglobulin-like adhesion molecules, as a potential target in epithelial cancers. We conducted immunohistochemical analysis of 2,394 patient specimens from bladder, breast, lung, pancreatic, ovarian, head/neck, and esophageal tumors and found that 69% of all specimens stained positive for nectin-4. Moderate to strong staining was especially observed in 60% of bladder and 53% of breast tumor specimens, whereas the expression of nectin-4 in normal tissue was more limited. We generated a novel antibody-drug conjugate (ADC) enfortumab vedotin comprising the human anti-nectin-4 antibody conjugated to the highly potent microtubule-disrupting agent MMAE. Hybridoma (AGS-22M6E) and CHO (ASG-22CE) versions of enfortumab vedotin (also known as ASG-22ME) ADC were able to bind to cell surface-expressed nectin-4 with high affinity and induced cell death in vitro in a dose-dependent manner. Treatment of mouse xenograft models of human breast, bladder, pancreatic, and lung cancers with enfortumab vedotin significantly inhibited the growth of all four tumor types and resulted in tumor regression of breast and bladder xenografts. Overall, these findings validate nectin-4 as an attractive therapeutic target in multiple solid tumors and support further clinical development, investigation, and application of nectin-4-targeting ADCs. Cancer Res; 76(10); 3003-13. ©2016 AACR.

AGS16F Is a Novel Antibody Drug Conjugate Directed against ENPP3 for the Treatment of Renal Cell Carcinoma.

PURPOSE: New cancer-specific antigens are required for the design of novel antibody-drug conjugates (ADC) that deliver tumor-specific and highly potent cytotoxic therapy. EXPERIMENTAL DESIGN: Suppression subtractive hybridization identified ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3 or CD203c) as a potential human cancer-specific antigen. Antibodies targeting the extracellular domain of human ENPP3 were produced and selected for specific binding to ENPP3. Expression of ENPP3 in normal and cancer tissue specimens was evaluated by immunohistochemistry (IHC). ADCs comprising anti-ENPP3 Ab conjugated with maleimidocaproyl monomethyl auristatin F via a noncleavable linker (mcMMAF) were selected for therapeutic potential using binding and internalization assays, cytotoxicity assays, and tumor growth inhibition in mouse xenograft models. Pharmacodynamic markers were evaluated by IHC in tissues and ELISA in blood. RESULTS: ENPP3 was highly expressed in clear cell renal cell carcinoma: 92.3% of samples were positive and 83.9% showed high expression. By contrast, expression was negligible in normal tissues examined, with the exception of the kidney. High expression was less frequent in papillary renal cell carcinoma and hepatocellular carcinoma samples. AGS16F, an anti-ENPP3 antibody-mcMMAF conjugate, inhibited tumor growth in three different renal cell carcinoma (RCC) xenograft models. AGS16F localized to tumors, formed the active metabolite Cys-mcMMAF, induced cell-cycle arrest and apoptosis, and increased blood levels of caspase-cleaved cytokeratin-18, a marker of epithelial cell death. CONCLUSIONS: AGS16F is a promising new therapeutic option for patients with RCC and is currently being evaluated in a phase I clinical trial.

AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody-Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML.

CD37 is a tetraspanin expressed on malignant B cells. Recently, CD37 has gained interest as a therapeutic target. We developed AGS67E, an antibody-drug conjugate that targets CD37 for the potential treatment of B/T-cell malignancies. It is a fully human monoclonal IgG2 antibody (AGS67C) conjugated, via a protease-cleavable linker, to the microtubule-disrupting agent monomethyl auristatin E (MMAE). AGS67E induces potent cytotoxicity, apoptosis, and cell-cycle alterations in many non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) cell lines and patient-derived samples in vitro. It also shows potent antitumor activity in NHL and CLL xenografts, including Rituxan-refractory models. During profiling studies to confirm the reported expression of CD37 in normal tissues and B-cell malignancies, we made the novel discovery that the CD37 protein was expressed in T-cell lymphomas and in AML. AGS67E bound to >80% of NHL and T-cell lymphomas, 100% of CLL and 100% of AML patient-derived samples, including CD34(+)CD38(-) leukemic stem cells. It also induced cytotoxicity, apoptosis, and cell-cycle alterations in AML cell lines and antitumor efficacy in orthotopic AML xenografts. Taken together, this study shows not only that AGS67E may serve as a potential therapeutic for B/T-cell malignancies, but it also demonstrates, for the first time, that CD37 is well expressed and a potential drug target in AML.

Discoidin domain receptor 1 contributes to tumorigenesis through modulation of TGFBI expression.

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFß-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.

In vitro and in vivo evaluation of cysteine and site specific conjugated herceptin antibody-drug conjugates.

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC. The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities. In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody. We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.

[Fungi microbiot of Melipona subnitida Ducke (Hymenoptera: Apidae)]. / Microbiota fúngica de Melipona subnitida Ducke (Hymenoptera: Apidae).

This paper reports on the occurrence of filamentous fungi found on the surface of the bees body from the specie Melipona subnitida Ducke that inhabits rocky places on the semi-arid Northeastern Brazil. Bees with cause of natural death were collected of beehives belonging to the Centro de Multiplicação de Animais Silvestres of the Universidade Federal Rural do Semi-Arido. We found the fungi: Aspergillus sp. 6 (37.5%); Aspergillus niger 2 (12.5%); Penicilium sp. 2 (12.5%); Aspergillus terreus 1 (6.3%); Curvularia sp. 1 (6.35%); Monilia sp. 1 (6.3%); Nigrospora sp. 1 (6.3%); Cladosporium sp. 1 (6.3%); Tricoderma sp. 1 (6.3%).

Microbiota fúngica de Melipona subnitida Ducke (Hymenoptera: Apidae) / Fungi microbiot of Melipona subnitida Ducke (Hymenoptera: Apidae)

O presente trabalho descreve a ocorrência de fungos filamentosos sobre a superfície do corpo de abelhas da espécie Melipona subnitida Ducke, que habita regiões pedregosas do semi-árido nordestino. Abelhas com causa de morte natural foram coletadas de colméias pertencentes ao Centro de Multiplicação de Animais Silvestres da Universidade Federal Rural do Semi-Árido. Foram encontrados os fungos: Aspergillus sp. 6 (37,5 por cento); Aspergillus niger 2 (12,5 por cento); Penicilium sp. 2 (12,5 por cento); Aspergillus terreus 1 (6,3 por cento); Curvularia sp. 1 (6,3 por cento); Monilia sp. 1 (6,3 por cento); Nigrospora sp. 1 (6,3 por cento); Cladosporium sp. 1 (6,3 por cento); Tricoderma sp. 1 (6,3 por cento).

This paper reports on the occurrence of filamentous fungi found on the surface of the beeÆs body from the specie Melipona subnitida Ducke that inhabits rocky places on the semi-arid Northeastern Brazil. Bees with cause of natural death were collected of beehives belonging to the Centro de Multiplicação de Animais Silvestres of the Universidade Federal Rural do Semi-Árido. We found the fungi: Aspergillus sp. 6 (37.5 percent); Aspergillus niger 2 (12.5 percent); Penicilium sp. 2 (12.5 percent); Aspergillus terreus 1 (6.3 percent); Curvularia sp. 1 (6.35 percent); Monilia sp. 1 (6.3 percent); Nigrospora sp. 1 (6.3 percent); Cladosporium sp. 1 (6.3 percent); Tricoderma sp. 1 (6.3 percent).

Tyrosine kinase receptor RON in human pancreatic cancer: expression, function, and validation as a target.

BACKGROUND: Specific tyrosine kinase receptors such as c-MET mediate epithelial-mesenchymal (EMT) transition, leading to phenotypic alterations associated with increased cell motility. It was hypothesized that RON, a tyrosine kinase receptor related to c-MET, would be expressed in human pancreatic cancer cells, induce EMT, and would thus serve as a target for therapy in a preclinical model. METHODS: RON expression in human pancreatic cancer specimens was assessed by immunohistochemistry. In pancreatic cancer cell lines, RON expression was assessed by reverse-transcriptase polymerase chain reaction (PCR) and Western blot analysis. The human pancreatic cancer cell line L3.6pl, with high RON expression, was exposed to macrophage stimulating protein (MSP), the RON ligand, and assessed for cell migration, invasion, and changes associated with EMT. Western blot analysis and immunofluorescent staining were used to assess alterations in protein expression and cellular location, respectively. A RON monoclonal antibody (MoAb) was used to block ligand-induced activation of RON. RESULTS: Immunohistochemical staining revealed RON overexpression in 93% of human pancreatic cancer specimens relative to nonmalignant ductal tissue. RON mRNA and protein was expressed in 9 of 9 human pancreatic cancer cell lines. Treatment of L3.6pl cells with MSP increased Erk phosphorylation, cell migration, and invasion (P < .001). RON activation led to a decrease in membrane-bound E-cadherin in association with nuclear translocation of beta-catenin. RON MoAb inhibited downstream signaling as well as cell migration and invasion. In nude mice, RON MoAb inhibited subcutaneous and orthotopic tumor growth by about 60%. CONCLUSIONS: RON activation induced molecular and cellular alterations consistent with EMT. Inhibition of RON activation inhibited tumor growth in vivo. Novel antineoplastic therapies designed to inhibit RON activity may hinder mechanisms critical for pancreatic tumor progression.

Therapeutic implications of a human neutralizing antibody to the macrophage-stimulating protein receptor tyrosine kinase (RON), a c-MET family member.

RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in approximately 100 cancer cell lines and approximately 300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers.

Targeting the platelet-derived growth factor receptor alpha with a neutralizing human monoclonal antibody inhibits the growth of tumor xenografts: implications as a potential therapeutic target.

Platelet-derived growth factor receptor alpha (PDGFRalpha) is a type III receptor tyrosine kinase that is expressed on a variety of tumor types. A neutralizing monoclonal antibody to human PDGFRalpha, which did not cross-react with the beta form of the receptor, was generated. The fully human antibody, termed 3G3, has a Kd of 40 pmol/L and blocks both PDGF-AA and PDGF-BB ligands from binding to PDGFRalpha. In addition to blocking ligand-induced cell mitogenesis and receptor autophosphorylation, 3G3 inhibited phosphorylation of the downstream signaling molecules Akt and mitogen-activated protein kinase. This inhibition was seen in both transfected and tumor cell lines expressing PDGFRalpha. The in vivo antitumor activity of 3G3 was tested in human glioblastoma (U118) and leiomyosarcoma (SKLMS-1) xenograft tumor models in athymic nude mice. Antibody 3G3 significantly inhibited the growth of U118 (P=0.0004) and SKLMS-1 (P <0.0001) tumors relative to control. These data suggest that 3G3 may be useful for the treatment of tumors that express PDGFRalpha.

Conservation of receptor antagonist anti-tumor activity by epidermal growth factor receptor antibody expressed in transgenic corn seed.

Recombinant protein production in plants such as corn is a promising means to generate high product yields at low comparable production cost. The anti-EGFR monoclonal antibody C225, cetuximab, is a well-characterized receptor antagonist antibody recently approved for the treatment of refractory colorectal cancer. We initiated a study to test and compare the functional activity of glycosylated and aglycosylated C225 produced in stable transgenic corn seed. Both corn antibodies were shown to be functionally indistinguishable from mammalian-derived C225 in demonstrating high-affinity binding to the EGF receptor, blocking of ligand-dependent signaling, and inhibiting cell proliferation. In addition, consistent with cetuximab, both corn antibodies possessed strong anti-tumor activity in vivo. Acute dose primate pharmacokinetic studies, however, revealed a marked increase in clearance for the glycosylated corn antibody, while the aglycosylated antibody possessed in vivo kinetics similar to cetuximab. This experimentation established that corn-derived receptor blocking monoclonal antibodies possess comparable efficacy to mammalian cell culture-derived antibody, and offer a cost effective alternative to large-scale mammalian cell culture production.