CN100365015C - 12-mer peptide, compound containing the same, its preparation method and application in cancer treatment drug - Google Patents

Abstract

The present invention relates to a 12-peptide, a guiding compound containing the 12-peptide, a preparation method and an application for preparing medicines for curing liver cancer. The 12-peptide has an amino acid sequence shown in a sequence 1 in a sequence list and has the specific guiding function to liver cancer cells, the guiding compound containing the 12-peptide for guiding and curing liver cancer is composed of two parts comprising the 12-peptide used as a navigational system for biologic guiding therapy and super antigen as a warhead for the biologic guiding therapy or other biologic toxin molecules or chemotherapeutic medicines, and the two parts are combined in the way of chemical interlinkage or gene fusion. The 12-peptide and the guiding compound containing the 12-peptide in the present invention can be used for preparing medicines for curing the liver cancer, and have important significance in curing the liver cancer.

Description

A kind of dodecapeptide, contain the mixture of this dodecapeptide, its preparation method and the application in the preparation cancer treatment drug

Technical field

The present invention relates to a peptide species, contain the mixture of this dodecapeptide, also relate to its preparation method and the application in preparation liver cancer targeted therapy medicine thereof.

Background technology

Hepatocellular carcinoma (HCC) be the most common, also be one of the most fatal malignant tumour, have the title of " cancer king ", be characterized in the morbidity concealment, shift early the case fatality rate height.

The traditional treatment of liver cancer is based on excision and chemotherapy.Operation thoroughly excision provides best healing chance, is the prefered method for the treatment of liver cancer at present, yet for most tumors, too greatly or too disperse, can't excise more than 75% when detecting." X ray of gamma-rays or linear electron accelerator, energetic ray etc.; traditional route of administration has been improved in the chemotherapy aspect; for the primary hepatocarcinoma that can't excise is not very sensitive; it is to the destruction of body immune system in addition; treatment back patient's The average survival time only 4 months are seldom above 9 months though the radiotherapy aspect has adopted.

Topmost restriction for the patient that can not carry out surgical blanking is that general toxicity and levels of drugs do not reach effective concentration at traditional chemotherapy and radiation, and targeted therapy has good application prospects because of its highly selective and high-affinity for inoperable patient of treatment or the tumour that extensively shifts.At present the liver cancer targeted therapy is to utilize a kind ofly to have the antibody of special avidity to make carrier to liver cancer, make the commissure thing with the bullet that the killing tumor cells effect is arranged (superantigen, chemotherapeutics, biotoxin albumen radionuclide), reduce the purpose of infringement healthy tissues to reach more killing tumor cells.Usefulness such as Chen Zhinan ¹³¹The liver cancer monoclonal antibody Hepama-1 of I mark injects 23 routine patients by artery in the liver, has obtained curative effect preferably, and 75% (12/16) patient AFP value descends, and patient's knurl piece of 78% (18/23) dwindles.II phase clinical study is the result show: complete remission rate 4.1%, part complete remission rate are that survival rate is 46.2% more than 53.1%, two year.

Although antibody targeted treatment is most active one of the cancer strategy of controlling, some problems that exist have limited their practical application effect.Treatment antibody at liver cancer is mouse source property at present, is applied to human body, is prone to human antimouse antibody (HAMA), ¹³¹In the therapeutic process of I-Hepama-1,43% (10/23) patient has produced the antibody at Hepama-1.Obviously, HAMA has limited multiple injection, and long-term treatment can't be carried out.The molecular weight of antibody is big in addition, and seepage force is poor, and being difficult for penetrating the tumour capillary vessel also is the major cause that causes liver cancer targeted therapy poor effect.Small molecular antibody have immunogenicity low, be easy to permeate destination organization, toxic side effect little, easily link to each other with effector molecule and constitute the advantages such as antibody molecule of new function, constantly carry out from the research work of single-chain antibody, single domain antibody, independence variable region of heavy chain, atom antibody fragments such as (MRU), but these antibody fragments are compared with parental antibody, there is decline in various degree in antigen-binding activity, and molecular weight is still bigger, with the single-chain antibody is example, molecular weight still reaches about 30Kda, and is relatively poor to the noumenal tumour penetrativity.

Arrange owing to contain all possible amino acid in the phage peptide library, thereby storage capacity is very big, is easy to screening and amplification.By the affinity screening, might obtain novel desired polypeptides than high specific and avidity, be used for the targeted therapy of liver cancer.Do not see at present the bibliographical information that adopts polypeptide liver cancer to be carried out targeted therapy.

Summary of the invention

For overcoming the deficiency of current liver cancer treatment, the present invention seeks to seek a kind of micromolecule polypeptide molecule that combines with liver-cancer cell specific and have high affinity.

The present invention by phage display peptide library screening obtained a kind of can with liver cancer cell specificity bonded 12 peptides, its aminoacid sequence is shown in sequence in the sequence table 1.

The invention also discloses a kind of nucleotide sequence of the above-mentioned dodecapeptide of coding, shown in sequence in the sequence table 2.

The liver cancer targeted therapy mixture that utilizes dodecapeptide of the present invention to prepare to contain this dodecapeptide.Wherein dodecapeptide is as " navigationsystem " of bio-guide treatment, and " bullet " can adopt chemotherapeutics, superantigen or other biotoxin molecule or chemotherapeutics, and both can be by the mode combination of chemical interlinkage or gene fusion.Dodecapeptide provided by the invention combines with the mode of superantigen by gene fusion, can conveniently prepare the fusion rotein of dodecapeptide and superantigen.Superantigen can be to derive from staphylococcus and streptococcic superantigen, for example staphylococcal intoxication shock toxin T SST-1, staphyloentero-toxin SEA, SEB, SEC, SED and SEE etc., SPE A, B, C etc.Other biotoxin molecule can be Pseudomonas aeruginosa extracellular toxin PEA, diphtheria toxin DT etc.In above " bullet " molecule, preferred staphylococcal intoxication shock toxin T SST-1, because in above-mentioned " bullet " molecule, TSST-1 molecular weight minimum, and have the superantigen activity stronger, thereby be suitable as " bullet " of 12 peptide targets than other staphylotoxin molecule.

The liver cancer targeted therapy mixture of dodecapeptide of the present invention can be by gene engineering method or the preparation of chemical interlinkage method.When preparing by gene engineering method, generally need through following steps: the PCR legal system is equipped with the fusion gene of 12 peptides and " bullet " molecule; Be connected into expression vector; Expressing fusion protein and purifying.By pcr amplification, the nucleotide sequence of above-mentioned dodecapeptide is connected with " bullet " molecular gene, express then.The present invention is merged dodecapeptide and toxic shock toxin (TSST-1) gene by the PCR method, in upstream primer, introduce 12 peptide sequences, adopt connection peptides Gly-Gly-Ser between 12 peptide sequences and the TSST-1, make up the liver cancer targeted therapy mixture that contains dodecapeptide.Be connected with expression vector then, make up recombinant expression plasmid.Through induce, after the expression, purifying, obtained special target protein, be solubility expression, after expressing protein is purified, through experiment confirm: the fusion rotein that contains this polypeptide can combine with liver-cancer cell specific, this fusion rotein of experiment in vitro can suppress the propagation of human liver cancer cell, rat liver cancer transplanted tumor test confirmation fusion rotein is compared with independent TSST-1 group, and toxicity reduces, and curative effect improves, play synergism and attenuation, confirm that this polypeptide has guide effect to liver cancer cell.The molecular weight of the fusion rotein of this 12 peptide sequence and TSST-1 has only about 24Kda, is the target superantigen molecule of current molecular weight minimum.

The present invention adopts brand-new targeted therapy thinking, discloses with liver-cancer cell specific and has combined the also dodecapeptide of tool high-affinity, can be used as " navigationsystem ", and this dodecapeptide molecular weight is little, with the normal liver cell debond.Internal and external test has confirmed this dodecapeptide and the guide effect that contains the fusion rotein of this peptide, for the further immunotherapy of liver cancer provides theoretical foundation, might become potential liver cancer targeted therapy medicine.

Description of drawings

Fig. 1: positive transformant Screening and Identification result, the about 750bp of amplification segment size

Fig. 2: the recombinant plasmid enzyme is cut qualification result, wherein 1,3 cuts the result for the vector plasmid enzyme, the 2 vector plasmid contrasts of cutting for enzyme not

Fig. 3: 12 peptides-TSST-1 recombinant protein purification collection of illustrative plates

Fig. 4: 12 peptides-TSST-1 recombinant protein product expression and purification product S DS-PAGE analyzes

Fig. 5: 12 peptides-TSST-1 recombinant protein combines specific activity with the liver cancer cell of Hab25SCFV-TSST-1

Fig. 6: the liver cancer cell killing activity of 12 peptides-TSST-1 recombinant protein, initial concentration are 10 times of serial dilutions.

Fig. 7: each organizes the gross tumor volume changing trend diagram.

Embodiment

Embodiment one contains the structure and the expression of high-affinity small peptide and toxic shock toxin Prokaryotic Expression plasmid

One, material

1. bacterial strain: bacillus coli DH 5 alpha, prokaryotic expression carrier: PBV220 is made up by domestic Virology Inst., China Academy of Preventive Medicine Sciences.

2. main agents

Restriction enzyme, sec.-propyl-β-D-sulfo-semi-lactosi glucosides (IPTG), 5-bromo-4-chloro-3-indoles galactoside (X-gal), T4 DNA ligase is available from the precious biotech firm in Dalian; The Taq polysaccharase, PCR reagent such as dNTP are given birth to the worker available from Shanghai; Tryptones (Bacto trypton), yeast extract powder (Bacto yeast-extract) is available from Difco company; Sodium lauryl sulphate (SDS), acrylamide, N,N methylene bis acrylamide is available from Amersham company; Agarose is available from Gibco company; Other reagent are analytical pure;

3. main solution

3.1 LB liquid and solid medium

Two, method

1, primer design and synthetic

By linker (Gly-Gly-Ser), that will obtain by phage selection and the small peptide nucleotide sequence liver cancer specific combination and TSST-1 (toxic shock toxin) coupling mutually:

The upstream primer sequence that contains wire 12 peptide specific short peptide sequences contains the EcoRI restriction enzyme site referring to sequence in the sequence table 3;

Downstream primer is seen sequence 4 in the sequence table, contains the HindIII restriction enzyme site, and primer is synthetic by Shanghai Bo Ya Bioisystech Co., Ltd.

2, PCR reaction

With streptococcus aureus FRI 1169 DNA is template (serving as to produce the TSST-1 type strain), makes up the fusion gene of 12 peptides and TSST-1 by the PCR reaction.

PCR reaction system (50 μ L): H ₂O 39 μ l, 10xPCR buffer 5 μ l, dNTP 2 μ l, primer 2 μ l, Taq 1 μ l, template 1 μ l.

The PCR reaction parameter: 96 ℃ of pre-sex change 5min, 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 45s, and 72 ℃ are extended 10min.The recovery of PCR reaction product is carried out according to ordinary method.

3, being connected of target DNA and T carrier, conversion

The PCR reaction product that reclaims is connected with PMD-18 T vector, and the ligation thing consists of: P1 4 μ l, PMD-18 T carrier 1 μ l, Solution I 5 μ l.16 ℃ connect 2 hours.

The preparation of competent cell connects the product conversion and carries out according to a conventional method.

The positive bacterium colony of picking carries out pcr amplification, and the PCR product is observed through 1% agarose electrophoresis, and positive colony checks order, and extracts the positive colony plasmid.

4. the structure of recombinant expression plasmid

The positive colony plasmid that filters out is carried out enzyme with restriction enzyme cut after order-checking confirms, be connected with the prokaryotic expression carrier PBV220 that enzyme of the same race is cut.Endonuclease reaction consists of 10 * buffer, 2 μ l, EcoRI2 μ l, HindIII 2 μ l, Vector Plasmid 14 μ l.37 ℃ digested 4 hours.After enzyme is cut end, connect conversion with ordinary method.Identify recombinant plasmid, picking list bacterium colony, carrying out volume is the PCR evaluation of 25 μ L; And extract positive bacterium colony plasmid and do enzyme and cut evaluation.Confirm as male and carry out abduction delivering.

5. abduction delivering and SDS-PAGE electrophoresis are identified

5.1 carry out temperature-induced to PBV220 and the segmental recombinant expression plasmid of purpose

Picking positive colony list bacterium colony, incubated overnight.Transferred in 5mL LB (contain 200mg/mL penbritin) with 1: 100 next day, 30 ℃, after 250rpm is cultured to logarithmic phase, is warming up to 42 ℃ immediately and cultivated 4 hours.

Get cultured bacterium liquid 1.5mL, with centrifugal 5 minutes collection of 8000rpm room temperature bacterium, abandon supernatant, 200 μ L pure water have hanged precipitation, get 25 μ L suspension and add 25 μ L, 2 * sds gel sample loading buffer, boil 10 minutes, get 20 μ L and carry out the SDS-PAGE analysis.

Three, result

1. polypeptide and wild-type TSST-1 gene Fusion

By Linker (Gly-Gly-Ser), with specificity small peptide nucleotide sequence (wire 12 peptides) and wild-type TSST-1 coupling connection mutually, cut the evaluation positive colony through blue hickie screening of T carrier and enzyme, equal then subclone is gone into expression vector PBV220 and is expressed.PCR and enzyme are cut the evaluation collection of illustrative plates and are seen Fig. 1,2.

2. the non-fusion expression of recombinant protein

Adopt temperature-induced expression, promptly bacterium is cultured to logarithmic phase for 30 ℃, is warming up to 42 ℃ immediately, cultivates 4 hours, and SDS-PAGE identifies expression, and 12 peptide couplings connection TSST-1 has obtained the expression (see figure 4) in intestinal bacteria.

The proteic purifying of embodiment two dodecapeptides and TSST-1 gene fusion is identified and is detected with active

One, material

Clone: hepatocellular carcinoma SMMC7721, BEL-7402, normal liver cell are HL-02.

Engineering bacteria: the intestinal bacteria (embodiment one makes up) that can efficiently express 12 peptide couplings connection TSST-1

ACTA FPLC protein purification system, purification column CM-Sepharose FF are the AMESHAM product.Anti-TSST-1 antibody is SIGMA company product, HRP mark two anti-(goat anti-rabbit antibody)

Two, method:

1. the purifying of expressing protein

By aforementioned condition, induce engineering bacteria in a large number, all in broken supernatant, this just makes protein purification comparatively easy to expressed proteins, after further diluting, last sample, and collection penetrates liquid; Expression product is in supernatant, and its purifying adopts CM column purification method, and SDS-PAGE evaluation collection of illustrative plates is seen figure three and figure four behind purifying collection of illustrative plates and the purifying.Purification process is referring to " Jiang Yongqiang etc., the preparation of staphylococcus Type B enterotoxin and anti-tumor activity analysis, cell and molecular immunology magazine, 2002,18 (3) "

2. the ELISA of fusion rotein and competitive ELISA are identified:

2.1 the ELISA of recombinant expression protein identifies

Get the good Tissue Culture Plate of sealing, seal, abandon confining liquid next day preceding a whole night, with 0.5%PBST washing 5 times, each 5 minutes at interval; Add the purifying protein solution of gradient dilution, room temperature left standstill 1 hour; Abandon not conjugated protein, with 0.5%PBST washing 5 times, each 5 minutes at interval; Add 200 μ L anti-TSST-1 antibody (dilution in 1: 500), room temperature left standstill 1 hour; Abandon not binding antibody, with 0.5%PBST washing 5 times, each 5 minutes at interval; Add HRP mark two anti-(goat anti-rabbit antibody was with dilution in 1: 1000), every hole 200 μ L, room temperature left standstill 1 hour; Abandon two and resist, with 0.5%PBST washing 5 times, each 5 minutes at interval; Add TMB colour developing liquid, after treating fully to develop the color, add 2N H ₂SO ₄Termination reaction is in the 450m value of reading.

2.2 the competitive ELISA of recombinant expression protein and dsFv-TSST-1 is identified

The single-chain antibody target TSST-1 fusion rotein (ScFv-TSST-1) that this chamber makes up, with liver cancer cell affinity and specificity are preferably arranged, competition by recombinant expression protein and dsFv-TSST-1 is compared, and can react the specificity and the avidity of 12 peptide TSST-1 recombinant proteins.The SCFv-TSST-1 recombinant protein is done gradient dilution, mix with recombinant protein respectively, survey A _450nmVariation.

3, the tumor cell in vitro killing activity of recombinant expression protein

The recombinant expression protein cell killing activity detects employing MTS assay kit, and (CellTiter 96TMAQUEOUS Promega) measures.Concrete grammar is that 100 μ l contain 5 * 10 ³Add the human peripheral lymphocyte (effector cell) of 80 μ l in the RPMI1640 perfect medium of individual SMMC-7721 liver cancer cell (target cell) with the RPMI1640 substratum dilution that does not contain serum, imitating the target ratio is 20: 1, every then hole adds the sample of 20 μ l gradient dilutions, 37 ℃ of cell incubators that contain 5%CO2 are cultivated more than 48 hours, discard substratum then, wash 3 times with not containing serum RPMI1640 substratum, add 100 μ l RPMI1640 perfect mediums and 20 μ lMTS and continue to cultivate 2h, 490nm surveys the OD value.

4, the interior tumor cell killing activity of recombinant expression protein

With male BABL/C mouse (18-22g) is experimental animal, and the H22 liver cancer cell is the inoculation oncocyte, and concrete grammar is: get H22 tumour ascites, with physiological saline dilution in 1: 4, be configured to tumor cell suspension, cell count is (2～3) * 10 ⁵/ ml, it is subcutaneous to be inoculated in BABL/C mouse forelimb armpit, male body weight 18～22g, every injected in mice 0.1ml (contains H22 cell 2*10 ⁶～4*10 ⁶Individual), every group of 8 animals.0 day of experiment, inoculated tumour.The 1st day, animal is weighed and be divided into fusion rotein high dose group (2.5mg/kg), middle dosage group (0.5mg/kg), low dose group (0.1mg/kg), TSST-1 control group (0.47mg/kg) and physiological saline control group at random, intravenously administrable then, 1 time/2 days, totally three times.Each is prepared before organizing administering medical liquids.Weighed every other day from the 1st day, survey knurl long (a), wide (b), press V=0.5*a*b2 and calculate the knurl volume.To the 9th day, animal was weighed, and cutd open the tumour of getting each treated animal, weigh and add up each the group tumour inhibiting rate.

Two, result

1, the expressing protein after the separation and purification has higher purity, and the SDS-PAGE electrophoresis result is seen Fig. 4.

2, the proteic liver cancer affinity of expression and purification dilution experiment (cell ELISA result)

After the recombinant protein that will contain wire 12 peptides and TSST-1 sequence is done serial gradient dilution, carry out the cell ELISA test, ELISA the results are shown in Table 1:

Table 1 ELISA method detects PBV220 and the segmental recombinant protein result of purpose

The dilution gradient	Stoste		10 -1	10 -2	10 -3	10 -4	10 -5	10 -6	10 -7	Negative control
											Sample	2.946	0.781	0.668	0.498	0.436	0.347	0.264	0.330	0.132

3, doing the fusion protease joint inspection with fixing sealing good 7402,7721 cell, HL-02 cell cell surveys, establish negative control simultaneously, fusion rotein and liver cancer cell bonded A value are significantly higher than normal liver cell, and be very low with the bonded OD value of normal liver cell, has specificity preferably.The results are shown in Table 2.

Table 2 12 peptide TSST-1 fusion roteins combine situation with liver cancer (7721,7402) and normal liver cell (HL-02 cell)

Cell type	-	-	1	2	3	4	5	6	7	8
											7721	0.097	0.112	0.860	1.024	0.917	0.699	0.670	0.764	0.825	0.913
7402	0.098	0.132	0.824	0.707	1.262	0.580	0.618	0.684	0.652	0.790
											HL-02	0.011	0.043	0.112	0.176	0.091	0.205	0.137	0.108	0.155	0.203

4, for further estimate recombinant protein in conjunction with active, we have done HAb25 ScFv-TSST-1 albumen (it has the very stable activity that combines with SMMC-7721) that 12 peptide TSST-1 fusion roteins and this chamber make up with liver cancer cell SMMC-7721 in conjunction with specific activity.The result is as follows: combining of 12 peptides-TSST-1 fusion rotein and Hab25 ScFv-TSST-1 is quite active, illustrate that 12 peptides and TSST-1 coupling join after, itself and liver cancer cell have very high avidity.(Fig. 5)

5,12 peptide TSST-1 fusion roteins that give expression to and dsFV-TSST-1 recombinant protein competition result

Utilize dsFV-TSST-1 serial dilution gradient recombinant protein and the above-mentioned albumen that gives expression to make competitive assay.Competition suppresses experimental result and shows: along with the increase of dsFV-TSST-1 recombinant protein extension rate, inhibiting rate is also increasing, this epi-position that just dsFV-TSST-1 is described and is constituted is identical with the epi-position of the polypeptide that filters out, and is promptly identical with the site of liver cancer cell 7721 specific combination.The results are shown in Table 3.

Table 3 dsFV-TSST-1 serial dilution gradient recombinant protein and expressing protein competitive assay result

Numbering/extension rate	Negative	Stoste		10 1	10 2	10 3	10 4	10 5	10 -6
										12 peptide protein	0.089	1.397	0.652	0.829	0.914	0.938	0.941	0.952
Inhibiting rate (%)			53.3	40.7	34.6	32.9	32.6	31.9

5, the tumor cytotoxicity effect of 12 peptide TSST-1 fusion roteins: 12 peptide TSST-1 fusion roteins have lethal effect to liver cancer cell, and dosage is high more, and fragmentation effect is obvious more, has clear and definite dose effect curve.Concrete fragmentation effect is seen Fig. 6.

6, the interior tumor cell fragmentation effect of 12 peptide TSST-1 fusion roteins:

(1) gross tumor volume changes

With the length of vernier caliper measurement tumour and wide, calculate gross tumor volume the 1st, 3,5,7 and 9 day of experiment as follows, the tumor growth of medication group is considerably slower than the physiological saline control group, has clear and definite its variation tendency of tumor suppression effect and sees Fig. 7.

(2) be subjected to reagent thing influence and the inhibiting rate heavy to knurl

Take off vertebra when experiment finishes and put to death mouse, strip tumour, weigh and calculate the fusion rotein of various dose and TSST-1 inhibiting rate tumour.The tumor control rate of high, normal, basic three the dosage groups of 12 peptide TSST-1 fusion roteins all is higher than the TSST-1 control group, and toxicity has reduction trend, the TSST-1 control group has 2 death in 8 animals in experimentation, and the dosage group does not have dead mouse in the 12 peptide TSST-1 fusion roteins, and growth conditions is good, demonstrates certain synergism and attenuation.Tumor suppression the results are shown in Table 4.

Table 4. is respectively organized tumor weight (mean value SD) and the inhibiting rate of laboratory animal

Group	High dosage (2.50mg/kg)	Middle dosage (0.50mg/kg)	Low dosage (0.10mg/kg)	TSST-1 control group (0.47mg/kg)	The N.S control group
						Heavy (g) tumor control rate of knurl	0.3249±0.1934 69.35％ ***	0.3615±0.1812 65.90.％ ***	0.4075±0.1798 61.55％ ***	0.4828±0.2152 54.45％ **	1.0600±0.3566 -

Sequence table

＜110〉Inst. of Epidemiology and Microbiology, Academy of Military Medical Sciences, PL

＜120〉a kind of dodecapeptide, contain the mixture of this dodecapeptide, its preparation method and the application in the preparation cancer treatment drug

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Claims (5)

1. liver cancer targeted therapy mixture that contains dodecapeptide, wherein the aminoacid sequence of dodecapeptide is shown in sequence in the sequence table 1, it is characterized in that dodecapeptide by " navigationsystem " for the treatment of as bio-guide, form with the superantigen or the chemotherapeutics two portions of " bullet " for the treatment of as bio-guide, these two portions adopt the mode combination of chemical interlinkage or gene fusion.

2. according to the described liver cancer targeted therapy mixture that contains dodecapeptide of claim 1, it is characterized in that described superantigen is staphylococcal intoxication shock toxin T SST-1, dodecapeptide is connected by gene fusion with TSST-1, and connection peptides is Gly-Gly-Ser.

3. claim 1 or the 2 described preparation methods that contain the liver cancer targeted therapy mixture of dodecapeptide is characterized in that may further comprise the steps:

(1) the PCR legal system is equipped with the fusion gene of 12 peptides and TSST-1;

(2) be connected into expression vector pBV220;

(3) expressing fusion protein and purifying.

4. claim 1 or the 2 described application of liver cancer targeted therapy mixture in the preparation cancer treatment drug that contain dodecapeptide.

5. the application of the dodecapeptide of its aminoacid sequence shown in sequence in the sequence table 1 in the preparation cancer treatment drug.

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