CN109748975A - A kind of bispecific chimeric antigen receptor and its application - Google Patents

Abstract

A kind of bispecific chimeric antigen receptor and its application, for the bispecific chimeric antigen receptor by CD8 α, the anti-Trop2 of humanization and anti-PD-L1 single-chain antibody, transmembrane region CD8TM and intracellular region CD28, CD137 and CD3 ζ are in series.Chimeric antigen receptor CD8 α-Trop2/PD-L1 scFv-CD8TM-CD28-CD137-CD3 ζ provided by the invention uses Retroviral technology, it can Infection in Vitro human T lymphocyte, CAR-T cell obtained can pass through the tumour cell of the single chain antibody portion specificity of CAR identified while expressing Trop2 and PD-L1 antigen, CAR-T cell is activated simultaneously, so that it is discharged cytokine profiles, realizes the specific killing action to tumour cell.

Description

A kind of bispecific chimeric antigen receptor and its application

Technical field

The invention belongs to cellular immunity technologies, and in particular to a kind of Trop2/PD-L1 bispecific chimeric antigen receptor and It is applied.

Background technique

Chimeric antigen receptor T cell immunization therapy (Chimeric Antigen Receptor T-cell Immunotherapy, CAR-T) oneself is treated through obtaining very big progress, especially in hematological system for tumor vaccine cells Exciting curative effect is achieved in terms of oncotherapy.CAR-T cell is the killing by the high-affinity of antibody and T lymphocyte Effect combines, by constructing specific chimeric antigen receptor carrier, through gene modification make T cell express this chimeric antigen by Body, can specific recognition target antigen to killing tumor cell.The structure of CAR includes that can identify that the single-stranded of tumour antigen resists Body sequence (scFv) and immunoreceptor tyrosine activating motif intracellular (ITMA, usually CD3 ζ) and costimulatory molecules signal (CM, usually CD28, CD134, CD137 etc.), the CAR-T cell being prepared on the one hand can be thin by scFv target tumor Born of the same parents, on the other hand by molecular activation T cell intracellular to the lethality of tumour cell.T cell is treated as tumor vaccine cells Effector cell, main cause have t cell responses have specificity, secrete various cell factors, killing tumor cell；CAR-T Cell can be proliferated in vivo, once 1000 times of clonal expansion can be carried out by being activated；CAR-T cell reaches site where antigen And remove cancer stove；CAR-T cell effect has Memorability, can long period maintenance therapy effect etc..It clinically uses at present CAR-T cell is the mixed cellularity group of various T cells, including CD4+, CD8+, memory T cell, effector T cell etc., different T Cell subsets may adopt in T cell plays synergistic effect in therapeutic process.

Although CD19-CAR-T cell therapy acute leukemia has obtained important breakthrough, to the treatment of solid tumor there are still Many problems, so as to cause unsatisfactory curative effect.CAR-T chimeric dreamboat antigen is only to express in tumour cell height, and just Often do not expressed in tissue.CAR-T cell utilizes the single-chain antibody molecules energy specific recognition tumour cell of targeting antigen, Jin Ertong Immune costimulatory signal molecule stimulation is crossed, specifically killing tumor cell, and avoid killing normal tissue, reduction poison is secondary to be made With.The CAR-T cell of one single-chain antibody still has the risk of on-target/off tumor toxicity, bispecific Single-chain antibody can then substantially reduce this risk.In addition, T cells with antigenic specificity will receive negativity regulatory factor in patient's body Regulation, make its anti-tumor effect lose or be weakened.It has been investigated that intracorporal this inhibitive ability of immunity environment mainly from Tumor microenvironment, a large amount of inhibitive ability of immunity molecules and cell can partially or completely inhibit the Proliferative Activated of tumor specific T cells, Anti-tumor effect is played, and then is significantly weakened the effect of T cells with antigenic specificity adoptive therapy, is such as studied at present more It is programmed death receptor -1(programmed death-1, PD-1) and its ligand PD-L1(programmed death Ligand 1, PD-L1).

PD-1 is negativity costimulatory molecules receptor, and cytoplasmic domain contains an immunity receptor Tyrosine Inhibitory Motifs (ITIM) and an immunity receptor tyrosine converts motif (ITSM).PD-1 mainly induces table on T, B and NK cell of activation It reaches.PD-1 has two ligands of PD-L1 and PD-L2, and PD-L1 wide expression is on multiple types tumour cell, and PD-L2 is only expressed In the macrophage of activation, Dendritic Cells, the stroma cell of derived from bone marrow and individual tumor cell strain.Studies have shown that PD-1 It is gradually raised with T cell activation degree, PD-1 and PD-L1 cause the generation for inhibiting signal after be combineding with each other, in tumour micro-loop Key effect is played in border, not only can blocking t cell activate the first and second signal, ameliorating effect T cell activation proliferation, hair Wave anti-tumor effect, and can also auxiliary adjustment T cell (reg μ Latory cell, Treg) play and inhibit sexual function, very It is converted to induction helper T lymphocyte (T helper cells, Th) to Treg.PD-1/PD-L1 is deposited extensively in a variety of solid tumors , it may be possible to one of the main reason for CAR-T technology is ineffective in solid tumor.PD-1/PD-L1 monoclonal antibody is being treated The curative effect of highly significant is had been achieved in the clinical test of solid tumor.It has been reported that PD-L1 is drenched in stomach organization and Metastasis of Gastric Cancer Significantly high expression in bar tissue is and related to the survival region of patient.

Human trophocyte cell surface antigen 2(human trophoblast cell surface antigen 2, TACSTD2/Trop2/M1S1/GA733-1) it is a film surface antigen, is located at chromosome 1q32.Trop2 is had found earliest in people Trophocyte surface, molecular weight are about 36 kDa, are single span memebrane proteins, are divided into film outskirt, transmembrane region, film inner region and a phosphorus The cytoplasm tail of acidification.Trop2 is cracked into extracellular domain and Intracellular domain by TNF-α invertase, and Intracellular domain is left after transmembrane region can be into Enter and plays a role in endochylema or core.Trop2 is often overexpressed in different epithelial neoplasms, especially gastric adenocarcinoma cells film table There is facilitation in face to the malignant behaviors of tumour.It can be used as the target spot of many targeted therapy of malignant.

Summary of the invention

The technical issues of solution: technical problems based on background technology, the purpose of the present invention is to provide a kind of double Specific chimeric antigen receptor and its application.

Technical solution: a kind of bispecific chimeric antigen receptor, the bispecific chimeric antigen receptor is by CD8 α, humanization Anti- Trop2 and anti-PD-L1 single-chain antibody, transmembrane region CD8TM and intracellular region CD28, CD137 and CD3 ζ are in series.

The amino acid sequence of above-mentioned bispecific chimeric antigen receptor is encoded as shown in SEQ ID NO.1.

Nucleic acid sequence containing encoding bispecific Chimeric antigen receptor is as shown in SEQ ID NO.2.

The heavy chain amino acid sequence of the anti-Trop2 single-chain antibody of above-mentioned people is encoded as shown in SEQ ID NO.3, light chain amino acid Sequence is as shown in SEQ ID NO.4.

The heavy chain nucleic acid sequence of the anti-Trop2 single-chain antibody of above-mentioned people is encoded as shown in SEQ ID NO.5, light chain nucleic acid sequence As shown in SEQ ID NO.6.

The heavy chain amino acid sequence of the anti-PD-L1 single-chain antibody of above-mentioned people is encoded as shown in SEQ ID NO.7, light chain amino acid Sequence is as shown in SEQ ID NO.8.

The heavy chain nucleic acid sequence of the anti-PD-L1 single-chain antibody of above-mentioned people is encoded as shown in SEQ ID NO.9, light chain nucleic acid sequence As shown in SEQ ID NO.10.

The intracellular region of above-mentioned CD8 α, transmembrane region CD8TM and intracellular region CD28, CD137 and CD3 ζ knot in series Fruit is signal transduction structural domain, and wherein amino acid sequence is respectively such as SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, shown in SEQ ID NO.19.

The nucleic acid sequence of above-mentioned signal transduction structural domain is encoded respectively such as SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, shown in SEQ ID NO.20.

The T lymphocyte of above-mentioned bispecific chimeric antigen receptor modification.

The T lymphocyte application in preparation of anti-tumor drugs of above-mentioned bispecific chimeric antigen receptor modification.

A kind of anti-gastric cancer medicament, effective component are the T lymphocyte of above-mentioned bispecific chimeric antigen receptor modification.

The utility model has the advantages that anti-Trop2 monoclonal antibody that the present invention is filtered out from human antibody library according to this laboratory and anti- The Fab of PD-L1 antibody, codon optimization add signal peptide in 5 ' sections, and full genome synthesizes Chimeric antigen receptor CD8 α- Trop2/PD-L1 scFv-CD8TM- CD28-CD137-CD3 ζ, and the sequence of the synthesis is cloned into retroviral vector In, anti-human Trop2/PD-L1 CAR expression plasmid is constructed, that is, obtains Chimeric antigen receptor CD8 α-Trop2/PD- of the invention L1 scFv-CD8TM- CD28-CD137-CD3ζ。

Using the packaging plasmid RD114env of the Chimeric antigen receptor and retrovirus in 293T cell to virus into Row packaging, using this retroviral infection T lymphocyte, makes T cell express the Chimeric antigen receptor.By the CAR-T of acquisition Cell is co-cultured with tumour cell in vitro, passes through Flow cytometry CAR-T cell surface antigen expression, CCK-8 Method detects the CAR-T cell to the specific killing action of the stomach cancer cell of Trop2/PD-L1 difference expression, to confirm to be somebody's turn to do The T lymphocyte of bispecific chimeric antigen receptor modification to the specific killing action of stomach cancer cell, and effector cell (E) and For target cell (T) in 20:1, bispecific Trop2/PD-L1 CAR-T cellkilling capacity (88.12% ± 6.08) is higher than Dan Te Anisotropic CAR-T cell (Trop2 CAR-T (67.07% ± 6.05) and PD-L1 CAR-T (51.03% ± 2.99)) and separate control Group (CD19-CAR-T (36.97% ± 4.07) and CIK (33.12% ± 3.15)).Therefore chimeric antigen of the present invention by Body CD8 α-Trop2/PD-L1 scFv-CD8TM- CD28- CD137- CD3 ζ can be applied in related neoplasms treatment.

Chimeric antigen receptor CD8 α-Trop2/PD-L1 scFv-CD8TM- CD28- CD137-CD3 ζ provided by the invention Using Retroviral technology, Infection in Vitro human T lymphocyte, ELISA experiment detection bispecific Trop2/PD-L1 CAR-T cell releases IFN-γ and IL- 2, and compared to list in the stomach cancer cell of the Trop2 and PD-L1 of killing expression Specific C AR-T groups of cells and separate control group, the CAR-T cell of bispecific can release most during killing Cell factor (IFN-γ and IL- 2).

Chimeric antigen receptor CD8 α-Trop2/PD-L1 scFv-CD8TM- CD28- CD137-CD3 ζ provided by the invention Using Retroviral technology, can Infection in Vitro human T lymphocyte, CAR-T cell obtained can pass through the single-stranded of CAR The recognition expression of antibody moiety specificity while the tumour cell of Trop2 and PD-L1, while CAR-T cell is activated, make its release Cytokine profiles realize the specific killing action to tumour cell.

Detailed description of the invention

Fig. 1 is synthesis Trop2 of the present invention, the electrophoresis of PD-L1 scFv and transmembrane region and intracellular region sequence band Figure, A figure swimming lane 1 are 10000bp nucleic acid molecular weight standard, and swimming lane 2 is Trop2 scFv, and swimming lane 3 is transmembrane region and intracellular signal Area；B figure swimming lane 1 is PD-L1 scFv, and swimming lane 2 is 2000bp nucleic acid molecular weight standard.

Fig. 2 is that Trop2/PD-L1 CAR of the present invention is cloned into retroviral vector, is usedBamH IWithXho IDouble digestion identification, release segment are CD8 α-Trop2/PD-L1 scFv-CD8TM-CD28-CD137-CD3 ζ, and size is about 1.8kb, swimming lane 1 are the nucleic acid molecular weight standard of 2000bp, and swimming lane 2 is the CAR plasmid of non-digestion, and swimming lane 3 is warpBamH IWithXho IThe plasmid of double digestion.

Fig. 3 is the CD8 α-Trop2/PD-L1 scFv- CD8TM- CD28- that the western blot detection present invention constructs CD137-CD3 ζ transfects the expression figure after 293T cell, is detected with anti-human CD3 ζ monoclonal antibody.Swimming lane 1 is to have transfected Trop2/ The 293T cell of PD-L1-CAR carrier；Swimming lane 2 is the 293T cell for having transfected CD19-CAR carrier；Swimming lane 3 is untransfected CAR The 293T cell of carrier.

Fig. 4 is the expression figure of CAR molecule in the T cell for the Trop2/PD-L1-CAR modification that the flow cytometer detection present invention constructs.

Fig. 5 is the expression figure of Trop2 and PD-L1 on western blot detection stomach cancer cell line BGC823, on normal gastric Chrotoplast strain GES-1 is control cell strain.

Fig. 6 is Trop2/PD-L1-CAR bispecific chimeric antigen receptor modification T cell of the present invention to Trop2 and PD-L1 The lethal effect of the stomach cancer cell of different expressions detects figure.Cell after transfection is transferred in no IL-12 culture medium for 24 hours, Respectively with expression Trop2⁺And PD-L1⁺BGC823, BGC823 shRNA-Trop2 and shRNA-PD-L1, BGC823-shRNA- Trop2, BGC823-shRNA-PD-L1 cell are individually incubated for 4h.Means standard deviation is by three independent experimental calculations.(A) with The target cell (T) that the BGC823 cell of expression Trop2 and PD-L1 cell is detected as CCK-8.Add in the ratio indicated in x-axis Add effector cell (E).On each E:T ratio, the difference between different groups is statistically significant (P < 0.0 5).(2) with The target cell that Trop2+/PD-L1-BGC823 cell is detected as CCK-8.Effector cell is added in the ratio indicated in x-axis. (C) with Trop2^-/PD-L1⁺The target cell that BGC823 cell is detected as CCK-8.Effector is added in the ratio indicated in x-axis Cell.(D) with Trop2^-/PD-L1^-The target cell that BGC823 cell is detected as CCK-8.In the ratio addition effect indicated in x-axis Answer device cell.* is compared with monospecific CAR-T group, and ## is compared with bispecific CAR-T group, P < 0.001.

Fig. 7 is Trop2/PD-L1-CAR bispecific chimeric antigen receptor modification T cell of the present invention to Trop2 and PD- The detection figure of cytokine release during the stomach cancer cell killing of L1 difference expression.(A) ELISA detects Trop2/PD- L1 bispecific CAR-T cell and Trop2⁺/PD-L1⁺BGC823 cell is incubated for the interferon-generated afterwards for 24 hours with the ratio of 10:1 γ, and be compared with other kinds of CAR-T cell.(B) with ELISA method detection Trop2/PD-L1 CAR-T cell with Trop2⁺/PD-L1^-The IFN-γ that BGC823 cell is generated afterwards for 24 hours in the ratio culture of E:T=10:1, and with it is other kinds of CAR-T cell is compared.(C) ELISA method detection bispecific Trop2/PD-L1 CAR-T cell and Trop2 are used^-/PD-L1⁺ BGC823 cell cultivates the IFN-γ generated afterwards for 24 hours under conditions of E:T=10:1, and carries out with other types of CAR-T cell Compare.(D) ELISA method detection Trop2/PD-L1-CAR-T cell and Trop2 are used^-/PD-L1^-BGC823 cell presses E:T=10:1 The IFN-γ that generates afterwards for 24 hours of ratio culture, and be compared with other kinds of CAR-T cell.(5) bispecific Trop2/ PD-L1 CAR-T cell and Trop2⁺/PD-L1⁺After BGC823 cell is in the ratio culture for 24 hours of E:T=10:1, examined with ELISA method The IF-2 of its release is surveyed, and is compared with other types of CAR-T cell.(F) bispecific Trop2/PD-L1 CAR-T is thin Born of the same parents and Trop2⁺/PD-L1^-After BGC823 cell is incubated for for 24 hours in the ratio of E:T=10:1, with the release of ELISA method detection IF-2, And it is compared with other types of CAR-T cell.(G) bispecific Trop2/PD-L1 CAR-T cell and Trop2^-/PD- L1⁺After BGC823 cell is incubated for for 24 hours in the ratio of E:T=10:1, with the release of ELISA method detection IF-2, and with it is other types of CAR-T cell is compared.(H) bispecific Trop2/PD-L1 CAR-T cell and Trop2^-/PD-L1^-BGC823 cell is pressed After the ratio of E:T=10:1 is incubated for for 24 hours, with the release of ELISA method detection IF-2, and compared with other types of CAR-T cell Compared with.* is compared with monospecific CAR-T group, and ## is compared with bispecific CAR-T group, P < 0.001.

Specific embodiment

The technical scheme of the invention is described in detail through specific implementation examples.

Embodiment 1

The building of Trop2/PD-L1 bispecific chimeric antigen receptor retroviral plasmid:

1. human Fab's sequence of display technique of bacteriophage screens according to this laboratory anti-Trop2 and anti-PD-L1 extracellular region V_HAnd V_LAmino acid sequence, Trop2: V_H(SEQ ID NO.3), V_L(SEQ ID NO.4)；PD-L1:V_H(SEQ ID NO.7), V_L(SEQ ID NO.8) optimizes Codon sequences using Optimum Gene TM gene design software, is not changing It is set to be more suitable for the expression system of people in the case where amino acid sequence.In V_HAnd V_LBetween be added link peptide (G₄S)₃, before add Signal peptide CD8 α, the scFv part-structure for constructing the bispecific chimeric antigen receptor of acquisition is CD8 α-Trop2 V_H-(G₄S)₃- Trop2V_L-(G₄S)₃-PD-L1 V_H-(G₄S)₃- Trop2V_L, junction introducesBamH IWithXho IRestriction enzyme site.Similarly Method digestion Psfg- CD8TM- CD28-CD137-CD3 ζ carrier recycles purpose band, as a result such as with agarose gel electrophoresis Shown in Fig. 1.

2. the carrier after the segment and digestion of recycling passes through In-Fusion PCR(Takara company) it is attached, it connects System is as follows, and target gene digestion products 100ng, 1 μ L of carrier digestion products 300ng, In-Fusion add water to complement to 20 μ L system, 37 DEG C of 15min, 50 DEG C of 15min, connection product conversion take 10 μ L products to be added in DH5 α competent cell, and 37 DEG C Overnight incubation, picking single bacterium colony expand culture, extract positive colony plasmid with plasmid extraction kit (Axygene company), Through digestion and sequencing detection, correct carrier is named as CD8 α-Trop2/PD-L1 scFv-CD8TM-CD28-CD137-CD3 ζ.Nucleic acid sequence is as shown in SEQ ID NO.2.

Bispecific chimeric antigen receptor retroviral plasmid restriction enzymeBamH IWithXho ICarry out double enzymes It cuts, digestion system is as follows,BamH I 2μL、Xho I 2 μ L, 10 × K buffer, 5 μ L, 5 BSA μ L, 5 μ g of CAR plasmid, add Water is supplied to 20 μ L, and 37 DEG C of water-baths are overnight, and 1% agarose gel electrophoresis of digestion products cuts purpose band under ultraviolet, adopts Target fragment is recycled with DNA gel QIAquick Gel Extraction Kit (Axygene company), result is as shown in Figure 2.

Embodiment 2

Bispecific chimeric antigen receptor CD8 α-Trop2/PD-L1 scFv-CD8TM- CD28- CD137- CD3 ζ expression mirror It is fixed:

The bispecific chimeric antigen is extracted referring to the operational manual of (Tiangeng biology) in the big extraction reagent kit of endotoxin-free plasmid Receptor retroviral plasmid is transfected into human embryonic kidney cells 293T cell with PI transfection reagent, after 48h, is washed one time with PBS, Reagent RIPA lytic cell is extracted with cell protein, the albumen of the 293T cell after extracting transfection is carried out through 10% SDS-PAGE Gel electrophoresis, transferring film overnight, then with the secondary antibody that horseradish crosses the anti-mouse that oxygenase marks are incubated with 4 DEG C of the anti-human CD3 ζ antibody incubation of mouse Educate 1h, ECL colour developing.As a result as shown in figure 3, anti-human CD3 ζ antibody can detect CAR developed by molecule, protein molecular size and theory It is consistent, is 75kDa(such as Fig. 3).

Embodiment 3

Bispecific chimeric antigen receptor CD8 α-Trop2/PD-L1 scFv-CD8TM- CD28- CD137- CD3 ζ modification The preparation of T lymphocyte:

1. expressing the packaging preparation of the retrovirus of CAR molecule

Plasmid extraction kit extracts packaging plasmid pRD114 and the Peg-pam3 plasmid of retrovirus, with CD8 α-Trop2/ PD-L1 scFv-CD8TM- CD28-CD137-CD3 ζ retroviral plasmid cotransfection 293T cell is collected carefully after being incubated for 48h Born of the same parents' supernatant, 4000rpm are centrifuged 10min, and then with 0.45 μm of membrane filtration, -80 DEG C of packing are frozen.

2. the preparation of T lymphocyte

The fresh anticoagulation for taking 30mL healthy volunteer is thin with lymphocyte separation medium (GE company) separation peripheral blood mononuclear Born of the same parents (PBMC).The isolated cell orifice plate stimulation 48h for being coated with CD3 and CD28, with T lymphocyte culture medium GT-T551 (Takara company) plus 3% self blood plasma carry out Fiber differentiation, obtain T lymphocyte.

3. the preparation of CAR-T cell

Takara company is purchased from the RetroNectin(of 50 μ g/mL) non-24 orifice plate of tissue cultures of coating, 500 μ L of every hole, 4 DEG C overnight, then 1.5mL retrovirus is added in every hole, and 32 DEG C of placements 1h abandon supernatant, again the reverse transcription of addition 1.5mL T lymphocyte and retrovirus are added in hole again by virus, 32 DEG C of placement 1h after discarding supernatant, to obtain expression CD8 α- Trop2/PD-L1 scFv-CD8TM- CD28- CD137- CD3 ζ T cell, that is, CAR-T cell.

4. the expression of CAR molecule in flow cytometer detection CAR-T cell

CD8 α-Trop2/PD-L1 scFv-CD8TM- CD28-CD137-CD3 ζ T cell, that is, CAR-T cell PE will be expressed After 37 DEG C of anti-human Fc antibodies' (being purchased from Jackson company) of label are incubated for 1h, progress flow cytometer detection, testing result such as Fig. 4, as a result Show that the CAR-T cell efficiency of this method preparation is greater than 90%.

Embodiment 4

The T lymphocyte of Chimeric antigen receptor CD8 α-Trop2/PD-L1 scFv-CD8TM- CD28-CD137-CD3 ζ modification To the lethal effect of the stomach cancer cell of expression Trop2 and PD-L1:

1. the expression that Western blot detects Trop2 and PD-L1 in stomach cancer cell

BGC823 is stomach cancer cell, people gastric epithelial cell GES-1(Kai Ji company), cell with RIPA cell pyrolysis liquid ( Add the proteinase inhibitor C ocktail Tablets of Roche) crack 30 min on ice after, extract cell protein, carry out Western blot detection, testing result such as Fig. 5.

3. CAR-T cell detects stomach cancer cell lethality

Tumour cell is adjusted to 4 × 10 with culture medium⁶/ mL, places 30min on ice, after washing three times with PBS, with 1640 trainings It supports base weight and hangs cell, count.Adjust separately stomach cancer cell (Trop2⁺/PD-L1⁺、Trop2⁺/PD-L1^-、Trop2^-/PD-L1⁺、 Trop2^-/PD-L1^-) to 4 × 10⁶/ mL takes 250 μ L to be added in 24 orifice plates, is 20:1 by E:T；10:1；5:1；2:1 difference CAR-T cell 5 × 10 is added⁵；2×10⁵；1×10⁵；5×10⁴, 4h is co-cultured, 3 multiple holes are arranged in every hole, after cell mixes, It is placed in 96 orifice plates and is put into 37 DEG C, 5%CO₂Incubator in be incubated for 24 hours.Cell conditioned medium is sucked, PBS solution cleaning 3 is used Time, the adherent cell of Kong Zhongwei is washed, the mixing of 10+90 μ L RPMI of μ L CCK-8 solution, 1640 culture medium is added in every hole Liquid, 37 DEG C, 5%CO₂Incubator in be incubated for 4 hours.CCK-8 testing result (such as Fig. 6) shows CAR-T cell to Trop2/PD- L1, which expresses higher BGC823 cell, preferable lethality, and kills ability and be higher than monospecific CAR-T cell (Trop2 CAR-T and PD-L1 CAR-T) and separate control group (CD19-CAR-T and CIK).

4. the release conditions of ELISA detection CAR-T born of the same parents cell factor IFN-γ and IL-2 during killing.

IFN-γ antibody (1:250 dilution) and IL-2(1:250 dilution are diluted with sterile coating buffer), in elisa plate It is upper that every 100 μ L of hole is added, 4 DEG C of refrigerator overnights are placed on, are coated with.Coating buffer is sopped up from plate within second day.With PBS board-washing two It is secondary, 200 holes μ L/.200 μ L Dliuent containing ELISA are added in every hole, place 1 hour at 37 DEG C.Supernatant is sucked, is washed again with PBS 3 times.

By E:T=10:1, effector cell (E) and target cell (T) are added in 24 orifice plates, are placed in 37 DEG C, 5%CO₂Culture 24 hours.After 24 hours, cell is washed off, three times with PBS board-washing.Anti- IFN-γ antibody and the IL-2 for diluting biotin labeling are anti- Body, every hole add 100 μ L, are incubated at room temperature 1 hour.Antibody is washed off, then is washed 4 times with PBS.Prepare Avidin-HRP, add 100 μ L into Every hole.It is placed at room temperature for 1 hour.Avidin-HRP is washed off, is washed 3 times with PBS-tween20, is then washed 3 times with PBS.Prepare TMB Solution is added the 100 every holes μ L, room temperature 15 minutes, 50 μ L 1M H is added₃PO₄Solution terminates reaction.Microplate reader measures each Kong Yu Absorbance value (result such as Fig. 7) at 450nm, the results showed that bispecific Trop2/PD-L1 CAR-T cell is in killing expression When the stomach cancer cell line BGC823 of Trop2 and PD-L1, IFN-γ and IL- 2 are released, and thin compared to monospecific CAR-T Born of the same parents' group and separate control group, the CAR-T cell of bispecific can release most cell factor (IFN- during killing γ and IL- 2).

Sequence table

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

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

20 25 30

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

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

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Gly Leu Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr

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

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Ala Val Tyr Tyr Cys Ala Arg Asp Pro Gly Trp Gly Phe Asp Tyr Trp

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

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Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu

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

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Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala

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

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

225 230 235 240

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

245 250 255

Gly Pro Gly Thr Lys Val Asp Ile Lys Gly Gly Gly Gly Ser Gly Gly

260 265 270

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala

275 280 285

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

290 295 300

Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val

305 310 315 320

Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr

325 330 335

Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser

340 345 350

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

355 360 365

Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser Ser Thr Arg

370 375 380

Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gly Gly Gly Ser

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

405 410 415

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

420 425 430

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ile

435 440 445

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

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Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Ser Val Lys

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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

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

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Lys Asp Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln Gly

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Thr Leu Val Thr Val Ser Ser Phe Trp Val Leu Val Val Val Gly Gly

530 535 540

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

545 550 555 560

Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn

565 570 575

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

580 585 590

Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Thr Thr Thr Pro Ala

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

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

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

785 790 795 800

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

805 810 815

Ala Leu His Met Gln Ala Leu Pro Pro Arg

820 825

<210> 2

<211> 4177

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

ttgtctgctg cagcatcgtt ctgtgttgtc tctgtctgac tgtgtttctg tatttgtctg 60

aaaatatggg cccgggctag cctgttacca ctcccttaag tttgacctta ggtcactgga 120

aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta 180

ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta 240

accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc 300

cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg 360

tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc 420

cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt 480

ctctaggcgc ccccatatgg ccatatgaga tcttatatgg ggcacccccg ccccttgtaa 540

acttccctga ccctgacatg acaagagtta ctaacagccc ctctctccaa gctcacttac 600

aggctctcta cttagtccag cacgaagtct ggagacctct ggcggcagcc taccaagaac 660

aactggaccg accggtggta cctcaccctt accgagtcgg cgacacagtg tgggtccgcc 720

gacaccagac taagaaccta gaacctcgct ggaaaggacc ttacacagtc ctgctgacca 780

cccccaccgc cctcaaagta gacggcatcg cagcttggat acacgccgcc cacgtgaagg 840

ctgccgaccc cgggggtgga ccatcctcta gggatccatg gccttaccag tgaccgcctt 900

gctcctgccg ctggccttgc tgctccacgc cgccaggccg caggtgcagc tggtggagtc 960

tgggggaggc gtggtccagc ctgggaggtc cctgagactc tcctgtgcag cgtctggatt 1020

caccttcagt agctatggca tgcactgggt ccgccaggct ccaggcaagg ggctggagtg 1080

ggtggcagtt atatggtatg atggaagtaa taaatactat gcagactccg tgaagggccg 1140

attcaccatc tccagagaca attccaagaa cacgctgtat ctgcaaatga acagcctgag 1200

agccgaggac acggctgtgt attactgtgc gagagatccg ggctggggat ttgactactg 1260

gggccaggga accctggtca ccgtctcctc aggtggtggt ggttctggtg gtggtggttc 1320

tggcggcggc ggctccggtg gtggtggttc ggagctccag atgacccagt ctccatcctc 1380

cctgtctgca tctgtaggag acagagtcac catcacttgc cgggcaagtc agagcattag 1440

cagctattta aattggtatc agcagaaacc agggaaagcc cctaagctcc tgatctatgc 1500

tgcatccagt ttgcaaagtg gggtcccatc aaggttcagt ggcagtggat ctgggacaga 1560

tttcactctc accatcagca gtctgcaacc tgaagatttt gcaacttact actgtcaaca 1620

gagttacagt accccattca ctttcggccc tgggaccaaa gtggatatca aaggtggtgg 1680

tggttctggt ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt cgcagtctgc 1740

cctgactcag cctgcctccg tgtctgggtc tcctggacag tcgatcacca tcagctgcac 1800

tggcacctcc agtgacgttg gcggctataa ctacgtctcc tggtaccaac agcacccagg 1860

caaggccccc aaactcatga tctacgaggt gtccaaccgg ccctcagggg tttctaacag 1920

attctctggc tccaagtccg gcaacacggc ctccctgacc atcagcggac tgcaggctga 1980

ggacgaggct gattattact gctcctcata tacctcctcc agcactagag tgttcggcac 2040

cggcacaaaa gtgaccgtgc tgggtggtgg tggttctggt ggtggtggtt ctggcggcgg 2100

cggctccggt ggtggtggtt cggaggtgca gctgttggag tctgggggag gactggtaca 2160

gcctgggggg tccctgagac tctcctgtgc agcctccgga ttcacctttt ccagctacat 2220

catgatgtgg gtccgccagg ctccagggaa gggcctggaa tgggtgtcat ccatttaccc 2280

ctccggcggc atcaccttct acgccgactc cgtgaagggc cggttcacca tctcccggga 2340

caattccaag aacacgctgt acctgcaaat gaactccctg cgggccgagg acacggccgt 2400

atattactgc gcgaaagaca agctgggcac cgtgaccacc gtggactact ggggccaggg 2460

caccctggtg acagtgtcct ccttttgggt gctggtggtg gttggtggag tcctggcttg 2520

ctatagcttg ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag 2580

gctcctgcac agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca 2640

ttaccagccc tatgccccac cacgcgactt cgcagcctat cgctccacca cgacgccagc 2700

gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag cccctgtccc tgcgcccaga 2760

ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga 2820

tatctacatc tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat 2880

caccctttac tgcaaacggg gcagaaagaa actcctgtat atattcaaac aaccatttat 2940

gagaccagta caaactactc aagaggaaga tggctgtagc tgccgatttc cagaagaaga 3000

agaaggagga tgtgaactga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacaa 3060

gcagggccag aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt 3120

tttggacaag agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc 3180

tcaggaaggc ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat 3240

tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag 3300

tacagccacc aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgctaaac 3360

gcgttagcat gcacctcgag atcgatccgg attagtccaa tttgttaaag acaggatatc 3420

agtggtccag gctctagttt tgactcaaca atatcaccag ctgaagccta tagagtacga 3480

gccatagata aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc 3540

cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat ggaaaaatac 3600

ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag ctgaatatgg 3660

gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg 3720

aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg 3780

gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag agaaccatca 3840

gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa 3900

tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc 3960

acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg tacccgtgta 4020

tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct tgggagggtc 4080

tcctctgagt gattgactac ccgtcagcgg gggtctttca catgcagcat gtatcaaaat 4140

taatttggtt ttttttctta agtatttaca ttaaatg 4177

<210> 3

<211> 117

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 3

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Asp Pro Gly Trp Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 4

<211> 107

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 5

<211> 351

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagatccg 300

ggctggggat ttgactactg gggccaggga accctggtca ccgtctcctc a 351

<210> 6

<211> 321

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

gagctccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ccccattcac tttcggccct 300

gggaccaaag tggatatcaa a 321

<210> 7

<211> 110

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

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

35 40 45

Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 8

<211> 120

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Lys Asp Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 9

<211> 330

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60

agctgcactg gcacctccag tgacgttggc ggctataact acgtctcctg gtaccaacag 120

cacccaggca aggcccccaa actcatgatc tacgaggtgt ccaaccggcc ctcaggggtt 180

tctaacagat tctctggctc caagtccggc aacacggcct ccctgaccat cagcggactg 240

caggctgagg acgaggctga ttattactgc tcctcatata cctcctccag cactagagtg 300

ttcggcaccg gcacaaaagt gaccgtgctg 330

<210> 10

<211> 360

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

gaggtgcagc tgttggagtc tgggggagga ctggtacagc ctggggggtc cctgagactc 60

tcctgtgcag cctccggatt caccttttcc agctacatca tgatgtgggt ccgccaggct 120

ccagggaagg gcctggaatg ggtgtcatcc atttacccct ccggcggcat caccttctac 180

gccgactccg tgaagggccg gttcaccatc tcccgggaca attccaagaa cacgctgtac 240

ctgcaaatga actccctgcg ggccgaggac acggccgtat attactgcgc gaaagacaag 300

ctgggcaccg tgaccaccgt ggactactgg ggccagggca ccctggtgac agtgtcctcc 360

<210> 11

<211> 21

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

His Ala Ala Arg Pro

20

<210> 12

<211> 63

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 13

<211> 68

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 13

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser

20 25 30

Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly

35 40 45

Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala

50 55 60

Ala Tyr Arg Ser

65

<210> 14

<211> 204

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 120

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180

cgcgacttcg cagcctatcg ctcc 204

<210> 15

<211> 69

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 15

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> 16

<211> 207

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

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> 17

<211> 42

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 17

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> 18

<211> 126

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 19

<211> 112

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 19

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 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> 20

<211> 336

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca 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 cctcgc 336

Claims (10)

1. a kind of bispecific chimeric antigen receptor, it is characterised in that the bispecific chimeric antigen receptor is by CD8 α, humanization Anti- Trop2 and anti-PD-L1 single-chain antibody, transmembrane region CD8TM and intracellular region CD28, CD137 and CD3 ζ are in series.

2. bispecific chimeric antigen receptor according to claim 1, it is characterised in that it is anti-to encode the bispecific chimeric The amino acid sequence of original receptor is as shown in SEQ ID NO.1.

3. bispecific chimeric antigen receptor according to claim 1, it is characterised in that contain encoding bispecific inosculating antibody The nucleic acid sequence of original receptor is as shown in SEQ ID NO.2.

4. bispecific chimeric antigen receptor according to claim 1, it is characterised in that it is single-stranded anti-to encode the anti-Trop2 of the people The heavy chain amino acid sequence of body is as shown in SEQ ID NO.3, and light-chain amino acid sequence is as shown in SEQ ID NO.4；Described in coding The heavy chain amino acid sequence of the anti-PD-L1 single-chain antibody of people is as shown in SEQ ID NO.7, light-chain amino acid sequence such as SEQ ID Shown in NO.8.

5. bispecific chimeric antigen receptor according to claim 4, it is characterised in that it is single-stranded anti-to encode the anti-Trop2 of the people The heavy chain nucleic acid sequence of body is as shown in SEQ ID NO.5, and light chain nucleic acid sequence is as shown in SEQ ID NO.6；It is anti-to encode the people The heavy chain nucleic acid sequence of PD-L1 single-chain antibody is as shown in SEQ ID NO.9, and light chain nucleic acid sequence is as shown in SEQ ID NO.10.

6. bispecific chimeric antigen receptor according to claim 1, it is characterised in that the CD8 α, transmembrane region CD8TM, with And the intracellular region result in series of intracellular region CD28, CD137 and CD3 ζ are signal transduction structural domain, wherein amino acid sequence Column are respectively as shown in SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19.

7. bispecific chimeric antigen receptor according to claim 6, it is characterised in that encode the signal transduction structural domain Nucleic acid sequence respectively such as SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID Shown in NO.20.

8. the T lymphocyte of any one of the claim 1-7 bispecific chimeric antigen receptor modification.

9. T lymphocyte the answering in the preparation of antitumor drugs of the modification of bispecific chimeric antigen receptor described in claim 8 With.

10. a kind of anti-gastric cancer medicament, it is characterised in that effective component is that bispecific chimeric antigen receptor described in claim 8 is repaired The T lymphocyte of decorations.