CN109912718A

CN109912718A - Isolated binding protein, nucleic acid, carrier, CAR-T cell and its application of B7-H3 antigen-binding domains

Info

Publication number: CN109912718A
Application number: CN201910210679.XA
Authority: CN
Inventors: 陈模江; 王刚; 刘金超
Original assignee: Beijing Shanke Biotechnology Co Ltd
Current assignee: Beijing Shanke Biotechnology Co Ltd
Priority date: 2019-03-20
Filing date: 2019-03-20
Publication date: 2019-06-21
Anticipated expiration: 2039-03-20
Also published as: CN109912718B

Abstract

The present invention relates to CAR-T cell fields, in particular to the isolated binding protein of B7-H3 antigen-binding domains, nucleic acid, carrier, CAR-T cell and its application.The present invention is directed to the scFV segment that B7-H3 antigen obtains, design construction is with second generation B7-H3-CAR that CD28 or 4-1BB are costimulatory molecules, and its ability for killing cancer cell is demonstrated in the non-small cell lung cancer of the B7-H3 positive, the result shows that when T cell and cancer cell ratio are 1:5, B7-H3-CAR-T still is able to efficiently kill very much the non-small cell lung cancer cell of the B7-H3 positive, and a large amount of I type interferon is released, such as IFN γ and TNF α.

Description

Isolated binding protein, nucleic acid, carrier, the CAR-T of B7-H3 antigen-binding domains Cell and its application

Technical field

The present invention relates to CAR-T cell fields, in particular to the isolated combination of B7-H3 antigen-binding domains Albumen, nucleic acid, carrier, CAR-T cell and its application.

Background technique

Lung cancer is the primary tumour of global cancer mortality, can increase about 1,300,000 new cases, about 80- every year 85% lung cancer newly diagnosed is non-small cell lung cancer, such as gland cancer, squamous carcinoma and large cell carcinoma.China had about 800,000 in 2017 New hair cases of lung cancer, 700,000 dead cases of lung cancer, 40% or so of the world morbidity and mortality Jun Zhan lung cancer, significantly larger than its Its country.In China, lung cancer is also the cancer of the double rates first of morbidity and mortality, not with air pollution problems inherent in recent years It is disconnected to aggravate, it is contemplated that the quantity of the following patients with lung cancer will be further increased, and drastically influence health and the service life of compatriots.

Although the early stage of lung cancer tumor resection, some patientss can realize healing by way of operation, big most absolutely In several cases, when patient assessment has been advanced cancer, belongs to unresectable lesion or has already appeared transfer, belongs to The morbid state that can not be cured.Synchronous chemoradiotherapy can be received for the Patients with Non-small-cell Lung of local advanced, joint or Not combined surgical treatment can be obtained average 8 months in this way and be survived without progression of disease, but 5 years overall survivals still less than 15%.The patient that advanced lung cancer has been diagnosed as when medical is subjected to novel cytotoxic treatments drug, such as pemetrexed, in Position life span is about 17 months.

CAR-T (english abbreviation of Chimeric Antigen Receptor T Cells) is a kind of based on genetic modification Autologous T cells immunotherapy.The therapy is after the T cell for extracting patient, by genetic modification, by a kind of specific egg It is white ----Chimeric antigen receptor (i.e. CAR) expression identifies the specific antigen of cancer cell surfaces on the surface of T cell whereby, and leads to The antigen Scavenging activity for crossing these T cells kills cancer cell.CAR-T treatment achieves in the multinomial clinical test carried out at present Huge success especially targets the CAR-T of CD19 and BCMA.Although it is swollen that CAR-T treats the hematological systems such as lymthoma, leukaemia Tumor has obtained extraordinary clinical therapeutic efficacy, but progress of the CAR-T in solid tumor is relatively slow, one of them is important The reason of be no suitable target spot because highly expressed antigen also can be in some normal tissue expressions usually in solid tumor.

CAR-T has been realized in surprising treatment effect as state-of-the art revolutionary immunotherapy method in leukaemia How fruit is applied to solid tumor and also needs further to research and develop.

In view of this, the present invention is specifically proposed.

Summary of the invention

The present invention is directed to the obtained scFV segment of B7-H3 antigen, and design construction is costimulation point with CD28 or 4-1BB The second generation B7-H3-CAR of son, and its ability for killing cancer cell is demonstrated in the non-small cell lung cancer of the B7-H3 positive, The result shows that B7-H3-CAR-T still is able to efficiently kill very much the B7-H3 positive when T cell and cancer cell ratio are 1:5 Non-small cell lung cancer cell, and a large amount of I type interferon is released, such as IFN γ and TNF α.

In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:

On the one hand, the present invention provides the isolated binding protein comprising B7-H3 antigen-binding domains, light chain variable region And/or the amino acid sequence of heavy chain variable region successively as shown in SEQ ID NO.1-2 and with ammonia shown in SEQ ID NO.1-2 Base acid sequence has the amino acid sequence of at least 95% sequence identity.

It should be clear that having the amino acid of at least 95% sequence identity with amino acid sequence shown in SEQ ID NO.1-2 Sequence is can be deduced still to have preferably by some or certain several amino acid sequence transformation by those skilled in the art With the effect of B7-H3 antigen binding.As according to the actual situation, at least 95% sequence identity can for 95%, 96%, 97%, 98%, 99% etc., the changed number of amino acid can be 1,2,3,4,5,6 etc..

Further, the binding protein is sc FV segment.

Further, the light chain variable region is connect with the heavy chain variable region by bonding pad, the ammonia of the bonding pad Base acid sequence is as shown in SEQ ID NO.3.

B7-H3 is an I type memebrane protein, the high expression in a variety of solid tumors, for example, 93.7% cancer of pancreas, 90.6% Breast cancer, 83% oophoroma, 86% carcinoma of the rectum, 93.8% liver cancer and 70% non-small cell lung cancer are a tumours The novel targets of immunization therapy.But B7-H3 is not in minds such as normal tissue expressions, such as heart, lung, kidney, liver and brain Through cell.In recent years, the antibody drugs of multiple targeting B7-H3, the antibody of antibody, isotope labelling including Fc enhancing and anti- Body-drug conjugates etc. are shown preferable in multiple Phase I clinical trials and preclinical mouse tumor model research Antitumous effect.These results of study further demonstrate that, the CAR of design targeting B7-H3 for treatment of solid tumors be it is reasonable, It is feasible.

The present invention obtains anti-B7-H3 monoclonal antibody 2D6 through screening, is capable of the antibody of the anti-B7-H3 of stably excreting.Through dividing Bonding pad clone of the sub- biological means by the heavy chain variable region of the monoclonal antibody and light chain variable region and between the two, Nucleic acid sequence is successively as shown in SEQ ID NO.4-6, and amino amino acid sequence is successively as shown in SEQ ID NO.1-3.

Second aspect, the present invention provides a kind of isolated nucleic acid, the above-mentioned binding proteins of the nucleic acid encode.

Further, the nucleic acid sequence of the light chain variable region is as shown in SEQ ID NO.4.

Further, the nucleic acid sequence of the heavy chain variable region is as shown in SEQ ID NO.5.

Further, the nucleic acid sequence of the bonding pad is as shown in SEQ ID NO.6.

Further, the nucleic acid is also connected with signal peptide nucleic acid sequence.

Further, the nucleic acid sequence of the signal peptide is as shown in SEQ ID NO.7.The corresponding amino acid sequence of signal peptide As shown in SEQ ID NO.8.

Further, the signal peptide is located at the nitrogen end of the light chain variable region.

Signal peptide sequence in the present invention is included in the end N- primer used in PCR, in the sequence that amplification obtains in this way Contain signal peptide.

The third aspect, the present invention provide a kind of carrier, and it includes above-mentioned nucleic acid.

Further, the carrier contains the intracellular knot of the hinge area of CD8 α, the transmembrane domain of CD8 α and CD3 ζ Structure domain, and include CD28 or 4-1BB costimulation structural domain.

Further, the hinge area of CD8 α, CD8 α transmembrane domain nucleic acid sequence as shown in SEQ ID NO.9, Corresponding amino acid sequence is as shown in SEQ ID NO.10.

Further, the nucleic acid sequence of the intracellular domain of CD3 ζ is as shown in SEQ ID NO.11, corresponding amino Acid sequence is as shown in SEQ ID NO.12.

Further, the nucleic acid sequence of CD28 is as shown in SEQ ID NO.13, corresponding amino acid sequence such as SEQ ID Shown in NO.14.

Further, the nucleic acid sequence of 4-1BB is as shown in SEQ ID NO.15, corresponding amino acid sequence such as SEQ ID Shown in NO.16.

Further, the carrier is slow virus carrier；The slow virus carrier be preferably pCDH-CMV-CAR-28 or pCDH-CMV-CAR-BB。

Further, the connection type of each nucleic acid sequence is as follows in the carrier:

PCDH-CMV-B7-H3-CAR-28 or pCDH-CMV-B7-H3-CAR-BB；

Wherein, the nucleic acid sequence of B7-H3-CAR-28 is followed successively by signal peptide-VL-Linker-VH-CD8 ɑ-CD28-CD3 ζ；

Preferably, the nucleic acid sequence of B7-H3-CAR-28 is as shown in SEQ ID NO.17, corresponding amino acid sequence As shown in SEQ ID NO.18.

The nucleic acid sequence of B7-H3-CAR-BB is followed successively by signal peptide-VL-Linker-VH-CD8 ɑ -4-1BB-CD3 ζ.

Preferably, the nucleic acid sequence of B7-H3-CAR-BB is as shown in SEQ ID NO.19, corresponding amino acid sequence As shown in SEQ ID NO.20.

In carrier provided by the invention, the second generation B7-H3- of CD28 or 4-1BB as costimulatory molecules is constructed CAR, and its ability for killing cancer cell is demonstrated in the non-small cell lung cancer of the B7-H3 positive, it can efficiently kill very much Hurt the non-small cell lung cancer cell of the B7-H3 positive.

Fourth aspect, the present invention provide a kind of host cell, and the host cell includes above-mentioned nucleic acid or above-mentioned load Body.

Further, the host cell is for expressing the nucleic acid or carrier.I.e. the host cell is for expressing external source The cell strain of gene.

Further, the host cell is 293 cells.

293 cells are human renal epithelial cell lines, and there are many derivative strains, such as HEK293,293T/17 etc..

Host cell provided by the invention, to be expanded and be enriched with slow virus either plasmid.For subsequent The preparation of CAR-T cell.

In actual operation, after plasmid-infected host cells, the culture supernatant containing slow virus is collected, and with 0.45 μm Then filter filtering freezes stand-by.

5th aspect, the present invention provides a kind of preparation methods of CAR-T cell, include the following steps:

Above-mentioned host cell is obtained to contain virulent Supernatant infection T cell, isolated CAR-T cell.

T cell is prepared using following methods: separating PBMC lymphocyte in peripheral blood, CD3 and CD28 antibody is then added In coated culture plate, after 20-30 hours, be added IL2 (10 μ g/mL), stimulation 40-60 hour after collection T cell to get.

Further, PBMC lymphocyte is added in coated 24 orifice plate of CD3 and CD28 antibody, and 1 × 10⁶A cell is every Hole.

Peripheral blood is obtained from the volunteer of health.

The T cell stimulated is used for above-mentioned recombinant slow virus infection.

Specifically, T cell is carried out for the infection of above-mentioned recombinant slow virus using following methods:

5 × 10 are added in 24 porocyte culture plates⁵Then a T cell stimulated is added 1mL and expresses B7-H3-CAR- The slow virus of 28 or B7-H3-CAR-BB, 2000g are centrifuged 1.5 hours, and cell is placed in 37 DEG C of cell incubators after centrifugation After 24 hours, the culture medium containing slow virus is removed, is changed to fresh culture for middle culture, and hereafter, replacement in every two days is primary Culture medium after 12 days, collects CAR-T cell, detects the killing ability of CAR-T cell.

6th aspect, the present invention provide CAR-T cell made from above-mentioned preparation method.

7th aspect, the present invention provide application of the above-mentioned CAR-T cell in preparation treatment antineoplastic agents.

Further, the tumour includes the tumour of all B7-H3 positives, including blood tumor and solid tumor, such as but not Be limited to lung cancer, cancer of pancreas, gastric cancer, breast cancer, oophoroma, cervical carcinoma, brain tumor, head and neck cancer, colorectal cancer, melanoma, liver cancer, Gastric cancer, cancer of the esophagus, carcinoma of testis, prostate cancer, kidney, glioma, neuroblastoma, myeloma, appointing in lymthoma It is a kind of.

The CAR-T cell that the present invention demonstrates offer has efficient lethality to non-small cell lung cancer.Obviously It can deduce, efficient lethality is also had to other solid tumors such as cancer of pancreas, breast cancer, oophoroma, the carcinoma of the rectum, liver cancer.

Compared with prior art, the invention has the benefit that

(1) the scFV segment that the present invention is obtained for B7-H3 antigen, design construction CD28 or 4-1BB are thorn altogether Swash the second generation B7-H3-CAR of molecule, and demonstrates its energy for killing cancer cell in the non-small cell lung cancer of the B7-H3 positive Power.

(2) it when CAR-T cell provided by the invention and cancer cell ratio are 1:5, still is able to efficiently kill very much B7-H3 Positive non-small cell lung cancer cell, and a large amount of I type interferon is released, it is the treatment of solid tumor such as IFN γ and TNF α Good basis is provided.

(3) CAR-T cell provided by the invention can be used for treating solid tumor, and solid tumor includes non-small cell lung cancer, pancreas Cancer, breast cancer, oophoroma, the carcinoma of the rectum, liver cancer etc..

Detailed description of the invention

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described.

Fig. 1 is the structural schematic diagram of carrier in the embodiment of the present invention 1；

Fig. 2 is that the slow-virus infection of the Plasmid DNA of B7-H3-CAR-28 or B7-H3CAR-BB in the embodiment of the present invention 1 swashs In CAR-T cell made from T cell living, the expression figure of B7-H3-CAR-28 and B7-H3-CAR-BB；

After Fig. 3 co-cultures for cells different in the embodiment of the present invention 1 from different Lines, fluidic cell Instrument detects T cell (CD3⁺) and remaining cancer cell ratio (B7-H3⁺) result figure；

The expression that Fig. 4 is B7-H3 after B7-H3 antibody infectious difference Lines in the embodiment of the present invention 1 Situation map；

Fig. 5 is that different cytosiies secrete IFN γ and TNF α amount when non-small cell lung cancer cell in the embodiment of the present invention 1 Column diagram；

Fig. 6 is the monitoring figure that B7-H3-CAR-T removes lung cancer tumor situation in Mice Body in the embodiment of the present invention 2.

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is The conventional products that can be obtained by commercially available purchase.

Embodiment 1

1, B7-H3 antibody screening

It is mixed with the B7-H3-His albumen of purifying with Freund's adjuvant, by the way that immune BALB/c mouse is subcutaneously injected, every two weeks It is immune primary, it is immunized altogether three times.35th day, take a blood sample and by ELISA detection serum in anti-B7-H3 antibody, when 4000 times it is dilute When OD value after releasing is greater than 1.0, show the anti-B7-H3 antibody in serum containing higher concentration.39th day, mouse is immunized again, 42nd day collection Mouse spleen cells, prepare Single cell suspensions, and merge by PEG with myeloma cell F0.Culture 7 It after it, is screened by supernatant of the ELISA to the hybridoma clone of fusion, detects their specificity and sensibility, passed through After secondary screening and subclone, the antibody that monoclonal 2D6 is capable of the anti-B7-H3 of stably excreting is obtained.

2, plasmid construction

In order to construct the CAR of targeting B7-H3, we have selected the 2D6 monoclonal antibody of anti-B7-H3, can by encoding antibody Become area DNA sequence dna (scFv) be cloned into slow virus carrier, with comprising encode B7-H3-CAR-28 or B7-H3CAR-BB matter The T of the slow-virus infection activation of grain DNA makes CAR-T cell.

Wherein, slow virus carrier selects pCDH-CMV-CAR-28 or pCDH-CMV-CAR-BB, the two carriers all contain The hinge area and transmembrane domain of CD8 α and the intracellular domain of CD3 ζ, and separately include CD28 or 4-1BB costimulation knot Structure domain, for constructing B7-H3-CAR-28 and B7-H3-CAR-BB (Fig. 1).

The variable region fragment (VL and VH) of the light chain of anti-B7-H3 monoclonal antibody 2D6 and heavy chain is amplified by PCR Come, signal peptide (SP) sequence is included in the end N- primer used in PCR, then uses XbaI and BamHI restriction enzyme enzyme After cutting, by T4 ligase be cloned into slow virus carrier pCDH-CMV-CAR-28 (pCDH-CMV-B7-H3-CAR-28) or pCDH-CMV-CAR-BB(pCDH-CMV-B7-H3-CAR-BB)。

Wherein, the DNA sequence dna (scFv) of 2D6 monoclonal antibody encoding antibody variable includes light chain variable region (nucleic acid sequence Column are as shown in SEQ ID NO.4) and heavy chain variable region (nucleic acid sequence is as shown in SEQ ID NO.5), and between bonding pad (nucleic acid sequence is as shown in SEQ ID NO.6)；The nucleic acid sequence of signal peptide is as shown in SEQ ID NO.7, corresponding amino acid Sequence is as shown in SEQ ID NO.8；The nucleic acid sequence of CD8 α is as shown in SEQ ID NO.9, corresponding amino acid sequence such as SEQ Shown in ID NO.10；The nucleic acid sequence of CD3 ζ is as shown in SEQ ID NO.11, corresponding amino acid sequence such as SEQ ID Shown in NO.12；The nucleic acid sequence of CD28 is as shown in SEQ ID NO.13, corresponding amino acid sequence such as SEQ ID NO.14 institute Show；The nucleic acid sequence of 4-1BB is as shown in SEQ ID NO.15, and corresponding amino acid sequence is as shown in SEQ ID NO.16；B7- H3-CAR-28, that is, B7-H3-CAR-CD8 α-CD28-CD3 ζ complete sequence is as shown in SEQ ID NO.17, corresponding amino acid Sequence is as shown in SEQ ID NO.18；B7-H3-CAR-BB, that is, B7-H3-CAR-CD8 α -4-1BB-CD3 ζ complete sequence such as SEQ Shown in ID NO.19, corresponding amino acid sequence is as shown in SEQ ID NO.20.

3, the slow virus preparation of B7-H3-CAR is expressed

By 7 × 10⁶293T cell inoculation is in the Tissue Culture Dish of 10cm, after 16 hours, by 4 μ g pCDH-CMV- B7-H3-CAR-28 or pCDH-CMV-B7-H3-CAR-BB plasmid, 4 μ g psPAX2 plasmids and the mixing of 2 μ g pMD2G plasmids, so Plasmid mixture and GeneJuice transfection reagent (Merck Millipore) are mixed afterwards, 10 minutes is stood, then transfects 293T cell.After transfection 48 hours, the culture supernatant containing slow virus is collected, and is filtered with 0.45 μm of filter, then It freezes stand-by.

4, the preparation of CAR-T cell and amplification in vitro

PBMC lymphocyte is separated from volunteer's peripheral blood of health, CD3 and CD28 antibody coated 24 is then added In orifice plate, 1 × 10⁶A cell per well.It after 24 hours, is added IL2 (10 μ g/mL), T cell is collected in stimulation two days later, is counted For use.

The above-mentioned recombinant slow virus of the T cell stimulated is infected, steps are as follows for specific infection: in 24 porocyte culture plates It is middle to be added 5 × 10⁵Then the slow disease of 1mL expression B7-H3-CAR-28 or B7-H3-CAR-BB is added in a T cell stimulated Poison, 2000g are centrifuged 1.5 hours, cell are placed in 37 DEG C of cell incubators after centrifugation and is cultivated, after 24 hours, will be contained There is the culture medium of slow virus to remove, be changed to fresh culture, hereafter, every two days one subcultures of replacement after 12 days, collect CAR- T cell detects the killing ability of CAR-T cell.After infection 4 days, 5 × 10 are taken⁵A cell is thin with the antibody dye CAR-T of anti-Fab The CAR of cellular surface, and with the expression of flow cytomery B7-H3-CAR, as a result as shown in Figure 2.

I.e. with the T cell of the slow-virus infection activation of the Plasmid DNA comprising encoding B7-H3-CAR-28 or B7-H3CAR-BB To make CAR-T cell, the results showed that B7-H3-CAR-28 and B7-H3-CAR-BB can be expressed efficiently in CAR-T cell table Face, 95% or more CAR-T cell are B7-H3-CAR positive (Fig. 2).

5, flow cytometer detection

T cell and cancer cell are marked with the antibody of CD3-APC-H7 and B7-H3-APC (BD bioscience) respectively, The expression of B7-H3-CAR is the antibody by Anti-Fab (Jackson ImmunoResearch Laboratories INC.) It marks, the sample of label passes through BD FACSCanto II flow cytometer BD Diva software (BD Biosciences it) detects, each sample at least collects 10,000 living cells, the knot collected with the analysis of 10 software of Flowjo Fruit.

NT, B7-H3-CAR-28-T or B7-H3-CAR-BB-T cell and three kinds of Lines HCC827, NCI-H522 and NCI-H1299 is co-cultured, and the ratio of T cell and cancer cell is that 1:5 is examined after co-culturing 5 days with flow cytometer Survey T cell (CD3⁺) and remaining cancer cell ratio (B7-H3⁺)。

It detects B7-H3-CAR-28 and B7-H3-CAR-BB and kills B7-H3⁺The ability of cancer cell, by HCC827, NCI- These three B7-H3 of H522 and NCI-H1299⁺Lung carcinoma cell (Fig. 3) with compare T cell (not by the T cell of slow-virus infection, NT), B7-H3-CAR-28-T or B7-H3-CAR-BB-T cell co-cultures, and T cell and cancer cell ratio are 1:5, co-cultures 5 days Afterwards, collect suspension T cell and adherent cancer cell, two kinds of cells are combined, respectively use CD3 and B7-H3 antibody mark Remember T cell and cancer cell, then uses the ratio of flow cytomery residual cancer cell.As a result as shown in Figure 3.

From figure 3, it can be seen that B7-H3-CAR-28-T and B7-H3-CAR-BB-T cell can be completely after co-culturing 5 days These three lung carcinoma cells are removed.It demonstrates B7-H3-CAR-28-T and B7-H3-CAR-BB-T cell and can identify and target and kill Hurt these three lung carcinoma cells.

With antibody infectious Lines HCC827, NCI-H522 and NCI-H1299 of B7-H3, then use The expression of flow cytomery B7-H3, as a result as shown in Figure 4.

From fig. 4, it can be seen that the high expression B7-H3 of non-small cell lung cancer.

6, ELISA is detected

B7-H3-CAR-T secretes a large amount of IFN γs and TNF α when killing non-small cell lung cancer cell.IFN is detected using ELISA The amount of γ and TNF α.It is specific as follows:

By non-small cell lung cancer cell with 5 × 10⁵Every hole is added in 24 porocyte culture plates, after 16 hours, addition 5 × 10⁵The ratio of NT, B7-H3-CAR-28 or B7-H3-CAR-BB CAR-T cell, T cell and cancer cell is 1:5, and every hole 2mL is thin Born of the same parents' culture medium.Co-culture 24 hours after, collect 1mL supernatant, with the ELISA kit (R&D system) of IFN γ and TNF α come Detection co-cultures the amount of both I type interferon in supernatant.As a result as shown in Figure 5.

From fig. 5, it can be seen that B7-H3-CAR-28-T and B7-H3-CAR-BB-T cell releases a large amount of I type interference Element-IFN γ and TNF α.

Embodiment 2

1, killing experiments are co-cultured

By non-small cell lung cancer cell with 5 × 10⁵Every hole is added in 24 porocyte culture plates, after 16 hours, addition 5 × 10⁵The ratio of NT, B7-H3-CAR-28 or B7-H3-CAR-BB CAR-T cell, T cell and cancer cell is 1:5.It co-cultures 5 days Afterwards, the T cell of suspension is collected and adherent cancer cell is all collected, respectively with the antibody label T cell of CD3 and B7-H3 and Then cancer cell uses the ratio of flow cytomery residual cancer cell, removed with Zombie Aqua Dye (Biolegend) Dead cell.

2, the effect of B7-H3-CAR-T killing tumour is assessed in Mice Body with H522 lung cancer metastasis model

The H522 lung carcinoma cell for being overexpressed luciferase is entered in NSG Mice Body by tail vein injection, every mouse note Penetrate 2 × 10⁶A H522 cell constructs the lung cancer metastasis model of mouse with this.After 14 days, with NT, B7-H3-CAR-28-T or B7- H3-CAR-BB-T cell handles tumor-bearing mice, every mouse injection 10 × 10⁶A T cell, weekly by IVIS instrument take pictures come Monitor the variation of tumor size.

As a result as shown in fig. 6, after treatment in one week, in B7-H3-CAR-28-T or B7-H3-CAR-BB-T cell In processing group, the cancer cell of mouse lung is completely disappeared, but still has a part cancer cell survival by caudad in mouse, two weeks it Afterwards, the cancer cell of mouse systemic was fully erased by B7-H3-CAR-28-T or B7-H3-CAR-BB-T cell, at subsequent more than 60 days Tracking and monitoring in, the mouse of all B7-H3-CAR-T processing does not observe Lung Cancer Recurrence phenomenon.The above result shows that B7- H3-CAR-28-T and B7-H3-CAR-BB-T cell can in Mice Body Efficient killing effect B7-H3 positive lung cancer cells, and prevent Only recur.

All solid tumor cancer cells, such as 93.7% cancer of pancreas, 90.6% cream are expressed in since B7-H3 is almost high Gland cancer, 83% oophoroma, 86% carcinoma of the rectum, 93.8% liver cancer and 70% non-small cell lung cancer etc..Therefore, it targets The CAR-T of B7-H3 can be widely applied to all B7-H3 of targeted therapy⁺Solid tumor or myeloma.

Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims Including belonging to all such changes and modifications in the scope of the invention.

SEQUENCE LISTING

<110>Beijing Shan Ke Biotechnology Co., Ltd

<120>the isolated binding protein of B7-H3 antigen-binding domains, nucleic acid, carrier, CAR-T cell and its application

<130> 2019

<160> 20

<170> PatentIn version 3.3

<210> 1

<211> 106

<212> PRT

<213> Mus musculus

<400> 1

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

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ile Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Ser Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Ala Ser Gly Ala Ser Tyr Ser Leu Ser Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Glu Ile Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 2

<211> 118

<212> PRT

<213> Mus musculus

<400> 2

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Val His Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Gly Tyr

20 25 30

Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Ser Leu Glu Trp Leu

35 40 45

Gly Met Ile Trp Cys Asp Gly Arg Thr Asp Tyr Asn Ser Gly Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala

85 90 95

Arg Gly Tyr Gly Asn His Ala Trp Leu Ala Tyr Trp Cys Gln Gly Thr

100 105 110

Leu Val Ser Val Ser Ala

115

<210> 3

<211> 15

<212> PRT

<213> Homo sapiens

<400> 3

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 4

<211> 318

<212> DNA

<213> Mus musculus

<400> 4

caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60

atgacctgca gtgccagctc aagtgtaatt tacatgcact ggtaccagca gaagtcaggc 120

acctcctcca aaagatggat ctatgacaca tccaaactgg cttctggagt ccctgctcgc 180

ttcagtggca gtgcgtctgg cgcctcttac tctctctcaa tcagcagcat ggaggctgaa 240

gatgctgcca cttattactg ccagcagtgg gaaattaacc cgctcacgtt cggtgctggg 300

accaagctgg agctgaaa 318

<210> 5

<211> 354

<212> DNA

<213> Mus musculus

<400> 5

caggtgcagc tgcaggagtc aggacctggc ctggtggtgc actcacagag cctgtccatc 60

acatgcaccg tttcagggtt ctcattaatc ggctatggtg taaactgggt tcgccagcct 120

ccaggaaaga gtctggagtg gctcggaatg atatggtgtg atggaaggac agactataat 180

tcaggtctca aatccagact gagcatcagc aaggacaact ccaagagcca agttttctta 240

aaaatgaaca gtctgcaaac tgatgacaca gccaggtatt actgtgccag agggtatggt 300

aaccacgcct ggcttgctta ctggtgccaa gggactctgg tcagtgtctc tgca 354

<210> 6

<211> 45

<212> DNA

<213> Homo sapiens

<400> 6

ggcggcggag gatctggcgg aggcggaagt ggcggagggg gctct 45

<210> 7

<211> 57

<212> DNA

<213> Homo sapiens

<400> 7

atggaattcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgc 57

<210> 8

<211> 19

<212> PRT

<213> Homo sapiens

<400> 8

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys

<210> 9

<211> 207

<212> DNA

<213> Homo sapiens

<400> 9

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180

ctgtcactgg ttatcaccct ttactgc 207

<210> 10

<211> 69

<212> PRT

<213> Homo sapiens

<400> 10

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> 11

<211> 339

<212> DNA

<213> Homo sapiens

<400> 11

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339

<210> 12

<211> 112

<212> PRT

<213> Homo sapiens

<400> 12

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

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

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 13

<211> 123

<212> DNA

<213> Homo sapiens

<400> 13

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 14

<211> 41

<212> PRT

<213> Homo sapiens

<400> 14

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 15

<211> 126

<212> DNA

<213> Homo sapiens

<400> 15

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 16

<211> 42

<212> PRT

<213> Homo sapiens

<400> 16

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 Leu

35 40

<210> 17

<211> 1443

<212> DNA

<213>artificial sequence

<400> 17

atggaattcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgccaa 60

attgttctca cccagtctcc agcaatcatg tctgcatctc caggggagaa ggtcaccatg 120

acctgcagtg ccagctcaag tgtaatttac atgcactggt accagcagaa gtcaggcacc 180

tcctccaaaa gatggatcta tgacacatcc aaactggctt ctggagtccc tgctcgcttc 240

agtggcagtg cgtctggcgc ctcttactct ctctcaatca gcagcatgga ggctgaagat 300

gctgccactt attactgcca gcagtgggaa attaacccgc tcacgttcgg tgctgggacc 360

aagctggagc tgaaaggcgg cggaggatct ggcggaggcg gaagtggcgg agggggctct 420

caggtgcagc tgcaggagtc aggacctggc ctggtggtgc actcacagag cctgtccatc 480

acatgcaccg tttcagggtt ctcattaatc ggctatggtg taaactgggt tcgccagcct 540

ccaggaaaga gtctggagtg gctcggaatg atatggtgtg atggaaggac agactataat 600

tcaggtctca aatccagact gagcatcagc aaggacaact ccaagagcca agttttctta 660

aaaatgaaca gtctgcaaac tgatgacaca gccaggtatt actgtgccag agggtatggt 720

aaccacgcct ggcttgctta ctggtgccaa gggactctgg tcagtgtctc tgcaaccacg 780

acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 840

cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 900

gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 960

ctggttatca ccctttactg caggagtaag aggagcaggc tcctgcacag tgactacatg 1020

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 1080

cgcgacttcg cagcctatcg ctccagagtg aagttcagca ggagcgcaga cgcccccgcg 1140

taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 1200

gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 1260

aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1320

gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1380

ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1440

taa 1443

<210> 18

<211> 480

<212> PRT

<213>artificial sequence

<400> 18

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala

20 25 30

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

35 40 45

Ile Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Ser Lys Arg

50 55 60

Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe

65 70 75 80

Ser Gly Ser Ala Ser Gly Ala Ser Tyr Ser Leu Ser Ile Ser Ser Met

85 90 95

Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Glu Ile Asn

100 105 110

Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly

115 120 125

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

130 135 140

Gln Glu Ser Gly Pro Gly Leu Val Val His Ser Gln Ser Leu Ser Ile

145 150 155 160

Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Gly Tyr Gly Val Asn Trp

165 170 175

Val Arg Gln Pro Pro Gly Lys Ser Leu Glu Trp Leu Gly Met Ile Trp

180 185 190

Cys Asp Gly Arg Thr Asp Tyr Asn Ser Gly Leu Lys Ser Arg Leu Ser

195 200 205

Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser

210 215 220

Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg Gly Tyr Gly

225 230 235 240

Asn His Ala Trp Leu Ala Tyr Trp Cys Gln Gly Thr Leu Val Ser Val

245 250 255

Ser Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys

340 345 350

His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

355 360 365

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

370 375 380

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

385 390 395 400

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

405 410 415

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

420 425 430

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

435 440 445

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

450 455 460

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

465 470 475 480

<210> 19

<211> 1446

<212> DNA

<213>artificial sequence

<400> 19

atggaattcg gcctgagctg gctgttcctg gtggccatcc tgaagggcgt gcagtgccaa 60

attgttctca cccagtctcc agcaatcatg tctgcatctc caggggagaa ggtcaccatg 120

acctgcagtg ccagctcaag tgtaatttac atgcactggt accagcagaa gtcaggcacc 180

tcctccaaaa gatggatcta tgacacatcc aaactggctt ctggagtccc tgctcgcttc 240

agtggcagtg cgtctggcgc ctcttactct ctctcaatca gcagcatgga ggctgaagat 300

gctgccactt attactgcca gcagtgggaa attaacccgc tcacgttcgg tgctgggacc 360

aagctggagc tgaaaggcgg cggaggatct ggcggaggcg gaagtggcgg agggggctct 420

caggtgcagc tgcaggagtc aggacctggc ctggtggtgc actcacagag cctgtccatc 480

acatgcaccg tttcagggtt ctcattaatc ggctatggtg taaactgggt tcgccagcct 540

ccaggaaaga gtctggagtg gctcggaatg atatggtgtg atggaaggac agactataat 600

tcaggtctca aatccagact gagcatcagc aaggacaact ccaagagcca agttttctta 660

aaaatgaaca gtctgcaaac tgatgacaca gccaggtatt actgtgccag agggtatggt 720

aaccacgcct ggcttgctta ctggtgccaa gggactctgg tcagtgtctc tgcaaccacg 780

acgccagcgc cgcgaccacc aacaccggcg cccaccatcg cgtcgcagcc cctgtccctg 840

cgcccagagg cgtgccggcc agcggcgggg ggcgcagtgc acacgagggg gctggacttc 900

gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 960

ctggttatca ccctttactg caaacggggc agaaagaaac tcctgtatat attcaaacaa 1020

ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 1080

gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc 1140

gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1200

tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1260

aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1320

agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1380

ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1440

cgctaa 1446

<210> 20

<211> 481

<212> PRT

<213>artificial sequence

<400> 20

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala

20 25 30

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

35 40 45

Ile Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Ser Lys Arg

50 55 60

Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe

65 70 75 80

Ser Gly Ser Ala Ser Gly Ala Ser Tyr Ser Leu Ser Ile Ser Ser Met

85 90 95

Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Glu Ile Asn

100 105 110

Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly

115 120 125

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

130 135 140

Gln Glu Ser Gly Pro Gly Leu Val Val His Ser Gln Ser Leu Ser Ile

145 150 155 160

Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Gly Tyr Gly Val Asn Trp

165 170 175

Val Arg Gln Pro Pro Gly Lys Ser Leu Glu Trp Leu Gly Met Ile Trp

180 185 190

Cys Asp Gly Arg Thr Asp Tyr Asn Ser Gly Leu Lys Ser Arg Leu Ser

195 200 205

Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser

210 215 220

Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg Gly Tyr Gly

225 230 235 240

Asn His Ala Trp Leu Ala Tyr Trp Cys Gln Gly Thr Leu Val Ser Val

245 250 255

Ser Ala Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

340 345 350

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

355 360 365

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

370 375 380

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

385 390 395 400

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

405 410 415

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

420 425 430

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

435 440 445

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

450 455 460

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

465 470 475 480

Arg

Claims

1. including the isolated binding protein of B7-H3 antigen-binding domains, which is characterized in that its light chain variable region and/or again The amino acid sequence of chain variable region successively as shown in SEQ ID NO.1-2 and with amino acid sequence shown in SEQ ID NO.1-2 Arrange the amino acid sequence of the sequence identity at least 95%；

Further, the binding protein is scFv；

Further, the light chain variable region is connect with the heavy chain variable region by bonding pad, the amino acid of the bonding pad Sequence is as shown in SEQ ID NO.3.

2. a kind of isolated nucleic acid, which is characterized in that the nucleic acid encode binding protein described in claim 1；

Further, the nucleic acid sequence of the light chain variable region is as shown in SEQ ID NO.4；

Further, the nucleic acid sequence of the heavy chain variable region is as shown in SEQ ID NO.5；

Further, the nucleic acid sequence of the bonding pad is as shown in SEQ ID NO.6；

Further, the nucleic acid is also connected with signal peptide nucleic acid sequence；

Further, the nucleic acid sequence of the signal peptide is as shown in SEQ ID NO.7；

3. a kind of carrier, which is characterized in that it includes nucleic acid as claimed in claim 2.

4. carrier according to claim 3, which is characterized in that the carrier contains the cross-film of the hinge area of CD8 α, CD8 α The intracellular domain of structural domain and CD3 ζ, and include CD28 or 4-1BB costimulation structural domain；

Further, the hinge area of CD8 α, CD8 α transmembrane domain nucleic acid sequence as shown in SEQ ID NO.9；

Further, the nucleic acid sequence of the intracellular domain of CD3 ζ is as shown in SEQ ID NO.11；

Further, the nucleic acid sequence of CD28 is as shown in SEQ ID NO.13；

Further, the nucleic acid sequence of 4-1BB is as shown in SEQ ID NO.15.

5. carrier according to claim 3, which is characterized in that the carrier is slow virus carrier；The slow virus carrier Preferably pCDH-CMV-CAR-28 or pCDH-CMV-CAR-BB；

PCDH-CMV-B7-H3-CAR-28 or pCDH-CMV-B7-H3-CAR-BB；

Preferably, the nucleic acid sequence of B7-H3-CAR-28 is as shown in SEQ ID NO.17；

The nucleic acid sequence of B7-H3-CAR-BB is followed successively by signal peptide-VL-Linker-VH-CD8 ɑ -4-1BB-CD3 ζ；

Preferably, the nucleic acid sequence of B7-H3-CAR-BB is as shown in SEQ ID NO.19.

6. a kind of host cell, which is characterized in that the host cell includes nucleic acid as claimed in claim 2 or claim 3- 5 described in any item carriers.

7. host cell according to claim 6, the host cell is for expressing the nucleic acid or carrier, the host Cell is preferably 293 cells.

8. a kind of preparation method of CAR-T cell, which comprises the steps of:

Host cell described in claim 6 or 7 is obtained to contain virulent Supernatant infection T cell, and isolated CAR-T is thin Born of the same parents.

9. CAR-T cell made from preparation method according to any one of claims 8.

10. application of the CAR-T cell as claimed in claim 9 in preparation treatment antineoplastic agents；Further, the tumour packet Include the tumour of all B7-H3 positives, including blood tumor and solid tumor, such as, but not limited to lung cancer, cancer of pancreas, gastric cancer, breast cancer, Oophoroma, cervical carcinoma, brain tumor, head and neck cancer, colorectal cancer, melanoma, liver cancer, gastric cancer, cancer of the esophagus, carcinoma of testis, prostate cancer, Any one of kidney, glioma, neuroblastoma, myeloma, lymthoma.