Targeted prodrug cytosine deaminase fusion carrier and application thereof

Abstract

The present invention provides a targeted prodrug enzyme fusion carrier comprising a targeted molecule and a prodrug enzyme. The targeted prodrug enzyme fusion carrier can effectively identify and bind to tumor cells and tumor-induced tumor angiogenesis. The targeted prodrug enzyme fusion carrier also has a targeted prodrug enzyme fusion protein and a theranostic system utilizing the method of in vivo nuclear medicine for the clinical diagnosis and treatment of individual patients with tumors.

Claims

1. A targeted prodrug enzyme fusion carrier for identifying and binding a tumor cell or/and an angiogenic endothelial cell of tumor tissues, and diagnosing and treating cancer, comprising a prodrug enzyme at least, wherein the prodrug enzyme is selected from the group consisting of yeast cytosine deaminase (yCD) (SEQ ID NO: 1), E. coli. cytosine deaminase (EcCD) (SEQ ID NO: 17), and EcCD_D314A (SEQ ID NO: 18), when the prodrug enzyme is yCD (SEQ ID NO: 1), the targeted prodrug enzyme fusion carrier is consisting of RGD4C-yCD (SEQ ID NO: 3) and RGD4C_yCD_yUPRT (SEQ ID NO: 4), when the prodrug enzyme is EcCD (SEQ ID NO: 17), the targeted prodrug enzyme fusion carrier is consisting of RGD4C_EcCD_WT (SEQ ID NO: 19), when the prodrug enzyme is EcCD_D314A (SEQ ID NO: 18), the targeted prodrug enzyme fusion carrier is consisting of RGD4C_EcCD_D314A (SEQ ID NO: 20), wherein the targeted prodrug enzyme fusion carrier can convert a 5-fluorocytosine (5-FC) into a 5-fluorouracil (5-FU), wherein the targeted prodrug enzyme fusion carrier is administered a therapeutically effective amount to a subject in need thereof, wherein the subject is diagnosed with cancer.

2. A targeted prodrug enzyme fusion carrier for identifying and binding a tumor cell or/and an angiogenic endothelial cell of tumor tissues, and diagnosing and treating cancer, comprising at least a prodrug enzyme, wherein the prodrug enzyme is selected from the group consisting of yeast cytosine deaminase (yCD) (SEQ ID NO: 1), E. coli. cytosine deaminase (EcCD) (SEQ ID NO: 17), and EcCD_D314A (SEQ ID NO: 18), when the prodrug enzyme is yCD (SEQ ID NO: 1), the targeted prodrug enzyme fusion carrier is selected from the group consisting of RGD_EGF_yCD (SEQ ID NO: 5), RGD4C_EGF_yCD (SEQ ID NO: 7), yCD_RGD_EGF (SEQ ID NO: 9), yCD_RGD4C_EGF (SEQ ID NO: 11), RGD_VEGF_yCD (SEQ ID NO: 13), RGD4C_VEGF_yCD (SEQ ID NO: 15), RGD_EGF_yCD_yUPRT (SEQ ID NO: 6), RGD4C_EGF_yCD_yUPRT (SEQ ID NO: 8), yCD_yUPRT_RGD_EGF (SEQ ID NO: 10), yCD_yUPRT_RGD4C_EGF (SEQ ID NO: 12), RGD_VEGF_yCD_yUPRT (SEQ ID NO: 14), and RGD4C_VEGF_yCD_yUPRT (SEQ ID NO: 16), when the prodrug enzyme is EcCD (SEQ ID NO: 17), the targeted prodrug enzyme fusion carrier is further selected from the group consisting of RGD_EGF_EcCD_WT (SEQ ID NO: 21), RGD4C_EGF_EcCD_WT (SEQ ID NO: 23), EcCD_WT_RGD_EGF (SEQ ID NO: 25), EcCD_WT_RGD4C_EGF (SEQ ID NO: 27), RGD_VEGF_EcCD_WT (SEQ ID NO: 29), RGD4C_VEGF_EcCD_WT (SEQ ID NO: 31), when the prodrug enzyme is EcCD_D314A (SEQ ID NO: 18), the targeted prodrug enzyme fusion carrier is further selected from the group consisting of RGD_EGF_EcCD_D314A (SEQ ID NO: 22), RGD4C_EGF_EcCD_D314A (SEQ ID NO: 24), EcCD_D314A_RGD_EGF (SEQ ID NO: 26), EcCD_D314A_RGD4C_EGF (SEQ ID NO: 28), RGD_VEGF_EcCD_D314A (SEQ ID NO: 30), and RGD4C_VEGF_EcCD_D314A (SEQ ID NO: 32), wherein the targeted prodrug enzyme fusion carrier can convert a 5-fluorocytosine (5-FC) into a 5-fluorouracil (5-FU), wherein the targeted prodrug enzyme fusion carrier is administered a therapeutically effective amount to a subject in need thereof, wherein the subject is diagnosed with cancer.

3. The targeted prodrug enzyme fusion carrier according to claim 1, further comprises a marker molecule.

4. The targeted prodrug enzyme fusion carrier according to claim 3, wherein the marker molecule is selected from the group consisting of a radionuclide and a fluorescent protein.

5. The targeted prodrug enzyme fusion carrier according to claim 4, wherein the radionuclide is selected from the indium-111 (.sup.111In), gallium-67, gallium-68, yttrium-90 and lutetium-177.

6. The targeted prodrug enzyme fusion carrier according to claim 5, further comprises a metal chelating agent.

7. The targeted prodrug enzyme fusion carrier according to claim 6, wherein the metal chelating agent is selected from the group consisting of DTPA, NOTA and DOTA.

8. The targeted prodrug enzyme fusion carrier according to claim 7, wherein the molar ratio of the prodrug enzyme and the metal chelating agent is less than 20%.

9. A kit comprising the targeted prodrug enzyme fusion carrier of claim 1 and a prodrug, wherein the prodrug is 5 fluorocytosine (5-FC).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIGS. 1A-1C show the recombinant protein design, expression and purification of RGD4C-yCD, yCD, RGD4C-yCD/UPRT, yCD/UPRT, RGD-VEGF-yCD, RGD4C-VEGF-yCD, RGD-EGF-yCD, RGD4C-EGF-yCD, yCD-RGD-EGF, yCD-RGD4C-EGF, RGD-VEGF-yCD/UPRT, RGD4C-VEGF-yCD/UPRT, RGD-EGF-yCD/UPRT, RGD4C-EGF-yCD/UPRT, yCD/UPRT-RGD-EGF as well as yCD/UPRT-RGD4C-EGF. FIG. 1A is a schematic diagram for the carrier and the gene; FIG. 1B is the purified protein analyzed by SDS-PAGE; and FIG. 1C shows the purified protein analyzed by western blot method and detected by anti-His6-tag antibody (Lane 1: yCD (18.7 kDa), lane 2: RGD4C-yCD (19.6 kDa), lane 3: yCD/UPRT (43.1 kDa), lane 4: RGD4C-yCD/UPRT (44.1 kDa)).

(3) FIGS. 2A-2B show the enzyme activities of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT proteins to convert 5-FC to 5-FU. 50 nM of RGD4C-yCD, yCD, RGD4C-yCD/UPRT or yCD/UPRT protein reacted with increasing concentrations of 5-FC in phosphate buffered saline solutions. FIG. 2A shows the consumption of 5-FC and FIG. 2B shows the formation amount of 5-FU. The measured data was used to plot the enzyme kinetic curve that takes the substrate concentration [S] as the x-axis and takes the initial reaction rate V.sub.0 as the y-axis,

(4) FIGS. 3A-3B show the the binding affinity and specificity of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT proteins to integrin .sub.v.sub.3. The binding affinities of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT proteins to integrin .sub.v.sub.3 were determined by ELISA assay using horseradish peroxidase (HRP)-tagged anti-His6 antibody (FIG. 3A). The dissociation constant K.sub.d is obtained by prism software with curve fitting method. FIG. 3B shows that RGD4C-yCD and RGD4C-yCD/UPRT are respectively added into the competing objects (RGDfv peptide) with different concentrations, to observe the inhibition of RGD4C-yCD and RGD4C-yCD/UPRT bound to integrin .sub.v.sub.3. (*compared with untreated RGDfv group, P<0.05)

(5) FIGS. 4A-4C shows that the expression level of integrin .sub.v.sub.3 in different cell lines analyzed by western blotting method and quantified by Image J software. FIG. 4A shows the expression quantity of v integrin protein in various cell lines; FIG. 4B shows the expression quantity of .sub.3 integrin protein in various cell lines; and FIG. 4C shows the expression quantity of -actin protein in various cell lines, respectively.

(6) FIGS. 5A-5C shows the binding affinity of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT to integrin .sub.v.sub.3 in high expression and low expression cell lines. FIG. 5A, FIG. 5B, and FIG. 5C are respectively represented HUVEC cells, U87MG cells, MCF-7 cells seeded in 96-well plates for 24 hours, and then RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT with different concentrations are added for cells binding assay. After reacted with anti-His-HRP antibody and TMB substrate, ELISA reader (OD450 nm) is used to detect the amount of recombinant protein bound to the cells, and the binding curve is drawn. The nonlinear curve of the prism software is used to make a diagram and calculate the dissociation constant (K.sub.d).

(7) FIGS. 6A-6B show the in-vitro competitive binding assay. For observing the ability of the competing objects to inhibit the fusion protein bound with integrin .sub.v.sub.3, the U87MG cells (FIG. 6A) and HUVEC cells (FIG. 6B) were incubated with RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT and then added competing objects (RGDfv peptide) with different concentrations. The RGDfv peptide competing objects with 10-fold relative concentration can inhibit the RGD4C-yCD and RGD4C-yCD/UPRT bound with integrin .sub.v.sub.3 (approximately 50% reduction), (*compared with untreated RGDfv group, P<0.05).

(8) FIGS. 7A-7C shows that RGD4C-yCD/5-FC and RGD4C-yCD/UPRT/5-FC combination treatment significantly reduces cell viability of high-integrin .sub.v.sub.3-expressing cells. The cell viability was determined by MTT assay. The seeded HUVEC cells shown in FIG. 7A, the seeded U87MG cells shown in FIG. 7B and the 96 wells of seeded MCF-7 cells shown in FIG. 7C are respectively added with 500 nM RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT recombinant protein, after removing the recombinant protein not bound with the cells and washing the cells, add 5-FC with different concentrations and observe the cell killing effect. Cells of the control groups were only incubated with different concentrations of 5-FU or 5-FC without protein treatment.

(9) FIGS. 8A-8D show that the fluorescence microscopy is used to observe the binding and distribution of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT fusion proteins in HUVEC and U87MG cells. Wherein, FIGS. 8A-B show that the HUVEC cells incubated with culture medium containing RGD4C-yCD and RGD4C-yCD/UPRT protein at 4 C. (FIG. 8A) or 37 C. (FIG. 8A) for 2 hours, and then the fluorescent staining is performed for the cells, and the confocal fluorescence microscope is used to observe the distribution of protein in cells; FIGS. 8C-D show that the U87MG cells incubated with culture medium containing RGD4C-yCD and RGD4C-yCD/UPRT protein at 4 C. (FIG. 8C) or 37 C. (FIG. 8D) for 2 hours, and then the fluorescent staining is performed for the cells, and the confocal fluorescence microscope is used to observe the distribution of protein in cells. (Green signal: .sub.3 integrin; red signal: anti-His6-tag (also represents the signal of the fusion protein); blue signal: Hoechst 33342 cell nuclear stain) (Calibration scale: 20 m).

(10) FIGS. 9A-9D show the cell apoptosis assay of the yCD series fusion protein combined with 5-FC in HUVEC and U87MG cells. Wherein, FIG. 9A and FIG. 9C show that the HUVEC and U87MG cells were treated with 500 nM yCD series proteins and combined with 5-FC at different concentrations. And then TUNEL assay kit was used to detect the cell apoptosis (red signal: DNA fragment detected by TUNEL assay kit; blue signal: Hoechst 33342 cell nuclear stain). Cells of the control groups were only incubated with different concentrations of 5-FU or 5-FC without protein treatment. FIG. 9B and FIG. 9D show that the TUNEL fluorescence image was quantified by MetaMorph software. (*P value<0.05; Calibration scale: 20 m)

(11) FIGS. 10A-10C show the design, expression and purification of EcCD series fusion protein (EcCD_WT, RGD4C-EcCD_WT, EcCD_D314A, RGD4C-EcCD_D314A, RGD-VEGF-EcCD_WT, RGD4C-VEGF-EcCD_WT, RGD-EGF-EcCD_WT, RGD4C-EGF-EcCD_WT, EcCD_WT-RGD-EGF, EcCD_WT-RGD4C-EGF, RGD-VEGF-EcCD_D314A, RGD4C-VEGF-EcCD_D314A, EcCD_D314A-RGD-EGF, EcCD_D314A-RGD4C-EGF, RGD-EGF-EcCD_D314A and RGD4C-EGF-EcCD_D314A). Wherein, FIG. 10A is a schematic diagram for carrier and gene; FIG. 10B shows the analysis of purified protein by SDS-PAGE; and FIG. 10C shows the analysis of purified protein by western blot method using horseradish peroxidase (HRP)-tagged anti-His6 antibody (Lane 1: EcCD_WT (48.8 kDa), Lane 2: EcCD_D314A (48.7 kDa), Lane 3: RGD4C-EcCD_WT (49.9 kDa), Lane 4: RGD4C-EcCD_D314A (49.9 kDa)).

(12) FIGS. 11A-11D show the activity and kinetics analysis of EcCD series fusion protein enzymes, and the EcCD series proteins are combined with 5-FC at different concentrations for reaction. Respectively calculate the 5-FU formation reaction rate shown in FIG. 11A, 5-FC consumption reaction rate shown in FIG. 10B, uracil formation reaction rate shown in FIG. 10C and cytosine consumption reaction rate shown in FIG. 10D, substitute Michaelis-Menten formula, and calculate the base mass (K.sub.cat) and K.sub.m value catalyzed by each mol enzyme in unit time.

(13) FIGS. 12A-12C show an enzyme stability analysis of EcCD series fusion protein and yCD protein. FIG. 12A shows that the EcCD series fusion protein and yCD protein are cultured in 100% FBS for 0-24 hours, then the enzyme kinetics is determined, and K.sub.cat/K.sub.m is used to show the catalytic efficiency and specificity of the enzyme to express the enzyme activity, culture for 0 hours to take the control group as a denominator, culture for 1 to 24 hours to take the experimental group as a numerator. FIG. 12B shows that the EcCD fusion protein and yCD protein are cultured in 10% FBS for 0-24 hours, then the enzyme kinetics is determined, and K.sub.cat/K.sub.m is used to show the catalytic efficiency and specificity of the enzyme to express the enzyme activity; and FIG. 12C shows that the EcCD fusion protein and yCD protein are cultured in 3 mg/ml transferrin for 0-24 hours, then the enzyme kinetics is determined, and K.sub.cat/K.sub.m is used to show the catalytic efficiency and specific enzyme activity.

(14) FIGS. 13A-13B show the binding activity and specificity test of EcCD fusion protein and v3 integrin receptor. Wherein, FIG. 13A shows that ELISA is used to analyze the binding of the EcCD fusion protein and the .sub.v.sub.3 integrin receptor, and calculate the K.sub.d value of the binding; and FIG. 13B shows that the competing objects at different concentrations (RGDfv peptide) are added to the EcCD fusion protein, and observe the binding inhibition of EcCD fusion protein and .sub.v.sub.3 integrin. (***compared with untreated RGDfv group, P value<0.001, indicating a statistically significant difference)

(15) FIGS. 14A-14E show the binding activity and specificity test of EcCD fusion protein and U87MG, HUVEC and MCF-7 cell strain. Wherein, FIGS. 14A-14C show that an ELISA method is used to analyze the binding of EcCD fusion protein and U87MG, HUVEC and MCF-7 cells, and calculate the K.sub.d value of the binding; FIG. 14D and FIG. 14E show that the competing objects at different concentrations (RGDfv peptide) are respectively added to the EcCD fusion protein, and observe the binding inhibition of EcCD fusion protein and cell strain U87MG, HUVEC of high expression of .sub.v.sub.3 integrin. (***compared to the untreated RGDfv group, P value<0.001, indicating a statistically significant difference)

(16) FIGS. 15A-15C show the binding and distribution of EcCD fusion proteins in U87MG, HUVEC and MCF-7 cell strain. The immunofluorescence staining is used to observe and experiment the EcCD fusion protein, which are respectively acted with U87MG cells in FIG. 15A, HUVEC cells in FIG. 15B and MCF-7 cells in FIG. 15C for 2 hours at 37 C., then fixed with 2% para-formadehyde, the fixed cells are first permeabilized with 0.1% Triton X-100 for 10 minutes, after that, cultured with 1% BSA blocking (dissolved in PBS containing Ca.sup.2+, Mg.sup.2+) for 1 hour at room temperature, then add a 1st antibody (Rabbit anti-His.sup.6tag, Mouse anti-human CD51/61.sub.v.sub.3 integrin), and then add a 2nd antibody (Anti-rabbit IgG-CF640R, Anti-mouse IgG-Alexa488), the protein distribution is observed by conjugated fluorescence microscope. (Green signal: .sub.v.sub.3 integrin; red signal: anti-His.sup.6tag; blue signal: Hoechst 33342 cell nucleus stain) (Scale bars: 20 m)

(17) FIGS. 16A-16E show that in vitro cell killing effect of the EcCD fusion protein combined with 5-FC. Relative cell viability was measured by MTT assay. The 50 nM EcCD series fusion protein is applied to U87MG, HUVEC and MCF-7 cells at 37 C. for 2 hours, after washing the unbound protein with PBS. In combination with 5-FC at different concentrations, separately add the group of 5-FU and 5-FC at different concentrations as the positive control group and the negative control group, the cell survival proportion is expressed by the amount of live cell metabolite formazan in MTT, and calculate the IC.sub.50 value of the 5-FC concentration.

(18) FIGS. 17A-17D show the elution curve of the protein reacted with DTPA dianhydride purified by AKTA FPLC desalting column. FIG. 17A shows yCD protein, FIG. 17B shows RGD4C-yCD protein, FIG. 17C shows DTPA-yCD protein, and FIG. 17D shows DTPA-RGD4C-yCD protein.

(19) FIG. 18 shows the enzyme kinetic curve of DTPA-yCD and DTPA-RGD4C-yCD. The enzyme activity of yCD is not affected by DTPA modification. Compared with the original protein, DTPA-RGD4C-yCD is slightly reduced to 80%.

(20) FIG. 19 shows a specific binding affinity analysis of DTPA-yCD and DTPA-RGD4C-yCD protein to integrin v3 receptor by ELISA.

(21) FIG. 20 shows the protein electrophoresis analysis of DTPA-yCD and DTPA-RGD4C-yCD. The molecular weights of DTPA-yCD (lane 2) and DTPA-RGD4C-yCD (lane 4) are higher than that of the original protein yCD (lane 1) and RGD4C-yCD (lane 3). DTPA-yCD formation dimer ratio is 8%. DTPA-RGD4C-yCD formation dimer ratio is 4%.

(22) FIG. 21 shows the molecular weight and corresponding strength of the protein determined by using MALDI-TOF MS. The value indicated on the diagram is molecular weight, and the average yCD is modified by 1.16 DTPA.

(23) FIG. 22 shows the molecular weight and corresponding strength of the protein determined by using MALDI-TOF MS. The value indicated on the diagram is molecular weight, and the average RGD4C-yCD is modified by 0.94 DTPA.

(24) FIGS. 23A-23J shows a instant thin-layer chromatography (ITLC) of .sup.111In-DTPA-yCD and .sup.111InDTPA-RGD4C-yCD. FIG. 23A and FIG. 23B show the indium-111 labeled DTPA-yCD and DTPA-RGD4C-yCD, as well as the indium-111 reacted with DTPA-yCD and DTPA-RGD4C-yCD at 37 C. for 1 hour at a specific activity of 30 Ci/g; FIG. 23C and FIG. 23D show that DTPA added with a 10-fold molar excess of DTPA-yCD and DTPA-RGD4C-yCD is reacted at room temperature for 1 hour to remove free indium-111 which is not chelated by the DTPA; FIG. 23E and FIG. 23F show that .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD are purified and concentrated by centrifugation for 2 times. FIG. 23G and FIG. 23H show that .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD are purified and concentrated by centrifugation for 4 times. FIG. 23I and FIG. 23J shows that .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD are purified by centrifugation for 5 times, and the solution is replaced from pH 5.5 to pH 7 HEPES buffer. Thin layer analysis conditions: the stationary phase is ITLC-SG, the mobile phase is 0.5M, pH 4.6 citrate buffer solution.

(25) FIGS. 24A-24B show the kinetic curve of yCD series protein modified, labeled and purified by DTPA. The enzyme activity of the two proteins is not affected by the labeling conditions. After adding EDTA with a 10-fold molar excess of protein, and it is competitively purified, In-DTPA-yCD compared with the original enzyme activity. The Vmax in FIG. 24A is reduced to 47%; and the In-DTPA-RGD4C-yCD in FIG. 24B compared with the original enzyme activity, the Vmax is reduced to 40%.

(26) FIG. 25 shows the specific binding ability analysis of the DTPA modified and In-labeled yCD series protein to the integrin .sub.v.sub.3 receptor by ELISA assay. In-DTPA-RGD4C-yCD under the condition of label, K.sub.d is 25.41.5 nM. After adding EDTA with a 10-fold molar excess of protein, the Kd of In-DTPA-RGD4C-yCD to the integrin .sub.v.sub.3 receptor is 34.65.4 nM, indicating that the fusion protein is slightly affected by the reaction conditions. Wherein yCD can not be bound with the integrin .sub.v.sub.3 receptor, with or without reaction conditions.

(27) FIG. 26 shows the stability of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD in HEPES buffer solution (4 C.) and fetal bovine serum (37 C.), part of the samples are respectively taken for radiochemical purity analysis at 1, 2, 4, 14, 24 and 48 hours.

(28) FIG. 27 shows the integrin expression level of different cell lines that identified by using flow cytometry. HUVEC and U87MG show high expression amount of integrin .sub.v.sub.3, MCF-7 shows low expression level; MCF-7 shows moderate expression level of integrin .sub.v.sub.5, HUVEC and U87MG show less expression level; all of three cell lines show a great expression level of integrin .sub.1.

(29) FIG. 28 shows a competitive cell uptake experiment of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD in integrin .sub.v.sub.3 high expressed (HUVEC, U87MG) or low expressed (MCF-7) cells. The uptake of .sup.111In-DTPA-RGD4C-yCD in HUVEC and U87MG cells (high integrin .sub.v.sub.3 expression) is significantly higher than that of MCF-7 (low integrin .sub.v.sub.3 expression). The uptake of .sup.111In-DTPA-yCD is low in the all three cell lines. After adding cRGDfk with a 1000-fold molar excess of protein, the uptake of .sup.111In-DTPA-RGD4C-yCD in HUVEC, U87MG and MCF-7 cells is respectively decreased by about 65%, 55% and 45%. The decreased part after competition indicated that .sup.111In-DTPA-RGD4C-yCD is specifically bound with the integrin receptor on the cell membrane. The uptake amount of .sup.111In-DTPA-RGD4C-yCD by each cell strain is used as a control group, and analyzed by two sample t-test statistical methods, *P-value<0.05, **P-value<0.01, ***P-value<0.001, ****P-value<0.0001.

(30) FIG. 29 shows the expression amount of integrin in different ovarian cancer cells that identified by flow cytometry. ES-2-luc and SKOV3ip1 express the integrin .sub.v.sub.3 moderately and slightly, and OVCAR-3 shows no expression amount; ES-2-luc and OVCAR-3 moderately express the integrin .sub.v.sub.5, SKOV3ip1 has less expression amount; all three cell strains have a great expression amount of integrin .sub.1.

(31) FIG. 30 shows a competitive cell uptake assay of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD in ES-2-luc cells (moderate integrin .sub.v.sub.3 expression). The uptake of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD in ES-2-luc cells is significantly different. After adding cRGDfk with a 1000-fold molar excess of protein, the uptake of .sup.111In-DTPA-RGD4C-yCD in ES-2-luc cells is decreased by about 70%. The decreased part after competition indicated that the .sup.111In-DTPA-RGD4C-yCD is specifically bound with the integrin receptor on the cell membrane. The uptake amount of .sup.111In-DTPA-RGD4C-yCD by cell is used as a control group, and analyzed by two sample t-test statistical methods, *P-value<0.05, **P-value<0.01, ***P-value<0.001, ****P-value<0.0001.

(32) FIG. 31 shows microSPECT/CT angiography at 1, 4, 24 and 48 hours after intratumoral injection of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD in U87MG tumor-bearing nude mice. The arrow is marked as a tumor.

DETAILED DESCRIPTION OF THE INVENTION

(33) The present invention provides a targeted prodrug enzyme fusion carrier, comprising a target molecule and a prodrug enzyme. The target molecule is selected from, for example, but not limited to, arginine-glycine-aspartic acid (RGD), RGD4C, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) arginine-glycine-aspartic acid-epidermal growth factor (RGD-EGF), arginine-glycine-aspartic acid-vascular endothelial growth factor (RGD-VEGF), RGD4C-EGF and RGD4C-VEGF.

(34) In one embodiment, the prodrug enzyme is selected from, for example, but not limited to, deaminase, pyrimidine deaminase, cytosine deaminase, yeast cytosine deaminase (yCD) (SEQ ID NO: 1), E. coli. cytosine deaminase (EcCD), E. coli. cytosine deaminase (EcCD) mutants, purine deaminase, adenine deaminase, guanine deaminase, 8-oxoguanine deaminase, 5-deoxyadenosine deaminase, uracil phosphoribosyltransferase, thymidine kinase, penicillin amidase, alkaline phosphatase, alcohol dehydrogenase, -lactamase, -glucoronidase, carboxyesterases, carboxypeptidase A, carboxypeptidase G2, glycosidases and nitroreductase.

(35) In one embodiment, the targeted prodrug enzyme fusion carrier further comprises a marker molecule, wherein the marker molecule is selected from, for example, but not limited to, a radionuclide, indium-111 (.sup.111In), gallium-67, gallium-68, yttrium-90, lutetium-177, and a fluorescent protein. The metal chelating agent is selected from, for example, but not limited to, DTPA, NOTA and DOTA. The molar ratio of the prodrug enzyme and the metal chelating agent is less than 20%.

(36) Moreover, the targeted prodrug enzyme fusion protein for example, but not limited to, RGD4C-yCD (SEQ ID NO: 3), RGD4C_yCD_yUPRT (SEQ ID NO: 4), RGD_EGF_yCD (SEQ ID NO: 5), RGD_EGF_yCD_yUPRT (SEQ ID NO: 6), RGD4C_EGF_yCD (SEQ ID NO: 7), RGD4C_EGF_yCD_yUPRT (SEQ ID NO: 8), yCD_RGD_EGF (SEQ ID NO: 9), yCD_yUPRT_RGD_EGF (SEQ ID NO: 10), yCD_RGD4C_EGF (SEQ ID NO: 11), yCD_yUPRT_RGD4C_EGF (SEQ ID NO: 12), RGD_VEGF_yCD (SEQ ID NO: 13), RGD_VEGF_yCD_yUPRT (SEQ ID NO: 14), RGD4C_VEGF_yCD (SEQ ID NO: 15), RGD4C_VEGF_yCD_yUPRT (SEQ ID NO: 16), RGD4C_EcCD_WT (SEQ ID NO: 19), RGD4C_EcCD_D314A (SEQ ID NO: 20), RGD_EGF_EcCD_WT (SEQ ID NO: 21), RGD_EGF_EcCD_D314A (SEQ ID NO: 22), RGD4C_EGF_EcCD_WT (SEQ ID NO: 23), RGD4C_EGF_EcCD_D314A (SEQ ID NO: 24), EcCD_WT_RGD_EGF (SEQ ID NO: 25), EcCD_D314A_RGD_EGF (SEQ ID NO: 26), EcCD_WT_RGD4C_EGF (SEQ ID NO: 27), EcCD_D314A_RGD4C_EGF (SEQ ID NO: 28), RGD_VEGF_EcCD_WT (SEQ ID NO: 29), RGD_VEGF_EcCD_D314A (SEQ ID NO: 30), RGD4C_VEGF_EcCD_WT (SEQ ID NO: 31), RGD4C_VEGF_EcCD_D314A (SEQ ID NO: 32).

(37) As mentioned above, the targeted prodrug enzyme fusion carrier of the present invention can effectively identify and bind the tumor cells, tumor-induced tumor angiogenesis and tumor microenvironment. The targeted prodrug enzyme fusion carrier also has a targeted prodrug enzyme fusion protein and a theranostic system utilizing the method of in vivo nuclear medicine for the clinical diagnosis and treatment of individual patients with tumors.

(38) The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE

(39) 1. Preparation, purification and identification of RGD4C-yCD, yCD, RGD4C-yCD/UPRT, yCD/UPRT, RGD-VEGF-yCD, RGD4C-VEGF-yCD, RGD-EGF-yCD, RGD4C-EGF-yCD, yCD-RGD-EGF, yCD-RGD4C-EGF, RGD-VEGF-yCD/UPRT, RGD4C-VEGF-yCD/UPRT, RGD-EGF-yCD/UPRT, RGD4C-EGF-yCD/UPRT, yCD/UPRT-RGD-EGF and yCD/UPRT-RGD4C-EGF recombinant proteins. After the expression carrier construction is completed by pET28a(+) yCD, pET28a(+)-RGD4C-yCD, pET28a(+)-yCDUPRT, and pET28a(+) RGD4C-yCD/UPRT, pET28a(+)-RGD4C-VEGF-yCD, pET28a(+)-RGD-EGF-yCD, pET28a(+)-RGD4C-EGF-yCD, pET28a(+)-yCD-RGD-EGF, pET28a(+)-yCD-RGD4C-EGF, pET28a(+)-RGD-VEGF-yCD/UPRT, pET28a(+)-RGD4C-VEGF-yCD/UPRT, pET28a(+)-RGD-EGF-yCD/UPRT, pET28a(+)-RGD4C-EGF-yCD/UPRT, pET28a(+)-yCD/UPRT-RGD-EGF and pET28a(+)-yCD/UPRT-RGD4C-EGF (see FIG. 1A), the E. coli BL21 (DE3) strain is introduced by heat shock mode, and the protein expression is induced by IPTG. After purification, the purified protein is analyzed by SDS-PAGE and Western blot (see FIG. 1B and FIG. 1C). From the results of SDS-PAGE, it was observed that all proteins are consistent with the corresponding molecular weight, and then the protein detected by anti-His6-tag-HRP antibody is also consistent with the result of SDS-PAGE. The purified yields of yCD and RGD4C-yCD are about 56.1 mg/L and 25.6 mg/L respectively. The purified yields of yCD/UPRT and RGD4C-yCD/UPRT are about 22.0 mg/L and 21.6 mg/L respectively.

(40) 2. Enzyme Activity and Kinetic Analysis of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT Recombinant Proteins

(41) The enzyme activity of 5-FC converted into 5-FU by RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT is performed. The recombinant protein concentration (50 nM) added with 5-FC at different concentrations. The 5-FC consumption and the generated amount of 5-FU is calculated. The measured data is taken as the x-axis by the substrate concentration [S]; the initial reaction rate V.sub.0 is the kinetic curve by making a diagramthe with y-axis (see FIG. 2A and FIG. 2B); the non-linear regression is made by the prism software according to the Michaelis-Menten equation; calculate the enzyme kinetic parameter K.sub.m and V.sub.max. Calculated by the 5-FC consumption, K.sub.m and V.sub.max values of yCD and RGD4C-yCD are similar, the values are 0.70.1 mM and 0.60.1 mM and 175.84.7 and 179.54.7 M/min, respectively (see FIG. 2A). Calculated by 5-FU, the values are not significantly different from the calculated enzyme kinetic parameters of 5-FC consumption. The K.sub.m values are 1.00.1 mM and 0.90.1 mM, respectively, and V.sub.max was 164.24.7 and 167.74.6 M/min respectively (see FIG. 1B); the kinetic parameters are calculated by 5-FC consumption for the yCD/UPRT and RGD4C-yCD/UPRT, the K.sub.m and V.sub.max of both are similar, and the values are 1.20.1 mM and 1.20.2 mM and 137.23.6 and 147.05.9 M/min respectively; When calculated by 5-FU, the K.sub.m values are 1.20.1 mM and 1.20.2 mM respectively, similar to the parameters calculated by 5-FC consumption. V.sub.max values are 112.78.1 and 109.210.1 M/min respectively, which is smaller than the V.sub.max calculated by 5-FC consumption. In summary, the catalytic rate of yCD/UPRT and RGD4C-yCD/UPRT for 5-FC is slightly slower than that of yCD and RGD4C-yCD, while the K.sub.m values are not significantly different from each other. It is speculated that the yCD function in each fusion protein is similar, but yUPRT may affect the effects of yCD and 5-FC. The results also show that yCD and yCD/UPRT have little effect on the activity of RGD4C short-chain peptide fusion.

(42) 3. Binding Activity and Specificity Test of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT Recombinant Protein and Integrin .sub.v.sub.3 Receptor

(43) The specific binding ability of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT recombinant protein to integrin v3 receptor is analyzed by ELISA binding assay, plotted with prism software and made with curve fitting to find the dissociation constant K.sub.d value. The experiment results show that yCD and yCD/UPRT with circular RGD4C sequence have good binding ability to integrin .sub.v.sub.3 receptor (K.sub.d is about 39.55.1 nM and 41.73.9 nM respectively) (see FIG. 3A and Table 1). The yCD and yCD/UPRT cannot be bound to integrin .sub.v.sub.3 due to be not similar RGD4C as short-chain peptide. However, the RGDfv peptide at same concentration can inhibit the RGD4C-yCD and RGD4C-yCD/UPRT bound to integrin .sub.v.sub.3 (decreased by 50%) (see FIG. 3B), which shows that RGD4C-yCD and RGD4C-yCD/UPRT are specifically bound with integrin .sub.v.sub.3 through the RGD4C peptide sequence.

(44) TABLE-US-00001 TABLE 1 The dissociation constant K.sub.d value of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT recombinant protein and integrin v3 receptor. Protein K.sub.d yCD N/A RGD4C-yCD 39.5 5.1 nM yCD/UPRT N/A RGD4C-yCD/UPRT 41.7 3.9 nM

(45) 4. The Expression Amount of the Integrin .sub.v.sub.3 Receptor in Different Cell Strains Analyzed by Western Blotting Method

(46) The protein lysates of each cell lines are subjected to protein electrophoresis, and then stained with anti-.sub.v and .sub.3 integrin antibodies and corresponding secondary fluorescent antibodies, and the expression levels of .sub.v and .sub.3 integrin receptors of each cell lines are analyzed by western blotting method (see FIG. 4A). The results show that the HUVEC and U87MG cells express higher .sub.v and 3 integrin than other strain cells, while MCF-7 cells almost do not express .sub.v and .sub.3 integrin. FIG. 4B and FIG. 4C show that the results of ROI selected and quantified by Image J image analysis software. Normalization is performed through -actin (internal control) quantitative values divided by the .sub.v and .sub.3 integrin quantitative values, and the ratio is defined as 1.0 after normalization is obtained by HUVEC cells. The results show that the expression amount of v and .sub.3 integrin in HUVEC and U87MG cells is about 10 times higher than that in MCF-7 cells. This study selects these three cells for subsequent experiments related to RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT recombinant protein.

(47) 5. Specific Binding Assay of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT Recombinant Protein and Cell

(48) As demonstrated above, the three cell strains are respectively seeded in a 96-well plate for 24 hours, while comparison of different protein concentration of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT is added and co-cultured with the cells for binding assay. The anti-His6-Tag-HRP antibody and TMB show the color reagent to detect the amount of recombinant protein bound to the cell, then the dissociation constant K.sub.d value of the recombinant protein and the integrin .sub.v.sub.3 receptor is calculated and a binding curve is drawn. The results show (see FIG. 5 and Table 2) that yCD and yCD/UPRT only show weak binding to the three cells. The RGD4C-yCD and RGD4C-yCD/UPRT are able to bind with highly expressing the integrin .sub.v.sub.3 receptor of HUVEC (dissociation constant K.sub.d is 47.57.4 nM and 48.39.8 nM respectively) and U87MG tumor cells (dissociation constant K.sub.d is 53.75.6 nM and 57.810.5 nM respectively). However, MCF-7 tumor cells with low integrin v3 receptor expression have no obvious binding ability (dissociation constants K.sub.d is 365.9104.1 nM and 335.193.2 nM respectively) with RGD4C-yCD and RGD4C-yCD/UPRT. The cell binding ability of RGD4C-yCD for HUVEC and U87MG are about 7.7 folds and 6.8 folds higher than MCF-7 respectively. The cell binding ability of RGD4C-yCD/UPRT for HUVEC and U87MG are about 6.9 folds and 5.8 folds higher than MCF-7 respectively. Competition with RGDfv peptide at 10-fold relative concentration can inhibit RGD4C-yCD and RGD4C-yCD/UPRT can be bound with integrin .sub.v.sub.3 (approximately decreased by 50%) (see FIG. 6). It is shown that RGD4C-yCD and RGD4C-yCD/UPRT are specially bound with integrin .sub.v.sub.3 through RGD4C sequence.

(49) TABLE-US-00002 TABLE 2 Dissociation constant K.sub.d values of RGD4C-yCD and RGD4C-yCD/UPRT and integrin .sub.v.sub.3 receptor. Protein (K.sub.d) RGD4C-yCD RGD4C-yCD/UPRT HUVEC vascular cell 47.5 7.4 nM 48.3 9.8 nM U87MG tumor cell 53.7 5.6 nM 57.8 10.5 nM MCF-7 tumor cell 365.9 104.1 nM 335.1 93.2 nM

(50) 6. Cytotoxicity Assay of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT Combined with 5-FC

(51) In this experiment, the MTT assay is applied to measure cytotoxicity induced by RGD4C-yCD/5-FC and RGD4C-yCD/UPRT/5-FC of the targeted prodrug enzyme fusion protein. In 96-well plate of seeded cells is respectively added with recombinant proteins such as 500 nM RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT, and co-cultured at 37 C. for 2 hours. The recombinant proteins not bound with cells are discarded. The cells were washed, and then were treated with different concentrations of 5-FC for observing the effect of cytotoxicity. On the other hand, the cells only treated with different concentrations of 5-FC and 5-FU separately as the control group. The experimental results show that the significant cytotoxic ability to HUVEC, U87MG and MCF-7 cells are treated with 5-FU (IC.sub.50: 1.60.2 M, 1.60.3 M and 2.10.3 M, respectively). There is not significant cytotoxic ability to HUVEC, U87MG and MCF-7 cells treated with 5-FC (shown in FIG. 7 and Table 3). Also, there is not significant cytotoxic ability to HUVEC, U87MG and MCF-7 cells treated with yCD coupled with 5-FC or yCD/UPRT coupled with 5-FC (the survival rate is higher than 80%, see FIG. 7). In other word, the results of yCD or yCD/UPRT respectly coupled with 5-FC are similar to the group treated with 5-FC. The cytotoxic effect of RGD4C-yCD coupled with 5-FC treatment to HUVEC and U87MG cells is significantly better than that of MCF-7 cells with low expression of integrin .sub.v.sub.3 (IC.sub.50 of the three group is 23.63.5, 37.115.4 and 5130.71002.4 M, respectively). The cytotoxic effect of the group of RGD4C-yCD/UPRT with 5-FC treatment to HUVEC and U87MG cells (IC.sub.50 is 8.61.5 and 5.11.3 M, respectively) is better than that of RGD4C-yCD with 5-FC treatment, which shows that yUPRT can improve the cytotoxic ability by accelerating the efficiency of converting 5-FU to 5-FUMP (see FIG. 7A and FIG. 7B). There is not significant cytotoxic ability of MCF-7 cells with low expression of integrin .sub.v.sub.3 treated by RGD4C-yCD/UPRT coupled with 5-FC (IC.sub.50 is 4341.31120.9 M). This result suggests that there is a specific cytotoxic ability of cells with high expression of integrin .sub.v.sub.3 treated by RGD4C-yCD coupled with 5-FC or RGD4C-yCD/UPRT coupled with 5-FC. However, the cytotoxic effect of RGD4C-yCD coupled with 5-FC or RGD4C-yCD/UPRT coupled with 5-FC are not as good as 5-FU. Conversely, there is more cell selectivity than 5-FU. The results also confirm that 5-FC is not highly toxic to cells, so 5-FC is suitable for using with a targeted prodrug enzyme fusion protein applied to a theranostic system.

(52) TABLE-US-00003 TABLE 3 Cytotoxic ability test of RGD4C-yCD plus 5-FC treatment, RGD4C-yCD/UPRT plus 5-FC treatment and 5-FU treatment to HUVEC, U87MG and MCF-7 cells respecyly. Protein (500 nM) with 5-FC or 5-FU(IC.sub.50) RGD4C-yCD/UPRT + RGD4C-yCD + 5-FC 5-FC 5-FU Vascular 23.6 3.5 M 8.6 1.5 M 1.6 0.2 M cell HUVEC U87MG 37.1 15.4 M 5.1 1.3 M 1.6 0.3 M tumor cell MCF-7 5130.7 1002.4 M 4341.3 1120.9 M 2.2 0.3 M tumor cell

(53) 7. Binding and Distribution of RGD4C-yCD, yCD, RGD4C-yCD/UPRT and yCD/UPRT Fusion Proteins with HUVEC and U87MG Cells

(54) HUVEC and U87MG cells are co-cultured with culture medium containing 500 nM RGD4C-yCD and RGD4C-yCD/UPRT protein at 4 C. and 37 C. for 2 hours, then the cells are stained with fluorescence, and the distribution in the cell is observed by conjugated fluorescence microscopy. RGD4C-yCD and RGD4C-yCD/UPRT will be only bound to HUVEC and U87MG cell surface (red fluorescence) at 4 C., and at colocalization with .sub.3 integrin (green fluorescence) (see FIG. 8A and FIG. 8C), it is shown that both proteins can be adhered on the cell membrane through specific binding with integrin, and the weak fluorescence signals can only be detected due to that yCD and yCD/UPRT can not be effectively bound with HUVEC and U87MG cells. Under co-culturing conditions at 37 C., RGD4C-yCD and RGD4C-yCD/UPRPT can enter the inside of HUVEC and U87MG cells through endocytosis (see FIG. 8B and FIG. 8D). The study results show that RGD4C-yCD and RGD4C-yCD/UPRPT can enter the cell by binding with integrin .sub.v.sub.3.

(55) 8. Analysis of Apoptosis Induced by RGD4C-yCD/5-FC and RGD4C-yCD/UPRT/5-FC Targeted Prodrug Enzyme Fusion Protein of Theranostic System

(56) Previous literature pointed out that 5-FU can promote apoptosis by inhibiting the synthesis of cellular DNA. This experiment is performed on apoptosis analysis to evaluate whether the enzyme precursor treatment system of RGD4C-yCD/5-FC and RGD4C-yCD/UPRT/5-FC can kill the cells by apoptotic mechanism. HUVEC and U87MG cells are co-cultured with culture medium containing 500 nM RGD4C-yCD and RGD4C-yCD/UPRT protein for 2 hours at 37 C., remove and wash the unbound or endocytic proteins, add 100 M 5-FC or 5-FU, after acting for 72 hours, the DNA fragment of the apoptosis is stained with fluorescence by TUNEL assay analysis set, and the apoptosis is observed by conjugated fluorescence microscope. The results show that the groups treated with 5-FU, RGD4C-yCD/5-FC and RGD4C-yCD/UPRT/5-FC will cause a large amount of apoptosis in HUVEC and U87MG cells (see FIG. 9A and FIG. 9C, the red fluorescence position indicates the DNA fragments produced by apoptosis, and its signal mainly exists in the nucleus). Furtherly, TUNEL fluorescence signals are quantified by image analysis software MetaMorph. The degree of apoptosis induced by the groups treated with RGD4C-yCD/5-FC and RGD4C-yCD/UPRT/5-FC respectly are significantly higher than that of the yCD/5-FC and yCD/UPRT/5-FC treatment group (*P<0.05) (see FIG. 9B and FIG. 9D). Moreover, the tendency of apoptosis for treated with RGD4C-yCD/UPRT/5-FC treatment is higher than that of RGD4C-yCD/5-FC (but no statistically significant difference). The results show that RGD4C-yCD/5-FC and RGD4C-yCD/UPRT/5-FC targeted prodrug enzyme fusion protein of theranostic system can induce death of the cells through apoptosis pathway.

(57) 9. EcCD series fusion protein (EcCD_WT, RGD4C-EcCD_WT, EcCD_D314A, RGD4C-EcCD_D314A, RGD-VEGF-EcCD_WT, RGD4C-VEGF-EcCD_WT, RGD-EGF-EcCD_WT, RGD4C-EGF-EcCD_WT, EcCD_WT-RGD-EGF, EcCD_WT-RGD4C-EGF, RGD-VEGF-EcCD_D314A, RGD4C-VEGF-EcCD_D314A, EcCD_D314A-RGD-EGF, EcCD_D314A-RGD4C-EGF, RGD-EGF-EcCD_D314A and RGD4C-EGF-EcCD_D314A) design, expression and purification

(58) After the carrier construction is completed by pET28a(+)-EcCD_WT, pET28a(+)-RGD4C-EcCD_WT, pET28a(+)-EcCD_D314A, pET28a(+)-RGD4C-EcCD_D314A, pET28a(+)-RGD-VEGF-EcCD_WT, pET28a(+)-RGD4C-VEGF-EcCD_WT, pET28a(+)-RGD-EGF-EcCD_WT, pET28a(+)-RGD4C-EGF-EcCD_WT, pET28a(+)-EcCD_WT-RGD-EGF, pET28a(+)-EcCD_WT-RGD4C-EGF, pET28a(+)-RGD-VEGF-EcCD_D314A, pET28a(+)-RGD4C-VEGF-EcCD_D314A, pET28a(+)-RGD-EGF-EcCD_D314A, pET28a(+)-RGD4C-EGF-EcCD_D314A, pET28a(+)-EcCD_D314A-RGD-EGF and pET28a(+)-EcCD_D314A-RGD4C-EGF expression (see FIG. 10A), the E. coli RG2 (DE3) strains are fed by the heat shock, the protein expression is induced by IPTG. After purification, the purified protein is analyzed by SDS-PAGE and Western blot (see FIG. 10B and FIG. 10C). It can be observed from the SDS-PAGE results that all proteins meet the corresponding size positions. Then, the protein detected by anti-His6tag-HRP mab also meets the size position of the SDS-PAGE result.

(59) 10. Enzyme Kinetic Analysis of EcCD Series Fusion Proteins

(60) The EcCD series fusion protein is identified by purification and liquid chromatography mass spectrometry (LC/MS/MS) to confirm the correct protein, and the enzyme activity of cytosine deaminase is determined, that is, the ability that the protein converts the matrix (5-FC, cytosine) into (5-FU, uracil). The reaction rate of 5-FU generation and 5-FC consumption at different substrate concentrations is separately detected (see FIG. 11A, FIG. 11B, Table 4 and Table 5), and it can be found in the part detected by 5-FU generation that the K.sub.m, K.sub.cat and K.sub.cat/K.sub.m values of EcCD_WT are similar to that of RGD4C-EcCD_WT. The K.sub.m is 2.90.8 mM and 2.30.5 mM respectively, the K.sub.cat is 8.80.9 sec.sup.1 and 10.80.6 sec.sup.1 respectively, K.sub.cat/K.sub.m is 3.0 and 4.7 respectively, the part of 5-FC consumption by detection is not much different from the result generated by 5-FU. K.sub.m is 2.80.7 mM and 2.30.4 mM respectively, K.sub.cat is 9.20.8 sec.sup.1 and 10.70.5 sec.sup.1 respectively, K.sub.cat/K.sub.m is 3.3 and 4.7 respectively. And the K.sub.m, K.sub.cat and K.sub.cat/K.sub.m values of EcCD_D314A and RGD4C-EcCD_D314A in the 5-FU generation part by detection are also similar. K.sub.m is 3.90.6 mM and 3.50.3 mM respectively, K.sub.cat is 36.21.8 sec.sup.1 and 39.41.1 sec.sup.1 respectively, K.sub.cat/K.sub.m is 9.3 and 11.3 respectively, the part of 5-FC consumption by detection is not much different from the result generated by 5-FU. K.sub.m is 3.80.6 mM and 3.50.2 mM respectively, K.sub.cat is 35.11.7 sec.sup.1 and 38.61.0 sec.sup.1 respectively, K.sub.cat/K.sub.m is 9.2 and 11.0 respectively. The K.sub.cat/K.sub.m of EcCD_D314A is about 3.1 times higher than that of EcCD_WT. The K.sub.cat/K.sub.m of RGD4C-EcCD D314A is about 2.4 times higher than that of RGD4C-EcCD_WT, which increase the activity of 5-FC.

(61) TABLE-US-00004 TABLE 4 The generation reaction rate of 5-FU calculated by EcCD series proteins with 5-FC at different concentrations K.sub.cat/K.sub.m Protein K.sub.m (mM) K.sub.cat (sec.sup.1) (sec.sup.1 mM.sup.1) yCD 1.0 0.1 47.2 0.9 47.2 EcCD_D314A 3.9 1.5 35.4 0.8 9.1 RGD4C-EcCD_D314A 3.4 0.8 38.4 0.8 11.3 EcCD_WT 2.9 0.6 9.2 0.6 3.2 RGD4C-EcCDWT 2.4 0.5 10.8 0.5 4.5

(62) TABLE-US-00005 TABLE 5 The consumption reaction rate of 5-FU calculated by EcCD series proteins with 5-FC at different concentrations K.sub.cat/K.sub.m Protein K.sub.m (mM) K.sub.cat (sec.sup.1) (sec.sup.1 mM.sup.1) yCD 1.0 0.1 48.5 1.3 48.5 EcCD_D314A 4.0 10.5 36.2 1.5 9.1 RGD4C-EcCD_D314A 3.5 0.3 39.1 1.0 11.2 EcCD_WT 2.7 0.9 8.8 0.8 3.3 RGD4C-EcCDWT 2.4 0.4 10.9 0.5 4.5

(63) The reaction rate of uracil generation and cytosine consumption at different substrate concentrations is separately detected (see FIG. 11C, FIG. 11D, Table 6 and Table 7), and it can be found in the part detected by uracil generation that the K.sub.m, K.sub.cat and K.sub.cat/K.sub.m values of EcCD_WT are similar to that of RGD4C-EcCD_WT. K.sub.m is 1.60.1 mM and 1.50.2 mM respectively, K.sub.cat is 147.43.4 sec.sup.1 and 141.75.5 sec.sup.1 respectively, K.sub.cat/K.sub.m is 92.1 and 94.5 respectively, the cytosine consumption part by detection is not much different from the result generated by uracil. K.sub.m is 1.70.1 mM and 1.70.2 mM respectively, K.sub.cat is 156.52.7 sec.sup.1 and 148.94.7 sec.sup.1 respectively, K.sub.cat/K.sub.m is 92.1 and 87.6 respectively. And the K.sub.m, K.sub.cat and K.sub.cat/K.sub.m values of EcCD_D314A and RGD4C-EcCD_D314A in the uracil generation part by detection are also similar. K.sub.m is 3.00.4 mM and 2.90.6 mM respectively, K.sub.cat is 22.11.9 sec.sup.1 and 17.71.6 sec.sup.1 respectively, K.sub.cat/K.sub.m is 7.4 and 6.1 respectively, the cytosine consumption part by detection is not much different from the result generated by uracil. K.sub.m is 2.80.5 mM and 3.00.6 mM respectively, K.sub.cat is 24.31.3 sec.sup.1 and 21.61.8 sec.sup.1 respectively, K.sub.cat/K.sub.m is 8.7 and 7.2 respectively. The K.sub.cat/K.sub.m of EcCD_D314A is about 12.5 times lower than that of EcCD_WT. The K.sub.cat/K.sub.m of RGD4C-EcCD_D314A is about 15.5 times lower than that of RGD4C-EcCD_WT, which decrease the activity of cytosine and increase the selectivity to 5-FC. Evaluation of the relative substrate selectivity of cytosine deaminase enzyme for 5-FC and Cytosine can be expressed by the following formula 1,

(64) $\begin{array}{cc}\frac{K_{\mathrm{cat}}/K_m\left(5-\mathrm{FC}\right)}{\left[K_{\mathrm{cat}}/K_m\left(5-\mathrm{FC}\right)\right]+\left[K_{\mathrm{cat}}/K_m\left(\mathrm{Cyosine}\right)\right]}& \mathrm{formula}1\end{array}$

(65) By substituting the previously measured enzyme K.sub.m, K.sub.cat and K.sub.cat/K.sub.m of 5-FC and cytosine into the above formula, you can obtain a value (see Table 8). The higher the value, the higher the selectivity of the enzyme to the 5-FC substrate. The relative substrate selectivity of EcCD_WT and RGD4C-EcCD_WT to 5-FC is similar from the results, 0.032 and 0.047 respectively are not much different from 0.027 in the literature. The relative substrate selectivity of EcCD_D314A and RGD4C-EcCD_D314A to 5-FC is also similar, 0.558 and 0.648 respectively are not much different from 0.513 in the literature. EcCD_D314A is significantly higher than EcCD_WT and the relative substrate selectivity to 5-FC is about 17.4 times. The relative substrate selectivity of RGD4C-EcCD_WT and RGD4C-EcCD_D314A is about 13.8 times significantly higher than that of 5-FC. It is confirmed by the above experiment that the EcCD series fusion protein to be produced has the enzyme activity of converting 5-FC into 5-FU, and also confirmed that RGD4C short peptide chains do not affect the enzyme activity.

(66) TABLE-US-00006 TABLE 6 The generation reaction rate of uracil calculated by EcCD series protein with 5-FC at different concentrations K.sub.cat/K.sub.m Protein K.sub.m (mM) K.sub.cat (sec.sup.1) (sec.sup.1mM.sup.1) yCD 3.9 0.2 54.6 1.3 14.0 EcCD_D314A 3.1 0.3 24.5 1.1 7.9 RGD4C-EcCD_D314A 3.8 1.7 21.7 4.8 5.7 EcCD_WT 1.7 0.1 157.3 2.4 92.5 RGD4C-EcCDWT 1.7 0.1 150.8 4.4 88.7

(67) TABLE-US-00007 TABLE 7 The consumption reaction rate of cytosine calculated by EcCD series protein with 5-FC at different concentrations K.sub.cat/K.sub.m Protein K.sub.m (mM) K.sub.cat (sec.sup.1) (sec.sup.1mM.sup.1) yCD 4.7 1.3 55.6 8.5 11.8 EcCD_D314A 2.6 0.4 22.3 1.6 8.6 RGD4C-EcCD_D314A 2.9 0.5 17.7 1.3 6.3 EcCD_WT 1.3 0.1 132.0 5.0 101.5 RGD4C-EcCDWT 1.6 0.2 143.9 5.1 89.9

(68) TABLE-US-00008 TABLE 8 Comparing the relative substrate selectivity of enzyme to 5-FC and cytosine, the greater the value converted by formula 1, the higher the relative substrate selectivity of the enzyme to 5-FC [K.sub.cat/K.sub.m(5-FC)]/ {[K.sub.cat/K.sub.m(cytosine)] + Protein [K.sub.cat/K.sub.m(5-FC)]} yCD 0.771 EcCD_D314A 0.535 RGD4C- EcCD_D314A 0.664 EcCDWT 0.033 RGD4C- EcCDWT 0.048 Biochemistry 2004, 43, 0.027 8957-8964EcCDWT Biochemistry 2004, 43, 0.513 8957-8964D314A

(69) 11. Comparative Analysis of Enzyme Stability Between EcCD Series Fusion Protein and yCD Protein

(70) Previous literature reports indicate that the thermostability of E. coli cytosine deaminase (EcCD) is better than that of yCD. In order to evaluate the stability of the enzymes of the EcCD series fusion protein and yCD protein expressed in this study and evaluate its stability in serum, in the experiment, EcCD_WT, EcCD_D314A, RGD4C-EcCD_WT, RGD4C-EcCD_D314A and yCD are diluted to 100% FBS (fetal bovine serum), after culturing at 37 C. for 0, 1, 2, 4, 24 hours, determine the enzyme kinetics, evaluate the enzyme stability of the EcCD series fusion protein and yCD protein by the K.sub.cat/K.sub.m for showing the catalytic efficiency and specificity of the enzyme. It can be observed from the experimental results that the yCD protein is cultured in 100% FBS for 1 hour, the enzyme activity rapidly decreases to near inactivation, and the EcCD series fusion protein can maintain a relatively high enzyme activity (enzyme activity is close to 100%) (see FIG. 12A). In order to simulate the environment of the protein in the cell killing test, the EcCD series fusion protein and the yCD protein are cultured in 10% FBS at 37 C. for 0, 1, 2, 4, 24 hours, and then determine the enzyme kinetics. It can be observed from the results that the yCD protein is cultured in 10% FBS for 1 hour, the enzyme activity is reduced to 80%, and when cultured for 2 and 4 hours, the enzyme activity is reduced to 50% and 20%, respectively, and the enzyme activity is lost after culturing for 24 hours, while the EcCD series fusion protein is similar to the culture in 100% FBS, which can maintain the original enzyme activity (see FIG. 12B). The literature points out that the metal ion of the enzyme catalytic center will affect the stability of the enzyme. In the yCD protein involved in the enzyme reaction, the metal ion of the enzyme catalytic center has a considerable relationship with the enzyme activity. The protein associated with transporting metal ions in serum is transferrin. It is speculated that transferrin may be related to the enzyme activity of yCD protein. Therefore, the EcCD series fusion protein and yCD protein are co-cultured respectively with transferrin at a concentration of 3 mg/ml (normal concentration 2.1-3.4 mg/mL) at 37 C. for 0, 1, 2, 4, 24 hours, then determine the enzyme kinetics (see FIG. 12C), it is known from the results that the longer the yCD protein is cultured in the transferrin, the lower the enzyme activity will be. The enzyme activity will be close to the loss of activity after culturing for 24 hours. This trend is similar to the culture at 10% in FBS. It is speculated that the factor of reducing the activity of yCD protein in FBS has a great relationship with transferrin. The stability of yCD protein in FBS is poor, and the stability of EcCD series fusion protein is better, so the industrial use of EcCD series fusion protein in both sexual and therapeutic applications is better than that of yCD protein, and it is also confirmed that the mutation of RGD4C peptide and D314A will not affect the enzyme stability of EcCD protein.

(71) 12. Binding Activity and Specificity Test of EcCD Series Fusion Protein and .sub.v.sub.3 Integrin

(72) To confirm the binding ability of RGD4C peptide and .sub.v.sub.3 integrin, the affinity of the protein and .sub.v.sub.3 integrin is confirmed by ELISA method. It is confirmed that RGD4C-EcCD_WT and RGD4C-EcCD_D314A have the ability to bind to .sub.v.sub.3 integrin relative to unfused EcCD_WT and EcCD_D314A (see FIG. 13A and Table 9), K.sub.d values are approximately 7.00.9 nM and 9.11.1 nM respectively. The RGDfv peptide at same concentration can inhibit about 30% binding capacity, while the RGDfv peptide at 10-fold concentration can inhibit about 80% binding capacity (see FIG. 13B). This experiment confirms that RGD4C-EcCD_WT and RGD4C-EcCD_D314A have the binding activity of .sub.v.sub.3 integrin, and this binding has the specificity of RGD4C peptide .sub.v.sub.3 integrin.

(73) TABLE-US-00009 TABLE 9 The binding of EcCD series fusion protein and .sub..sub.3 integrin receptor is analyzed by ELISA, and the bound K.sub.d value is calculated. Protein K.sub.d (nM) EcCD_WT N/A RGD4C- EcCDWT 7.0 0.9 EcCD_D314A N/A RGD4C- EcCD_D314A 9.1 01.1

(74) 13. Binding Activity and Specificity Test of EcCD Series Fusion Protein and U87MG, HUVEC and MCF-7 Cell Strain

(75) To confirm whether RGD4C-EcCD_WT and RGD4C-EcCD D314A are bound to the .sub.v.sub.3 integrin cell strain, ELISA is used to experiment that U87MG and HUVEC cells with high expression of .sub.v.sub.3 integrin and MCF-7 cells with low expression of .sub.v.sub.3 integrin are seeded in 96-well ELISA microplate, after the cells are fixed, add EcCD series fusion protein (0250 nM) at different concentrations, then detect the protein with anti-His6tag-HRP Mab and color with TMB, stop the reaction and then measure the absorbed light reading value of OD450 nm/OD570 nm to quantify the amount of protein bound to the cell, and calculate the dissociation constant K.sub.d value of the protein-cell binding. The results show that RGD4C-EcCD_WT and RGD4C-EcCD_D314A have the ability to bind to U87MG and HUVEC cells relative to unfused EcCD_WT and EcCD_D314A of RGD4C peptide (see FIG. 14A, FIG. 14B and Table 10), and calculate the K.sub.d value of RGD4C-EcCD_WT bound with U87MG and HUVEC cells, which is 13.81.9 nM and 20.33.3 nM respectively, while the results of RGD4C-EcCD_D314A bound to U87MG and HUVEC cells are similar, the K.sub.d values are 15.11.8 nM and 23.33.4 nM respectively. As for the poor binding of RGD4C-EcCD_WT and RGD4C-EcCD_D314A to the negative control MCF-7 cells (see FIG. 14C and Table 10), the K.sub.d values are 327.1119.5 nM and 299.9126.2 nM respectively. It can be observed that the binding ability of RGD4C-EcCD_WT to U87MG and HUVEC cells is 23.7 and 16.1 times higher than that of MCF-7 respectively, and the results of RGD4C-EcCD_D314A are similar, which are 19.9 and 12.9 times higher than that of MCF-7 respectively, indicating that the binding ability of RGD4C to cells depends on the expression amount of .sub.v.sub.3 integrin, RGDfv peptide competition at 10-fold relative concentration can inhibits about 50% binding (see FIG. 14D and FIG. 14E). It is confirmed from the above experimental results that the EcCD series protein fused to the RGD4C peptide has the binding activity with the expressing .sub.v.sub.3 integrin cell strain, and the binding has the specificity of the RGD4C peptide and .sub.v.sub.3 integrin of the cell expression, and also confirmed that the D314A mutation will not affect the binding activity of the RGD4C peptide and .sub.v.sub.3 integrin.

(76) TABLE-US-00010 TABLE 10 Binding activity and specificity test of EcCD series fusion protein with U87MG, HUVEC and MCF-7 cell strain RGD4C- RGD4C- Cell (K.sub.d value, nM) EcCD_WT EcCD_D314A RGD4C-yCD U87MG (.sub..sub.3 13.8 1.9 15.1 1.8 53.7 5.6 integrin(+++)) HUVEC(.sub..sub.3 20.3 3.3 23.3 3.4 47.5 7.4 integrin(+++)) MCF-7(.sub..sub.3 327.1 119.5 299.9 126.2 365.9 104.1 integrin())

(77) 14. EcCD Series Fusion Protein Bound with U87MG, HUVEC and MCF-7 Cell Strain and Distributed in the Cells

(78) Immunofluorescence is used to observe the distribution of the protein bound with cell in the cell. U87MG, HUVEC and MCF-7 cells act with culture medium containing EcCD_WT, RGD4C-EcCD_WT, EcCD_D314A and RGD4C-EcCD_D314A proteins at 37 C. for 2 hours, stain the cell, and observe protein distribution by the conjugated fluorescence microscopy. It is known from the resulted that EcCD_WT and EcCD_D314A are not observed on the cell due to no red light signal (red light: anti-His6tag), only the green light signal of .sub.v.sub.3 integrin is observed, which indicates that EcCD_WT and EcCD_D314A do not have the ability of binding with U87MG and HUVEC cells. Relative to EcCD_WT and EcCD_D314A, RGD4C-EcCD_WT and RGD4C-EcCD_D314A have the ability of binding with U87MG and HUVEC cells (see FIG. 15A and FIG. 15B). The binding of RGD4C-EcCD_WT and RGD4C-EcCD_D314A (red light: anti-His6tag) can be observed on the cell surface, and it is superimposed with the .sub.v.sub.3 integrin (green light) detected by the antibody (yellow light), which indicates that its binding is specific. At the same time, it can be observed that RGD4C-EcCD_WT and RGD4C-EcCD_D314A will enter the internalization. It is proved from the above experiment that RGD4C-EcCD_WT and RGD4C-EcCD_D314A will enter the internalization by binding to .sub.v.sub.3 integrin on U87MG and HUVEC cells, and this binding is specific; for MCF-7 cells, EcCD series fusion protein is not observed on the cells (due to that no red light signal is observed), and no green signal is observed, and MCF-7 cell is also shown to have low expression of .sub.v.sub.3 integrin (see FIG. 15C).

(79) 15. Cytotoxic Test of EcCD Series Fusion Protein Coupled with 5-FC to Treat with U87MG, HUVEC and MCF-7 Cells

(80) To confirm whether RGD4C-EcCD_WT and RGD4C-EcCD_D314A can kill the cells by the principle of enzyme prodrug system, so it is further tested by MTT assay. After the EcCD series fusion protein (50 nM) is dissolved in the cell culture medium and cultured at 37 C. for 2 hours, the protein of the unbound or un-endocytosed cells are washed out with PBS, and 5-FC and 5-FU at different concentrations are added and cultured at 37 C. for 72 hours, and the cell survival ratio is observed by MTT assay, and its IC50 (half maximal inhibitory concentration) is obtained (see FIG. 16A, FIG. 16B, FIG. 16C and Table 11). The results show that the survival rate of U87MG, HUVEC and MCF-7 cells in the negative control group treated separately with 5-FC is close to 100%, which indicates that 5-FC will not affect the cells, and EcCD_WT and EcCD_D314A treated with 5-FC also have no obvious killing effect (survival rate is higher than 75%), while the positive control group treated separately with 5-FU has the best effect. The IC.sub.50 for U87MG, HUVEC and MCF-7 cells is: 6.50.2 M, 6.51.3 M and 25.60.3 M respectively. As for the group of RGD4C-EcCD_WT treated with 5-FC, the IC.sub.50 of U87MG and HUVEC cells is 44.11.5 M and 35.92.6 M respectively, although the effect is not as good as that of the positive control group directly treated with 5-FU, but there is also good killing ability, and the IC.sub.50 of the group of RGD4C-EcCD D314A treated with 5-FC for U87MG and HUVEC cells is 5.01.4 M and 8.21.5 M respectively. The effect is significantly higher than that of RGD4C-EcCD_WT. The IC.sub.50 is less 8.8 times and 4.4 times respectively. It showed that the 5-FC enzyme activity increased by D314A mutation may have the effect of increasing cell killing of EcCD/5-FC, the killing effect of RGD4C-EcCD_WT and RGD4C-EcCD_D314A for MCF-7 cell strain of .sub.v.sub.3 integrin with low expression is worst, and IC.sub.50 is greater than 10 mM. HUVEC is compared with U87MG the difference is more than 200 times. In terms of EcCD series fusion protein, although the killing effect of RGD4C-EcCD_WT combined with 5-FC does not meet the effect of 5-FC to be directly treated, but compared with the previous RGD4C-yCD, the effect is close, and RGD4C-EcCD_WT protein treatment concentration is 10 times lower than RGD4C-yCD, which indicates that RGD4C-EcCD_WT combined with 5-FC is better than RGD4C-yCD. The cell killing effect of RGD4C-EcCD_D314A combined with 5-FC is similar to that of 5-FU to be directly treated, which indicates that 5-FC enzyme activity increased by D314A mutation may improve the cell killing effect on EcCD/5-FC, while RGD4C-EcCD_WT and RGD4C-EcCD_D314A co-treated with 5-FC is safer than 5-FU, which indicates that this enzyme pro-system is safer.

(81) TABLE-US-00011 TABLE 11 The EcCD series fusion protein is combined with 5-FC for U87MG cell cytotoxic test, HUVEC and MCF-7 cells, and the cell viability is detected by MTT assay for killing test, and find oud its IC.sub.50 (half maximal inhibitory concentration). Protein (50 nM) and RGD4C- RGD4C- RGD4C-yCD 5-FC or 5-FU (IC.sub.50) EcCD_WT EcCD_D314A (500 nM) 5-FU U87MG 44.1 1.5 M 5.0 1.4 M 37.1 15.4 M 6.5 0.2 M HUVEC 35.9 2.6 M 8.2 1.5 M 23.6 3.5 M 6.2 1.3 M MCF-7 >10 mM >10 mM 5130.7 1002.4 M 25.6 0.3 M

(82) In order to confirm the protein reaction concentration IC.sub.50 of killing effect of RGD4C-EcCD_WT and RGD4C-EcCD_D314A against U87MG and HUVEC cells (see FIG. 16D, FIG. 16E and Table 12), the proteins at different concentrations are dissolved in cell culture medium and cultured at 37 C. for 2 hours, and then the unbound or un-endocytosed cells are washed with PBS, and cultured at 37 C. for 72 hours in 100 M 5-FC. The cell survival ratio is observed by MTT assay. It was known from the results that RGD4C-EcCD_WT and RGD4C-EcCD_D314A have specific killing ability for U87MG and HUVEC cells of expressing v3 integrin. The IC.sub.50 of RGD4C-EcCD_WT for U87MG and HUVEC cells is 38.58.2 nM and 22.91.2 nM respectively. The IC.sub.50 of RGD4C-EcCD_D314A is 4.01.4 nM and 9.81.5 nM respectively. The killing effect is obvious compared with RGD4C-EcCD_WT. As with the previous experimental results, the D314A mutation may improve the killing ability of EcCD/5-FC. The IC.sub.50 of RGD4C-EcCD_WT and RGD4C-EcCD D314A relative to the RGD4C-yCD protein concentration is about 4.652.4 times, which shows that the killing ability of RGD4C-EcCD_WT and RGD4C-EcCD_D314A is better than RGD4C-yCD against U87MG and HUVEC cells. It is proved from the above experimental results that RGD4C-EcCD_WT and RGD4C-EcCD_D314A have specific killing ability for cells of expressing .sub.v.sub.3 integrin, and are affected by the expression amount of .sub.v.sub.3 integrin.

(83) TABLE-US-00012 TABLE 12 Cytotoxic test of EcCD series fusion protein coupled with 5-FC for U87MG, HUVEC and MCF-7 cells. The protein reaction concentration IC.sub.50 of cytotoxic effect of RGD4C-EcCD_WT and RGD4C-EcCD_D314A treated with U87MG and HUVEC cell are evaluated. Protein (IC.sub.50) and RGD4C- RGD4C- RGD4C-yCD 100 M 5-FC EcCD_WT EcCD_D314A (500 nM) U87MG 38.5 8.2 nM 4.0 1.4 nM 177.0 12.4 nM HUVEC 22.9 1.2 nM 9.8 1.5 nM 209.4 16.8 nM

(84) 16. Preparation of yCD Series Protein Modified by Bifunctional Base Metal Chelating Agent (DTPA)

(85) The protein and DTPA dianhydride are dissolved in 20 mM HEPES buffer (pH 7) solution at a molar ratio of 1:10, and reacted at room temperature for 1 hour. After the reaction is completed, the protein is separated by AKTA FPLC Desalting Column (Desalting HiPrep 26/10 column), and purified with small molecular weight salts. The tubular column material is G25 sephadex, the elution phase is 20 mM HEPES buffer (pH 7) solution, and the elution rate is set as 4.0 mL/min. FIG. 17A and FIG. 17B show yCD and RGD4C-yCD respectively. The two protein samples are eluted out at about 4 minutes when entering the tubular column (red vertical line); FIG. 17C and FIG. 17D respectively show DTPA-yCD and DTPA-RGD4C-yCD for modifying the metal chelating agent (DTPA). The two modified protein samples are eluted out at about 4.sup.th to 5.sup.th minute when entering the tubular column (red vertical line), and the light absorption value measured at 7.sup.th to 8.sup.th minute is DTPA salts. The protein modified by metal chelating agent (DTPA) can be successfully purified by AKTA FPLC Desalting Column (Desalting HiPrep 26/10 column).

(86) 17. Enzyme Kinetic Analysis of yCD Series Proteins Modified by Metal Chelating Agent (DTPA)

(87) The curve and enzyme kinetic parameters (see FIG. 18 and Table 13) are fitted by the DNPA-modified protein enzyme kinetics through the Michaelis-Menten formula in the Graph prism program. The V.sub.max of DTPA-yCD is 286.810.80 M/min, K.sub.m is 839.871.94 M, which indicate that the enzyme activity of yCD is not affected by DTPA modification; the V.sub.max of DTPA-RGD4C-yCD is 224.68.408 M/min, K.sub.m is 1163107.8 M; compared with the original protein, the enzyme activity is slightly affected by DTPA modification, and the V.sub.max of DTPA-RGD4C-yCD is reduced to 80%.

(88) TABLE-US-00013 TABLE 13 Enzyme kinetic parameters V.sub.max and K.sub.m Protein V.sub.max (M/min) K.sub.m (M) yCD 264.1 8.256 839.8 71.94 DTPA-yCD 286.8 10.80 1038 96.19 RGD4C-yCD 288.7 8.883 1004 73.19 DTPA-RGD4C-yCD 224.6 8.408 1163 107.8

(89) Note: V.sub.max is the maximum initial velocity of the enzyme; K.sub.m is the concentration of the substrate required to reach half of V.sub.max.

(90) 18. Binding Ability Analysis of yCD Series Protein and Integrin .sub.v.sub.3 Receptor

(91) The curve and receptor binding ability parameters (see FIG. 19 and Table 14) are fitted by the binding ability of DTPA-modified protein and integrin .sub.v.sub.3 receptor through the One site-Specific binding formula in the Graph prism program. Compared with RGD4C-yCD, the K.sub.d of DTPA-RGD4C-yCD is increased slightly to 47.65.9 nM, but it still has good receptor binding ability; yCD can not be bound with integrin .sub.v.sub.3 receptor before and after modification.

(92) TABLE-US-00014 TABLE 14 Binding ability parameters of DTPA-yCD and DTPA-RGD4C- yCD proteins and integrin .sub..sub.3 receptor Protein K.sub.d (nM) yCD N/A DTPA-yCD N/A RGD4C-yCD 20.7 2.2 DTPA-RGD4C-yCD 47.6 5.9 Note: K.sub.d is the protein concentration required to reach half of B.sub.max.

(93) 19. Physical Characterization Identification of yCD Series Proteins Modified by Metal Chelating Agent (DTPA)

(94) DTPA is mainly modified on the primary amine of the protein. Since this DTPA is a di-anhydride type, the modification reaction has a chance to occur on the primary amine of two different proteins. If this reaction occurs, the molecular weight of the protein will increase by about twice, and the effectiveness of DTPA chelated metals (such as: Indium-111) may be affected. it can be seen from the results of protein electrophoresis (see FIG. 20), the molecular weights of DTPA-yCD (lane 2) and DTPA-RGD4C-yCD (lane 4) are larger than the original proteins yCD (lane 1) and RGD4C-yCD (lane 3). The ROI (red box) at twice the molecular weight (dimer) is selected by the software (Image J), and calculate its ratio in all the molecular weights, the ratio of DTPA-yCD forming dimer can be 8%; the ratio of DTPA-RGD4C-yCD forming dimer is 4%, both of which are less than 10%, which indicate that the DTPA simultaneous modification has no much reaction on the two proteins. In order to know the number of DTPA modifications on the protein, the molecular weight of the protein before and after the modification is measured by MALDI-TOF MS (Microflex MALDI-TOF MS (Bruker Daltonics), Mingxin Biotechnology Co., Ltd.) (see FIG. 21, Table 15, FIG. 22 and Table 16), by using the formula: (molecular weight after modificationmolecular weight before modification)DTPA molecular weight after reaction (375 Da)the ratio of the molecular weight intensity after modification accounting for all the intensities. After the modified protein signal is calculated by the formula, the modification number can be obtained. After calculation, yCD is modified to 1.16 DTPA on average; RGD4C-yCD is modified to 0.94 DTPA on average.

(95) TABLE-US-00015 TABLE 15 Molecular weight and strength of yCD and DTPA-yCD determined by MALDI-TOF MS yCD DTPA-yCD Molecular weight Molecular weight m/z Strength m/z Strength 18521.926 7227 18520.542 1658 18662.807 4777 18898.391 3924 18877.983 1292 19029.247 3143 37180.566 384 19274.798 2277 37970.307 253

(96) TABLE-US-00016 TABLE 16 Molecular weight and strength of RGD4C-yCD and DTPA-RGD4C-yCD determined by MALDI-TOF MS RGD4C-yCD DTPA-RGD4C-yCD Molecular weight Molecular weight m/z Strength m/z Strength 19478.775 10302 19479.223 1127 19662.162 4186 19828.504 2121 38950.150 905 20164.539 1206 39676.233 65

(97) 20. DTPA-yCD and DTPA-RGD4C-yCD Protein Labeled with Indium-111

(98) In the case of radiolabeling, the indium-111 was added to DTPA-yCD and DTPA-RGD4C-yCD protein (specific activity: 30 Ci/g), and after reacting at 37 C. for 1 hour, the thin layer analysis method of ITLC-SG (Silica Gel) Media) is used to determine the radiochemical purity: the stationary phase is ITLC-SG (Silica Gel media), and the mobile phase is 0.5M, pH 4.6 citrate buffer solution. The indium-111 labeled protein will stay at origin, and the free indium-111 will be bound with citrate in the developing solution to form .sup.111In-citrate and spread to solvant front. The indium-111 labeling efficiency of DTPA-yCD and DTPA-RGD4C-yCD is 54% and 73% respectively (see FIG. 23A and FIG. 23B). After that, EDTA added with a 10-fold molar excess of protein acts at room temperature for 1 hour to compete for indium-111 that un-chelated with DTPA. It can be seen that the radiochemical purity of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD does not decrease significantly (see FIG. 23C and FIG. 23D). Finally, the centrifuge tube (3 kDa) of the membrane filtration concentration is used to separate the radioactive mark protein above 18 kDa and .sup.111In-EDTA below 3 kDa. After 2.sup.nd centrifugation, the radiochemical purity of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD is 84% and 89% respectively (see FIG. 23E and FIG. 23F); after 4.sup.th centrifugation, the radiochemical purity of both radioactive mark proteins can be more than 95% (see FIG. 23G and FIG. 23H); in the 5.sup.th centrifugation, in addition to improving the radiochemical purity, it is mainly necessary that the solution is replaced from pH 5.5 to pH 7 HEPES buffer; and after analysis, .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD with high purity can be obtained. The final radiochemical purity is 96% and 97% respectively (see FIG. 23I and FIG. 23J). After purification, the protein is quantified and its specific activity is calculated. The recoveries are 64% and 57% respectively; the specific activity is 21 Ci/g and 27 Ci/g respectively.

(99) 21. Biochemical Characteristics of DTPA Modified and Radiolabeled yCD Series Proteins

(100) Before the protein is labeled by radioisotope, its enzyme function must be determined, and based on the original enzyme function, it should be examined whether it will be affected during the radioactive marking process. The curve and enzyme kinetic parameters of protein are fitted by the Michaelis-Menten formula in the Graph prism program. The V.sub.max of yCD and RGD4C-yCD is 264.18.256 M/min and 288.78.883 M/min respectively, K.sub.m is 839.871.94 M and 100473.19 M respectively (see FIG. 24 and Table 17). Whether the yCD and RGD4C-yCD protein detected by the same method will be affected under the conditions of DTPA modification, marking and purification. Since the radioactive metal marking conditions will change the pH value to 5.5 and heat up to 37 C. for 1 hour, in order to evaluate the effect on the enzyme activity under these conditions, after simulating the same marking conditions, the stable isotope of indium is removed by membrane centrifugation, and the solution is replaced by pH 7.0 for enzyme activity analysis, and the V.sub.max and K.sub.m of In-DTPA-yCD and In-DTPA-RGD4C-yCD are obtained, which are similar to the enzyme activity after modification of DTPA, which indicates that this marking condition will not destroy the enzyme activity (see Table 24 and Table 17). After the completion of the radioactive metal mark, it is necessary to add EDTA of 10-fold protein Moire number and react at room temperature for 1 hour to compete for the radioactive metal isotope indium which is not chelated on DTPA. The EDTA in this step also has the chance to chelate the metal zinc ion of the enzyme catalytic center, the enzyme activity is impaired, so it is necessary to analyze the enzyme activity of In-DTPA-yCD and In-RGD4C-yCD protein after competition with EDTA. After the EDTA competitive reaction is completed, the enzyme kinetics analysis can be carried out by reducing the EDTA molecule weight through membrane centrifugation method and by replacing the solution with pH 7.0. As a result, the V.sub.max of In-DTPA-yCD and In-RGD4C-yCD is significantly reduced to 47% and 40%, and K.sub.m increases (see FIG. 24 and Table 17 below). The curve and receptor binding ability parameters of the protein and integrin .sub.v.sub.3 are fitted under the conditions of DTPA modification, marking and purification by the One site-Specific binding formula in the Graph prism program (see FIG. 25 and Table 18). The K.sub.d of In-DTPA-RGD4C-yCD is 25.41.5 nM under simulated conditions. After challenged by the EDTA with a 10-fold molar excess of protein, the K.sub.d of In-DTPA-RGD4C-yCD is 34.65.4 nM, it still has good integrin .sub.v.sub.3 receptor binding ability; Regardless of before and after modifying, marking and purifying DTPA, yCD can not be bound with integrin v3 receptor. Although the two proteins after marking are greatly affected by the enzyme activity, they still have good binding ability to the integrin .sub.v.sub.3 receptor. In consideration of the indispensability of EDTA for radiochemical purity, it retains a considerable amount of enzyme activity. In addition, the effect of integrin .sub.v.sub.3 receptor binding ability is minimal, in combination of cell uptake, biodistribution and angiography experiment, focusing on the evaluation of target results, and the subsequent biological experiment is still carried out by the marking and purification conditions.

(101) TABLE-US-00017 TABLE 17 yCD series protein enzyme kinetic parameters V.sub.max and K.sub.m after DTPA modification, marking and purification Protein V.sub.max (M/min) K.sub.m (M) yCD 264.1 8.256 839.8 71.94 DTPA-yCD 286.8 10.80 1038 96.19 In- DTPA-yCD 270.0 6.165 1125 58.72 In- DTPA-yCD 10x EDTA 125.2 9.652 1932 286.1 RGD4C-yCD 288.7 8.883 1004 73.19 DTPA- RGD4C-yCD 224.6 8.408 1163 107.8 In-DTPA- RGD4C-yCD 226.8 6.918 956.4 76.79 In-DTPA- RGD4C-yCD 10x EDTA 117.7 9.510 1627 273.1 Note: V.sub.max is the maximum initial velocity of the enzyme; K.sub.m is the concentration of the substrate required to reach half of V.sub.max.

(102) TABLE-US-00018 TABLE 18 Binding ability parameters of yCD series protein and integrin .sub..sub.3 receptor after DTPA modification, marking and purification Protein K.sub.d (nM) yCD N/A DTPA-yCD N/A In- DTPA-yCD N/A In- DTPA-yCD 10x EDTA N/A RGD4C-yCD 20.7 2.2 DTPA- RGD4C-yCD 47.6 5.9 In-DTPA- RGD4C-yCD 25.4 1.5 In-DTPA- RGD4C-yCD 10x EDTA 34.6 5.4 Note: K.sub.d is the protein concentration required to reach half of B.sub.max.

(103) 22. In-Vitro Radiochemical Stability Test of In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD

(104) The radiochemical purity of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD are greater than 95% after marking and purification. Both are stored in HEPES buffer liquid (4 C.) for 48 hours, and the radiochemical purity is still higher than 85% (see FIG. 26). Both are placed in fetal bovine serum (37 C.) to simulate their stability in vivo. After 48 hours, the radiochemical purity of both is about 80%. It is shown that radioactive metal indium-111 is stably chelated on DTPA.

(105) 23. Identification of Integrin Expression Amount in Different Cell Strains

(106) Referring to FIG. 27, the three cells are the high-expression or low-expression cell strains of integrin .sub.v.sub.3 frequently mentioned in the literature. HUVEC is the normal cell of human umbilical vein epithelium (high expression), and U87MG is human glioma cell strain (high expression). MCF-7 is a human breast adenoma cell (low expression). In order to know the integrin expression amount of other types, the flow cytometry is used to identify the expression amount of integrin .sub.v.sub.3, integrin .sub.v.sub.5 and integrin .sub.1 on three cells. The integrin .sub.v.sub.3 expression amount of HUVEC and U87MG is high, MCF-7 has no expression; MCF-7 moderately expresses the integrin .sub.v.sub.5, HUVEC and U87MG show less expression amount; all three cells greatly express the integrin .sub.1.

(107) 24. Competitive Cell Uptake Results of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD by Different Integrin .sub.v.sub.3 Expression Cells

(108) .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD (20 Ci/50 pmol/mL) are separately added to the 24-well plate of the seeded cells (2.510.sup.5 cells/well) and cultured for 4 hours, and then the radioactivity of the cells and culture medium are separately measured, calculate the uptake amount of the drug by the cells (shown as % AD/10.sup.6 cells). The uptake amount of HUVEC and U87MG cells of high expression integrin .sub.v.sub.3 to .sup.111In-DTPA-RGD4C-yCD is significantly higher than that of .sup.111In-DTPA-yCD, although MCF-7 cells of integrin .sub.v.sub.3 low expression have significant difference on uptake amount of both drugs, but its drug uptake amount is the lowest among the three cells (see FIG. 28). In the competitive cell uptake assay, .sup.111In-DTPA-RGD4C-yCD (20 Ci/50 pmol/mL) is mixed with cRGDfk of the 1-fold, 10-fold, 100-fold or 1000-fold molar excess of protein, and cultured in 24-well plate added with the seeded cells (2.510.sup.5 cells/well) for 4 hours, the results show that the uptake amount of .sup.111In-DTPA-RGD4C-yCD by HUVEC and U87MG cells with high expression of integrin .sub.v.sub.3 is gradually decreased along with the increase of the molar excess of the competitor cRGDfk, the uptake amount of .sup.111In-DTPA-RGD4C-yCD by MCF-7 with low expression of integrin .sub.v.sub.3 has no significant decrease. After adding the competition of cRGDfk with 1000-fold molar excess of protein, the uptake amount of .sup.111In-DTPA-RGD4C-yCD by HUVEC, U87MG and MCF-7 cells is decreased by about 65%, 55% and 45% respectively (see FIG. 28), the decreased part after competition is the specific binding of .sup.111In-DTPA-RGD4C-yCD and the integrin receptor on the cell.

(109) 25. Identification of Integrin Expression Amount in Ovarian Cancer Cells

(110) The three cells are of the ovarian cancer cells as shown in FIG. 29. In order to screen out the cell strain with high expression amount of integrin, the expression amount of integrin .sub.v.sub.3, integrin .sub.v.sub.5 and integrin .sub.1 on the three cells is identified by flow cytometry. ES-2-luc and SKOV3ip1 express the integrin .sub.v.sub.3 moderately and slightly, and OVCAR-3 has no expression; ES-2-luc and OVCAR-3 moderately expresses integrin .sub.v.sub.5, SKOV3ip1 has less expression; all three cells greatly express integrin .sub.1. By summing the integrin expression amount of all kinds, the screened ES-2-luc is the cell strain of ovarian cancer peritoneal metastasis.

(111) 26. Competitive Cell Uptake Assay of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD by ES-2-luc Cells

(112) .sup.11In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD (20 Ci/50 pmol/mL) are separately added to a 24-well plate of the seeded ES-2-luc cells (2.510.sup.5 cells/well) for 4 hours, and then the radioactivity in cells and culture medium is measured separately, and the uptake amount of the drug by the cells is calculated (shown as % AD/10.sup.6 cells). The uptake amount of .sup.111In-DTPA-RGD4C-yCD by cells is significantly higher than that of .sup.111In-DTPA-yCD (see FIG. 30). In competitive cell uptake experiment, .sup.111In-DTPA-RGD4C-yCD (20 Ci/) 50 pmol/mL) is mixed with cRGDfk of the 1-fold, 10-fold, 100-fold or 1000-fold molar excess of protein, and cultured in 24-well plate added with the seeded cells (2.510.sup.5 cells/well) for 4 hours. The results show that the uptake amount of .sup.111In-DTPA-RGD4C-yCD by ES-2-luc cells is gradually decreased along with the increase of the molar excess of the competitor cRGDfk. After adding the competition of cRGDfk with with a 1000-fold molar excess of protein, the uptake amount of .sup.111In-DTPA-RGD4C-yCD by ES-2-luc cells is decreased by about 70% (see FIG. 30), the decreased part after competition is the specific binding of .sup.111In-DTPA-RGD4C-yCD and the integrin receptor on the cell.

(113) 27. Intratumoral Injection of .sup.111in-DTPA-yCD and .sup.111in-DTPA-RGD4C-yCD into U87MG Tumor Nude Mice was Performed by Single-Photon and Computed Tomography

(114) In in-vitro cell experiment, the binding ability of .sup.111In-DTPA-RGD4C-yCD and the integrin .sub.v.sub.3 receptor on the cell membrane has been verified. In order to know whether the drug can show its target effect in vivo, integrin .sub.v.sub.3 is seeded subcutaneously in the right side of nude mice. After the high-expression U87MG tumor cells are grown to 100 mm.sup.3, 60-70 Ci (3-3.5 g) of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD are injected directly into the tumor, the images are collected by microSPECT/CT after 1, 4, 24, 48 hours. It can be seen from the image (see FIG. 31) that the drug retention in the .sup.111In-DTPA-RGD4C-yCD group (orange arrow) is better than that in the .sup.111In-DTPA-yCD, and the target ligand RGD4C is missing. .sup.111In-DTPA-yCD enters the systemic blood circulation system within 1 hour and is metabolized by normal organs. From FIG. 31, it can be seen that the kidney is the main metabolic organ of these two drugs. The amount of .sup.111In-DTPA-yCD accumulated in the tumor is gradually decreased with time. After 24-48 hours, the amount of accumulation in the tumor is running out. On the contrary, .sup.111In-DTPA-RGD4C-yCD is bound with integrin v3 on tumor cells, then the ratio of drug entering the systemic blood circulation is reduced, and the metabolic rate is slow. At 48.sup.th hour, there is still considerable drug accumulation in the tumor.

(115) 28. Biodistribution Experiment of Intraperitoneal Injection of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD in ES-2-luc Tumor Nude Mice

(116) ES-2-luc tumor nude mice is intraperitoneally injected with 50 Ci (2.5 g) .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD for 1, 4 and 24 hours, then organs are collected and their radioactivity is measured to obtain biodistribution, as shown in Table 19 and Table 20. It can be seen from the results that the radioactivity of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD in the peritoneal cavity is decreased rapidly with time, and there are 2.180.62% ID and 4.201.05% ID at 1 hour after injection, which has been reduced to and in 4 hours after injection, which shows that the drug stays in the abdominal cavity for a short time. .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD are injected in the abdominal cavity, a part is accumulated on tumor cells after 1 hour, the radioactivity is 10.503.57% ID/g and 24.322.58% ID/g respectively. After 4 hours, the radioactivity of .sup.111In-DTPA-RGD4C-yCD in tumor is approximately 3 times higher than that of .sup.111In-DTPA-yCD (10.484.33% ID/g), which indicatinges that .sup.111In-DTPA-RGD4C-yCD is assuredly bound with integrin .sub.v.sub.3 on tumor cells; after 24 hours, the accumulation amount of both drugs in tumor cells is decreased. In addition to the part accumulated in the tumor, another part of the drug is absorbed back into the blood circulation system by the peritoneal blood vessels. After 1 hour of injection of .sup.111In-DTPA-yCD and .sup.111In-DTPA-RGD4C-yCD, the radioactivity in the kidney is 55.733.57% ID/g and 48.602.98% ID/g respectively, it is maintained at 5060% ID/g after 24 hours of injection, which indicates that once the two drugs return to the blood, they are quickly metabolized by the kidneys; after 24 hours of injection, the blood radioactivity is less than 1% ID/g. When the organs are collected, it is found that the appearance of the pancreas is erosive and swollen. The pancreatic radioactivity of the two drugs is observed after 1 hour of injection. The pancreatic radioactivity of .sup.111In-DTPA-RGD4C-yCD (8.060.88% ID/g) is higher than that of .sup.111In-DTPA-yCD (3.910.36% ID/g), it is suspected that tumor cells invade the pancreas.

(117) TABLE-US-00019 TABLE 19 Biodistribution of ES-2-luc tumor nude mice after intraperitoneal injection of .sup.111In-DTPA-yCD 1 hour 4 hours 24 hours Blood 1.07 0.20 1.30 0.74 0.28 0.23 Heart 0.57 0.07 0.51 0.05 0.51 0.01 Lung 1.19 0.25 0.96 0.25 1.18 0.30 Liver 4.27 0.91 4.22 0.31 3.51 0.21 Stomach 2.27 0.61 2.31 0.30 2.97 0.04 S. intestine 2.39 1.05 2.00 0.44 1.98 0.21 L. intestine 3.35 0.76 3.54 0.96 3.39 2.17 Pancreas 3.91 0.36 5.72 0.57 3.82 0.69 Spleen 2.43 0.50 2.31 0.30 2.97 0.04 Muscle 0.29 0.04 0.25 0.05 0.21 0.01 Bone 0.43 0.10 0.45 0.20 0.34 0.18 Bladder 3.61 0.80 5.35 1.27 3.17 0.96 Kidney 55.73 3.57 47.40 4.22 68.84 6.61 Urine 75.03 10.81 48.48 34.28 17.17 1.86 Feces 0.62 0.63 1.94 1.68 1.50 0.65 Tumor 10.50 3.57 10.48 4.33 7.38 0.47 Ascites (% ID) 2.18 0.62 0.97 0.15 0.13 0.02

(118) TABLE-US-00020 TABLE 20 Biodistribution of ES-2-luc tumor nude mice after intraperitoneal injection of .sup.111In-DTPA- RGD4C-yCD 1 hour 4 hours 24 hours Blood 1.53 0.87 1.07 0.48 0.85 0.32 Heart 0.44 0.03 0.40 0.01 0.35 0.05 Lung 0.89 017 0.94 0.10 1.91 0.54 Liver 4.77 0.35 5.59 0.17 3.71 0.92 Stomach 2.31 0.48 1.87 0.24 1.84 0.09 S. intestine 3.37 0.80 3.05 0.87 2.28 0.53 L. intestine 2.73 0.45 2.47 0.12 1.87 0.42 Pancreas 8.06 0.88 6.85 0.29 6.10 0.70 Spleen 2.81 0.61 2.89 0.16 3.30 0.40 Muscle 0.34 0.15 0.23 0.01 0.14 0.03 Bone 0.30 0.17 0.21 0.15 0.13 0.03 Bladder 3.03 0.26 3.73 0.87 3.52 0.79 Kidney 48.60 2.98 56.73 0.02 51.16 2.63 Urine 167.93 16.37 70.60 57.04 16.19 5.47 Feces 1.32 0.71 1.94 0.27 1.00 0.07 Tumor 24.32 2.58 30.48 9.64 17.63 3.78 Ascites (% ID) 4.20 1.05 0.70 0.12 0.76 0.22

(119) The technical features of the invention disclosed in all specifications can be combined in any manner. Each of the technical features disclosed in the specifications can be replaced by other means of the same, equivalent or similar purpose. Therefore, unless otherwise stated, all the features disclosed herein are only the example of the general series of equivalent or similar features.

(120) It is known from the above that those skilled in the art can readily understand the essential features of the present invention, and can make many changes and adjustments to the present invention for different uses and conditions without departing from the spirit and scope thereof.

(121) Although the present invention has been described in terms of specific exemplary embodiments and examples, it will be appreciated that the embodiments disclosed herein are for illustrative purposes only and various modifications and alterations might be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the following claims.