CN106519037B - Activable Chimerical receptor - Google Patents

Abstract

The present invention relates to activatable chimeric receptors. The activatable chimeric receptor of the present invention can target the corresponding antigen only in the presence of pathological tissue-specifically expressed or highly expressed protease to achieve the killing effect of pathological tissue or cells; and in the absence of protease It does not work under the conditions of on-target-off-tumor, which provides a feasible solution for reducing on-target-off-tumor and other problems.

Description

activatable chimeric receptor

technical field

The present invention belongs to the field of tumor immunotherapy, and more particularly, the present invention relates to an activatable chimeric receptor, its preparation method and its application.

Background technique

The role of immune effector cells in tumor immune responses has received increasing attention. Adoptive immunotherapy based on immune effector cells has achieved certain effects in some tumors, and this immunotherapy method can overcome the above-mentioned defects of antibody therapy, but the efficacy in most tumors is still unsatisfactory [Grupp SA, et al. al. Adoptive cellulartherapy. Curr Top Microbiol Immunol., 2011;344:149-72.]. In recent years, according to the discovery that the recognition specificity of cytotoxic T lymphocytes (CTL) to target cells depends on the T Cell Receptor (TCR), the scFv of antibodies against tumor cell-associated antigens are combined with T cells. Intracellular signal activation motifs such as CD3ζ or FcεRIγ of lymphocyte receptors are fused into chimeric antigen receptors (Chimeric antigen receptors, CAR; now also known as chimeric receptors (CR)), and they are passed through e.g. Lentiviral infection and other methods are genetically modified on the surface of T lymphocytes. Such CAR T lymphocytes can selectively direct T lymphocytes to tumor cells and specifically kill tumors in a non-limiting manner by the Major Histocompatibility Complex (MHC). CAR T lymphocytes are a new immunotherapy strategy in the field of tumor immunotherapy [Schmitz M, et al. Chimeric antigen receptor-engineered T cells for immunotherapy of Cancer. J Biomed Biotechnol, 2010, doi: 10.1155/2010/956304.]. In addition, CAR-modified NK cells (Klingemann H.Challenges of cancer therapy with naturalkiller cells.Cytotherapy.2014Dec 18.pii:S1465-3249(14)00791-9) or NKT cells also showed good resistance to Tumor activity (Heczey A1, Liu D1, Tian G2, Courtney AN1, WeiJ1, Marinova E1, Gao X1, Guo L1, Yvon E3, Hicks J2, Liu H4, Dotti G5, MetelitsaLS6. Invariant NKT cells with chimeric antigen receptor provide a novel platform for safe and effective cancer immunotherapy. Blood. 2014;124(18):2824-33).

Chimeric antigen receptors include an extracellular binding domain, a transmembrane domain and an intracellular signaling domain. Usually the extracellular region contains scFv that can recognize tumor-associated antigens, the transmembrane region adopts the transmembrane region of CD8, CD28 and other molecules, and the intracellular signal region adopts the immunoreceptor tyrosine activation motif (ITAM) CD3ζ or FcεRIγ and co-stimulatory Intracellular signaling region of signaling molecules CD28, CD27, CD137, CD134, etc.

The first-generation CAR T lymphocytes that only contain ITAM in the intracellular signal region, in which each part of the chimeric antigen receptor is linked in the following form: scFv-TM-ITAM. This CAR T can stimulate anti-tumor cytotoxic effects, but the secretion of cytokines is relatively low, and it cannot stimulate lasting anti-tumor effects in vivo [Zhang T. et al. Chimeric NKG2D-modified T cells inhibit systemic T-cell lymphoma growth In a manner involving multiple cytokines and cytotoxic pathways, Can Res 2007, 67(22): 11029-11036.].

The second generation of CAR T lymphocytes subsequently developed added the intracellular signaling region of CD28 or CD137 (aka 4-1BB), in which the chimeric antigen receptor moieties were linked as follows: scFv-TM-CD28-ITAM or scFv -TM-/CD137-ITAM. The co-stimulation of B7/CD28 or 4-1BBL/CD137 in the intracellular signaling region causes the continuous proliferation of T lymphocytes, and can increase the levels of cytokines such as IL-2 and IFN-γ secreted by T lymphocytes, and at the same time increase the level of CAR T lymphocytes. In vivo survival cycle and antitumor effect [Dotti G. et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor modified T cells in lymphomapatients. J Clin Invest, 2011, 121(5): 1822-1826.].

The third generation of CAR T lymphocytes developed in recent years, in which each part of the chimeric antigen receptor is connected as follows: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, which further improves the CAR The survival cycle of T in vivo and its antitumor effect [Carpenito C., et al. Control of large established tumorxenografts with genetically retargeted human T cells containing CD28 and CD137 domains. PNAS, 2009, 106(9): 3360-3365.].

Although CAR T lymphocytes have an attractive prospect in tumor immunotherapy, their higher risks also need to be considered. For example, due to the low expression of specific antigens recognized by CAR in some/kind of normal tissues, CAR T lymphocytes may damage normal tissues expressing antigens. For example, targeting the antigen carbonic anhydrase IX (CAIX) expressed on tumor cells of patients with renal cell carcinoma is the first case of adoptive therapy of CAR T lymphocytes for clinical use, and the first case to report off-target effects of CAR cells . The patient developed grade 2-4 hepatotoxicity after multiple infusions of CAR T lymphocytes. The reason for the analysis was the low expression of CAIX in hepatic bile duct epithelial cells, and the original clinical trial had to be interrupted and any evaluation of the patient's treatment effect was excluded [StoterG.et al.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: firstclinical experience. J clin oncol, 2006, 24(13): e20-e22.; Ngo MC., et al. Ex vivogene transfer for improved adoptive immunotherapy of cancer. Human Molecular Genetics, 2011, R1-R7]. In addition, excessive co-stimulatory signals in CARs reduce the threshold required for effector cell activation, allowing genetically modified T lymphocytes to be activated even in the presence of low levels of antigen or no antigen triggering, resulting in the release of large amounts of cytokines This may lead to the so-called "cytokine storm". This signalleakage can lead to off-target cytotoxicity, resulting in nonspecific tissue damage. For example, in the clinical treatment of a patient with advanced colon cancer with liver and lung metastases using a third-generation CAR targeting Her2, the so-called "cytokine storm" caused sudden death due to low expression of Her2 in normal lung tissue [Morgan RA., et al. Report of aserious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing Erbb2. Molecular Therapy, 2010, 18(4):843-851.].

Therefore, it is necessary to find a suitable method to enable CAR-based immune effector cells to act on tumors more precisely and avoid the higher risks when applied to tumor immunotherapy.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an activatable chimeric receptor, its preparation method and its application.

In a first aspect of the present invention, an activatable chimeric receptor (Activatable Chimeric Receptor, ACR) is provided, comprising:

Chimeric Receptor (CR), which can target (bind) antigens highly expressed in pathological tissues in an activated state;

A blocking element (BE) that inhibits the binding of the chimeric receptor to the highly expressed antigen of the pathological tissue; and

Cleavable element (CE), which is located between the chimeric receptor and the blocking element.

In a preferred embodiment, the chimeric receptor comprises sequentially linked: an extracellular antigen binding region, a transmembrane region and an intracellular signal region; the intracellular signal region is selected from the group consisting of: CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134, the intracellular signal region sequence of CD40 or Myd88, or a combination thereof.

In another preferred embodiment, the extracellular antigen-binding region is an antibody that specifically binds to an antigen highly expressed in the pathological tissue.

In another preferred example, the antibody that specifically binds to the highly expressed antigen in the pathological tissue may be: single chain antibody (scFV), monoclonal antibody, single domain antibody, Fab fragment, Fd fragment, Fv Fragments, F(ab')2 fragments and derivatives thereof, or other forms of antibodies.

In another preferred embodiment, the antibody that specifically binds to the highly expressed antigen of the pathological tissue is selected from (but not limited to): a single-chain antibody or a single-domain antibody.

In another preferred example, the chimeric receptor can effectively recognize the highly expressed antigen in the pathological tissue in the absence of the blocking element, and the blocking element can interfere with or compete with the chimeric receptor and the highly expressed antigen in the pathological tissue antigen binding.

In another preferred embodiment, the activatable chimeric receptors, in order from the amino terminus to the carboxyl terminus, include: a blocking element, a cleavable element, and a chimeric receptor.

In another preferred embodiment, between the chimeric receptor, the blocking element and the cleavable element, a linker peptide is also included.

In another preferred embodiment, the activatable chimeric receptor contains a first linking polypeptide (LP1) and a second linking polypeptide (LP2); wherein the activatable chimeric receptor is arranged according to the sequence from the amino terminus to the carboxyl group The sequence of the ends, in turn, includes: a blocking element, a first linking polypeptide, a cleavable element, a second linking polypeptide, and a chimeric receptor.

In another preferred example, the blocking element is selected from, but not limited to: a polypeptide that directly binds to the chimeric receptor; or a polypeptide that sterically hinders the binding of the chimeric receptor to an antigen.

In another preferred embodiment, the blocking element is 2-100aa (eg 10aa, 20aa, 30aa, 40aa, 50aa, 60aa, 70aa, 80aa); preferably 2-40aa polypeptides.

In another preferred embodiment, the cleavable element is an element that can be cleaved, reduced or decomposed by a protease specifically expressed or highly expressed in the pathological tissue, and the protease and the highly expressed protease in the pathological tissue Antigens co-localize in the same pathological tissue.

In another preferred embodiment, the pathological tissue includes but is not limited to: tumor, tissue of autoimmune disease, tissue infected by virus (such as HIV virus).

In another preferred example, the pathological tissue is a tumor, and the proteases specifically expressed or highly expressed in the pathological tissue include (but are not limited to): urokinase-type plasminogen activator (urokinase-type plasminogen activator) plasminogenactivator, uPA), legumain protease or matriptase (MT-SP1).

In another preferred embodiment, the pathological tissue-specifically expressed or highly expressed protease is urokinase-type plasminogen activator or matriptase, and the cleavable element is the amino acid shown in SEQ ID NO: 2 sequence of polypeptides.

In another preferred example, the highly expressed antigens in pathological tissues include but are not limited to: GPC3, EGFR, HER2, EphA2, Claudin18.1, Claudin18.2, Claudin 6, GD2, EpCAM, mesothelin, CD19, CD20 or ASGPR1.

In another preferred example, the highly expressed antigen in the pathological tissue is GPC3, and the blocking element is the binding polypeptide of the GC33 antibody; preferably, the blocking element is the amino acid shown in SEQ ID NO: 1 sequence of polypeptides.

In another preferred example, when the highly expressed antigen in the pathological tissue is GPC3, the pathological tissue is a tumor, including liver cancer, melanoma, ovarian clear cell carcinoma, yolk sac tumor, and neuroblastoma.

In another aspect of the present invention, there is provided a polynucleotide encoding the activatable chimeric receptor.

In another aspect of the present invention, there is provided an expression vector comprising a nucleic acid encoding the activatable chimeric receptor.

In another preferred embodiment, the expression vector is derived from the lentiviral plasmid pWPT.

In another aspect of the present invention, there is provided a virus comprising the vector.

In another aspect of the present invention, the use of any of the aforementioned activatable chimeric receptors, or a nucleic acid encoding the same, or an expression vector or virus comprising the nucleic acid, is provided for the preparation of a target pathological tissue. Chimeric receptor immune effector cells.

In another aspect of the present invention, there is provided a chimeric receptor immune effector cell transduced with a nucleic acid encoding any of the aforementioned activatable chimeric receptors, or the aforementioned expression vector or the aforementioned virus; or its surface expresses any of the activatable chimeric receptors described above.

In another preferred embodiment, the immune effector cells include: T lymphocytes, NK cells or NKT cells, and Treg cells.

In another aspect of the present invention, the use of the chimeric receptor immune effector cells is provided for preparing a drug targeting pathological tissue, and the pathological tissue highly expresses the antigen that the chimeric receptor can bind.

In a preferred example, the pathological tissue is a tumor, and the drug targeting the pathological tissue is a tumor-inhibiting drug.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising: the aforementioned chimeric receptor immune effector cells.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. Schematic diagram of the structure of the activatable chimeric receptor ACR.

In order of N-terminal→C-terminal: Sp: secretion signal peptide, BE: blocking element, LP1: linker 1; CE1: cleavable element; LP2: linker 2, Ab: antigen-binding region; LP3: linker 3; TM: Transmembrane domain; SD: intracellular signaling domain.

Figure 2. Schematic representation of GC33-28BBZ-ACR.

According to the direction of N-terminal → C-terminal, each module in the figure is CD8 secretion signal, BE (blocking element), LP1, CE (cleavable element, UP1 enzyme cleavage substrate), LP2, CR (GC33-28BBZ), F2A, eGFP.

Figure 3. FACS analysis of GC33-28BBZ-ACR positive rate of T cells.

The positive rate of GFP was used as an indicator of the positive rate of GC33-28BBZ-ACR in T cells after lentivirus infection.

Figure 4. In vitro toxicity test. The liver cancer cell line Huh-7 was used as target cells, and the effector cells were T cells expressing GC33-28BBZ-ACR cultured in vitro for 11 days. The effector-target ratios were 3:1, 1:1 and 1:3, respectively. is 10,000 cells/well, corresponding to effector cells according to different effector-target ratios. Add uPA and MTSP1 respectively, and set up a low-dose group, a high-dose group and a blank group. Each group was set with 5 duplicate wells, and the average value of the 5 duplicate wells was taken. The detection time is the 18th hour.

Detailed ways

After extensive research, the inventors have revealed an activatable chimeric receptor based on chimeric antigen receptor (CAR) technology, which is only specifically expressed or highly expressed in pathological tissues. Only in the presence of protease can target the corresponding antigen to achieve the killing effect of pathological tissue or cells; but it does not play a role in the absence of protease. The present invention provides a feasible solution for avoiding on-target-off-tumor.

As used herein, the term "chimeric receptor" is used interchangeably with "chimeric antigen receptor".

As used in the present invention, the "antigen highly expressed in pathological tissue" refers to the antigen targeted by the activated chimeric receptor, and the antigen is highly expressed in pathological tissue or cells. In the present invention, the "antigen highly expressed in pathological tissues" may also be expressed in normal tissues or cells other than pathological tissues or cells. Preferably, the "highly expressed antigen in pathological tissue" is a tumor-associated antigen, such as selected from (but not limited to): GPC3, EGFR, HER2, EphA2, Claudin18.1, Claudin18.2, Claudin 6, GD2, EpCAM , mesothelin, CD19, CD20 or ASGPR1.

As used in the present invention, the "pathological tissue-specifically expressed or highly expressed protease" refers to a proteolytic (hydrolytic) enzyme that is only expressed in pathological tissue or cells, or is highly expressed in pathological tissue or cells. Capable of hydrolyzing its specific substrates.

As used in the present invention, the "blocking element" refers to a polypeptide capable of blocking the binding of the chimeric receptor to its corresponding antigen, either by directly binding to the chimeric receptor or by sterically blocking the chimeric receptor. The chimeric receptor is combined with the antigen to play the role of blocking the binding of the chimeric receptor to the target site.

As used in the present invention, the "cleavable element" is a polypeptide located between the chimeric receptor and the blocking element, which is the substrate of the "pathological tissue-specifically expressed or highly expressed protease" , in the presence of the protease, the "cleavable element" can be cleaved, reduced or cleaved, so that the blocking element no longer blocks the binding of the chimeric receptor to its corresponding antigen.

As used in the present invention, the "pathological tissue" includes (but is not limited to): tumor, tissue of autoimmune disease, tissue infected by virus (eg HIV virus) and the like.

In the present invention, the pathological tissue can be various harmful tissues or lesions in the body that are not conducive to health, and must be removed from the body. The pathological target tissue includes tumors. Any tumor known in the art can be included in the present invention, as long as the tumor is capable of expressing tumor-associated antigens that are lowly expressed in normal tissues. For example, the tumors include (but are not limited to): liver cancer, lung cancer, glioma, breast cancer, gastric cancer, prostate cancer, brain tumor, ovarian cancer, bone tumor, colon cancer, thyroid tumor, mediastinal tumor, intestinal tumor, Kidney tumor, adrenal tumor, bladder tumor, testicular tumor, malignant lymphoma, multiple myeloma, nervous system tumor, esophageal cancer, thymic mesothelioma, pancreatic cancer, leukemia, head and neck tumor, cervical cancer, skin cancer, melanoma tumor, vaginal epithelial cancer, gallbladder cancer, malignant fibrous histiocytoma. For example, the tumor-associated antigens include (but are not limited to): GPC3, EGFR, HER2, EphA2, Claudin18.1, Claudin18.2, Claudin 6, GD2, EpCAM, mesothelin, CD19, CD20, ASGPR1, EGFRvIII, de4 EGFR , CD19, CD33, IL13R, LMP1, PLAC1, NY-ESO-1, MAGE4, MUC1, MUC16, LeY, CEA, CAIX (carbonic anhydrase IX), CD123.

The term "chimeric receptor immune effector cell" is well known in the art, which is an immune effector cell that expresses an antigen (eg, tumor antigen) specific chimeric receptor using genetic modification technology, and can play a targeted killing effect. The immune effector cells include, for example, T cells, NK cells, NKT cells, and regulatory T cells (Treg for short). Conventional methods for preparing "chimeric receptor immune effector cells" are known to those skilled in the art, including allowing them to express intracellular co-stimulatory cell molecule intracellular domains, such as CD28 (preferably including CD28a, CD28b), One or more of CD137, CD27, CD3ζ (preferably CD3ζ intracellular domain), CD8, CD19, CD134, CD20, FcεRIγ. Through their binding with corresponding ligands, the second signal of immune effector cells is activated, the proliferation ability of immune cells and the secretion function of cytokines are enhanced, and the survival time of activated immune cells is prolonged.

Given that there are very few absolute tumor-specific targets, most CAR-modified immune cells (such as CAR T cells) target antigens (such as CD19, CD20, Her2, EGFR, EpCAM, etc.) It is expressed in tissues, so on-target-off-tumor side effects are inevitable. How to reduce or reduce the effect of this On-target-off-tumor becomes very important. The inventors extensively compared the differences between the microenvironment of tumor tissue and normal tissue, and for the first time, based on the fact that some proteolytic enzymes such as uPA, MT-sp1, Legumain protease, etc. are highly expressed in tumor tissue but not expressed or low in normal tissue The specificity of these hydrolases is used to transform immune cells modified by chimeric receptors, and the obtained immune cells can only play a more effective anti-tumor function after the action of these hydrolases, thereby effectively improving immunity. Cell safety.

Therefore, the present invention provides an activatable chimeric receptor (Activatable Chimeric Receptor, ACR), which comprises a chimeric receptor (Chimeric Receptor, CR), a cleavable element (Cleavable element, CE) and a blocking element ( Blocking element, BE) is connected, and the three can be connected by connecting polypeptide. After the CE antigen is cleaved, reduced, optically decomposed, or otherwise modified, ACRs can display an activated conformation, making it easier for CR to bind to the target.

As a preferred mode of the present invention, a schematic diagram of the structure of the activatable chimeric receptor ACR of the present invention is shown in FIG. 1 . Arranged from left to right, that is, from the N-terminus to the C-terminus of the protein, a transmembrane receptor can be activated to obtain a schematic diagram of the chimeric receptor ACR. In the inactive state, BE will bind to the Ab region, or sterically hinder the binding of Ab to the target antigen; and once CE1 is cleaved by proteases, the Ab can bind to the target antigen.

The blocking element can be any block that can block the binding of the chimeric receptor to the target site by directly binding to the chimeric receptor or sterically hindering the binding of the chimeric receptor to the antigen. the role of peptides. The blocking element can be selected according to the type of antigen binding region of the chimeric receptor. For example, the antigen binding region is an antibody, and the blocking element can be a binding polypeptide of the antibody. In a specific embodiment of the present invention, the antigen-binding region of the chimeric receptor adopts anti-GPC3 antibody GC33, and its binding polypeptide (NSQQATPKDNEISTFH) is used as a blocking element.

The cleavable element is a substrate that can be cleaved, reduced or decomposed by a pathological tissue-specific or highly expressed protease. The specific or highly expressed protease can be selected according to the corresponding indication, and its substrate can be used as the cleavable element. The higher the expression specificity of the protease, the more preferred it is. In a preferred mode of the present invention, the indication is tumor, the microenvironment of tumor tissue is different from that of normal tissue, and proteolytic enzymes such as uPA, MT-sp1, Legumain protease, etc. are highly expressed in tumor tissue, while the microenvironment of tumor tissue is different from that of normal tissue. Normal tissues do not express or have low expression, so the inventors used the specificity of these hydrolases to transform immune cells modified by chimeric receptors, and the immune cells can only play an anti-tumor function more effectively after the action of these hydrolases, Thus, the safety of CAR-modified immune cells can be effectively improved. In a specific embodiment of the present invention, the substrate polypeptide LSGRSDNH of uPA and MT-SP1 protease is used as the cleavable element.

Between the chimeric receptor, the blocking element and the cleavable element, a linking peptide can also be included. The connecting peptide is not particularly limited, and can be any polypeptide that can provide flexible connection between the chimeric receptor, blocking element, and cleavable element without affecting the function of each element itself. Preferably, the linker includes 2-40 amino acids; preferably 3-30 amino acids, such as 5, 8, 10, 15, 20, 25 amino acids.

The present invention also includes nucleic acids encoding such activatable chimeric receptors. The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention.

The present invention also provides a vector comprising the nucleic acid of the activatable chimeric receptor described above. In a specific embodiment, the vector used in the present invention is a lentiviral plasmid vector pWPT. It will be appreciated that other expression vectors are also useful.

The present invention also includes viruses comprising the above-described vectors. The virus of the present invention includes the packaged infectious virus, and also includes the virus to be packaged containing the necessary components for packaging as the infectious virus. Other viruses and their corresponding plasmid vectors known in the art that can be used for the transduction of exogenous genes into immune effector cells can also be used in the present invention.

The present invention also provides a genetically modified immune effector cell transduced with a nucleic acid encoding the activatable chimeric receptor or transduced with the above-mentioned recombinant plasmid comprising the nucleic acid, or comprising the plasmid virus. Conventional nucleic acid transduction methods in the art, including non-viral and viral transduction methods, can be used in the present invention. Non-viral-based transduction methods include electroporation and transposon methods. Recently, the Nucleofector nucleotransfection instrument developed by Amaxa can directly introduce foreign genes into the nucleus to obtain efficient transduction of target genes. In addition, the transduction efficiency of transposon systems based on SleepingBeauty system or PiggyBac transposon is greatly improved compared with ordinary electroporation. It has been reported [Davies JK., et al. Combining CD19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies. Cancer Res, 2010, 70(10): OF1-10.], this method has both Higher transduction efficiency can also achieve site-directed integration of target genes. In one embodiment of the present invention, the method of transduction of immune effector cells that achieve genetic modification of chimeric receptors (in the present invention, activatable chimeric receptors) is based on transduction of viruses such as retroviruses or lentiviruses method. The method has the advantages of high transduction efficiency, stable expression of exogenous genes, and shortening the time for in vitro cultured immune effector cells to reach clinical level. On the surface of the transgenic immune effector cell, the transduced nucleic acid is expressed on its surface through transcription and translation. It is proved by in vitro cytotoxicity experiments on various cultured tumor cells that the immune effector cells of the present invention have a highly specific tumor cell killing effect (also known as cytotoxicity). Therefore, the nucleic acid encoding the chimeric receptor protein, the plasmid containing the nucleic acid, the virus containing the plasmid and the transgenic immune effector cells transduced with the nucleic acid, plasmid or virus of the present invention can be effectively used for tumor immunotherapy.

The immune cells of the present invention can also carry the coding sequences of exogenous cytokines; the cytokines include but are not limited to: IL-12, IL-15 or IL-21 and the like. These cytokines have immunomodulatory or anti-tumor activities, can enhance the function of effector T cells and activated NK cells, or directly exert anti-tumor effects. Therefore, those skilled in the art can understand that the use of these cytokines helps the immune cells to function better.

The immune cells of the present invention can also express another chimeric receptor other than the above-mentioned chimeric receptor, the receptor does not contain CD3ζ, but contains the intracellular signaling domain of CD28 and the intracellular signaling domain of CD137 or a combination of the two.

The immune cells of the present invention can also express chemokine receptors; the chemokine receptors include but are not limited to CCR2. Those skilled in the art can understand that the CCR2 chemokine receptor can make CCR2 in vivo competitively bind with it, which is beneficial for blocking tumor metastasis.

The immune cells of the present invention can also express siRNA that can reduce the expression of PD-1 or a protein that blocks PD-L1. Those skilled in the art can understand that competitive blocking of the interaction of PD-L1 with its receptor PD-1 is beneficial to restore anti-tumor T cell responses, thereby inhibiting tumor growth.

The immune cells of the present invention can also express a safety switch; preferably, the safety switch includes: iCaspase-9, Truancated EGFR or RQR8.

The chimeric receptor immune effector cells of the present invention can be used to prepare pharmaceutical compositions or diagnostic reagents. In addition to comprising an effective amount of the immune cells, the composition may also comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.

Specific examples of some substances which may be pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxymethyl cellulose Sodium, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycols; alginic acid; emulsifiers such as Wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions and phosphate buffers, etc.

The composition of the present invention can be prepared into various dosage forms according to needs, and can be administered by a physician at a dose beneficial to the patient according to factors such as the type, age, weight and general disease state of the patient, and the mode of administration. The mode of administration can be, for example, by injection or other treatment.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, 2002, or according to the conditions described by the manufacturer. the proposed conditions.

Example 1. Construction of anti-GPC3 third-generation chimeric antigen receptors

Using the previously constructed anti-glypican-3 (GPC3) third-generation chimeric antigen receptor (Chimericantigen receptor, CAR), the single-chain antibody used in the CAR is derived from the GC33 antibody. For the construction of the CAR T, see In the application number CN201310164725.X, its GPC3-28BBZ is used in this embodiment, which is also called GC33-28BBZ. The amino acid sequence of the single chain antibody described therein is shown in SEQ ID NO: 13 (VL _GPC3- (G ₄ S) ₃ -VH _GPC3 ).

The inventors used the binding polypeptide of GC33 antibody (NSQQATPKDNEISTFH (SEQ ID NO: 1)) as the BE element, and the substrate polypeptide LSGRSDNH (SEQ ID NO: 2) of uPA and MT-SP1 protease as the CE element. In addition, the ribosomal skipping sequence 2A (F2A) from the foot-and-mouth disease virus (FMDV) is used to link eGFP to the C-terminus of ACR to realize the co-expression of eGFP and ACR. The expression of eGFP indirectly indicates the expression of ACR.

(1) Obtaining the fragment of Sp+BE+LP1+CE+LP2

Primers were designed as shown in Table 1.

Table 1

Wherein the fragment of Sp+BE+LP1+CE+LP2 adopts the primers as shown in Table 1, and is obtained by Overlap PCR, and the amplification conditions are:

Pre-denaturation: 94℃, 4min;

25 cycles as follows: Denaturation: 94°C, 30s,

Annealing: 50℃, 30s,

Extension: 68℃, 20s;

Then, it was extended for another 10 min at 68°C.

Subsequently, the PCR amplification products were recovered by gel using conventional methods to obtain DNA fragments connected to each other by Sp+BE+LP1+CE+LP2.

(2) Acquisition of LP2-GC33-28BBZ fragment

Design primers as in 2.

Table 2

Take 1 microliter (100ng) of GC33-28BBZ plasmid (see CN201310164725.X patent for this plasmid) as a template, use GPC3scfv-F and 3z-F2A-R as upstream and downstream primers respectively, and carry out PCR amplification in a 50 microliter system to obtain The PCR product was recovered by gel cutting to obtain the LP2-GC33-28BBZ fragment.

(3) Acquisition of F2A-EGFP

Design primers as in 3.

table 3

Take 1 microliter (100 ng) of the GC33-28BBZ plasmid as a template, use F2A-EGFP-F and pwpt-EGFP-R as upstream and downstream primers, respectively, carry out PCR amplification, and the obtained PCR products are recovered by cutting gel to obtain F2A- EGFP.

(4) Acquisition of lentiviral plasmid pWPT-GC33-28BBZ-ACR

Carry out overlap PCR with the three fragments prepared in the above (1) to (3). Specifically, the three fragments are mixed according to an equimolar ratio, and the overlap PCR is carried out as described in the above step (1). The upstream and downstream primers are CD8sp. -F with EGFP-R. The obtained PCR products were recovered by gel cutting.

MluI and SalI were double digested with restriction endonucleases and then ligated into the corresponding site of the lentiviral plasmid pWPT vector.

The obtained recombinant plasmid was transformed into Escherichia coli Top10 competent cells, and cultured for about 12 h for verification by picking clones. Clones containing recombinant plasmids were selected for sequencing. Sequence-correct clones were selected for virus preparation and packaging.

The obtained vector was named pWPT-GC33-28BBZ-ACR.

Example 3. Preparation of CAR-T

1. Packaging of lentivirus of GC33-28BBZ-ACR

HEK-293T cells (ATCC: CRL-11268) cultured to passages 6 to 10 were seeded at a density of 6×10 ⁶ in 10 cm dishes at 37° C., 5% CO ₂ overnight to prepare for transfection. The medium was DMEM (purchased from gibco company) containing 10% fetal bovine serum (purchased from gibco company).

The steps of transfection are as follows:

(1) Dissolve 10 μg of the target gene plasmid pWPT-GC33-28BBZ-ACR, 6.5 μg of the packaging plasmid PAX2: and 3.5 μg of the envelope plasmid pMD2.G into 800 μL of serum-free DMEM medium, and mix well.

(2) Add 60 μl of PEI (polyethyleneimine, purchased from Polysciences, into a working solution with a concentration of 1 μg/μL), into the above-mentioned 800 μL of serum-free DMEM medium with plasmids, vortex and mix, and let stand at room temperature Incubate for 25min.

(3) 800 μL of the transfection complex was added to the HEK-293T cells to be transfected. After 6-8 hours, the medium was changed to the transfected 293T cells with DMEM medium with 10% FBS.

(4) About 24 hours after transfection, the transfection efficiency (ie, the proportion of cells with green fluorescence) was observed. 72h after transfection, the virus was collected by filtration using a 0.45 μm filter (purchased from Millipore), and the virus was concentrated by ultracentrifugation (Beckman Optima L-100XP ultracentrifuge at 28000rpm, 4°C for 2 hours). The pellet obtained by centrifugation was resuspended with 1/30 stock volume of AIM-V medium (purchased from Gibco Company), and aliquoted in 100 μL/tube and stored at -80° C. to infect T lymphocytes. At the same time, the concentrated virus obtained is titrated.

2. Infection of GC33-28BBZ-ACR

Human peripheral blood mononuclear cells (provided by Shanghai Blood Center) were obtained from healthy human peripheral blood by density gradient centrifugation. CD4+ and CD8+ positive primary human T lymphocytes were obtained from peripheral blood mononuclear cells by negative sorting with CD4+/CD8+ cell magnetic beads (purchased from Stem Cell Technologies). The purity of the sorted T cells is tested by flow cytometry, and the next step is appropriate when the positive rate of the target cells is ≥95%. AIM-V lymphocyte culture medium (purchased from Gibco, containing 2% human AB serum) was added at a density of 1 × 10 ⁶ /mL for culture and added at a cell:magnetic bead ratio of 1:1 and coated with anti-CD3 and Magnetic beads of CD28 antibody (Invitrogen) and recombinant human IL-2 (purchased from Shanghai Huaxin Biotech Co., Ltd.) with a final concentration of 300 U/mL were stimulated and cultured for 24 h. Before infection, CD4+CD8+ T lymphocytes were mixed at a ratio of 1:1, then the virus concentrate was mixed with the T cells to be infected at MOI ≈ 5-10, and polybrene was added at a final concentration of 6 μg/mL. After infection, the medium was changed every other day, and the medium without virus was changed. The cells were passaged at a density of 5×10 ⁵ /mL every other day, and the lymphocyte culture medium was supplemented with recombinant human IL-2 at a final concentration of 300 U/mL.

The expression of chimeric antigen receptor GC33-28BBZ-ACR was detected by flow cytometry in primary T cells after infection on days 9-11 (i.e., the end of the first expansion cycle). -ACR co-expression, the positive cells for the detection of eGFP are the positive cells expressing the chimeric antigen receptor, and the uninfected T lymphocytes are used as the negative control.

The results are shown in Figure 3. The positive rate of GFP was used as an indicator of the positive rate of GC33-28BBZ-ACR in T cells after lentivirus infection, and the positive rate was 61.0%.

Example 4. Exogenous recombinant uPA promotes the activation of BE-GPC3-CAR T

The materials used in the in vitro toxicity experiments are as follows:

The liver cancer cell line Huh-7 was used as the target cells, and the effector cells were T cells (positive rate 61%) expressing GC33-28BBZ-ACR cultured in vitro for 11 days. The effector-target ratios were 3:1, 1:1 and 1, respectively. : 3, the number of target cells is 10,000 cells/well, corresponding to effector cells according to different effector-target ratios. Add uPA and MTSP1 respectively, and set up a low-dose group (uPA: 0.08μg/mL; MT-SP1: 0.04μg/mL) and a high-dose group (uPA: 0.4μg/mL; MT-SP1: 0.2μg/mL) ) and blank group (uPA: 0 μg/mL; MT-SP1: 0 μg/mL). Each group was set with 5 duplicate wells, and the average value of the 5 duplicate wells was taken. The detection time is the 18th hour.

The experimental groups and the control groups are as follows:

Experimental group (blank group): target cells Huh-7+GC33-28BBZ-ACR T cells+different concentrations of enzymes,

Control group 1: target cells release LDH+ enzymes at different concentrations at the maximum,

Control group 2: target cells spontaneously release LDH+ enzymes at different concentrations,

Control group 3: Effector cells spontaneously release LDH+ enzymes at different concentrations.

Detection method: CytoTox 96 non-radioactive cytotoxicity detection kit (Promega company) was used for detection.

The formula for calculating cytotoxicity is:

It can be seen from Figure 4 that the antitumor activity of GC33-28BBZ-ACR T cells (T cells infected with GC33-28BBZ-ACR) is low, but can be effectively activated under the action of uPA or MT-SP1, thereby Kill tumor cells.

The above results show that the design of the present invention is reasonable, that is, through the action of the blocking element, the killing effect of T cells can indeed be weakened; but in the local environment of the tumor, if there is a sufficient amount of uPA or MT-SP1 and other corresponding proteases. ACR can be activated and kill tumor cells, thereby exerting local anti-tumor activity and reducing damage to normal tissues.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (17)

1. An activatable chimeric receptor comprising: Chimeric receptors, which target highly expressed antigens in pathological tissues in an activated state; said chimeric receptors comprise an extracellular antigen-binding region, which is a single-chain antibody; said chimeric receptors are highly expressed in pathological tissues The antigen is GPC3; the amino acid sequence of the single-chain antibody is shown in SEQ ID NO: 13; A blocking element, which inhibits the binding of the chimeric receptor to the highly expressed antigen of the pathological tissue; the blocking element is a binding polypeptide of the GC33 antibody, which is a polypeptide of the amino acid sequence shown in SEQ ID NO: 1; and A cleavable element located between the chimeric receptor and the blocking element. 2. The activatable chimeric receptor of claim 1, wherein the chimeric receptor comprises sequentially connected: an extracellular antigen binding region, a transmembrane region and an intracellular signal region; the Said intracellular signal region is selected from: CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134, the intracellular signal region sequence of CD40 or Myd88, or a combination thereof. 3. The activatable chimeric receptor according to claim 1, wherein the activatable chimeric receptor comprises, in order from the amino terminus to the carboxyl terminus: a blocking element, a cleavable element , chimeric receptors. 4. The activatable chimeric receptor according to claim 1, wherein the chimeric receptor, the blocking element and the cleavable element further comprise a linker peptide. 5. The activatable chimeric receptor of claim 1, wherein the cleavable element is an element that is cleaved, reduced or decomposed by a protease specifically expressed or highly expressed in pathological tissues , the protease and the highly expressed antigen of the pathological tissue co-localize in the same pathological tissue. 6. The activatable chimeric receptor of claim 5, wherein the pathological tissue comprises: a tumor. 7. The activatable chimeric receptor according to claim 6, wherein the pathological tissue-specifically expressed or highly expressed protease comprises: urokinase-type plasminogen activator, legumain protease or matriptase. 8. The activatable chimeric receptor of claim 7, wherein the pathological tissue-specifically expressed or highly expressed protease is urokinase-type plasminogen activator or matriptase, and the The cleavable element is the polypeptide of the amino acid sequence shown in SEQ ID NO:2. 9. A polynucleotide encoding the activatable chimeric receptor of any one of claims 1-8. 10. An expression vector comprising the polynucleotide of claim 9. 11. A virus, wherein the virus comprises the expression vector of claim 10. 12. Use of the activatable chimeric receptor of any one of claims 1-8, or a nucleic acid encoding the same, or an expression vector or virus comprising the nucleic acid, for the preparation of a chimeric receptor targeting pathological tissue. immune effector cells. 13. A chimeric receptor immune effector cell transduced with a nucleic acid encoding the activatable chimeric receptor of any one of claims 1-8, or the expression vector of claim 10 or claim 11 The virus; or the surface expression of the activatable chimeric receptor according to any one of claims 1-8. 14. The chimeric receptor immune effector cell of claim 13, wherein the immune effector cell comprises: T lymphocytes, NK cells or NKT cells, and Treg cells. 15. Use of the chimeric receptor immune effector cell according to claim 13 or 14, for the preparation of a drug targeting pathological tissue, the pathological tissue highly expresses the antigen to which the chimeric receptor can bind, the The antigen is GPC3. 16. The use of claim 15, wherein the pathological tissue is a tumor, and the drug targeting the pathological tissue is a tumor-inhibiting drug. 17. A pharmaceutical composition, comprising: the chimeric receptor immune effector cell of claim 13 or 14.