CN106987631A - A kind of immune group sequencing technologies for the adjoint diagnosis of PD 1/PD L1 blocking treatments - Google Patents
A kind of immune group sequencing technologies for the adjoint diagnosis of PD 1/PD L1 blocking treatments Download PDFInfo
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Abstract
The invention discloses a kind of for immune group sequence measurement of the PD 1/PD L1 blocking treatments with diagnosis.This method extracts RNA and/or DNA from the PMBC of cancer patient and the cancerous tissue (borderline tumor and core) of patient, φt cell receptor specific primer and universal primer is recycled to expand the variable section of φt cell receptor β chain genes, multiple sample PCR primers are mixed again and are sequenced, utilize the joint on specific primer, and then analyze the frequency of V J genetic fragments in each sample, CDR3 sequences are analyzed, immune library spectrum is set up.And application immunity receptor diversity and Clonal sign carry out the immunocompetence assessment of patient, determine that patient includes but is not limited to the possibility of PD 1/PD L1 paths blocking treatment reaction to immunotherapy, this method is selecting PD 1/PD L1 path blocking treatments medicine to carry out the diagnosis that treatment provides predictability for cancer patient.
Description
Technical field
The present invention relates to molecular Biological Detection field, more particularly to a kind of PD-1/PD-L1 blocking treatments that are used for are with examining
Disconnected immune group sequencing technologies.
Background technology
The immunotherapy of tumour is a kind of emerging, with significant curative effect tumor treatment model, is a kind of autoimmunity
The new bio treatment method of anticancer.Immunotherapy of tumors is by swashing intravital immunocyte, specifically removing tumour
MRD or a kind for the treatment of method for substantially suppressing tumor cell proliferation.This treatment method has action period length and pair
The advantages of acting on small, is referred to as the 4th kind of pattern of modern oncotherapy.Immunologic test point therapy (immune
Checkpointtherapy) to be a class improve the treatment side of anti tumor immune response by adjusting in patient's body T cell activity
Method, immunologic test point can suppress the function of T cell under normal circumstances, be formed while may be utilized in tumor tissues by tumour
Immunologic escape, common immunologic test point includes CTLA4, PD-1, LAG-3, TIM-3 etc., can be with by blocking immunity checkpoint
Discharge repressed T cell activity.And wherein PD-1/PD-L1 antibody blockings are treated this tumour immunotherapy and achieved significantly
Break through.
PD-1 is immunoglobulin superfamily CD28 family members, the I type transmembrane glycoprotein for being 50~55kD of molecular weight, by
Similar IgGV domain, membrane spaning domain and cytoplasmic tail domain composition.PD-L1 is B7 family members, is PD-1
Part, belongs to 1 type transmembrane protein.PD-L1 parts or the high expression under IFN-γ induction can be expressed on kinds of tumor cells surface
PD-L1 parts, after the PD-1 on the PD-L1 and lymphocyte of tumour cell is largely combined, can suppress T cell function so that
Immune clearance in tumor escape body.And utilize the PD-1 acceptors and cancer of T cell expression in PD-1/PD-L1 antibody blocking bodies thin
Combination between the part PD-L1 of cellular expression, can make T cell recover to play its cell killing work(under immunosuppressive condition
Can, and then eliminated tumour cell using self immune system.The PD-1 antibody of currently acquired U.S. FDA approval listing has silent sand
East Keytruda and Shi Guibao Opdivo, including to malignant mela noma, non-small cell lung cancer, clear-cell carcinoma, oophoroma,
The PD-1 antibody such as carcinoma of urinary bladder, stomach cancer, incidence and esophageal squamous cell carcinoma all shows good therapeutic effect.The PD- of Roche
L1 antibody new drugs Atezolizumab also obtains FDA approval listings, for treating most common carcinoma of urinary bladder.But PD-1/PD-L1
Antibody therapy not has a positive role to all patients, and related data shows only 20% or so patient to PD-1/PD-L1
Antibody drug has active responding, for carrying out treating to optimize patient rights and interestses, minimum with diagnosis before PD-1/PD-L1 medications
When toxic side effect and instruct therapeutic alliance to seem important.Examined in vitro as a kind of associated with Personalized medicine with diagnosis
Disconnected technology, mainly by detect people's vivo protein, mutator expression, filtered out in different types of sick people
Optimal medication crowd, targetedly carries out Personalized medicine.The security and validity of concomitant drugs can be improved with diagnosis
And patient's treatment prognosis and reduction health cost can be improved, help quickly whether judge anti-cancer drug regimens with diagnostic techniques
It is adapted to specific patient.In order that the reaction for including but is not limited to PD-1/PD-L1 antibody therapies to immunization therapy is optimized, need
To understand the immune state of patient to design a kind of effective immunization therapy side to carrying out before patient medication accurately with diagnosis
Case, filter out it is most possible to PD-1/PD-L1 benefit to obtain patient, we design a kind of based on immune group sequencing technologies point herein
The PD-1/PD-L1 of patient's immune state is analysed with diagnostic method.
In addition, having occurred in that the PD-1/PD-L1 antibody mediated immunities treatment of some clinic approvals with diagnosis in the market
Product, it resists mainly by being detected to the PD-L1 protein expression levels in patient's cancerous tissue to PD-1/PD-L1
Before body treatment there is patient's cancerous tissue PD-L1 expression heterogeneities in immunohistochemistry technique used in detection, and PD-L1 antibody used is known
The problems such as positive threshold value disunity of other epitope heterogeneity and the PD-L1 expressions used, (and have document report PD-
The reactivity of L1 expression rate and PD-1 blocking antibodies and without good correlation), other reports are directed to immunologic test
Diagnostic method before point inhibitor medication, including dystopy lymph node spline structure is detected by biopsy specimen and then neoplasm invasiveness is detected
The density of cell, builds the related gene expression spectrum of gene expression profile especially beta interferon induction, and MHCII (HLA-DR)
The high expression immunogene marking relevant with clinical response increase detect, be also that can make relatively low standard under certain condition
The prediction of the PD-1 antibody positive effect patients of true rate.Now, high throughput sequencing technologies (NGS) of new generation are then a preferable solutions
Certainly scheme, the method that a series of its tumour atlas analysis pattern that can detect single-genes changes into multiple analytes, bring more
For accurately oncology.In the diversity of detection vivo immuning system, immune group sequencing technologies undoubtedly have unique advantage.
Immune group sequencing (Immune Repertoire sequencing (REP-seq)) is using T/B lymphocytes to grind
Study carefully target, determine that B-cell receptor (BCR) or φt cell receptor (TCR) are multifarious mutually with multiplex PCR or the amplification of 5 ' RACE technologies
Mend and determine area (CDR region), in conjunction with high throughput sequencing technologies (NGS), the diversity of comprehensive assessment immune system is deeply excavated and exempted from
The technology of the relation of epidemic disease group storehouse and disease.T/B cell receptors are in during dynamic reorganization in human body, to what is occurred in body
Range of pathogen and antigen can be carried out identification.Detection TCR is increasingly recognized in the importance of human health and disease.
The patient's immune group storehouse data obtained by immune group sequencing technologies are analyzed according to the comparison of the database to different tumours, can be with
T/B cells in tumor patient immune system are comprehensively analyzed, patient's in-vivo tumour specificity T/B are quickly detected thin
Presence, diversity and the clone's distribution of born of the same parents.
It is an object of the invention to provide a kind of for immune group sequencing skill of the PD-1/PD-L1 blocking treatments with diagnosis
Art, by being detected and analyzed to the immune system status in patient's body, determines effectiveness and reaction of the patient to immunization therapy
Possibility.
The content of the invention
The present invention provides a kind of for immune group sequencing technologies of the PD-1/PD-L1 blocking treatments with diagnosis, of the invention
What technical scheme was realized in：
A kind of immune group sequencing technologies for the adjoint diagnosis of PD-1/PD-L1 blocking treatments, comprise the following steps：(1) receive
Collect the RNA or genomic DNA sample of blood samples of patients lymphocyte or cancerous tissue；(2) using the labeling method of DNA molecular to institute
Obtain DNA and carry out the PCR amplifications based on primer bar code；(3) TCR high pass measurements are carried out using the sequence measuring joints in random primer bar code
Sequence；(4) the high-flux sequence data based on resulting subject, analysis TCR sequence polymorphisms and TCR sequences distribution score with
And TCR is Clonal, determine that subject is reacted the possibility of PD-1 or PD-L1 immunization therapies.
Further, the samples sources in the step (1) in one or more patient in one or more time
The sample of point.
Further, it is cDNA by the RNA reverse transcriptions that the cancerous tissue extracted is collected in the step (1).
Further, molecule labelling method is that excessive random primer bar code is added at DNA two ends in the step (2),
DNA molecular two ends are made respectively to mark corresponding random primer bar code.
Further, described random primer bar code be the specific primer by being designed according to DNA molecular anti-sense primer,
What random bar code and sequence measuring joints were constituted in series.
Further, the concrete operation method of the step (3) is that excessive random primer bar code is added into the step
Suddenly in the PCR amplification system of (2), high-flux sequence is carried out using the sequence measuring joints in random primer bar code, specific steps are sequenced
Carried out according to Illumina platforms step, sequencing data carries out VDJ comparisons, analyze CDR3 sequences, obtain single complete be immunized
Compose in storehouse.
Further, sequence measuring joints are compatible with Illumina platform in the step (3).
Further, the multifarious determination method of TCR sequences is in the step (4)：According in the step (3)
TCR high-flux sequence data determine the total number of retracing sequence and unique total number for resetting DNA sequence dna in the sample, so that
The diversity fraction of the TCR sequences of quantitative one or more samples；The determination method of TCR distribution numbers is root in the step (4)
Account for and observed according to each unique rearrangement DNA sequence dna in each unique frequency of occurrences and one or more samples for resetting DNA sequence dna
To the percentage of total number of retracing sequence determine；It is according to specific TCR DNA that TCR is Clonal in the step (4)
The quantity of sequence and the number of the frequency of occurrences are determined.
Further, TCR sequence polymorphisms and TCR sequence distribution scores are the immune shapes of subject in the step (4)
The determination foundation of state, described TCR sequence polymorphisms and TCR it is Clonal be diagnosis basis before PD-1/PD-L1 medications.
Further, the immune group sequencing technologies are any being directed in PD-1 Antybody therapies and PD-L1 Antybody therapies
Kind.
Beneficial effects of the present invention：The present invention provides a kind of for immune group of the PD-1/PD-L1 blocking treatments with diagnosis
Sequencing technologies, efficiently solve problem present in PD-1/PD-L1 antibody tumor immunotherapies：PD-1/PD-L1 antibody is used
The evaluation of patient's immune state before medicine, filters out most possibly benefited patient.
Brief description of the drawings
Technical scheme in order to illustrate the embodiments of the present invention more clearly, makes required in being described below to embodiment
Accompanying drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for
For those of ordinary skill in the art, on the premise of not paying creative work, other can also be obtained according to these accompanying drawings
Accompanying drawing.
Fig. 1 present invention is for flow chart of the PD-1/PD-L1 blocking treatments with the immune group sequencing technologies of diagnosis
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole embodiments.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
In terms of sample of the present invention is collected with patient's immune group storehouse structure, comprise the following steps：(1) from least one patient
Blood or cancerous tissue separation WBC sub-population, separate RNA from the cell subsets, RT-PCR profits are used in the first amplified reaction
With nested primers reverse transcription RNA and expanded to produce amplicon, at least a portion of nested primers includes additional nucleotides
It is so that the binding site for consensus primer is incorporated in gained amplicon, the amplicon of the first amplified reaction and the first amplification is anti-
The one or more untapped primer separation answered, the first amplification is expanded by adding consensus primer in the second amplified reaction
The amplicon for having at least one for the binding site of consensus primer reacted, and the amplicon of the second amplified reaction is sequenced
Reset with the antibody and/or acceptor that recognize in cell subsets；(2) then the same sample of analysis CDR3 sequences result, obtain
Single complete immune library spectrum.
Method for producing immune state report comprises the following steps：(1) identification is deposited in subject immune's spectrum with coming from
The one or more unique CDR3 sequences being had between the immune spectrum of accumulation in the disease storehouse being stored in database, to corresponding to that
The sum of the detected sequence of the subject of shared unique CDR3 sequences is summed and calculates generation in subject immune's spectrum a bit
The percentage of those unique CDR3 being had between subject and disease storehouse of table detected sequence sum, to produce one
Or multiple indexes initially having.(2) diversity to TCR in patient tissue and Clonal analyze.Specifically, to patient
The high-flux sequence sequence information that nucleic acid samples are produced, includes multiple unique sequences for resetting nucleotide sequence, each unique core
Acid sequence is single TCR sequences and polypeptide.For one or more samples, according to nucleotide sequence sequence information, it is determined that
Unique total number for resetting DNA sequence dna in the total number and sample of the retracing sequence observed in the sample；According to it
Uniqueness resets the total number of DNA sequence dna, the TCR sequence polymorphisms of quantitative one or more samples；According to each unique rearrangement DNA
The frequency of occurrences of sequence calculates the percentage of the total number of the retracing sequence observed in one or more samples, so as to determine
Measure the TCR distribution scores of one or more samples；And the TCR sequence polymorphisms fraction according to patient and the distribution point of TCR sequences
Count the immune state to determine patient and the possibility to PD-1/PD-L1 antibody drugs is reacted.
The technical scheme of adjoint diagnosis before being treated below in conjunction with the accompanying drawings with metastasis melanin tumor patient PD-1 is done into one
Step is introduced.The technical scheme of the present embodiment is broadly divided into four parts：Sample is collected, patient's immune group storehouse is built, biological information
Credit analysis and analysis model are built.
1. sample is collected
(1) sample is collected
Collect 10 milliliters of patient peripheral's blood sample and puncturing tissue sample is a.The venous blood of patient is extracted, in blood plasma point
From single blood sampling composition separation that center carries out 12-15 milliliters.The PBMC of patient is obtained during the program, it is therefore an objective to obtain extremely
Few 50 × 109Individual leucocyte is in case follow-up builds storehouse, by the baseline blood cell freezing guarantor for meeting FDA requirements and research of acquisition
Deposit.
(2) blood lymphocytes separation and RNA are extracted
Take 2ml to be layered liquid to be placed in centrifuge tube, dilute blood is slowly superimposed on layering liquid along tube wall, clear boundary is formed
Face.The volume fraction of dilute blood and layering liquid is advisable with 2: 1~3: 1.Put 2000r/min in horizontal centrifuge and centrifuge 20min.
It is divided into four layers after centrifugation from the bottom of centrifuge tube to liquid level, is followed successively by red blood cell and granulocyte layer, layering liquid layer, mononuclearcell
Layer, plasma layer (containing blood platelet and smudge cells).Mononuclearcell layer is directly suctioned out with dropper or is sucked and inhales this again after plasma layer
Layer, is placed in another centrifuge tube.The PBS liquid of 4 times of amount above is added, is fully mixed, 1000r/min centrifugations l0min.Centrifuge hypsokinesis
Supernatant is abandoned, then is washed 2 times with PBS liquid.Cell is prepared with PBS liquid or nutrient solution containing 10%~20% inactivation calf serum to hang
Liquid.Count, and count granulocyte and mononuclearcell number respectively, while detecting cell viability with trypan blue, finally empirically
Ask and cell suspension is adjusted to debita spissitudo.The PBMC cell One-step RT-PCR (QINGEN) separated are in RLT
Cracked in buffer, and RNA extractions are carried out according to One-step RT-PCR (QINGEN) operating procedure.The RNA's extracted
Concentration by2.0 detection.
(3) tissue RNA is extracted
Sample treatment：Take it is fresh or -70 DEG C freeze 100mg tissue plus 1ml lysates, it is even with tissue grinder pestle or homogenizer
Slurry processing.Sample after processing is placed 5 minutes in room temperature so that nucleic acid-protein compound is kept completely separate.Into homogenised sample
Plus 0.2ml chloroforms, lid is covered, acutely vibration 15 seconds, room temperature is placed 3-5 minutes.2-8 DEG C of 12000rpm is centrifuged 10 minutes.RNA
Mainly in the colourless aqueous phase in upper strata, aqueous phase is transferred in new pipe, precipitation should not be drawn onto.Adsorption column pre-treatment：In adsorption column
Middle addition 500ul washes post liquid, and room temperature is placed 2 minutes, and 2-8 DEG C 12,000rpm centrifugation 2min abandon waste liquid.The supernatant that 4th step is collected
Middle addition 200ul absolute ethyl alcohols are mixed, and are added adsorption column and are stood 2 minutes, and 2-8 DEG C of 12000rpm centrifuges 2min, abandons waste liquid.To suction
600 μ L rinsing liquids (please first checked whether before use and added absolute ethyl alcohol), 2-8 DEG C 12,000rpm centrifugations are added in attached column
2min, abandons waste liquid.600 μ L rinsing liquids are added into adsorption column, 2-8 DEG C 12,000rpm centrifugation 2min abandon waste liquid.Then
12000rpm centrifuges 2min, discards collecting pipe, and adsorption column is placed in into room temperature places several minutes by rinsing liquid remaining in adsorption column
Remove.Adsorption column is put into new pipe, 50-100ul RNase free ddH2O are added dropwise to film center, room temperature places 5min,
12000rpm room temperatures centrifugation 2min is to obtain RNA.
(4) genomic dna sequence
In some case study on implementation, the nucleic acid extracted includes the blood of patient and the genomic dna sequence of cancerous tissue.Tool
The extracting genome DNA process of body is according to the human tissues of Qiagen 51306 and cell DNA extracts kit, Qiagen 51104
The operating instruction of blood DNA extracts kit is operated.Researcher in this field is it is to be understood that the above is to RNA and gene
Group DNA extraction process, can not be confined to the extraction process of the kit and method, any can extract reaches this patent
It is required that RNA and DNA methods can serve as extract process.
2. patient's immune group storehouse is built
In some case study on implementation, the nucleic acid extracted includes genomic DNA, in other case study on implementation, is extracted
Nucleic acid include cDNA, in some case study on implementation, the nucleic acid extracted include mRNA.CDNA synthesis uses One-
Step RT-PCR (QINGEN) subsequent step is carried out.For that can cover the diversity of all φt cell receptors, we will design
Multiple PCR primer.Forward primer design will cover φt cell receptor V genetic fragments, by design 11 forward primers with including
All V genetic fragments.5 reverse primer designs will cover J genetic fragments, and (specific primer sequence refers to national inventing patent
201310230809.9).Then the PCR in 16 cycles is carried out to cDNA or genomic DNA to expand for the first time, then carries out magnetic bead pure
Change.Magnetic beads for purifying PCR-1 products (AgencourtAmpure XP system Becman Coulter) carry out 35 cycles afterwards
PCR2 to be expanded to TCR DNA fragmentations.PCR primer by QIAquickPCR Purification Kit (Qiagen) and
QIAquick gel extraction kit (Qiagen) are purified.DNA concentration by2.0 fluorescence photometers are determined.Purification
DNA afterwards, to build library, and will be compiled fully according to Illumina Hiseq 2000 flow to the addition identification of each sample
Code (barcode).All samples are sequenced on Illumina Hiseq 2000.
3. bioinformatic analysis
Sample is sequenced after basic sub-sieve choosing processing in patient, each sequence all with V, D in IMGT databases
It is compared with the embryonal system reference sequences of J genetic fragments, come the V, D and the J that determine it classification.Sort out after summarizing, draw VDJ classes
Other number distribution.Advantage TCR clones are found out, and are compareed with the TCR storehouses before corrective surgery, tumour specific antigen is found out
T cell.Then TCR is classified into clone with the cluster analysis (clustering) in statistics.Define a measurement
The index of TCR storehouses diversity and clone's dominance, by Shannon entropy (entropy), is presented TCR storehouses Clonal (based on by accounting for
The number of uniqueness TCR sequences present in each sample, the modification for being normalized to the TCR sequence Distribution Entropies of (0-1) is measured) put down
Equal and standard variance, this index of patient and inhibition response the PD-1 model index treated is associated, creation analysis PD-1 treatments
Immune state to determine patient is compared and right in preceding adjoint diagnostic model, clone's distribution and model by the TCR of patient
The possibility of PD-1 treatments.
In some case study on implementation, of the invention is used for immune group sequencing skill of the PD-1/PD-L1 blocking treatments with diagnosis
Art also includes the Repertoire sequences distribution for being used for the quantitative subject, and specific method is：There is provided a kind of for determining to suffer from
The computer implemented method of person's immune state, to being stored in the sequencing data that multiple time points obtain from multiple Patient Sample As, really
Surely combination the frequency of occurrences and CDR3 it is Clonal.Including quantitatively determining CDR3 region sequence diversity fractions and determining uniqueness in sample
The total number of clone.
In some case study on implementation, of the invention is used for immune group sequencing skill of the PD-1/PD-L1 blocking treatments with diagnosis
Art also includes resetting average value and mark that displaying patient T cells invade profit by the φt cell receptor for measuring each diploid gene group
Quasi- variance.
In one embodiment, the reaction is the positive immune response to immunotherapy.
4. analysis model is built
The standard that the feature construction in the immune group storehouse of PD-1/PD-L1 antibody drugs is evaluated, and and depth are responded based on patient
Particular patient tumor specific T cells are Clonal obtained by sequencing combination bioinformatic analysis compares, and sees whether both accord with
Close.Effect of Immuno Suppressive Therapy is independently estimated by the clinical criterion of standard tumor.Subject has following sign：
(part reaction indicates that patient tumors load is reduced to reactor, and stable disease indicates to lack progress and tumor burden does not subtract
It is few) or non-reactor (disease remain unchanged progress).Show that the immune system of each patient includes but is not limited to PD-1 to immunotherapy
Antibody therapy, the relative ability of beneficial reaction calculates to predict by the modification entropy that TCR storehouses are distributed before immunotherapy.
(Clonal) is calculated using modification entropy, wherein what the unique TCR observed by accounting in the tumor sample was reset
Number, scope (0-1) is normalized to by the TCR sequence Distribution Entropies of each tumor sample, and takes it reciprocal so that high standardization
Entropy Changes is into low Clonal, and vice versa.Before immunotherapy is started, by lymphocyte present in patient tumors biopsy samples
Whether the distribution of TCR sequences is Clonal compares with model or compare with control group Clonal with higher distribution, that is, lacks
The TCR storehouses of the T cell clone of amount height amplification, have more preferable PD-1 antibody responses with the Clonal patients of higher TCR.
There is the patient compared with increased resistance invasion humidity T lymphocytes to be more likely to respond PD-1 Antybody therapies simultaneously.
Claims (10)
1. it is a kind of for immune group sequencing technologies of the PD-1/PD-L1 blocking treatments with diagnosis, it is characterised in that：Including following
Step：(1) blood samples of patients lymphocyte, the RNA of cancerous tissue or genomic DNA sample are collected；(2) mark of DNA molecular is utilized
Method carries out the PCR based on primer bar code to gained DNA and expanded；(3) TCR is carried out using the sequence measuring joints in random primer bar code
High-flux sequence；(4) the high-flux sequence data based on resulting subject, analysis TCR sequence polymorphisms and TCR sequences point
Cloth fraction and TCR are Clonal, determine the possibility reaction that subject treats to PD-1/PD-L1 antibody mediated immunities.
2. it is according to claim 1 a kind of for immune group sequencing technologies of the PD-1/PD-L1 blocking treatments with diagnosis,
It is characterized in that：Samples sources in the step (1) in one or more patient one or more time point sample
This.
3. it is according to claim 1 a kind of for immune group sequencing technologies of the PD-1/PD-L1 blocking treatments with diagnosis,
It is characterized in that：It is cDNA sequence by the cancerous tissue RNA reverse transcriptions being collected into the step (1).
4. it is according to claim 1 a kind of for immune group sequencing technologies of the PD-1/PD-L1 blocking treatments with diagnosis,
It is characterized in that：Molecule labelling method is that excessive random primer bar code is added at DNA two ends in the step (2), makes DNA points
Sub- two ends respectively mark corresponding random primer bar code.
5. it is according to claim 4 a kind of for immune group sequencing technologies of the PD-1/PD-L1 blocking treatments with diagnosis,
It is characterized in that：Described random primer bar code is the anti-sense primer of the specific primer by being designed according to DNA molecular, random bar
What code and sequence measuring joints were constituted in series.
6. it is according to claim 1 a kind of for immune group sequencing technologies of the PD-1/PD-L1 blocking treatments with diagnosis,
It is characterized in that：The concrete operation method of the step (3) is that excessive random primer bar code is added into the step (2)
In PCR amplification system, using in random primer bar code sequence measuring joints carry out high-flux sequence, sequencing specific steps according to
Illumina platforms step is carried out, and sequencing data carries out VDJ comparisons, analyzes CDR3 sequences, obtains single complete immune library spectrum.
7. it is according to claim 1 a kind of for immune group sequencing technologies of the PD-1/PD-L1 blocking treatments with diagnosis,
It is characterized in that：Sequence measuring joints are compatible with Illumina platform in the step (3).
8. it is according to claim 1 a kind of for immune group sequencing technologies of the PD-1/PD-L1 blocking treatments with diagnosis,
It is characterized in that：The multifarious determination method of TCR sequences is in the step (4)：According to TCR high passes in the step (3)
Amount sequencing data determines the total number of retracing sequence and unique total number for resetting DNA sequence dna in the sample, so that quantitative one
The diversity fraction of the TCR sequences of individual or multiple samples；The determination method of TCR distribution numbers is according to each in the step (4)
Each unique DNA sequence dna of resetting accounts for observed weight in the frequency of occurrences and one or more samples of uniqueness rearrangement DNA sequence dna
The percentage of the total number of Sorted list is determined；It is according to specific TCR DNA sequence dna that TCR is Clonal in the step (4)
The number of quantity and the frequency of occurrences is determined.
9. it is according to claim 8 a kind of for immune group sequencing technologies of the PD-1/PD-L1 blocking treatments with diagnosis,
It is characterized in that：Described TCR sequence polymorphisms and TCR sequence distribution scores is the determination foundation of the immune state of subject,
Described TCR sequence polymorphisms and TCR it is Clonal be diagnosis basis before PD-1/PD-L1 Antybody therapy medications.
10. it is according to claim 1 a kind of for immune group sequencing technologies of the PD-1/PD-L1 blocking treatments with diagnosis,
It is characterized in that：The immune group sequencing technologies are to be directed to any of PD-1 Antybody therapies and PD-L1 Antybody therapies.
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| CN201710212710.4A CN106987631A (en) | 2017-04-01 | 2017-04-01 | A kind of immune group sequencing technologies for the adjoint diagnosis of PD 1/PD L1 blocking treatments |
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Inventor after: Han Junfeng Inventor after: Liu Huiying Inventor after: Liu Cong Inventor after: Lei Jing Inventor after: Zhao Shuangshuang Inventor before: Liu Cong Inventor before: Lei Qing Inventor before: Zhao Shuangshuang |
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Application publication date: 20170728 |