CN106350578A - Application of NY-ESO-1 in diagnosis and treatment of microsatellite instability intestinal cancer - Google Patents
Application of NY-ESO-1 in diagnosis and treatment of microsatellite instability intestinal cancer Download PDFInfo
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- CN106350578A CN106350578A CN201510410087.4A CN201510410087A CN106350578A CN 106350578 A CN106350578 A CN 106350578A CN 201510410087 A CN201510410087 A CN 201510410087A CN 106350578 A CN106350578 A CN 106350578A
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Abstract
The invention provides application of NY-ESO-1 in diagnosis and treatment of the microsatellite instability intestinal cancer. Specifically, the invention provides application of an NY-ESO-1 gene, a protein or a detection reagent in preparation of a kit for (a) diagnosing the microsatellite instability intestinal cancer, and/or (b) judging prognosis of an intestinal cancer patient. Furthermore, an experiment shows that by the use of DC cells stimulated by NY-ESO-1, generation of T cells can be effectively stimulated, and the T cells have the effect of inhibiting the tumor of the microsatellite instability intestinal cancer. By the use of the gene and the protein, a treatment scheme for the intestinal cancer patient and forecasting of prognosis can be guided.
Description
Technical field
The present invention relates to biology and medical domain, relate more specifically to application in microsatellite instability immunotherapy of Colorectal Cancer for the ny-eso-1.
Background technology
Colorectal cancer (colorectal cancer, crc) is one of global modal malignant tumour, and its incidence of disease is respectively at the 3rd and the 2nd of malignant entity tumor in the male sex and women, ranks cancer related mortality reason the 4th.In recent years, the incidence of disease of colorectal cancer constantly rises, and the rate of climb of China is 2 times of international average speedup (2%), especially in the southeastern coast such as Shanghai prosperity city, the incidence of disease reaches the second (30/,100,000) of entity tumor, seriously threatens the Health and Living quality of the mankind.
At present, treatment for colorectal cancer is still confined to perform the operation, chemotherapy and radiation, metastatic colorectal carcinoma treatment method main at present is still chemotherapy, but explicitly point out microsatellite instability (microsatellite instability in " the nccn colorectal cancer clinical practice guideline " of 2011 editions, msi) colorectal cancer patients will not benefit from the NACT of 5 FU 5 fluorouracil (5-fu), therefore, advise that the patient of all the right side of fiftys all should consider to carry out msi detection, there is msi-h (high-frequency microsatellite instability, msi-h patient) is it is not recommended that adopt chemotherapy.Because, in colorectal carcinoma chemotherapy scheme main at present, 5-fu is main basic chemotherapeutics, the new clinical treatment means therefore inquiring into this kind of patient are significant for the curative effect improving msi colorectal cancer patients.
Content of the invention
First aspect present invention, there is provided the purposes of ny-eso-1 gene, albumen or its detection reagent, for preparing (a) diagnosis microsatellite instability intestinal cancer；And/or (b) judges the kit of patients with bowel cancer prognosis.
In another preference, described ny-eso-1 gene source is in mammal, it is preferred that deriving from mouse, rat or people.
In another preference, described kit includes: ny-eso-1 albumen or mrna are carried out with detection reagent and corresponding label or the specification of quantitative determination.
In another preference, described kit also includes ny-eso-2 albumen or mrna are carried out with the detection reagent of quantitative determination.
In another preference, described detection reagent includes ny-eso-1 specific primer, specific antibody, probe or chip.
In another preference, described detection reagent is ny-eso-1 specific primer.
In another preference, above-mentioned reagent includes detection chip, including nucleic acid chip and protein-chip.
In another preference, described nucleic acid chip includes the specific oligonucleotide probe of the substrate and point sample microsatellite instability intestinal cancer related gene on substrate, and the specific oligonucleotide probe of described cancer related gene includes the probe with ny-eso-1 gene or mrna specific binding.
In another preference, described protein-chip includes the specific antibody of the substrate and point sample cancer-associated proteins on substrate, and the specific antibody of described microsatellite instability intestinal cancer GAP-associated protein GAP includes the specific antibody of anti-ny-eso-1 albumen.
In another preference, described prognosis includes predicting the life cycle making a definite diagnosis patients with bowel cancer.
In another preference, dated herein below in described label or specification:
When detection object ny-eso-1 relative to the mrna expression of reference gene and the ny-eso-1 of cancer beside organism relative to ratio >=1 of the mrna expression of reference gene, then point out the probability that this detection object suffers from microsatellite instability intestinal cancer to be higher than general population, and/or
When make a definite diagnosis the patient ny-eso-1 with intestinal cancer relative to the mrna expression of reference gene with the ny-eso-1 of cancer beside organism relative to ratio >=1 of the mrna expression of reference gene, then point out this to make a definite diagnosis the patient's good prognosis with intestinal cancer.
In another preference, described intestinal cancer includes colon cancer or the carcinoma of the rectum.
In another preference, described kit is used for detecting people's intestinal cancer tissue sample or blood sample.
Present invention also offers a kind of kit of detection microsatellite instability intestinal cancer, wherein, described kit contains ny-eso-1 gene, albumen or its detection reagent, and corresponding label or specification.
In another preference, also contain ny-eso-1 gene, albumen in described kit as positive control.
A kind of second aspect present invention, there is provided the purposes of ny-eso-1 gene, albumen or its activator, for the pharmaceutical composition of preparation treatment intestinal cancer.
In another preference, described pharmaceutical composition is vaccine combination.
In another preference, described activator includes 5- azepine -2 ' deoxycytidine (dac), anti-pd-1 antibody, anti-ctla-4 antibody etc..
In another preference, described intestinal cancer is microsatellite instability intestinal cancer.
In another preference, the preparation of described pharmaceutical composition can be as described below:
(a1) provide ny-eso-1 antigen；
(b1) ny-eso-1 antigen and antigen presenting cell are incubated altogether, thus obtaining the antigen presenting cell through ny-eso-1 sensitization；
(c1) product in (b1) is mixed with pharmaceutically acceptable carrier, thus obtaining described pharmaceutical composition.
In another preference, step (b1) also includes, and the antigen presenting cell through ny-eso-1 sensitization is incubated further altogether with t cell, thus obtaining the t cell with antineoplastic immune activity through ny-eso-1 activation.
In another preference, described antigen presenting cell is BMDC.
In another preference, the preparation of described pharmaceutical composition also can be as described below:
(a2) carrier of be co-expressed anti-ny-eso-1 antibody gene and t cell activation gene is provided；
(b2) use the carrier transfection t cell in (a2), thus obtaining Chimeric antigen receptor t cell (car-t)；
(c2) product in (b2) is mixed with pharmaceutically acceptable carrier, thus obtaining described pharmaceutical composition.
Third aspect present invention, there is provided a kind of method that the candidate compound of intestinal cancer is treated in screening, including step:
In (i) test group, add test compound in the cultivating system of cell, and observe the expression of ny-eso-1 and/or activity in the colon-cancer cell of described test group；In control group, without test compound in isocellular cultivating system, and observe the expression of ny-eso-1 and/or activity in the described cell of control group；
Wherein, if the expression of the ny-eso-1 of cell and/or activity are more than control group in test group, indicate that this test compound be the expression to ny-eso-1 and/or activity have facilitation treatment intestinal cancer candidate compound.
In another preference, step can also be included:
(ii) compare the product of interpolation or the test group without the compound or control group stimulation to the antineoplastic immune of t cell activity.
Fourth aspect present invention, the method providing a kind of suppression microsatellite instability colon-cancer cell of external non-therapeutic, including step: there is activate through ny-eso-1 the t cell of antineoplastic immune activity and/or express the t cell of anti-ny-eso-1 antibody and/or expression is had the t cell of tcr of identification ny-eso-1 epi-position and is incubated altogether with colon-cancer cell, thus suppressing microsatellite instability colon-cancer cell.
Present invention also offers a kind of method treating microsatellite instability intestinal cancer, including step: apply to the object of needs, through what ny-eso-1 activated, there is the t cell of antineoplastic immune activity and/or the t cell expressed the t cell of anti-ny-eso-1 antibody and/or express the tcr with identification ny-eso-1 epi-position, thus treating microsatellite instability intestinal cancer.
In another preference, the object of described needs is the object with microsatellite instability intestinal cancer.
In another preference, the object of described needs is mammal, including mouse, rat or people.
It should be understood that within the scope of the present invention, can be combined with each other between above-mentioned each technical characteristic of the present invention and each technical characteristic specifically describing in below (eg embodiment), thus constituting new or preferred technical scheme.As space is limited, here is no longer tired out one by one and is stated.
Brief description
Fig. 1 shows in retrospective analysis, and in msi-h patient (Fig. 1 a) and mss patient (Fig. 1 b), ny-eso-1 differential expression is for the impact of 3 years life spans.
Fig. 2 a shows in gene microarray analysis result, ny-eso-1 notable rise of ny-eso-1 expression in ls180-msi-h/s model msi-h cell；Fig. 2 b shows in qpcr analysis result, ny-eso-1 notable rise of ny-eso-1 expression in ls180-msi-h/s model msi-h cell；Fig. 2 c is to be detected using the average level of its expression ny-eso-1/2 in 7 plants of msi-h colorectal cancer cell systems to document report for the flow cytometry technique.
Fig. 3 a-c shows the expression in patient's cancerous tissue for the ny-eso-1.
Fig. 4 shows the identification of ny-eso-1 albumen.
Fig. 5 a shows that the dc cell of ny-eso-1/2 sensitization is higher to the t cytositimulation effect secreting ifn- γ in msi-h intestinal cancer, and then poor in mss intestinal cancer；Fig. 5 b shows that the dc cell of ny-eso-1/2 sensitization is higher to the killing rate of msi-h colon-cancer cell, then poor to the killing rate of mss colon-cancer cell.
Fig. 6 a shows ls180-msi-h tumor bearing nude mice and carries out the growth curve of tumour after the dc adoptive therapy of polypeptide sensitization；Fig. 6 b shows the survivorship curve of tumor bearing nude mice.
Specific embodiment
The present inventor is through extensively in-depth study, first it was unexpectedly observed that ny-eso-1 height in microsatellite instability intestinal cancer is expressed, by the use of ny-eso-1 as the molecular marked compound of intestinal cancer, contribute to carrying out parting to intestinal cancer, thus instructing the therapeutic scheme of different types of intestinal cancer；Additionally, the present inventors have additionally discovered that, the prognosis of patients with bowel cancer and the expression of ny-eso-1 have strong correlation, and that is, the patient of the high expression of ny-eso-1 all can have more preferable prognosis.The present inventor finds also by experiment, and the dc cell after ny-eso-1 sensitization can more effectively activate the anti-tumor immunity of t cell in vivo, thus reaching the purpose for the treatment of microsatellite instability intestinal cancer.On this basis, complete the present invention.
Microsatellite instability intestinal cancer
Colorectal cancer (colorectal cancer, crc) is one of global modal malignant tumour, and its incidence of disease is respectively at the 3rd and the 2nd of malignant entity tumor in the male sex and women, ranks cancer related mortality reason the 4th.In recent years, the incidence of disease of colorectal cancer constantly rises, and the rate of climb of China is 2 times of international average speedup (2%), especially in the southeastern coast such as Shanghai prosperity city, the incidence of disease reaches the second (30/,100,000) of entity tumor, seriously threatens the Health and Living quality of the mankind.
Microsatellite refers to the < simple repeated sequence of 10 nucleotides in genome, the abundantest with the repetitive sequence of two nucleotides compositions, number of repetition is 10～50 times, it is mainly included in the noncoding region of gene, its sequence is short, it is most that < 200bp, the change of its recurring unit's quantity can cause at a relatively high polymorphism.The increase of simple repeated sequence or loss are referred to as msi.By detecting the stability of microsatellite, crc can be divided into microsatellite instability (msi) crc and microsatellite stability (mss) crc.
Wherein, microsatellite instability intestinal cancer is confirmed to be and cannot benefit from chemotherapy.In the present invention, using ny-eso-1 as molecular marked compound, can assist to diagnose microsatellite instability intestinal cancer, and ny-eso-1 and the life cycle of patients with bowel cancer there is also significant correlation, be can aid in using ny-eso-1 and prognosis is carried out to patients with bowel cancer, be i.e. ny-eso-1 high expression patient's prognosis is better than ny-eso-1 low expression patient.
Ny-eso-1 and its coded polynucleotide
Ny-eso-1 is the important a member in cancer-testis (cancer-testis) antigen gene family, the protein relative molecular weight of coding is 18kd, amino acid number is 180, and there is a glycine rich region at its n end, and c end contains a hydrophobic amino acid tail.It has stronger immunogenicity in tumour antigen, but expression in the normal tissue is only limitted to testis and embryonic tissue it is known that ny-eso-1 expresses in malignant mela noma, hepatocellular carcinoma, oophoroma etc..However, in intestinal cancer, the expression of ny-eso-1 is but uncertain, expression frequency has the quite poor opposite sex.
The present inventor is by finding, the expression of ny-eso-1 has good correlation with the microsatellite stability parting of intestinal cancer to the retrospective study of a large amount of difference colon-cancer cell systems and experimental verification.Being expressed in microsatellite instability intestinal cancer ny-eso-1 more.Thus, it is possible to parting be carried out to intestinal cancer using the expression of ny-eso-1, contribute to the guidance of therapeutic scheme.
In addition, the inventors discovered that, using ny-eso-1 as antigenic stimulus antigen presenting cell, thus the t cell stimulating, or express the t cell of anti-ny-eso-1 antibody or the t cell of the tcr of expression identification ny-eso-1 has good microsatellite instability intestinal cancer inhibitory activity.And this tumors inhibition activity is then weaker to microsatellite stability intestinal cancer.
The anti-ny-eso-1 antibody that can be used for the present invention is not particularly limited, and can be the monoclonal antibody of specific binding ny-eso-1.Described monoclonal can be obtained by conventional method, produces for example with hybridoma.
The t cell of the tcr of the t cell producing anti-tumor immunity or the Chimeric antigen receptor t cell expressing anti-ny-eso-1 antibody or expression identification ny-eso-1 can produce stronger tumor inhibition effect to microsatellite instability intestinal cancer.
Ny-eso-1 antigen used by the present invention or its code nucleic acid are detached albumen or its code nucleic acid.As used herein, term " ny-eso-1 albumen ", " ny-eso-1 antigen ", " ny-eso-1 polypeptide ", " molecular marker of the present invention " can be with used interchangeably, refer to albumen or the polypeptide with ny-eso-1 amino acid sequence, described sequence is as shown in seq id no.:2.The polynucleotide sequence such as seq id no.:1 (dna) of coding ny-eso-1 amino acid.
As used herein, " detached " refers to that material separates (if crude, primal environment is natural surroundings) from its primal environment.As the polynucleotides under the native state in active somatic cell and polypeptide do not isolate and purify, but same polynucleotides or polypeptide are as separately, then isolated and purified with other materials existing from native state.
As used herein, " detached ny-eso-1 albumen or polypeptide " refers to that ny-eso-1 albumen is substantially free of natural relative other albumen, lipid, carbohydrate or other materials.Those skilled in the art can purify ny-eso-1 albumen with the purified technology of protein of standard.Substantially pure polypeptide can produce single master tape in non-reducing polyacrylamide gel.In the present invention, ny-eso-1 albumen includes fusion protein and non pregnant women.
The polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide, preferably recombinant polypeptide.The polypeptide of the present invention may also include or not include initial methionine residues.Additionally, albumen of the present invention can also be co-expressed with t Cell Activating Molecule, and transfecting t cell, thus obtaining Chimeric antigen receptor t cell, carrying out the killing of tumour cell with ny-eso-1 for target.
The polynucleotides of the present invention can be dna form or rna form.Dna form includes cdna, genome dna or artificial synthesized dna.Dna can be single-stranded or double-strand.Dna can be coding strand or noncoding strand.
The polynucleotides of the mature polypeptide of coding ny-eso-1 include: the coded sequence of an encoding mature polypeptide；The coded sequence of mature polypeptide and various additional coding sequence；The coded sequence (and optional additional coding sequence) of mature polypeptide and non-coding sequence.Term " polynucleotides of coded polypeptide " can be polynucleotides or the polynucleotides also including additional code and/or non-coding sequence including encoding such peptides.
The invention still further relates to the variant of above-mentioned polynucleotides, its coding and the present invention have the polypeptide of identical amino acid sequence or the fragment of polypeptide, analogs and derivatives.The variant of this polynucleotides can be the allelic variant of natural generation or the variant of non-natural generation.These nucleotide variants include substitution variants, Deletion variants and insert variation.As known in the art, allelic variant is the alternative forms of polynucleotides, and it is probably replacement, disappearance or the insertion of one or more nucleotides, but will not be from the function of substantially changing its coded polypeptide.
The invention still further relates to the nucleic acid fragment with above-mentioned sequence hybridization, including the nucleic acid fragment of justice and antisense.As used herein, the length of " nucleic acid fragment " at least contains 15 nucleotides, preferably at least 30 nucleotides, and more preferably at least 50 nucleotides, more than preferably at least 100 nucleotides.Nucleic acid fragment can be used for the amplification technique (as pcr) of nucleic acid to determine and/or to separate the polynucleotides encoding ny-eso-1 albumen.
People's ny-eso-1 nucleotides full length sequence of the present invention or its fragment generally can be obtained with pcr TRAP, recombination method or artificial synthesized method.For pcr TRAP, can be according to published relevant nucleotide sequence, especially open reading frame sequence is designing primer, and the cdna storehouse with commercially available cdna storehouse or as prepared by conventional method well known by persons skilled in the art is as template, amplification and relevant sequence.When sequence is longer it is often necessary to carry out twice or multiple pcr amplification, the fragment then amplifying each time again is stitched together by proper order.
Once obtaining relevant sequence it is possible to obtain relevant sequence in large quantity with recombination method.This is typically cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by conventional method and obtains relevant sequence.
Additionally, also relevant sequence can be synthesized with artificial synthesized method, when especially fragment length is shorter.Generally, by first synthesizing multiple small fragments, then it is attached obtaining the very long fragment of sequence again.
The method that application pcr technology expands dna/rna is optimized for obtaining the gene of the present invention.Primer for pcr can properly select according to the sequence information of invention disclosed herein, and available conventional method synthesis.Available conventional method as separated and purifying the dna/rna fragment of amplification by gel electrophoresis.
The present invention also relates to comprising the carrier of the polynucleotides of the present invention, and the host cell that the carrier with the present invention or ny-eso-1 albumen coded sequence produce through genetic engineering, and the method producing polypeptide of the present invention through recombinant technique.
By conventional restructuring dna technology, the polynucleotide sequence of the available present invention can be used to express or produce the ny-eso-1 albumen of restructuring.In general there are following steps:
(1). with the polynucleotides (or variant) of the encoding human ny-eso-1 albumen of the present invention, or with the conversion of the recombinant expression carrier containing this polynucleotides or suitable host cell of transduceing；
(2). the host cell of culture in suitable culture medium；
(3). separation, protein purification from culture medium or cell.
Method well-known to those having ordinary skill in the art can be used for building the expression vector that ny-eso-1 containing people encodes dna sequence and suitable transcription/translation control signal.These methods include vitro recombination dna technology, dna synthetic technology, In vivo recombination technology etc..Described dna sequence can be effectively connected in the suitable promoter in expression vector, to instruct mrna to synthesize.Expression vector also includes ribosome bind site and the transcription terminator of translation initiation.
In addition, expression vector preferably comprises one or more selected markers, to provide the phenotypic character of the host cell for selecting conversion, as the dihyrofolate reductase of eukaryotic culture, neomycin resistance and green fluorescent protein (gfp), or it is used for colibacillary tetracycline or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence and suitable promoter or control sequence, can be used for converting suitable host cell, allow it to marking protein.
Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells；Or higher eucaryotic cells, such as mammalian cell.Representative example has: Escherichia coli, the bacterial cell of streptomyces；Fungal cell's such as yeast；Plant cell；The insect cell of fruit bat s2 or sf9；Zooblast of cho, cos or 293 cells etc..
Can be carried out with routine techniques well known to those skilled in the art with restructuring dna transformed host cell.When host is prokaryotes such as Escherichia coli, the competent cell that can absorb dna can harvest after exponential phase of growth, uses cacl2Method is processed, and step used is generally well-known in the art.Another kind of method is to use mgcl2.If necessary, conversion also can be carried out with the method for electroporation.When host is eucaryote, can be selected for following dna transfection method: calcium phosphate precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging etc..
The transformant obtaining can be cultivated with conventional method, the polypeptide of the coded by said gene of the expression present invention.According to host cell used, in culture, culture medium used is selected from various conventional mediums.Cultivated under conditions of being suitable to host cell growth.After host cell growth is to suitable cell density, induces the promoter of selection with suitable method (as temperature transition or chemical induction), cell is further cultured for a period of time.
Recombinant polypeptide in the above methods can be expressed in the cell or on cell membrane or is secreted into extracellular.If necessary, its physics, chemistry and other characteristics can be utilized to separate by various separation methods and purification of Recombinant albumen.These methods are well-known to those skilled in the art.The example of these methods includes but is not limited to: conventional renaturation process, processes (salting-out method), centrifugation, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, the combination of ion-exchange chromatography, high performance liquid chroma- tography (hplc) and other various LC technology and these methods with protein precipitant.
Antigen presenting cell
Antigen presenting cell (antigen presenting cell, apc) refers to have picked-up, processes antigen and offer, to a class cell of t lymphocyte, to be also called accessory cell by antigenic information.The medical research of early stage finds, during thymus dependent antigen induction b lymphocyte produces antibody, not only need the synergy of t, b lymphocyte in addition it is also necessary to the assistance of another kind of cell, then such cell is referred to as accessory cell (accessory cells).The Immune discrimination of body, immune response and immunological regulation play an important role.
The antigen presenting cell that can be used for the present invention is not particularly limited, and is that any antigen cross that plays in immune system is offered (or crossed sensitization) and stimulated the antigen presenting cell of t cell generation immune response (especially tumor immune response).Preferably, described antigen presenting cell is BMDC, and it, as the apc the strongest of function in immune system, preferably becomes the antigen presenting cell using ny-eso-1 antigen sensibilization of the present invention.
Activator and pharmaceutical composition
Using albumen of the present invention, by various conventional screening assays, the material that interaction occurs, especially activator etc. can be filtered out with ny-eso-1 albumen.
The activator of ny-eso-1 albumen of the present invention, when being administered (administration) in treatment, expression and/or the activity of ny-eso-1 albumen can be promoted, ny-eso-1 antigen using high expression, and then stimulate t cell to produce anti-tumor immunity, thus suppressing growth or the propagation of microsatellite instability intestinal cancer.
Pharmaceutical composition of the present invention can be prepared by multiple methods:
Method 1:
(a1) provide ny-eso-1 antigen；
(b1) ny-eso-1 antigen and antigen presenting cell are incubated altogether, thus obtaining the antigen presenting cell through ny-eso-1 sensitization；
(c1) product in (b1) is mixed with pharmaceutically acceptable carrier, thus obtaining described pharmaceutical composition.
In this pharmaceutical composition y1, containing the antigen presenting cell through ny-eso-1 sensitization and pharmaceutically acceptable carrier.Described pharmaceutical composition is applied to the patient with microsatellite instability intestinal cancer, permissible
Generally, but by these activators be formulated in nontoxic, in inert and pharmaceutically acceptable aqueous carrier medium, wherein ph ordinarily be about 5-8, and preferably ph is about 6-8, although ph value can be varied from the property being formulated material and illness to be treated.The pharmaceutical composition preparing can be administered by conventional route, including (but being not limited to): in knurl, intramuscular, intraperitoneal, intravenous, subcutaneous, intracutaneous or local be administered.
Present invention also offers a kind of pharmaceutical composition, it contains ny-eso-1 albumen of the present invention or its activator and pharmaceutically acceptable carrier or the excipient of safe and effective amount.This kind of carrier includes (but being not limited to): salt solution, buffer solution, glucose, water, glycerine, ethanol, and combinations thereof.Pharmaceutical preparation should be matched with administering mode.The pharmaceutical composition of the present invention can be made into injection form, for example, be prepared by conventional method with physiological saline or the aqueous solution containing glucose and other assistant agents.The pharmaceutical composition of such as tablet and capsule etc, can be prepared by conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule preferably aseptically manufacture.The dosage of active component is therapeutically effective amount, for example daily about 1 microgram -10 mg/kg body weight.
Detection method and kit
The invention still further relates to quantitative and detection and localization people's ny-eso-1 protein level or mrna level diagnostic testing process.These tests are known in the art.The people's ny-eso-1 protein level being detected in test, can be used for diagnosing intestinal cancer, liver cancer.
A kind of method that whether there is ny-eso-1 albumen in detection sample is to be detected, it includes using the specific antibody of ny-eso-1 albumen: sample is contacted with ny-eso-1 protein specific antibody；See whether to form antibody complex, define antibody complex and mean that in sample, there is ny-eso-1 albumen.
Ny-eso-1 albumen or its polynucleotides can be used for diagnosis and the treatment of ny-eso-1 protein related diseases.Part or all of the polynucleotides of the present invention can be fixed on microarray or dna chip as probe, the Differential expression analysis of gene and gene diagnosis in organizing for analysis.The antibody of anti-ny-eso-1 can be fixed on protein-chip, for the ny-eso-1 albumen in detection sample.
Present invention also offers a kind of kit of detection liver cancer, it contains primer pair and/or the ny-eso-1 specific antibody of specific amplification ny-eso-1.
Screening technique
Present invention also offers the method that drug screening is carried out based on ny-eso-1.A kind of method is the compound of first screening impact (promotion) ny-eso-1 expression or activity, then tests it further to cancer cell to the compound filtering out.A kind of screening technique can mrna based on ny-eso-1 expression.
Wherein, representational cancer cell includes (but being not limited to): microsatellite instability colon-cancer cell.
Excellent effect of the present invention:
1. utilize the positive expression of ny-eso-1, parting can be carried out to the microsatellite stability of intestinal cancer, sensitivity is higher.
The expression of 2.ny-eso-1 has significant correlation to the prognosis of patients with bowel cancer, and that is, the life span of ny-eso-1 positive cases is longer.
3. by can be used for stimulating t cell to produce antitumor activity using the dc cell of ny-eso-1 sensitization, but this antitumor activity is more effective to microsatellite instability intestinal cancer.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, such as sambrook et al., molecular cloning: laboratory manual (new york:cold spring harbor laboratory press, 1989) condition described in, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and number are percentage by weight and parts by weight.
Under embodiment 1 reviewing analysis difference microsatellite state, the differential expression of ny-eso-1 is for the impact of survival of patients time
The present embodiment have collected 186 clinical samples of 2005-2009, by microsatellite phenotypic analysis, determine the state of its microsatellite, wherein 123 mss, 63 msi-h patients, analyze the expression of ny-eso-1 in its tumor tissues using the method for SABC, it is found that the positive ratio of wherein ny-eso-1 expression is 57.14% in msi-h, be significantly higher than expression ratio in mss.
Using retrospective analysis difference microsatellite state under ny-eso-1 differential expression for the survival of patients time impact.In msi-h group, positive patient three annual survival rate of ny-eso-1 expression is 74.18%, and negative patient three annual survival rate of ny-eso-1 expression is 55.71% Fig. 1 a)；And in mss group, positive patient three annual survival rate of ny-eso-1 expression is also above expression negative patient (Fig. 1 b).
High expression checking in msi colorectal cancer for embodiment 2ny-eso-1
Adherent tumour cell pancreatin digestion, washes 2 times with pbs after being collected by centrifugation, and 1 × 106Cell, rna extracts kit, extract the total rna of cell, after finally carrying out concentration and purity testing, be stored in -80 DEG C.Take the total rna of 2 μ g cells to synthesize cdna according to reverse transcription reagent box specification reverse transcription, act on 30 minutes in 42 DEG C, add 16 μ l distilled water dilutions after 99 DEG C of effects inactivation in 5 minutes, the template of qpcr reaction can be made.Use mage-a4, quantitative pcr primer (the f:5 '-cagaccaccgccaactgca-3 ' (seq id no.:3) of ny-eso-1, ny-eso-2 equimolecular and actin respectively；R:5 '-tgagggaggctgagccaaa-3 ' (seq id no.:4)), adoptGreen realtime pcr master mix carries out quantitative pcr amplification.As shown in Figure 2 a.
Adherent tumour is with, after pancreatin digestion, making single cell suspension, adjusting cell concentration 1 × 106/ ml, takes 100ul to be placed in 1.5ml centrifuge tube, 2000r/min, 4 DEG C, centrifugation 5min, washes cell twice with cold pbs, is eventually adding the pbs re-suspended cell of 200-400ul, carry out antibody labeling according to antibody specification, ultimately join 200ul pbs re-suspended cell, machine in preparation.As shown in Figure 2 b.Average level using its expression ny-eso-1/2 in 7 plants of msi-h colorectal cancer cell systems to document report for the flow cytometry technique detects, as shown in Figure 2 c, result shows that the expression of ny-eso-1/2 in msi-h colorectal cancer cell system is higher, and in mss colorectal cancer cell, the expression of ny-eso-1/2 is relatively low.
Embodiment 3 carries out parting using ny-eso-1 expression to intestinal cancer microsatellite
Intestinal cancer sample using microsatellite parting known in embodiment 1 carries out double blinding mensure, thus expressing positive events according to ny-eso-1 to carry out parting to intestinal cancer sample.Result shows, judges that the sensitivity of microsatellite instability intestinal cancer is 90% according to ny-eso-1 expression, and specificity is 78%.Therefore, microsatellite stability parting can be carried out to intestinal cancer according to the expression of ny-eso-1.
The preparation of embodiment 4ny-eso-1/2 albumen
Pet28a/ny-eso-1 recombinant plasmid is proceeded in bl21-de3 competent cell, 37 DEG C, 220rpm is expanded to exponential phase, i.e. 540nm absorbance od=0.6-0.8.Iptg is added to mix, final concentration of 0.1mm/l.20 DEG C, 220rpm inducible protein is expressed, 16-24 hour.5000rpm is centrifuged 15 minutes, collects bacterium solution.Buffer solution resuspended bacterium solution, ultrasonic 60-70hz crushes Escherichia coli 10-15 minute, and 15000rpm is centrifuged 30 minutes, collects supernatant.Using affinity column histrap, bacterial supernatant is carried out with preliminary purification, 400-600mm/l imidazoles elutes to destination protein.Using ion exchange column sepharose qff, upper step eluent is further purified, collects and flow through and gradient eluent, concentrate, -20 DEG C of freezen protective are stand-by.As shown in fig. 3 a-c.
Ny-eso-2 albumen is prepared using same procedure.
The identification of embodiment 5ny-eso-1/2 albumen
After sds-page gel electrophoresis, on transferring film pvdf film, after Ponceaux dyeing, clip film to be measured bar tbst cleans 3 times, 5min/ time.5% skimmed milk power closing 2h, tbst cleaning 3 times, 5min/ time.One is added to resist in suitable ratio, 4 DEG C of overnight incubation, tbst cleaning 3 times, 5min/ time.The two of horseradish peroxidase is added to resist in suitable ratio, incubated at room 2h.Tbst cleans 3 times, 5min/ time, adds nitrite ion, lucifuge develops the color to occurring putting into terminating reaction in distilled water during band.As shown in Figure 4.
Embodiment 6msi-h Cell tumor antigen sensitization dc extracorporeal anti-tumor effect
Dislocation of cervical vertebra method puts to death hla-a2.1/kb mouse, aseptic take femur, serum-free rpmi1640 culture medium flushes out bone marrow cell, after the centrifugation of 1000g × 5 minute, careful suction abandons culture medium supernatant, the tris-nh4cl solution of 3-5ml dissolves red blood cell, adds anti-ia, b220, cd4, cd8 monoclonal antibody (final concentration is 10 μ g/ml) and complement (10:1 dilution), and 37c water-bath removes t, b and ia+ cell in 45 minutes, after washing twice, with 1 × 106Cells/well adds 24 orifice plate cultures, plus 10ng/ml mgm-csf, 1ng/ml mil-4, after culture 3 days, culture medium and suspension cell are abandoned in suction, rejoin fresh rpmi1640 complete medium and mgm-csf, mil-4, after continuing culture 3 days, loose adherent proliferative cell aggregation under piping and druming, collect rearmounted new blake bottle culture, after 3 days, collect the BMDC (bone marrow-derived dendritic cell, bmdc) that suspension cell is the derived from bone marrow of enrichment.
Collect the mouse dc of above-mentioned culture to the 6th day, be 2 × 10 with mouse dc culture medium (rpmi1640 complete medium, 10ng/ml mgm-csf, 1ng/ml mil-4) adjustment cell concentration6Cell/ml concentration, is divided into 24 orifice plates, every hole 1ml, hole count is identical with the number of elements of the mouse needing immunity, it is separately added into 10 μ g/ml ny-eso-1 polypeptides, ny-eso-2 polypeptide, mage-a4 polypeptide according to experiment packet, ova polypeptide, and arrange identical hole count not plus any stimulation dc as blank control group, after 48 hours collect cell, abandon culture medium supernatant, twice to remove stimulus present in former culture medium, finally adjust cell concentration with pbs is 1 × 10 to pbs centrifuge washing cell6Cell/100 μ l concentration, totally 200 μ l (wherein 100 μ l are to fill syringe dead volume in the future to ensure the cell number of immunity), is used for immune mouse immediately.
After final immunization 7 days, put to death each group mouse, mouse spleen is won in sterile working, rinse after residual blood with aseptic pbs, it is immersed in rpmi1640 culture medium, is gently ground on 400 mesh steel meshes with asepsis injector nook closing member and obtain single cell suspension, and the big tissue block of removal is filtered by steel mesh.After the single cell suspension 1000 × g of filtration is centrifuged 5 minutes, discards culture medium supernatant, be suspended in hypotonic nh4Cl solution (0.15m nh4Cl, 1m khco3, 0.1mm na2Edta, ph 7.2) in 3 minutes, destroy red blood cell, then use twice of rpmi 1640 culture medium centrifuge washing.The mouse boosting cell removing red blood cell is suspended in the rpmi1640 culture medium containing 10% hyclone, counts.
The mouse boosting cell that said method obtains is suspended in the rpmi1640 culture medium containing 10% hyclone, with ls180-msi-h, ls180-mss, two kind of cell mitomycin final concentration 1mg/ml, 37 DEG C, and 5%co2Incubation 1h, as stimulating cell after going propagation, as control stimulation group, it is 2 × 10 that adjustment stimulates cell concentration to the ova polypeptide of final concentration 10ug/ml5/ ml, then cell suspension is separately added in the detection hole containing reacting cells, 100 μ l/ holes, with reference to the t cell colony of the method detection secretion ifn- γ of ifn- γ elispot detection kit specification.Each of the above detection is all provided with 3 multiple holes.As shown in Figure 5 a.It can be seen that the dc cell of ny-eso-1/2 sensitization is higher to the t cytositimulation effect secreting ifn- γ in msi-h intestinal cancer, and then poor in mss intestinal cancer.
By ls180-msi-h, ls180-mss, t2 cell, as target cell, is 4 × 10 with physiological saline adjustment target cell concentration7/ ml, 500ul, add cfse, final concentration 2.5um, 37 DEG C, add 1640 culture medium 5ml terminating reactions, pbs cleaning cell three times, adjust target cell concentration 2 × 10 after 10min6/ ml, add in 96 hole round bottom plates, 100ul/ hole, take the mouse lymphocyte after antigenic stimulus as effector cell, add lymphocyte by effect target than 5:1,10:1,20:1, after incubated overnight, collect cell, pbs cleaning cell three times, 200ul pbs re-suspended cell, add pi final concentration 2ug/ml, the double positive cell proportion of cfse-pi is analyzed using streaming technology, not add the double positive cell proportion of the target cell cfse-pi stimulating cell as blank, calculate the kill rate of ctl, computing formula is as follows.(detection of each of the above gradient is all provided with three secondary orifices)
Killing rate %=(the double positive cell %- blank % of experimental group cfse-pi)/(1- blank %).As shown in Figure 5 b.It can be seen that the dc cell of ny-eso-1/2 sensitization is higher to the killing rate of msi-h colon-cancer cell, then poor to the killing rate of mss colon-cancer cell.
GVT in embodiment 7msi-h Cell tumor antigen sensitization dc body
The Female nude mice of 6-8 week old totally 40, is divided into 5 groups, every group 8, and in the good ls180-msi-h tumour cell of the vigor of the right abdomen of every mouse more subcutaneous inoculation cultured and amplified in vitro, dosage is 2 × 106Cell/mouse.Carry out after 5 days in above-mentioned nude inoculation tumour cell.Experiment packet is according to the packet of above-mentioned immunity hla-a2.1/kb transgenic mice.As previously mentioned through collecting the splenocyte of each group immune mouse, pbs adjusts concentration, and tail vein is fed back in tumor bearing nude mice body, and quantity is 1 × 108Cell/200 μ l mouse, feeds back 3 times, every minor tick one week.Observe tumour growth, draw tumor growth curve and mouse survival curve.As shown in Figure 6 it is seen that after the dc cell therapy of ny-eso-1/2 sensitization, the mouse survival rate with msi-h intestinal cancer is higher.
However, identical tumor killing effect is but unable to reach to mss using identical dc cell.
The all documents referring in the present invention are all incorporated as reference in this application, are individually recited as with reference to like that just as each document.In addition, it is to be understood that after the above-mentioned instruction content having read the present invention, those skilled in the art can make various changes or modifications to the present invention, these equivalent form of values equally fall within the application appended claims limited range.
Claims (10)
- The purposes of 1.ny-eso-1 gene, albumen or its detection reagent it is characterised in that be used for prepare (a) diagnose micro- Satellite unstability intestinal cancer；And/or (b) judges the kit of patients with bowel cancer prognosis.
- 2. purposes as claimed in claim 1 is it is characterised in that described kit includes: to ny-eso-1 egg White or mrna carries out detection reagent and corresponding label or the specification of quantitative determination.
- 3. purposes as claimed in claim 1 it is characterised in that described detection reagent to include ny-eso-1 special Property primer, specific antibody, probe or chip.
- 4. purposes as claimed in claim 1 is it is characterised in that indicate in following in described label or specification Hold:Ny-eso-1 when the mrna expression relative to reference gene for the ny-eso-1 and the cancer beside organism of detection object Ratio >=1 of the mrna expression of relative reference gene, then point out this detection object to suffer from microsatellite instability intestinal cancer Probability is higher than general population, and/orWhen making a definite diagnosis the patient ny-eso-1 with intestinal cancer relative to mrna expression and the cancer beside organism of reference gene Ny-eso-1 relative to ratio >=1 of the mrna expression of reference gene, then points out the patient that this makes a definite diagnosis with intestinal cancer pre- Good afterwards.
- 5. purposes as claimed in claim 1 is it is characterised in that described intestinal cancer includes colon cancer or the carcinoma of the rectum.
- 6. the purposes of a kind of ny-eso-1 gene, albumen or its activator is it is characterised in that treat intestines for preparation The pharmaceutical composition of cancer.
- 7. purposes as claimed in claim 6 is it is characterised in that described intestinal cancer is microsatellite instability intestinal cancer.
- 8. a kind of method of the candidate compound of screening treatment intestinal cancer is it is characterised in that include step:In (i) test group, add test compound in the cultivating system of cell, and observe the intestinal cancer of described test group The expression of ny-eso-1 and/or activity in cell；In control group, in isocellular cultivating system without Test compound, and observe the expression of ny-eso-1 and/or activity in the described cell of control group；Wherein, if the expression of the ny-eso-1 of cell and/or activity are more than control group in test group, indicate that this Test compound is the expression to ny-eso-1 and/or activity have facilitation treatment intestinal cancer candidate compound.
- 9. the method for candidate compound as claimed in claim 8 is it is characterised in that step can also be included:(ii) compare interpolation or the product of the test group without compound or control group is lived to the antineoplastic immune of t cell The stimulation of property.
- 10. a kind of method of the suppression microsatellite instability colon-cancer cell of external non-therapeutic is it is characterised in that wrap Include step: there is the t cell of antineoplastic immune activity and/or express activate through ny-eso-1 anti-ny-eso-1 and resist The t cell of tcr that the t cell of body and/or expression have identification ny-eso-1 epi-position is incubated altogether with colon-cancer cell, thus Suppression microsatellite instability colon-cancer cell.
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| PCT/CN2016/089112 WO2017008680A1 (en) | 2015-07-13 | 2016-07-07 | Application of ny-eso-1 in diagnosis and treatment of microsatellite instable colorectal cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111328420A (en) * | 2017-07-12 | 2020-06-23 | Nouscom股份公司 | Universal vaccine based on consensus tumor neoantigens for the prevention and treatment of microsatellite unstable (MSI) cancers |
| CN111599410A (en) * | 2020-05-20 | 2020-08-28 | 深圳市新合生物医疗科技有限公司 | Method for extracting new antigen for microsatellite unstable immunotherapy by integrating multiple sets of mathematical data and application |
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| CN103874770A (en) * | 2011-08-08 | 2014-06-18 | 卡里斯生命科学卢森堡控股有限责任公司 | Biomarker compositions and methods |
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| CN103874770A (en) * | 2011-08-08 | 2014-06-18 | 卡里斯生命科学卢森堡控股有限责任公司 | Biomarker compositions and methods |
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| 黄晓辉: "MSI-H/S结直肠癌差异表达分子的筛选及其在免疫治疗中的意义", 《中国博士学位论文全文数据库：医药卫生科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111328420A (en) * | 2017-07-12 | 2020-06-23 | Nouscom股份公司 | Universal vaccine based on consensus tumor neoantigens for the prevention and treatment of microsatellite unstable (MSI) cancers |
| CN111599410A (en) * | 2020-05-20 | 2020-08-28 | 深圳市新合生物医疗科技有限公司 | Method for extracting new antigen for microsatellite unstable immunotherapy by integrating multiple sets of mathematical data and application |
| CN111599410B (en) * | 2020-05-20 | 2023-06-13 | 深圳市新合生物医疗科技有限公司 | Method for extracting microsatellite unstable immunotherapy new antigen by integrating multiple sets of chemical data and application |
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