Preparation and Stability of N-Terminal Mono-PEGylated

Bioconjugate Chem. 2006, 17, 995−999

995

Preparation and Stability of N-Terminal Mono-PEGylated Recombinant Human Endostatin Yongjun Nie, Xin Zhang, Xinchang Wang, and Junhui Chen* State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, P.R. China. Received December 14, 2005; Revised Manuscript Received May 27, 2006

Endostatin can specifically inhibit endothelial proliferation and potently inhibit angiogenesis and tumor growth. N-Terminal site-specific mono-PEGylation of recombinant human endostatin (mPEG-rhES) was accomplished by using methoxy poly-ethylene glycol (mPEG) propionaldehyde with an average molecular weight of 5000 Da through a reactive terminal aldehyde group. The site-specific mPEG conjugation was conducted under optimal conditions, which were identified through a statistical L9(34) orthogonal test. In this study, we have investigated the stability and antitumor activity of mPEG-rhES. SDS-PAGE, RP-HPLC, and UV spectrophotometric analysis were used to identify the purity and stability of mPEG-rhES. When incubated with protease or placed in an extreme environment, mPEG-rhES was more stable than rhES. The unmodified and PEGylated rhES were tested for their ability to inhibit the tumor growth of mouse H22 liver cancer in male mice. In a multiple versus single doses comparison study, daily administration of 0.25, 0.50, and 1.00 µmol/kg of unmodified rhES for 7 days resulted in 26.9%, 43.0%, and 64.9% reductions in tumor weight, respectively, while single doses of 0.13, 0.25, and 0.50 µmol/kg of the PEGylated protein per day resulted in 24.8%, 38.0%, and 64.5% reductions, respectively. Both treatments resulted in statistically significant reductions in mean tumor weight as compared to the physiological saline solution (control)-treated mice, with the dose of mPEG-rhES being a half of rhES, respectively, while the tumor inhibition rates were similar. Therefore, it is suggested that PEGylation enhances the stability of rhES and improves its antitumor activity.

INTRODUCTION Protein and peptide drugs have great potential as therapeutic agents. However, many of them are degraded by proteolytic enzymes in vivo, and they are rapidly cleared by the kidneys. In addition, B cells can generate neutralizing antibodies against these proteins and peptides. Therefore, these drugs usually have a short circulating half-life (1). Poly(ethylene glycol) (PEG) is a widely investigated polymer used for the covalent modification of biological macromolecules for many pharmaceutical and biotechnical applications, especially of peptides and proteins (2). By increasing the molecular mass of proteins and peptides and shielding them from proteolytic enzymes, PEGylation improves pharmacokinetics (1). A wide range of therapeutic proteins, including human growth hormone, interferon, insulin, granulocyte-colony stimulating factor (G-CSF), and interleukin II, have been PEGylated (3). The first few PEG-protein products, now on the market (Adagen, Oncospar, and PEGIntron), were developed using first generation of PEG chemistry. The second generation of PEGylation was designed to avoid the problems of first generation chemistry, notably diactivated PEG impurities, restriction to low molecular weight mPEG, unstable linkages, and lack of selectivity in modification (1). Recently, site-specific PEGylation of proteins has been attempted using a special class of functionalized PEG derivatives under specific conjugation conditions. For example, N-terminal specific PEGylation of G-CSF was obtained by conjugating methoxy-PEG derivatives at acidic pH conditions (4). This strategy was based on the fact that primary amine residues in protein have different pKa values: pKa 7.8 for the N-terminal R-amino group and 10.1 for the -amino group in lysine residues (5). When the propionaldehyde or N-hydroxysuccinimide PEG * To whom correspondence should be addressed. Phone: 086-2583592331. Fax: 086-25-83592331. E-mail: [email protected].

was conjugated at lower pH conditions, PEGylation at the N-terminus site occurred preferentially due to the different pKa values between the two kinds of  and R primary amines (6). Endostatin, a 20 kDa carboxy-terminal fragment of collagen XVIII, was isolated from the conditioned media of hemangioendothelioma (EOMA) cells. It specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth (7). Endostatin is derived from elastase-mediated cleavage (8). Ongoing angiogenesis is essential for the rapid growth of solid tumors, and it appears that successful tumors actively influence the ‘angiogenic switch’ to sustain continuous cell proliferation (9). Interestingly, endostatin does not seem to induce drug resistance. Furthermore, repeated cycles of systemic endostatin administration in tumor-bearing mice caused sustained tumor dormancy in the absence of further treatment (10). Therefore, endostatin will become the new and important antitumor agent. In this study, we PEGylated the N-terminus of rhES in a sitespecific manner with PEG-propionaldehyde derivative (4, 6). Various characterization methods, such as fast protein liquid chromatography (FPLC) and reversed phase high-performance liquid chromatography (RP-HPLC) techniques were used to reveal that the mono-PEGylation reaction occurred site-specifically at the N-terminal R-amine group. In vitro stability experiments such as incubating with trypsin or chymotrypsin and being kept at an extreme pH or temperature and in vivo antitumor activity experiments investigated in mice with H22 liver cancer were performed to examine the therapeutic potential of mono-PEGylated endostatin.

EXPERIMENTAL PROCEDURES Materials. Recombinant human endostatin (rhES) was donated by ZhongKai Bio-pharma (Suzhou, Jiangsu, China). The 5 kDa mPEG-propionaldehyde was purchased from SunBio

10.1021/bc050355d CCC: $33.50 © 2006 American Chemical Society Published on Web 06/23/2006

996 Bioconjugate Chem., Vol. 17, No. 4, 2006

Nie et al.

Table 1. Factors and Levels of the Orthogonal Tests factor level

pH

molar ratio mPEG/rhES

temperature (°C)

time (h)

1 2 3

5.0 5.5 6.0

2 5 10

4 20 30

10 16 24

(Anyang city, Seoul, Korea); trypsin, chymotrypsin, sodium cyanoborohydride, and dialysis membranes (MW cutoff 1000) were from Sigma (St. Louis, MO); CM Sepharose FF was form Amersham Pharmacia Biotech (Buckinghamshire, UK); 5 µm pore size BDS C18 reversed phase column (150 × 4.6 mm) was from Hypersil (Shandon HPLC, Runcorn, Cheshire, UK); and mouse H22 liver cancer cells and KunMing mice were form SIPI (Shanghai, CN). PEGylation of rhES with PEG-Propionaldehyde Derivatives. The optimal condition of the reaction was achieved through a statistical L9(34) orthogonal test. The four factors were pH, molar ratio of mPEG propionaldehyde to rhES, temperature, and time. Table 1 reports the three levels of each factor. The reactions were terminated by adjusting pH to 3.5 with 1 M HCl, and samples were analyzed by SDS-PAGE and Gelworks 1D intermediate software system. Separation and Purification by A 2 CTA FPLC. The reaction mixture was loaded onto a CM Sepharose FF cation exchange column (1.0 cm × 20 cm) preequilibrated with 20 mM sodium acetate, pH 4.5 (buffer A), at a flow rate of 1.0 mL/min using a Pharmacia LCC 501 Plus FPLC system. The column was washed with 300 mL of buffer A before a ladder gradient to 20%, 40%, and 60% buffer B (buffer A + 1 M NaCl) was applied about 5 column volumes per gradient. The fractions were analyzed by nonreducing SDS-PAGE and Gelworks 1D intermediate software system. The samples containing monoPEGylated and unmodified rhES were collected sequentially. Samples were stored at -20 °C after dialysis and lyophilization. UV Spectrophotometry. The protein content of PEGylated rhES can be determined by comparing the UV absorbance of pure rhES and mPEG-rhES at 280 nm. The purity and PEGresidue count of PEGylated rhES can be identified by the protein content. Reversed Phase HPLC (RP-HPLC). PEGylated rhES or rhES was analyzed on a Hypersil BDS C18 column equilibrated with buffer C (H2O containing 0.1% TFA) with a flow rate of 1.0 mL/min and monitored at 280 nm, and eluted with a linear gradient to 60% buffer D (CH3CN containing 0.1% TFA) over 20 min. In Vitro Proteolysis. 400 µL of mPEG-rhES (1.25 mg/mL, pH 7.4 phosphate buffer (PBS)) was incubated with 4 µL trypsin (1.0 mg/mL) or 20 µL chymotrypsin (2.0 mg/mL) at 37 °C. 20 µL of each sample was loaded onto a C18 column at different intermediate incubation time and identified by RP-HPLC. The peak area with mPEG-rhES characteristic retention time indicated the remaining mPEG-rhES. Meanwhile the rhES was taken as compared with mPEG-rhES. Stability of mPEG-rhES at Extreme pH or Temperature. To obtain the effects of temperature on rhES and mPEG-rhES, the mPEG-rhES (1.25 mg/mL, pH 7.4 PBS, 400 µL) or rhES (1.0 mg/mL, pH 7.4 PBS, 400 µL) was incubated at 4, 25, 37, 45, 50, 55, 60, 65 °C, respectively. After 24 h of incubation, all samples were centrifuged at 15000 rpm for 10 min. The absorbance of supernatant was measured at 280 nm to determine the remaining protein. Similarly, to obtain the effects of acid or alkali, the pH of mPEG-rhES (1.25 mg/mL, pH 7.4 PBS, 400 µL) or rhES (1.0 mg/mL, pH 7.4 PBS, 400 µL) was adjusted to 2, 3, 4, 5, 6 using acetic solution and 8, 9, 10, 11 using sodium hydroxide

Figure 1. SDS-PAGE analysis of the orthogonal samples stained with Coomassie Blue. Lane 1-9: nine samples in the orthogonal tests corresponding to the test number. Lane 10: the original unmodified rhES solution.

solution. The samples were incubated at 37 °C for 24 h and collected by centrifugation. The remaining protein in supernatant was measured by UV spectrophotometry. Antitumor Activity of mPEG-rhES in Mouse H22 Liver Cancer Allografts. Well-growth ascites of mouse H22 liver cancer, obtained from SIPI (Shanghai Institute of Pharmaceutical Industry, Shanghai, CN), were aspirated and diluted with 4-fold volumes physiological saline solution. 0.2 mL of the diluted ascites was subcutaneously inoculated with a trocar needle into the axillary flank of each male KunMing mouse (age 4 weeks; weight 19-21 g). The next day after inoculation, the mice were randomly allocated to drug groups of 10 animals each and 0.2 mL drug injections were administered, respectively. The rhES was administered intravenously (i.v.) to the tail at doses of 0.25, 0.50, and 1.00 µmol/kg, respectively, once a day for 7 consecutive days, and mPEG-rhES at doses of 0.13, 0.25, and 0.50 µmol/kg, and physiological saline solution at a dose of 12.5 mL/kg in the control group. After 10 days, the mice were sacrificed, and tumors were harvested for weight-measuring. The following formula was used to calculate the antitumor effect: tumor inhibition rate (TIR%) ) (1 - mean tumor weight of treatment group/mean tumor weight of untreated group) × 100. All animal experiments were carried out in accordance with the Guidelines for the Welfare of Animals in Experimental Neoplasia.

RESULTS PEGylation of rhES. The statistical L9(34) orthogonal test analyzed by SDS-PAGE indicated mono-PEGylation was better in Lane 1 or 4 (Figure 1). The ratios of mono-PEGylated rhES were 66.80% and 63.08% in Lane 1 and 4, respectively. The higher PEG concentration led to a higher ratio of multiPEGylated rhES (Lane 3, 6, 9 in Figure 1). After orthogonal analysis on the four factors, we found the factor which played the most important role in the reaction was the molar ratio of mPEG to rhES, followed by temperature, time, and pH (Table 2). The mono-PEGylated rhES produced in the PEGylation acylation reaction was separated from multi-PEGylated rhES and the unreacted rhES molecules by cation exchange chromatography (Figure 2). After dialysis and lyophilization, the purity of the mono-PEGylated rhES was measured by SDS-PAGE (Figure 3), UV spectrophotometry, and RP-HPLC (Figure 4). The results of SDS-PAGE and PR-HPLC chromatography indicated that the mPEG-rhES were pure. The experimental protein content of PEGylated rhES was 80.21%, and its PEGresidue count was 1. This result not only indicated that mPEGrhES was pure, but also exactly showed that the product was unitarily PEGylated. Stability of mPEG-rhES. Quantitations of percent remaining rhES following incubation with trypsin or chymotrypsin, by C18 column RP-HPLC, showed that the PEGylated rhES was more

Bioconjugate Chem., Vol. 17, No. 4, 2006 997

Mono-PEGylated Recombinant Human Endostatin Table 2. Results of Orthogonal Testsa test no.

pH

molar ratio mPEG/rhES

temperature (°C)

time (h)

1 2 3 4 5 6 7 8 9 K1c K2 K3 K1/3d K2/3 K3/3 Re

1 1 1 2 2 2 3 3 3 136.11 150.48 139.56 45.37 50.16 46.52 4.79

1 2 3 1 2 3 1 2 3 179.23 144.18 102.74 59.74 48.06 34.25 25.50

1 2 3 2 3 1 3 1 2 155.26 151.97 118.92 51.75 50.66 39.64 12.11

1 2 3 3 1 2 2 3 1 155.94 136.50 133.71 51.98 45.50 44.57 7.41

RMPES%b 66.80 47.54 21.78 63.08 47.79 39.61 49.36 48.85 41.35

a The factor which played the most important role in the reaction was found out by the statistical orthogonal test. b The ratios of mono-PEGylated rhES (RMPES%) were worked out from the gel by Gelwoks 1D intermediate software system. c K value was the sum of the RMPES% of a certain factor with the same level. d K1/3 meant that K1 value was divided by 3. e To a certain factor, the minimal Kx/3 (x ) 1, 2, or 3) subtracted from the maximal one gave the R value. The larger the R value, the more important the factor’s role in the reaction. In a degressive order, the most important factor was the molar ratio of mPEG to rhES, followed by temperature, time, and pH.

Figure 3. SDS-PAGE analysis of the two purified fractions on a nonreducing 17% gel and stained with Coomassie brilliant Blue. Lane A, molecular weight markers; Lane B, unmodified rhES (Peak 2 in Figure 2); Lane C, mono-PEGylated rhES (Peak 1 in Figure 2).

Figure 4. The HPLC profile of mono-PEGylated rhES after purification.

Figure 2. The profile of cation exchange fast protein liquid chromatogram (A ¨ CTA FPLC) of the PEGylated rhES.

resistant to trypsin or chymotrypsin proteolysis than rhES. After 40 min of incubation with trypsin, only 4% mono-PEGylated rhES in contrast to 90% of rhES was degraded (Figure 5). When mPEG-rhES and rhES were incubated with chymotrypsin, the results showed that the half-life of mPEG-rhES was about 60 min while that of rhES was about 24 min (Figure 6). Therefore, PEGylation distinctly increased the antiproteolytic stability of rhES. Recombinant hES was stable during 4 °C to 40 °C just like mPEG-rhES. However when the temperature was increased to 50 °C, there was only 20% rhES remaining while almost 100% mPEG-rhES remaining. When the denaturalization ratio of rhES was 50% the temperature was about 47.6 °C, whereas the corresponding temperature of mPEG-rhES was about 62.6 °C (Figure 7). Therefore, PEGylation significantly increased the heat stability of endostatin. Recombinant hES was stable during pH 2 to pH 7 as well as mPEG-rhES. When the pH exceeded 9, mPEG-rhES was significantly more stable than rhES. Even the pH rose to 10, mPEG-rhES was still stable while the denaturalization ratio of

Figure 5. The relative resistance of PEG-rhES (9) and rhES (]) to trypsin proteolysis. Each data value represents means ( SD (n ) 3).

rhES reached at 84.4% (Figure 8). Therefore, the N-terminus site-specific PEGylation also enhanced the acid-base stability of rhES significantly. In Vivo Activity of Unmodified and PEGylated rhES. In a multiple versus single doses comparison study, daily administration of 0.25, 0.50, and 1.0 µmol/kg of unmodified rhES for 7 days resulted in 26.9%, 43.0%, and 64.9% reductions in tumor weight respectively, while single doses of 0.13, 0.25, and 0.50 µmol/kg of the PEGylated protein per day resulted in

998 Bioconjugate Chem., Vol. 17, No. 4, 2006

Nie et al. Table 3. Antitumor Activities of RhES and MPEG-rhES against Mouse H22 Liver Cancer Allografts drug PSS rhES mPEG-rhES

Figure 6. The relative resistance of PEG-rhES (9) and rhES (]) to chymotrypsin proteolysis. Each data value represents means ( SD (n ) 3).

Figure 7. The effect of PEGylation on the relative susceptibility of PEG-rhES (9) and rhES (]) to temperature. Each data value represents means ( SD (n ) 3).

Figure 8. The effect of PEGylation on the relative susceptibility of PEG-rhES (9) and rhES (]) to pH. Each data value represents means ( SD (n ) 3).

24.8%, 38.0%, and 64.5% reductions respectively (Table 3). Both treatments resulted in statistically significant reductions in mean tumor weight as compared to the physiological saline solution (control)-treated mice at P-value < 0.001. The dose of mPEG-rhES was a half of rhES, repectively, while the tumor inhibition rate (TIR%) was rather closer.

DISCUSSION The conjugation of PEG derivative to primary amine groups of endostatin takes place primarily via a nucleophilic substitution reaction: the attack of unprotonated amine group to the carbonyl groups of aldehyde. This indicated that all the primary amines

dose (µmol/kg)

administration

tumor weight (g)a

TIR%b

12.5 mL/kg 0.25 0.50 1.00 0.13 0.25 0.50

iv × 7 iv × 7 iv × 7 iv × 7 iv × 7 iv × 7 iv × 7

2.42 ( 0.28 1.77 ( 0.32c 1.38 ( 0.14c 0.85 ( 0.30c 1.82 ( 0.29c 1.50 ( 0.17c 0.86 ( 0.22c

26.9 ( 13.2 43.0 ( 5.8 64.9 ( 12.4 24.8 ( 12.0 38.0 ( 7.0 64.5 ( 9.1

a After 10 days, the mice were sacrificed, and the tumors were harvested for weight-measuring. Values represent the means and standard deviations of results from 10 tumors. b Tumor inhibition rate (TIR%) ) (1 - mean tumor weight of treatment group/mean tumor weight of untreated group) × 100. c Significantly different from the PSS control group at P < 0.001.

(R and ) were equally reactive to PEGylation in basic conditions, so a heterogeneous mixture of multi-PEGylated species may be produced. In contrast, in the acidic condition, selective unprotonation of the N-terminal R-amine group could occur, making it more reactive than the -amine groups in lysine residues because of the difference of the pKa values: 7-8 for R-amine, and 10-11 for -amine (5). This reductive amination reaction with the N-terminal R-amine group, in the presence of sodium cyanoborohydride as a reducing agent, possibly resulted in the site-specific mono-PEGylated CSF and EGF (4, 6). MonoPEGylated protein conjugates, such as endostatin, were similarly obtained through a selective conjugation at the N-terminal R-amine residue by a statistical orthogonal test. After the simple purification steps of cation exchange chromatography and dialysis, the pure mono-PEGylated rhES conjugate could be obtained in the absence of rhES as evidenced by the SDS-PAGE and RP-HPLC analysis. The content of rhES protein in the mono-PEGylated rhES was measured by UV 280 nm. Through calculation, the protein content of mPEG-rhES was 80.21% and the PEG-residue count (n) was 1. This result not only indicated that mPEG-rhES was pure, but also adequately revealed that the molecular weights were in good agreement with the endostatin conjugate containing a single 5000 Da PEG derivative. In the United States, phase I clinical trials of rhES therapy were performed with doses ranging from 15 to 600 mg/m2, and the doses were generally well tolerated (11, 12). In October 2002, rhES entered phase II clinical trials for neuroendocrine tumors and metastatic melanoma (13). Recently rhES therapy passed Phase III clinical trials in non small cell lung cancer in China (14), and rhES developed by Shandong Yantai Medgenn Co., Ltd., was approved by China State Food and Drug Administration in March 2005. The dosage of rhES is 7.5-60 mg/m2 per day (14, 15). Such a dosage is a huge challenge to a recombinant gene product. However, the results of this study indicate that PEGylated rhES develops significant resistance to proteolysis and exhibits high stability to changes of temperature or pH. On the other hand, when we tested a 1 µmol/kg (20 mg/kg) dosage of rhES against mouse H22 liver cancer, the tumor inhibition rate (TIR%) was 64.9%. While the TIR% of mPEG-rhES was equivalent to rhES (64.5%), the dose of mPEG-rhES was just a half of rhES. Therefore, the PEGylation did not diminish the antitumor efficacy of rhES and also showed the possibility of reducing the administerial dosage in tumor treatments. In summary, it is suggested that PEGylation enhances the stability of rhES and improves its antitumor activity. Our data reveal the possibility to use a lower dosage of rhES in the clinic by the use of monoPEGylation.

Bioconjugate Chem., Vol. 17, No. 4, 2006 999

Mono-PEGylated Recombinant Human Endostatin

CONCLUSIONS In this study, we showed that mPEG-propionaldehyde was specifically conjugated at the N-terminal R-amine group of rhES in a weak acidic pH environment. The stability of monoPEGylated rhES, analyzed by protease degradation and an extreme environment in vitro, was enhanced significantly. Moreover, endostatin’s antitumor activity, investigated in mice with H22 liver cancer, was also improved by PEGylation.

ACKNOWLEDGMENT The authors thank Suzhou ZhongKai Bio-Pharma. Co., Ltd. for the generous donation of recombinant human endostatin.

LITERATURE CITED (1) Harris, J. M., and Chess, R. B. (2003) Effect of pegylation on pharmaceuticals. Nat. ReV. Drug DiscoVery 2, 214-221. (2) Roberts, M. J., Bentley, M. D., and Harris. J. M. (2002) Chemistry for peptide and protein PEGylation. AdV. Drug DeliVery ReV. 54, 459-476. (3) Harris, J. M., and Zilpsky, S. (1997) Poly(ethylene glycol): Chemistry and Biological Applications. ACS Symposium Series 680, American Chemical Society, Washington, DC. (4) Kinstler, O. B., Brems, D. N., Lauren, S. L., Paige, A. G., Hamburger, J. B., and Treuheit, M. J. (1996) Characterization and stability of N-terminally PEGylated rhG-CSF. Pharm. Res. 13, 9961002. (5) Wong, S. S. (1991) Chemistry of protein conjugation and crosslinking. CRC Press, Boca Raton, FL. (6) Lee, H., Jang, I. H., Ryu, S. H., and Park, T. G. (2003) N-terminal site-specific mono-PEGylation of epidermal growth factor. Pharm. Res. 20, 818-825. (7) O’Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J. (1997) Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277-285.

(8) Wen, W., Moses, M. A., Wiederschain, D., Arbiser, J. L., and Folkman, J. (1999) The generation of endostatin in mediated by elastase. Cancer Res. 59, 6052-6056. (9) Hanahan, D., and Folkman, J. (1996) Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353-364. (10) Boehm, T., Folkman, J., Browder, T., and O’Reilly, M. S. (1997) Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390, 404-407. (11) Herbst, R. S., Hess, K. R., Tran, H. T., Tseng, J. E., Mullani, N. A., Charnsangavej, C., Madden, T., Davis, D. W., McConkey, D. J., O’Reilly, M. S., Ellis, L. M., Pluda, J., Hong, W. K., and Abbruzzese, J. L. (2002) Phase I study of recombinant human endostatin in patients with advanced solid tumors. J. Clin. Oncol. 20, 3792-3803. (12) Eder, J. P., Jr., Supko, J. G., Clark, J. W., Puchalski, T. A., Carbonero, R. G., Ryan, D. P., Shulman, L. N., Proper, J., Kirvan, M., Rattner, B., Connors, S., Keogan, M. T., Janicek, M. J., Fogler, W. E., Schnipper, L., Kinchla, N., Sidor, C., Phillips, E., Folkman, J., and Kufe, D. W. (2002) Phase I clinical trial of recombinant human endostatin administered as a short intravenous infusion repeated daily. J. Clin. Oncol. 20, 3772-3784. (13) Rehman, S., and Jayson, G. C. (2005) Molecular imaging of antiangiogenic agents. Oncologist 10, 92-103. (14) Sun, Y., Wang, J., Liu, Y., Song, X., Zhang, Y., Li, K., Zhu, Y., Zhou, Q., You, L., and Yao, C. (2005) Results of Phase III trial of EndostarTM (rh-endostatin, YH-16) in advanced nonsmall cell lung cancer (NSCLC) patients. ASCO Annual Meeting Summaries, No. 7138. (15) Kulke, M., Bergsland, E., Ryan, D. P., Clark, J. W., Enzinger, P. C., Michelini, A., Kinsella, K., Fogler, W., Venook, A., and Fuchs, C. (2003) A phase II, open-label, safety, pharmacokinetic, and efficacy study of recombinant human endostatin in patients with advanced neuroendocrine tumors. ASCO Annual Meeting Summaries, No. 958. BC050355D