CN109161532A - The engineering immunocyte of PSMA and PD-L1 is targeted simultaneously - Google Patents

Abstract

The present invention relates to the engineering immunocytes for targeting PSMA and PD-L1 simultaneously.Specifically, the present invention relates to a kind of engineering immunocytes for targeting PD-L1 and prostate-specific membrane antigen (PSMA) simultaneously, there is good lethal effect to the tumour of double positives, the tumours of the double positives for the treatment of PD-L1 and prostate-specific membrane antigen that can be specific, and do not have lethal effect to the normal cell of the mono- positive of PSMA, toxic side effect of missing the target is greatly reduced, safety is enhanced.

Description

The engineering immunocyte of PSMA and PD-L1 is targeted simultaneously

Technical field

The present invention relates to immunotherapy fields, immune thin more particularly to the engineering for targeting PSMA and PD-L1 simultaneously Born of the same parents.

Background technique

Prostate cancer is the highest cancer of male cancer deaths rate.It is cut currently, prostate cancer therapy means are mainly performed the operation It removes, radiotherapy, chemotherapy, but these common treatment means there is no conspicuousness extend the life cycle of prostate cancer patient. Therefore, prostate cancer therapy is badly in need of new therapeutic scheme.Nearly 2 years, Chimeric antigen receptor T cell (CAR-T) was used as tumor target To immunization therapy, good therapeutic effect, the especially CAR- to target CD19 molecule are all shown with clinical trial in vitro CD19T cell obtains soul-stirring result in blood tumor.But CAR-T does not obtain good treatment in solid tumor Effect, mainly lacks the antigen of real tumour-specific, undershooting-effect occur in a variety of CAR-T, to normal cell in solid tumor There is also lethal effects.

Therefore, this field forwardly needs to develop that specificity is good, curative effect is stable, the treatment prostate cancers of Small side effects Cellular immunotherapy.

Summary of the invention

That the object of the present invention is to provide a species specificity is good, curative effect is stable, the cell of the treatment prostate cancer of Small side effects Immunotherapy.

It is a further object of the present invention to provide one kind can target simultaneously PD-L1 and PSMA engineering immunocyte and its Purposes.

The first aspect of the present invention, provides a kind of immunocyte of engineering, and the immunocyte expresses the first CAR With the 2nd CAR；And

The structure of first CAR is as shown in following formula I: L1-scFv1-H1-TM1-C (I)；

The structure of 2nd CAR is as shown in Formula Il: L2-scFv2-H2-TM2-CD3 ζ (II)；

In formula,

Each "-" independently is link peptide or peptide bond；

L1 and L2 is each independently optional signal peptide sequence；

One is the antigen-binding domains for targeting PSMA in both scFv1 and scFv2, another is targeting PD-L1 Antigen-binding domains；

H1 and H2 is each independently optional hinge area；

TM1 and TM2 are each independently transmembrane domain；

C is costimulatory signal molecule；

CD3 ζ is the endochylema signal transduction sequence derived from CD3 ζ.

In another preferred example, scFv1 is the antigen-binding domains for targeting PSMA, and scFv2 is to target resisting for PD-L1 Former binding structural domain.

In another preferred example, the first CAR targets PSMA, and the 2nd CAR targets PD-L1.

In another preferred example, the amino acid sequence such as SEQ ID NO.:1 of the antigen-binding domains of the targeting PSMA In shown in 21-260.

In another preferred example, the nucleotide sequence such as SEQ ID NO.:1 of the antigen-binding domains of the targeting PSMA In shown in 61-780.

In another preferred example, the amino acid sequence such as SEQ ID NO. of the antigen-binding domains of the targeting PD-L1: In 2 shown in 21-171.

In another preferred example, the nucleotide sequence such as SEQ ID NO. of the antigen-binding domains of the targeting PD-L1: In 2 shown in 61-513.

In another preferred example, the immunocyte of the engineering is selected from the group: Chimeric antigen receptor T cell (CAR- T cell), Chimeric antigen receptor NK cell (CAR-NK cell), or combinations thereof.

In another preferred example, the immunocyte of the engineering is CAR-T cell.

In another preferred example, the first CAR and the 2nd CAR is positioned at the cell membrane of the immunocyte.

In another preferred example, expression has the first CAR and the 2nd CAR on the cell membrane of the immunocyte.

In another preferred example, described L1, L2 are each independently the signal peptide of albumen selected from the group below: CD8, CD28, GM-CSF, CD137, or combinations thereof.

In another preferred example, the L1 is the signal peptide of CD8 albumen, preferably such as 1- in SEQ ID NO.:1 Shown in 20.

In another preferred example, the L2 is the signal peptide of CD8 albumen, preferably such as 1- in SEQ ID NO.:3 Shown in 20.

In another preferred example, described H1, H2 are each independently the hinge area of albumen selected from the group below: CD8, CD28, CD137, or combinations thereof, preferably, being each independently the hinge area of CD8 albumen.

In another preferred example, described TM1, TM2 are each independently the transmembrane region of albumen selected from the group below: CD28, CD8, CD9, CD16, CD22, CD137, CD154, or combinations thereof.

In another preferred example, the TM1 and TM2 is each independently the transmembrane region in the source CD8.

In another preferred example, 261-329 in the amino acid sequence of the TM1 or TM2 such as SEQ ID NO.:5 It is shown.

In another preferred example, the C is the costimulatory signal molecule of albumen selected from the group below: CD28,4-1BB (CD137), OX40, ICOS, or combinations thereof.

In another preferred example, the C is the costimulatory signal molecule in the source 4-1BB.

In another preferred example, the amino acid sequence of the 4-1BB such as 330-371 institutes in SEQ ID NO.:5 Show.

In another preferred example, the amino acid sequence of the CD3 ζ is as shown in 632-743 in SEQ ID NO.:5.

In another preferred example, the amino acid sequence of the first CAR is as shown in 1-371 in SEQ ID NO.:5.

In another preferred example, the amino acid sequence of the 2nd CAR such as 392-743 institutes in SEQ ID NO.:5 Show.

The second aspect of the present invention provides a kind of method of preparation engineering immunocyte, comprising the following steps:

(A) immunocyte to be rebuilt is provided；With

(B) immunocyte is transformed, so that the immunocyte expresses the first CAR and second CAR, to obtain the immunocyte of engineering described in first aspect present invention.

In another preferred example, in step (B), the first expression cassette for being used to express the first CAR is imported including (B1) To the immunocyte；The second expression cassette for being used to express the 2nd CAR is imported into the immunocyte by (B2), wherein institute Stating step (B2) can be before step (B1), later, either simultaneously or alternately carries out.

In another preferred example, when the immunocyte to be rebuilt in step (A) has expressed the first CAR or the 2nd CAR When, then step (B1) or step (B2) can be omitted.

In another preferred example, the immunocyte is T cell or NK cell.

In another preferred example, first expression cassette and the second expression cassette are located on identical or different carrier.

In another preferred example, first expression cassette and the second expression cassette are located at identical carrier.

In another preferred example, the carrier is viral vectors.

In another preferred example, the carrier is selected from the group: DNA, RNA, plasmid, slow virus carrier, adenovirus carry Body, retroviral vector, transposons, other gene transfer systems, or combinations thereof.

In another preferred example, the method further includes that function and effectively is carried out to the engineering immunocyte of acquisition Property detection the step of.

The third aspect of the present invention, provides a kind of preparation, and the preparation contains engineering described in first aspect present invention Immunocyte and pharmaceutically acceptable carrier, the diluent or excipient of change.

In another preferred example, the preparation is liquid formulation.

In another preferred example, the dosage form of the preparation is injection.

In another preferred example, the concentration of the immunocyte of engineering described in the preparation is 1 × 10³-1×10⁸It is a Cell/ml, preferably 1 × 10⁴-1×10⁷A cell/ml.

The fourth aspect of the present invention provides a kind of immunocyte of engineering as described in the first aspect of the invention Purposes is used to prepare the drug or preparation of prevention and/or treating cancer or tumour.

In another preferred example, it is used to prepare prevention and/or treats and express the positive with the PSMA expression positive and PD-L1 The drug or preparation of relevant disease.

In another preferred example, the tumour is selected from the group: neoplastic hematologic disorder, solid tumor, or combinations thereof.

In another preferred example, the solid tumor is selected from the group: gastric cancer, gastric cancer peritoneum transfer, liver cancer, leukaemia, kidney Tumour, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, cervical carcinoma, oophoroma, lymph cancer, Nasopharyngeal carcinoma, adrenal tumor, tumor of bladder, non-small cell lung cancer (NSCLC), glioma, carcinoma of endometrium, or combinations thereof.

In another preferred example, the solid tumor is prostate cancer.

The fifth aspect of the present invention provides a kind of kit for being used to prepare cell described in first aspect present invention, institute It states kit and contains container, and in container:

(1) first nucleic acid sequence, first nucleic acid sequence contain the first expression cassette for expressing the first CAR； With

(2) second nucleotide sequence, the second nucleotide sequence contain the second expression cassette for expressing the 2nd CAR.

In another preferred example, first and second nucleic acid sequences are located in identical or different container.

In another preferred example, first and second nucleic acid sequences are located at same expression vector.

In the sixth aspect of the present invention, a kind of method for treating disease is provided, including is applied to object in need for the treatment of Preparation described in cell described in suitable first aspect present invention or third aspect present invention.

In another preferred example, the disease is cancer or tumour.

In another preferred example, the disease is to express the positive relevant disease of positive and PD-L1 expression to PSMA.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, Not repeated them here.

Detailed description of the invention

Fig. 1 shows the structural schematic diagram of the CAR of targeting prostate gland cancer cell surface antigen PSMA.

Fig. 2A shows that pLVX- α PSMA-CAR plasmid construct schematic diagram, Fig. 2 B show successfully building pLVX- α PSMA- CAR plasmid.

Fig. 3 shows the structural schematic diagram of the CAR of targeting PD-L1.

Fig. 4 A shows that the structural schematic diagram of pLVX-PD1-CAR plasmid, Fig. 4 B show successfully to construct the pLVX-PD1- CAR plasmid.

Fig. 5 shows the structural schematic diagram of α PSMA-PD1-CAR.

Fig. 6 A shows that the structural schematic diagram of pLVX- α PSMA-PD1-CAR plasmid, Fig. 6 B show successfully to construct described PLVX- α PSMA-PD1-CAR plasmid.

Fig. 7 shows the flow cytometer showed result of JurCAR- α PSMA, JurCAR-PD1, JurCAR- α PSMA-PD1 cell.

Fig. 8 shows that α PSMA-PD1-CAR-T specific killing prostate gland cancer cell PC3 is normal without killing prostate Cell PNTIA.

Specific embodiment

The present inventor is by depth studying extensively, being surprised to find that for the first time a kind of while targeting PD-L1 and prostate The engineering immunocyte of specific membrane antigen (PSMA) has good lethal effect to the tumour of double positives, only can specificity Double positive tumor cells for all expressing of killing PD-L1 and PSMA, and do not have killing to the normal cell of the mono- positive of PSMA and make With greatly reducing toxic side effect of missing the target, enhance safety.And the study found that the first CAR targets PD-L1, the 2nd CAR When targeting PSMA, engineering immunocyte still has faint lethal effect, may damage normal prostatic cell；And When the first CAR targets PSMA, the 2nd CAR targeting PD-L1, the safety for being engineered immunocyte is higher, hardly kills just Normal prostatic cell.On this basis, the present invention is completed.

Present invention firstly provides the undershooting-effect that CAR-T cell is reduced with the bis- target spots of PD-L1 and PSMA, this α PSMA- PD1-CAR-T killing prostate cancer cell, does not kill normal prostate cell, this is a kind of safety-type CAR-T.The present invention The scFv for targeting PSMA (abbreviation PSMA scFv) is connected with costimulatory molecules 4-1BB, by PD-1 extracellular fragment and CD3 ζ sequence Column are connected, and are connected between the two segments with T2A, construct the second generation CAR matter of scFv containing PSMA and PD-1 extracellular fragment , PSMA scFv and costimulatory molecules 4-1BB amalgamation and expression, PD-1 extracellular fragment after the slow-virus infection Jurkat cell of packaging With CD3 ζ amalgamation and expression, since costimulatory molecules 4-1BB and CD3 ζ are separately expressed, which only has while identifying PSMA With can just be activated after PD-L1 molecule, and then can kill target cell (such as PSMA and PD-L1 coexpression prostate cancer it is thin Born of the same parents), and the normal prostate cell for only expressing PSMA cannot activate the CAR-T cell, from without lethal effect.Therefore, should Double targeting CAR-T cells have good safety, and the undershooting-effect of CAR-T cell can be effectively reduced.

Term

In order to which the disclosure can be more easily to understand, certain terms are defined first.As used in this application, unless Otherwise herein clearly stipulate that otherwise each of following term should have meaning given below.It is illustrated in entire application Other definition.

Term " about " can refer to the acceptable error model of the particular value or composition that determine in those of ordinary skill in the art Value or composition in enclosing, will depend partially on how measuring or measured value or composition.

Term " giving " refers to will using any one of various methods well known by persons skilled in the art and delivery system Product physics of the invention introduces subject, including intravenous, intramuscular, subcutaneously, in peritonaeum, spinal cord or other parenteral administrations Approach, such as by injecting or being transfused.

As used herein, " PD-L1 " refers to programmed death ligand 1, the high expression in a variety of cancer cells, and before normal It is not expressed in column gland cell.

Prostate-specific membrane antigen (PSMA)

Prostate-specific membrane antigen (PSMA) is a kind of newly to be formed in prostatic epithelium cancer cell and a variety of solid tumors The transmembrane protein of (new vessels of normal tissue are expressed without PSMA) overexpression in blood vessel, it is non-in metastatic and androgen Expression highest on dependence prostate gland cancer cell, while it also has a little expression in prostate normal cell.

The killing prostate specific membrane antigen of engineering immunocyte energy specificity in the present invention expresses the swollen of the positive Oncocyte, to achieve the effect that treat tumour.

Chimeric antigen receptor (CAR)

Chimeric immunizing antigen receptor (Chimeric antigen receptors, CARs) by extracellular antigen recognition region, Usually scFv (single-chain variable fragment), transmembrane region and costimulatory signal region intracellular composition. The design of CARs experienced following procedure: only one intracellular signal component C D3 ζ or Fc γ RI molecule of first generation CAR, by In only one activation domain intracellular, therefore it can only cause of short duration T cell proliferation and less cytokine secretion, And prolonged T cell proliferation signal and lasting internal anti-tumor effect can not be provided, so there is no obtain well Clinical efficacy.Second generation CARs introduces a costimulatory molecules on the basis of original structure, as CD28,4-1BB, OX40, ICOS, function improves a lot compared with generation CARs, further strengthens the duration of CAR-T cell and to tumour cell Killing ability.Connect some new co-stimulators such as CD27, CD134 on the basis of two generation CARs, develops into three generations With four generation CARs.

The extracellular fragment of CARs can recognize a special antigen, is then transduceed the signal, is caused carefully by intracellular domain Activation and proliferation, cell dissolution toxicity and the secrete cytokines of born of the same parents, and then remove target cell.Patient's autogenous cell is separated first (or allogeneic donor), activate and carry out genetic modification generate CAR immunocyte, be subsequently injected into it is same in patient body.It is this It is extremely low that mode suffers from graft versus host disease(GVH disease) probability, and antigen is identified in a manner of non-MHC limitation by immunocyte.

CAR- immune cell therapy achieves very high clinical response rate in hematologic malignancies treatment, such High reactivity is that previous any treatment means are all unable to reach, and has respectively caused the upsurge of clinical research in the world.

Specifically, Chimeric antigen receptor of the invention (CAR) includes extracellular domain, transmembrane domain and intracellular Structural domain.Extracellular domain includes target-specific binding members (also referred to as antigen-binding domains).Intracellular domain packet Include costimulatory signal conducting region and/or ζ chain part.Costimulatory signal conducting region refers to the intracellular structure including costimulatory molecules The a part in domain.Costimulatory molecules are cell surface molecule required for effective response of the lymphocyte to antigen, rather than anti- Original receptor or their ligand.

Between the extracellular domain and transmembrane domain of CAR, or CAR cytoplasmic domain and transmembrane domain it Between, it may be incorporated into connector.As used herein, term " connector " is often referred to play the born of the same parents that transmembrane domain is connected to polypeptide chain Extracellular portion or any oligopeptides or polypeptide of cytoplasmic domain effect.Connector may include 0-300 amino acid, preferably 2 to 100 amino acid and most preferably 3 to 50 amino acid.

CAR of the invention can carry out antigen recognizing based on antigen-binding specificity when expressing in T cell.When it When in conjunction with its associated antigen, tumour cell is influenced, causes tumour cell not grow, be prompted to death or otherwise by shadow It rings, and the tumor load of patient is caused to reduce or eliminate.Antigen-binding domains are preferably and from costimulatory molecules and/or ζ chain One or more of intracellular domain fusion.Preferably, antigen-binding domains and 4-1BB signal transduction structural domain And/or the intracellular domain fusion of CD3 ζ signal domain combination.

As used herein, " antigen-binding domains " " single chain antibody fragments " refer both to the Fab piece with antigen-binding activity Section, Fab ' segment, F (ab ')₂Segment or single Fv segment.Fv antibody contains antibody heavy chain variable region (VH), light chain variable region (VL), but there is no constant region, and there is the minimum antibody fragment of whole antigen binding sites.In general, Fv antibody also includes VH Peptide linker between VL structural domain, and structure needed for being capable of forming antigen binding.Antigen-binding domains are usually scFv(single-chain variable fragment).Be typically of size of a complete antibody 1/6 of scFv.It is single-stranded Antibody is preferably the amino acid chain sequence encoded by a nucleotide chain.As preferred embodiment of the invention, the scFv Antibody comprising specific recognition tumour high-expression antigen PD-L1 and/or PSMA, preferably single-chain antibody.

In the present invention, scFv of the invention further includes its conservative variant, is referred to and the amino acid sequence of scFv of the present invention Column are compared, and have at most 10, and preferably at most 8, more preferably at most 5, most preferably at most 3 amino acid are similar by property Or similar amino acid is replaced and forms polypeptide.

In the present invention, the addition, missing, modification and/or the amino acid quantity replaced, preferably no more than initially The 40% of amino acid sequence total amino acid quantity, more preferably less than 35%, more preferably 1-33%, more preferably 5- 30%, more preferably 10-25%, more preferably 15-20%.

In the present invention, the addition, missing, modification and/or the amino acid quantity replaced are usually 1,2,3,4 or 5 It is a, preferably 1-3, it is more preferably 1-2, is most preferably 1.

For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include the extracellular structure for being fused to CAR The transmembrane domain in domain.In one embodiment, using naturally with the associated transmembrane structure of one of structural domain in CAR Domain.In some instances, transmembrane domain may be selected, or modified by amino acid replacement, to avoid by such structure Domain is bound to the transmembrane domain of identical or different surface membrane protein, to minimize other members with receptor complex Interaction.

Intracellular domain in CAR of the invention includes that the signal transduction structural domain of 4-1BB and/or the signal of CD3 ζ pass Transduction domain.

In the present invention, the first CAR and the 2nd CAR be as described in the first aspect of the invention.

Chimeric antigen receptor T cell (CAR-T cell)

As used herein, term " CAR-T cell ", " CAR-T ", " CAR-T cell of the present invention " refer both to first party of the present invention CAR-T cell described in face, CAR-T cell of the present invention can target PD-L1 and prostate-specific membrane antigen (PSMA) simultaneously. The present invention targets the Chimeric antigen receptor T cell energy specific killing prostate gland cancer cell of the bis- target spots of PSMA-PD-L1, without killing Hurt prostate normal cell.

After first CAR and the 2nd CAR expression of the present invention, cell membrane can be passed through and be located on cell membrane.

CAR-T cell is more other, and based on the therapeutic modality of T cell, there are following advantages: (1) CAR-T cell make it is used Journey is not limited by MHC；(2) in view of the identical tumour antigen of many tumor cells expressions, for a certain tumour antigen It completes, can be widely used once CAR is gene constructed；(3) CAR not only can use oncoprotein matter antigen, but also available Glycolipid class nonprotein antigen expands the target spot range of tumour antigen；(4) rejection is reduced instead using autologous patient cell The risk answered；(5) CAR-T cell has the function of immunological memory, can survive in vivo for a long time.

Carrier

The nucleic acid sequence of coding expectation molecule is obtained using the recombination method being known in the art, such as logical It crosses and screens library from the cell of expressing gene, by obtaining the gene from the known carrier including the gene, or pass through benefit With the technology of standard, it is directly separated from cell and tissue comprising the gene.Optionally, interested gene can be synthesized Production.

Present invention provides the carriers for being wherein inserted into expression cassette of the invention.Derived from retrovirus such as slow virus Carrier be the suitable tools for realizing long-term gene transfer because they allow long-term, the stable integration of transgenosis and its It is proliferated in daughter cell.It is more than the carrier from oncogenic retrovirus such as murine leukemia virus that slow virus carrier, which has, Advantage, because of their transducible non-proliferative cells, such as liver cell.They also have the advantages that low immunogenicity.

Simplified summary typically operatively connects expression cassette or nucleic acid sequence of the invention to promoter, and is incorporated into Expression vector.The carrier is suitable for replicating and integrating eukaryocyte.Typical cloning vector includes that can be used for adjusting desired nucleic acid Transcription and translation terminator, initiation sequence and the promoter of sequence expression.

The gene delivery protocols of standard can also be used in expression construct of the invention, are used for nucleic acid immunization and gene therapy. The method of gene delivery is well known in the art.See such as U.S. Patent number 5,399,346,5,580,859,5,589, 466, it is incorporated to by reference of text herein.In another embodiment, the present invention provides gene therapy vectors.

The nucleic acid can be cloned into the carrier of many types.For example, the nucleic acid can be cloned into such carrier comprising But it is not limited to plasmid, phasmid, phage-derived object, animal virus and clay.Specific carrier interested includes that expression carries Body, replicating vector, probe generation vectors and sequencing vector.

Further, expression vector can be supplied to cell in the form of viral vectors.Viral vector technology is in the art It is well known and in (2001, Molecular Cloning:A the Laboratory Manual, Cold such as such as Sambrook Spring Harbor Laboratory, New York) and other virology and molecular biology manual in be described. The virus that can be used as carrier includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpesviral and slow virus.It is logical Often, suitable carrier includes the replication orgin to work at least one organism, promoter sequence, convenient restriction enzyme Site and one or more selectable labels are (for example, WO01/96584； WO01/29058；With U.S. Patent number 6,326, 193)。

Many systems based on virus are developed, for gene transfer to be entered mammalian cell.For example, reverse transcription Virus provides the convenient platform for gene delivery system.Using the technology that is known in the art by the base of selection Because being inserted into carrier and being packaged into retroviral particle.The recombinant virus then can be separated and be transferred to internal or external Subject cell.Many retroviral systems are well known in the art.In some embodiments, it is carried using adenovirus Body.Many adenovirus vectors are well known in the art.In one embodiment, using slow virus carrier.

Additional promoter element, such as enhancer, the frequency that adjustable transcription starts.Normally, these are located at In the region 30-110bp of beginning site upstream, although having shown that many promoters recently also and including the function in initiation site downstream It can element.Interval between promoter element is often flexible, to be squeezed or move relative to another when element When, keep promoter function.In thymidine kinase (tk) promoter, the interval between promoter element, which can be increased, to be separated 50bp, activity are just begun to decline.Depending on promoter, showing discrete component can cooperate or independently work, to play turn Record.

One example of suitable promoter is instant early stage cytomegalovirus (CMV) promoter sequence.The promoter sequence The strong constitutive promoter sequence for being operably coupled to any polynucleotide sequence high level expression thereon can be driven by being classified as Column.Another example of suitable promoter is -1 α of the elongation growth factor (EF-1 α).It is also possible, however, to use other composing types Promoter sequence, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary cancer viral (MMTV), people exempt from The long end of epidemic disease defective virus (HIV) repeats (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Ai Bai Immediately early promoter, Rous sarcoma virus promoter and people's gene open Si Tan-Ba Er (Epstein-Barr) virus Mover, such as, but not limited to actin promoter, Myosin promoter, ferroheme promoter and creatine kinase promoter. Further, the present invention should not limited to the application of constitutive promoter.Inducible promoter is also contemplated as of the invention one Part.The use of inducible promoter provides molecular switch, can be when such expression is desired, and opening can be grasped Make the expression of the polynucleotide sequence of ground connection inducible promoter, or closes expression when expression is undesirable.Induction type The example of promoter includes but is not limited to metallothionein promoter, Glucocorticoid promoter, progesterone promoter and tetracycline Promoter.

In order to assess the expression of CAR polypeptide or part thereof, the expression vector for being introduced into cell also may include selectable mark Any of gene or reporter or both are remembered, in order to from the cell for seeking to be transfected or infect by viral vectors Expression cell is identified and selected in group.In other respects, selectable label can be carried on independent section of DNA and be used for Cotransfection program.The flank of selectable label and both reporters can all have adjusting sequence appropriate, so as to It is expressed in host cell.Useful selectable marker includes such as antibiotics resistance gene, such as neo etc..

Reporter is used to identify the cell of potential transfection and for evaluating the functionality for adjusting sequence.Normally, it reports Gene is following gene: it is not present in recipient organism or tissue or is expressed by recipient organism or tissue, and its Polypeptide is encoded, the expression of the polypeptide is clearly showed that by some property such as enzymatic activitys for being easy detection.Drawn in DNA After entering recipient cell, the expression of reporter is measured under the suitable time.Suitable reporter may include that coding is glimmering Gene (the example of light element enzyme, beta galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or Green Fluorescent Protein Such as, Ui-Tei etc., 2000FEBS Letters479:79-82).Suitable expression system is well known and using known skill Art is prepared or is commercially obtained.In general, the structure with minimum 5 flanking regions of the reporter expression of display highest level It builds body and is accredited as promoter.Such promoter region can be connected to reporter and adjust promoter-for evaluating reagent Drive the ability of transcription.

Gene is introduced into cell and is well known in the art the method that gene expression enters cell.In expression vector Content in, carrier can be easily introduced into host cell by any method in the art, for example, mammal, thin Bacterium, yeast or insect cell.For example, expression vector can be transferred to host cell by physics, chemistry or biological means.

By polynucleotides introduce host cell physical method include calcium phosphate precipitation, lipofection, particle bombardment, Microinjection, electroporation etc..Production includes that the method for the cell of carrier and/or exogenous nucleic acid is well known in the present art.See (2001, Molecular Cloning:A Laboratory Manual, Cold the Spring Harbor such as such as Sambrook Laboratory,New York).It is calcium phosphate transfection by the preferred method that polynucleotides introduce host cell.

It include using DNA and RNA carrier by the biological method that interested polynucleotides introduce host cell.Virus Carrier, especially retroviral vector have become most widely used by gene insertion mammal such as people's cell Method.Other viral vectors may originate from slow virus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus etc..See Such as U.S. Patent number 5,350,674 and 5,585,362.

It include dispersion system of colloid by the chemical means that polynucleotides introduce host cell, such as macromolecular complex is received Rice glue capsule, microballoon, pearl；With the system based on lipid, including oil in water emulsion, micella, mixed micelle and liposome.As body Outer and internal tool for transmitting (delivery vehicle) exemplary colloid system is liposome (for example, artificial membrane vesicle).

Using non-viral delivery system, exemplary tool for transmitting is liposome.Consider to use lipid system Nucleic acid is introduced host cell (external, in vitro (ex vivo) or in vivo) by agent.On the other hand, which can be with lipid It is associated.Nucleic acid associated with lipid can be encapsulated into the aqueous interior of liposome, be dispersed in the lipid bilayer of liposome It is interior, through both associated connection molecule is attached to liposome with liposome and oligonucleotides, liposome is fallen into, with lipid Bluk recombination is dispersed in the solution comprising lipid, mixes with lipid, combine with lipid, is included in lipid as suspension, It is compound or otherwise associated with lipid included in micella or with micella.Lipid associated with composition, lipid/ DNA or lipid/expression vector are not limited to any specific structure in solution.For example, they may be present in bilayer structure In, as micella or there is " (collapsed) of collapse " structure.They can also simply be distributed in the solution, possible shape At the aggregation that size or shape are inhomogenous.Lipid is fatty material, can be the natural lipid occurred or synthesize.For example, rouge Matter includes lipid droplet, it is natural occur cytoplasm and comprising long-chain fat race hydrocarbon and their derivative it is such as fatty Acid, alcohols, amine, alkamine and aldehydes such compound in.

It is preferably carried out in mode at of the invention one, the carrier is slow virus carrier.

Preparation

The present invention provides a kind of immunocytes of engineering described in first aspect present invention, and can pharmaceutically connect Carrier, diluent or the excipient received.In one embodiment, the preparation is liquid formulation.Preferably, the preparation For injection.Preferably, the concentration of CAR-T cell described in the preparation is 1 × 10³-1×10⁸A cell/ml, more preferably 1 ×10⁴-1×10⁷A cell/ml.

In one embodiment, the preparation may include buffer such as neutral buffered saline, sulfate buffered saline Etc.；Carbohydrate such as glucose, mannose, sucrose or glucan, mannitol；Protein；Polypeptide or amino acid are such as Glycine；Antioxidant；Chelating agent such as EDTA or glutathione；Adjuvant (for example, aluminium hydroxide)；And preservative.The present invention Preparation be preferably formulated for intravenously applying.

Therapeutic application

The present invention include the cell (for example, T cell) transduceed with the slow virus carrier (LV) for encoding expression cassette of the present invention into Capable therapeutic application.The marker PD-L1 and PSMA of the T cell targets neoplastic cells of transduction, synergistic activation T cell cause Immunocyte immune response, to significantly improve its killing-efficiency to tumour cell.

Therefore, present invention provides stimulation is immune to the target cell group of mammal or T cell-mediation of tissue The method of response comprising following steps: CAR- cell of the invention is applied to mammal.

In one embodiment, the present invention includes a kind of cell therapy, separation patient's Autologous T cells (or heterologous confession Body), activate and carry out genetic modification generate CAR-T cell, be subsequently injected into it is same in patient body.It is anti-that this mode suffers from graft Host disease probability is extremely low, and antigen is identified in a manner of no MHC limitation by T cell.It is somebody's turn to do in addition, a kind of CAR-T can treat expression All cancers of antigen.Unlike antibody therapy, CAR-T cell can replicate in vivo, generate the length that can lead to continued tumor control Phase persistence.

In one embodiment, CAR- immunocyte of the invention (such as CAR-T cell) can undergo firm internal T Cell expansions and sustainable extended time quantum.In addition, the immune response that CAR is mediated can be the one of adoptive immunotherapy step Part, wherein CAR- modifies induced t cell to the immune response of the antigen-binding domains specificity in CAR.

Although data disclosed herein are specifically disclosed including anti-PD-L1scFv ,-PSMA scFv, hinge and cross-film The slow virus carrier in area and 4-1BB and CD3 ζ signal transduction structural domain, but this invention generally should be construed as including to construct group At any amount of variation of each of part.

Medicable cancer includes that not by vascularization or substantially there are no by the tumour and vascularization of vascularization Tumour.Cancer may include non-physical knurl (such as haematological tumours, such as leukaemia and lymthoma) or may include solid tumor. It include but is not limited to cancer, enblastoma and sarcoma and certain leukaemia or lymph with the cancer types that CAR of the invention is treated Malignant tumour, benign and malignant tumour and malignant tumor, such as sarcoma, cancer and melanoma.It also include adult lesion/cancer disease and youngster Virgin lesion/cancer disease.

Hematologic cancer is the cancer of blood or marrow.The example of hematology (or hematogenous) cancer includes leukaemia, packet Include acute leukemia (such as acute lymphoblastic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and pulpefaction Cellularity, promyelocyte, grain-monocyte type, monocarpotic cellularity and erythroleukemia), chronic leukemia (such as chronic marrow Cell (granulocytic) leukaemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, Lymthoma, hodgkin's disease, non Hodgkin lymphom (painless and high-grade form), Huppert's disease, Walden this Special Lun Shi macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Solid tumor is the abnormal mass of the tissue usually not comprising tumour or fluid zone.Solid tumor can be benign or malignant 's.Different types of solid tumor names (such as sarcoma, cancer and lymthoma) with the cell type for forming them.Solid tumor is such as The example of sarcoma and cancer includes fibrosarcoma, myxosarcoma, embryonal-cell lipoma celiothelioma, lymphoid malignancy, cancer of pancreas and ovary Cancer.Preferably, solid tumor is prostate cancer.

CAR- modification T cell of the invention also is used as the vaccine to mammal Ex vivo immunization and/or in vivo Type.Preferably, mammal is behaved.

For Ex vivo immunization, at least one of the following occurs in vitro before cell application is entered mammal: i) Amplifying cells, ii) nucleic acid that will encode CAR introduces cell and/or iii) Cell Cryopreservation.

In vitro program is well known in the present art, and is being discussed more fully below.Briefly, cell from Separate in mammal (preferably people) and with the carrier of expression CAR disclosed herein carry out gene modification (that is, ex vivo transduction or Transfection).The cell of CAR- modification can be administered to mammalian subject, to provide treatment benefit.Mammalian subject can It can be self relative to recipient for people and the CAR- cell modified.Optionally, cell can be of the same race relative to recipient Allogene, isogenic (syngeneic) or xenogenesis.

Other than for Ex vivo immunization using based on the vaccine of cell, present invention provides vivo immunizations to draw Play the composition and method for the immune response of antigen in patient.

The present invention provides the methods for the treatment of tumour comprising is administered to subject a effective amount of hair for needing it The T cell of bright CAR- modification.

The T cell of CAR- modification of the invention can be administered alone or as pharmaceutical composition and diluent and/or and its His component such as IL-2, IL-17 or other cell factors or cell mass combine application.Briefly, pharmaceutical composition of the invention Object may include target cell group as described herein, with one or more pharmacy or physiologically acceptable carriers, diluent or tax Shape agent combines.Such composition may include buffer such as neutral buffered saline, sulfate buffered saline etc.；Carbon aquation Close object such as glucose, mannose, sucrose or glucan, mannitol；Protein；Polypeptide or amino acid such as glycine；Antioxygen Agent；Chelating agent such as EDTA or glutathione；Adjuvant (for example, aluminium hydroxide)；And preservative.Composition of the invention is excellent Choosing is formulated for intravenously applying.

The mode that pharmaceutical composition of the invention can be suitable for the disease of (or prevention) to be treated is applied.The number of application Amount and frequency will be determined by such factor, although such as the illness of patient and the type of patient disease and severity --- it is appropriate Dosage can be determined by clinical test.

When pointing out " effective quantity in immunology ", " antitumor effective quantity ", " tumour-inhibition effective quantity " or " therapeutic dose ", The precise volume of the present composition to be administered can be determined that the age of consideration patient (object), weight, tumour are big by doctor The individual difference of small, infection or metastasis degree and illness.It can usually point out: the pharmaceutical composition including T cell described herein It can be with 10⁴To 10⁹A cell/kg weight dosage, preferably 10⁵To 10⁶A cell/kg weight dosage (including those models Enclose interior all integer values) application.T cell composition can also be with these dosage multiple applications.Cell can be by using immune Well known injection technique in therapy (see such as Rosenberg etc., NewEng.J.of Med.319:1676,1988) application.It is right It can be by monitoring the disease indication of patient and therefore adjustment for the treatment of is led by medicine in the optimal dose and therapeutic scheme of specific patient Field technique personnel are readily determined.

The application of object composition object can carry out in any convenient manner, including pass through spray-on process, inject, swallow, is defeated Liquid, implantation or transplanting.Compositions described herein can by subcutaneous, intradermal, tumor, in knot, in spinal cord, intramuscular, pass through vein Patient is administered in interior (i.v.) injection or peritonaeum.In one embodiment, T cell composition of the invention passes through intradermal Or subcutaneous injection is administered to patient.In another embodiment, T cell composition of the invention is preferably infused by i.v. Penetrate application.The composition of T cell can be injected directly into tumour, lymph node or infection position.

In some embodiments of the present invention, using method described herein or it is known in the art other by T cell The cell for extending to the method activation and extension of therapeutic level, in conjunction with any amount of related form of therapy (for example, it Before, simultaneously or after) be administered to patient, the form of therapy includes but is not limited to be treated with following reagent: the reagent Such as antiviral therapy, cidofovir and interleukin 2, cytarabine (also being known as ARA-C) or that MS patient His pearl monoclonal antibody treats or to the method pearl monoclonal antibody in distress treatment of psoriatic or to the other treatment of PML patient.Further real Apply in mode, T cell of the invention can with below in conjunction with using: chemotherapy, radiation, immunosuppressor, such as, cyclosporin, sulphur Azoles purine, methopterin, mycophenolate and FK506, antibody or other immunotherapeutic agents.In further embodiment, originally The cell composition and bone-marrow transplantation of invention utilize chemotherapeutics such as fludarabine, external beam radiation therapy (XRT), ring phosphorus Amide is administered to patient in conjunction with (prior to, concurrently with, or after for example).For example, in one embodiment, object can be undergone The standard care of high dose chemotherapy carries out autologous peripheral blood stemcell transplant later.In some embodiments, after the transfer, right Injection as receiving the immunocyte of extension of the invention.In an additional embodiment, the cell of extension is in surgery hand The application of preoperative or surgical site infections.

The dosage for being administered to the above treatment of patient will become with the exact properties for the treatment of illness and the recipient for the treatment of Change.The practice that people's applied dose ratio can receive according to this field is implemented.In general, each treatment or each course for the treatment of, it can be by 1 ×10⁶It is a to 1 × 10¹⁰A modified engineering immunocyte of the present invention is applied to for example, by the mode of venous re-transfusion Patient.

Main advantages of the present invention include:

1. engineering immunocyte of the invention can target PD-L1 and PSMA simultaneously, and only to PD-L1 and PSMA simultaneously Positive cell has strong lethal effect, and positive to PD-L1 or the PSMA positive the cell does not kill or lethal effect is very weak, Normal cell (such as prostate normal cell) will not be killed, " the off- of immunocyte (such as CAR-T cell) is greatly reduced Tumor, on target " effect.

2. the first CAR of the present invention targets the engineering immunocyte of PSMA, the 2nd CAR targeting PD-L1, safety is higher, Hardly kill normal prostatic cell.

3. the antigen-binding domains of present invention targeting PD-L1 are PD-1 extracellular fragment sequence, rather than for PD-L1's Single-chain antibody.PD-1 extracellular fragment sequence is source of people, and immunogenicity is low.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate this hair It is bright rather than limit the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to routine Condition, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, Otherwise percentage and number are weight percent and parts by weight.

Embodiment 1

The present invention mainly constructs three kinds of slow virus packaging plasmids, be respectively pLVX- α PSMA-CAR, pLVX- PD1-CAR and pLVX-αPSMA-PD1-CAR。

The building of 1.1 pLVX- α PSMA-CAR plasmids

As shown in Figure 1, the structure of the CAR (abbreviation α PSMA-CAR) of targeting prostate gland cancer cell surface antigen PSMA is L- α PSMA-CD8TM-4-1BB-CD3 ζ, amino acid sequence is as shown in SEQ ID NO.:1.

METDTLLLWVLLLWVPGSTGEVQLVQSGAEVKKPGASVKISCKTSGYTFTEYTIHWVKQASGKGLEWIGNINPNNGG TTYNQKFEDRATLTVDKSTSTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTTVTVSSGSTSGGGSGGGSGGGGSSD IVMTQSPSSLSASVGDRVTITCKASQDCGTAVDWYQQKPGKAPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISSL QPEDFADYFCQQYNSYPLTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR(SEQ ID NO.:1)

Wherein PSMA scFv (i.e. α PSMA) is the antigen-binding domains (scFv) for targeting PSMA, amino acid sequence As shown in underscore part in SEQ ID NO.:1, nucleic acid coding sequence is as shown in 61-780 in SEQ ID NO.:2:

ATGGAAACCGACACCCTGCTGCTGTGGGTGCTGCTGCTCTGGGTCCCAGGCTCCACCGGTGAAGTGCAGCTGGTGCA GTCTGGCGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGATCTCCTGCAAGACCTCCGGCTACACCTTCACCGAGT ACACCATCCACTGGGTGAAACAGGCCTCCGGCAAGGGCCTGGAATGGATCGGCAACATCAACCCTAACAACGGCGGC ACCACCTACAACCAGAAGTTCGAGGACCGGGCCACCCTGACCGTGGACAAGTCCACCTCCACCGCCTACATGGAACT GTCCTCCCTGCGGTCTGAGGACACCGCCGTGTACTACTGCGCCGCTGGCTGGAACTTCGACTACTGGGGCCAGGGCA CCACAGTGACAGTCTCGAGCGGCTCTACCTCTGGCGGAGGCTCTGGGGGAGGAAGCGGCGGAGGCGGCTCCTCTGAC ATCGTGATGACCCAGTCCCCCTCCTCCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCACATGCAAGGCCTCCCA GGATTGTGGCACCGCCGTGGACTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTGGGCCTCCA CCAGACACACCGGCGTGCCTGACAGATTCACCGGCTCCGGCTCTGGCACCGACTTCACCCTGACCATCTCCAGCCTG CAGCCTGAGGACTTCGCCGACTACTTCTGCCAGCAGTACAACTCCTACCCTCTGACCTTCGGCGGAGGCACCAAGCT GGAAATCAAA(SEQ ID NO.:2)

Construct coded sequence pLVX- α PSMA-CAR plasmid, the plasmid contain above-mentioned α PSMA-CAR, IRES element and ZsGreen element, structural schematic diagram are as shown in Figure 2 A.Fig. 2 B shows successfully to construct the pLVX- α PSMA-CAR plasmid.

The building of 1.2 pLVX-PD1-CAR plasmids

As shown in figure 3, the structure of the CAR (abbreviation PD1-CAR) of targeting PD-L1 is L-PD-1-CD8TM- 4-1BB-CD3 ζ, amino acid sequence is as shown in SEQ ID NO.:3.

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTD KLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTA HPSPSPRSAGQFQTLVV TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVL LLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELN LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR(SEQ ID NO.: 3)

Wherein PD-1 extracellular fragment (abbreviation PD-1) is the antigen-binding domains for targeting PD-L1, and amino acid sequence is such as In SEQ ID NO.:3 shown in underscore part, nucleic acid coding sequence is as shown in 61-513 in SEQ ID NO.:4:

ATGCAGATCCCACAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAGA CTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGACAACGCCACCT TCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGAC AAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGG GCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCATCTCCCTGG CCCCCAAGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGAGAGAAGGGCAGAAGTGCCCACAGCC CACCCCAGCCCCTCACCCAGGTCAGCCGGCCAG TTCCAAACCCTGGTGGTT(SEQ ID NO.:4)

PLVX-PD1-CAR plasmid is constructed, which contains the volume of above-mentioned PD1-CAR, IRES element and ZsGreen element Code sequence, structural schematic diagram are as shown in Figure 4 A.Fig. 4 B shows successfully to construct the pLVX-PD1-CAR plasmid.

The building of 1.3 pLVX- α PSMA-PD1-CAR plasmids

As shown in figure 5, L- α PSMA is connected with CD8TM, costimulatory molecules 4-1BB, the first CAR, amino are obtained Acid sequence is as shown in 1-371 in SEQ ID NO.:5；L-PD-1 extracellular fragment is connected with CD8TM, CD3 ζ sequence, is obtained To the 2nd CAR, amino acid sequence is as shown in 392-743 in SEQ ID NO.:5.It is used between first CAR and the 2nd CAR T2A connection (is expressed as α PSMA-PD1-CAR).

METDTLLLWVLLLWVPGSTGEVQLVQSGAEVKKPGASVKISCKTSGYTFTEYTIH WVKQASGKGLEWIGNINPNNGGTTYNQKFEDRATLTVDKSTSTAYMELSSLRSEDTA VYYCAAGWNFDYWGQGTTVTVSSGSTSGGGSGGGSGGGGSSDIVMTQSPSSLSASVG DRVTITCKASQDCGTAVDWYQQKPGKAPKLLIYWASTRHTGVPDRFTGSGSGTDFTLT ISSLQPEDFADYFCQQYNSYPLTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP VQTTQEEDGCSCRFPEEEEGGCELRAEGRGSLLTCGDVEENPGPMQIPQAPWPVVWAV LQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSP SNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAP KAQIKESLRAELRVTERRAEVPTAHPSPSPRSAGQFQTLVVTTTPAPRPPTPAPTIASQPL SLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.:5)

Construct pLVX- α PSMA-PD1-CAR plasmid, the plasmid contain above-mentioned α PSMA-PD1-CAR, IRES element and The coded sequence of ZsGreen element, structural schematic diagram is as shown in Figure 6A, and Fig. 6 B shows successfully to construct the pLVX- α PSMA- PD1-CAR plasmid.

Embodiment 2

The slow virus that tri- kinds of pLVX- α PSMA-CAR, pLVX-PD1-CAR, pLVX- α PSMA-PD1-CAR plasmids are packed It is named as Lenti- α PSMA-CAR, Lenti-PD1-CAR, Lenti- α PSMA-PD1-CAR.

2.1 plasmid transfection

2.1.1 interior for 24 hours before transfection to pass on 293T cell, cell density, transfection are adjusted according to cell density and state When cell need completely it is adherent and it is adherent uniformly, stand density ensures that cell state is good up to 60~90%, and incubation time is not More than for 24 hours；

2.1.2 according to transfection cell quantity, by following three plasmid mixed liquor of proportional arrangement, of short duration to be vortexed 5 times, room temperature is quiet Set 5min；

2.1.3 according to transfection cell quantity, transfection reagent polyethyleneimine (PEI) dilution is prepared by 64 μ L/mL/ wares, It is of short duration to be vortexed 5 times, it is stored at room temperature 5min；

2.1.4 1.5mL EP pipe mixing plasmid mixed liquor and PEI dilution are used: it is mixed that 500 μ L plasmids being first added into pipe Liquid to be closed, then is slowly added to 500 μ L PEI dilutions along tube wall, the concussion that is vortexed immediately is sufficiently mixed solution, of short duration vortex 2s, It is continuous to be vortexed 10 times；

2.1.5 being stored at room temperature polymerization 20min；

2.1.6 culture dish is jiggled in front and back on one side, and mixed liquor is added dropwise to cells and supernatant on one side, when dropwise addition, needs Nearly liquid level is softly slowly added dropwise, and prevents rate of addition too fast or firmly excessive leads to cells float；

2.1.7 gently shaking culture dish is uniformly mixed transfection liquid with culture supernatant, 37 DEG C of 5% carbon dioxide incubator transfection 6~10h；

2.1.8 whole culture supernatants are discarded, 1640 complete medium of 10mL is added, continue culture to 72h；

2.1.9 change after liquid for 24 hours, 48h, 72h, using inverted phase contrast fluorescence microscope, follow-up observation cell growth state and Transfection efficiency；

2.1.10 the culture supernatant of 48h and 72h is usually collected respectively, if cell proliferation rate is fast, culture supernatant turns yellow, Culture supernatant can be collected in time replace fresh medium again and continue to cultivate, supernatant be purpose viral solution, can be in 4 DEG C in short-term Save a couple of days.

2.2 supercentrifugation concentrating virus

2.2.1 be unable to autoclave sterilization since the ultracentrifugation pipe that uses is concentrated, before concentration, need to will hypervelocity from Heart pipe impregnates 72h or more in 75% ethyl alcohol, guarantees that centrifuge tube is sterile；

2.2.2 the ultracentrifugation pipe for impregnating 72h or more is taken out, 5mL DMEM culture solution is added, by centrifuge tube rinse one It is secondary, rinse liquid is discarded, centrifuge tube is buckled on sterile blotting paper dry；

2.2.3 viral solution is filled it up with into centrifuge tube, tightens centrifuge tube lid, should have no or only pole in centrifuge tube at this time A small amount of bubble；

2.2.4 after stringent trim, 40,000rpm, 4 DEG C of low temperature ultracentrifugation 3h；

2.2.5 supernatant is abandoned, if virus stock solution used volume is larger, cannot can add phase into same centrifuge tube once from complete Virus stock solution used is answered, according to the above method repeated centrifugation；If virus stock solution used volume is not enough to fill a centrifuge tube, can be added appropriate DMEM culture solution fills；

2.2.6 after virus liquid is all centrifuged, last pipe waste liquid is discarded, according to virus stock solution used volume difference, Xiang Chao From 0.5~3mL DEME complete culture solution is added in pipe, by 25~100 times of viral concentration, piping and druming is until precipitating is completely molten in pipe Solution, 200 μ L/ pipes are dispensed to sterile EP tube, are saved in -80 DEG C.In use, paying attention to avoiding concentrating virus liquid multigelation.

2.3 slow virus titer determinations

2.3.1 the good HEK-293T cell of growth conditions is taken, is counted after digestion, it is by 2x105 cells/well that cell is equal It is adherent to cell to cultivate 6~10h to 24 porocyte culture plates for even paving；

2.3.2 the concentrating virus liquid of various concentration is added in cells and supernatant into 24 orifice plates, gently pats 24 orifice plates Edge is uniformly mixed virus liquid with culture solution, and 48h is infected in incubator；

2.3.3 after infecting 48h, digest and collect cell, Flow cytometry positive cell percentage, and according to Lower formula calculates virus titer:

As a result as shown in the table:

Titre	PD1-CAR	αPSMA-CAR	αPSMA-PD1-CAR
				Stoste	4.0*106U/mL	1.48*106U/mL	7.3*105U/mL
Concentrate (100 times of concentration)	4.0*108U/mL	1.48*108U/mL	7.3*107U/mL

The preparation of 2.4 JurCAR-T cells

2.4.1 by concentrating virus liquid Lenti- α PSMA-CAR of previous step preparation, Lenti-PD1-CAR, Lenti- α PSMA-PD1-CAR infects Jurkat cell by MOI=10 respectively, while 1 μ g/mL being added into infection system Polybrene is to improve efficiency of infection.

2.4.2 liquid is changed in centrifugation after infecting 12h, continues to cultivate 48h, the cell of positive infection is that purpose JurCAR-T is thin Born of the same parents.

2.4.3 cell, flow cytomery ZsGreen positive cell percentage are collected.

As a result as shown in fig. 7, three kinds of viral efficiency of infection are higher, three kinds of aim cell JurCAR- α PSMA of gained, JurCAR-PD1, JurCAR- α PSMA-PD1 positive rate be respectively 92.7%, 99.3%, 98.5%.The result shows that with JurCAR-T (JurCAR- α PSMA, JurCAR-PD1, JurCAR- α PSMA-PD1) the cell preparation of Jurkat cell system building Success.

3 α PSMA-PD1-CAR-T specific killing prostate gland cancer cell PC3 of embodiment is normally thin without killing prostate Born of the same parents PNTIA

As shown in figure 8, with positive JurCAR- α PSMA cell killing prostate gland cancer cell PC3 and prostate in embodiment 2 Normal cell PNTIA, discovery JurCAR- α PSMA cell have an obvious fragmentation effect to prostate gland cancer cell PC3, effect target ratio (E: T) be 10:1 when, killing rate reaches 62%, but JurCAR- α PSMA cell also has 24% to prostate normal cell PNTIA Killing rate.

Prostate cancer table can be combined by carrying out prostate cancer PC3 killing verifying PD-1-CAR with JurCAR-PD1 cell first Face PD-L1 plays killing ability.It is finally normally thin with JurCAR- α PSMA-PD1 killing prostate cancer cell PC3 and prostate Born of the same parents PNTIA, JurCAR- α PSMA-PD1 has obvious fragmentation effect to prostate gland cancer cell PC3 as the result is shown, and just to prostate Normal cell PNTIA is not killed, it was demonstrated that α PSMA-PD1-CAR be a safety-type specific killing prostate gland cancer cell and Not double CAR systems of killing prostate normal cell.

All references mentioned in the present invention is incorporated herein by reference, just as each document coverlet It is solely incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Fixed range.

Sequence table

<110>East China Normal University

Shanghai Bang Yao Biotechnology Co., Ltd

<120>the engineering immunocyte of PSMA and PD-L1 is targeted simultaneously

<130> P2018-0744

<160> 5

<170> PatentIn version 3.5

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

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

20 25 30

Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr

35 40 45

Phe Thr Glu Tyr Thr Ile His Trp Val Lys Gln Ala Ser Gly Lys Gly

50 55 60

Leu Glu Trp Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr

65 70 75 80

Asn Gln Lys Phe Glu Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr

85 90 95

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

100 105 110

Val Tyr Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly

115 120 125

Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly Ser Gly

130 135 140

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

145 150 155 160

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

165 170 175

Lys Ala Ser Gln Asp Cys Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys

180 185 190

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His

195 200 205

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

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

225 230 235 240

Cys Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

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Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

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

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Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

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

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

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

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

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

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Pro Pro Arg

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tcctgcaaga cctccggcta caccttcacc gagtacacca tccactgggt gaaacaggcc 180

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atggaactgt cctccctgcg gtctgaggac accgccgtgt actactgcgc cgctggctgg 360

aacttcgact actggggcca gggcaccaca gtgacagtct cgagcggctc tacctctggc 420

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Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

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Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

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Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

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

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ala Leu His Met Gln Ala Leu Pro Pro Arg

385 390

<210> 4

<211> 513

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 4

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggtcagccg gccagttcca aaccctggtg gtt 513

<210> 5

<211> 743

<212> PRT

<213>artificial sequence (artificial sequence)

<400> 5

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

1 5 10 15

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

20 25 30

Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr

35 40 45

Phe Thr Glu Tyr Thr Ile His Trp Val Lys Gln Ala Ser Gly Lys Gly

50 55 60

Leu Glu Trp Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr

65 70 75 80

Asn Gln Lys Phe Glu Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr

85 90 95

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

100 105 110

Val Tyr Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly

115 120 125

Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Gly Gly Ser Gly

130 135 140

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

145 150 155 160

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

165 170 175

Lys Ala Ser Gln Asp Cys Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys

180 185 190

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His

195 200 205

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

210 215 220

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

225 230 235 240

Cys Gln Gln Tyr Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

340 345 350

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

355 360 365

Cys Glu Leu Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

370 375 380

Val Glu Glu Asn Pro Gly Pro Met Gln Ile Pro Gln Ala Pro Trp Pro

385 390 395 400

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

405 410 415

Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu

420 425 430

Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser

435 440 445

Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser

450 455 460

Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro

465 470 475 480

Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp

485 490 495

Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr

500 505 510

Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser

515 520 525

Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr

530 535 540

Ala His Pro Ser Pro Ser Pro Arg Ser Ala Gly Gln Phe Gln Thr Leu

545 550 555 560

Val Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Leu Val Ile Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

Met Gln Ala Leu Pro Pro Arg

740

Claims (10)

1. a kind of immunocyte of engineering, which is characterized in that the immunocyte expresses the first CAR and the 2nd CAR；And

The structure of first CAR is as shown in following formula I: L1-scFv1-H1-TM1-C (I)；

The structure of 2nd CAR is as shown in Formula Il: L2-scFv2-H2-TM2-CD3 ζ (II)；

In formula,

Each "-" independently is link peptide or peptide bond；

L1 and L2 is each independently optional signal peptide sequence；

One is the antigen-binding domains for targeting PSMA in both scFv1 and scFv2, another is the antigen for targeting PD-L1 Binding structural domain；

H1 and H2 is each independently optional hinge area；

TM1 and TM2 are each independently transmembrane domain；

C is costimulatory signal molecule；

CD3 ζ is the endochylema signal transduction sequence derived from CD3 ζ.

2. immunocyte as described in claim 1, which is characterized in that scFv1 is the antigen-binding domains for targeting PSMA, ScFv2 is the antigen-binding domains for targeting PD-L1.

3. immunocyte as described in claim 1, which is characterized in that the amino of the antigen-binding domains of the targeting PSMA Acid sequence is as shown in 21-260 in SEQ ID NO.:1；And/or

The amino acid sequence of the antigen-binding domains of the targeting PD-L1 is as shown in 21-171 in SEQ ID NO.:2.

4. immunocyte as described in claim 1, which is characterized in that the amino acid sequence such as SEQ ID of the first CAR In NO.:5 shown in 1-371.

5. immunocyte as described in claim 1, which is characterized in that the amino acid sequence such as SEQ ID of the 2nd CAR In NO.:5 shown in 392-743.

6. a kind of method of preparation engineering immunocyte, which comprises the following steps:

(A) immunocyte to be rebuilt is provided；With

(B) immunocyte is transformed, so that the immunocyte expresses the first CAR and the 2nd CAR, from And obtain the immunocyte of engineering described in claim 1.

7. method as claimed in claim 6, which is characterized in that in step (B), including (B1) will be used for table s up to the first CAR The first expression cassette imported into the immunocyte；The second expression cassette for being used to express the 2nd CAR imported into described by (B2) Immunocyte, wherein the step (B2) can be before step (B1), later, either simultaneously or alternately carry out.

8. a kind of preparation, which is characterized in that the preparation contains the immunocyte and medicine of engineering described in claim 1 Acceptable carrier, diluent or excipient on.

9. the purposes of immunocyte that one kind is engineered as described in claim 1, which is characterized in that be used to prepare prevention and/ Or the drug or preparation for the treatment of cancer or tumour, preferably, the cancer is prostate cancer.

10. a kind of kit for being used to prepare cell described in claim 1, which is characterized in that the kit contains container, with And in container:

(1) first nucleic acid sequence, first nucleic acid sequence contain the first expression cassette for expressing the first CAR；With

(2) second nucleotide sequence, the second nucleotide sequence contain the second expression cassette for expressing the 2nd CAR.