Process for preparing a metastasis-inhibiting protein and uses thereof.

Abstract: Disclosed is a novel protein which has a molecular weight of 45,000±5,000 and pI 5.7±0.5 and exhibits cancer metastasis-inhibitory activity. The protein can be prepared by culturing human cells, animal cells and microorganisms capable of producing the protein in a nutrient culture medium while stimulating them with an inducer such as Bacille Calmette-Guérin and lipopolysaccharide.

Description: The present invention relates to a protein for use as a medicament and to uses of the protein Nowadays, the treatment of cancers is mainly attained by surgical operations, chemotherapies and radiotherapies. Although most of cancers may be cured by such a treatment, a part of viable cancer cells remaining after such a treatment may be scattered throughout the body of a cancer patient, and may cause a more serious cancer-metastasis and even shorten the patient's life span. If the metastasis of cancers can be inhibited, cancer patients's pain would be relieved, and their life spans would be extended much more. Therefore, the development of agents which effectively inhibit the metastasis of cancers has been in a great demand. In general, the metastasis of cancers, however, has been considered to occur via a complicated process, and this hinders the realization of satisfiable cancer metastasis-inhibitory agents. A variety of proteins possessing cancer metastasis-inhibitor activity were reported. Examples of such a conventional protein are interferons and interleukin 2 which have been reported to have cancer metastasis-inhibitor activity. The protein preparable according to the present invention clearly differs from such a conventional protein in molecular weight and amino acid sequence. None of conventional proteins have not yet been realized as a cancer metastasis-inhibitory agent. In Japanese Patent Laid-Open No.308,799/90, a cancer metastasis-inhibitory factor produced by cells derived from human hematopoietic tissues is reported and its structure and physicochemical properties are, however, far from substantial elucidation because the description in the patent is vague and it only teaches the molecular weight ranging 10,000-450,000. One object of the present invention is to provide a protein for use as a medicament, or a diagnostic agent for cancer metastasis. Another object of the present invention is to provide uses of said protein. A further object of the present invention is to provide a method for making a medicament, or a diagnostic agent for cancer metastasis. A final object of the present invention is to provide uses of a DNA encoding a protein according to the present invention. In order to attain the aforesaid objects, the present inventors studied substances which inhibit the metastasis of cancers. The present inventors continued studies in order to obtain a novel cancer metastasis-inhibitory substance, and, as a result eventually found cancer metastasis-inhibitory activity in a culture supernatant of HPB-MLT cell (FERM BP-2430), an established cell line derived from human T-cell leukemia, which had been stimulated in a nutrient culture medium with BCG and LPS. The present inventors revealed that the entity of the activity is a protein having the following physicochemical properties: (1) Molecular weight 45,000±5,000; (2) Isoelectric point pI=5.7±0.5; (3) Partial amino acid sequence Possessing a partial amino acid sequence of Asp-Ser-Glu-Gly-Tyr-Ile-Tyr-Ala-Arg-Gly-Ala-Gln-Asp-Met-Lys or Glu-His-Trp-Ser-His-Asp-Pro-Phe-Glu; (4) Solubility in solvent Soluble in water, physiological saline and phosphate buffer; (5) Biological activity Exhibiting a metastasis-inhibitory activity on RPMI 4788 cell (FERM BP-2429), an established cell line derived from human colon cancer; and (6) Stability Inactivated in water at pH 7.2 and 100°C for 30 minutes. Stable in water at pH 7.2 and 4°C for one month. The present inventors isolated a protein possessing cancer metastasis-inhibitory activity from a culture of HPB-MLT cell (FERM BP-2430) stimulated with BCG and LPS. The present inventors revealed the physicochemical properties of said protein and found that it has the amino acid sequence as shown in Chemical formula 1. The wording "substantially has the amino acid sequence as shown in Chemical formula 1" as referred to in the invention means that it should not be restricted to that as shown in Chemical formula 1 and shall include all the homologous variants thereof. In other words, the present invention includes any protein having a partial amino acid sequence in the amino acid sequence as shown in Chemical formula 1 as long as it possesses the same physicochemical properties as the above-mentioned protein. Based on the above-mentioned amino acid sequence, the present inventors screened DNAs which might code the present protein from HPB-MLT cell, and found that the present protein contained the base sequence as shown in Chemical formula 2. The DNA according to the present invention is not restricted to that as shown in Chemical formula 2. The wording "substantially has the base sequence as shown in Chemical formula 2" as referred to in the invention means that it has the whole or a part of the base sequence of Chemical formula 2. The base sequences usable in the invention are, for example, those formed by a genetic code degeneracy wherein one or more bases in Chemical formula 2 are replaced with other bases, those which code the aforesaid homologous variants, and those which are complemental to the base sequence as shown in Chemical formula 2. The complementary base sequences may be wholly or partially complemental to that as shown in Chemical formula 2. The present invention provides a protein for use as a medicament, said protein having the following physicochemical properties: (1) Molecular weight 45,000±5,000 as determined by SDS - PAGE ; (2) Isoelectric point pI=5.7±0.5; (3) Partial amino acid sequence Possessing a partial amino acid sequence of Asp-Ser-Glu-Gly-Tyr-Ile-Tyr-Ala-Arg-Gly-Ala-Gln-Asp-Met-Lys or Glu-His-Trp-Ser-His-Asp-Pro-Phe-Glu; (4) Solubility in solvent Soluble in water, physiological saline and phosphate buffer; (5) Biological activity Exhibiting metastasis-inhibitory activity on RPMI 4788 cell (FERM BP-2429), an established cell line derived from human colon cancer; and (6) Stability Inactivated in water at pH 7.2 and 100°C for 30 minutes. Stable in water at pH 7.2 and 4°C for one month. Futhermore, the present invention provides the use of a protein for the manufacture of a prophylactic-, therapeutic, or diagnostic agent for cancer metastasis, said protein having the following physicochemical properties: (1) Molecular weight 45,000±5,000, as determined by SDS-PAGE ; (2) Isoelectric point pI=5.7±0.5; (3) Partial amino acid sequence Possessing a partial amino acid sequence of Asp-Ser-Glu-Gly-Tyr-Ile-Tyr-Ala-Arg-Gly-Ala-Gln-Asp-Met-Lys or Glu-His-Trp-Ser-His-Asp-Pro-Phe-Glu; (4) Solubility in solvent Soluble in water, physiological saline and phosphate buffer; (5) Biological activity Exhibiting metastasis-inhibitory activity on RPMI 4788 cell (FERM BP-2429), an established cell line derived from human colon cancer; and (6) Stability Inactivated in water at pH 7.2 and 100°C for 30 minutes. In addition, the present invention provides a process to prepare the above-mentioned protein, comprising culturing a human T-cell leukaemia capable of producing said protein in a nutrient culture medium to form said protein, and recovering the resultant protein from the culture. Examples of such human T-cell leukaemia are HPB-MLT cell and MOLT-4 cell (ATCC CRL 1582). In addition, those cells into which had been introduced the present DNA by conventional cell fusion or genetic engineering techniques can be advantageously used in the invention as long as the present protein is recovered from their cultures. If necessary the present protein can be prepared by culturing any one of the cells in a nutrient culture medium while stimulating it with an adequate stimulant such as BCC and LPS, and recovering the resultant protein having a metastasis-inhibitory activity from the resultant cells or supernatant. The cultivation of such a cell is carried out according to conventional techniques for animal cells and microorganisms. Conventional nutrient culture media containing vitamins, minerals, carbohydrates and the like can be employed in the invention. The recovering methods suitably used in the invention are two or more methods usually used in the purification of physiologically-active proteinous substances: For example, salting out, dialysis, centrifugation, gel filtration chromatography, ion-exchange chromatography, affinity chromatography, electrophoresis, isoelectrofocusing and isoelectric fractionation are suitably used in combination. The present invention attains the aforesaid object, and encompasses the preparation of a protein possessing a metastasis-inhibitory activity and uses of said protein. The following experiments will explain the present invention. Experiment 1 Assay for metastasis-inhibitory activity In accordance with the method of Y. Naomoto et al. described in Journal of Cancer Research and Clinical Oncology , Vol.113, pp.544-549 (1987), a metastasis-inhibitory activity was assayed with a model wherein RPMI 4788 cells (FERM BP-2429) are transplanted to nude mice so as to induce lung metastasis. In a test group, 5 or more BALB/c nude mice were injected through their tail veins with 0.2ml of phosphate buffer containing a test specimen 3 times in total before the cell transplantation, i.e. at 2 day, 1 day and 3 hours before the cell transplantation. From the next day of the transplantation of 2x10 6 RPMI 4788 cells per mouse, the mice were successively injected similarly as above with a test specimen one shot per day over a period of 7 days. In a control group, mice were similarly treated as in the test group except for using phosphate buffer free of the test specimen. On the 21st day after the cell transplantation, nude mice were sacrificed, and the number of metastatic nodules formed on the surfaces of the lungs was macroscopically counted. It was judged that a test specimen had a positive activity when the following requirements were fulfilled: (i) The mean value of numbers of lung metastatic nodules formed in the mice in the control group was 50 or more; (ii) the mean value of those in the test group was lowered to 1/2 or lower against that in the control group; and (iii) the reduction level in (ii) was evaluated as statistically significant. Experiment 2 Preparation of supernatant from culture of HPB-MLT cell stimulated with BCG and LPS A new born hamster was first injected with a serum of anti-hamster thymus prepared from rabbits in usual manner, then subcutaneously transplanted with HPB-MLT cells, and bred for 4 weeks in usual manner. About 20g weight tumor subcutaneously formed in the hamster was cut into pieces, dispersed, washed with serum-free RPMI 1640 medium, and suspended in a fresh preparation of the same medium to give a concentration of 5x10 6 cells/ml. The cell suspension was added with 10µg/ml BCG and incubated at 37°C for one day. Thereafter, the resultant cell suspension was added with one µg/ml LPS, incubated for one day, and centrifuged to obtain a supernatant. Experiment 3 Purification and physicochemical properties of the present protein The supernatant in Experiment 2 was concentrated by about 20-fold on "AIL 3013", a membrane module commercialized by Asahi Chemical Ind., Tokyo, Japan, and the concentrate was dialyzed against 25mM imidazol-HCl buffer (pH 7.4). The resultant solution in a dialytic bag was fed to a column packed with "PBE-94", a product of Pharmacia LKB, Uppsala, Sweden, preequilibrated with 25mM imidazol-HCl buffer (pH 7.4). The column was fed with "Polybuffer® 74 (pH 4.0)", commercialized by Pharmacia LKB, Uppsala, Sweden, to fractionate the supernatant, and the resultant fractions were respectively dialyzed against phosphate-buffered saline (PBS), followed by assaying each fraction for cancer metastasis-inhibitory activity. As a result, it was revealed that the fractions eluted between a pH range of 5.0-6.5 had cancer metastasis-inhibitory activity. The active fractions were pooled, and refractionated on a column packed with "Sephacryl S-200", a product commercialized by Pharmacia LKB Uppsala, Sweden. The resultant fractions were assayed for their cancer metastasis-inhibitory activity to test whether or not that the fractions, eluted with a ratio of (Elution volume)/(Void volume) in the range of 0.5-0.65, might have the activity. The active fractions were pooled and dialyzed against 10mM potassium phosphate buffer (pH 7.4). The solution in a dialytic bag was fed to a column packed with "DEAE-5PW", a product of Tosoh Corporation, Tokyo, Japan, preequilibrated with 10mM potassium phosphate buffer (pH 7.4), and eluted with a liner gradient of 10-500mM potassium phosphate buffer (pH 7.4). In this case, a substance with cancer metastasis-inhibitory activity was eluted in fractions with about 70mM potassium phosphate. The fractions thus obtained were pooled and dialyzed against 10mM sodium phosphate buffer (pH 6.8), and fed to a hydroxyapatite column commercialized by Toa Nenryo Kogyo K.K., Tokyo, Japan, preequilibrated with 10mM sodium phosphate buffer (pH 6.8). In this case, cancer metastasis-inhibitory activity was found in non-adsorbed fractions which were then fed to a column packed with "Mono-P", a product of Pharmacia LKB, Upssala, Sweden, preequilibrated with 25mM bis-tris-iminodiacetate buffer (pH 7.1), and eluted with "Polybuffer® 74 (pH 4.0)", a product of Pharmacia LKB, Uppsala, Sweden, followed by isolating the present protein with cancer metastasis-inhibitory activity. Thus, about 70µg of the present protein was isolated from 50L of the culture supernatant of HPB-MLT cells stimulated with BCG and LPS. The physicochemical properties of the present protein were studied with the isolated protein. (1) Molecular weight In accordance with the method of U. K. Laemmli described in Nature, Vol.227, pp.680-685 (1970), the protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (abbreviated as "SDS-PAGE" hereinafter), and the molecular weight was determined to be 45,000±5,000 based on the relative mobility of the protein against marker proteins. (2) Isoelectric point The isoelectric point of the protein was estimated to be 5.7±0.5 based on the pHs of eluates on a column chromatography using Mono-P column; (3) Partial amino acid sequence The protein was subjected to SDS-PAGE, and a band corresponding to the molecular weight of about 45,000 in the resultant gel was isolated by cutting. The resultant gel piece was soaked in 100mM Tris-HCl buffer (pH 9.0) containing 0.1% sodium dodecyl sulfate (SDS) at 37°C for one hour, and digested at 37°C overnight by the addition of 5µg/ml lysyl endopeptidase, an enzyme specimen commercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan. The supernatant obtained from the resultant was fractionated on a reverse-phase chromatography using a column of "C-18", commercialized by VYDAC, Hesperia, USA, equipped with a precolumn of DEAE, followed by recovering peptide fragments which were then analyzed on "470A", an amino acid sequencer commercialized by Applied Biosystems Inc., Foster City, USA. The results were as shown in Chemical formulas 3, 4, 5 and 6. (4) Solubility in solvent The protein was soluble in water, physiological saline and phosphate buffer. (5) Biological activity The protein was tested for cancer metastasis-inhibitory activity with the method in Experiment 1. As a result, the number of metastatic nodules was 314±169 in a control group consisting of 5 nude mice, while that in a test group, wherein 5 nude mice were administered with a solution containing 250µg/ml of the protein, was 100±43. These confirmed that the protein exhibited a strong cancer metastasis-inhibitory activity. (6) Stability The protein was dissolved in phosphate buffer (pH 7.2) and allowed to stand at 100°C for 30 minutes, followed by determining the residual activity with the method in Experiment 1 to give no activity. Thus, it was revealed that the protein was inactivated under the conditions.While the protein was treated by dissolving it in phosphate buffer (pH 7.2), and allowing the resultant solution to stand at 4°C for one month, followed by assaying the residual activity similarly as above. As a result, no substantial loss of activity was found, and this revealed that the protein was stable under the conditions. Experiment 4 Acute toxicity By using 7 week-old mice, the present protein was tested for acute toxicity. As a result, the LD 50 in mice of the protein was 50mg/kg or higher when intravenously administered to the mice, and this revealed that the toxicity was extremely low. Experiment 5 Base sequence coding the present protein In this experiment, the base sequence of the present protein was determined by conventional method as described by T. Maniatis et al. in Molecular Cloning, A Laboratory Manual , 2nd edition, published by Cold Spring Harbor Laboratory Press (1989), New York, USA. Experiment 5-1 Construction of cDNA library of HPB-MLT cell HPB-MLT cells obtained by the method in Experiment 2 were suspended in a serum-free RPMI 1640 medium to give a concentration of 5x10 6 cells/ml. The cell suspension was added with 10µg/ml BCG and incubated at 37°C for one day. The culture thus obtained was added with one µg/ml LPS and incubated for 4.5 hours. The resultant culture was centrifuged to obtain cells which were then solubilized with 4M guanidium isocyanate and homogenized. The resultant homogenate was overlaid on 5.7M cesium chloride, and the mixture was centrifuged at 25,000rpm for 17 hours to obtain the whole RNAs of the cells. Poly (A) + RNA was purified from the whole RNAs on "Oligotex® -dT30<Super>", a bead for purification of poly (A) + RNA commercialized by Daiichi Pure Chemicals, Tokyo, Japan. The purified poly (A) + RNA was treated with "cDNA synthesis system plus", a product of Amersham International plc, Buckinghashire, England, to synthesize a cDNA under the direction of the appended manual. The cDNA thus obtained was ligated with a λgt10 phage DNA by using "cDNA cloning system λgt10", a product of Amersham International plc, Buckinghashire, Englnad. The resultant recombinant phage DNA was packaged by "Lambda(λ) in vitro packaging kit", a product of Amersham International plc, Buckinghashire, Englnad, to obtain a cDNA library of HPB-MLT cell. Experiment 5-2 Construction of radiolabeled DNA probe Based on the partial amino acid sequence as shown in Chemical formula 3 in Experiment 3, base sequences estimable from the amino acid sequence of Glu-Gly-Tyr-Ile-Tyr-Ala in Chemical formula 3 were synthesized by a DNA synthesizer commercialized by Applied Biosystems, Inc., Foster City, USA. Ninety-six base sequences consisting of 17 synthesized bases are as shown in Table 1. As for the partial amino acid sequence as shown in Chemical formula 5, complementary chains of base sequences, estimable from Asn-Pro-His-Leu-Lys in the partial amino acid sequence in Chemical formula 5, were synthesized similarly as above. Ninety-six base sequences consisting of 14 synthesized bases are as shown in Table 2. The DNAs thus obtained were radiolabeled with [γ- 32 P]ATP and T4 polynucleotide kinase. Experiment 5-3 Screening with radiolabeled DNA probes A solution of the recombinant λgt10, a cDNA library of HPB-MLT cell prepared in Experiment 5-1, was mixed with an overnight culture of microorganisms of E . coli strain NM 514 in L-broth, and the mixture was incubated at 37 C for 15 minutes. The resultant was admixed with a soft agar, and the resultant mixture was overlaid on a hard-agar plate and solidified. The resultant agar plate containing the microorganisms were incubated at 37°C for 8 hours, cooled and overlaid with "Hybond-N filter", a membrane filter of Amersham International plc, Buckinghamshire, England, to transfer the phage and to fix the phage DNA on the membrane filter. In order to prevent a non-specific bonding of the radiolabeled DNA probes with DNAs except for the objective complementary DNA, the membrane filter was soaked in a solution of a salmon sperm DNA commercialized by Sigma Chemical company, St., Louis, USA, to effect prehybridization, followed by screening positive clones by the southern hybridization with the radiolabeled DNA probes in Experiment 5-2. The results in the first screening test with the probe 1 in Table 1 and the second screening test with the probe 2 in Table 2 revealed that 3 positive clones were present among about 600,000 clones. Phage DNAs isolated from the positive clones were digested with a restriction enzyme EcoRI to remove them from vector DNAs, and the length of the inserted fragments were studied on agarose electrophoresis, followed by analyzing a clone having the longest inserted-fragment of about 1.5kbp. Experiment 5-4 Base sequence of gene coding the present protein From the positive clones obtained in Experiment 5-3, a phage DNA was isolated and digested with a restriction enzyme EcoRI, and the resultant fragments were separated on SDS-PAGE to obtain an inserted DNA fragment of about 1.5kbp which was then ligated with a pUC18 plasmid with a ligation kit commercialized by Amersham International plc, Buckinghashire, England, to obtain a recombinant plasmid. The recombinant plasmid thus obtained was introduced in usual manner into E . coli to obtain a recombinant microorganism, and from which a plasmid DNA was prepared.. The dideoxy chain termination method was applied to the resultant plasmid DNA to reveal the base sequence of the present protein. Based on the base sequence, the amino acid sequence of the present protein was estimated. As a result, it was revealed that the present DNA consisted of 1,224 bases and coded a protein consisting of 408 amino acids. The estimated amino acid sequence encompassed the amino acid sequences as shown in Chemical formulas 3, 4, 5 and 6 in Experiment 3. The following is an example of the preparation of the present protein. Example Similarly as the method in Experiment 2, MOLT-4 cell (ATCC CRL 1582), an established cell line derived from human T-cell leukemia commercialized by Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan, were cultured in a nutrient culture medium while stimulating the cells with BCG and LPS. Similarly as in Experiment 3, a supernatant prepared from the resultant culture was treated to obtain the present protein possessing cancer metastasis-inhibitory activity. The yield was about 40µg per 50L of the culture supernatant. The protein had the same physicochemical properties as the one in Experiment 3. The protein preparable according to the present invention effectively inhibits the metastasis of cancers, and this renders it advantageously useful as prophylactic-, therapeutic- and diagnostic-agents for cancer metastases. The toxicity of the present protein is satisfactorily low, because of this it can be systematically administered to patients in the form of an injection, sublingual agent, and the like. The protein having the aforesaid advantages is prepared on an industrial scale by the present invention. The DNA coding the protein is useful in the preparation of the protein preparable according to the present invention by genetic engineering techniques.

Claims: A protein for use as a medicament, or a diagnostic agent for cancer metastasis said protein having the following physicochemical properties: (1) Molecular weight 45,000 ± 5,000 as determined by SDS - PAGE ; (2) Isoelectric point PI = 5.7 ± 0.5 (3) Partial amino acid sequence Possessing a partial amino acid sequence of Asp-Ser-Glu-Gly-Tyr-Ile-Tyr-Ala-Arg-Gly-Ala-Gln-Asp-Met-Lys or Glu-His-Trp-Ser-His-Asp-Pro-Phe-Glu; (4) solubility in solvent Soluble in water, physiological saline and Phosphate buffer; (5) Biological activity Exhibiting metastasis-inhibitory activity on RPMI 4788 cell (FERM BP-2429), an established cell ine derived from human colon cancer; and (6) Stability Inactivated in water at pH 7.2 and 100°C for 30 minutes. Stable in water at pH 7.2 and 4°C for one month. A protein according to claim 1, which substantially has the amino acid sequence as shown in chemical formula 1: Use of a protein for the manufacture of a prophylactic, therapeutic, or diagnostic agent for, cancer metastasis, said protein having the following physicochemical properties: (1) Molecular weight 45,000 ± 5,00 as determined by SDS - Page ; (2) Isoelectric point pI=5.7±0.5; (3) Partial amino acid sequence Possessing a partial amino acid sequence of Asp-Ser-Glu-Gly-Tyr-Ile-Tyr-Ala-Arg-Gly-Ala-Gln-Asp-Met-Lys or Glu-His-Trp-Ser-His-Asp-Pro-Phe-Glu; (4) Solubility in solvent Soluble in water, physiological saline and Phosphate buffer; (5) Biological activity Exhibiting metastasis-inhibitory activity on RPMI 4788 cell (FERM BP-2429), an established cell line derived from human colon cancer; and (6) Stability Inactivated in water at pH 7.2 and 100°C for 30 minutes. Stable in water at pH 7.2 and 4°C for one month. Use of a protein according to claim 3, wherein the protein substantially has the amino acid sequence as shown in chemical formula 1 as defined in claim 2. A process for making a medicament, or a diagnostic agent for cancer metastasis, which process comprises providing a protein as defined in claim 1 or claim 2; and formulating the medicament or diagnostic agent for cancer metastasis therefrom. A process according to claim 5, wherein the step of providing the protein comprises the steps of: (a) culturing a human T-cell leukaemia capable of producing a protein in a nutrient culture medium to form the protein; and (b) recovering the resultant protein. A process according to claim 6, wherein the human cell derived from human T-cell leukaemia is HPB-MLT cell (FERM BP-2430 or MOLT-4 cell (ATCC CRL 1582). A process according to claim 6 or claim 7, wherein step (a) contains a step of allowing the cell to contact with Bacille Calmette-Guerin and lipopolysaccharide as an inducer of the protein. A process according to claim 5, wherein the step of providing a protein as defined in claim 1 comprises the steps of: (a) culturing a cell capable of producing a protein as defined in claim 1 in a nutrient culture medium with or without an adequate stimulant to produce the protein; and (b) recovering the resulting protein. A process according to claim 9, wherein the cell has been introduced with DNA substantially having a nucleotide sequence as shown in chemical formula 2. Use of a DNA encoding a protein as defined in claim 1 or claim 2 for the manufacture of a medicament, or a diagnostic agent comprising a protein as defined in claim 1 or claim 2 for the inhibition of cancer metastasis. Use of a DNA according to claim 11, wherein the DNA substantially has a nucleotide sequence as shown in chemical formula 2.

Inventors: IWAKI KANSO, KURIMOTO MASASHI, OHTA TSUNETAKA, IWAKI, KANSO, KURIMOTO, MASASHI, OHTA, TSUNETAKA
Applicants: HAYASHIBARA BIOCHEM LAB, KABUSHIKI KAISHA HAYASHIBARA SEIBUTSU KAGAKU KENKYUJO
Application year: 1993
Main classification: C12N15
IPC codes: C07K14, A61K38, C12N5, C12P21, A61P35, C12N15, C12R1