CN108484777A

CN108484777A - The Chimeric antigen receptor construct and nucleic acid molecules of humanization RP215 monoclonal antibodies and application

Info

Publication number: CN108484777A
Application number: CN201810275467.5A
Authority: CN
Inventors: 李吉祐
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-03-31
Filing date: 2018-03-30
Publication date: 2018-09-04
Anticipated expiration: 2038-03-30
Also published as: CN108484777B

Abstract

The present invention provides the Chimeric antigen receptor construct of humanization RP215 monoclonal antibodies and nucleic acid molecules and applications, the construct has nucleotide sequence shown in sequence table 7, the nucleic acid includes the construct, can be applied to immunocyte, has significant application value for the treatment of tumour.

Description

The Chimeric antigen receptor construct and nucleic acid molecules of humanization RP215 monoclonal antibodies With application

Technical field

The present invention relates to biotechnology, especially a kind of Chimeric antigen receptor of humanization RP215 monoclonal antibodies Construct and nucleic acid molecules and application.

Background technology

RP215 is the monoclonal antibody developed in 1987, then shows that RP215 can be expressed with most of human cancer cell Immunoglobulin on the relevant antigenic determinant of carbohydrate (CA215) react (these cancer cells include come From in ovary, cervix, lung and liver), however, it is special not to be found RP215 in the normal immunoglobulin that B cell generates The antigenic determinant of property.Evidence show the RP215 of mouse source or humanization form can induce withering for nearly all cancer cell It dies and complement-dependent cytotoxicity.Therefore the various cancers of the mankind are directed to, RP215 is anti-as high degree of specificity monoclonal Body has treatment potentiality.

The U.S. Patent number 8974786 for authorizing Lee discloses the nucleotide and ammonia of mouse source RP215 and humanization RP215 Base acid sequence.Biochemistry and immunological experiment prove mouse sourceization and the RP215 of humanization to the CA215 on cancer cell surfaces (mainly cancerous immune globulin) has high degree of specificity and affinity.RP215 (including its scFv of mouse sourceization and humanization Segment) apoptosis and complement-dependent cytotoxicity that cancer cell can be induced, the cytotoxicity for eventually leading to cancer cell is dead.

As disclosed in Lee in United States Patent (USP) 8974786, humanization RP215 monoclonal antibodies are in identification cancer cell table There is bioequivalence with mouse source RP215 when antigenic determinant relevant with carbohydrate on the CA215 of face expression.

Chimeric antigen receptor (CAR)-T cell therapy techniques combine T cell immunotherapy, gene therapy and immune treatment Method.CAR-T cell therapies have been used for treatment of cancer.CAR-T technologies include modifying the T cell of patient, the T cell table after modification It, can be with the surface antigen on tumor cell up to Chimeric antigen receptor (CARs).It is obtained using the trial of CAR-T treating cancers Some successes.Go to treat different types of hematologic cancers by using the relevant Chimeric antigen receptor platforms of CD19 be exactly Successful CAR-T cell therapy examples.United States Patent (USP) 8916381 discloses a kind of method for treating leukaemia with CAR-T technologies. Brentjens et al. treated with CAR-T the clinical test of chronic lymphocytic leukemia, the T cell energy of modified Enough identify the CA19 expressed in most of B cell malignant tumours.

Nevertheless, these selectively targetings and kill cancer cell construct and method still it is in need improvedly Side.

Invention content

Technical problem to be solved by the present invention lies in provide a kind of chimeric antigen of humanization RP215 monoclonal antibodies by Body construct.

Another technical problem to be solved by this invention is to provide the nucleic acid molecules with above-mentioned construct.

Another technical problem to be solved by this invention is to provide the application of above-mentioned nucleic acid molecules.

In order to solve the above technical problems, the technical scheme is that：

Chimeric antigen receptor (CAR) construct of humanization RP215 monoclonal antibodies, signal peptide (Signal Peptide)-hRP215scFv-CD8-4-1BB-CD3Z-2A-IL7, nucleotides sequence are classified as sequence described in Fig. 2 D, have sequence Nucleotide sequence shown in table 7.

Preferably, Chimeric antigen receptor (CAR) construct of above-mentioned humanization RP215 monoclonal antibodies, amino acid are Sequence described in Fig. 2 E, amino acid sequence shown in sequence table 8；

After above-mentioned Chimeric antigen receptor construct is transfected into T cell, which can express humanization ScFv (single variable domain) segment of RP215, by this scFV and the humanization for carrying out Validation in vitro by cell culture RP215 compares, they have similar affinity and specificity with the CA215 expressed on cancer cell surfaces.

A kind of nucleic acid molecules of separation, it includes in vitro or in vivo, immunocyte transfect and express people source Change RP215 monoclonal antibodies or the construct of its antigen-binding fragment；This construct includes encoding humanized RP215 monoclonals The nucleotide sequence of antibody or its segment.

Preferably, the nucleic acid molecules of above-mentioned separation, the humanization RP215 monoclonal antibodies or its segment contain tool sequence The heavy chain of amino acid sequence shown in table 1.

Preferably, the heavy chain of the nucleic acid molecules of above-mentioned separation, the encoding humanized RP215 monoclonal antibodies or its segment Nucleotide sequence tool sequence table 2 shown in nucleotide sequence.

Preferably, the nucleic acid molecules of above-mentioned separation, the humanization RP215 monoclonal antibodies or its segment contain tool sequence The light chain of amino acid sequence shown in table 3.

Preferably, the core of the light chain of the nucleic acid molecules of above-mentioned separation, encoding humanized RP215 monoclonal antibodies or its segment Nucleotide sequence has nucleotide sequence shown in sequence table 4.

Preferably, the nucleic acid molecules of above-mentioned separation, the humanization RP215 include amino acid sequence shown in polynucleotide 5 The heavy chain variable region of row.

Preferably, the nucleic acid molecules of above-mentioned separation, the humanization RP215 include amino acid sequence shown in polynucleotide 6 The light chain variable region of row.

Preferably, the nucleic acid molecules of above-mentioned separation, the humanization RP215 monoclonal antibodies or its antigen-binding fragment with Compatibility and the specificity that CA215 is combined are substantially equivalent to the humanization RP215 disclosed in U.S. Patent No. 8974786 Monoclonal antibody or its segment.

Preferably, the nucleic acid molecules of above-mentioned separation, including nucleotide sequence shown in the sequence table 9 of encoded signal structural domain, For guiding expressed protein to cell membrane, wherein signal domain signal sequence containing interleukin 2.

Preferably, the nucleic acid molecules of above-mentioned separation, including nucleosides shown in the sequence table 10 of coding hinge and transmembrane domain Acid sequence, the extracellular humanization RP215 monoclonal antibodies or its antigen-binding fragment for connecting expressed protein and institute The intracellular costimulation structural domain and signal domain of protein are expressed, wherein hinge and transmembrane domain can contain the hinge of CD8 And transmembrane domain.

Preferably, the nucleic acid molecules of above-mentioned separation, including nucleotides sequence shown in the sequence table 11 of coding costimulation structural domain Row, for costimulation T cell to improve the internal persistence of Chimeric antigen receptor construct, wherein costimulation structural domain can contain 4- The costimulation structural domain of 1BB.

Preferably, the nucleic acid molecules of above-mentioned separation, including nucleotide sequence shown in the sequence table 12 of encoded signal structural domain, For signal transduction in T cell, wherein signal domain can contain the ζ subunits (TCR) of CD3 molecules.

Preferably, the nucleic acid molecules of above-mentioned separation, including nucleotide sequence shown in the sequence table 13 of coding self cleavage peptide, Middle self cleavage peptide can contain 2A self cleavage peptides.

Preferably, the nucleic acid molecules of above-mentioned separation, including nucleotide shown in the sequence table 14 in Codocyte factor structure domain Sequence, wherein cytokine domain can contain IL7 structural domains.

Preferably, the nucleic acid molecules of above-mentioned separation have universal architecture shown in Fig. 3.

Preferably, the nucleic acid molecules of above-mentioned separation, including nucleotide sequence shown in sequence table 7.

Preferably, the nucleic acid molecules of above-mentioned separation, the polypeptide of amino acid sequence shown in coding tool sequence table 8.

Preferably, the nucleic acid molecules of above-mentioned separation, encoded polypeptide have nucleotide sequence institute table shown in sequence table 7 The protein amino acid sequence reached.

Preferably, the nucleic acid molecules of above-mentioned separation, it includes the nucleotide sequence of coding polypeptide, the polypeptide has：

Signal domain；

Transmembrane domain；

CD3-zeta signal domains；

Cytokine domain；

Wherein as many as described peptide can contain from the ends N- to C-terminal signal domain, humanization RP215 monoclonal antibodies or its Segment, transmembrane domain, CD3-zeta signal domains and cytokine domain；

The wherein described cytokine domain can contain IL-7 or its segment.

Preferably, the nucleic acid molecules of above-mentioned separation, signal domain include signal peptide amino acid sequence shown in Fig. 2 E, With amino acid sequence shown in sequence table 15.

Preferably, the nucleic acid molecules of above-mentioned separation, the transmembrane domain include CD8, have amino acid shown in Fig. 2 E Sequence has amino acid sequence shown in sequence table 16.

Preferably, the nucleic acid molecules of above-mentioned separation also include self cleavage peptide 2A, with self cleavage peptide 2A shown in Fig. 2 E Amino acid sequence has amino acid sequence shown in sequence table 17.

Preferably, the nucleic acid molecules of above-mentioned separation also include costimulatory signal structural domain 4-1BB, have shown in Fig. 2 E Amino acid sequence has amino acid sequence shown in sequence table 18.

Preferably, the nucleic acid molecules of above-mentioned separation, the cytokine domain include cell factor knot shown in Fig. 2 E The amino acid sequence of structure domain IL7 has amino acid sequence shown in sequence table 19.

A kind of carrier for transfection and expression humanization RP215 monoclonal antibodies or its segment in immunocyte, this Carrier includes any one aforementioned nucleic acid molecules.

Preferably, above-mentioned carrier also includes plasmid.

Preferably, above-mentioned carrier, including Chimeric antigen receptor construct.

A kind of slow virus carrier being suitable for being transfected into T cell or natural killer cells, has any one aforementioned nucleic acid point Son.

Preferably, above-mentioned slow virus carrier, including nucleotide sequence shown in sequence table 7.

Preferably, above-mentioned slow virus carrier encodes a kind of polypeptide, has amino acid sequence shown in sequence table 8.

A kind of immunocyte, including T cell or natural killer cells, including defined in any one preceding claims Nucleic acid molecules, carrier or slow virus carrier.

Preferably, above-mentioned immunocyte, including T cell or natural killer cells, nucleic acid molecule have passed through inosculating antibody Short palindrome repetitive sequence (CRISPR) the gene editing technology of original receptor T cell immunotherapy or the regular intervals of cluster is embedded in In immunocyte, T cell or natural kill immunocyte.

Using above-mentioned immunocyte (including T cell or natural killer cells), preparing cancer, (including to be related to CA215 immune Any illness of globulin height expression) application in terms of medicine.

Preferably, the application of above-mentioned immunocyte, CA215 is carcinous exempts from the expression of cancer cell surfaces height for cancer treatment drugs Epidemic disease globulin.

Preferably, the application of above-mentioned immunocyte, the medicine are utilized by institute in aforementioned any one claim Express humanization in vitro or in vivo in the immunocyte surface that nucleic acid molecules, carrier or the slow virus carrier of definition are transfected RP215 monoclonal antibodies or its antigen-binding fragment, wherein immunocyte can contain T cell or natural killer cells.

Preferably, the application of above-mentioned immunocyte, humanization RP215 monoclonal antibodies or its antigen-binding fragment are immune Expression on cell surface can lead to the selective killing to cancer cell, can be by cancer cell specific induction of apoptosis or cytotoxicity Killing.

Preferably, the application of above-mentioned immunocyte, the cancer include breast cancer, oophoroma, carcinoma of endometrium, cervix Cancer, cancer of pancreas, colon cancer, lung cancer, liver cancer or kidney.

Preferably, the application of above-mentioned immunocyte further includes the immunocyte that will be transfected, T cell or natural killer cells It separates, and by cell storage in freezing state, can be long-term storage；The frozen cell can may be sent out in future It thaws for unique individual in raw spontaneous cancer, and carries out emergency treatment.

Above-mentioned T cell or natural killer cells include mammalian cell, can also be human cell；Patient includes lactation Animal can also be the mankind.

The method for expressing associated disease with CA215 high using said medicine treatment, the method includes modification T cells with table Up to Chimeric antigen receptor, wherein Chimeric antigen receptor includes

Signal domain；

Transmembrane domain；

CD3-zeta signal domains；

Antigen-binding domains, wherein this antigen-binding domains include a kind of polypeptide, the affinity combined with CA215 Have substantially quite with RP215；

With the human T cells for giving modified.

The beneficial effects of the invention are as follows：

The present invention provides the Chimeric antigen receptor construct of humanization RP215 monoclonal antibodies and nucleic acid molecules, can answer For immunocyte, there is significant application value for the treatment of tumour.

Description of the drawings

Figure 1A -1D show the humanization RP215 genes derived from antibody stabilization cell strain (UY463-RP215) (SEQIDNOs.:Nucleotide 1-4) and amino acid sequence.In order to obtain stable cell strain DNA vector, the weight of RP215 antibody Chain and light chain are cloned into the exclusive all LakePharma carriers designed exclusively for production stable cell line.By plasmid During DNA amplifications transfect, and the DNA library for being stored in -20 DEG C is deposited；A part of this DNA is used in exploitation stable cell line.

Fig. 2A shows the structure signal of humanization RP215 genes (hRP215BBZ7) Chimeric antigen receptor slow virus carrier Figure.The heavy chain and light chain of RP215 antibody are cloned into the exclusive proprietary LakePharma designed for production stable cell line In carrier.Plasmid DNA is amplified and is transfected, and during the DNA library for being stored in -20 DEG C is deposited；A part of this DNA is used in exploitation and stablizes Cell strain.Fig. 2 B and 2C respectively illustrate the protein sequence (sequence table 5 and 6) of VH and VL.According to the frame of foundation, RP215 Chimeric antigen receptor expression cassette construct overall length is synthesized, and is then subcloned into lentio-Puro carriers.This embedded chip Section is proved that Fig. 2 D show the nucleotide sequence (sequence of RP215 Chimeric antigen receptor expression cassette constructs by Sanger sequencings List 7).Fig. 2 E show the protein sequence of RP215 Chimeric antigen receptor expression cassette constructs (sequence table 8).

Fig. 3 is shown in the RP215 Chimeric antigen receptors in lentivirus construct and is expressed with open RP215 Chimeric antigen receptors The gene order of frame construction body (RP215-scFv-4-1BB-CD3Z-2A-IL7).In this example, RP215 Chimeric antigen receptors The gene order of expression cassette construct exists in the form of plasmid, this contains insert plasmid and is referred to as transferring plasmid, it will It is used to transfect 293T cells to generate virus with together with three kinds of plasmids of the coding other different pieces of slow virus.Wherein,

(1) abbreviation of Chimeric antigen receptor component

EF-1 α promoters:Promoter for starting the expression of T cell Chimeric antigen receptor.

IL2ss:Interleukin 2 signal sequence, for guiding the Chimeric antigen receptor on cell membrane to express.

RP215scFv:The single variable domain of RP215 monoclonal antibodies.ScFv is by heavy-chain variable domains and light The single-stranded peptide that chain variable domains are formed.This is the core of Chimeric antigen receptor structure, it assigns the spy of Chimeric antigen receptor It is anisotropic.

CD8hinge&TM:The hinge and transmembrane domain of CD8 molecules, for connecting extracellular scFv and intracellular thorn altogether Swash structural domain and signal domain.

4-1BB costimulation structural domains:One structural domain of 4-1BB molecules, for co-stimulatory T cell to improve in vivo The persistence of CAR-T.

CD3zeta:The ζ subunits of CD3 (TCR) molecule, the signal transduction being responsible in T cell.

2A:One short self cleavage peptide sequence makes two kinds of protein of an identical rna expression.After translation, contain The long peptide of two kinds of protein carries out Self cleavage in 2A sequence areas.

IL7:It is a kind of that the cell factor having a major impact is developed to T cell.

(2) abbreviation of construct other components

WPRE:Controlling element after groundhog hepatitis virus transcription.By increasing neclear export to stimulate transgene expression Sequence.

3’LTR:By adding polyadenylic acid after R sequences to terminate the transcription since 5'LTR.

RSV:Constitutive promoter.

5’LTR:There is identical function with RNApolII promoters.According to definition, transcript is capped since R, and is passed through U5 and provirus are elsewhere.

Gag:Containing matrix, the structural protein precursor of the lentiviral particle of capsid and nucleocapsid.

RRE:Rev response elements；Its sequence and Rev protein bindings.

env:VSV-G envelope proteins.

cppt:Polypurine tract center；The recognition site of proviral DNA synthesis, can be improved transduction efficiency and transgene expression.

Fig. 4 shows the hRP215BBZ constructs verified by endonuclease digestion.

Fig. 5 A and 5B show the qPCR standard curves for being titrated by slow virus and showing titre.Fig. 5 A show the standard of WPRE Curve, Fig. 5 B show the standard curve of ALB.

Fig. 6 A and 6B show the standard curve determination copy number formulated using Ct values, and then verify Chimeric antigen receptor Expression.Fig. 6 A show that the standard curve of ALB, Fig. 6 B show the standard curve of LTR.

Fig. 7 A-C show the cracking that target tumour cell is measured with LDH measuring methods, to the CAR-T (BID4G- of verification CAR-IL15=RP215-CAR-T；BIG4G-CAR-IL15=GHR106-CAR-T) feasibility.LDH measurement is repeated three times, As a result Fig. 7 A-C are shown in.

Fig. 8 A-C are shown measures falling off for cell factor with ELISA measuring methods, to verify CAR-T constructs (BID4G-CAR-IL15=RP215-CAR-T；BIG4G-CAR-IL15=GHR106-CAR-T feasibility).Fig. 8 D-F are shown Falling off for cell factor is measured with ELISA measuring methods, to the CAR-T constructs (BID4G-CAR-IL15=of verification RP215-CAR-T；BIG4G-CAR-IL15=GHR106-CAR-T feasibility).

Specific implementation mode

Technical solution of the present invention is further described with reference to specific embodiment.

The present invention relates to the fields of the slow virus Chimeric antigen receptor construct with humanization RP215 genes.Humanization RP215 monoclonal antibodies and mouse source RP115 have bioequivalence, can specifically recognition expression in cancer cell surfaces On CA215 with the relevant antigenic determinant of carbohydrate.

It is embedding when being transfected into the Chimeric antigen receptor construct in the T cell detached from individual patient or NK cells The scFv chains of humanization RP215 can be expressed in vitro or in vivo by closing the immunocyte of antigen receptor transfection.RP215 genes or its piece The expression of section can lead to the cytotoxic killer of tumour cell.

It can be utilized with the relevant Chimeric antigen receptors of RP215-application of the T cell technology in cancer immunotherapy chimeric T cell that antigen receptor transfected is realized.The T cell transfected can express the scFv of RP215, and with cancer cell surfaces CA215 reacts, and then inducing cell apoptosis and complement-dependent cytotoxicity and other cytotoxic effects are thin to kill cancer Born of the same parents.

The relevant Chimeric antigen receptor constructs of RP215 can be transfected to the T cell separated from individual patient.These come It can be expanded from particular patient, the T cell of modified by vitro culture, then defeated return to identical patient to carry out cancer Treatment.After the effect of this treatment of cancer carried out with the relevant Chimeric antigen receptor constructs of RP215, can be by diagnosis and treatment Tumor growth inhibition confirm or clinical observation.

There are expression carcinoma cell immunization globulin or CA215 in view of nearly all cancer cell surfaces, it is contemplated that RP215 phases The Chimeric antigen receptor construct of pass can be widely used in the treatment with the high CA215 all human cancers expressed On.

Some embodiments are related to and relevant Chimeric antigen receptor T cell technology the answering in cancer immunotherapy of RP215 With.In some embodiments, with the application of the relevant Chimeric antigen receptor T cell technologies of RP215 can by chimeric antigen by T cell that body transfected is realized.The T cell transfected can express the scFv of RP215, and with the CA215 of cancer cell surfaces Reaction, and then inducing cell apoptosis and complement-dependent cytotoxicity and other cytotoxic effects kill cancer cell.

In some embodiments, it is transfected into from individual patient and detaches with the relevant Chimeric antigen receptor constructs of RP215 T cell out.These can be expanded from particular patient, the T cell of modified by vitro culture, then it is defeated return to it is identical Patient to carry out treatment of cancer.The treatment of this treatment of cancer carried out with the relevant Chimeric antigen receptor constructs of RP215 Effect can be confirmed by the Tumor growth inhibition after diagnosis and treatment or clinical observation.

In other examples, other gene therapies other than Chimeric antigen receptor T cell technology can be used, make to exempt from Epidemic disease cell is expressed RP215 monoclonal antibodies or its antigen-binding fragment and then is combined with CA215 on immunocyte surface.For example, In some embodiments, suitable carrier can be embedded in by short palindrome repetitive sequence (CRISPR) technology of the regular intervals of cluster In immunocyte, make immunocyte expression RP215 monoclonal antibodies or its antigen-binding fragment, so immunocyte surface with CA215 is combined.In some embodiments, available immunocyte can be T cell or natural kill (NK) cell.

Lee discloses the nucleotide sequence of humanization RP215 genes in United States Patent (USP) 8974786, and passes through repetition Sequencing and analysis of molecules demonstrate the sequence.The stable cell line for producing this antibody has been established and openly (see Fig. 2A -2E), institute The antibody sequence mouse sourceization that bioequivalence is studied compared with being previously used for and humanization RP215 of the stable cell line production of foundation Sequence have been found to be identical.Chimeric antigen receptor overall length can be synthesized according to established frame, then be subcloned into In lentio-Puro carriers.This insertion segment is proved by Sanger sequencings.

Embodiment 1

Final RP215 Chimeric antigen receptor expression cassette constructs

Fig. 3 shows coding RP215 Chimeric antigen receptor expression cassette constructs (RP215-scFv-4-1BB-CD3Z-2A- IL7 gene order).In this example, the gene order of RP215 Chimeric antigen receptors expression cassette construct is in the form of plasmid In the presence of this plasmid contains insert, is referred to as transferring plasmid.It by with coding the other different pieces of slow virus three kinds of plasmids one It rises for transfecting 293T cells to generate virus

The final nucleotide sequence of hRP215 genes is that the antibody produced by stable cell line derives and goes out.Such as Figure 1A- Shown in 1D, the hRP215 genes (U463 and Y463, the sequence table that are generated by CHO stable cell lines：4) nucleotide sequence with pass through The nucleotide sequence for the RP215 genes that transient expression generates is consistent.ScFv chains are by the list in U463 and Y463 variable domains Chain is linked by known technology and is formed thoroughly.HRP215BBZ constructs are to be verified by endonuclease digestion, and be shown in figure In 4.

HRP215-4-1BB-CD3Z-2A-1L7 (hRP215BBZ7) is standby by a large amount of extracts kits of Qiagen plasmids System.RP215 Chimeric antigen receptor constructs are verified by a series of external activity test, this includes that (1) is embedding Antigen receptor expression test is closed to confirm the expression of Chimeric antigen receptor, (2) Chimeric antigen receptor binding assay is chimeric to confirm Antigen receptor can be combined with the CA215 on immunocyte surface, and (3) are killed with the cell that the T cell that Chimeric antigen receptor transfects carries out Wound is measured to confirm that the cell killing function of the T cell of Chimeric antigen receptor transfection is complete, and (4) cytotoxicity test is to confirm The cytotoxicity function of the T cell of Chimeric antigen receptor transfection is complete, and (5) Apoptosis is tested to confirm Chimeric antigen receptor The Apoptosis function of the T cell of transfection is complete, and (6) degranulation tests the T cell to confirm Chimeric antigen receptor transfection Degranulation function is complete, and the test of (7) cytokine release is to confirm that the cell factor of the T cell of Chimeric antigen receptor transfection is released Complete and (8) proliferation test of playing function is to confirm the quantity of the T cell of Chimeric antigen receptor transfection.

Validation test result meets the expected results of hRP215-4-1BB-CD3Z-1L7 Chimeric antigen receptor systems.It is described The validity of Chimeric antigen receptor expression cassette has been identified.

Embodiment 2

RP215 Chimeric antigen receptors are verified by the test of cytotoxic killer cancer cell

After successfully structure RP215 Chimeric antigen receptors lenti-B1D4G-CAR-IL15, it is necessary to carry out slow virus drop Step is determined to verify slow virus carrier.RP215-scRv-4-1BB-CD3Z-2A-IL7 is known as in slow virus titration test lenti-B1D4G-CAR-IL15.Slow virus is to build to complete via the following steps：Polyethyleneimine and slow virus are carried first After DNA needed for body is sufficiently mixed, then cell culture is added in DNA/polyethyleneimine amine compound In ware.The culture medium containing lentiviral particle is detached later and is stored in -80 DEG C.Slow virus titration results confirm lenti- The success of B1D4G-CAR-IL15 structures, as shown in Figure 5 A and 5B, qPCR standard curves show high-titer.This slow virus titrates Result be also summarised in table 1.

According to the standardization program of the separation of the primary T cells provided from donor, the copy number of Chimeric antigen receptor expression Verification is determined by making the standard curve of ALB and LTR.As a result Fig. 6 A, Fig. 6 B and table 2 are shown in.Chimeric antigen receptor T cell In average Chimeric antigen receptor gene copy number be measured as 3.6/ cell.These qPCR are the results show that the B1D4G- built The gene of CAR-IL15 is successfully transfected into T cell.The specific Chimeric antigen receptor expression of these result verifications.

Table 1

Table 2

Embodiment 3

Cracking of the Chimeric antigen receptor T cell to target tumour cell

Using ATCC cervical cancer cell lines, C33A tumour cells are as target tumour cell, in standard cell culture conditions Under with three kinds of different E/T ratios and Chimeric antigen receptor T cell co-incubation.In C33A tumour cells and Chimeric antigen receptor After T cell co-incubation 8 hours, supernatant is collected to measure the amount of LDH reductions.This tests in triplicate and is compared (such as Shown in Fig. 7 A-C).The result of cracking test proves that Chimeric antigen receptor T cell can kill target tumour cell, and it is killed Efficiency has very strong association with dosage.Compared with the T cell (lenti-B1D4G-CAR-IL15T cells) of transfection, the T of untransfected Cell also shows that the cell cracking effect of low degree.Without being bound by theory, this high background may be because of measurement system Middle AntiCD3 McAb and anti-CD28 antibody have activated the T cell of untransfected.Chimeric antigen receptor T cell cell caused by tumour cell Cracking effect obviously has statistical significance (P<0.05~0.001).

Embodiment 4

Chimeric antigen receptor T cell co-cultures the release of verification cell factor with tumour cell

For Chimeric antigen receptor T cell with tumour cell C33A co-incubations after 8 hours, the secretion of different cytokines is logical Typical enzyme immunoassay (EIA) is crossed to quantitative determine, and is repeated 3 times, these cell factors include IL2, IL7 and IFN-Y. Fig. 8 A-F show and compare the enzyme immunoassay (EIA) result of cytokine release.Cytokine release measurement result shows and tumour The Chimeric antigen receptor T cell of cell co-culture discharges more cell factors than the T cell of untransfected.

It draws a conclusion from these examples：(T transfected with lenti-B1D4G-CAR-IL15 is thin for Chimeric antigen receptor T cell Born of the same parents) cytotoxic killer of the tumour cell co-cultured in vitro can be effectively resulted in, and eventually lead to and show in vitro or in vivo The anticancer function of work.1) slow virus titration the result shows that, the present inventor is successfully prepared the slow virus carrier of high titre.2) QPCR results prove that slow virus carrier is transduceed into T cell.3) cracking measurement result proves that Chimeric antigen receptor T cell can kill Dead target cell, and its effect is relevant with dosage.The T cell that do not transduce also shows that cytolysis, this may be by resisting CD3 and anti-CD28 antibody activate the T cell that do not transduce and generate.4) cytokine release measurement result proves, chimeric antigen The cell factor secreted after recipient T cells and target cell co-incubation is more than the T cell that do not transduce.

Above-mentioned reference embodiment is to a kind of the Chimeric antigen receptor construct and nucleic acid of humanization RP215 monoclonal antibodies The detailed description that molecule is carried out with application is illustrative without being restrictive, and can be enumerated according to limited range several A embodiment, therefore the change and modification in the case where not departing from present general inventive concept, should belong within protection scope of the present invention.

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tccgtgaagg tgagctgcaa ggcatcaggc tacaccttca ctgattattg gatgcactgg 180

gtgagacagg cacccggtca ggggctcgaa tggatgggcg ccatcgatac tagcgattcc 240

tataccagat acgcacagaa gtttcaggga agagttacca tgactgtcga tgaatctaca 300

agcaccgtct acatggagct gagcagcctg cggtctgagg acaccgctgt ttactactgt 360

gcccgctcca tctatgattg gggtcaagga accttggtca cagtgagttc tgctagcacc 420

aagggcccca gcgtgttccc tctggccccc agcagcaaga gcaccagcgg cggaaccgcc 480

gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgtc ctggaacagc 540

ggcgctctga ccagcggagt gcacaccttc cctgccgtgc tgcagagcag cggcctgtac 600

tccctgagca gcgtggtgac cgtgcccagc agcagcctgg gcacccagac ctacatctgc 660

aacgtgaacc acaagccctc caacaccaag gtggacaaga aggtggagcc taagagctgc 720

gacaagaccc acacctgccc tccctgcccc gcccccgagc tgctgggcgg acccagcgtg 780

ttcctgttcc ctcccaagcc caaggacacc ctgatgatca gccgcacccc cgaggtgacc 840

tgcgtggtgg tggacgtgag ccacgaggac cccgaggtga agttcaactg gtacgtggac 900

ggcgtggagg tgcacaacgc caagaccaag cctcgggagg agcagtacaa ctccacctac 960

cgcgtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 1020

tgcaaggtga gcaacaaggc cctgcccgct cccatcgaga agaccatcag caaggccaag 1080

ggccagcccc gggagcctca ggtgtacacc ctgcccccca gccgcgacga gctgaccaag 1140

aaccaggtga gcctgacctg cctggtgaag ggcttctacc cctccgacat cgccgtggag 1200

tgggagagca acggccagcc tgagaacaac tacaagacca cccctcccgt gctggacagc 1260

gacggcagct tcttcctgta cagcaagctg accgtggaca agtcccggtg gcagcagggc 1320

aacgtgttca gctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 1380

ctgagcctga gccccggata g 1401

<210> 3

<211> 240

<212> PRT

<213>RP215 light-chain amino acid sequences (Synthetic Construct)

<220>

<221> CHAIN

<222> (1)..(240)

<400> 3

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Asn Ser Ser Asn Gln Lys Ser Tyr Leu Ala Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Pro Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg

65 70 75 80

Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr

100 105 110

Phe Cys Gln Gln His Tyr Ser Thr Pro Ser Thr Phe Gly Gly Gly Thr

115 120 125

Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

130 135 140

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

145 150 155 160

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

165 170 175

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

180 185 190

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

195 200 205

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

210 215 220

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235 240

<210> 4

<211> 723

<212> DNA

<213>RP215 light chain nucleotide sequences (Synthetic Construct)

<220>

<221> protein_bind

<222> (1)..(723)

<400> 4

atggagaccg acaccctgct gctctgggtg ctgctgctct gggtgcccgg ctccaccgga 60

gatatcgtga tgacccagtc ccccgacagc ctggccgtct ctctgggtga gcgggcaacc 120

atcaactgta agtctagcca gtccctgttg aacagtagca atcaaaagag ctatcttgcc 180

tggtatcagc aaaagcctgg ccagccacca aaactgcttg tctatttcgc ttccactcgg 240

gaaagcggtg taccagaccg cttttctggc tcaggttccg gcacagactt taccttgacc 300

attagctccc ttcaggcaga ggacgtggca gtctactttt gccagcaaca ctactccact 360

ccatcaacct ttggaggtgg cacaaaactg gagattaagc ggaccgtggc cgcccccagc 420

gtgttcatct tccctcccag cgacgagcag ctgaagtctg gcaccgccag cgtggtgtgc 480

ctgctgaaca acttctaccc ccgcgaggcc aaggtgcagt ggaaggtgga caacgccctg 540

cagagcggca acagccagga gagcgtgacc gagcaggact ccaaggacag cacctacagc 600

ctgagcagca ccctgaccct gagcaaggcc gactacgaga agcacaaggt gtacgcctgc 660

gaggtgaccc accagggact gtctagcccc gtgaccaaga gcttcaaccg gggcgagtgc 720

taa 723

<210> 5

<211> 113

<212> PRT

<213>RP215 heavy chain amino acid sequences (Synthetic Construct)

<220>

<221> CHAIN

<222> (1)..(113)

<400> 5

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ala Ile Asp Thr Ser Asp Ser Tyr Thr Arg Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Glu Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ile Tyr Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser

<210> 6

<211> 113

<212> PRT

<213>RP215 light-chain amino acid sequences (Synthetic Construct)

<220>

<221> CHAIN

<222> (1)..(113)

<400> 6

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Ser Asn Gln Lys Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

His Tyr Ser Thr Pro Ser Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 7

<211> 2067

<212> DNA

<213>RP215 Chimeric antigen receptor constructs nucleotide sequence (Synthetic Construct)

<220>

<221> protein_bind

<222> (1)..(2067)

<400> 7

gaattcgccg ccaccatggc cctccctgtc accgccctgc tgcttccgct ggctcttctg 60

ctccacgccg ctcggcccca ggttcagctg gttcagtctg gcgccgaagt gaagaaacct 120

ggcgcctctg tgaaggtgtc ctgcaaggcc agcggctaca cctttaccga ctactggatg 180

cactgggtcc gacaggctcc aggacaaggc ttggaatgga tgggcgccat cgacaccagc 240

gacagctaca caagatacgc ccagaaattc cagggcagag tgaccatgac cgtggacgag 300

agcaccagca ccgtgtacat ggaactgagc agcctgagaa gcgaggacac cgccgtgtac 360

tactgcgcca gatccatcta cgattggggc cagggcacac tggtcacagt ttctagcgga 420

ggcggaggtt ctggtggcgg aggaagtggc ggcggaggat ctgatatcgt gatgacacag 480

agccccgaca gcctggccgt gtctcttgga gaaagagcca ccatcaactg caagagcagc 540

cagagcctgc tgaacagcag caaccagaag tcctacctgg cctggtatca gcagaagcct 600

ggccagcctc ctaagctgct ggtgtacttt gccagcacca gagaaagcgg cgtgcccgat 660

agattttctg gctctggcag cggcaccgac ttcaccctga caatttctag cctgcaagcc 720

gaggacgtgg ccgtgtattt ctgccagcag cactacagca cccctagcac atttggcgga 780

ggcaccaagc tggaaatcaa gaccactacc ccagcaccga ggccacccac cccggctcct 840

accatcgcct cccagcctct gtccctgcgt ccggaggcat gtagacccgc agctggtggg 900

gccgtgcata cccggggtct tgacttcgcc tgcgatatct acatttgggc ccctctggct 960

ggtacttgcg gggtcctgct gctttcactc gtgatcactc tttactgtaa gcgcggtcgg 1020

aagaagctgc tgtacatctt taagcaaccc ttcatgaggc ctgtgcagac tactcaagag 1080

gaggacggct gttcatgccg gttcccagag gaggaggaag gcggctgcga actgcgcgtg 1140

aaattcagcc gcagcgcaga tgctccagcc tacaagcagg ggcagaacca gctctacaac 1200

gaactcaatc ttggtcggag agaggagtac gacgtgctgg acaagcggag aggacgggac 1260

ccagaaatgg gcgggaagcc gcgcagaaag aatccccaag agggcctgta caacgagctc 1320

caaaaggata agatggcaga agcctatagc gagattggta tgaaagggga acgcagaaga 1380

ggcaaaggcc acgacggact gtaccaggga ctcagcaccg ccaccaagga cacctatgac 1440

gctcttcaca tgcaggccct gccgcctcgg gagggcagag gcagcctgct gacatgtggc 1500

gacgtggaag agaaccctgg ccccatgttc catgtttctt ttaggtatat ctttggactt 1560

cctcccctga tccttgttct gttgccagta gcatcatctg attgtgatat tgaaggtaaa 1620

gatggcaaac aatatgagag tgttctaatg gtcagcatcg atcaattatt ggacagcatg 1680

aaagaaattg gtagcaattg cctgaataat gaatttaact tttttaaaag acatatctgt 1740

gatgctaata aggaaggtat gtttttattc cgtgctgctc gcaagttgag gcaatttctt 1800

aaaatgaata gcactggtga ttttgatctc cacttattaa aagtttcaga aggcacaaca 1860

atactgttga actgcactgg ccaggttaaa ggaagaaaac cagctgccct gggtgaagcc 1920

caaccaacaa agagtttgga agaaaataaa tctttaaagg aacagaaaaa actgaatgac 1980

ttgtgtttcc taaagagact attacaagag ataaaaactt gttggaataa aattttgatg 2040

ggcactaaag aacactaata atctaga 2067

<210> 8

<211> 683

<212> PRT

<213>RP215 Chimeric antigen receptor constructs amino acid sequence (Synthetic Construct)

<220>

<221> DNA_BIND

<222> (1)..(683)

<400> 8

Ala Ala Thr Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala

1 5 10 15

Leu Leu Leu His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly

20 25 30

Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala

35 40 45

Ser Gly Tyr Thr Phe Thr Asp Tyr Trp Met His Trp Val Arg Gln Ala

50 55 60

Pro Gly Gln Gly Leu Glu Trp Met Gly Ala Ile Asp Thr Ser Asp Ser

65 70 75 80

Tyr Thr Arg Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Val

85 90 95

Asp Glu Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser

100 105 110

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Ile Tyr Asp Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro

145 150 155 160

Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys

165 170 175

Ser Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Ser Tyr Leu Ala

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Val Tyr Phe

195 200 205

Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly

210 215 220

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp

225 230 235 240

Val Ala Val Tyr Phe Cys Gln Gln His Tyr Ser Thr Pro Ser Thr Phe

245 250 255

Gly Gly Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg

325 330 335

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

340 345 350

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

355 360 365

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

420 425 430

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

435 440 445

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

450 455 460

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg Glu Gly Arg Gly Ser Leu Leu Thr

485 490 495

Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Phe His Val Ser Phe

500 505 510

Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile Leu Val Leu Leu Pro Val

515 520 525

Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu

530 535 540

Ser Val Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu

545 550 555 560

Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His

565 570 575

Ile Cys Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg

580 585 590

Lys Leu Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu

595 600 605

His Leu Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr

610 615 620

Gly Gln Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro

625 630 635 640

Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu

645 650 655

Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys

660 665 670

Trp Asn Lys Ile Leu Met Gly Thr Lys Glu His

675 680

<210> 9

<211> 73

<212> DNA

<213>Encoded signal structural domain (Synthetic Construct)

<220>

<221> protein_bind

<222> (1)..(73)

<400> 9

gccgccacca tggccctccc tgtcaccgcc ctgctgcttc cgctggctct tctgctccac 60

gccgctcggc ccc 73

<210> 10

<211> 134

<212> DNA

<213>Encode hinge and transmembrane domain (Synthetic Construct)

<220>

<221> protein_bind

<222> (1)..(134)

<400> 10

ggcatgtaga cccgcagctg gtggggccgt gcatacccgg ggtcttgact tcgcctgcga 60

tatctacatt tgggcccctc tggctggtac ttgcggggtc ctgctgcttt cactcgtgat 120

cactctttac tgta 134

<210> 11

<211> 123

<212> DNA

<213>Encode costimulation structural domain (Synthetic Construct)

<220>

<221> protein_bind

<222> (1)..(123)

<400> 11

agcgcggtcg gaagaagctg ctgtacatct ttaagcaacc cttcatgagg cctgtgcaga 60

ctactcaaga ggaggacggc tgttcatgcc ggttcccaga ggaggaggaa ggcggctgcg 120

aac 123

<210> 12

<211> 339

<212> DNA

<213>Encoded signal structural domain (Synthetic Construct)

<220>

<221> protein_bind

<222> (1)..(339)

<400> 12

tgcgcgtgaa attcagccgc agcgcagatg ctccagccta caagcagggg cagaaccagc 60

tctacaacga actcaatctt ggtcggagag aggagtacga cgtgctggac aagcggagag 120

gacgggaccc agaaatgggc gggaagccgc gcagaaagaa tccccaagag ggcctgtaca 180

acgagctcca aaaggataag atggcagaag cctatagcga gattggtatg aaaggggaac 240

gcagaagagg caaaggccac gacggactgt accagggact cagcaccgcc accaaggaca 300

cctatgacgc tcttcacatg caggccctgc cgcctcggg 339

<210> 13

<211> 54

<212> DNA

<213>Encode self cleavage peptide (Synthetic Construct)

<220>

<221> protein_bind

<222> (1)..(54)

<400> 13

agggcagagg cagcctgctg acatgtggcg acgtggaaga gaaccctggc ccca 54

<210> 14

<211> 536

<212> DNA

<213>Codocyte factor structure domain (Synthetic Construct)

<220>

<221> protein_bind

<222> (1)..(536)

<400> 14

tgttccatgt ttcttttagg tatatctttg gacttcctcc cctgatcctt gttctgttgc 60

cagtagcatc atctgattgt gatattgaag gtaaagatgg caaacaatat gagagtgttc 120

taatggtcag catcgatcaa ttattggaca gcatgaaaga aattggtagc aattgcctga 180

ataatgaatt taactttttt aaaagacata tctgtgatgc taataaggaa ggtatgtttt 240

tattccgtgc tgctcgcaag ttgaggcaat ttcttaaaat gaatagcact ggtgattttg 300

atctccactt attaaaagtt tcagaaggca caacaatact gttgaactgc actggccagg 360

ttaaaggaag aaaaccagct gccctgggtg aagcccaacc aacaaagagt ttggaagaaa 420

ataaatcttt aaaggaacag aaaaaactga atgacttgtg tttcctaaag agactattac 480

aagagataaa aacttgttgg aataaaattt tgatgggcac taaagaacac taataa 536

<210> 15

<211> 24

<212> PRT

<213>Signal domain (Synthetic Construct)

<220>

<221> DNA_BIND

<222> (1)..(24)

<400> 15

Ala Ala Thr Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala

1 5 10 15

Leu Leu Leu His Ala Ala Arg Pro

20

<210> 16

<211> 69

<212> PRT

<213>Transmembrane domain CD8 (Synthetic Construct)

<220>

<221> DNA_BIND

<222> (1)..(69)

<400> 16

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 17

<211> 18

<212> PRT

<213>Self cleavage peptide 2A (Synthetic Construct)

<220>

<221> DNA_BIND

<222> (1)..(18)

<400> 17

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 18

<211> 41

<212> PRT

<213>Costimulatory signal structural domain 4-1BB (Synthetic Construct)

<220>

<221> DNA_BIND

<222> (1)..(41)

<400> 18

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu

35 40

<210> 19

<211> 177

<212> PRT

<213>Cytokine domain IL7 (Synthetic Construct)

<220>

<221> DNA_BIND

<222> (1)..(177)

<400> 19

Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile

1 5 10 15

Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys

20 25 30

Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu

35 40 45

Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe

50 55 60

Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe

65 70 75 80

Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser

85 90 95

Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr

100 105 110

Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro Ala Ala

115 120 125

Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu

130 135 140

Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu

145 150 155 160

Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu

165 170 175

His

Claims

1. the Chimeric antigen receptor construct of humanization RP215 monoclonal antibodies, it is characterised in that：For Signal peptide- HRP215 scFv-CD8-4-1BB-CD3Z-2A-IL7 have amino acid sequence shown in sequence table 8.

2. the Chimeric antigen receptor construct of humanization RP215 monoclonal antibodies according to claim 1, feature exist In：With nucleotide sequence shown in sequence table 7.

3. a kind of nucleic acid molecules of separation, it is characterised in that：Including construct described in claims 1 or 2.

4. the nucleic acid molecules of separation according to claim 3, it is characterised in that：The humanization RP215 monoclonal antibodies Or its segment contains the heavy chain of amino acid sequence shown in tool sequence table 1.

5. the nucleic acid molecules of separation according to claim 3, it is characterised in that：The encoding humanized RP215 monoclonals The nucleotide sequence of antibody or the heavy chain of its segment has nucleotide sequence shown in sequence table 2.

6. the nucleic acid molecules of separation according to claim 3, it is characterised in that：The humanization RP215 monoclonal antibodies Or its segment contains the light chain of amino acid sequence shown in tool sequence table 3.

7. the nucleic acid molecules of separation according to claim 3, it is characterised in that：Encoding humanized RP215 monoclonal antibodies Or nucleotide sequence shown in the nucleotide sequence tool sequence table 4 of the light chain of its segment.

8. the nucleic acid molecules of separation according to claim 3, it is characterised in that：The humanization RP215 includes code sequence The heavy chain variable region of amino acid sequence shown in list 5.

9. the nucleic acid molecules of separation according to claim 3, it is characterised in that：The humanization RP215 includes code sequence The light chain variable region of amino acid sequence shown in list 6.

10. the nucleic acid molecules of separation according to claim 3, it is characterised in that：Include the sequence of encoded signal structural domain Nucleotide sequence shown in table 9, for guiding expressed protein to cell membrane, wherein signal domain is containing interleukins 2 signal sequences.

11. the nucleic acid molecules of separation according to claim 3, it is characterised in that：Including coding hinge and transmembrane domain Sequence table 10 shown in nucleotide sequence, extracellular humanization RP215 monoclonal antibodies for connecting expressed protein or The intracellular costimulation structural domain and signal domain of its antigen-binding fragment and expressed protein, wherein hinge and cross-film knot Structure domain can contain the hinge and transmembrane domain of CD8.

12. the nucleic acid molecules of separation according to claim 3, it is characterised in that：Include the sequence of coding costimulation structural domain Nucleotide sequence shown in list 11, for costimulation T cell to improve the internal persistence of Chimeric antigen receptor construct, wherein Costimulation structural domain can contain the costimulation structural domain of 4-1BB.

13. the nucleic acid molecules of separation according to claim 3, it is characterised in that：Include the sequence of encoded signal structural domain Nucleotide sequence shown in table 12, for signal transduction in T cell, wherein signal domain can contain the ζ subunits of CD3 molecules.

14. the nucleic acid molecules of separation according to claim 3, it is characterised in that：Include the sequence table of coding self cleavage peptide Nucleotide sequence shown in 13, wherein self cleavage peptide can contain 2A self cleavage peptides.

15. the nucleic acid molecules of separation according to claim 3, it is characterised in that：Including Codocyte factor structure domain Nucleotide sequence shown in sequence table 14, wherein cytokine domain can contain IL7 structural domains.

16. the nucleic acid molecules of separation according to claim 3, it is characterised in that：It includes the nucleotides sequences of coding polypeptide Row, the polypeptide have：

Signal domain；

Transmembrane domain；

CD3-zeta signal domains；

Cytokine domain；

Wherein as many as described peptide can contain signal domain, humanization RP215 monoclonal antibodies or its piece from the ends N- to C-terminal Section, transmembrane domain, CD3-zeta signal domains and cytokine domain；

The wherein described cytokine domain contains IL-7 or its segment.

17. the nucleic acid molecules of separation according to claim 16, it is characterised in that：Its signal domain includes sequence table 15 Shown amino acid sequence.

18. the nucleic acid molecules of separation according to claim 16, it is characterised in that：The transmembrane domain includes CD8, tool Amino acid sequence shown in ordered list 16.

19. the nucleic acid molecules of separation according to claim 16, it is characterised in that：Also include self cleavage peptide 2A, there is sequence Amino acid sequence shown in list 17.

20. the nucleic acid molecules of separation according to claim 16, it is characterised in that：Also include costimulatory signal structural domain 4- 1BB has amino acid sequence shown in sequence table 18.

21. the nucleic acid molecules of separation according to claim 16, it is characterised in that：The cytokine domain includes sequence Amino acid sequence shown in list 19.

22. it is a kind of for humanization RP215 monoclonal antibodies or the carrier of its segment to be transfected and expressed in immunocyte, it is special Sign is：Including Chimeric antigen receptor construct.

23. according to carrier described in claim 22, it is characterised in that：This carrier includes any one aforementioned nucleic acid molecules.

24. according to carrier described in claim 22, it is characterised in that：It also include plasmid.

25. a kind of slow virus carrier being suitable for being transfected into T cell or natural killer cells, it is characterised in that：With any one Aforementioned nucleic acid molecules.

26. a kind of immunocyte, it is characterised in that：Including T cell or natural killer cells, including the aforementioned right of any one is wanted Nucleic acid molecules defined in asking, carrier or slow virus carrier.

27. immunocyte according to claim 26, it is characterised in that：Including T cell or natural killer cells, center Acid molecule passes through the short palindrome repetitive sequence CRISPR bases of Chimeric antigen receptor T cell immunotherapy or the regular intervals of cluster Because editing technique is embedded in immunocyte, in T cell or natural kill immunocyte.

28. utilizing application of the immunocyte in terms of preparing cancer treatment drugs described in claim 26.

29. according to the application of immunocyte described in claim 28, it is characterised in that：Cancer treatment drugs are in cancer cell surfaces height Degree expression CA215 cancerous immune globulin.

30. according to the application of immunocyte described in claim 29, it is characterised in that：The medicine is utilized by aforementioned any The immunocyte surface that nucleic acid molecules, carrier or slow virus carrier defined in one claim are transfected, in vitro or In vivo, T cell can be contained or kill naturally by expressing humanization RP215 monoclonal antibodies or its antigen-binding fragment, wherein immunocyte Hinder cell.

31. according to the application of immunocyte described in claim 29, it is characterised in that：Humanization RP215 monoclonal antibodies or its Expression of the antigen-binding fragment on immunocyte surface can lead to the selective killing to cancer cell, can be by inducing cancer Apoptosis or cytotoxic killer.

32. according to the application of immunocyte described in claim 28, it is characterised in that：The cancer includes breast cancer, oophoroma, Carcinoma of endometrium, cervix cancer, cancer of pancreas, colon cancer, lung cancer, liver cancer or kidney.

33. according to the application of immunocyte described in claim 30, it is characterised in that：Further include the immunocyte that will be transfected, T is thin Born of the same parents or natural killer cells are separated, and by cell storage in freezing state, can be long-term storages；The frozen cell It can thaw for unique individual in the spontaneous cancer that future may occur, and carry out emergency treatment.

34. using the method for immunocyte drug therapy described in claim 26 and CA215 high expression associated diseases, feature exists In：The method includes modification T cells to express Chimeric antigen receptor, and wherein Chimeric antigen receptor includes：

Signal domain；

Transmembrane domain；

CD3-zeta signal domains；

Antigen-binding domains, wherein this antigen-binding domains include a kind of polypeptide, the affinity combined with CA215 and RP215 has substantially quite；

With the human T cells for giving modified.