Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis by stimulating the proangiogenic signaling of endothelial cells via activation of VEGF receptor (VEGFR) tyrosine kinases. Therefore, VEGFRs are an attractive therapeutic target for cancer treatment. In the present study, we show that a quinoline-urea derivative, KRN951, is a novel tyrosine kinase inhibitor for VEGFRs with antitumor angiogenesis and antigrowth activities. KRN951 potently inhibited VEGF-induced VEGFR-2 phosphorylation in endothelial cells at in vitro subnanomolar IC 50 values (IC 50 = 0.16 nmol/L). It also inhibited ligand-induced phosphorylation of plateletderived growth factor receptor-B (PDGFR-B) and c-Kit (IC 50 = 1.72 and 1.63 nmol/L, respectively). KRN951 blocked VEGFdependent, but not VEGF-independent, activation of mitogenactivated protein kinases and proliferation of endothelial cells. In addition, it inhibited VEGF-mediated migration of human umbilical vein endothelial cells. Following p.o. administration to athymic rats, KRN951 decreased the microvessel density within tumor xenografts and attenuated VEGFR-2 phosphorylation levels in tumor endothelium. It also displayed antitumor activity against a wide variety of human tumor xenografts, including lung, breast, colon, ovarian, pancreas, and prostate cancer. Furthermore, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) analysis revealed that a significant reduction in tumor vascular hyperpermeability was closely associated with the antitumor activity of KRN951. These findings suggest that KRN951 is a highly potent, p.o. active antiangiogenesis and antitumor agent and that DCE-MRI would be useful in detecting early responses to KRN951 in a clinical setting. KRN951 is currently in phase I clinical development for the treatment of patients with advanced cancer. (Cancer Res 2006; 66(18): 9134-42)

Engineered cysteine residues are particularly convenient for site-specific conjugation of antibody-drug conjugates (ADC), because no cell engineering and additives are required. Usually, unpaired cysteine residues form mixed disulfides during fermentation in Chinese hamster ovarian (CHO) cells; therefore, additional reduction and oxidization steps are required prior to conjugation. In this study, we prepared light chain (Lc)-Q124C variants in IgG and examined the conjugation efficiency. Intriguingly, Lc-Q124C exhibited high thiol reactivity and directly generated site-specific ADC without any pretreatment (named active thiol antibody: Actibody). Most of the cysteine-maleimide conjugates including Lc-Q124C showed retro-Michael reaction with cysteine 34 in albumin and were decomposed over time. In order to acquire resistance to a maleimide exchange reaction, the facile procedure for succinimide hydrolysis on anion exchange resin was employed. Hydrolyzed Lc-Q124C conjugate prepared with anion exchange procedure retained high stability in plasma. Recently, various stable linkage schemes for cysteine conjugation have been reported. The combination with direct conjugation by the use of Actibody and stable linker technology could enable the generation of stable site-specific ADC through a simple method. Actibody technology with Lc-Q124C at a less exposed position opens a new path for cysteine-based conjugation, and contributes to reducing entry barriers to the preparation and evaluation of ADC.

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We assessed the antitumor efficacy of KRN951, a novel tyrosine kinase inhibitor of vascular endothelial growth factor receptors, using a rat colon cancer RCN-9 syngeneic model in which the tumor cells are transplanted into the peritoneal cavity of F344 rats. KRN951 treatments that commenced 4 days after tumor transplantation (day 4) significantly inhibited tumor-induced angiogenesis, the formation of tumor nodules in the mesenteric windows, and the accumulation of malignant ascites. Moreover, KRN951 treatments initiated on day 14, by which time angiogenesis and malignant ascites have already been well established, resulted in the regression of newly formed tumor vasculatures with aberrant structures and also in the apparent loss of malignant ascites by the end of the study period. Quantitative analysis of the vessel architecture on mesenteric windows revealed that KRN951 not only regressed, but also normalized the tumor-induced neovasculature. Continuous daily treatments with KRN951 significantly prolonged the survival of rats bearing both early stage and more advanced-stage tumors, compared with the vehicle-treated animals. The results of our current study thus show that KRN951 inhibits colon carcinoma progression in the peritoneal cavity by blocking tumor angiogenesis, ascites formation, and tumor spread, thereby prolonging survival. Moreover, these studies clearly demonstrate the therapeutic effects of KRN951 against established tumors in the peritoneal cavity, including the regression and normalization of the tumor neovasculature. Our findings therefore suggest that KRN951 has significant potential as a future therapeutic agent in the treatment of peritoneal cancers with ascites. (Cancer Sci 2008; 99: 623-630) V ascular endothelial growth factor and VEGFR are promising targets for antitumor therapies as they play major roles during tumor angiogenesis, which is an essential process for tumor growth and metastasis. A number of studies have now demonstrated a clear correlation between VEGF expression, microvessel density, and a poorer prognosis.(1-4) VEGF mediates angiogenic signaling to the endothelium via two receptor tyrosine kinases, VEGFR-1 and VEGFR-2. In particular, VEGFR-2 in vascular endothelial cells is the main transmitter of the activation signals that induce the proliferation and migration of endothelial cells upon their binding of VEGF.(5) In addition, it seems that the signaling pathways mediated via VEGFR-1 and -2 are also involved in the VEGF-induced hyperpermeability of tumor vasculatures. (6) As a result of these previous findings, there is now great interest in developing blocking antibodies and small molecules that target the VEGF and VEGFR pathway as such agents are good candidate anticancer therapeutics. In this regard, several agents that target angiogenesis through VEGF function, including those targeting VEGF itself or the VEGFR, are now in advanced stages of development.(7-13) Bevacizumab, a VEGF-neutralizing monoclonal antibody, is the first antiangiogenic agent to be approved by the...

KRN633 is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases. However, it is poorly water-soluble; consequently, relatively high doses are required to achieve substantial in vivo tumor growth suppression after oral administration. We subjected KRN633 to the solid dispersion technique to improve its solubility, absorption, and antitumor efficacy after oral administration. This technique transformed the drug into an amorphous state and dramatically improved its dissolution rate. It also enhanced the bioavailability of the drug in rats by f7.5-fold. The solid dispersion form of KRN633 also dramatically inhibited human tumor growth in murine and rat xenograft models: similar rates of tumor growth inhibition were obtained with 10-to 25-fold lower doses of the solid dispersion preparation relative to the pure drug in its crystalline state. Histologic analysis of tumors treated with the solid dispersion preparation revealed a significant reduction in microvessel density at much lower doses when compared with the crystalline form preparation. In addition, a dose-finding study using the solid dispersion form in a rat xenograft model revealed that there was a substantial range of doses at which KRN633 in the solid dispersion form showed significant antitumor activity but did not induce weight loss or elevate total urinary protein levels. These data suggest that the solid dispersion technique is an effective approach for developing

Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 play a central role in angiogenesis, which is necessary for solid tumors to expand and metastasize. Specific inhibitors of VEGFR-2 tyrosine kinase are therefore thought to be useful for treating cancer. We showed that the quinazoline urea derivative KRN633 inhibited tyrosine phosphorylation of VEGFR-2 (IC50 = 1.16 nmol/L) in human umbilical vein endothelial cells. Selectivity profiling with recombinant tyrosine kinases showed that KRN633 was highly selective for VEGFR-1, -2, and -3. KRN633 also blocked the activation of mitogen-activated protein kinases by VEGF, along with human umbilical vein endothelial cell proliferation and tube formation. The propagation of various cancer cell lines in vitro was not inhibited by KRN633. However, p.o. administration of KRN633 inhibited tumor growth in several in vivo tumor xenograft models with diverse tissue origins, including lung, colon, and prostate, in athymic mice and rats. KRN633 also caused the regression of some well-established tumors and those that had regrown after the cessation of treatment. In these models, the trough serum concentration of KRN633 had a more significant effect than the maximum serum concentration on antitumor activity. KRN633 was well tolerated and had no significant effects on body weight or the general health of the animals. Histologic analysis of tumor xenografts treated with KRN633 revealed a reduction in the number of endothelial cells in non-necrotic areas and a decrease in vascular permeability. These data suggest that KRN633 might be useful in the treatment of solid tumors and other diseases that depend on pathologic angiogenesis.

432 Background: KRN330 is a novel recombinant human IgG1 monoclonal antibody (mAb) targeting A33 surface differentiation antigen that is uniformly expressed on the surface of 95% of colorectal cancer (CRC) cells. In this study, we characterized the activity of KRN330 for its in vitro properties, as well as for its in vivo antitumor activity. Methods: A kinetic analysis of the interaction between KRN330 and recombinant human A33 was conducted using a Biacore 3000. Western blot analysis was conducted using A33 expressing COLO205 lysates under reducing and non-reducing conditions. Binding of KRN330 to human colorectal cancer tissues were investigated using FITC-labeled KRN330. We also developed more conventional staining methods of A33 and investigated A33 expression using human colon cancer tissue microarray (TMA). ADCC and CDC activities of KRN330 were assessed using a standard 51Cr release assay. A33 expression levels of 14 CRC cell lines were analyzed using flow cytometer. In vivo antitumor activities of KRN330 alone or in combination with chemotherapeutic agents against subcutaneous or intraperiotoneal human CRC (COLO205 and LS174T) models were investigated using mice and rats xenograft model. Results: A kinetic analysis revealed that KRN330 showed a high binding affinity to A33. Western blot analysis also showed that antibody recognized not any protein under reducing condition, but non-reducing condition. A33 staining of TMA with 204 different samples revealed the majority of tumor expressed A33. KRN330 exhibited ADCC activity against A33 expressing human colorectal cancer cell lines which include both K-ras wild and mutated types. KRN330 showed dose-dependent antitumor activities in vivo. KRN330 also significantly prolonged survival of human colon tumor bearing mice. In addition, combination treatment of KRN330 with irinotecan showed increased antitumor activitiy and prolongation of survival, compared to either irinotecan or KRN330 alone. Conclusions: These results suggest that KRN330 is a promising candidate of novel therapy for CRC. The phase I/II study of KRN330 plus irinotecan in patients with second line metastatic CRC is ongoing. [Table: see text]

Purpose: Many antibody therapies have been introduced into clinical practice in the past decade. Although pharmacokinetic data provide information regarding serum concentration, there are few reports that show the interaction of antibodies with the tumor in vivo. KRN330 is a novel recombinant human IgG1 monoclonal antibody (mAb) targeting the A33 surface differentiation antigen that is uniformly expressed on the surface of 95% of colorectal cancer (CRC) cells. We investigated whether KRN330 binds and stays bound to tumors established in a xenograft rodent model as well as biopsied tumors from CRC patients treated with KRN330 on a phase I trial. Methods: Time-course of KRN330 binding was analyzed using a rodent xenograft tumor model bearing human LS174 colon cancer cells. Tumors were collected at specified time points, ranging from the end of infusion on Day 1 to Day 36 after a single dose of KRN330 (1 mg/kg). KRN330 on the tumor was detected with either flow cytometry or an immunohistochemical method (IHC) for each time point. Clinical KRN330 binding data were analyzed using A33-positive tumor biopsies from 4 patients with advanced CRC who consented to be enrolled into the Phase 1 monotherapy study of KRN330. The biopsy was conducted on Day 8-9, or Day 14, after an initial 3 mg/kg dose of KRN330. KRN330 binding on human CRC tissues was assessed using IHC with the anti KRN330 idiotype antibody. Serum concentrations of KRN330 were obtained at several time points in both studies. Results: Xenograft KRN330 binding analyses indicate that KRN330 rapidly bound to the tumor after dosing and remained bound throughout the study period. Although the serum concentration decreased to 15% of that originally observed at 10 minutes post administration by Day 8, based on flow cytometry data, the KRN330 bound on tumors on Days 8 and 15 was 100% and 70%, respectively, of the maximum level bound (Day 2). Xenograft tumors in animals that were infused with a non-specific human immunoglobulin G control showed there was very minimal binding on Day 2. IHC results demonstrated that staining intensity supported the flow cytometry analysis and remained at a similar level for up to Day 15. Clinical binding data confirmed KRN330 bound and remained bound to the CRC tumor cells even at Day 14 as well as Days 8 and 9. On the other hand, serum concentrations of KRN330 decreased to 12% and 4% of the value at the end of infusion on Day 8-9 and Day 15, respectively. Conclusion: These data are the first to show that KRN330 binds rapidly and stays bound to CRC tumors for an extended period in rodent xenograft models as well as CRC patients. This suggests that KRN330 might be able to remain active on tumor even after the serum concentration drops to very low levels. Further investigation of this mAb in clinical studies is currently underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2431.