CN106519037A - Chimeric receptor capable of being activated - Google Patents

Abstract

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

Description

activatable chimeric receptor

technical field

The invention belongs to the field of tumor immunotherapy, and more specifically, the invention relates to an activatable chimeric receptor, a preparation method and application thereof.

Background technique

The role of immune effector cells in the tumor immune response has been increasingly recognized. 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 curative effect in most tumors is still unsatisfactory [Grupp SA, et al. al.Adoptive cellular therapy.Curr Top Microbiol Immunol., 2011;344:149-72.]. In recent years, based on the discovery that cytotoxic T lymphocytes (cytotoxic lymphocytes, CTLs) recognize target cells specifically depends on T cell receptors (T Cell Receptors, TCRs), the scFv of antibodies against tumor cell-associated antigens was combined with Intracellular signal activation motifs such as CD3ζ or FcεRIγ of T lymphocyte receptors are fused into chimeric antigen receptors (Chimeric antigen receptors, CAR; also known as chimeric receptors (CR) now), and passed through such as Lentiviral infection and other ways of genetically modifying the surface of T lymphocytes. This CAR T lymphocyte can selectively direct T lymphocytes to tumor cells in a major histocompatibility complex (Major Histocompatibility Complex, MHC) non-restricted manner and specifically kill tumors. 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. JBiomed Biotechnol, 2010, doi:10.1155/2010/956304.]. In addition, CAR-modified NK cells (Klingemann H. Challenges of cancer therapy with natural killer cells. Cytotherapy. 2014 Dec 18.pii: S 1465-3249(14) 00791-9) or NKT cells have also shown good performance in preclinical studies. Antitumor activity (Heczey A1, Liu D1, Tian G2, CourtneyAN1, Wei J1, Marinova E1, Gao X1, Guo L1, Yvon E3, Hicks J2, Liu H4, Dotti G5, Metelitsa LS6.Invariant NKT cells with chimeric antigen receptorprovide a novel platform for safe and effective cancer immunotherapy. Blood. 2014; 124(18):2824-33).

Chimeric antigen receptors include an extracellular binding region, a transmembrane region and an intracellular signaling region. 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 immunoreceptor tyrosine activation motif (ITAM) CD3ζ or FcεRIγ and co-stimulatory Intracellular signaling regions of signaling molecules CD28, CD27, CD137, CD134, etc.

The first-generation CAR T lymphocytes containing only ITAM in the intracellular signal region, in which each part of the chimeric antigen receptor is connected in the following form: scFv-TM-ITAM. This kind of CAR T can stimulate anti-tumor cytotoxic effect, but the secretion of cytokines is relatively small, and it cannot stimulate long-lasting anti-tumor effect 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 subsequent development of the second-generation CAR T lymphocytes added the intracellular signaling region of CD28 or CD137 (also known as 4-1BB), in which the parts of the chimeric antigen receptor were connected in the following form: scFv-TM-CD28-ITAM or scFv -TM-/CD137-ITAM. The B7/CD28 or 4-1BBL/CD137 co-stimulation in the intracellular signal region causes the continuous proliferation of T lymphocytes, and can increase the levels of cytokines secreted by T lymphocytes such as IL-2 and IFN-γ, and at the same time increase the CAR T In vivo survival cycle and anti-tumor effect [Dotti G. et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor modified T cells in lymphoma patients. J Clin Invest, 2011, 121(5): 1822-1826.].

In the third-generation CAR T lymphocytes developed in recent years, the parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, which further improves the CAR T lymphocytes. The survival cycle of T in vivo and its anti-tumor effect [Carpenito C., et al. Control of large established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. PNAS, 2009, 106(9): 3360–3365.].

Although CAR T lymphocytes have attractive prospects 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 normal tissues, CAR T lymphocytes may damage normal tissues expressing antigens. For example, targeting the antigen carbonic anhydrase IX (CAIX) expressed on the tumor cells of patients with renal cell carcinoma is the first case of adoptive therapy of CART lymphocytes used in clinical practice, and it is also the first case to report the off-target effect 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, the original clinical trial was forced to be interrupted and any evaluation of the treatment effect of the patient was excluded [Stoter G. et al. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J clin oncol, 2006, 24(13): e20-e22.; Ngo MC., et al. Ex vivo gene transfer for improved adopted immunotherapy of cancer. Human Molecular Genetics, 2011, R1-R7]. In addition, excessive co-stimulatory signals in CAR will reduce the threshold required for effector cell activation, so that genetically modified T lymphocytes may also be activated under low-level or no antigen-triggered conditions, resulting in the release of a large number of cytokines As a result, the so-called "cytokine storm" may be triggered. This signal leakage 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 the third-generation CAR targeting Her2, the so-called "cytokine storm" caused the sudden death of the patient due to the low expression of Her2 in normal lung tissue[Morgan RA., et al. Report of a serious 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 to avoid the high risks of applying to tumor immunotherapy.

Contents of the invention

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

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

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

Blocking element (Blocking element, BE), which can inhibit the binding of the chimeric receptor to the antigen highly expressed in the pathological tissue; and

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

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

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

In another preferred example, the antibody specifically binding to an antigen highly expressed 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 example, the antibody specifically binding to the antigen highly expressed in the pathological tissue is selected from (but not limited to): single chain antibody or single domain antibody.

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

In another preferred example, the activatable chimeric receptor comprises, in sequence from the amino terminal to the carboxyl terminal: a blocking element, a cleavable element, and a chimeric receptor.

In another preferred example, the chimeric receptor, the blocking element, and the cleavable element further include a connecting peptide.

In another preferred example, the activatable chimeric receptor contains a first linking polypeptide (LP1) and a second linking polypeptide (LP2); wherein the activatable chimeric receptor is sequenced from the amino terminal to the carboxyl The order of the end includes: blocking element, first linking polypeptide, cleavable element, second linking polypeptide, 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 example, the blocking element is 2-100aa (such as 10aa, 20aa, 30aa, 40aa, 50aa, 60aa, 70aa, 80aa); preferably a polypeptide of 2-40aa.

In another preferred example, the cleavable element is an element that can be cleaved, reduced or decomposed by a protease specifically expressed or highly expressed in pathological tissues, and the protease is related to the highly expressed protein in the pathological tissue. Antigens colocalize in the same pathological tissue.

In another preferred example, the pathological tissues include but are not limited to: tumors, autoimmune disease tissues, and tissues infected by viruses (such as HIV virus).

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

In another preferred example, 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 antigens highly expressed 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 antigen highly expressed 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 antigen highly expressed 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, a polynucleotide encoding the activatable chimeric receptor is provided.

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

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

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

In another aspect of the present invention, there is provided any one of the aforementioned activatable chimeric receptors, or the nucleic acid encoding it, or the use of an expression vector or virus comprising the nucleic acid, for preparing pathological tissue-targeted Chimeric receptor immune effector cells.

In another aspect of the present invention, a chimeric receptor immune effector cell is provided, which is 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 one of the aforementioned activatable chimeric receptors.

In another preferred example, 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 the preparation of drugs targeting pathological tissues, and the pathological tissues highly express the antigens to which the chimeric receptors can bind.

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

In another aspect of the present invention, a pharmaceutical composition is provided, which comprises: 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.

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

Figure 2. Schematic diagram 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, enzyme-cleavable substrate of UP1), 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 to indicate the positive rate of GC33-28BBZ-ACR of 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 effect-to-target ratios were 3:1, 1:1 and 1:3, respectively. 10,000 cells/well, corresponding to effector cells according to different effector-target ratios. Add uPA and MTSP1 respectively, set up a low-dose group, a high-dose group and a blank group. Each group had 5 replicate holes, and the average value of the 5 replicate holes was taken. The detection time is the 18th hour.

detailed description

After extensive research, the inventors have revealed an activatable chimeric receptor based on chimeric antigen receptor (CAR) technology, which can only be specifically expressed or highly expressed in pathological tissues Only when the protease exists can it target the corresponding antigen and realize the killing effect of pathological tissues or cells; it will 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" and "chimeric antigen receptor" are used interchangeably.

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 "antigen highly expressed in pathological tissue" is a tumor-associated antigen, for example 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 protein (hydrolytic) enzyme expressed only in pathological tissues or cells, or highly expressed in pathological tissues or cells, which Able to hydrolyze 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, by directly binding to the chimeric receptor or sterically hindering the The combination of the above-mentioned chimeric receptor and antigen can play a role in 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" , when the protease exists, the "cleavable element" can be cleaved, reduced or decomposed, 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 not limited to): tumor, autoimmune disease tissue, virus (such as HIV virus) infected tissue, etc.

In the present invention, the pathological tissue may be various harmful tissues or lesions in the body that are not conducive to health, and must be removed from the body. Said pathological target tissues include tumors. Any tumor known in the art can be included in the present invention, as long as the tumor can express tumor-associated antigens that are lowly expressed in normal tissues. For example, the tumors include (but 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 tumors, adrenal tumors, bladder tumors, testicular tumors, malignant lymphoma, multiple myeloma, nervous system tumors, esophageal cancer, thymic mesothelioma, pancreatic cancer, leukemia, head and neck tumors, cervical cancer, skin cancer, melanoma tumor, vaginal epithelial carcinoma, gallbladder carcinoma, malignant fibrous histiocytoma. For example, the tumor-associated antigens include (but not limited to): GPC3, EGFR, HER2, EphA2, Claudin18.1, Claudin18.2, Claudin 6, GD2, EpCAM, mesothelin, CD19, CD20, ASGPR1, EGFRvIII, de4EGFR, CD19, CD33, IL13R, LMP1, PLAC1, NY-ESO-1, MAGE4, MUC1, MUC16, LeY, CEA, CAIX (carbonic anhydrase IX), CD123.

The term "chimeric receptor immune effector cells" is well known in the art, which are immune effector cells expressing antigen (such as tumor antigen)-specific chimeric receptors by genetic engineering 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 (Regulatory cell, 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 molecular 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 combination 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 absolutely specific targets for tumors, the antigens (such as CD19, CD20, Her2, EGFR, EpCAM, etc.) targeted by most CAR-modified immune cells (such as CAR T cells) are more or less normal Tissue expression, so on-target-off-tumor side effects are unavoidable. How to reduce or reduce the effect of this on-target-off-tumor becomes very important. The inventors extensively compared the difference between the microenvironment of tumor tissue and the microenvironment of normal tissue. For the first time, some proteolytic enzymes such as uPA, MT-sp1, and Legumain protease were highly expressed in tumor tissue but not or low in normal tissue. The expression characteristics of these hydrolytic enzymes are used to transform the immune cells modified by chimeric receptors. The obtained immune cells can only exert anti-tumor functions more effectively after the action of these hydrolytic enzymes, thereby effectively improving the immune system. 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 (Cleavableelement, CE) and a blocking element (Blocking element, BE) connection, the three can be connected by linking polypeptide. After the CE antigen is cleaved, reduced, optically decomposed or otherwise modified, the ACRs can display an activated conformation, making it easier for the 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-terminal to the C-terminal of the protein, a transmembrane receptor can be activated to obtain a schematic diagram of the chimeric receptor ACR. In the unactivated 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 function 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 the polypeptide. The blocking element can be selected according to the type of antigen binding region of the chimeric receptor. For example, if the antigen binding region is an antibody, the blocking element can be the binding polypeptide of the antibody. In a specific embodiment of the present invention, the anti-GPC3 antibody GC33 is used for the antigen-binding region of the chimeric receptor, 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 proteases specifically expressed or highly expressed in pathological tissues. The protease specifically expressed or highly expressed 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. In the preferred mode of the present invention, the indication is tumor, and the microenvironment of tumor tissue is different from that of normal tissue, wherein proteolytic enzymes such as uPA, MT-sp1, Legumain protease, etc. are highly expressed in tumor tissue, while in No or low expression in normal tissues, so the inventors used the specificity of these hydrolytic enzymes to engineer chimeric receptor-modified immune cells, and the immune cells could more effectively exert anti-tumor functions only after the action of these hydrolytic enzymes. Thereby effectively improving the safety of CAR-modified immune cells. In a specific embodiment of the present invention, the substrate polypeptide LSGRSDNH of uPA and MT-SP1 protease is used as the cleavable element.

A linker peptide may also be included among the chimeric receptors, blocking elements, and cleavable elements. The connecting peptide is not particularly limited, and can be any polypeptide that can provide flexible connections among the chimeric receptors, blocking elements, and cleavable elements without affecting the functions 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 invention also includes nucleic acids encoding said activatable chimeric receptors. The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention.

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

The present invention also includes viruses comprising the vectors described above. The viruses of the present invention include packaged infectious viruses, as well as viruses to be packaged that contain the components necessary for packaging as infectious viruses. Other viruses and their corresponding plasmid vectors known in the art that can be used to transfer foreign genes into immune effector cells can also be used in the present invention.

The present invention also provides genetically modified immune effector cells, which are transduced with the nucleic acid encoding the activatable chimeric receptor or transduced with the above-mentioned recombinant plasmid containing 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 nucleofector developed by Amaxa can directly introduce exogenous genes into the nucleus to obtain high-efficiency transduction of target genes. In addition, the transduction efficiency of transposon systems based on Sleeping Beauty system or PiggyBac transposon is greatly improved compared with ordinary electroporation, and the combined application of nucleofector transfection instrument and Sleeping Beauty transposon system It has been reported [Davies JK., et al.Combining CD19redirection and alloanergization to generate tumor-specifichuman T cells for allogeneic cell therapy of B-cell malignancies.Cancer Res, 2010, 70(10):OF1-10.], the method It not only has a high transduction efficiency but also can realize the site-specific integration of the target gene. In one embodiment of the invention, the method of transduction of immune effector cells genetically modified to achieve chimeric receptors (in the present invention activatable chimeric receptors) is based on the transduction of viruses such as retroviruses or lentiviruses method. This method has the advantages of high transduction efficiency, stable expression of exogenous genes, and can shorten the time for culturing immune effector cells in vitro to reach clinical-grade quantities. On the surface of the transgenic immune effector cells, the transduced nucleic acid is expressed on the surface through transcription and translation. In vitro cytotoxicity experiments on various cultured tumor cells prove that the immune effector cells of the present invention have highly specific tumor cell killing effects (also known as cytotoxicity). Therefore, the nucleic acid encoding the chimeric receptor protein of the present invention, the plasmid containing the nucleic acid, the virus containing the plasmid, and the transgenic immune effector cells transduced with the above nucleic acid, plasmid or virus can be effectively used in the immunotherapy of tumors.

The immune cells of the present invention can also carry coding sequences of exogenous cytokines; said cytokines include but not limited to: IL-12, IL-15 or IL-21 and so on. These cytokines have immunoregulatory 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, which 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 not limited to CCR2. Those skilled in the art can understand that the CCR2 chemokine receptor can make CCR2 in the body competitively bind to 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 competitively blocking the interaction between PD-L1 and its receptor PD-1 is beneficial to restore the anti-tumor T cell response, 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 applied to the preparation of pharmaceutical compositions or diagnostic reagents. In addition to the effective amount of the immune cells, the composition may also contain 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 that can be used as 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 carboxymethylcellulose sodium, ethylcellulose, and methylcellulose; 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, glycerin, sorbitol, mannitol, and polyethylene glycol; 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 solution and phosphate buffer, etc.

The composition of the present invention can be made into various dosage forms according to needs, and can be administered by the doctor according to the patient's type, age, body weight and general disease condition, administration method and other factors to determine the dosage beneficial to the patient. The mode of administration may be, for example, injection or other therapeutic means.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as edited by J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the conditions described in the manufacturer suggested conditions.

Example 1, Construction of the third-generation chimeric antigen receptor against GPC3

Using the previously constructed anti-phosphatidylinositol proteoglycan-3 (GPC3) third-generation chimeric antigen receptor (Chimeric antigen receptor, CAR), the single-chain antibody used in the CAR is derived from the GC33 antibody, and the construction of the CAR T See application number CN201310164725.X, in which GPC3-28BBZ is applied in this embodiment, also known as GC33-28BBZ.

The inventors used the binding polypeptide of the 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, eGFP is connected to the C-terminus of ACR by the ribosomal skipping sequence 2A (ribosomal skipping sequence 2A) (F2A) from food-and-mouth disease virus (FMDV) to realize the co-expression of eGFP and ACR. The expression of eGFP can indirectly explain the expression of ACR.

(1) Acquisition of fragments of Sp+BE+LP1+CE+LP2

Primers were designed as shown in Table 1.

Table 1

The fragments of Sp+BE+LP1+CE+LP2 are obtained by Overlap PCR using the primers in Table 1, and the amplification conditions are as follows:

Pre-denaturation: 94°C, 4min;

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

Annealing: 50℃, 30s,

Extension: 68℃, 20s;

Then, extend at 68°C for another 10 min.

Subsequently, the PCR amplification product was gel-recovered by a conventional method to obtain a DNA fragment in which Sp+BE+LP1+CE+LP2 were interconnected.

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

Design primers as in 2.

Table 2

Take 1 microliter (100ng) of GC33-28BBZ plasmid (see patent CN201310164725.X 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 (100ng) of GC33-28BBZ plasmid as a template, use F2A-EGFP-F and pwpt-EGFP-R as upstream and downstream primers respectively, and carry out PCR amplification, and the obtained PCR products are recovered by gel cutting to obtain F2A- EGFP.

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

Perform overlap PCR on the three fragments prepared in the above (1)-(3), specifically, mix the three fragments in an equimolar ratio, and perform overlap PCR 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 sites of the lentiviral plasmid pWPT vector.

The obtained recombinant plasmid was transformed into Escherichia coli Top10 competent cells, and cultured for about 12 hours to verify clones. Clones containing recombinant plasmids were selected for sequencing. Clones with the correct sequence were selected for virus preparation and packaging.

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

Embodiment 3, preparation of CAR-T

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

Inoculate HEK-293T cells (ATCC: CRL-11268) from passage 6 to passage 10 at a density of 6×10 ⁶ in a 10 cm dish, and culture overnight at 37°C in 5% CO ₂ for transfection. The culture medium is 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 culture medium, and mix well.

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

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

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

2. Infection of GC33-28BBZ-ACR

Human peripheral blood mononuclear cells (provided by Shanghai Blood Center) were obtained from peripheral blood of healthy people by density gradient centrifugation. CD4+ and CD8+ positive primary human T lymphocytes were obtained from peripheral blood mononuclear cells by CD4+/CD8+ cell magnetic beads (purchased from Stem Cell Technologies) negative sorting method. The purity of the sorted T cells was tested by flow cytometry, and the next step should be carried out when the positive rate of the target cells was ≥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 the ratio of cells: magnetic beads was added at a 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.) at a final concentration of 300 U/mL stimulated the culture for 24 hours. Before infection, mix CD4+CD8+T lymphocytes at a ratio of 1:1, then mix the above virus concentrate with T cells to be infected at MOI≈5-10, and add polybrene at a final concentration of 6 μg/mL. For infected cells, change the medium every other day, and change the culture medium that does not contain the virus. Subculture was carried out at a density of 5×10 ⁵ /mL every other day, and at the same time, recombinant human IL-2 was added to the lymphocyte culture medium at a final concentration of 300 U/mL.

The expression of chimeric antigen receptor GC33-28BBZ-ACR was detected by flow cytometry on days 9-11 (that is, the end of the first round of expansion cycle) of the primary T cells after infection. Since eGFP and GC33-28BBZ - ACR co-expression, the positive cells that detect eGFP are the positive cells expressing the chimeric antigen receptor, and uninfected T lymphocytes are used as negative controls.

The results are shown in Figure 3. The positive rate of GFP is used as an indicator of the positive rate of GC33-28BBZ-ACR of T cells after lentivirus infection. It can be seen that the positive rate is 61.0%.

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

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

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

Wherein each experimental group and each control group 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,

Control group 2: target cells spontaneously released LDH + enzymes of different concentrations,

Control group 3: effector cells spontaneously release LDH+enzyme 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 anti-tumor activity of GC33-28BBZ-ACR T cells (T cells infected with GC33-28BBZ-ACR) is low, but they 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, the killing effect of T cells can indeed be weakened by the function of the blocking element; but if there is a sufficient amount of corresponding proteases such as uPA or MT-SP1 in the local tumor environment ACR can be activated and kill tumor cells, thereby exerting local anti-tumor activity and reducing damage to normal tissues.

All documents mentioned in this application are incorporated by reference in this application as if each 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 (22)

1. An activatable chimeric receptor comprising: Chimeric receptors, which can target antigens highly expressed in pathological tissues in the activated state; a blocking element that inhibits the binding of the chimeric receptor to an antigen highly expressed in said pathological tissue; and A cleavable element positioned between the chimeric receptor and the blocking element. 2. The activatable chimeric receptor according to claim 1, characterized in that, said chimeric receptor comprises sequentially connected: an extracellular antigen-binding region, a transmembrane region and an intracellular signal region; The intracellular signal region is selected from: CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134, CD40 intracellular signal region sequence or Myd88, or a combination thereof. 3. The activatable chimeric receptor according to claim 2, wherein the extracellular antigen-binding region is an antibody that specifically binds to an antigen highly expressed in the pathological tissue. 4. The activatable chimeric receptor according to claim 3, characterized in that, the antibody specifically binding to the antigen highly expressed in the pathological tissue is selected from: single chain antibody or single domain antibody. 5. The activatable chimeric receptor as claimed in claim 1, characterized in that, the activatable chimeric receptor comprises in sequence from the amino terminal to the carboxyl terminal: a blocking element, a cleavable element , a chimeric receptor. 6. The activatable chimeric receptor according to claim 1, characterized in that, the chimeric receptor, the blocking element and the cleavable element further include a connecting peptide. 7. The activatable chimeric receptor of claim 1, wherein said blocking element is selected from the group consisting of: A polypeptide that directly binds to said chimeric receptor; or A polypeptide that sterically blocks said chimeric receptor from binding to an antigen. 8. The activatable chimeric receptor according to claim 1, wherein the cleavable element can be cleaved, reduced or decomposed by proteases specifically expressed or highly expressed in pathological tissues element, the protease colocalizes with the antigen highly expressed in the pathological tissue in the same pathological tissue. 9. The activatable chimeric receptor according to claim 8, wherein said pathological tissues include: tumors, autoimmune disease tissues, and virus-infected tissues. 10. The activatable chimeric receptor according to claim 9, wherein the pathological tissue is a tumor, and the protease specifically expressed or highly expressed in the pathological tissue comprises: urokinase-type cellulose Lysinogen activator, legumain protease or matriptase. 11. The activatable chimeric receptor as claimed in claim 10, characterized in that, the protease specifically expressed or highly expressed in the pathological tissue is urokinase-type plasminogen activator or matriptase, and the The cleavable element of is the polypeptide of the amino acid sequence shown in SEQ ID NO:2. 12. The activatable chimeric receptor according to claim 9, wherein the antigens highly expressed in pathological tissues include: GPC3, EGFR, HER2, EphA2, Claudin18.1, Claudin18.2, Claudin 6. GD2, EpCAM, mesothelin, CD19, CD20 or ASGPR1. 13. The activatable chimeric receptor according to claim 12, wherein the antigen highly expressed in the pathological tissue is GPC3, and the blocking element is a binding polypeptide of the GC33 antibody; preferably, The blocking element is a polypeptide of the amino acid sequence shown in SEQ ID NO:1. 14. A polynucleotide encoding the activatable chimeric receptor of any one of claims 1-13. 15. An expression vector comprising a nucleic acid encoding the activatable chimeric receptor of claim 14. 16. A virus, characterized in that the virus comprises the vector of claim 15. 17. Use of the activatable chimeric receptor according to any one of claims 1-13, or the nucleic acid encoding it, or the expression vector or virus comprising the nucleic acid, for preparing chimeric receptors targeting pathological tissues immune effector cells. 18. A chimeric receptor immune effector cell transduced with the nucleic acid encoding the activatable chimeric receptor of any one of claims 1-13, or the expression vector of claim 15 or the expression vector of claim 16 The virus; or its surface expresses the activatable chimeric receptor according to any one of claims 1-13. 19. The chimeric receptor immune effector cell according to claim 18, characterized in that, the immune effector cell comprises: T lymphocyte, NK cell or NKT cell, Treg cell. 20. The use of the chimeric receptor immune effector cells according to claim 18 or 19, for the preparation of drugs targeting pathological tissues, and the pathological tissues highly express the antigens to which the chimeric receptors can bind. 21. The use according to claim 20, wherein the pathological tissue is a tumor, and the drug targeting the pathological tissue is a tumor suppressing drug. 22. A pharmaceutical composition, characterized in that it comprises: the chimeric receptor immune effector cell according to claim 18 or 19.