CN109096405A - Using GD2 as the Chimeric antigen receptor of target spot and pharmaceutical composition - Google Patents
Using GD2 as the Chimeric antigen receptor of target spot and pharmaceutical composition Download PDFInfo
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Abstract
The present invention relates to fields of biomedicine, in particular to a kind of using GD2 as the Chimeric antigen receptor of target spot.The Chimeric antigen receptor sequentially includes the Runx3 excitement factor of antigen binding domain, cross-film costimulation structural domain, T cell signal transduction area's functional domain and coexpression by N-terminal to C-terminal.The Runx3 excitement factor of coexpression can significantly increase the therapeutic effect of CAR-T.The invention further relates to a kind of pharmaceutical composition, the composition includes cell, CpG oligodeoxynucleotide and the sustained-release hydrogel of CAR modification；Pharmaceutical composition provided by the present invention has slow releasing function, and the curative effect of CAR-T can be further increased with the cooperation of CpG oligodeoxynucleotide.
Description
Technical field
The present invention relates to fields of biomedicine, in particular to using GD2 as the Chimeric antigen receptor of target spot and medicine group
Close object.
Background technique
CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy), i.e. Chimeric antigen receptor T
Cellular immunotherapy.The therapy be it is a kind of occur many years but in recent years just be modified use the novel cell in clinic to treat
Method.There is significant curative effect in the treatment of acute leukemia and non-Hodgkin lymphoma, it is considered to be most promising tumour
One of therapeutic modality.Technology as in all is the same, and CAR-T technology also undergoes a very long evolutionary process, exactly at this
In the evolutionary process of series, CAR-T technology gradually moves to maturity.
This new therapeutic strategy it is critical that identification target cell referred to as Chimeric antigen receptor (CAR)
Artificial receptors, and after gene modification, patient's T cell can express this CAR.In human clinical trial, science
Family some T cells in patient body are gone out by a kind of procedure extraction of similar dialysis, then in laboratory to their progress bases
Because of modification, the channel genes of this CAR will be encoded, these T cells in this way can express this new receptor.These pass through
The T cell of gene modification is proliferated in laboratory, and then they are perfused back in patient body.These T cells utilize their tables
The CAR receptor reached is integrated to the molecule on target cell surface, and this combination triggers a kind of internal signal generation, then this interior
Portion's signal so activates these T cells so that they rapidly destroy target cell potently.
In recent years, CAR-T immunotherapy is other than being used to treatment acute leukemia and non-Hodgkin lymphoma, through changing
Into rear, the diseases such as treatment solid tumor, autoimmune disease, HIV infection and graft rejection are also used to, there is broader practice
Space.
Bifunctional sialyltransferase gangliosides GD2 is considered as neuroblastoma, and osteosarcoma, melanoma and sarcoma etc. have very much
The marker of potentiality.2017, the research on " Journal of Clinical Investigation " magazine passed through modification
CAR-T cell realizes effectively treatment entity tumor.The nutriment that researcher wraps up CAR-T cell and boiomacromolecule
The combination preparation of formation is added in Mice Body, and the size of mouse pancreas cancer and melanoma, treatment effect can be effectively reduced
Fruit is more more significant than individually injection T cell.
However, being not mature enough in the prior art by the CAR-T of target spot of GD2, it is not also prepared into slow releasing pharmaceutical
Record.
Summary of the invention
The present invention relates to a kind of using GD2 as the Chimeric antigen receptor of target spot, sequentially includes antigen binding by N-terminal to C-terminal
The Runx3 excitement in area, transmembrane structure area, costimulatory signal conducting region, T cell signal transduction area's functional domain and coexpression
The factor；
The sequence of complementary determining region of heavy chain CDR-VH1, CDR-VH2, CDR-VH3 that the antigen binding domain has are successively
As shown in NO:1~3 SEQ ID, the sequence of complementary determining region of light chain CDR-VL1, CDR-VL2, CDR-VL3 are successively such as SEQ ID
Shown in NO:4~6；
The amino acid sequence of the Runx3 excitement factor is as shown in SEQ ID NO:7.
The invention further relates to a kind of pharmaceutical composition, the composition includes cell, the CpG of CAR modification as described above
Oligodeoxynucleotide and sustained-release hydrogel；
Compared with prior art, the invention has the benefit that
1, compared with traditional tumour treatment method, this method Small side effects.
2, the Runx3 excitement factor co-expressed can significantly increase the therapeutic effect of CAR-T.Runx3, can drive T cell from
Lymphoid tissue is opened, is gathered in tumor tissues.The gene expression is improved in mouse, can significantly increase CAR-T in solid tumor
Aggregation.
3. pharmaceutical composition provided by the present invention has slow releasing function, and can be into the cooperation of CpG oligodeoxynucleotide
The curative effect of one step increase CAR-T.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is each group Cytotoxicity in vitro comparison in one embodiment of the invention；ANOVA, * * p < 0.01, * * * p < 0.001,
Vs T cell group；#p < 0.05, vs experimental group；
Fig. 2 is the result of the internal verifying 4th week of CAR-T cell in one embodiment of the invention；ANOVA, * p <
0.05, * * p < 0.01, * * * p < 0.001, vs blank group；#p < 0.05, vs experimental group.
Specific embodiment
The present invention relates to a kind of using GD2 as the Chimeric antigen receptor of target spot, sequentially includes antigen binding by N-terminal to C-terminal
Area, cross-film costimulation structural domain, T cell signal transduction area's functional domain and coexpression the Runx3 excitement factor；
The sequence of complementary determining region of heavy chain CDR-VH1, CDR-VH2, CDR-VH3 that the antigen binding domain has are successively
As shown in NO:1~3 SEQ ID, the sequence of complementary determining region of light chain CDR-VL1, CDR-VL2, CDR-VL3 are successively such as SEQ ID
Shown in NO:4~6；
The amino acid sequence of the Runx3 excitement factor is as shown in SEQ ID NO:7；
Preferably, Chimeric antigen receptor as described above, the cross-film costimulation structural domain are selected from CD27, CD28,4-
1BB, OX40 (CD134), CD30, CD40, PD-1, LFA-1, CD2, CD7, LIGHT, NKG2C, the total thorn of B7-H3 protein molecular
The ligand or any combination thereof of energizing signal conducting region and CD3 ζ specific binding；
Preferably CD28 and 4-1BB.
Preferably, Chimeric antigen receptor as described above, the antigen binding domain are selected from human antibody, humanized antibody or embedding
Close antibody；Or the function fragment with antigen-binding activity of above-mentioned Antibody types.
Preferably, the heavy chain framework region sequence of the antigen binding domain is successively as shown in NO:8~11 SEQ ID, light chain bone
Frame region sequence is successively as shown in NO:12~15 SEQ ID；
Preferably, the antigen binding domain is selected from scFv；
ScFV is connected to obtain by VI with VH, and the sequence of link peptide (linker) used is (GGGGS) 3.
Preferably, Chimeric antigen receptor as described above, T cell signal transduction area's functional domain be selected from PKC θ,
Fc ε RI γ, ZAP70 or CD3 ζ or any combination thereof；
Preferably, T cell signal transduction area's functional domain is selected from CD3 ζ.
According to an aspect of the present invention, the invention further relates to a kind of isolated nucleic acid molecules, the nucleic acid molecules be DNA or
RNA encodes Chimeric antigen receptor as described above.
According to another aspect of the present invention, the invention further relates to a kind of carriers, and it includes nucleic acid molecules as described above；
Preferably, the carrier is Retroviral Vector, more preferably slow virus.
According to another aspect of the present invention, the invention further relates to a kind of method of cell for preparing CAR modification, the methods
It include: by be finished intracellular of nucleic acid molecules as described above or vector introduction as described above, to obtain the CAR
The cell of modification；
According to another aspect of the present invention, the CAR modification being prepared the invention further relates to method as described above it is thin
Born of the same parents；
Preferably, the cell of the CAR modification is T cell, NK cell, CIK cell, DC-CIK cell.
According to another aspect of the present invention, the invention further relates to carriers as described above or cell as described above to make
The application being ready for use in the therapeutic agent for the treatment of GD2 positive solid tumor；
Preferably, the GD2 positive solid tumor includes neuroblastoma, melanoma, sarcoma, brain tumor, osteosarcoma, rouge
Fat sarcoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, spindle cell sarcoma, brain tumor, Small Cell Lung Cancer,
Retinoblastoma.
According to another aspect of the present invention, the invention further relates to a kind of pharmaceutical composition, the composition includes institute as above
Cell, CpG oligodeoxynucleotide and the sustained-release hydrogel for the CAR modification stated；
Preferably, in the sustained-release hydrogel, the additive amount of the cell of the CAR modification is 1 × 106~8/mL；It is described
The additive amount of CpG oligodeoxynucleotide is 10~16 μ g/mL.
Preferably, pharmaceutical composition as described above, the sequence of the CpG oligodeoxynucleotide such as SEQ ID NO:
Shown in 16.
Preferably, pharmaceutical composition as described above, the sustained-release hydrogel are that the mixing of sodium alginate and α cyclodextrin is molten
The gel that liquid is formed, wherein the concentration of sodium alginate is 1.5-2% (w/v), and the concentration of α cyclodextrin is 0.5-0.8% (w/v).
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
The preparation of embodiment 1sc-Fv
1. the preparation of animal immune and monoclonal antibody
It using the extracellular domain part of polypeptide segment of people's GD2 albumen as immunogene, mixes, is immunized with Freund's complete adjuvant
6 week old female Balb/c mouse (immunogene dosage is 100 μ g/ mouse), immunization ways are immune for subcutaneous multiple spot；After two weeks,
Immunogene is mixed with incomplete Freund's adjuvant, booster immunization (immunogene dosage is 50 μ g/ mouse) is carried out to the mouse, is exempted from
Epidemic disease mode is immune for subcutaneous multiple spot；Carry out booster immunization in the same way every two weeks, altogether booster immunization 3 times.For the last time
The 7th day after booster immunization, eyeball of mouse is extractd, carry out the blood sampling of mouse orbit veniplex and is centrifugated serum, ELISA detection
The antibody titer of serum.If (P refers to that immune serum sample OD450 value subtracts blank control OD450 value, and N refers to feminine gender for P/N >=2.1
Control OD450 subtracts blank control OD450 value), then it is judged as positive.
The high mouse of antibody titer is selected from positive serum, takes the spleen of mouse under aseptic conditions, splenocyte is made
Suspension, the myeloma cell SP2/0 of logarithmic growth phase, is merged with splenocyte, and fused cell suspension is dispensed to 96 holes
Tissue culture plate is placed in HAT Selective agar medium and is cultivated.More wheel repeated screenings are carried out with ELISA method, and carry out Dan Ke
Longhua culture finally obtains positive cell strain through screening, is GA6 by the antibody, is prepared respectively using method is induced in Mice Body
Ascitic type monoclonal antibody, it is spare by antibody described in Protein G affinitive layer purification.
2. monoclonal antibody activity and affinity analysis
Coating buffer dilutes GD2, GD1a, GD1b, GD3 recombinant protein antigen and carries out microwell plate coating, every hole to 0.5ug/ml
100uL, 4 DEG C overnight；Next day, cleaning solution are cleaned 2 times, are patted dry；It is added confining liquid (20%BSA+80%PBS), every hole 120uL,
37 DEG C, 1h is patted dry；GD2 monoclonal antibody (GA6) after dilution is added, the hole 100uL/, 37 DEG C, 30min (part supernatant 1h)；
Cleaning solution cleans 5 times, pats dry；Be added sheep anti-mouse igg-HRP, every hole 100uL, 37 DEG C, 30min；Cleaning solution cleans 5 times, claps
It is dry；It is added in developing solution A liquid (hole 50uL/), is added in developing solution B liquid (hole 50uL/), 10min；Terminate liquid, the hole 50uL/ is added；Enzyme
It marks and reads OD value at 450nm on instrument (referring to 630nm).As a result as follows:
1 antibody activity of table analyzes data
| Extension rate | GD2 | GD1a | GD1b | GD2 |
| Former times | 2.103 | 1.065 | 1.132 | 1.104 |
| 5 times of dilution | 2.048 | - | - | - |
| 25 times of dilution | 1.879 | - | - | - |
| 125 times of dilution | 1.752 | - | - | - |
| 625 times of dilution | 1.546 | - | - | - |
| 3125 times of dilution | 1.239 | - | - | - |
| 15625 times of dilution | - | - | - | - |
| Blank well | - | - | - | - |
Note :-activity is represented lower than 1.0
As known from Table 1, GA6 antibody activity with higher provided by the invention, and do not sent out with the same family protein of CEA
Raw cross reaction.
Affinity analysis
Using AMC sensor, the antibody being purified is diluted to 10ug/ml, people's GD2 recombinant protein PBST with PBST
Carry out gradient dilution: 2000nmol, 1000nmol, 500nmol, 250nmol, 125nmol, 62.5nmol, 31.25nmol,
15.625nmol,0nmol；Operational process: 60s, curing antibody 300s in antibody-solutions, buffering are balanced in buffer 1 (PBST)
It is incubated for 180s in liquid 2 (PBST), 420s is combined in antigenic solution, 800s is dissociated in buffer 2, with 1.69 GLY of 10mM pH
Solution and buffer 3 carry out sensor regeneration, output data.
2 affinity analysis data of table
As known from Table 2, CG3 antibody has very excellent affinity of antibody.
3. the sequencing of monoclonal antibody
The GA6 antibody monoclonal that screening is obtained carries out the measurement of antibody dna sequence.Cell mRNA is extracted first, is used
RNAprep Pure kit (Tiangen, DP430).Steps are as follows: the collection 1 × 10 of suspension cell7, 300 × g centrifugation
Cell is collected into centrifuge tube by 5min, carefully absorbs all culture medium supernatants.Cleavage step is carried out immediately.Flick centrifuge tube
Bottom keeps cell precipitation loose, and appropriate lysate RL600uL, vortex concussion is added.All solution are transferred to Filter column CS
Upper (Filter column CS is placed in collecting pipe), 12,000rpm (~13,400 × g) are centrifuged 2min, collect filtrate.It is added into filtrate
1 times of 70% ethyl alcohol of volume (usually 350 μ l or 600 μ l) mixes, and obtained solution and precipitating is transferred to together in adsorption column CR3
(adsorption column CR3 is put into collecting pipe), 12,000rpm (~13,400 × g) are centrifuged 30~60sec, outwell useless in collecting pipe
Liquid puts back to adsorption column CR3 in collecting pipe.Be added into adsorption column CR3 350 μ l protein liquid removal RW1,12,000 rpm (~
13,400 × g) 30~60sec of centrifugation, outwells the waste liquid in collecting pipe, adsorption column CR3 is put back in collecting pipe.To adsorption column
The DNase I working solution of 80 μ l is added in the center CR3, is placed at room temperature for 15min.350 μ l protein liquid removals are added into adsorption column CR3
RW1,12,000rpm (~13,400 × g) are centrifuged 30~60sec, outwell the waste liquid in collecting pipe, adsorption column CR3 is put back to receipts
In collector.500 μ l rinsing liquid RW (please first check whether before use and ethyl alcohol has been added) are added into adsorption column CR3, are stored at room temperature
2min, 12,000rpm (~13,400 × g) are centrifuged 30-60sec, outwell the waste liquid in collecting pipe, adsorption column CR3 is put back to receipts
In collector.12,000rpm (~13,400 × g) are centrifuged 2min, outwell waste liquid.Adsorption column CR3 is placed in and is placed at room temperature for several minutes,
Thoroughly to dry rinsing liquid remaining in adsorbent material.Adsorption column CR3 is transferred in a new RNase-Free centrifuge tube, is added
Enter 30~100 μ l RNase-Free ddH2O is placed at room temperature for 2min, and 12,000rpm (~13,400 × g) are centrifuged 2min, obtain
RNA solution.
The first chain of cDNA is synthesized using QuantScript RT kit (Tiangen, KR103).Steps are as follows, by template
RNA thaws on ice；Primer, 10 × RT mix (wherein include RNasin and DTT), Super pure dNTP mixed liquor,
RNase-Free ddH2O thaws at (15~25 DEG C) of room temperature, is immediately placed on ice after defrosting.Every kind of solution is vortexed before use
Oscillation mixes, and brief centrifugation remains in the liquid of tube wall to collect.Mixed liquor is prepared according to the reverse transcription system of table 1, it is thoroughly mixed
Even, the vortex oscillation time is no more than 5min.Brief centrifugation is placed on ice, is finally added to template ribonucleic acid (μ g of 50ng~2)
It in mixed liquor, thoroughly mixes, the vortex oscillation time is no more than 5sec, and brief centrifugation is to collect tube wall residual liquid.37 DEG C incubate
Educate 60min.The first chain of cDNA of the generation of reverse transcription is used for subsequent PCR reaction (expanding using high mutant primer).It will
The purpose band that PCR amplification obtains is cloned into carrier.It send to business microarray dataset and is sequenced, obtain its CDR region amino acid
Sequence.The sequence of complementary determining region of heavy chain CDR-VH1, CDR-VH2, CDR-VH3 are measured successively such as SEQ ID NO:1~3 institute
Show, the sequence of complementary determining region of light chain CDR-VL1, CDR-VL2, CDR-VL3 are successively as shown in NO:4~6 SEQ ID.
4. the preparation of humanization GA6 antibody MAb
The anti-human GA6 antibody MAb of humanization is according to Leung et al. (1995, Molecule Immunol 32:1413-
27) what method obtained.It is chosen and mouse antibody variable region sequences match best source mould in Germline database
Plate.Source of mouse antibody CDR region is transplanted in the humanization template of selection, the CDR region of people's template, the humanization transplanted are replaced
Antibody variable region.It screens to obtain available humanized sequence by affinity and stability.The heavy chain framework area of antigen binding domain
Sequence is successively as shown in NO:8~11 SEQ ID, and light chain framework region sequence is successively as shown in NO:12~15 SEQ ID.
Heavy chain variable region and light chain variable region are attached with (GGGGS) 3linker, obtain sc-Fv sequence.
The building of 2 Chimeric antigen receptor of embodiment
Pass through full genome composite signal peptide, GD2 antigen-binding domains (sc-Fv that embodiment 1 is prepared), CD28
Cross-film costimulation structural domain, the Runx3 excitement of 4-1BB cross-film costimulation structural domain, CD3 ζ T cell signal transduction area, coexpression
Factor structure domain:
GD2-CAR: signal peptide-GD2scFv-CD28-4-1BB-CD3 ζ-Runx3；
Wherein signal peptide sequence is as shown in SEQ ID NO:17；
The gene order number of CD28 is XM_006712862.1；The gene order number of 4-1BB is U03397.1；CD3 ζ's
Gene order number is AF228312.1；The selection of sequence refers to Sadelain M, Nature biotechnology, 2013,31
(1): the structure of the Chimeric antigen receptor of 71-5 design.
Comparison sequence GD2-CAR-control is set simultaneously, and compared with GD2-CAR, difference, which is only that, has lacked the area Runx3
Section.
The building of 3 GD2-CAR-T slow virus of embodiment
GD2-CAR segment and GD2-CAR-control are cloned into slow virus carrier pc DNATMIn 3.1 (+), selection
Being inserted into restriction enzyme site is Nhe I/Bam HI.Sequence is provided by applicant, and assembly program has Suzhou Ji Ma gene Co., Ltd complete
At.
The preparation of 4 CAR-T cell of embodiment
T cell is source of people, (contains IL-2, IL-7, IL- using by CD3 positive T cell 15 culture medium of Lonza x-vivo
The cell factors such as 15 and inactivation AB blood plasma) it is cultivated.After 48 hours, cell-stimulating is in good condition while cell number is basic
It remains unchanged, with the slow virus containing CAR, (virus is pc DNATMThe slow virus of 3.1 (+)-CAR recombinant vectors, with certain
MOI (2-4) infects the cell of culture.After 12 hours, carry out full dose change liquid, to remove the virus of infection, then after
Continuous culture；Cell is carried out in subsequent incubation according to cell growing way and partly changes liquid, needed for supplementing in cell growth process
Nutriment.After cell culture, packing is sent to third party into probe tube, after label and carries out Quality Control detection after sampling.
Chlamydia, mycoplasma, endotoxin and microorganism in CAR-T cell obtained are detected, CAR-T obtained
It is feminine gender that Chlamydia, mycoplasma, endotoxin and microorganism, which carry out testing result, in cell；By CAR-T cell obtained into
The detection of row CAR expression rate, the results showed that expression rate is 65% or more.
The functional verification of 5 CAR-T cell of embodiment
1. cell culture
A.LAN-1 cell (Human Neuroblastoma Cell Line of the GD2 positive) cultivates (DMEM+15%FBS)
Motility rate is counted and measured, is resuspended after centrifugation with physiological saline, adjusting its viable cell concentrations is 3 × 108A/mL, always
It counts up to 1.8 × 109It is a.
B.T cell culture
The motility rate for counting and measuring T cell, is resuspended after centrifugation with physiological saline, and adjusting its concentration is 5 × 105A/mL, always
Number reaches 6 × 105It is a.
C.CAR-T cell culture
The CAR percentage composition for measuring CAR-T cell, counts and measures motility rate, be resuspended after centrifugation with physiological saline, adjusts
(living cells and CAR is expressed as the positive, the CAR-T cell being previously mentioned in this experimental example refers both to effective CAR-T for effective CAR-T cell
Cell) concentration and prepare CAR-T cell.
2. prepared by Cellular gels:
GD2-CAR gel:
The CAR-T cell, CpG oligodeoxynucleotide and 0.5-0.8% (w/v) α cyclodextrin of GD2-CAR will be transfected,
The mixing of 1.5-2% (w/v) sodium alginate (800~1000kD of average molecular weight) solution, is added drop-wise in 200mM calcium chloride solution,
30min is reacted, the calcium alginate micro gel bead of the cell containing CAR-T is formed.Wherein CAR-T cell in gel final concentration of 1 ×
107/mL；Final concentration of 14 μ g/mL of the CpG in gel.
Control group 1: preparation method is to replace in the CAR-T cell for transfecting GD2-CAR with GD2-CAR gel, difference
Transfect the CAR-T cell of GD2-CAR-control.
Control group 2: preparation method is with GD2-CAR gel, the glucan for the quality such as difference is to replace with α cyclodextrin
(800~1000kD of average molecular weight).
Control group 3: preparation method with GD2-CAR gel, difference be to add CpG negative control sequence (SEQ ID NO:
18)。
3. Cytotoxicity in vitro Contrast on effect.
Step 1: Calcein-AM marks target cell
1) Calcein-AM is diluted to 1mg/mL with DMSO；
2) the full culture medium of target cell is resuspended at 1 × 106The density of/mL；
3) 15 μM of Calcein-AM, 37 DEG C, 5%CO are added230min is cultivated, every 10min is mixed gently；
4) 1500rpm is centrifuged, and removes supernatant, is resuspended with full culture medium, is repeated twice；
Step 2: killing
1) target cell marked is resuspended according to the density of 5000-50000/mL, 100 μ L is taken to be added to 96 orifice plates
In；
2) 100 μ L effector cell's gels are added according to ET ratio appropriate, are added by above-mentioned grouping, every group 3 parallel；
Meanwhile having that individual A group 6 is parallel, only target cell (spontaneous release)；There is individual B group 6 in parallel, only
There is target cell+2%Triton X-100 (maximum release)；
Step 3:
1) 37 DEG C, 5%CO2After culture 4 hours, centrifugation takes 75 μ L supernatants, is transferred on a new culture plate；
2) sample utilizes pectramax Gemini dual-scanning microplate
Spectrofluorimeter detects (excitation filter:485 ± 9nm；band-pass filter:530±9
Nm), data are shown in the form of AFU；
3) percentage of cell cracking: [(test release-spontaneous release)/(maximum is calculated
release–spontaneous release)]*100。
Experimental result is as shown in Figure 1.
It will be seen from figure 1 that experimental group and 1~3 killing ability of control group are all remarkably higher than T cell；And experimental group is killed
The ability of wound is significantly higher than control group 1 and control group 3.
The internal verifying of 3.CAR-T cell
1. cell inoculation
LAN-1 cell is resuspended with physiological saline, adjusting its viable cell concentrations is 3 × 108A/mL, on ice by its with
Matrigel is mixed well according to the volume ratio of 2:1.It is inoculated with and (is inoculated in nude mice) by hypodermic mode.
Successfully to grow 300mm3Tumour as the successful criterion of mouse neuroblastoma model construction.Wherein
Gross tumor volume calculation formula are as follows: gross tumor volume (mm3)=tumour major diameter (mm) × 2 (mm of tumour minor axis2)×0.5；
2. mouse tumor model is administered.It is D0 on the day of recording tumor inoculation, PBS or CAR-T is given when D7.It is logical
The mode for crossing the minimally invasive injection of ultrasonic wave added CAT cell and comparison liquid are directly injected by tumor tissues, and all mouse are single
Secondary administration, specific administration are as shown in table 3.
Specifying information is administered in table 3
Two, evaluating drug effect:
1, mouse is observed
From buying mouse, until 4 weeks continuous survival states for observing mouse daily after administration, the stringent physique for monitoring animal
Situations such as feature, unfavorable condition such as 10% or more weight loss, depilation, diarrhea, conjunctivitis and paralysis that record may occur.
It is observed that learning, no significant difference between five groups.
2, body weight determination
It is grouped at random after measuring mouse weight when grouping；Mouse weight is measured respectively before inoculation and before administration；It is every after administration
All 2 measurement mouse weights.Weight can reflect mouse health status and tumor proliferation situation.Mouse is measured with electronic balance
Weight retains an effective digital after decimal point.It learns after measured, no significant difference between five groups.
3, the measurement of gross tumor volume
The tumour major diameter and minor axis for measuring mouse after inoculation 2 times a week, thus calculate gross tumor volume.Gross tumor volume can reflect
The proliferative conditions of mouse interior tumor out.With vernier caliper measurement tumour major diameter and minor axis, gross tumor volume (mm3)=tumour major diameter
(mm) × tumour minor axis2(mm2)×0.5.Compare the difference between the difference and each group and negative control group between each group.Its
In, at first week, each group gross tumor volume reached 300mm3More than, it was demonstrated that it models successfully.The knurl weight of each group in 4th week
Situation is as shown in Figure 2.As can be seen from Figure 2, experimental group has the effect of optimal inhibition tumour growth.
4, the measurement of survival time of mice
The survival state of observation mouse daily, when mouse tumor volume reaches 3000mm3It is denoted as death, after record inoculation
It the time of every dead mouse and group and is counted in 4 weeks, gross tumor volume is not up to 3000mm after 4 weeks3Then it is denoted as survival mice.
Count the mean survival time of every group of mouse.Life cycle reflects the proliferative conditions of mouse interior tumor.Use vernier caliper measurement
Tumour major diameter and minor axis, gross tumor volume (mm3)=tumour major diameter (mm) × tumour minor axis2(mm2)×0.5.It learns after measured, phase
For other two groups, CAR-T (GD2-CAR) cell therapy group can significantly extend life cycle.
The ratio of our initial mouse of survival mice when 4 4th week of table
As can be known from the above table, different gel cross-linkage degree are different, can be influenced on the release efficiency of CAR-T cell, Jin Erzao
At the reduction of mouse survival rate.
It is raw that the above results can be seen that the tumour that CAR-T cell provided by the invention can significantly inhibit in animal pattern
It is long, have antitumor function.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features
It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
SEQUENCE LISTING
<110>Hangzhou Pu Lve Biotechnology Co., Ltd
<120>using GD2 as the Chimeric antigen receptor of target spot and pharmaceutical composition
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 10
<212> PRT
<213>artificial sequence
<400> 1
Cys Lys Val Cys Ile Cys Gln Val Pro Glu
1 5 10
<210> 2
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<212> PRT
<213>artificial sequence
<400> 2
Ser Asp Lys Ser Tyr Tyr Thr Met Ser
1 5
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<211> 9
<212> PRT
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<400> 3
Ser Leu Thr Cys Met Ile Thr Thr Ile
1 5
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<211> 7
<212> PRT
<213>artificial sequence
<400> 4
Thr Gly Pro Lys Ser Arg Tyr
1 5
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<212> PRT
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<400> 5
Ser Leu Ser Gln Cys Met Lys Ile Thr
1 5
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<400> 6
Asp Ser Ile Lys Gly Leu Phe Ser Asp
1 5
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<211> 239
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<213> Homo sapiens
<400> 7
Pro Arg Glu Pro Arg Arg His Arg Gln Lys Leu Glu Asp Gln Thr Lys
1 5 10 15
Pro Phe Pro Asp Arg Phe Gly Asp Leu Glu Arg Leu Arg Met Arg Val
20 25 30
Thr Pro Ser Thr Pro Ser Pro Arg Gly Ser Leu Ser Thr Thr Ser His
35 40 45
Phe Ser Ser Gln Pro Gln Thr Pro Ile Gln Gly Thr Ser Glu Leu Asn
50 55 60
Pro Phe Ser Asp Pro Arg Gln Phe Asp Arg Ser Phe Pro Thr Leu Pro
65 70 75 80
Thr Leu Thr Glu Ser Arg Phe Pro Asp Pro Arg Met His Tyr Pro Gly
85 90 95
Ala Met Ser Ala Ala Phe Pro Tyr Ser Ala Thr Pro Ser Gly Thr Ser
100 105 110
Ile Ser Ser Leu Ser Val Ala Gly Met Pro Ala Thr Ser Arg Phe His
115 120 125
His Thr Tyr Leu Pro Pro Pro Tyr Pro Gly Ala Pro Gln Asn Gln Ser
130 135 140
Gly Pro Phe Gln Ala Asn Pro Ser Pro Tyr His Leu Tyr Tyr Gly Thr
145 150 155 160
Ser Ser Gly Ser Tyr Gln Phe Ser Met Val Ala Gly Ser Ser Ser Gly
165 170 175
Gly Asp Arg Ser Pro Thr Arg Met Leu Ala Ser Cys Thr Ser Ser Ala
180 185 190
Ala Ser Val Ala Ala Gly Asn Leu Met Asn Pro Ser Leu Gly Gly Gln
195 200 205
Ser Asp Gly Val Glu Ala Asp Gly Ser His Ser Asn Ser Pro Thr Ala
210 215 220
Leu Ser Thr Pro Gly Arg Met Asp Glu Ala Val Trp Arg Pro Tyr
225 230 235
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<213> Homo sapiens
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Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Ile Ser Gly
1 5 10 15
Gly Ser Lys Asp Ser Thr Ala Cys
20
<210> 9
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<212> PRT
<213> Homo sapiens
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Met Tyr Gln Leu Leu Leu Lys Arg Leu Glu Trp Ser Gln Ser Ile Ser
1 5 10 15
Leu Ser
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Leu Phe Ile Gly Arg Asp Asn Ala Lys Asn Thr Ser Ile Ser Asn Asn
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20 25
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Gly Gly Thr Lys Thr Lys Leu Glu Gln
1 5
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Glu Met Asn Leu Ile Glu Ser His Lys Phe Met Ser Tyr Arg Leu Ser
1 5 10 15
Cys Gly Ala Ser Gly
20
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<213> Homo sapiens
<400> 13
Trp Gly Trp Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Phe Lys Gly
1 5 10 15
Leu Glu Thr Ser
20
<210> 14
<211> 33
<212> PRT
<213> Homo sapiens
<400> 14
Asp Leu Tyr Thr Leu Lys Gly Thr Leu Ser Ile Phe Pro Gly Glu Tyr
1 5 10 15
Leu Gln Met Gln Gln Lys Pro Glu Trp Glu Ala Gly Asn Thr Phe Val
20 25 30
Cys
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<213> Homo sapiens
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Arg Gly Tyr Ile Tyr Thr Leu Thr Ser Thr Val Ser
1 5 10
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tccatgtcgt tcctgacgtt 20
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<212> PRT
<213> Homo sapiens
<400> 17
Met Glu Cys Ser Cys Val Met Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser
<210> 18
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<212> DNA
<213>artificial sequence
<400> 18
tccatggctt tcctgacgtt 20
Claims (10)
1. it is a kind of using GD2 as the Chimeric antigen receptor of target spot, it sequentially include antigen binding domain, cross-film costimulation by N-terminal to C-terminal
The Runx3 excitement factor of structural domain, T cell signal transduction area's functional domain and coexpression；
The sequence of complementary determining region of heavy chain CDR-VH1, CDR-VH2, CDR-VH3 that the antigen binding domain has are successively such as SEQ
Shown in NO:1~3 ID, the sequence of complementary determining region of light chain CDR-VL1, CDR-VL2, CDR-VL3 successively as SEQ ID NO:4~
Shown in 6；
The amino acid sequence of the Runx3 excitement factor is as shown in SEQ ID NO:7；
Preferably, the cross-film costimulation structural domain is selected from CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, LFA-1,
The costimulatory signal conducting region of CD2, CD7, LIGHT, NKG2C, B7-H3 protein molecular, with CD3 ζ specific binding ligand or
Any combination thereof；More preferably CD28 and 4-1BB；
Preferably, T cell signal transduction area's functional domain is selected from PKC θ, Fc ε RI γ, ZAP70 or CD3 ζ or it is any
Combination；It is furthermore preferred that T cell signal transduction area's functional domain is selected from CD3 ζ.
2. Chimeric antigen receptor according to claim 1, which is characterized in that the antigen binding domain is selected from human antibody, people
Source antibody or chimeric antibody；Or the function fragment with antigen-binding activity of above-mentioned Antibody types；
Preferably, the heavy chain framework region sequence of the antigen binding domain is successively as shown in NO:8~11 SEQ ID, light chain framework area
Sequence is successively as shown in NO:12~15 SEQ ID；
Preferably, the antigen binding domain is selected from scFv.
3. a kind of isolated nucleic acid molecules, which is characterized in that the nucleic acid molecules are DNA or RNA, encode claims 1 or 2
The Chimeric antigen receptor.
4. a kind of carrier, it includes nucleic acid molecules as claimed in claim 3；
Preferably, the carrier is Retroviral Vector, more preferably slow virus.
5. a kind of method for the cell for preparing CAR modification, which is characterized in that the described method includes: by core as claimed in claim 3
Acid molecule or to be finished intracellular of vector introduction as claimed in claim 4, to obtain the cell of the CAR modification.
6. the cell for the CAR modification that method described in claim 5 is prepared；
Preferably, the cell of the CAR modification is T cell, NK cell, CIK cell, DC-CIK cell.
7. carrier as claimed in claim 4 or the cell of CAR as claimed in claim 6 modification are in preparation for treating the GD2 positive
Application in the therapeutic agent of solid tumor；
Preferably, the GD2 positive solid tumor includes neuroblastoma, melanoma, sarcoma, brain tumor, osteosarcoma, fatty meat
Tumor, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, spindle cell sarcoma, brain tumor, Small Cell Lung Cancer, view
Film blastoma.
8. a kind of pharmaceutical composition, the composition includes cell, the CpG oligomerization deoxidation core of CAR modification as claimed in claim 6
Thuja acid and sustained-release hydrogel；
Preferably, in the sustained-release hydrogel, the additive amount of the cell of the CAR modification is 1 × 106~8/mL；The CpG is few
The additive amount of poly- deoxynucleotide is 10~16 μ g/mL.
9. pharmaceutical composition according to claim 8, which is characterized in that the sequence of the CpG oligodeoxynucleotide is such as
Shown in SEQ ID NO:16.
10. pharmaceutical composition according to claim 8, which is characterized in that the sustained-release hydrogel is sodium alginate and α ring
The gel that the mixed solution of dextrin is formed, wherein the concentration of sodium alginate is 1.5-2% (w/v), and the concentration of α cyclodextrin is 0.5-
0.8% (w/v).
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| WO2023165517A1 (en) * | 2022-03-02 | 2023-09-07 | Beijing Meikang Geno-Immune Biotechnology Co., Ltd. | Gd2 chimeric antigen receptor and use thereof |
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