the present invention provides an endostatin product which has a longer half - life . it shows metabolic stability and longer half - life in vivo than unmodified endostatin , while maintaining the activity in preventing new blood vessel formation . preferably , the endostatin used in this invention is human endostatin . one embodiment of the present invention relates to a conjugate formed by a modifying agent and endostatin . the term “ conjugate ” in this invention refers to a modified endostatin , in which the endostatin and a modifying agent are linked by either a covalent bond or a non - covalent bond to form a stable complex . therefore , the “ modification ” can be achieved via either a covalent or non - covalent linkage . the modifying agent can be either chemically coupled to the endostatin or expressed as a part of a fusion with endostatin . in a preferred embodiment , the modification is site specific and will not influence the bioactivity of endostatin , e . g ., binding to its receptors . this modified product maintains the antiangiogenic activity and shows higher metabolic stability and longer half - life in blood than the unmodified endostatin , and therefore it can be used as an antiangiogenic or anti - tumor conjugate . the modifying agent can be one or more macromolecule polymers , proteins or its fragments , peptides or other chemical compounds , which is biocompatible and able to increase the half - life of endostatin . the macromolecule polymers as mentioned above can either have its own bioactivity or not . the suitable macromolecule polymers include , but not limited to , polyethylene alcohols , polyether compounds , polyvinylpyrrolidone , poly amino acids , copolymer of divinyl ether and maleic anhydride , n -( 2 - hydroxypropyl )- methacrylamide , polysaccharide , polyoxyethylated polyol , heparin or its fragment , poly - alkyl - ethylene glycol and its derivatives , copolymers of poly - alkyl - ethylene glycol and its derivatives , poly ( vinyl ethyl ether ), a , p - poly (( 2 - hydroxyethyl )- dl - aspartamide , polycarboxylates , poly oxyethylene - oxymethylenes , polyacryloyl morpholines , copolymer of amino compounds and oxidative olefin , poly hyaluronic acid , polyoxiranes , copolymer of ethanedioic acid and malonic acid , poly 1 , 3 - dioxolane , ethylene and maleic hydrazide copolymer , poly sialic acid , cyclodextrin and etc . the polyethylene alcohols as mentioned above include , but not limited to , polyethylene glycol ( monomethoxy polyethylene glycol , monohydroxy polyethylene glycol ), polyvinyl alcohol , polyallyl alcohol , polybutene alcohol and their derivatives . in a preferred embodiment , the modifying agent is polyethylene glycol . in a more preferred embodiment , polyethylene glycol is monomethoxy polyethylene glycol . the polyethers as mentioned above include , but not limited to , poly alkylene glycol ( ho (( ch 2 ) x o ) n h ), polypropylene glycol , polyoxyethylene ( ho (( ch 2 ) 2 o ) n h ), polyvinyl alcohol (( ch 2 choh ) n ) and derivatives thereof . the poly amino acids as mentioned above include , but not limited to , polymers of one type of aminoacid , copolymers of two or more types of amino acids , for example , polyalanine . the polysaccharides as mentioned above include , but not limited to , glucosan and its derivatives , for example dextran sulfate , cellulose and its derivatives ( including methyl cellulose and carboxymethyl cellulose ), starch and its derivatives , polysucrose and etc . a preferred protein molecule used as a modifying carrier of the invention is a protein naturally occurring or a fragment thereof , which includes , but not limited to , thyroxine - binding protein , transthyretin , transferrin , fibrinogen , immunoglobulin , albumin and fragments thereof . preferred carrier proteins are human proteins . a fragment of a protein as mentioned above refers to a part of the protein that still has the activity to serve as a carrier protein . endostatin is directly or indirectly linked to the modifying agent via a covalent bond . direct linking means that one amino acid of endostatin is directly linked to one amino acid of the carrier protein , via peptide bond or a disulfide bridge . indirect linking means that endostatin is linked to the carrier protein via one or more chemical groups . there have been some reports on modifying endostatin with polyethylene glycol . guoying mou ( 2005 ) and shenglin yang ( 2005 ) reported modifying endostatin with peg molecules having an average molecule weight of 6 kda or 10 kda , and the modifying agents are linked to ε - amino groups on the side chains of lysine residues , and thus the product is a mixture of multi - sites modification products . although the product can be purified by molecular sieve or ion - exchange chromatography to obtain a product of certain range of molecule weight , there are still some drawbacks , such as the inhomogeneity in the modification sites and in the protein conformation , due to the reactive properties of such modifying agents . moreover , the multi - sites modification of a protein will cause some change of the conformation of internal amino acids , resulting in a decrease of the activity of the protein . for a biological product , the homogeneity of the product is very important for decreasing the toxicity as well as the immunogenicity . in addition , for a mixture , it is also different to ensure the same quality and the stability of efficacy between different batches . therefore , it is an important issue and a problem to be solved in drug design and preparation how to ensure the consistency of the ingredients , the homogeneity of conformation , and the stability of the activity . in one embodiment of the present invention , the polyethylene glycol ( peg )- modified endostatin is characterized in that one endostatin molecule is covalently coupled to one or more peg molecules . the coupling site of endostatin may be one of the n - terminal α - amino group , the ε - amino group on the side chain of a lysine residue , the mercapto group of a cysteine residue , the carboxyl group on the side chain of an aspartate residue , the carboxyl group on the side chain of a glutamate residue , or any combination thereof . the polyethylene glycol used for coupling may be either linear or branched , with an average molecular weight of from 1 , 000 to 100 , 000 daltons , preferably from 5 , 000 to 40 , 000 daltons . as used herein , the molecular weight of polyethylene glycol refers to the average molecular weight . although peg modification may improve the pharmacokinetic characteristics of the drug , such modification , however , may also reduce the biological activities of a macromolecule drug . the main reason for this is that different modification sites may result in different structural changes and may shield the active sites of the drug ( veronese f ., et al . drug discovery today , 10 , 21 , 2005 ). for example , after modification with different pegs , the activity of differently modified interferon β - 1b decreases , and the highest activity of the modified molecule only reached a level corresponding to 71 % of the activity of the unmodified , and some modified proteins almost showed no activity at all . so , although peg modification changes the pharmacokinetic characteristics of a drug , it may also impair the activity of the drug , and consequently . therefore , both sides are taken into account when selecting the modification process and modifying agents , so as to achieve the optimal overall results in vivo . in one embodiment of the present invention , an n - terminal specific pegylating reagent ( mpeg - butyrald , nektar ) is used to modify the recombinant human endostatin . this gives rise to a product which is formed by coupling one peg molecule to one recombinant endostatin at the n - terminal α - amino group of the protein . it was reported that the interferon β - 1b modified by using the same n - terminal pegylation reagent only showed 70 % of the activity of the unmodified protein in an in vitro test ( filpula d ., et al . bioconjugate chem . 2006 , 17 , 618 - 630 ). in the present invention , however , it is surprisingly found that efficacy of n - terminal modified endostatin reached two times or more that of unmodified protein in inhibiting endothelial cell migration , indicating a prominent increase in the biological activity of endostatin after modification , which has not been reported yet . however , it was reported by g mou et al ( 2005 ) that multiple sites modification of endostatin resulted in a decreased activity in an in vitro test . this implies a much better in vitro activity of the product of the present invention as compared to the modified endostatin in relevant literatures . on the other hand , as shown in structure analysis , a zinc binding site was found in the n - terminus of human wild - type endostatin , which is consisting of the 1st , 3rd , 11th , and 76th amino acids . it has been reported that these sites are critical for the stability and activity of human endostatin , and form the binding site of human endostatin through which endostatin binds to its receptor and exerts its activity ( ding , y . h ., proc . natl . acad . sci . u . s . a . 95 , 10443 - 10448 ; hohenester , e ., embo j . 17 , 1656 - 1664 ; boehm , t ., biochem . biophys . res . commun . 252 , 190 - 194 ; ricard - blum , s ., j . biol . chem . 279 , 2927 - 2936 ; tjin tham sjin , r . m ., cancer res . 65 ( 9 ): 3656 - 63 ). as peg has a large molecular weight and large spatial hindrance , it has been proposed that the modification to the protein should not take place at active sites . otherwise this may greatly destroy the protein structure , which in turn which may impair the activity of the protein . however , the product of the present invention not only avoids activity loss , but exhibits an unexpectedly higher activity . in one embodiment of the present invention , endostatin is coupled to a protein or peptide , characterized in that one endostatin molecule is directly or indirectly linked to one or more proteins or peptides . in the case that endostatin is coupled to more carrier proteins , the selected carrier proteins can be either the same or different . preferably , the carrier proteins are naturally occurring proteins or fragments thereof . in a preferred embodiment , the carrier protein selected for modification is human serum albumin or its fragments . in another preferred embodiment , the carrier protein is an fc fragment of human immunoglobulin igg . in another embodiment of the present invention , the modifying agent is a small molecule or a small peptide or other compound , which enables the conjugate comprising endostatin to react with or have high affinity to certain components in the blood . in a specific embodiment , the product has a reactively active group , capable of reacting with amino , carboxyl or mercapto group to form a covalent bond . in another specific embodiment , the active group of the product is maleimide , which is capable of reacting with a mercapto group of a blood component , such as albumin . in another specific embodiment , the product has strong affinity to some blood components such as albumin or immunoglobulin , allowing the formation of larger complex . in another embodiment of the present invention , the endostatin modified with a small molecule or a small peptide may be glycosylated , phosphorylated or acylated . the modification sites may be amino acid residues originally contained the protein or amino acid residues generated via mutation . although the molecular weight of the conjugate is not very large , the half - life of the conjugate becomes longer since the modification changes the characteristics of endostatin . in another embodiment of the present invention , the conjugate comprising endostatin is a conjugate formed via non - covalent interaction between endostatin and a modifying agent such as protein and small molecule . this conjugate has the activity to inhibit angiogenesis and has longer in vivo half - life than endostatin . another embodiment of the present invention relates to a sustained - release formulation comprising endostatin . the sustained - release formulation is consisting of endostatin or any of the aforementioned conjugate and a biocompatible substance . endostatin in this composition still has biological activity , and at the same time , its in vivo half - life is prolonged because of the carrier which changes the pharmacokinetic characteristics of the drug . the sustained - release formulation can be , but not limited to , a microcapsule , hydrogel , microbeads , micro - osmotic pump and liposome , et al . the present invention also provides a pharmaceutical composition comprising the aforementioned endostatin - containing conjugate or sustained - release formulation and a pharmaceutically acceptable carrier . furthermore , the present invention also provides a kit comprising the aforementioned endostatin - containing conjugate or sustained - release formulation and an instruction for use . the pharmaceutically acceptable carriers used in this invention include carriers , excipients , or stabilizers , which are nontoxic to the cells or mammals to be contacted at selected doses and concentrations . a commonly used physiologically acceptable carrier is an aqueous ph buffer solution . examples of physiologically acceptable carriers include solutions such as phosphate buffer solution , citrate buffer solution and other organic acid buffer solution ; anti - oxidants including ascorbic acid ; polypeptides with low molecular weight ( no more than 10 residues ); proteins such as serum albumin , glutin or immunoglobulin ; hydrophilic polymers such as polyvinylpyrrolidone ; amino acids such as glycine , glutamine , asparagine , arginine or lysine ; monosaccharide , disaccharide and other carbohydrates including glucose , mannose or dextrin ; chelating agents such as edta ; sugar alcohol such as mannitol or sorbitol ; salt - forming counter ion such as sodium ion ; and / or nonionic surfactant such as tween ®, peg and pluronics ®. preferably , the excipients are sterilized and usually free of undesired substance . these compositions can be sterilized by routine sterilizing techniques . the present invention also relates to methods of preparing the aforementioned products of endostatin . specifically , the present invention provides a method of preparing a pegylated endostatin comprising mixing the activated peg with endostatin , allowing reaction under proper conditions including solution , temperature , ph , and reaction molar ratio , and purifying the coupled product by using cation - exchange column or molecular sieve . preferably , the ph of this reaction is within 3 to 10 . the present invention also provides the use of the aforementioned endostatin - containing conjugate , sustained - release formulation , pharmaceutical composition or kit in the prevention , diagnosis and treatment of tumor . the tumors that are suitable to be treated with the method of the invention include , but not limited to , lung cancer , neuroendocrine tumor , colon cancer , bone cancer , liver cancer , gastric cancer , pancreatic cancer , oral cancer , breast cancer , prostate cancer , lymphoma , esophagus cancer , oral cancer , nasopharyngeal carcinoma , cervical cancer , sarcoma , renal carcinoma , biliary cancer , malignant melanoma , et al . the present invention also provides the use of the aforementioned endostatin - containing conjugate , pharmaceutical composition , or sustained - release formulation in preparation of an anti - tumor medicament . the present invention also provides the use of the aforementioned endostatin - containing conjugate , sustained - release formulation , pharmaceutical composition , or kit for the prevention , diagnosis , or treatment of a disease characterized by , but not limited to , tissue or organ lesions caused by abnormal neovascularization , or in preparation of a medicament for the treatment of such a disease . according to the present invention , the administration route is selected from the group consisting of intravenous injection , intravenous infusion , subcutaneous injection , intramuscular injection , intraperitoneal injection , percutaneous absorption , subcutaneous embedment , liver arterial injection , oral administration , nasal administration , oral mucosa administration , ocular administration , rectal administration , vaginal delivery , or other clinically acceptable routes . the present invention also provides a method for prolonging the half - life of endostatin , comprising the step of forming a conjugate between endostatin and a modifying agent capable of prolonging in vivo half - life of endostatin and the step of preparing a sustained - release formulation containing endostatin . our experimental data show that , a product of the present invention , i . e ., a conjugate formed by coupling peg to the n - terminus of endostatin , has the activity of inhibiting the migration of endothelial cell proliferation and tumor growth in mice . this conjugate showed significantly higher activity as compared with non - modified endostatin . furthermore , pharmacokinetic study revealed that the in vivo metabolism of this modified product was effectively slowed down , and its half - life was prolonged . unless otherwise specified , the endostatin as used herein can be of human , murine or other mammals origin . in a preferred embodiment , the endostatin is human endostatin . unless otherwise specified , the endostatin as used herein can be a wild - type endostatin , i . e ., the naturally occurring form ; or a biologically active mutant , fragment , isomer , or derivative thereof , or their combinations . endostatin can be obtained upon expression in animal cells , or can be obtained by fermentation and further purification from yeasts or e . coli . the amino acid sequence of the wild - type human endostatin expressed in animal cells is shown in seq id no . 1 . the recombinant human endostatin expressed in yeasts may either have an amino acid sequence as shown in seq id no . 1 , or have the sequence of seq id no . 1 with no more than 10 amino acids added to or deleted from its n - terminus . the amino acid sequence of the recombinant human endostatin expressed in e . coli is shown in seq id no . 2 , but sometimes the n - terminal met will be deleted after expression , and when this happens , the resultant recombinant human endostatin will have the sequence as shown in seq id no . 1 . a mutant of endostatin refers to an endostatin molecule obtained by introducing amino acid replacement , deletion , or addition . an endostatin fragment refers to a part of the sequence of seq id no . 1 or seq id no . 2 . the fragments may be obtained through , among others , enzymatic cleavage , genetic engineering expression , or polypeptide synthesis . for instance , the fragment with the sequence of seq id no . 3 , seq id no . 4 , or seq id no . 5 was reported to have the activity to inhibit solid tumor ( cattaneo m g et al . experimental cell research 283 ( 2003 ) 230 - 236 ; tjin tham sjin r m et al . cancer research ( 2005 ) 65 ( 9 ) 3656 - 3663 ). an isomer of endostatin refers to a molecule with the same amino acid sequence as the wild - type endostatin but with different conformations , including difference in the secondary or tertiary protein structure , or changed optical activity in regional amino acids . the isomer may be a naturally occurring mutant or obtained via artificial design . a derivative of endostatin refers to a product generated by modification of the wild - type endostatin . modification refers to covalently attaching one or more small molecules , such as phosphoric acid molecules or carbohydrate molecules , or oligopeptide with less than 20 amino acids , to the protein at any amino acid of the protein . in a preferred embodiment , a 9 - amino acid peptide chain containing a his - tag ( mggshhhhh ) is added to the n - terminus of human endostatin , and this derived endostatin has the sequence of seq id no . 6 ( luo yongzhang et al . us2004110671 ). when expressed in e . coli , its n - terminal met sometimes will be deleted , and when this happens , the resultant n - terminal met deleted derivative of recombinant human endostatin will have the sequence of seq id no . 7 . the combination of the active mutants , fragments , isomers or derivatives of endostatin means that the product simultaneously has two or more of the modifications mentioned above , for example without limitation , a mutant of a fragment or a modified mutant , et al . coupling of 20 kda peg to the n - terminus of recombinant human endostatin the recombinant human endostatins used in this example include recombinant human endostatins have the sequence of seq id no . 2 and seq id no . 6 . specifically , the recombinant human endostatin ( protgen ltd .) was dialyzed into 30 mm sodium acetate solution , ph 5 . 5 . protein concentration was determined by measuring absorbance at 280 nm using uv spectrophotometer ( agilent technologies ), and then was adjusted to 2 mg / ml . a 20 kda peg ( mpeg - butyrald 20 kda , nektar ) which specifically modifies the n - terminus of protein was used for covalently coupling . specifically , 80 mg of this 20 kda peg ( solid ) was added to 20 ml protein solution ( containing 40 mg protein ), and the mixed solution was stirred at room temperature until peg solid dissolved completely and the molar ratio of peg and endostatin was 2 : 1 . ch 3 bnna ( sigma ) was added as reductant to reach a final concentration of 20 mm , and finally the ph value of the solution was adjusted to 5 . after resting at room temperature for 10 hours , more than 80 % of endostatin was modified with mono - pegylation , which means one peg molecule was covalently linked to one endostatin molecule at n - terminal α - amino group of endostatin . only a small amount of endostatin was non - specifically modified at multi - sites . the reaction solution can be purified directly through column chromatography . electrophoresis analysis of solutions before and after the reaction is shown in fig7 and 8 . purification of recombinant human endostatin modified with 20 kda peg at n - terminus through cation - exchange sp column the recombinant human endostatins used in this example include recombinant human endostatins have the sequence of seq id no . 2 and seq id no . 6 . specifically , the human endostatin modified with 20 kda peg was purified through sp chromatography column ( amersham ). the ph value of mixed solution was adjusted to 6 after reaction . the sample was loaded onto the column pre - equilibrated by a buffer containing 10 mm phosphate , ph 6 . 0 . after loading the sample , gradient elution was performed with buffer containing 10 mm phosphate , 1 m nacl , ph 6 . 0 . the peak of peg which did not involved in the reaction appeared during penetration and washing due to its minimal charge . the elution peaks appeared in the following order : multi - modified endostatin , mono - modified endostatin , and non - modified endostatin . different fractions can be collected according to absorbance at 280 nm . the result of purification is shown in fig9 and 10 . coupling of 40 kda peg to n - terminus of the recombinant human endostatin the recombinant human endostatins used in this example include recombinant human endostatins have the sequence of seq id no . 2 and seq id no . 6 . specifically , the recombinant human endostatin was dialyzed into 30 mm sodium acetate solution , ph 5 . 5 . the protein concentration was measured by uv spectrophotometer at 280 nm wavelength , and then was adjusted to 2 . 5 mg / ml . a 40 kda peg ( mpeg - butyrald 40 kda , nektar ) which specifically modifies the n - terminus of protein was used for covalently coupling . specifically , 160 mg of this 40 kda peg ( solid ) was added to 20 ml protein solution ( containing 40 mg protein ), the solution was stirred at room temperature until peg completely dissolved and the molar ratio of peg and endostatin was 2 : 1 . after that , ch 3 bnna ( sigma ) was added as a reductant to a concentration of 20 mm , and finally , the ph value of solution was adjusted to 5 . after resting at room temperature for 10 hours , more than 80 % of the endostatin was modified by mono - pegylation , i . e . one molecule of peg was covalently coupled to one endostatin molecule at the n - terminal α - amino group of the protein . only a small amount of endostatin was nonspecifically modified at multiple sites . the reaction solution can be purified directly through column chromatography . purification of recombinant human endostatin modified with 40 kda peg at n - terminus through cation - exchange sp column the recombinant human endostatins used in this example include recombinant human endostatins have the sequence of seq id no . 2 and seq id no . 6 . specifically , the human endostatin modified with 40 kda peg was purified through sp chromatography column ( amersham ). the ph value of mixed solution was adjusted to 6 after reaction . the sample was loaded onto the column pre - equilabrated by a buffer containing 10 mm phosphate , ph 6 . 0 . after loading the sample , gradient elution was performed with buffer containing 10 mm phosphate , 1 m nacl , ph 6 . 0 . the peak of peg which did not involved in the reaction appeared during penetration and washing due to its minimal charge . the elution peaks appeared in the following order : multi - modified endostatin , mono - modified endostatin , and non - modified endostatin . different fractions can be collected according to absorbance at 280 nm . the result of purification of recombinant human endostatin having the sequence of seq id no . 2 is shown in fig1 . the result of purification of recombinant human endostatin having the sequence of seq id no . 6 is similar to that of seq id no . 2 ( data not shown ). prolonged efficacy of the recombinant human endostatin with 20 kda peg covalently coupled to its n - terminus in the blood the recombinant human endostatins used in this example include recombinant human endostatins have the sequence of seq id no . 2 and seq id no . 6 . the prolonged efficacy of the recombinant human endostatin with peg modification was evaluated by measuring the in vivo half - life of the protein recombinant human endostatin with or without peg modification , respectively . six healthy kunming mice ( about 25 g ) ( vitalriver experimental animal center ) were divided into two groups , 3 mice per group . then the mice were given an injection via tail vein of recombinant human endostatin ( protgen ltd .) either unmodified or n - terminus modified with 20 kda peg , respectively , in a dose of 15 mg / kg body weight . and then , blood samples were taken from tail vein at 2 , 10 , 30 minutes , 1 , 2 , 4 , 8 , 16 , 24 , 36 , 48 , and 72 hours . plasma was then collected and the concentration of endostatin with or without peg modification was measured by sandwich elisa , respectively . as revealed by the pharmacokinetic analysis , upon modification with 20 kda peg , the half - life of recombinant human endostatin with the sequence of seq id no . 2 was increased from an average of 4 . 8 hours to an average of 16 hours in mice , and the half - life of recombinant human endostatin with the sequence of seq id no . 6 was increased from an average of 7 . 5 hours to an average of 16 hours in mice . these results demonstrated that coupling of high molecular weight peg to the recombinant human endostatin can effectively increase the in vivo half - life of the endostatin and achieve a prolonged efficacy . the activity of recombinant human endostatin with 20 kda peg coupled to its n - terminus to inhibit the migration of vascular endothelial cells the recombinant human endostatins used in this example include recombinant human endostatins have the sequence of seq id no . 2 and seq id no . 6 . specifically , the human microvascular endothelial cells ( hmec ) were cultured in the dmem medium ( hyclone ) containing 10 % serum to exponential phase . then the cells were left starving for 12 hours . after digesting with trypsin ( sigma ), cells were added into plates at 10000 cells per well for measurement of cell migration . conditions for migration assay included dmem medium , 1 % serum , and 10 ng / ml bfgf ( sigma ) and antibiotics ( streptomycin and ampicillin , both at 10 μg / ml , sigma ). for the treatment groups , endostatin with or without modification was added to the cells at 30 μg / ml . after culturing for 8 hours at 37 ° c ., the cells in the plates were fixed with 1 % glutaraldehyde and then the upper layer of cells on the membranes , which did not migrate , were removed , and the lower layer of cells were stained with hematoxylin . the cell number was counted under microscope ( olympus ). cells in three different fields on one plate were counted and then the inhibition rate was calculated . the results revealed that the inhibition rate on cells migration was 42 % for recombinant human endostatin with the sequence of seq id no . 2 , while the inhibition rate on cells migration was 90 % for modified endostatin . the inhibition rate on cells migration was 45 % for the recombinant human endostatin of the sequence of seq id no . 6 , while the inhibition rate was 90 % for modified endostatin . the above results demonstrate that the modification of endostatin with peg will not only maintain but greatly enhance its biological activity . the experimental results are shown in fig1 . activity of the recombinant human endostatin with 20 kda peg coupled to its n - terminus in the treatment of mice tumor model the recombinant human endostatins used in this example include recombinant human endostatins have the sequence of seq id no . 2 and seq id no . 6 . experiments were performed to observe the in vivo inhibitory activity of endostatin modified by 20 kda peg on mice s180 tumor . kunming mice of about 20 g were used in the experiment . mice were inoculated at axillary with s180 sarcoma cells ( china cell bank qk10952 ), 2 × 10 6 cells per mouse . the mice were then randomly grouped on the following day , eight mice per group , including the negative control group ( normal saline ), the positive control group ( endostatin , 1 . 5 mg / kg body weight , daily ), three treatment groups ( endostatin modified with peg , 1 . 5 mg / kg body weight , daily , every other day , or every three days , respectively ). the treatment was administered by way of tail vein injection . administration lasted for 10 days . the mice were sacrificed on day 11 , the weight of tumor was measured , and the efficacy was evaluated by tumor inhibition rate calculated as follows : tumor inhibition rate =( tumor weight of negative control group − tumor weight of treatment group )/ tumor weight of negative control group × 100 %. for the recombinant human endostatin with the sequence of seq id no . 2 , the experimental results revealed that the tumor inhibition rate of the positive control group was 34 %, whereas the tumor inhibition rates were 47 %, 53 % and 40 % for the groups treated daily , every other day and every three days , respectively . for the recombinant human endostatin having the sequence of seq id no . 6 with n - terminal additional amino acids , the experimental results revealed that the tumor inhibition rate of positive control group was 40 %, whereas the tumor inhibition rates were 45 %, 57 % and 50 % for the groups treated daily , every other day and every three days , respectively . the results demonstrate that the recombinant human endostatin with modification indeed has the antitumor activity in vivo , and since it has a slow in vivo metabolic rate , the activity of the modified recombinant human endostatin is superior to that of the unmodified recombinant human endostatin . furthermore , still it can maintain its superior efficacy as compared with the unmodified recombinant human endostatin even administered at a prolonged interval . the experimental results are shown in fig1 . modification of ε - amino group on the side chain of lysine residue of the recombinant human endostatin with 20 kda peg the recombinant human endostatins used in this example include recombinant human endostatins have the sequence of seq id no . 2 and seq id no . 6 . specifically , the recombinant human endostatin is dialyzed into 30 mm phosphate buffer solution , ph 7 . 8 . the protein concentration is measured by uv spectrophotometer at 280 nm wavelength , and then is adjusted to 2 mg / ml . a 20 kda peg ( mpeg - spa 20 kda , nektar ) which modifies the ε - amino group on the side chain of lysine residue is added and the molar ratio of peg and the protein is 8 : 1 . the recombinant human endostatin can be modified quickly , and after placing at room temperature for 30 minutes , 80 % of the endostatin is modified . the amount of modification product increases with the time of reaction , but the reaction speed slows down after 1 hour . the products are mainly mono - pegylated and multi - pegylated endostatin . the reaction solution can be purified directly through column chromatography . molecular sieve purification of recombinant human endostatin with 20 kda peg modification on ε - amino group on the side chain of lysine residue the recombinant human endostatins used in this example include recombinant human endostatins have the sequence of seq id no . 2 and seq id no . 6 . the endostatin modified by 20 kda peg was purified by molecular sieve . after reaction the ph value of the mixture was adjusted to 7 . 4 . the sample was loaded onto the column pre - equilabrated by a buffer containing 20 mm phosphate , 100 mm nacl , ph 7 . 4 . different fractions can be collected according to the absorbance at 280 nm using a uv spectrophotometer . the purification result of the modified endostatin with the sequence of seq id no . 6 is shown in the fig1 . ion - exchange chromatography purification of the recombinant human endostatin with 20 kda peg modification on the ε - amino group on the side chain of lysine residue the recombinant human endostatins used in this example include recombinant human endostatins have the sequence of seq id no . 2 and seq id no . 6 . the 20 kda peg - endostatin conjugate was purified by cm chromatography column . after reaction , the ph value of the mixture was adjusted to 6 . 5 . the sample was loaded onto the column pre - equilabrated by a buffer containing 10 mm phosphate , ph 6 . 5 . after loading the sample , gradient elution was performed with buffer containing 10 mm phosphate , 1 m nacl , ph 6 . 5 . the peak of peg which did not involved in the reaction appeared during penetration and washing due to its minimal charge . the elution peaks appeared in the following order : multi - modified endostatin , mono - modified endostatin , and non - modified endostatin . different fractions can be collected according to absorbance at 280 nm . the result of purification of the modification product of endostatin having a sequence of seq id no . 6 is shown in fig1 . activity of the recombinant human endostatin with n - terminal modification with 20 kda peg in the treatment of mice tumor model the recombinant human endostatin used in this example has the sequence of seq id no . 6 . the experiment was to observe the in vivo inhibitory activity of endostatin modified with 20 kda peg ( mpeg - butyrald 20 kda , nektar ) administered at increasing intervals on the growth of s180 tumor in mice . kunming mice ( 20 g ) were used in the experiment . the mice were inoculated at axillary with s180 sarcoma cells ( china cell bank qk10952 ), 2 × 10 6 cells per mouse . the mice were then randomly grouped on the following day , eight mice per group , including the negative control group ( normal saline ), the positive control group ( endostatin , 1 . 5 mg / kg body weight , administered every five days ), four treatment groups ( endostatin modified with peg , 1 . 5 mg / kg body weight , administered every five days , every seven days , every ten days , or every fourteen days , respectively ). the treatment was administered by way of tail vein injection . administration lasted for 14 days . the mice were sacrificed on day 15 , the weight of tumor was measured , and the efficacy of treatment was evaluated by tumor inhibition rate calculated as follows : tumor inhibition rate =( tumor weight of negative control group − tumor weight of treatment group )/ tumor weight of negative control group × 100 %. for the recombinant human endostatin having the sequence of seq id no . 6 with n - terminal additional amino acids , the experimental results revealed that the tumor inhibition rate of the positive control group was 34 %, whereas the tumor inhibition rates were 56 . 4 %, 37 . 1 % and 31 . 8 %, and 0 % for the groups treated every five days , every seven days , every ten days , or every fourteen days , respectively . this suggests that the modified recombinant human endostatin can still exhibit in vivo inhibitory activity on tumor growth even administered at an interval as long as 10 days .
the present invention discloses an anti - tumor conjugate and pharmaceutical composition or kits comprising the conjugate , as well as a method of producing the anti - tumor conjugate . the anti - tumor conjugate of the present invention is metabolically stable in vivo , and is ultimately available for the treatment of tumors and production of anti - tumor medicaments .