CN110272874A - A kind of immunocyte drug comprising loading the B cell vaccine of neoantigen - Google Patents
A kind of immunocyte drug comprising loading the B cell vaccine of neoantigen Download PDFInfo
- Publication number
- CN110272874A CN110272874A CN201810212231.7A CN201810212231A CN110272874A CN 110272874 A CN110272874 A CN 110272874A CN 201810212231 A CN201810212231 A CN 201810212231A CN 110272874 A CN110272874 A CN 110272874A
- Authority
- CN
- China
- Prior art keywords
- cell
- neoantigen
- vaccine
- neob
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000003719 b-lymphocyte Anatomy 0.000 title claims abstract description 37
- 229940030156 cell vaccine Drugs 0.000 title claims abstract description 17
- 239000003814 drug Substances 0.000 title claims abstract description 16
- 229940079593 drug Drugs 0.000 title claims abstract description 13
- 101100228946 Streptomyces fradiae neoB gene Proteins 0.000 claims abstract description 64
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 63
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 39
- 229960005486 vaccine Drugs 0.000 claims abstract description 32
- 230000003321 amplification Effects 0.000 claims abstract description 23
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 23
- 201000011510 cancer Diseases 0.000 claims abstract description 12
- 238000001727 in vivo Methods 0.000 claims abstract description 7
- 238000000338 in vitro Methods 0.000 claims abstract description 6
- 238000002659 cell therapy Methods 0.000 claims abstract description 4
- 210000002865 immune cell Anatomy 0.000 claims abstract description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract 9
- 230000001225 therapeutic effect Effects 0.000 claims abstract 3
- WYFYSTBFFDOVJW-UHFFFAOYSA-L 2-[4-[4-(3,5-diphenyltetrazol-2-ium-2-yl)phenyl]phenyl]-3,5-diphenyltetrazol-2-ium;dichloride Chemical compound [Cl-].[Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC(=CC=2)C=2C=CC(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC=CC=2)=NN1C1=CC=CC=C1 WYFYSTBFFDOVJW-UHFFFAOYSA-L 0.000 claims description 33
- 210000004027 cell Anatomy 0.000 claims description 29
- 210000000612 antigen-presenting cell Anatomy 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 201000010099 disease Diseases 0.000 claims description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 8
- 230000002265 prevention Effects 0.000 claims description 8
- 238000001802 infusion Methods 0.000 claims description 7
- 229920001184 polypeptide Polymers 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 210000004369 blood Anatomy 0.000 claims description 5
- 239000008280 blood Substances 0.000 claims description 5
- 238000002474 experimental method Methods 0.000 claims description 5
- 210000005259 peripheral blood Anatomy 0.000 claims description 5
- 239000011886 peripheral blood Substances 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 239000000427 antigen Substances 0.000 claims description 2
- 230000030741 antigen processing and presentation Effects 0.000 claims description 2
- 108091007433 antigens Proteins 0.000 claims description 2
- 102000036639 antigens Human genes 0.000 claims description 2
- 230000001900 immune effect Effects 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 239000005482 chemotactic factor Substances 0.000 claims 8
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 claims 4
- 206010069754 Acquired gene mutation Diseases 0.000 claims 2
- 108700020796 Oncogene Proteins 0.000 claims 2
- 208000035269 cancer or benign tumor Diseases 0.000 claims 2
- 230000037439 somatic mutation Effects 0.000 claims 2
- 229940124597 therapeutic agent Drugs 0.000 claims 2
- 102100036842 C-C motif chemokine 19 Human genes 0.000 claims 1
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 claims 1
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 claims 1
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 claims 1
- 230000001413 cellular effect Effects 0.000 claims 1
- 238000005034 decoration Methods 0.000 claims 1
- 230000012202 endocytosis Effects 0.000 claims 1
- 238000012239 gene modification Methods 0.000 claims 1
- 230000036039 immunity Effects 0.000 claims 1
- 239000003112 inhibitor Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000012986 modification Methods 0.000 claims 1
- 230000004048 modification Effects 0.000 claims 1
- 238000012827 research and development Methods 0.000 claims 1
- 238000010361 transduction Methods 0.000 claims 1
- 230000026683 transduction Effects 0.000 claims 1
- 210000004881 tumor cell Anatomy 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 11
- 229940029030 dendritic cell vaccine Drugs 0.000 abstract description 4
- 229940021747 therapeutic vaccine Drugs 0.000 abstract 1
- 230000000638 stimulation Effects 0.000 description 13
- 230000004913 activation Effects 0.000 description 8
- 230000004083 survival effect Effects 0.000 description 7
- 230000007246 mechanism Effects 0.000 description 6
- 230000008901 benefit Effects 0.000 description 5
- 229940022399 cancer vaccine Drugs 0.000 description 5
- 238000009566 cancer vaccine Methods 0.000 description 5
- 230000007547 defect Effects 0.000 description 4
- 239000011324 bead Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 230000002459 sustained effect Effects 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 230000003442 weekly effect Effects 0.000 description 3
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 229920001917 Ficoll Polymers 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 229940022005 RNA vaccine Drugs 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 108700021021 mRNA Vaccine Proteins 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 241000700605 Viruses Species 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Oncology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention provides a kind of immune cell therapy drugs of B cell vaccine (neoB) comprising load tumour neoantigen, and the pharmaceutical composition that neoB and tumor specific T cells are combined.NeoB vaccine of the present invention can have stronger amplification in vitro than DC vaccine, and the stronger effect for continuing to activate T cell in vivo when being transfused repeatedly, the preventative or therapeutic vaccine of one kind or immunocyte drug can be become and be applied in the antineoplaston for not distinguishing cancer kind.The pharmaceutical composition that neoB and tumor specific T cells are combined, further stimulates tumor specific T cells using neoB in vivo, and making the latter, quantity expands and promotes therapeutic effect in vivo.
Description
Technical field
The invention belongs to biomedicine technical fields, and specifically the present invention provides a kind of B cell epidemic disease for loading neoantigen
Seedling, and the immune cell therapy drug comprising the vaccine, this vaccine or drug can be used for prevention and treatment to tumour.
Background technique
Auxiliary or main means with cancer vaccine as prevention and control of cancer are the directions worthy of expecting of prevention and control of cancer,
But there are three problem letter is to be solved for cancer vaccine:
Problem 1: being the neoantigen cancer vaccine for constructing the vaccine or individuation multiple target point of fixed target spot?
Compared to the cancer vaccine of fixed target spot, individuation neoantigen cancer vaccine is trend of the times.
Problem 2: it is building polypeptide or RNA vaccine, or using antigen presenting cell as the carrier of vaccine, constructs cell epidemic disease
Seedling?
Neoantigen vaccine mostly uses greatly polypeptide or RNA vaccine, however polypeptide or RNA are possible to be offered by normal cell,
To increase the danger attacked by T cell, there are safety risks.Polypeptide or RNA are loaded into antigen presenting cell, constructed
Cell vaccine is safer safe strategy.
Problem 3: if the quantity limitation of antigen presenting cell can be broken through, and energy continuous and effective offers neoantigen, thus
Continuous and effective activation and amplification neoantigen reaction-ive T cell?
Though cell vaccine is had it long ago, all it is the general antigen of load, not yet loads individuation neoantigen, and adopt mostly
With DC vaccine, and there are quantity bottleneck and mechanism defects for DC cell.
DC is to need to use a large amount of vaccines for disease because DC cell can not expand in vitro there are quantity bottleneck
When prevention and treatment, DC quantity will become bottleneck.
The mechanism defect of DC is that it is easy to be killed by T cell, when needs are repeated multiple times gives boosting vaccine, after
The T cell that the DC cell that the DC cell being transfused several times can be transfused for the first time is induced is killed, so after the effect that is transfused several times
It has a greatly reduced quality.
Neoantigen treatment is finally to activate neoantigen reaction-ive T cell by loading the antigen presenting cell (APC) of neoantigen
(neoantigen reactive T cells, neoT) finally identifies tumour neoantigen to kill tumour by neoT.For
Overcome tumor microenvironment to inhibit, immunologic test point-costimulation reverse conversion molecule can be added in neoT, the suppression to T cell
Signal processed switchs to activation signal, constructs enhanced neoantigen reaction-ive T cell (Enhanced neoantigen reactive T
Cells, ENT).But all there is bottleneck in either neoT or ENT, quantity, need a large amount of vaccines, in vivo repetitious stimulation
Expand its quantity.However DC vaccine is difficult to become effective hand of amplification neoT or ENT there are quantity bottleneck and mechanism defect
Section.
Summary of the invention
The present invention is by loading to B cell, the B cell vaccine of building load individuation neoantigen for individuation neoantigen
(neoB), and using the cell comprising neoB as drug, become the auxiliary or main means of prevention and control of cancer.
B cell can become good antigen presenting cell and construct vaccine, on the one hand because B cell can express MHC
I class and II class molecule and possess preferable antigen presentation function, another aspect B cell quantity can expand to than DC have number
Amount advantage, there are also B cells can escape from the attack of T cell to which there are advantages in mechanism than DC.
But individuation neoantigen is loaded to B cell by past never someone, constructs neoB.Past, more someone was not by neoB
It is combined with neoT or ENT, so that neoB becomes the means in patient's body amplification neoT or ENT.
The present invention can be used for the prevention and treatment of cancer using the cell comprising neoB as independent drug, compared with DC vaccine,
It is advantageous that neoB can be expanded, advantage is quantitatively formed, so as to activate more neoT, plays better cancer
Control efficiency.On the other hand, infusion neoB can play Cascaded amplification effect to neoT quantity repeatedly, and neoDC is because of itself
Quantity bottleneck and mechanism defect, be difficult very limited to the Cascaded amplification effect of neoT quantity.
In addition, neoB and neoT or ENT is combined by the present invention, so that neoB, which becomes, expands neoT or ENT in patient's body
Means, neoT or ENT can be made to break through quantity bottleneck in this way, obtain better prevention and control of cancer effect.
Specific embodiment
In order to be more fully understood and with the application of the invention, hereinafter with reference to embodiment the present invention is described in detail, the implementation
Example is only intended to illustrate the present invention, and limits the scope of the invention without being intended to.The scope of the present invention is by appended right
It is required that specific limit.
Embodiment one: the preparation of the B cell vaccine (neoB) and DC cell vaccine (neoDC) of neoantigen is loaded
B cell and DC cell are separated in blood of cancer patients mononuclearcell.
The separation and amplification of B cell: venous collection peripheral blood in patients is separated using Ficoll lymphocyte separation medium
PBMC sub-elects the B cell of the CD19 positive using CD19 antibody magnetic bead and is counted.Related culture medium and cell factor is added
Culture amplification B cell, collects B cell on the 12nd day, it can be achieved that 10~30 times of amplification.
The separation of DC cell and differentiation and maturation: venous collection singly adopts peripheral blood in patients, using Ficoll lymphocyte point
Chaotropic isolates PBMC and 2 hours adherent, takes adherent monocytes, be added relevant cell factor promote DC cell differentiation at
It is ripe, it cultivates 7-8 days and collects mature DC cell.
Gene sequencing analysis is carried out to specimens, outer-gene synthesis is carried out to candidate neoantigen mutation, and
Being transcribed in vitro becomes RNA, and electric shock is transferred to B cell and DC cell respectively, obtains the B cell vaccine (neoB) of load neoantigen, and
Load the DC cell vaccine (neoDC) of neoantigen.
Embodiment two: in experiment in vitro, the activation of neoB vaccine, neoDC vaccine is enriched with the comparison of neoT effect
Tumor-infiltrated T lymphocyte (TIL) is separated from tumor patient autologous tissue, or single from blood of cancer patients
T cell is separated in nucleus.It is trained altogether with the mixture of the neoB and neoDC that are prepared from the autologous patient cell with T cell respectively
It supports 1 day, flow cytometer detection CD137+T cell proportion in all T cells, i.e. neoantigen reaction-ive T cell (neoT), as a result
It was found that:
The ratio of the CD137+T cell of patient 1: neoB stimulation is that 21.3%, neoDC stimulation is 22.1%；
The ratio of the CD137+T cell of patient 2: neoB stimulation is that 6.3%, neoDC stimulation is 6.5%,
The ratio of the CD137+T cell of patient 3: neoB stimulation is that 3.6%, neoDC stimulation is 3.8%,
The ratio of the CD137+T cell of patient 4: neoB stimulation is that 33.7%, neoDC stimulation is 34.1%,
The ratio of the CD137+T cell of patient 5: neoB stimulation is that 5.7%, neoDC stimulation is 5.9%,
As it can be seen that very big difference is presented because of individual patients sex differernce in the ratio of CD137+T cell；Same patient with neoB and
The ratio of the CD137+T cell of neoDC stimulation is very close.Illustrate that neoB has to be enriched with neoDC very close activation
The ability of neoT.
Embodiment three: the preparation of plain edition and enhanced neoantigen reaction-ive T cell
Tumor-infiltrated T lymphocyte (TIL) is separated from tumor patient autologous tissue, or single from blood of cancer patients
T cell is separated in nucleus.1 is co-cultured with the mixture and T cell of the neoB and neoDC prepared from the autologous patient cell
It obtains neoantigen reaction-ive T cell (neoT) using the T cell of CD137 antibody magnetic bead screening and activating.
The slow virus carrier of the reverse conversion molecule of PD1/4-1BB is transferred to neoT, transfection efficiency is about 60%, streaming point
Choosing or magnetic bead sorting go out PD1+T cell constructs enhanced neoT (ENT), remaining PD1-T cell is then common neoT.
Example IV: neoB vaccine, neoDC vaccine compare the amplification enrichment effect and own survival of two kinds of T cells
Mouse experiment, using immunodeficient mouse, a point following groups give tail vein feedback:
ENT(A0)；
A group, neoB give two kinds of T cells: neoT+neoB (A1) respectively；ENT+neoB(A2)；
B group, neoDC give two kinds of T cells: neoT+neoDC (B1) respectively；ENT+neoDC(B2)；
NeoT and ENT give 105Dosage.NeoB and neoDC give 105Dosage.
After feedback in 20 minutes and after three days, the quantity of acquisition mouse peripheral blood monitoring neoB and neoDC cell.After feedback
In the 20 minutes and after a week quantity of acquisition mouse peripheral blood detection neoT and ENT cell.
As the result is shown:
Phenomenon 1: no matter A group or B group, for same vaccine, the ENT quantity after being activated is both greater than neoT quantity, than
In A group, the ENT quantity of A2 is 2.1 × 106, the neoT quantity of A1 is 7.2 × 105；In B group, the ENT quantity of B2 is 0.9 ×
106, the neoT quantity of B1 is 3.7 × 105.Illustrate that ENT can be activated preferably by antigen presenting cell.
Phenomenon 2: either giving neoT or ENT, and third day neoDC quantity is all remarkably decreased, third day B1 group DC number
Mesh is about 1.2 × 104, B2 group DC number is substantially not detectable；Third day neoB quantity is declined, but decline it is few, such as the
A1 group B cell number is about 0.83 × 10 within three days5, A2 group B cell number is about 0.61 × 105.Illustrate that neoB is offering neoantigen
While, it can preferably avoid being killed by T cell.
Phenomenon 3: either giving neoT or ENT, and neoB is better than neoDC to the activation amplification effect of T cell.
Five: neoB vaccine of embodiment, neoDC vaccine various dose the amplification enrichment effect of ENT cell is compared
Mouse experiment, using immunodeficient mouse, a point following groups give tail vein feedback:
ENT (control group)；
ENT+105Dosage neoB (A1)；ENT+3×105Dosage neoB (A2)；ENT+106Dosage neoB (A3)； ENT+3
×106Dosage neoB (A4)；
ENT+105Dosage neoDC (B1)；ENT+3×105Dosage neoDC (B2)；ENT+106Dosage neoDC (B3)； ENT
+3×106Dosage neoDC (B4)；
All groups of ENT give 106Dosage.NeoB or neoDC is fed back in ENT in advance.It monitors within continuous six weeks, monitors weekly
ENT, neoB, neoDC quantity.
As the result is shown:
The peak value of the ENT of each subgroup, component A are not 2.6 × 106(A1), 5.3 × 106(A2), 21.4 × 106(A3),
55.6×106(A4), B component is not 1.5 × 106(B1), 1.9 × 106(B2), 7.5 × 106(B3), 16.3 × 106(B4),
Control group is 1.2 × 106
Phenomenon 1: either neoB group (A group) or neoDC group (B group), ENT quantity all with vaccine infusion dosage positive
It closes.Illustrate that the dosage of vaccine is very crucial to the amplification and enrichment of neoantigen reaction-ive T cell.
Phenomenon 2:neoB group (A group) is compared with each dosage subgroup of neoDC group (B group), in final ENT quantity,
NeoB group (A group) is all significantly more than neoDC group (B group).
Phenomenon 3: in terms of the survival of neoB and neoDC, neoB is obviously higher than neoDC survival ratio, monitors several times after
When, DC almost monitored less than.This can explain phenomenon 2, and after neoDC is killed by T cell, quantity falls sharply, therefore cannot be further continued for
T cell is stimulated, and neoB survival rate is high, it can be with sustained activation T cell.
This example demonstrates that the antigen presenting cell quantity of high load neoantigen, it is thin can to increase tumour identity T
The amplification amplitude of the quantity of born of the same parents or neoantigen reaction-ive T cell.Therefore the B cell that can be expanded, can be thinner than the DC that cannot be expanded
Born of the same parents have more advantage in terms of Enrichment Amplification neoantigen reaction-ive T cell.
Embodiment six, continuous several times neoB and neoDC compare the amplification enrichment effect of ENT cell
Mouse experiment, a point following groups give tail vein feedback:
ENT (control group)；
neoB(A1)；ENT+ single neoB (A2)；ENT+2 neoB (A3)；ENT+3 neoB (A4)；ENT+4 times
neoB(A5)；
neoDC(B1)；ENT+ single neoDC (B2)；ENT+2 neoDC (B3)；ENT+3 neoDC (B4)； ENT+4
Secondary neoDC (B5)；
ENT gives single 105Quantity.First dose of neoB and neoDC is given 105Quantity is fed back on the same day with ENT.For multiple
The subgroup of vaccine is given, gives a vaccine infusion weekly, and dosage doubles every time, i.e., by second 2 × 105(A3~5, B3
~5), third time 4 × 105(A4~5, B4~5), the 4th time 8 × 105(A5, B5) Dosage fractionation infusion.
Blood drawing detection neoB, neoDC and ENT quantity weekly.
As the result is shown:
Phenomenon 1: in final ENT quantitative aspects, component A is not 23 × 105(A2), 127 × 105(A3), 639 × 105
(A4), 2130 × 105(A5), B component is not 9.2 × 105(B2), 32 × 105(B3), 83 × 105(B4), 194 × 105(B5),
Control group is 2.2 × 105.The quantity increasing degree of neoB vaccine amplification ENT is given every time in A group, and it is each to be significantly greater than B group
Give the quantity increasing degree of neoDC vaccine amplification ENT, it is repeated multiple times when giving vaccine, neoB to the amplification effect of ENT significantly
Better than neoDC.
Phenomenon 2: in terms of the survival of neoB and neoDC, neoB is obviously higher than neoDC survival ratio, in repeatedly infusion epidemic disease
The subgroup (A3~6, B3~6) of seedling, when monitoring several times after, DC has almost been monitored less than and B cell quantity and the number that is fed back
Magnitude is close.Illustrate that neoDC can only play the role of activating T cell when feeding back first time, to after second, T cell can be killed
Dead DC cell acts on to be difficult recurrence to sustained activation.
This can explain phenomenon 1, and after neoDC is killed by T cell, quantity falls sharply, therefore cannot be further continued for stimulation T cell, and
NeoB survival rate is high, can be with sustained activation T cell.
This example demonstrates that neoB and enhanced neoantigen reaction-ive T cell are combined, ENT effectively can be substantially expanded
Quantity.
In conclusion to allow the vaccine of load neoantigen to expand neoT or ENT in vivo, it is necessary to continue to be transfused epidemic disease repeatedly
Seedling, neoB vaccine of the present invention because in its amplifiable property and mechanism with neoDC vaccine advantage, in lasting amplification neoT or ENT
Effectiveness on, be better than neoDC vaccine.
Claims (5)
1. a kind of immunocyte preparation method, preparation " the B cell vaccine (neoB) of load neoantigen ", it is characterised in that:
1) using the B cell of tumor patient or donor as antigen presenting cell (APC), the individuation knubble of tumor patient is newly resisted
Former (neoantigen) loads to B cell, becomes the B cell vaccine (neoB) of load neoantigen；
2) B cell both can come from autologous patient, can be from allosome donor:
3) B cell can be simple B cell, is also possible to a variety of antigens including B cell, DC cell and offers carefully
The mixture of born of the same parents；
4) the individuation knubble neoantigen of the tumor patient loads to neoB and can be obtained by following either method or a variety of sides
Method joint is completed::
A. tumour neoantigen gene order is obtained through oncogene sequencing analysis somatic mutation, and synthesizes neoantigen gene order
It obtains DNA or is further transcribed in vitro to obtain RNA, then the DNA or RNA is passed through in carrier transduction to B cell；
B. tumour neoantigen gene order is obtained through oncogene sequencing analysis somatic mutation, and synthesizes neoantigen polypeptide, then
Polypeptide is loaded into B cell；
C. its is made to discharge albumen after tumour cell being crushed, mixing with B cell makes these albumen of its endocytosis and offer neoantigen.
5) process of the load neoantigen can carry out after B cell reaches quantity amplification by vitro culture, can also
To be done before；
6) the B cell vaccine (neoB) of the load neoantigen, can be added or be added without including but not limited to following gene and repair
Decorations；
A. the immunologic test point inhibitor including PD1 monoclonal antibody, PDL1 monoclonal antibody；
B. the one of the CC chemotactic factor (CF) including IL7, CCL19, Gro-beta-T, C chemotactic factor (CF) and CX3C chemotactic factor (CF)
Kind or a variety of chemotactic factor (CF)s.
2. a kind of preventative or therapeutic cells vaccine or the pharmaceutical composition comprising cell vaccine, which is characterized in that described thin
It has used during any one of born of the same parents' vaccine or pharmaceutical composition research and development, production, preparation, application and has exempted from described in claim 1
Epidemic disease cell preparation method, or the cell for using method described in claim 1 to prepare is expanded or is carried out other experiments
The working process of method, and its any one derivative application in therapeutic agent, treatment method.
3. preventative or therapeutic cells vaccine immunity cellular therapeutic agent as claimed in claim 2 or pharmaceutical composition, special
Sign is that the drug or pharmaceutical composition are in any cancer kind or disease, the prevention of any neoplasm staging or antineoplaston
Using, and repeated multiple times infusion is given in administration mode.
4. a kind of immune cell therapy drug or pharmaceutical composition, it is characterised in that:
1) drug or pharmaceutical composition include individuation knubble specific T-cells, and by individuation knubble specific T-cells and are weighed
Cell vaccine described in force request 2 is used in combination；
2) the individuation knubble specific T-cells can be prepared by following either method:
A. tumor-infiltrated T lymphocyte (TILs) is separated from tumor tissues and is obtained, or is added on this TILs and is helped it and overcome
Tumor microenvironment obstacle helps the gene modification of its quantity amplification and obtains；
B. by load tumour individuation neoantigen DC cell B cell or other have the cell of antigen presentation function, with periphery
The T cell in blood source or the TIL in tumour source are mixed or are co-cultured in vitro, the neoantigen specific T-cells of screening and activating
(neoT) it obtains, or is added on this neoT cell and helps its gene for overcoming tumor microenvironment obstacle or helping the amplification of its quantity
Modification, constructs enhanced neoantigen reaction-ive T cell (Enhanced neoantigen recognizing T cells, ENT)
And obtain, wherein the T cell of the derived from peripheral blood can come from by or without patient vaccine-induced described in claim 2
Or donor；
3) described that individuation knubble specific T-cells and cell vaccine described in claim 2 are used in combination, refer to that individuation is swollen
The action time window of both tumor specific T-cells and cell vaccine described in claim 2 in vivo is existed using neoB there are Chong Die
Be transfused this kind of individuation knubble specific T-cells of TIL or neoT or ENT are further activated in vivo, expand it further,
The two can be transfused simultaneously, infusion when can also be different.
5. immune cell therapy drug as claimed in claim 4 or pharmaceutical composition, which is characterized in that the drug or medicine group
Close the application in any cancer kind or disease, the antineoplaston of any neoplasm staging.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810212231.7A CN110272874A (en) | 2018-03-15 | 2018-03-15 | A kind of immunocyte drug comprising loading the B cell vaccine of neoantigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810212231.7A CN110272874A (en) | 2018-03-15 | 2018-03-15 | A kind of immunocyte drug comprising loading the B cell vaccine of neoantigen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110272874A true CN110272874A (en) | 2019-09-24 |
Family
ID=67957625
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810212231.7A Pending CN110272874A (en) | 2018-03-15 | 2018-03-15 | A kind of immunocyte drug comprising loading the B cell vaccine of neoantigen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110272874A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111303268A (en) * | 2020-03-10 | 2020-06-19 | 江苏兰恩生物治疗技术研究院有限公司 | Hysteromyoma neoantigen, application thereof and hysteromyoma vaccine |
| WO2021087839A1 (en) * | 2019-11-07 | 2021-05-14 | 武汉华大吉诺因生物科技有限公司 | Tumor-specific polypeptide sequence and application thereof |
| WO2021204204A1 (en) * | 2020-04-08 | 2021-10-14 | 北京卡替医疗技术有限公司 | Antigen gene transfection cell vaccine and related immune cell |
| CN113957045A (en) * | 2021-10-25 | 2022-01-21 | 北京卡替医疗技术有限公司 | HLA semi-compatible allogeneic immune cell for identifying self-antigen of subject |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106456724A (en) * | 2013-12-20 | 2017-02-22 | 博德研究所 | Combination therapy with neoantigen vaccine |
-
2018
- 2018-03-15 CN CN201810212231.7A patent/CN110272874A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106456724A (en) * | 2013-12-20 | 2017-02-22 | 博德研究所 | Combination therapy with neoantigen vaccine |
Non-Patent Citations (9)
| Title |
|---|
| CARSTEN LINNEMANN等: "High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma.", 《NATURE MEDICINE》 * |
| CYRILLE J. COHEN 等: "Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes", 《THE JOURNAL OF CLINICAL INVESTIGATION》 * |
| MACLEAN HALL等: "Expansion of tumor-infiltrating lymphocytes (TIL) from human pancreatic tumors.", 《JOURNAL FOR IMMUNOTHERAPY OF CANCER》 * |
| PATRICK A. OTT 等: "An immunogenic personal neoantigen vaccine for patients with melanoma", 《NATURE》 * |
| REJEAN LAPOINTE 等: "CD40-stimulated B Lymphocytes Pulsed with Tumor Antigens Are Effective Antigen-presenting Cells That Can Generate Specific T Cells", 《CANCER RESEARCH》 * |
| 万亚锋等: "负载肝癌肿瘤抗原的 B 淋巴细胞诱导特异性抗肿瘤免疫的实验研究", 《实用医学杂志》 * |
| 张昌菊主编: "《医学免疫学》", 31 January 2010, 世界图书出版公司 * |
| 郑杰编著: "《肿瘤的细胞和分子生物学》", 31 January 2011, 上海科学技术出版社 * |
| 韩明勇，窦明金，孙迎红等主编: "《肿瘤分子靶向治疗学》", 31 August 2008, 济南出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021087839A1 (en) * | 2019-11-07 | 2021-05-14 | 武汉华大吉诺因生物科技有限公司 | Tumor-specific polypeptide sequence and application thereof |
| CN111303268A (en) * | 2020-03-10 | 2020-06-19 | 江苏兰恩生物治疗技术研究院有限公司 | Hysteromyoma neoantigen, application thereof and hysteromyoma vaccine |
| WO2021204204A1 (en) * | 2020-04-08 | 2021-10-14 | 北京卡替医疗技术有限公司 | Antigen gene transfection cell vaccine and related immune cell |
| CN113957045A (en) * | 2021-10-25 | 2022-01-21 | 北京卡替医疗技术有限公司 | HLA semi-compatible allogeneic immune cell for identifying self-antigen of subject |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Snell et al. | CD8+ T cell priming in established chronic viral infection preferentially directs differentiation of memory-like cells for sustained immunity | |
| CN110272874A (en) | A kind of immunocyte drug comprising loading the B cell vaccine of neoantigen | |
| DE69435075T2 (en) | IMMUNOGENEOUS CHIMERAL COMPRISING NUCLEIC ACID SEQUENCES, THE ENDOPLASMATIC RETICULAR SIGNAL SEPARATIVE PEPTIDE AND AT LEAST ONE OTHER PEPTIDE, THEIR USE IN VACCINES, AND THE TREATMENT OF DISEASES | |
| CN101918544B (en) | Method for increasing immunoreactivity | |
| CN105296431B (en) | The α β T cells and its suppression cancer purposes of tumor combination specificity gamma delta T CR genetic modifications | |
| US20200384021A1 (en) | Hiv immunotherapy with no pre-immunization step | |
| DE10335833A1 (en) | Transfection of blood cells with mRNA for immune stimulation and gene therapy | |
| CN105985931A (en) | NK cell in-vitro amplification composition and NK cell amplification method | |
| CN107488235B (en) | A kind of preparation and application of new enhanced antigen joint polypeptid induction liver cancer-specific CTL cell | |
| JP2019520061A (en) | T cell proliferation | |
| Cousin et al. | Persistence of integrase-deficient lentiviral vectors correlates with the induction of STING-independent CD8+ T cell responses | |
| WO2009139413A1 (en) | Method for production of cell mass containing cytokine-induced killer cell | |
| BRPI0821998A2 (en) | immunomodulation compositions and uses thereof. | |
| WO2021178571A1 (en) | On demand expression of exogenous factors in lymphocytes to treat hiv | |
| Yang et al. | A Trichosanthin-derived peptide suppresses type 1 immune responses by TLR2-dependent activation of CD8+ CD28− Tregs | |
| CN110016465A (en) | A kind of immunocyte drug comprising B cell and the double identity T cells of tumour | |
| Teixeira et al. | Propionibacterium acnes enhances the immunogenicity of HIVBr18 human immunodeficiency virus-1 vaccine | |
| US20220025006A1 (en) | An Interleukin 21 Protein (IL21) Mutant and Use Thereof | |
| CN107952069A (en) | Recombinant vaccine and its application | |
| US20240115604A1 (en) | Methods of manufacturing genetically-modified lymphocytes | |
| CN111214650A (en) | Application of dissociation factor of HIV antigen-receptor trimer complex | |
| CN107106578A (en) | Treat the bisphosphonate compound of E Positan epstein-Barr virus relevant diseases and the therapy of gamma delta T cells mediation | |
| CN111000839A (en) | Use of dimethylarginine and its derivatives for inhibiting T, B cell proliferation | |
| US20230372398A1 (en) | Virus specific t-cells and methods of treating and preventing viral infections | |
| CN108441473A (en) | A kind of method of ex vivo enrichment CD8+* T cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190924 |