CN108047332A - Using CD19 as the specific antibody of target spot, CAR-NK cells and its preparation and application - Google Patents

Using CD19 as the specific antibody of target spot, CAR-NK cells and its preparation and application Download PDF

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CN108047332A
CN108047332A CN201810036245.8A CN201810036245A CN108047332A CN 108047332 A CN108047332 A CN 108047332A CN 201810036245 A CN201810036245 A CN 201810036245A CN 108047332 A CN108047332 A CN 108047332A
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sequence
antigen
car
cells
binding fragment
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CN108047332B (en
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李华顺
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Asclepius Suzhou Technology Company Group Co Ltd
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Zhejiang Askeli Biotechnology Co Ltd
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Priority to PCT/CN2019/071559 priority patent/WO2019137518A1/en
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    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Abstract

The present invention provides a kind of using CD19 as the anti-CD 19 antibodies of target spot or its antigen-binding fragment, wherein described antibody includes heavy chain variable region (VH) and/or light chain variable region (VL), wherein the heavy chain variable region includes heavy chain VHCDR1, VHCDR2, VHCDR3；The light chain variable region includes light chain VLCDR1, VLCDR2, VLCDR3.The present invention is provided using CD19 as the anti-CD 19 antibodies of target spot or its antigen-binding fragment, and as monoclonal cell strain culture and expands gained using the Anti CD19 CAR NK cells constructed by this, and character is stablized, and is prepared available for large-scale production.Anti CD19 CAR NK cells specifically can kill or kill lymphoma cell using CD19 molecules as target antigen, can be as the medicine of lymthoma class disease, for the treatment of the tumour of the high expression of CD19 molecules.

Description

Using CD19 as the specific antibody of target spot, CAR-NK cells and its preparation and application
Technical field
The present invention relates to biomedicine fields, and in particular to using CD19 as the specific antibody of target spot, using CD19 as target spot CAR-NK cells and its preparation and application.
Background technology
The immunocyte of Chimeric antigen receptor (chimeric antigen receptor, abridge CAR) modification uses heredity Engineering means modification immunocyte makes it express exogenous Antioncogene.CAR genes mainly include Extracelluar pathway domain and thin Intracellular signal transduction structural domain：The former divides for identifying tumor surface specific molecular and the latter for starting identification tumor surface Immune cell responses after son play cytotoxicity, mainly using T- cells as carrier.
CAR-T, full name are Chimeric AntigenReceptor T-Cell Immunotherapy, chimeric antigen by Body T cell immunotherapy.Chimeric antigen receptor T cell (CAR-T cells) be will identify certain tumour antigen antibody it is anti- The intracellulars element such as former engaging portion and CD3- ζ chains or 4-1BB is coupled in vitro as a mosaic gene, passes through the method for gene transfer The T cell of patient is transfected, it is made to express Chimeric antigen receptor (CAR).The T cell of patient after " recodification ", is generated a large amount of swollen The CAR-T cells of knurl specificity.CD19 is that 95kD wears membrane glycoprotein, its contactin and in B cell It adjusts and plays the role of a nucleus in response.Substantial amounts of research has shown that CD19 can be used for CAR-T's as the target spot for the treatment of B cell lymphoma Structure.
It proves by substantial amounts of clinical practice, is achieved significantly by the CAR-T drugs that target spot is developed of the CD19 at present Curative effect.Novartis announces that its CAR-T products C TL019 enters FDA and accelerates examination ＆ approval passage on March 29th, 2017.2015 December, FDA are granted by the breakthrough sex therapy certification (BTD) of the CAR-T therapies KTE-C19 of Kite Pharma, are diffused for treating The indication therapy of property large B cell lymphoid tumor (DLBCL).On April 1st, 2017, Kite Pharma also announce formally complete to FDA Permit application into the biological products of CAR-T therapies KTE-C19 (being renamed as afterwards " axicabtagene ciloleucel ") are submitted (BLA) roll and declare.
But current CD19 CAR-T researchs still have some problems.Such as：First, preparing the T cell of CAR-T can only come From patient itself, it is impossible to which allosome is fed back；Second, the ScFV sequences for the CAR-T CD19 antibody prepared are mostly mouse in the world Source antibody, there are larger immunological rejection risks.
Natural kill (natural killer, abridge NK) cell is the important component of non-specific immune systems, The key mediator cell of innate immune system reaction.NK cells are a kind of broad immune cells, have quick discovery and destroy The exceptional function of abnormal cell (such as cancer or the cell of virus infection), and sensitization or HLA distribution type need not be shifted to an earlier date, you can exhibition Show the activity of powerful dissolving abnormal cell.It is new trend in recent years to carry out (including NK cells) treating cancer using immunocyte, This new treatment, which is expected to provide new healing for the tumour invalid to traditional operation, chemotherapy and radiation, wishes.
The content of the invention
In view of this, the present invention provides a kind of using CD19 as the anti-CD 19 antibodies of target spot or its antigen-binding fragment, with And the CAR-NK cells of the structural domain and its preparation and application can be expressed, which stablizes, and can in high volume give birth to Production and preparation, can carry out allosome backtracking；On the other hand, for building its antibody sequence behaviour source antibody sequence, it is used as medicine Object is in application, significantly reduce the risk of immunological rejection.
One aspect of the present invention offer is a kind of using CD19 as the anti-CD 19 antibodies of target spot or its antigen-binding fragment, wherein described Antibody include heavy chain variable region (VH) and/or light chain variable region (VL), wherein the heavy chain variable region include heavy chain VHCDR1, VHCDR2、VHCDR3；The light chain variable region includes light chain VLCDR1, VLCDR2, VLCDR3, wherein the amino of the VHCDR1 Acid sequence such as SEQ ID NO：Sequence or its homologous sequence shown in 1；The amino acid sequence of the VHCDR2 such as SEQ ID NO：2 Shown sequence or its homologous sequence；The amino acid sequence of the VHCDR3 such as SEQ ID NO：Sequence shown in 3 or its is homologous Sequence；The amino acid sequence of the VLCDR1 such as SEQ ID NO：Sequence or its homologous sequence shown in 4；The ammonia of the VLCDR2 Base acid sequence such as SEQ ID NO：Sequence or its homologous sequence shown in 5；The amino acid sequence of the VLCDR3 such as SEQ ID NO：Sequence or its homologous sequence shown in 6.
Illustratively, the amino acid sequence of the VH chains such as SEQ ID NO：Sequence or its homologous sequence shown in 7.
Illustratively, the amino acid sequence of the VL chains such as SEQ ID NO：Sequence or its homologous sequence shown in 8.
Illustratively, the homology of the homologous sequence and former sequence is more than 60%, for example, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% Or more, 99.6% or more, 99.7% or more, 99.8% or more or 99.9% or more etc..
Illustratively, the VH chains and VL chains are directly connected to or are connected by connecting peptide.
In a specific embodiment provided by the invention, using CD19 as the anti-CD 19 antibodies of target spot or its antigen binding Segment includes VH chains and VL chains.
In a specific embodiment provided by the invention, the VH chains and VL chains are connected by connecting peptide.
In a specific embodiment provided by the invention, the nucleosides of the anti-CD 19 antibodies or its antigen-binding fragment Acid sequence such as SEQ ID NO：Sequence or its degenerate sequence shown in 9.
In a specific embodiment provided by the invention, the anti-CD 19 antibodies or its antigen-binding fragment and CD19 Affinity reach 8.473 × 10-8More than M.
Another aspect of the present invention provides a nucleotide sequence, can express above-mentioned anti-CD 33 antibody or its antigen binding fragment Section.
In a specific embodiment provided by the invention, the nucleotide sequence includes SEQ ID NO：Shown in 10 Sequence or its degenerate sequence and/or SEQ ID NO：Sequence or its degenerate sequence shown in 11.
In a specific embodiment provided by the invention, the nucleotide sequence such as SEQ ID NO：Shown in 12 Sequence or its degenerate sequence.
Illustratively, the homology of the degenerate sequence and former sequence is more than 60%, for example, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% Or more, 99.6% or more, 99.7% or more, 99.8% or more or 99.9% or more etc..
Another aspect of the present invention provides above-mentioned anti-CD 19 antibodies or the preparation method of its antigen-binding fragment, including：It builds Vertical phage antibody library simultaneously therefrom screens the antibody or antibody fragment that can be combined with CD19.Its detailed process includes：
1. prepare M13KO7 helper phages；
2. establish phage antibody library using M13KO7 helper phages；
3. using CD19 antigens from step 2. in antibody library in screen single stranded phage DNA；And optionally,
4. the single stranded phage DNA of step 3. middle acquisition is expressed, and purify.
Another aspect of the present invention provides a kind of Anti CD19 CAR-NK cells, the cell can express chimeric antigen by Body, the Chimeric antigen receptor include above-mentioned anti-CD 19 antibodies or its antigen-binding fragment.
In the specific embodiment of the present invention, the Chimeric antigen receptor is also comprising transmembrane domain and/or altogether Stimulus signal conducting region.
Illustratively, the transmembrane domain is selected from：CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、 One or more transmembrane domains in CD22, CD33, CD37, CD134, CD137, ICOS and CD154；Preferably, it is described Transmembrane domain be CD8 transmembrane domains；And/or
The costimulatory signal conducting region includes the intracellular domain of costimulatory molecules, costimulatory molecules choosing From：CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、 One or more in CD137, ICOS, CD154,4-1BB and OX40；Preferably, the costimulatory signal conducting region includes 4-1BB and CD3 ζ intracellular domain.
Preferably, the anti-CD 19 antibodies or its antigen-binding fragment can specifically bind tumour specific antigen CD19, and pass through transmembrane domain and costimulatory signal conducting region activates the NK cells.
Illustratively, the Chimeric antigen receptor be structure be SCFV-CD8-4-1BB-CD3 ζ fusion protein, the fusion Albumen being capable of specific recognition CD19 molecules.
In the specific embodiment of the present invention, the amino acid of the SCFV-CD8-4-1BB-CD3 ζ fusion proteins Sequence such as SEQ ID NO：Shown in 13 or its homologous sequence etc..
Illustratively, the sequence of the nucleotide of the SCFV-CD8-4-1BB-CD3 ζ fusion proteins such as SEQ ID NO：14 Shown or its degenerate sequence etc..
In the specific embodiment of the present invention, the Anti CD19 CAR-NK cells can effectively kill or Kill Raji cells etc..
Another aspect of the present invention provides the preparation method of above-mentioned Anti CD19 CAR-NK cells, includes the following steps：
(1) synthesize and expand SCFV-CD8-4-1BB-CD3 ζ antigen-4 fusion protein genes, by the SCFV-CD8-4-1BB-CD3 ζ antigen-4 fusion protein genes are cloned on Lentiviral；
(2) the Lentiviral plasmid infection 293T cells obtained using slow virus packaging plasmid and step (1), bag Dress and preparation slow virus；
(3) the slow-virus infection NK-92 cells obtained using step (2), obtain CD19 CAR-NK cells.
It is expressed the present invention also provides above-mentioned anti-CD 19 antibodies or its antigen-binding fragment and/or above-mentioned nucleotide sequence Anti-CD 19 antibodies or its antigen-binding fragment and/or above-mentioned Anti CD19 CAR-NK cells are preparing treatment and/or prevention height Express the application in the tumour of CD19 molecules and the drug of relevant disease.
Illustratively, Anti CD19 CAR-NK cells are in the drug for preparing the lymthoma for treating high expression CD19 molecules Application.
The present invention also provides a kind of pharmaceutical composition, including above-mentioned anti-CD 19 antibodies or its antigen-binding fragment and/ The expression of above-mentioned nucleotide sequence anti-CD 19 antibodies or its antigen-binding fragment and/or above-mentioned Anti CD19 CAR-NK it is thin Born of the same parents and optionally, pharmaceutically acceptable auxiliary material.
The present invention at least has one of following advantage：
The present invention is provided using CD19 as the anti-CD 19 antibodies of target spot or its antigen-binding fragment, and with the Anti constructed by this CD19 CAR-NK cells is monoclonal cell strain cultures and expand gained, and character is also stablized, and are prepared available for large-scale production. Anti CD19 CAR-NK cells specifically can kill or kill lymphoma cell using CD19 molecules as target antigen, can make For the medicine of lymthoma class disease, for the treatment of the lymthoma of the high expression of CD19 molecules.
Description of the drawings
Fig. 1 show pCDNA3.4-ScFV provided in an embodiment of the present invention (anti-CD19) expression vector structural representation Figure.
Fig. 2 show pRRSLIN-ScFV provided in an embodiment of the present invention (2-27)-CD8TM-4-1BB-CD3 ζ slow virus The structure diagram of transfection carrier.
Fig. 3 show the result of flow cytomery CD19 CAR-NK cell positive rates provided in an embodiment of the present invention Figure；Fig. 3 a are NK-92 control groups, and Fig. 3 b are CD19 NK-92 experimental groups.
Fig. 4 show the experimental result picture of CD19 CAR-NK cell killings Raji cells provided in an embodiment of the present invention.
Specific embodiment
Unless otherwise defined, all technical and scientific terms used in the present invention have and technical field of the present invention The normally understood identical meanings of those of ordinary skill.
Specifically, the sequence of the nucleotide of coding SCFV-CD8-4-1BB-CD3 ζ fusion proteins of the present invention is Any any DNA sequence dna that can encode the fusion protein, it is preferable that the sequence is SEQ ID NO：14 or its complementary series. On the other hand, the sequence of the nucleotide of coding SCFV-CD8-4-1BB-CD3 ζ fusion proteins of the present invention can be tight Under the conditions of with by SEQ ID NO：14 nucleotide sequence is hybridized and encodes polynucleotides or its complementation of the fusion protein Sequence；
" stringent condition " as described herein can be any in low stringent condition, middle stringent condition, high stringent condition Kind, it is preferably high stringent condition.Illustratively, " low stringent condition " can be 30 DEG C, 5 × SSC, 5 × Denhardt liquid, 0.5% The condition of SDS, 52% formamide；" middle stringent condition " can be 40 DEG C, 5 × SSC, 5 × Denhardt liquid, 0.5%SDS, 52% The condition of formamide；" high stringent condition " can be 50 DEG C, 5 × SSC, 5 × Denhardt liquid, 0.5%SDS, 52% formamide Condition.It should be understood by those skilled in the art that temperature, which gets over Gao Yueneng, obtains the polynucleotides of high homology.In addition, art technology Personnel can select temperature, concentration and probe concentration, probe length, ionic strength, time, the salinity of stringency of influence hybridization etc. more Synthesis result that a factor is formed realizes corresponding stringency.
In addition interfertile polynucleotides can also be to pass through the homology searches software such as FASTA, BLAST system The default parameters of system setting is when being calculated, with the polynucleotides of code sequence row number 6 have about 60% or more, about 70% or with It is upper, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or with It is upper, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or with It is upper, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or with It is upper, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or with It is upper, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, The polynucleotides of 99.6% or more, 99.7% or more, 99.8% or more or 99.9% or more homogeneity.
The homogeneity of nucleotide sequence can use the algorithmic rule BLAST of Karlin and Altschul (Proc.Natl.Acad.Sci.USA 87：2264-2268,1990；Proc.Natl.Acad.Sci.USA 90:5873, 1993) determine.Program BLASTN, BLASTX based on BLAST algorithm rule has been developed that (Altschul SF, et al:J Mol Biol 215:403,1990).Using BLASTN analyze base sequence when, such as make parameter for score=100, Wordlength=12；When using BLASTX analysis of amino acid sequence in addition, it is score=50, wordlength such as to make parameter =3；During using BLAST and Gapped blast programs, default parameter value can be set using the system of each program.
In the present invention, term " antibody " refers to the immunoglobulin molecules combined with antigentic specificity.Antibody can be source In natural source or the complete immunoglobulin in restructuring source is come from, and can be the immune response part of intact immunoglobulins.It is anti- Body is usually the tetramer of immunoglobulin molecules.Antibody in the present invention can exist in a variety of forms, including for example, more grams Grand antibody, monoclonal antibody, Fv, Fab and F (ab) 2 and single-chain antibody and humanized antibody etc. (Harlow etc., 1999, In: Using Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,NY； Harlow etc., 1989, In:Antibodies:A Laboratory Manual,Cold Spring Harbor,New York； Houston etc., 1988, Proc.Natl.Acad.Sci.USA 85:5879-5883；Bird etc., 1988, Science 242: 423-426)。
Term " antibody fragment " refers to a part for complete antibody, and refers to that the antigen decision of complete antibody is variable Area.The example of antibody fragment includes but not limited to Fab, Fab', F (ab') 2 and Fv segments, is formed by antibody fragment linear anti- Body, scFv antibody and multi-specificity antibody.
Unless otherwise prescribed, " coding nucleotide " includes for degeneracy version each other and encodes the institute of identical amino acid sequence There is nucleotide sequence.The nucleotide sequence of coding protein may include introne.
Term " slow virus " refers to the category of Retroviridae, after can effectively infecting aperiodicity and mitosis Cell；The hereditary information that they can transfer significant quantity enters the DNA of host cell, so as to they be gene delivery vector most One of effective method.
Term " carrier " is composition of matter, and including separated nucleic acid, and it can be used for transferring separated nucleic acid extremely Cell interior.Many carriers are well known in the art, and include but not limited to linear polynucleotides and ion or amphiphatic molecule The relevant polynucleotides of compound, plasmid and virus.Therefore, term " carrier " includes the plasmid or virus of autonomous replication.The art Language should also be interpreted as including nucleic acid being transferred to the non-plasmid of cell and non-viral compound, such as polylysine Compound, liposome etc..The example of viral vectors includes but not limited to, adenovirus vector, adeno-associated virus vector, reverse transcription Viral vectors etc..
Term " cancer " is defined as the disease characterized by the quick and uncontrolled growth for the cell that distorts.Cancer cell can office Spread or spread to by blood flow and lymphatic system the other parts of body in portion.The example of various cancers includes but not limited to mammary gland It is cancer, prostate cancer, oophoroma, cervix cancer, cutaneum carcinoma, cancer of pancreas, colorectal cancer, kidney, liver cancer, the cancer of the brain, lymthoma, white Blood disease, lung cancer etc..
As it is used herein, "comprising" and " comprising ", " containing " or " being characterized in that " it is synonymous, and in being included in Or open, and it is not excluded for the other element do not stated or method and step.Term "comprising" any table herein State, particularly in the method for the description present invention, purposes or during product, it is thus understood that including substantially by the component or element or Those products, method and the purposes that step is formed and is made of the component or element or step.The sheet of description exemplified here Invention suitably can be there is no the situations of any one or more of element not specifically disclosed herein, one or more limitation Under put into practice.
The term and statement used herein is used as descriptively rather than restrictive term, and in such term and statement Use in it is not expected exclude shown in and the feature or part thereof any equivalent, it is appreciated that various modifications are being asked It is possible in the scope of the present invention of protection.It is therefore understood that although the present invention by preferred embodiment and optionally Feature specifically discloses, but the modification and transformation of concept disclosed herein, and such modification may be employed in those skilled in the art It is considered as with variation in the scope of the present invention such as defined by accessory claim.
Herein presented English name case-insensitive；The meaning phase that CD19 CAR-NK, CD19-CAR NK are represented Together；Meaning identical with Anti CD19-CAR NK expressions CD19 CAR-NK represents the CAR-NK cells of anti-CD19 molecules； NK-92, NK92 represent NK92 cells.
" NK " of the present invention is human body normal NK cells or NKT cells or NK cell lines, including NK-92 cells, YT cells, NKL cells, HANK-1 cells, NK-YS cells, KHYG-1 cells, SNK-6 cells and IMC-1 cells etc..The present invention Specific embodiment in illustrated by taking NK-92 cells as an example.
It to be illustrated more clearly that the present invention, is described in detail in conjunction with following examples, but these embodiments are only To the exemplary description of the present invention, the limitation to the application should not be construed as.
The screening of 1 CD19 human antibodies of embodiment
2 × YT fluid nutrient mediums：16g peptones, 10g yeast extracts, 5g sodium chloride, 800mL water, with sodium hydroxide tune PH to 7.0 adds water to be settled to 1000mL, 121 DEG C of sterilizing 20min.
2×YT-G：2% glucose is added in 2 × YT culture mediums.
2×YT-AK：The ampicillin of 100g/mL and the kanamycins of 50g/mL are added in 2 × YT culture mediums.
First, M13KO7 helper phages are prepared
1. under the conditions of 37 DEG C, with the TG1 of the helper phage M13K07 infection exponential phases of different diluted concentrations Bacterial cell 30 minutes, is coated on agar plate afterwards.
It is cultivated 2. TG1 bacterial plaques clone is chosen in 3mL liquid 2YT culture mediums.When culture 2 is small at 37 DEG C.
3. the culture in step 2 is transferred in 1L 2YT culture mediums, kanamycins is added in 50 μ g/m, 37 DEG C of cultures 16 it is small when.
4. centrifuging (10min at 5000g) removal bacterial cell, adding in phages agent in supernatant collects phagocytosis Body.
5. calculating phage titre, 1 × 10 is diluted to afterwards13Pfu/mL is simultaneously stored in -20 degree refrigerators, spare.
2nd, phage antibody library is prepared
1. phage library glycerol stock is taken to be inoculated into 500ml 2YT-G culture mediums, in 37 DEG C, 250rpm conditions down toward OD values For 0.8-0.9.
2. M13KO7 prepared in step 1 is added in above-mentioned steps 1, final concentration of 5 × 109Pfu/mL, 37 DEG C Stand 30min, afterwards under 200rpm rotating speeds, constant-temperature table culture 30min.
3. centrifuging the culture solution in (2200g, 15min) step 2, bacterial cell is collected, bacterium is resuspended with 2YT-AK culture mediums Mud is incubated overnight under 300rmp rotating speeds under the conditions of 30 DEG C.
4. centrifuging the culture solution in (7000g, 15min, 4 DEG C) step 3, bacterial cell is removed, supernatant is collected into 1L bottles In.
5. adding in phages agent in the supernatant into step 4, phages are collected.
6. centrifuging (7000g, 15min), supernatant is removed, collects phages, phages are added in into 8ml PBS.
7. centrifuging (12,000g, 10min) removal bacterial debris again, retain bacteriophage in supernatant, it is spare.
3rd, antibody screening
1. CD19 antigens are coated with orifice plate 2h under the conditions of 4 DEG C, are washed afterwards three times with the concentration of 5 μ g/mL.Utilize envelope Liquid is closed to close under the conditions of 4 DEG C overnight.
2. outwelling confining liquid and washing, bacteriophage (being prepared in step 2) and the human IgG albumen of 100 μ g/mL are resuspended in (contain 2% milk) in confining liquid and add in and 1h is incubated at room temperature in orifice plate, wash afterwards.
3. bacteriophage on orifice plate is incorporated in by Trypsin Induced, for the follow-up second wheel screening.
4. repeat three-wheel screening by the flow in above-mentioned 1-3.
5. obtain single stranded phage DNA, i.e. single-chain antibody gene sequence, and sequencing identification by precipitating.
Embodiment 2：The expression and identification of CD19 antibody
By on the single stranded phage DNA clone that embodiment 1 obtains to pCDNA3.4 carriers, build as shown in Figure 1 The carrier wink is gone to Chinese hamster ovary celI and expressed by pCDNA3.4-ScFV (anti-CD19) expression vector, should by nickel strain purifying Single chain antibody sequence is used for subsequent analysis.
With the affinity of the single-chain antibody that SPR method evaluation and screenings arrive and CD19 albumen, therefrom select and CD19 albumen The highest single-chain antibody of affinity, and it is named as single-chain antibody 2-27.Single-chain antibody 2-27 and CD19 molecules affinity are such as Shown in table 1.
Table 1：Single-chain antibody 2-27 and CD19 molecule affinity
sequence Ka(1/Ms) Kd(1/s) KD(M)
2-27 4.984E+4 0.004223 8.473E-8
Wherein, the amino acid sequence of the VH chains of single-chain antibody 2-27 is as shown in SEQ ID NO.7, and nucleotide sequence is such as Shown in SEQ ID NO.10；The amino acid sequence of the VL chains of single-chain antibody 2-27 is as shown in SEQ ID NO.8, nucleotide sequence As shown in SEQ ID NO.11；The amino acid sequence of single-chain antibody 3-3 is as shown in SEQ ID NO.9, the core of single-chain antibody 2-27 Nucleotide sequence is as shown in SEQ ID NO.12.
The preparation of 3 Lentiviral of embodiment
Gene chemical synthesis SCFV (2-27)-CD8-4-1BB-CD3 ζ fusion gene sequences (its amino acid sequence such as SEQ ID NO：Shown in 13, gene order such as SEQ ID NO：Shown in 14).By digestion, converted and be connected in PRRSL IN carriers, Upstream region of gene is EP-1 α promoters.Carrier converts Stbl3 coli strains, and ampicillin screening obtains positive colony, Plasmid is extracted, digestion identification clone obtains PRRLSIN-SCFV (2-27)-CD8-4-1BB-CD3 ζ slow-virus transfections carrier (such as Shown in Fig. 2).
The preparation of 4 slow virus of embodiment
(1) transfect before 24 it is small when, with every ware about 8 × 106By 293T cell inoculations into 15cm culture dishes.When ensuring transfection Cell 80% or so degree of converging and be uniformly distributed in culture dish.
(2) prep solution A and solution B
Solution A：6.25ml 2 × HEPES buffer buffer solutions (amount that 5 big ware is packed together, effect are best).
Solution B：It is separately added into the mixture of following plasmid：112.5μg PRRLSIN-SCFV(2-27)-CD8-4-1BB- CD3ζ(target plasmid)；39.5μg pMD2.G(VSV-G envelop)；73 μ g pCMVR8.74 (gag, pol, tat, rev)；625 μ l 2M ionic calcium solns.Solution B total volume：6.25ml.
Abundant mixing solution B gently while vortex solution A, is added dropwise solution B, stands 5-15 minutes.Gently it is vortexed The mixed solution of above-mentioned A and B, is added dropwise in the culture dish of the cell containing 293T, gently sway forwards and backwards culture dish make DNA and calcium from The mixture of son is uniformly distributed.(should not rotating and culturing ware) be positioned in incubator cultivate 16-18 it is small when.Replace fresh cultured Base continues to cultivate, respectively when 48 is small and when 72 is small after collect containing virus supernatant.500g, 25 DEG C centrifuge 10 minutes.PES (0.45 μm) filtering of film.With 70% ethanol disinfection Beckman Kurt Ultra-clear SW28 centrifuge tubes, and It is placed under ultraviolet lamp and sterilizes 30 minutes.The filtered supernatant containing slow virus is transferred in centrifuge tube.In centrifugation bottom of the tube 20% sucrose of careful layer overlay (adds 1ml sucrose) per 8ml supernatants.With PBS equilibrium centrifugation pipes, 25000rpm (82,700g), When 4 DEG C of centrifugations 2 are small.It is careful to take out centrifuge tube, supernatant is outwelled, centrifuge tube is inverted and removes residual liquid.100 μ l PBS are added in, Centrifuge tube is sealed, when 4 DEG C of placements 2 are small, is gently vortexed once within every 20 minutes, 500g centrifugations 1 minute (25 DEG C) are collected in virus Clearly.After cooled on ice, -80 DEG C of preservations are placed in.
The preparation of 5 CD19 CAR-NK-92 cells of embodiment
NK-92 cell densities are adjusted to 2-3 × 105/ ml, by volume (V/V) viral vectors：Cell culture medium=1: The ratio addition viral vectors (prepared by embodiment 4) of 5-10, while add 8 μ g/ml of polybrene.After 4h, equivalent is added Fresh complete medium cell density is adjusted to 1 × 105/ ml continues to cultivate.All cells are centrifuged, added by next day Enter fresh culture medium, continue to cultivate.Fluid infusion was carried out every 1-2 days, makes maintenance cell density in 2-3 × 105/ml.72h is laggard Row CAR antibody dyes, while airflow classification CD19 CAR NK-92 positive cells and expands culture.The face of observation culture medium daily Color change, cell density, cellular morphology simultaneously make respective record.
Using flow cytometer detection CAR NK-92 cell positive rates, flow cytometer detection result is as shown in Figure 3a and Figure 3b shows.Fig. 3 a and figure In 3b, used antibody is APC fluorescent markers, is indicated on the horizontal scale, if NK92 cell successful expression CAR molecules, The signal value will be raised significantly.As can be seen that the signal value of APC fluorescent markers significantly raises from Fig. 3 a and Fig. 3 b, show NK- 92 cell successful expressions go out CAR molecules, and CAR-NK92 positive rates are 99.91%.
The assessment of 6 CD19 CAR-NK cells in vitro tumor-killing effects of embodiment
Fragmentation effect of the CD19 CAR-NK cells to Raji cells is detected using CCK-8 methods.Examination on experimental operation is such as Under：
(1) 1ml Raji cell suspensions (2X10^4/hole) are prepared in 24 orifice plates.By culture plate in incubator preculture 12h。
(2) culture supernatant of 24 orifice plates is discarded, the ratio of 1ml effector cell, effector cell and target cell population are added in per hole Example is 1:1 or 0.5:1.Culture medium control wells only add 1ml culture mediums, and three multiple holes are put in each experiment.Effector cell and target cell Altogether be incubated 4 it is small when or 2h, experiment packet such as the following table 2.
2 CD19 CAR-NK cells of table test grouping to the fragmentation effect of Raji cells
(3) 100ul CCK-8 solution is added in per hole, culture plate is incubated 2h in incubator.
(4) absorbance at 450nm is measured with microplate reader.
(5) killing rate=(As-Ab)/(Ac-Ab) X100%；
As：Test hole (culture medium, CCK-8, CAR-NK containing tumour cell)；
Ac：Control wells (culture medium, CCK-8 containing tumour cell)；
Ab：Blank control (culture medium, CCK-8 without cell and CAR-NK)；
The experimental result of CD19 CAR-NK cells in vitro tumor-killing recruitment evaluations is as shown in Figure 4.
Experimental result as shown in Figure 4 is shown, compared with NK92 control groups, the CD19 CAR NK-92 prepared by the present invention are thin Born of the same parents can significantly kill the strain of Raji target cells.
The CD19 CAR NK-92 of the present invention are that CD19 CAR molecules are infected NK92 cell lines and are obtained through overflow-type screening Monospecific polyclonal cell is obtained, character is stablized into the high CAR NK92 monoclonal cell strains cultures of killing activity and expands acquisition.This is thin Born of the same parents can be used for large-scale production and prepare, and can be used for different patients and will not generate GVHR repulsions.CD19 CAR NK-92 are thin Born of the same parents are compared with CAR-T cells, without separating patient's peripheral blood mononuclear cells (PBMC), without specific activation T cell and preparation CAR-T cells (process needs patient to wait 10 days or more time), are not required customization of individual character and can be used for multiple patients, shorten Time, CD19 CAR-NK92 cells can be prepared on a large scale culture, and patient is to use；On the other hand, routinely prepare CAR-T cells, the T cell due to being separation patient are prepared through virus infection, and T cell is not same monoclonal source, And the CAR-NK92 cell deriveds sub-elected are in same monoclonal, character and activity are homogeneous and stablize, convenient for large-scale production And Quality Control；Simultaneously compared with NK92 cells, due to having carried out CAR vector introductions, killing activity is special, and oncotherapy effect is bright It is aobvious.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention Within god and principle, any modification for being made, equivalent substitution etc. should all be included in the protection scope of the present invention.
Sequence table
<110>Zhejiang power bio tech ltd of Ah Cisco
<120>Using CD19 as the specific antibody of target spot, CAR-NK cells and its preparation and application
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223> VHCDR1
<400> 1
Ser Tyr Ala Met Ser
1 5
<210> 2
<211> 20
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223> VHCDR2
<400> 2
Ser Ile Asp Ala Gln Gly Leu Pro Thr Arg Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly Arg Phe Thr
20
<210> 3
<211> 7
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223> VHCDR3
<400> 3
Ser Gly Thr Pro Phe Asp Tyr
1 5
<210> 4
<211> 11
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223> VLCDR1
<400> 4
Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn
1 5 10
<210> 5
<211> 7
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223> VLCDR2
<400> 5
Ser Ala Ser His Leu Gln Ser
1 5
<210> 6
<211> 9
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223> VLCDR3
<400> 6
Gln Gln Ala Asn Thr Ser Pro Thr Thr
1 5
<210> 7
<211> 116
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 7
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Asp Ala Gln Gly Leu Pro Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Gly Thr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 8
<211> 109
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223> VL
<400> 8
Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
20 25 30
Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Ser Ala Ser His Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
65 70 75 80
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Thr Ser Pro
85 90 95
Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 9
<211> 240
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223> Anti CD19
<400> 9
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Asp Ala Gln Gly Leu Pro Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Gly Thr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
130 135 140
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
145 150 155 160
Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
165 170 175
Lys Leu Leu Ile Tyr Ser Ala Ser His Leu Gln Ser Gly Val Pro Ser
180 185 190
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
195 200 205
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn
210 215 220
Thr Ser Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
225 230 235 240
<210> 10
<211> 348
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> VH
<400> 10
gaggtgcagc tgctggaatc aggaggagga ctggtgcagc caggaggatc tctgagactg 60
tcttgcgccg cttccggctt caccttctct tcctacgcca tgtcttgggt ccgacaggct 120
ccaggaaagg gactggagtg ggtgtcctct atcgacgcac agggcctgcc taccagatac 180
gccgattccg tgaagggcag gttcaccatc tcccgggaca actccaagaa caccctgtac 240
ctgcagatga actccctgag ggccgaggat accgcagtgt actattgcgc caagagcggc 300
acccctttcg actattgggg ccagggaacc ctcgtgacag tgtctagc 348
<210> 11
<211> 327
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> VL
<400> 11
accgacatcc agatgaccca gtccccctct tctctgagcg cttccgtggg cgatagggtg 60
accatcactt gcagagcctc ccagtccatc tcctcctacc tgaattggta ccagcagaag 120
ccaggcaagg cccctaagct gctgatctac tccgcttctc atctgcagag cggcgtgcct 180
tctagatttt ccggctccgg atccggcacc gatttcaccc tgaccatctc ctccctgcag 240
ccagaggact tcgccaccta ctattgccag caggccaaca cctcccctac aaccttcgga 300
cagggcacca aggtggagat caagagg 327
<210> 12
<211> 795
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> Anti CD19
<400> 12
atggagaccg acacactgct cctgtgggtc ctgctcctct gggtgccagg aagtacagga 60
ggaggaggag gatctgaggt gcagctgctg gaatcaggag gaggactggt gcagccagga 120
ggatctctga gactgtcttg cgccgcttcc ggcttcacct tctcttccta cgccatgtct 180
tgggtccgac aggctccagg aaagggactg gagtgggtgt cctctatcga cgcacagggc 240
ctgcctacca gatacgccga ttccgtgaag ggcaggttca ccatctcccg ggacaactcc 300
aagaacaccc tgtacctgca gatgaactcc ctgagggccg aggataccgc agtgtactat 360
tgcgccaaga gcggcacccc tttcgactat tggggccagg gaaccctcgt gacagtgtct 420
agcggaggag gaggatctgg aggaggagga tccggaggag gaggatctac cgacatccag 480
atgacccagt ccccctcttc tctgagcgct tccgtgggcg atagggtgac catcacttgc 540
agagcctccc agtccatctc ctcctacctg aattggtacc agcagaagcc aggcaaggcc 600
cctaagctgc tgatctactc cgcttctcat ctgcagagcg gcgtgccttc tagattttcc 660
ggctccggat ccggcaccga tttcaccctg accatctcct ccctgcagcc agaggacttc 720
gccacctact attgccagca ggccaacacc tcccctacaa ccttcggaca gggcaccaag 780
gtggagatca agagg 795
<210> 13
<211> 464
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<220>
<223> ScFV(2-27)- CD8TM-4-1BB-CD3ζ
<400> 13
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Asp Ala Gln Gly Leu Pro Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Gly Thr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
130 135 140
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
145 150 155 160
Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
165 170 175
Lys Leu Leu Ile Tyr Ser Ala Ser His Leu Gln Ser Gly Val Pro Ser
180 185 190
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
195 200 205
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn
210 215 220
Thr Ser Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
225 230 235 240
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
245 250 255
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
260 265 270
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
275 280 285
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
290 295 300
Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
305 310 315 320
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
325 330 335
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
340 345 350
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
355 360 365
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
370 375 380
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
385 390 395 400
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
405 410 415
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
420 425 430
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
435 440 445
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Trp
450 455 460
<210> 14
<211> 1467
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<223> ScFV(2-27)- CD8TM-4-1BB-CD3ζ
<400> 14
atggagaccg acacactgct cctgtgggtc ctgctcctct gggtgccagg aagtacagga 60
ggaggaggag gatctgaggt gcagctgctg gaatcaggag gaggactggt gcagccagga 120
ggatctctga gactgtcttg cgccgcttcc ggcttcacct tctcttccta cgccatgtct 180
tgggtccgac aggctccagg aaagggactg gagtgggtgt cctctatcga cgcacagggc 240
ctgcctacca gatacgccga ttccgtgaag ggcaggttca ccatctcccg ggacaactcc 300
aagaacaccc tgtacctgca gatgaactcc ctgagggccg aggataccgc agtgtactat 360
tgcgccaaga gcggcacccc tttcgactat tggggccagg gaaccctcgt gacagtgtct 420
agcggaggag gaggatctgg aggaggagga tccggaggag gaggatctac cgacatccag 480
atgacccagt ccccctcttc tctgagcgct tccgtgggcg atagggtgac catcacttgc 540
agagcctccc agtccatctc ctcctacctg aattggtacc agcagaagcc aggcaaggcc 600
cctaagctgc tgatctactc cgcttctcat ctgcagagcg gcgtgccttc tagattttcc 660
ggctccggat ccggcaccga tttcaccctg accatctcct ccctgcagcc agaggacttc 720
gccacctact attgccagca ggccaacacc tcccctacaa ccttcggaca gggcaccaag 780
gtggagatca agaggaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 840
gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 900
cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 960
tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 1020
ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1080
ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1140
agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1200
aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1260
atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1320
gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380
gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1440
cacatgcagg ccctgccccc tcgctaa 1467

Claims (18)

1. anti-CD 19 antibodies or its antigen-binding fragment, wherein the antibody includes heavy chain variable region (VH) and/or light chain variable Area (VL), wherein the heavy chain variable region includes heavy chain VHCDR1, VHCDR2, VHCDR3；The light chain variable region includes light chain VLCDR1, VLCDR2, VLCDR3, wherein the amino acid sequence of the VHCDR1 such as SEQ ID NO：Sequence shown in 1 or it is same Source sequence；The amino acid sequence of the VHCDR2 such as SEQ ID NO：Sequence or its homologous sequence shown in 2；The VHCDR3's Amino acid sequence such as SEQ ID NO：Sequence or its homologous sequence shown in 3；The amino acid sequence of the VLCDR1 such as SEQ ID NO：Sequence or its homologous sequence shown in 4；The amino acid sequence of the VLCDR2 such as SEQ ID NO：Sequence shown in 5 or its Homologous sequence；The amino acid sequence of the VLCDR3 such as SEQ ID NO：Sequence or its homologous sequence shown in 6.
2. anti-CD 19 antibodies as described in claim 1 or its antigen-binding fragment, the amino acid sequence such as SEQ of the VH chains ID NO：Sequence or its homologous sequence shown in 7；And optionally, the amino acid sequence of the VL chains such as SEQ ID NO：8 institutes The sequence shown or its homologous sequence.
3. anti-CD 33 antibody as claimed in claim 1 or 2 or its antigen-binding fragment, the homologous sequence is same with former sequence Source property is more than 60%, it is preferable that the homology is more than 90%.
4. anti-CD 33 antibody as claimed in claim 1 or 2 or its antigen-binding fragment, the VH chains and VL chains be directly connected to or It is connected by connecting peptide, it is preferable that the amino acid sequence of the anti-CD 33 antibody or its antigen-binding fragment such as SEQ ID NO：9 Shown sequence or its homologous sequence.
5. anti-CD 19 antibodies as claimed in claim 1 or 2 or its antigen-binding fragment, the anti-CD 19 antibodies or its antigen knot The affinity for closing segment and CD19 reaches 8.473 × 10-8More than M.
6. express the nucleotide sequence of the anti-CD 19 antibodies or its antigen-binding fragment any one of claim 1-5.
7. nucleotide sequence as claimed in claim 6, wherein the nucleotide sequence includes SEQ ID NO：Shown in 10 Sequence or its degenerate sequence and/or SEQ ID NO：Sequence or its degenerate sequence shown in 11, it is preferable that the nucleotides sequence Row such as SEQ ID NO：Sequence or its degenerate sequence shown in 12.
8. the homology of nucleotide sequence as claimed in claim 7, the degenerate sequence and former sequence is more than 60%, preferably Ground, the homology are more than 90%.
It is 9. any in the anti-CD 19 antibodies or its antigen-binding fragment or claim 6-8 any one of claim 1-5 Described in nucleotide expressed by anti-CD 19 antibodies or its antigen-binding fragment preparation method, including：In host cell Nucleotide any one of middle expression claim 6-8.
10.Anti CD19 CAR-NK cells, can express Chimeric antigen receptor, and the Chimeric antigen receptor will including right It asks any one of anti-CD 19 antibodies or its antigen-binding fragment and/or the claim 6-8 any one of 1-5 Anti-CD 19 antibodies prepared by anti-CD 19 antibodies or its antigen-binding fragment and/or claim 9 expressed by nucleotide or its Antigen-binding fragment.
11. Anti CD19 CAR-NK cells as claimed in claim 10, the Chimeric antigen receptor also includes transmembrane structure Domain and/or costimulatory signal conducting region；Preferably, the transmembrane domain is selected from：CD28、CD3ε、CD45、CD4、CD5、 One or more transmembrane structures in CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154 Domain；Preferably, the transmembrane domain is CD8 transmembrane domains；And/or
The costimulatory signal conducting region includes the intracellular domain of costimulatory molecules, and the costimulatory molecules is selected from： CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、 One or more in ICOS, CD154,4-1BB and OX40；Preferably, the costimulatory signal conducting region include 4-1BB and CD3 ζ intracellular domain；Preferably, the anti-CD 19 antibodies or its antigen-binding fragment can specifically bind tumour-specific Antigens c D19, and pass through transmembrane domain and costimulatory signal conducting region activates the NK cells.
12. Anti CD19 CAR-NK cells as claimed in claim 11, the Chimeric antigen receptor is that structure is scFV- The fusion protein of CD8-4-1BB-CD3 ζ, wherein the scFV can specifically bind CD19 molecules, it is preferable that described The sequence of the amino acid of SCFV-CD8-4-1BB-CD3 ζ fusion proteins such as SEQ ID NO：13 or its homologous sequence.
13. Anti CD19 CAR-NK cells as claimed in claim 12, the SCFV-CD8-4-1BB-CD3 ζ fusion proteins Coding nucleotide sequence such as SEQ ID NO：14 or its degenerate sequence.
14. the Anti CD19 CAR-NK cells as any one of claim 10-13, can effectively kill and/ Or kill Raji cells.
15. the preparation method of Anti CD19 CAR-NK cells any one of claim 10-14, including following step Suddenly：
(1) synthesis and the nucleotide of the scFV-CD8-4-1BB-CD3 ζ fusion proteins described in amplification coding claim 12 or 13, And the nucleotide is cloned on Lentiviral；
(2) the Lentiviral plasmid infection cell obtained using slow virus packaging plasmid and step (1), packaging and preparation Slow virus；
(3) the slow-virus infection NK-92 cells obtained using step (2), obtain CAR-NK cells.
16. a kind of pharmaceutical composition, it includes the anti-CD 19 antibodies according to any one of claim 1-5 or its antigen knots Close segment and/or the encoded anti-CD 33 antibody of nucleotide sequence any one of claim 6-8 or its antigen binding Appoint in anti-CD 19 antibodies or its antigen-binding fragment and/or claim 10-14 prepared by segment and/or claim 9 The Anti CD19 CAR-NK cells prepared by Anti CD19 CAR-NK cells and/or claim 15 described in one.
17. pharmaceutical composition as claimed in claim 16 further includes pharmaceutically acceptable auxiliary material.
18. in the anti-CD 19 antibodies or its antigen-binding fragment and/or claim 6-8 any one of claim 1-5 Prepared by the encoded anti-CD 33 antibody of any one of them nucleotide sequence or its antigen-binding fragment and/or claim 9 Anti-CD 19 antibodies or its antigen-binding fragment and/or the Anti CD19 CAR-NK any one of claim 10-14 Anti CD19 CAR-NK cells prepared by cell and/or claim 15 are preparing treatment and/or the high expression CD19 of prevention Tumour and relevant disease drug in purposes；Preferably, the tumour of the high expression CD19 is lymthoma.
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CN112029729A (en) * 2020-09-09 2020-12-04 广东昭泰体内生物医药科技有限公司 CD19 and CD22 double-target chimeric antigen receptor NK cell and application thereof
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CN112195157B (en) * 2020-10-12 2023-03-31 汤朝阳 CD19 and CD22 double-target chimeric antigen receptor T cell and application thereof
CN112195157A (en) * 2020-10-12 2021-01-08 广东昭泰体内生物医药科技有限公司 CD19 and CD22 double-target chimeric antigen receptor T cell and application thereof
CN113461830B (en) * 2021-07-22 2022-04-26 徐州医科大学 Umbilical cord blood-derived CD 19-targeted CAR-NK cell and preparation method thereof
CN113461830A (en) * 2021-07-22 2021-10-01 徐州医科大学 Umbilical cord blood-derived CD 19-targeted CAR-NK cell and preparation method thereof

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