CN109432429A - Application of the PD1 molecule inhibitor in T lymphocyte leukemia treating - Google Patents
Application of the PD1 molecule inhibitor in T lymphocyte leukemia treating Download PDFInfo
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Abstract
The present invention provides application of the PD1 molecule inhibitor in T lymphocyte leukemia treating.Specifically, the present invention provides a kind of purposes of the inhibitor of PD-1, it is used to prepare the pharmaceutical composition for inhibiting T-ALL cell differentiation and being proliferated, and/or treat Pancytopenia.Present invention shows that anti-PD-1 Antybody therapy inhibits proliferation of the T-ALL cell in spleen, T-ALL is clinically treated for anti-PD-1 antibody and provides certain experimental basis.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to PD1 molecule inhibitor is in T lymphocyte leukemia treating
Using.
Background technique
Pancytopenia (T-cell acute lymphoblastic leukemia, T-ALL) is a kind of
The neoplastic hematologic disorder of Highly invasive is formed by the unordered proliferation of T cell progenitor cells.Account for about 15% children's case and 25%
Adult case.T-ALL current chemotherapeutic regimen is unsatisfactory, and the remission rate in children's case can reach 85%, and
Remission rate in adult case is no more than 50%.
Immunity inspection point Immune checkpoint refers mainly to co-suppression molecule and costimulation molecule in T cell,
Physiologically playing the role of maintaining self tolerance, tumor escape can be helped in tumor microenvironment.Wherein, programmed cell is dead
Dying receptor -1 (PD-1) is a kind of important immunity inspection point.PD-1 signal path can inhibit the cell cycle, promote Apoptosis,
And damage T cell anti-tumor function.It is stimulated when by the antigens sustained low dose such as chronic infection, T cell can be in a kind of function
The state that energy property is exhausted, and the high a variety of Inhibitory receptors of expression, such as PD-1.
Clinically, a variety of PD-1/PDL1 inhibiting antibodies are ratified to list by FDA, have been used to treatment melanoma, non-
The neoplastic hematologic disorders such as the solids such as Small Cell Lung Cancer tumor and B cell lymphoma, acute myeloid leukemia have the treatment of duration to imitate
Fruit and certain safety.But shown now with report, during PD-1/PDL1 treatment, it will appear with certain patients
Drug resistance even disease aggravation, such as adenocarcinoma of lung, Head and neck squamous cell carcinoma, adult T-cell leukemia-lymthoma.
Therefore, there is an urgent need in the art to develop a kind of more effective fruit, the less treatment method of toxicity.
Summary of the invention
It is an object of the invention to provide a kind of application of PD1 molecule inhibitor in T lymphocyte leukemia treating.
In the first aspect of the present invention, a kind of purposes of the inhibitor of PD-1 is provided, (i) is used to prepare and inhibits T-ALL
The pharmaceutical composition of cell differentiation and proliferation, and/or (ii) treatment Pancytopenia.
In another preferred example, the T-ALL cell is the positive T-ALL cell for expressing PD-1.
In another preferred example, the PD-1 derives from mammal；Preferably, people, mouse, rat or rabbit are derived from；
It is highly preferred that deriving from people.
In another preferred example, the PD-1 includes albumen, code nucleic acid, active fragment of PD-1 or derivatives thereof.
In another preferred example, the homology of the active fragment and/or derivative and PD-1 is at least 90%, preferably
95%, more preferably 98%, 99%.
In another preferred example, the active fragment and/or derivative at least have 80%, 85%, 90%, 95%,
100% PD-1 activity.
In another preferred example, the PD-1 albumen is as shown in SEQ ID NO.:2.
MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYR
MSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTE
RRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDY
GELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
In another preferred example, the code nucleic acid of the PD-1 albumen is as shown in SEQ ID NO.:1.
In another preferred example, the inhibitor of the PD-1 includes PD-1 antibody or the activity inhibitor of PD-1.
In another preferred example, the inhibitor of the PD-1 inhibits the activity and/or expression quantity of PD-1.
In another preferred example, the inhibitor of the PD-1 is selected from the group: PD1 antibody.
In the second aspect of the present invention, a kind of method of the inhibition T-ALL cell Proliferation of external non-therapeutic is provided,
It is characterized in that, comprising steps of
In the presence of PD-1 inhibitor, T-ALL cell is cultivated, to inhibit T-ALL cell Proliferation.
In the third aspect of the present invention, a kind of method for promoting intracellular IGFBP3 and SULT1A3 to express in vitro is provided,
Characterized in that it comprises the following steps:
In the presence of PD-1 inhibitor, T-ALL cell is cultivated, so that intracellular IGFBP3 and SULT1A3 be promoted to express.
In another preferred example, the method also includes detecting the expression quantity of intracellular IGFBP3 and SULT1A3.
In the fourth aspect of the present invention, a kind of method of candidate combinations object for screening PD-1 regulator, including step are provided
It is rapid:
(a) in test group, candidate combinations object is added in cultivating system, and observe the test group T-ALL cell
Expression quantity and/or activity；In control group, the candidate combinations object is not added in identical cultivating system, and described in observation
The expression quantity and/or activity of T-ALL cell in control group；
Wherein, if the expression quantity of T-ALL cell and/or activity P1 are significantly higher than control group in test group cultivating system
P0 then illustrates that the candidate combinations object is the inhibitor of PD-1；
If the expression quantity of T-ALL cell and/or activity P1 are substantially less than control group P0 in test group cultivating system, say
The bright candidate combinations object is PD-1 agonist.
In the fifth aspect of the invention, a kind of inhibition T-ALL cell differentiation and proliferation and/or the acute T leaching for the treatment of are provided
The method of bar chronic myeloid leukemia, comprising steps of PD-1 inhibitor to the object application safe and effective amount needed or containing PD-1
The pharmaceutical composition of inhibitor.
In another preferred example, the object of the needs includes the mammal with tumour, it is therefore preferable to people, mouse or
Rat.
In another preferred example, the application includes that PD-1 inhibitor is directly inputted in subject in need,
Such as vein, muscle or gastrointestinal route.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 shows the building and feature of primary T-ALL heteroplastic transplantation model.(A) structure of T-ALL heteroplastic transplantation model
It builds.By (n=6 mouse) in the T-ALL cell tail vein injection to B-NDG Mice Body in patient source, 5 × 106A cell/mouse,
In the spleen cell of 24-30 days harvest mouse.(B) Flow cytometry people T-ALL purity is used.Obtaining Mouse spleen cells
Afterwards, padding is carried out with anti-human CD5, CD7, CD45, CD33, CD19, CD56 antibody, with CD3 antibody and IgG2a, κ homotype
Control antibodies carry out dyeing intracellular, thereby confirm that the spleen cell obtained from mouse is mainly T-ALL cell.
Fig. 2 shows each immune inspection of the flow cytometer detection on primary T-ALL cell and healthy human peripheral blood CD3+T cell
Test point (PD-1, CD28, CTLA-4, ICOS, 4-1BB, BTLA, LAG-3, OX40, CD40L, GITR, TIGIT, TIM-3,
VISTA, CD200) expression (A) and average fluorescent strength (MFI) (B).Healthy People n=4.With mean in four independent experiments
± SD form shows data.The statistical difference of two groups of samples has shown that (P < 0.001 * P < 0.05, * * P < 0.01, * * *).
" T " represents healthy human peripheral blood CD3+T cell.
Fig. 3 shows the mRNA expression of the immunity inspection point on primary T-ALL cell and healthy human T-cell.(A)
With ICOS, PD-1, BTLA, the CD200 of Q-PCR detection on primary T-ALL cell and healthy human peripheral blood CD3+T cell
MRNA differential expression.Healthy People n=4.Show data in the form of mean ± SD in four independent experiments.The statistics of two groups of samples
It learns difference and has shown that (P < 0.001 * P < 0.05, * * *).(B) it is expressed with the mRNA of RT-PCR detection PD-1.Respectively in T-
When mRNA in ALL cell is reversed into cDNA is 2 negative controls without RNA reverse transcriptase and without template ribonucleic acid, to construct
PD-1 stablizes the 293T cell strain of expression as positive control, detects T-ALL cell and the PD-1 on Healthy People PBMC
Transcript." T " represents healthy human peripheral blood CD3+T cell." PBMC " represents healthy human peripheral blood monocyte.
Fig. 4 shows the SNP single nucleotide polymorphism sequencing of PD-1 gene on T-ALL cell.As the result is shown in PD-1 base
Because the cytimidine C of the monoallelic on the 5th exon of intracellular region is mutated into thymidine T, mutational site is in PD-1 function
Except motif ITIM, ITSM.
Fig. 5, which is shown, detects the adjusting PD- on primary T-ALL cell and healthy human peripheral blood CD3+T cell with Q-PCR
The transcription factor (T-bet, Blimp-1, IRF9, STAT3, STAT4, cFos, FoxO1, NOTCH1, NFATc1) of 1 expression
MRNA expression.Healthy People n=4.Show data in the form of mean ± SD in four independent experiments.The statistics of two groups of samples
It learns difference and has shown that (P < 0.001 * * *)." T " represents healthy human peripheral blood CD3+T cell.
Fig. 6 shows that anti-PD-1 antibody expresses water to the mRNA of some molecules on T-ALL cell in vitro with Q-PCR detection
Flat influence.By T-ALL cell seeding (2.5 × 10 in 12 orifice plates6/ hole), while the anti-of final concentration of 50ug/ml is added
It is small to be incubated for 48 altogether for PD-1 antibody (Bioxcel, BE0188) or isotype control Ab (ctrl Ab) (Bioxcel, BE0083)
When.Show data in the form of mean ± SD in the independent experiment more than or equal to 3 times.The statistical difference of two groups of samples is
It shows (P < 0.001 * P < 0.05, * * P < 0.01, * * *).
Fig. 7 shows effect of the anti-PD-1 antibody blocking in T-ALL heteroplastic transplantation model.(A) with anti-PD-1 antibody (this
Laboratory preparation) or the general introduction treated of isotype control Ab (ctrl Ab) (Bioxcel, BE0086).B-NDG mouse
(n=6 mouse/group) is in the primary T-ALL cell (5 × 10 of tail vein injection6A cell/mouse) on the day before start, every two days
Anti- PD-1 antibody or ctrl Ab (per injection 200ug) is injected intraperitoneally.(B) white to CD45+ in spleen with streaming when putting to death
Blood disease cell is quantified.(C) the T-ALL cell in the spleen of the mouse of anti-PD-1 Antybody therapy carries out CD45 and Ki67 dye
After color, with the proliferation of streaming assessment leukaemia cell.(D) the T-ALL cell in the spleen of the mouse of anti-PD-1 Antybody therapy
After carrying out CD45, Annexin V and 7-AAD dyeing, with the level of apoptosis of flow cytometer detection leukaemia cell.Data with mean ±
SEM form shows.The statistical difference of two groups of samples has shown that (P < 0.01 * *).
Fig. 8 shows that people's T-ALL cell expresses PD-1.(A) the transcript profile sequencing data of hundreds patient T-ALL.In cake
The relative percentage of patient T-ALL of expression PD-1 transcript is shown in the black portions of figure.(B) in the black portions of pie chart
The relative percentage of people's T-ALL cell line of display expression PD-1 transcript.
Fig. 9 show with flow cytometer detection in vitro anti-PD-1 antibody to the proliferation of T-ALL cell and the influence of apoptosis.(A)
After cell is dyed with CFSE, bed board, and anti-PD-1 antibody (Bioxcel, BE0188) or the Isotype control of 50ug/ml is added
Antibody (ctrl Ab) (Bioxcel, BE0083), respectively in 0h, 12h, for 24 hours, 48h, 72h flow cytometer detection CFSE fluorescence intensity.
(B) by T-ALL cell seeding in orifice plate, and anti-PD-1 antibody (Bioxcel, BE0188) or the homotype of 50ug/ml is added
Control antibodies (ctrl Ab) (Bioxcel, BE0083), after being incubated for 48 hours altogether, progress Annexin V and 7-AAD, which are dyed, to be used in combination
Its level of apoptosis of flow cytometer detection.Without apparent statistical difference between two groups.
Figure 10 shows the transcript profile on T-ALL cell after anti-PD-1 antibody (Bioxcel, BE0188) blocking in vitro
Sequencing result is as shown in the figure.(A-C) anti-PD-1 antibody blocking is showed (in fact with Venn figure (A), volcano figure (B), thermal map (C) respectively
Test group) and isotype control Ab (ctrl Ab) (Bioxcel, BE0083) processing two groups of (control group) between differential expression base
Cause.(D) thermal map represents fold differences between two groups and comes first 30 and the difference expression gene of functional report.Biology
Number of repetition be every group 3 times.
Figure 11 shows that anti-PD-1 antibody expresses the mRNA of some molecules on T-ALL cell in vitro with Q-PCR detection
Horizontal influence.By T-ALL cell seeding (2.5 × 10 in 12 orifice plates6/ hole), while the anti-of final concentration of 50ug/ml is added
It is small to be incubated for 48 altogether for PD-1 antibody (Bioxcel, BE0188) or isotype control Ab (ctrl Ab) (Bioxcel, BE0083)
When.Show data in the form of mean ± SD in being greater than or equal to secondary independent experiment.The statistical difference of two groups of samples is
It shows (P < 0.001 * P < 0.05, * * P < 0.01, * * *).
Figure 12 shows effect of the anti-PD-1 antibody blocking in T-ALL heteroplastic transplantation model.(A) streaming when putting to death
CD45+ leukaemia cell in marrow is quantified.(B) the T-ALL cell in the marrow of the mouse of anti-PD-1 Antybody therapy
After carrying out CD45 and Ki67 dyeing, with the proliferation of streaming assessment leukaemia cell.(C) in the bone of the mouse of anti-PD-1 Antybody therapy
After T-ALL cell in marrow carries out CD45, Annexin V and 7-AAD dyeing, with the apoptosis water of flow cytometer detection leukaemia cell
It is flat.N=6 mouse/group.Data are showed in the form of mean ± SEM.Apparent statistical difference is had no between two groups of samples.Anti- PD-
1 antibody (preparation of this laboratory), isotype control Ab (ctrl Ab) (Bioxcel, BE0086).
Figure 13 shows effect of the anti-PD-1 antibody blocking in T-ALL heteroplastic transplantation model.With streaming to marrow (A) and
CD5+ leukaemia cell (left side) and CD7+ leukaemia cell (right side) in spleen (B) quantify.N=6 mouse/group.Data with
Mean ± SEM form shows.The statistical difference of two groups of samples has shown that (P < 0.01 * P < 0.05, * *).Anti- PD-1 antibody
(preparation of this laboratory) or isotype control Ab (ctrl Ab) (Bioxcel, BE0086).
Specific embodiment
The present inventor after extensive and in-depth study, it is white in T lymphocyte to develop a kind of PD1 molecule inhibitor for the first time
Application in the treatment of blood disease.The experimental results showed that anti-PD-1 antibody influences the expression of the intracellular IGFBP3 and SULT1A3 of T-ALL
Level, the result of experiment in vivo further demonstrate that anti-PD-1 Antybody therapy inhibits proliferation of the T-ALL cell in spleen.PD-1
There is expression on T-ALL cell, and play the role of certain rush tumor, this is clinically to treat T-ALL using anti-PD-1 antibody
Provide certain experimental basis.The present invention is completed on this basis.
Main advantages of the present invention include:
1. the present invention reports PD1 molecule in the table of the Pancytopenia cell T-ALL in patient source for the first time
Up to situation.
2. the present invention reports adjusting function of the PD1 molecule on T lymphocyte leukaemia cell for the first time
3. the expression that the present invention reports Transcription Factor T-bet, Blimp-1 and NOTCH1 on T-ALL cell for the first time becomes
Change may be related to the unconventionality expression of PD-1.
4. the present invention reports for the first time inhibits proliferation of the T-ALL cell in spleen with PD-1 Antybody therapy.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no
Then percentage and number are weight percent and parts by weight.
Method
Patient data and T-ALL heteroplastic transplantation model
Such patient is diagnosed as Pancytopenia, later half in monoploid Allogeneic Hematopoietic Stem Cell Transplantation
Year recurrence, multiple chemotherapy cannot be alleviated, and supplied oil layer failure is the disease terminal phase at present, and the state of an illness is uncontrollable, in advance
It is very poor afterwards.T-ALL patient samples cell is provided by hematology, Jiangsu TCM Hospital, this obtains Jiangsu TCM Hospital's ethics committee member
The approval of meeting.In the preceding patient's signed Written informed consent of experiment.
The initial patient's immunophenotype of T-ALL: CD4, CD5, CD7, CD3, CD2, cCD3 are positive, CD13, CD117, CD22,
CD19, CD56, CD33, CD34, HLA-DR, CD8, CD10, CD20, CD79a, MPO are negative.
The female NOD-Prkdc of 5 week oldscid(mature T lymphocyte, bone-marrow-derived lymphocyte and NK are thin for IL2rg/Bcgen mouse
Born of the same parents' defect) (B-NDG mouse, Biocytogen, Beijing, China) by tail vein injection enter 100uLPBS be resuspended 5 × 106Patient
The T-ALL cell (mouse n=6) in source.At the 24-30 days or so, the spleen cell of mouse is ground, red blood cell is split
Solution, and its purity of flow cytometer detection is used, with anti-human CD5, CD7, CD45 antibody coloration result display T-ALL cell nearly 100%.
Cell and cell culture
40ml peripheral blood is obtained from each Healthy People, with Ficoll-Paque Plus (GE, Shanghai, China) gradient
PBMC is isolated, directly progress flow cytometer showed, or PBMC employment CD3 positive selective reagent box (Stemcell) is sub-elected
RNA is extracted after CD3+T cell (Healthy People n is more than or equal to 3).
Primary T-ALL cell is used and contains 10%FBS (Gibco), the MEM- α cell culture medium of 2.5%SUPERGROW is trained
Support, the IL-2 containing 100ng/mL, people SCF, 10ng/mL hIL-7, the hFL T3 of the insulin of 20nM, 20ng/ml matches
Body (Novoprotein), is cultivated in the incubator of 5%CO2 by 37 DEG C
People PD-1 produced by the invention stablizes the 293T cell strain (ATCC) of expression, with the Dulbecco for having added 10%FBS
Improvement Eagle culture medium (DMEM) (Hyclone) culture.
Flow cytometry
The single cell suspension of acquisition is with PD1-APC (Miltenyi, 130-100-440), CD28-APC, CTLA-4-PE,
ICOS-PE, 4-1BB-PE, BTLA-APC, LAG-3-PE, OX40-PECY7, CD40L-PE, GITR-APC, TIGIT-APC,
TIM-3-PECY7, VISTA-APC, CD200-PECY7, CD3-FITC, CD45-FITC, CD45-APC, CD5-FITC, CD7-
PE, CD7-PECY5, CD33-APC, CD19-FITC, CD56-APC (BD, R&D, Biolegend) are incubated for.Measure cCD3 in endochylema
Antigen presentation fixes/Permeablization kit (BD) with Cytofix/Cytoperm, carries out surface dye with CD45-APC
Color and after blocking surface C D3 antigen with the anti-cd 3 antibodies (BD) of purifying, fixed, rupture of membranes uses CD3-FITC and IgG2a, κ-respectively
FITC (BD) carries out dyeing intracellular.All data with FACS Calibur (BD) collect, with FlowJo software (Tree Star,
Inc. it) analyzes.
In vivo and in vitro anti-PD-1 antibody blocking
In vitro, mention the previous day by T-ALL cell recovery, after one day, by T-ALL cell seeding in 12 orifice plates (2.5 ×
106/ hole), and be added 50ug/ml anti-PD-1 antibody (Bioxcel, BE0188) or IgG1 control antibodies (Bioxcel,
BE0083), altogether be incubated for 48 hours, then collect cell extraction albumen perhaps RNA for being WB, QPCR or RNA
sequencing。
In vivo, it is given with anti-PD-1 antibody (preparation of this laboratory) or IgG2b control antibodies (Bioxcel, BE0086)
(each every diluted 200ug antibody of mouse injection 200ul PBS), injection one in every 2 days is injected intraperitoneally in B-NDG mouse
Secondary, injection of antibodies is from T-ALL cell inoculation (every tail vein injection 5 × 106Cell, 100ul PBS be resuspended) it is previous
It starts.When control group mice shows clinical leukaemia feature, all mouse are condemned to death (6 mouse/group).
Zooscopy is ratified by University Of Suzhou's animal care and using the committee.All zooperies are according to US National
The nursing of Institutes of Health Research experimental animal and guide for use (NIH, the 8th edition, 2011) are carried out.Through Ethics Committee, University Of Suzhou batch
It is quasi-.
RT-PCR and QPCR
About RT-PCR, Total RNAs extraction is come out with MicroElute total serum IgE kit (Omega), then uses
RevertAid First Strand cDNA synthetic agent box (Thermo scientific) is reversed into cDNA.Fluorescent quantitation
PCR is with Power SYBR Green PCR Master Mix (ABI) in QuantStudioTM 6Flex Real-Time PCR
(primer sequence is listed in SI table 2) is carried out on System (ABI).Thermal cycle carries out 2 minutes at 94 DEG C, then carries out at 94 DEG C
40 recycle 15 seconds, carry out 20 seconds at 60 DEG C, carry out 1 minute at 68 DEG C.All samples are 3 multiple holes every time, biology weight
Again number 3 times.Gene expression is calculated with respect to β-actin with 2 (- Δ Δ Ct) methods.
Detect cell Proliferation and apoptosis
When detecting proliferation with transcription factor buffer group (BD), with CD45-FITC padding, fixed, broken nuclear membrane is used
It is dyed in Ki67-647 or IgG1, κ -647 (BD) core.
Use CellTraceTMCFSE cell proliferation reagent box (Invitrogen) detects cell Proliferation, will be thin according to handbook
After born of the same parents are dyed with CFSE, bed board is counted, and anti-PD-1 antibody (Bioxcel) or IgG1 antibody (Bioxcel) (50ug/ is added
Ml), respectively in 0h, 12h, for 24 hours, 48h, 72h flow cytometer detection CFSE fluorescence intensity.
Apoptosis is detected with FITC Annexin V apoptosis detection kit (BD) and 7-AAD antibody (BD), is existed respectively
Cell and anti-PD-1 antibody (Bioxcel) or IgG1 antibody (Bioxcel) are incubated for for 24 hours, are detected after 48h, 72h, with 100ul 1
After cell is resuspended × buffer, 5ul 7-AAD and 5ul Annexin V is added, room temperature is protected from light incubation 15-20 minutes, then mends
Add 1 × buffer of 300-400ul, direct up flow type.
RNAseq
It will be incubated for altogether 48 hours T-ALL cells with anti-PD-1 antibody or IgG antibody (Bioxcel) to collect, extract
RNA.Library is established, and is sequenced by Beijing genomics (BGI) with BGISEQ-500.With filter software SOAPnuke into
Row data filtering, reading unknown base is more than 5%, low quality reading.Clear data (clear data (the clean obtained
Reads)) it is stored as FASTQ format.Clear data (clean reads) is compared to genome sequence is referred to HISAT, is made
Clear data (clean reads) is compared with Bowtie2 and arrives reference sequences, these sequences are the GRCh38.p11 from NCBI
(hg38) it is obtained in.The expression of gene and transcript is calculated with software RS EM.DEGseq method is for poor between screening two groups
The gene of different expression.
Data analysis
Data are analyzed with GraphPad Prism 6.Comparison among groups Student t-test.P value < 0.05 is considered as statistics
It is significant on.
Embodiment 1.
The building and feature of T-ALL heteroplastic transplantation model from leukemia patient
Because culture can not be such that primary T-ALL cell grows in vitro, in order to expand T-ALL cell for subsequent experimental,
Leukaemia heteroplastic transplantation model is constructed on B-B-NDG mouse.It will be (every after the T-ALL cell tail vein injection in patient source
Mouse 5 × 106Cell, 6 mouse), harvest the spleen cell (Figure 1A) of mouse.It is thin in order to verify the spleen obtained from mouse
Born of the same parents are T-ALL cells, in conjunction with the immunophenotype of the aforementioned middle initial T-ALL of patient, carry out cell table with the method for flow cytometry
Face marker dye and dyeing intracellular.As shown in Figure 1B, cell is people CD33, CD19, CD56 negative, eliminates this tumour cell
It is myeloid cell, B cell, the possibility of NK cell origin, cell is surface marker people CD5, CD7 expression up to 90% or more, born of the same parents
Interior molecule people cCD3 same high expression, it is thus determined that this disease is T-ALL, the spleen cell obtained from mouse is main
It is T-ALL cell.
Embodiment 2.
The differential expression of immunity inspection point on T-ALL cell and healthy human T-cell
Flow cytometry is carried out to the periphery blood T cell of T-ALL cell and many cases Healthy People source, detects each exempt from
The differential expression of epidemic disease check point.By Fig. 2A it is found that comparing Healthy People CD3+T cell, PD-1, CD28, CD200 on T-ALL cell
Expression obviously raise, and BTLA expression obviously lower, the expression of ICOS be it is slightly elevated, the expression of TIGIT is
Slightly reduce.When being analyzed with MFI the expression of these immunity inspection points, it is as a result also consistent (Fig. 2 B).
Embodiment 3.
The mRNA differential expression of ICOS, PD-1, BTLA, CD200 on T-ALL cell and healthy human T-cell
The above result shows that ICOS, PD-1, BTLA, CD200 are abnormal expressions on this T-ALL cell, next
Their expression is demonstrate,proved in the enterprising step of level of mRNA with the method for QPCR and RT-PCR.As shown in Figure 3A, compared to strong
Health people, the mRNA expression of ICOS, PD-1 are higher on T-ALL cell, and difference is statistically significant, and CD200 is in mRNA
Expression is significantly raised in level, and BTLA is expressed in mRNA level in-site and is almost missing from.PD-1 is constructed simultaneously to stablize
The 293T cell strain of expression detects the PD- on T-ALL and normal pbmc using the method for RT-PCR as positive control
1 transcript (Fig. 3 B).Therefore, ICOS, PD-1, CD200 have expression on the T-ALL cell in patient source.
Embodiment 4.
PD-1 is expressed in people T-ALL
Gene order-checking is carried out to hundreds patient T-ALL, sequencing result is shown in patient's 80.7%T-ALL tumour cell
Portion detects (> 0.5FPKM) and arrives PD-1 transcript (213/264), expression quantity up to 146FPKM (Fig. 8 A).The present inventor exists
Inquirer T-ALL cell line on EMBL-EBI expression map obtains 4 RNA sequencing data collection, finds 35.1% people T-ALL
Cell line has PD-1 transcript to express (13/37) (Fig. 8 B).
Embodiment 5.
The sequencing of T-ALL cell SNP single nucleotide polymorphism
The sequencing of SNP single nucleotide polymorphism is carried out to T-ALL cell, finds one of the 5th exon of PD-1 gene
Monoallelic mutation occurs for base, is mutated into thymidine T (Fig. 4) by cytimidine C.
Embodiment 6.
Regulate and control the expression of the transcription factor of PD-1 expression
In the immunity inspection point that Fig. 3 has differential expression, further research on T-ALL cell PD-1 express regulation because
Element, therefore have detected with the method for QPCR the mRNA expression of associated transcription factor.As shown in figure 5, thin compared to Healthy People T
Born of the same parents, in the inducible protein of T-ALL cell upregulation control PD-1 expression, the mRNA table of IRF9, STAT3, STAT4, cFos, FoxO1
Up to obviously lowering, the mRNA expression of NOTCH1 is obviously raised, and the mRNA of NFATc1 expresses no notable difference；?
In the inhibition albumen for regulating and controlling PD-1 expression, the mRNA expression of T-bet and Blimp-1 are significantly lowered.Therefore, exist for PD-1
Abnormal expression on T-ALL cell increases, inventors believe that inhibition the albumen T-bet and Blimp- of this and regulation PD-1 expression
1 mRNA expression is lowered and the mRNA of inducible protein NOTCH1 expression up-regulation is related.
Embodiment 7.
Influence of the anti-PD-1 antibody blocking to T-ALL intracellular molecules expression
In order to explore function of the PD-1 in T-ALL cell, distinguished with after the PD-1 of anti-PD-1 antibody blocking T-ALL cell
It is dyed and is detected with CFSE and Annexin V/7-AAD, as the result is shown the cell Proliferation and apoptosis of PD-1 blocking group and IgG control group
All without difference (Fig. 9).Then after anti-PD-1 antibody and IgG antibody being incubated for T-ALL cell altogether respectively, RNA is extracted, is carried out
Transcript profile sequencing.Sequencing result shows that correlation is up to the differential gene number between 98% or more, two group between biology repeat samples
There are 236 (Figure 10).Then it is more next than the progress of more significant, functional report gene therefrom to have chosen group difference result
Step is researched and analysed, and is verified with QPCR.QPCR the results show that anti-PD-1 antibody blocking induction of CD151, IGFBP3, CLU
MRNA expression, reduce TIAF1, MTPN, SULT1A3 mRNA expression, and to CD302, HHIPL2, MMP2, SULT1A4,
The mRNA expression of IGFBP7 is without influencing (Fig. 6, Figure 11, table 1).
Table 1
Embodiment 8.
Effect of the anti-PD-1 antibody blocking in T-ALL heteroplastic transplantation model
In order to study effect of the PD-1 blocking antibody in mouse interior tumor growth, by T-ALL cell tail vein injection
Enter B-NDG mouse, (every mouse 5 × 106Cell, 4 mouse), the anti-PD-1 antibody (experiment of the 200ug of intraperitoneal injection in every two days
Group) or IgG2b control antibodies (control group), injection of antibodies since the previous day of i.v. injection T-ALL until put to death mouse,
20-30 days (Fig. 7 A) after namely T-ALL injection.People's CD45+ cells ratio is without obvious poor in two groups of spleen and marrow
Not, illustrate that PD-1 blocking has not significant impact (Fig. 7 B, Figure 12 A) to infiltration of the T-ALL in spleen and marrow.To two groups
The detection of people CD5+ cell and people's CD7+ cell also demonstrates this result (Figure 13) in marrow and spleen.Ki67 coloration result is aobvious
Show that the splenic T-ALL cell Proliferation of experimental group significantly reduces, however two groups of marrow T-ALL cell Proliferation does not have notable difference
(Fig. 7 C, Figure 12 B).In addition, Annexin V/7-AAD expression analysis two groups of spleen and marrow T-ALL cell as the result is shown
Apoptosis and no difference of science of statistics (Fig. 7 D, Figure 12 C).Anti- PD-1 Antybody therapy only inhibits T-ALL cell as the result is shown for these
Proliferation in spleen but has no influence to the organ infiltration of T-ALL and apoptosis.
It discusses
The present inventor carries out the sequencing of SNP single nucleotide polymorphism to this T-ALL cell, finds the 5th of PD-1 gene
The cytimidine C of the monoallelic of exon is mutated into thymidine T, and the protein sequence that may cause herein is dashed forward by glycine
Become valine, both belong to nonpolar amino acid, and mutational site the intracellular region PD-1 function motif ITIM,
Except ITSM, thus this mutation may the structure and function on albumen do not influence.
Now in different immunocytes to the transcription factor of PD-1 expression regulation, including inhibit albumen T-bet and
Blimp-1, inducible protein ISGF3, STAT3, STAT4, cFos/AP-1, FoxO1, NOTCH, NFATc1, NF-KB.Chronic
In LCMV infection, the gene elmination experiment of the overexpression of external T-bet and internal T-bet all shows that T-bet can inhibit in vivo
PD-1 expression.The T cell isolated from the Blimp-1 knock-out mice of acute infection LCMV is the study found that after acute infection
The T cell that Blimp-1 lacks has higher PD-1 to express.Specifically inhibit Notch signal path in vitro experiment, activates T
PD-1 transcription and protein expression on cell all reduce.Therefore inventors believe that on T urgency lymphocyte inhibit albumen T-bet and
The expression of Blimp-1 is lowered and the expression of inducible protein NOTCH1 up-regulation may be the reason of PD-1 expression increases.
SULT1A3 belongs to SULT cytosol sulfotransferase superfamily, this family is catalyzed various exogenous materials, swashs
The sulfation of element and neurotransmitter.It has been reported that in liver cancer cells SULT1A3/4 can stimulate tumour cell migration,
Invasion, epithelial-mesenchymal conversion, and promote the activation of cancer stem cell, it may be possible to the potential treatment target and biology mark of liver cancer
Will.IGFBP3 is insulin-like growth factor binding protein 3 Insulin-like growth factor-binding
Protein 3, it has been reported that IGFBP3 is swollen to play by the proliferation of adjusting cell, apoptosis, migration in some tumour cells
Tumor inhibiting effect, such as lung cancer, T-ALL and B-ALL, breast cancer, wherein research finds marrow and periphery in patient ALL
IGFBP3 expression is to lower in blood.PD-1 blocking antibody causes in T-ALL cell in the experiment in vitro of the present inventor
The mRNA of SULT1A3 expresses decline, and the mRNA of IGFBP3, which is expressed, to be increased, inventors believe that PD-1 may on T-ALL cell
Promote cancer protein SULT1A3 by up-regulation, lower cancer suppressor protein IGFBP3, promotes the growth of T-ALL cell.
In B-NDG Mice Body, the use of anti-PD-1 antibody does not make infiltration of the T-ALL in spleen and marrow and withers
It dies and changes, inventors believe that PD-1 is to the transplanting of this T-ALL cell and apoptosis and has no significant effect；But anti-PD-1
Antibody reduces the T-ALL cell Proliferation in spleen, inventors believe that PD-1 has the function for the proliferation for promoting T-ALL cell
Can, it can accelerate tumour growth, and this is independent of adaptive immune system.It has been reported that thin in melanoma and hepatocellular carcinoma
The PD-1 in portion intracellular, which also has, promotees tumor effect, the result of study of this and the present inventor are consistent.Inventors believe that clinically resisting
PD-1 Antybody therapy not only can make them again with the Depletion T cell of function of the high expression PD-1 in target tumor microenvironment
Activation generates anti tumor immune response, can there is other immunocytes of PD-1 with targeted expression, can be on targets neoplastic cells
PD-1, inhibit PD-1 to act on the rush tumor of tumour cell, reduce the proliferation of tumour cell itself, thus various raisings
The effect of anti-PD-1 Antybody therapy.In fact, in the experiment of the T-ALL xenograft mouse model of the present inventor, anti-PD-1
Antibody inhibits the increment of T-ALL cell in spleen, discloses latent effect of the PD-1 in T-ALL treatment.
It summarizes
The present invention successfully constructs T-ALL heteroplastic transplantation model, has determined using flow cytometry from mouse spleen and has obtained
Cell be mainly PDT-ALL cell.It detects using flow cytometry, Q-PCR and RT-PCR in PDT-ALL cell and Healthy People
The expression of immunity inspection point on CD3+T cell, ICOS, PD-1, CD200 have expression on PDT-ALL cell as the result is shown.
It is compared with Healthy People CD3+T cell, the expression of albumen T-bet and Blimp-1 is inhibited to lower and induce egg on PDT-ALL cell
The expression up-regulation of white NOTCH1 may be the reason of PD-1 expression increases.In vitro, PD-1 blocking antibody leads to PDT-ALL cell
In the mRNA of SULT1A3 express decline, the mRNA of IGFBP3, which is expressed, to be increased.PD-1 may be by upper on PDT-ALL cell
It adjusts and promotees cancer protein SULT1A3, lowers cancer suppressor protein IGFBP3, promote the growth of PDT-ALL cell.In T-ALL heterograft mould
In type, anti-PD-1 Antybody therapy inhibits proliferation of the PDT-ALL cell in spleen, but soaks to the organ of PDT-ALL cell
Profit and apoptosis have no influence.PD-1 has the function of promoting PDT-ALL cell Proliferation, can accelerate tumour growth, and this is not depended on
In adaptive immune system.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
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<120>application of the PD1 molecule inhibitor in T lymphocyte leukemia treating
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Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg
85 90 95
Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg
100 105 110
Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu
115 120 125
Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val
130 135 140
Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro
145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly
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Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys
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Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro
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Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly
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Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro
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Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly
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Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg
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Claims (10)
1. a kind of purposes of the inhibitor of PD-1, which is characterized in that be used to prepare (i) inhibition T-ALL cell differentiation and be proliferated,
And/or the pharmaceutical composition of (ii) treatment Pancytopenia.
2. purposes as described in claim 1, which is characterized in that the T-ALL cell is that the positive T-ALL of expression PD-1 is thin
Born of the same parents.
3. purposes as described in claim 1, which is characterized in that the PD-1 derives from mammal；Preferably, it derives from
People, mouse, rat or rabbit；It is highly preferred that deriving from people.
4. purposes as described in claim 1, which is characterized in that the PD-1 includes the albumen, code nucleic acid, active tablet of PD-1
Section or derivatives thereof.
5. purposes as described in claim 1, which is characterized in that the inhibitor of the PD-1 includes PD-1 antibody or PD-1
Activity inhibitor.
6. purposes as described in claim 1, which is characterized in that the inhibitor of the PD-1 inhibits the activity and/or table of PD-1
Up to amount.
7. a kind of method of the inhibition T-ALL cell Proliferation of external non-therapeutic, which is characterized in that comprising steps of
In the presence of PD-1 inhibitor, T-ALL cell is cultivated, to inhibit T-ALL cell Proliferation.
8. the method that one kind promotes intracellular IGFBP3 and SULT1A3 to express in vitro, which is characterized in that comprising steps of
In the presence of PD-1 inhibitor, T-ALL cell is cultivated, so that intracellular IGFBP3 and SULT1A3 be promoted to express.
9. a kind of method for the candidate combinations object for screening PD-1 regulator, which is characterized in that comprising steps of
(a) in test group, candidate combinations object is added in cultivating system, and observe the expression of the test group T-ALL cell
Amount and/or activity；In control group, the candidate combinations object is not added in identical cultivating system, and observe the control
The expression quantity and/or activity of T-ALL cell in group；
Wherein, if the expression quantity of T-ALL cell and/or activity P1 are significantly higher than control group P0 in test group cultivating system,
Illustrate that the candidate combinations object is the inhibitor of PD-1；
If the expression quantity of T-ALL cell and/or activity P1 are substantially less than control group P0 in test group cultivating system, illustrate institute
Stating candidate combinations object is PD-1 agonist.
10. a kind of method for inhibiting T-ALL cell differentiation and proliferation and/or treating Pancytopenia, feature exist
In comprising steps of the PD-1 inhibitor to the object application safe and effective amount needed or the pharmaceutical composition containing PD-1 inhibitor
Object.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111575232A (en) * | 2020-05-15 | 2020-08-25 | 暨南大学 | Application of JQ1 in inhibiting expression of T lymphocyte PD-1 and/or Tim-3 |
| CN111751545A (en) * | 2019-03-28 | 2020-10-09 | 中国科学院上海药物研究所 | Method for screening PD-L1/PD-1 checkpoint inhibitor |
| CN114146097A (en) * | 2022-01-21 | 2022-03-08 | 大理大学 | Application of periplaneta americana extract and/or monomer in preparation of tumor microenvironment M2 polarization inhibition drugs |
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2018
- 2018-12-29 CN CN201811647912.2A patent/CN109432429A/en active Pending
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111751545A (en) * | 2019-03-28 | 2020-10-09 | 中国科学院上海药物研究所 | Method for screening PD-L1/PD-1 checkpoint inhibitor |
| CN111575232A (en) * | 2020-05-15 | 2020-08-25 | 暨南大学 | Application of JQ1 in inhibiting expression of T lymphocyte PD-1 and/or Tim-3 |
| CN114146097A (en) * | 2022-01-21 | 2022-03-08 | 大理大学 | Application of periplaneta americana extract and/or monomer in preparation of tumor microenvironment M2 polarization inhibition drugs |
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