Patent Application
20150284445
The present invention relates to an immune receptor modifier conjugate, its preparation and use thereof for antitumor, antivirus, diabetes, antibody induction and immunomodulation. In particular, the present invention relates to 9-position aminomethyl benzyl purine biotic coupling precursor for preparing the above-mentioned immune receptor modifier conjugate. In addition, the present invention relates to the compounds or salts thereof for synthesizing the coupling precursor.
Improved immunological effect can often be realized by combined application or conjugation of immune agonists and immune antigens such as polypeptides, proteins, glycoproteins, polysaccharides, polynucleotides, cell lysates, inactivated cells and inactivated microorganisms (World Chin J Digestol 2005 Sep. 15, 13(17):2078-2081; Vaccine, Volume 23, Issue 45, 1 Nov. 2005, Pages 5263-5270; The J. Clin. Invest. 2011, 121(5), 1782-1796).
The present invention prepares a series of small molecular immune agonist coupling precursors and conjugates synthesized from such immune agonist coupling precursors with biotic ligands or immune antigens. Also disclosed are the uses of such conjugates for anti-tumor, anti-virus, diabetes, antibody induction and immunomodulation.
The purpose of the present invention is to provide an immune receptor modifier conjugate with enhanced immunological effect and the preparation method thereof. The purpose of the present invention is to provide a coupling precursor for preparing such conjugate and to provide compounds or salts thereof for synthesizing such coupling precursor. A further purpose of the present invention is to provide the use of such immune receptor modifier conjugate in immunomodulation, antibody preparation, anti-virus, diabetes, tumor immunomodulation and tumor biological-immunotherapy.
According to one aspect of the present invention, provided is an immune receptor modifier conjugate which is obtained by reacting a coupling precursor with a biotic ligand, wherein the conjugate is a compound of general formula (I):
wherein R2 represents the biotic ligand, X1 represents OH or SH, R1 represents linear alkyl, branched alkyl, substituted alkyl, unsubstituted alkyl or alkoxyalkyl, X2 represents a coupling group; m and n each are an integer selected from 1 to 10, m is the number of small molecular agonist (m is defined as coupling degree), and n is the number of the biotic ligand.
When the coupling precursor is a compound of formula 1:
X2 represents the group thiocarbonyl
When the coupling precursor is a compound of formula 2:
X2 represents the group
When the coupling precursor is a compound of formula 3:
X2 represents the group
in which u is an integers selected from 0 to 12;
and
When the coupling precursor is a compound of formula (4):
X2 represents the group
in which PEG represents a polyethylene glycol group.
Among the above-mentioned immune receptor modifier conjugates, the biotic ligand is one or more selected from polypeptide, protein, glycoprotein, polysaccharide, polynucleotide, inactivated cells and inactivated microorganisms.
Among the above mentioned immune receptor modifier conjugates, PEG is a polyethylene glycol group
such as a di-polyethylene glycol group
a tri-polyethylene glycol group
and a tetra-polyethylene glycol group
When the coupling precursor is a compound of formula 3 or 4, by using the biotic ligand R2, such as OCT4, SOX2, NANOG, MUC1, MG7, POSTN, Twist, Anxa1, Akt, CD47, Sp17, PSMA, M2e (monomer and tetramer), NP366-374 (9-peptide epitope), FOX01, PEAK1, HER2, MMP-10, PD-L1, PD-1, and SGLT2, the typical representative structures of the protein conjugates formed are as follows:
In formulae (5) and (6), the expression “or R2” means that OCT4 and SOX2 can be substituted by other antigens, proteins or polypeptides, wherein the substitution by NANOG results in compounds 5-3 and 6-3 respectively; the substitution by MUC1 results in compounds 5-4 and 6-4 respectively; the substitution by MG7 results in compounds 5-5 and 6-5 respectively; the substitution by POSTN results in compounds 5-6 and 6-6 respectively; the substitution by Twist results in compounds 5-7 and 6-7 respectively; the substitution by Anxa1 results in compounds 5-8 and 6-8 respectively; the substitution by Akt results in compounds 5-9 and 6-9 respectively; the substitution by CD47 results in compounds 5-10 and 6-10 respectively; the substitution by Sp17 results in compounds 5-11 and 6-11 respectively; the substitution by PSMA results in compounds 5-12 and 6-12 respectively; the substitution by M2e (monomer and tetramer) results in compounds 5-13 and 6-13 respectively; the substitution by NP366-374 (9-peptide epitope) results in compounds 5-14 and 6-14 respectively; the substitution by SGLT2 results in compounds 5-15 and 6-15 respectively; the substitution by PEAK1 results in compounds 5-16 and 6-16 respectively; the substitution by HER2 results in compounds 5-17 and 6-17 respectively; the substitution by MMP-10 results in compounds 5-18 and 6-18 respectively; the substitution by PD-L1 results in compounds 5-19 and 6-19 respectively; and the substitution by PD-1 results in compounds 5-20 and 6-20 respectively.
The aforementioned compounds are shown in tables 1 and 2 below:
Among them, OCT4, SOX2 and NANOG are tumor stem cell antigen proteins (Chinese Medicine Herald, 2011, 8(8):17-20; Chinese Bulletin of Life Sciences, 2004, 16(3):129-134); MG7 is a stomach cancer-related antigen protein (China Oncology, 2010, 20(4): 312-313); MUC1 is an antigen related to various tumors (European Journal of Organic Chemistry, 2011, 20(21): 3685-3689); POSTN is a tumor-related protein (Nature, 2012, 481: 85-89); Twist and Akt are both tumor-related antigens (Journal of Shenyang Medical College, 2010, 12(3): 182-184); Anxa1 is specifically expressed in various tumor tissues (PNAS, 2011, Oct. 3, 19587-19592; Oncology Progress, 2010, Jan. 8(1):63-66); CD47 is highly expressed on the surface of almost every type of cancer cells (Science: Translational Medicine, 22 Dec. 2010, Vol 2, Issue 63 63ra94); Sp17 is a tumor antigen having very high expression in ovarian carcinoma cells (PloS ONE, 2010, 5(5), e10471, 1-13); PSMA is a special biomarker of prostate cancer (Cancer, 1998, 82(11): 2256-2261); M2e and NP366-374 are epitope polypeptides in the conserved protein of influenza virus A; SGLT2 is a functional protein related to sugar metabolism (Chinese Journal of Medicinal Chemistry, 2011, Vol. 21, No. 4, p 322); PEAK1 is a biomarker of early pancreatic cancer (Cancer Res. 2012 May 15; 72(10):2554-64); HER2 is a proto-oncogene human epidermal growth factor receptor 2 (MEDICAL JOURNAL OF CASC, 2002, 4 (1), 69-70); MMP-10 is a protein closely related to the occurrence and metastasis of lung carcinoma (Anticancer Res. 2007 July-August; 27(4C): 2791-5); and PD-L1 and PD-1 are a ligand and a receptor of tumor immune escape, respectively (N. Engl. J. Med., 2012, Jun. 2, 1-11).
When the coupling precursor is a compound of formula 1 or 2, typical representative synthesis and structure formulae of the polypeptide conjugates formed from the coupling precursor and biotic ligand R2 (such as MUC1
MG7 and M2e (monomer)) are as follows:
For the immune receptor modifier conjugates of this invention, suitable biotic ligands are not limited to the polypeptides or proteins as listed above, and can also be the tumor antigens as shown is table 3.
Preparation of the Coupling Precursor:
Synthesis of Coupling Precursor 1—Synthesis Protocol:
Method for preparing compound of formula 7 is described in WO 2009005687 for compound 1096159-02-2P.
Synthesis Process: Compound 7 (5 g) was dissolved into methanol (100 ml) and active nickel (0.5 g) was added. The mixture was reduced by hydrogenation at room temperature under 3 atm. for 24 h. After the catalyst nickel was filtered off, the residue was concentrated under reduced pressure to small volume, frozen at −10° C. for 12 h, and precipitated to give a solid product 8 (3 g, yield 60%). Melting point: 265-267° C. MS (ESI, M+1): 345.
Compound 8 (1 g, 2.9 mmol) was mixed with carbon disulfide (2 ml), triethylamine (0.5 ml), DMAP (0.1 g), (BOC)2O (640 mg) and dichloromethane (50 ml). The mixture was stirred at room temperature for 2 h, heated under reflux for 4 h, concentrated under reduced pressure, and separated by silica gel column chromatography (5% methanol-dichloromethane) to give compound 1 (0.7 g, yield 65%). Melting point: 221-223° C., MS (ESI, M+1): 373.
Synthesis of Coupling Precursor 2—Synthesis Protocol:
Synthesis Process: Compound 8 (1 g) was mixed with compound 9 (0.78 g) in DMF (30 ml), stirred under room temperature for 2 h, concentrated under reduced pressure, and separated by silica gel column chromatography (ethyl acetate) to give compound 2 (1.1 g, yield 82%). Melting point 203-205° C. MS (ESI, M+1): 496.
Synthesis of Coupling Precursor 3-1 (u=2)—Synthesis Protocol:
Synthesis Process: Compound 8 (1 g) was mixed with succinic anhydride (0.3 g) in DMF (50 ml) and stirred at room temperature for 12 h. N-hydroxy succinimide (0.34 g) and equal molar DCC were added. The mixture was stirred at room temperature for another 12 h to give a solution of compound 3 in DMF. The solution was distilled under reduced pressure to dryness. The resultant solid residue was extracted with ethyl acetate (50 ml), and then equal volume of diethyl ether was added. The mixture was frozen at −10° C. for 12 h and precipitated to give a solid 3 (u=2) (0.6 g, yield 38%). MS (ESI, M+1): 542.
Synthesis of Coupling Precursor 4-1—Synthesis Protocol
The reference for synthesis of compound 11: PCT patent application No. 2011134669, published on Nov. 3, 2011.
Note: 1) regioselective, scale up; 2) regioselective, scale up; 3) scale up, reactants: 3, reagents: 3, solvents: 3, steps: 3, stages: 3.
Synthesis of compound 12: the synthesis protocol is analogous to the synthesis of compound 8, yield: 88%, MS (ESI, M+1): 329.
Synthesis of compound 13: Compound 12 (1 g) was dissolved into dichloromethane (50 ml). Then bromine (10 ml) was added and the mixture was stirred at room temperature for 12 h. Air was blown through the mixture such that the excessive bromine was removed and absorbed into saturated sodium bicarbonate solution. The solid residue was filtered to give a pale yellow solid 13 (1 g), with a yield of 85% and mass spectra (ESI, M+1): 407.
Synthesis of compound 15: Compound 13 (0.5 g) was dissolved into anhydrous DMF (10 ml) and compound 14 (0.24 g) was added. The mixture was stirred homogenously, and then added with DCC (0.26 g) at 0° C. The mixture was stirred at 0° C. for 2 h and then at room temperature for another 12 h. DCU was filtered off and the remaining solution was distilled under reduced pressure to dryness. To the residue was added 50 ml of saturated sodium bicarbonate solution. The mixture was stirred at 40° C. for 4 h. Under cooling, the mixture was adjusted to pH 4 with concentrated hydrochloric acid. The precipitated solid was filtered, washed with water, and dried to give compound 15 (0.5 g, yield 72%). MS (ESI, M+1):567.
Synthesis of compound 16: Compound 15 (0.4 g) was dissolved into methanol (20 ml), and thiourea (1 g) was added. The mixture was heated under reflux for 12 h, cooled and filtered. The filtrate was concentrated under reduced pressure and the residue was dissolved into 5% sodium carbonate solution (10 ml). Under cooling, the mixture was adjusted to pH 4 with concentrated hydrochloric acid. The precipitated solid was filtered, washed with water and dried to give compound 16 (0.23 g, yield 65%). MS (ESI, M+1):521.
Synthesis of coupling precursor 4: Compound 16 (0.15 g) was dissolved into anhydrous DMF (5 ml), and N-hydroxy succinimide (0.04 g) was added. The mixture was stirred homogenously and then added with DCC (0.06 g) at 0° C. The resultant mixture was stirred at 0° C. for 2 h and then at room temperature for another 12 h. DCU was filtered off and the filtrate was distilled under reduced pressure to dryness. The remaining solid was extracted with ethyl acetate (10 ml). To the extraction was added equal volume of diethyl ether. The mixture was frozen at −10° C. for 12 h and precipitated to give a solid 4 (0.05 g, yield 32%). MS (ESI, M+1):618.
For immune therapy and immunomodulation of malignant tumor, the immune receptor modifier conjugate may be administered by intraperitoneal, subcutaneous, intramuscular or intravenous injection. Alternatively, the immune receptor modifier conjugate may be administered by in vivo re-transfusing the isolated immune cells after co-culturing the immune cells (for example, dendritic cells, natural killer (NK) cells, lymphocyte, monocyte/macrophage, granulocyte, etc.) with the immune receptor modifier conjugate of this invention.
The immune receptor modifier conjugate of the present invention can be used for immunomodulation, antibody preparation, anti-virus, diabetes, tumor immunomodulation and tumor biological-immunotherapy. The above-mentioned conjugates or salts thereof can be formulated into therapeutic drugs suitable for such therapies, or formulated into compound drugs with other pharmaceuticals, or formulated into complexes or combinations with pharmaceutically acceptable carriers. The present immune receptor modifier conjugates or salts thereof can be present at various ratios in the therapeutic preparations.
The present invention is further illustrated by the following figures and specific examples.
In the figures:
Source of the antigens: the molecular weight of all proteins may be found in the international protein database (http://www.uniprot.org/uniprot/P48432).
Source of proteins or polypeptides: Except the proteins or polypeptides for which the specific preparation or synthesis methods have been described, other polypeptides are synthesized by Hybio Pharmaceutical Co., Ltd.
Proteins are purchased from or synthesized by the following companies:
GenScript Inc., Nanjing, China (http://www.genscript.com.cn/index.html);
Novoprotein Scientific Inc., Shanghai, China (http://www.sinobio.net/);
Sino Biological Inc., Beijing, China (http://www.sinobiological.cn/);
Abnova Corporation (http://www.abnova.com/cn/).
Mass spectrometer for material identification: LDI-1700 MALDI-TOF mass spectroscopy (Linear Scientific Inc., USA).
The method for determining coupling degree by mass-spectrometry is, for example, as follows:
The average molecular weight of POSTN is 86 kDa (see, World J Gastroenterol, 2007 Oct. 21; 13(39); 5261-5266; http://www.sinobiological.cn/Periostin-Protein-g-465.html). The molecular weight of the resultant coupling product 5-6 is 88132, as determined by mass spectrum. The coupling degree is calculated as follows: (88132-86000)/427=4.99≈5 (the molecular weight 427 is obtained by subtracting one H2O molecular from molecular weight of compound 3). Thus, it can be determined that 5 monomers of compound 3 were coupled (coupling degree m=5).
The preparation of the present coupling conjugate is further illustrated by the following examples.
Synthesis of Compound 1-1:
MG7 (0.37 mmol) and compound 1 (0.88 mmol) were mixed and dissolved into absolute methanol, and then anhydrous triethylamine (1.12 mmol) was added. The mixture was reacted at 45° C. for 4 h. The solvent was removed by evaporating under reduced pressure and the residue was separated by silica gel column chromatography (10% methanol-dichloromethane) to give compound 1-1 (55 mg, yield 23%). MS (ESI): theoretical m/z 969.1516, found 970.1518 (M+H).
Synthesis of Compound 1-2:
Analogous to the synthesis of compound 1-1, MUC1 (epitope) was mixed with compound 1 at a molar ratio of 1:1, and then 1.5-fold molar of triethylamine was added. The resultant mixture was allowed to react in methanol at 20° C. for 12 h. For work up, please refer to “Synthesis of compound 1-1”. This gave compound 1-2 (yield 20%). MS (ESI): theoretical m/z 3341.72, found 3342.75 (M+H).
Synthesis of Compound 1-3:
Analogous to the synthesis of compound 1-1, M2e was mixed with compound 1 at a molar ratio of 1:1, and then 1.5-fold molar of triethylamine was added. The resultant mixture was allowed to in methanol at 20° C. for 12 h. For work up, please refer to “Synthesis of compound 1-1”. This gave compound 1-3 (yield 25%). MS (ESI): theoretical m/z 3303.32, found 3304.33 (M+H).
Synthesis of Compound 2-1:
Compound 2 (0.22 mmol) was mixed with M2e (0.1 mmol) in anhydrous DMSO (10 ml). The mixture was stirred at room temperature for 12 h. To the mixture H2O (100 ml) was added. The resultant mixture was lyophilized to remove solvents and the residue was separated by silica gel column chromatography (10% methanol-dichloromethane) to give compound 2-1 (56 mg, yield 15%). MS (ESI): theoretical m/z 3761.71, found 3761.75 (M+H).
Synthesis of Compound 5:
1) The expression and preparation of OCT4 are described in Progress in Modern Biomedicine, Vol. 10, NO. 9, May, 2010, 1610-1612).
2) Synthesis of Compound 5 (m=5):
OCT4 (10 mg, average molecular weight: 38216) was dissolved into PBS solution (10 ml). A solution of compound 3 (u=2, 50 mg) in 2 ml DMSO was mixed with equimolar NHS, and then equimolar EDC was added. The mixture was stirred at room temperature for 2 h. Subsequently, the solution of OCT 4 in PBS was added and the resultant mixture was stirred overnight at 10° C. The mixture was separated with PD-10 desalting column (Amersham disposable PD-1 desalting column). The eluates containing conjugate 5 were combined (detected by the absorptance at 320 nm) and lyophilized. The average molecular weight was 40348, as determined by MS. Thus, it was determined that the product 5 comprises five monomers of compound 3 (u=2), i.e. coupling degree m=5. Compound 5 (m=1, 2, 3, 4) was synthesized with compound 3 (u=2) at different molar ratios by the same method.
Synthesis of Compound 5-4:
1) Methods for preparing MUC1 (epitope) may be found in the references, such as, Proc Natl Acad Sci. 2011, 109(1): 261-266; Angew Chem Int Ed Engl, 2010, 49(21): 3688-3692; Angew Chem Int Ed Engl, 2011, 50(7): 1635-1639; European Journal of Organic Chemistry, 2011, 20(21): 3685-3689; and Chemistry, 2011, 17(23): 6396-6406. According to compound 11 shown in the reference Proc Natl Acad Sci. 2011, 109(1): 261-266, the molecular weight of MUC1 (epitope) is 2967.
2) Coupling precursor 3 (u=2, 10 mg, 0.019 mmol) was dissolved into DMSO (2 ml) and MUC1 (epitope) (59 mg, 0.02 mmol) was added. The mixture was stirred at room temperature for 2 h, and then H2O (20 ml) was added. The solvent was removed by lyophilization and the residue was separated by silica gel column chromatography (10% methanol-dichloromethane) to give compound 5-4 (16 mg, yield 25%). MS (ESI): 3396.6 (M+H). Coupling degree m=1.
Synthesis of Compound 5-14:
Analogous to the synthesis of compound 5-4, except that MUC1 (epitope) was replaced by NP366-374 (ASNENMDAM), other solvents and reactants are used in identical molar ratios to give compound 5-14, MS (ESI): 1424.54 (M+H). Coupling degree m=1.
Synthesis of the Following Conjugates:
If the antigen is a protein, the synthesis is analogous to the synthesis of compound 5. If the antigen is a polypeptide (with the number of amino acids being less than 50), then follow the synthesis method of compound 5-4 (the solvents and reactants were in identical molar ratios, and the determination of reaction time, temperature, molecular weight and coupling degree, as well as the steps and conditions of the method were also identical). The conjugates obtained are shown in the table below.
Wherein R2 represents polypeptide or protein
Preparation of Conjungate 6:
1) SOX2 is prepared according to Journal of Huazhong Normal University (Nat. Sci.), 2008, 42 (1), 102-105. Sp17 is prepared according to Chinese Journal of Pathophysiology, 2001, 17(10), 1019-1021. PSMA is prepared according to The Journal of Biomedical Research (Natural Science), 2010, 30(11): 1608-1611.
2) Synthesis of Compound 6 (m=3):
SOX2 (10 mg, average molecular weight: 34310) was dissolved into PBS solution (10 ml). A solution of compound 4 (50 mg) (PEG=ethylene glycol group) in DMSO (2 ml) was mixed with equimolar NHS, and then equimolar EDC was added. The mixture was stirred at room temperature for 2 h. Subsequently, the solution of SOX2 in PBS was added and the resultant mixture was stirred overnight at 10° C. The mixture was separated on PD-10 desalting column (Amersham disposable PD-1 desalting column). The eluates containing compound 6 (detected by the absorptance at 320 nm) were combined and lyophilized. The average molecular weight was 35817, as determined by MS. Thus, it was determined that the product 6 comprises three monomers of compound 4 (PEG=ethylene glycol group), i.e. coupling degree m=3. Compound 6 (m=1, 2, 4) was synthesized with compound 4 (PEG=ethylene glycol group) used at different molar ratios by the same method.
Synthesis of Compound 6-3 (m=4):
Synthesis of compound 6-3 (m=4) was analogous to the synthesis of compound 6. The molecular weight of NANOG was 34620. Compound 6-3 (m=4) was analyzed to have a molecular weight of 36630.24. Compound 6-3 (m=1, 2, 3, 5) was synthesized with compound 4 (PEG=ethylene glycol group) at different molar ratios by the same method.
Synthesis of Compound 6-14 (the Ligand in General Formula I is NP366-374, n=3):
Compound 4 (PEG=ethylene glycol group) (11 mg, 0.02 mmol) was dissolved into DMSO (2 ml), and then NP366-374 trimer (59.1 mg, 0.02mmol) was added. The mixture was stirred at room temperature for 2 h. To the mixture with 20 ml water was added. The resultant mixture was lyophilized to remove solvents, and then the residue was separated by silica gel column chromatography (10% methanol-dichloromethane) to give compound 6-14 (in which R2 is NP366-374 trimer) (17 mg, yield 25%). MS (ESI): 3460.8 (M+H). Coupling degree m=1.
Synthesis of the Following Conjugates:
If the antigen is a protein, synthesis is analogous to the synthesis of compound 6. If the antigen is a polypeptide (with the number of amino acids being less than 50), then follow the synthesis process of compound 6-14 (the solvents and reactants were in identical molar ratios, and the determination of reaction time, temperature, molecular weight and coupling degree, as well as the steps and conditions of the method were also identical). The conjugates obtained are shown in the table below.
R2 represents polypeptide or protein
Method for Assaying Bioactivity
1. Method for Assaying Immune Factors:
Method: ELISA
Reagents and Conditions:
Human peripheral blood mononuclear cells were separated by centrifugation sedimentation. The centrifugator was Ficoll-Hypaque. The separated cells were suspended in RPMI1640 medium, and then 10% of FBS, L-glutamine, and penicillin/streptomycin (RP10, Invitrogen) were added. The resultant mixture was placed into 96-well plate. The cells were stimulated by the compounds of the present invention at a concentration of 0.1-10 μM, and cultured at 37° C. with 5% CO2 for 24 h. The levels of γ-interferon and interleukin-12 were measured using Luminex (Austin, Tex.),
2. Method for Assaying Anti-Tumor Effect:
The method for assaying in vivo anti-tumor effect by the conjugates of this application such as conjugate 5-4 in mice was as follows:
Cell: 4T1, mouse breast carcinoma cells, 1×106 cells/mouse.
Animals: 5-week-old BALB/C mice (the number of male mice is identical with female mice).
Tumor transplantation: Tumor cells were injected subcutaneously on left/right sides of a mouse.
Administration: Intraperitoneal injection (0.125 mg/mouse/injection, PBS solution). Drugs were administered three times in total: one week before transplantation, on the day of transplantation and 7 days after transplantation.
The health state and tumor size of the mice were monitored (via visual observation). After 14 days, the mice were killed. Blood samples were collected from each mouse for testing cytokines, and the tumors were taken out to measure the weight, length and width so as to calculate their volumes. The method used for testing in vivo anti-tumor effect by, for example, compound 6-17, in mice was the same as those described above, except that the cells used were mouse lung carcinoma cells.
3. Antibody Induction Method (Taking Compounds 1-3 and 2-1 as the Examples):
36 female BALB/c mice (5 to 6-week-old, about 16 g) were randomly assigned into six cages, 6 mice each. The mice were provided with standard diet prepared by animal center and cooled boiled water on daily basis. After purchase, the mice were fed for one week to adapt the environment. After health examination, the experiments were started. The experimental animals were randomly divided into six groups, as shown in Table 4:
200 μl of the drugs were administered intraperitoneally to the above groups of mice, respectively. Booster immunization was performed with the same antigen and dosage on day 14 after initial immunization. Blood samples were collected by cutting rat tail for three times: before initial immunization, before booster immunization and 7 days after booster immunization. The blood samples were placed into 1.5 ml Eppendorf tubes at 4° C. for several hours, and then centrifuged at 3000 rpm for 15 min to separate serum. M2e-specific antibodies in the immune serum were assayed using standard ELISA method.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201210382202.8 | Oct 2012 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2013/077972 | 6/26/2013 | WO | 00 |
