WO2017041749A1

WO2017041749A1 - Activatable chimeric receptor

Info

Publication number: WO2017041749A1
Application number: PCT/CN2016/098617
Authority: WO
Inventors: 李宗海; 陈骋; 蒋华; 王华茂; 王鹏
Original assignee: 科济生物医药（上海）有限公司; 上海市肿瘤研究所
Priority date: 2015-09-11
Filing date: 2016-09-09
Publication date: 2017-03-16
Also published as: CN106519037A; CN106519037B

Abstract

Provided is an activatable chimeric receptor containing a closing element and a shearable element. The activatable chimeric receptor is in an unactivated state in normal tissues, and is activated in a pathological tissue which highly expresses a specific protease, thus making the chimeric receptor target a corresponding antigen, so that the killing of the pathological tissue or cells is achieved, and the effect of solving the problems that the on target/off tumour of a general chimeric receptor during the treatment of tumours is achieved.

Description

Activated chimeric receptor

Technical field

The present invention is in the field of tumor immunotherapy, and more particularly, the present invention relates to activatable chimeric receptors, methods for their preparation, and uses thereof.

Background technique

The role of immune effector cells in tumor immune response is receiving increasing attention. Adoptive immunotherapy based on immune effector cells has achieved certain effects in some tumors, and this immunotherapy method can overcome the above defects of antibody treatment, but the therapeutic effect in most tumors is still unsatisfactory [Grupp SA, et al.Adoptive cellular therapy. Curr Top Microbiol Immunol., 2011; 344: 149-72.]. In recent years, the recognition specificity of target cells based on cytotoxic lymphocytes (CTLs) depends on the discovery of T Cell Receptor (TCR), and the scFv of antibodies against tumor cell-associated antigens The intracellular signal-activating motif such as CD3ζ or FcεRIγ of the T lymphocyte receptor is fused to a chimeric antigen receptor (CAR; now also known as Chimeric receptor (CR)), and passed through Genetically modified on the surface of T lymphocytes, such as lentivirus infection. Such CAR T lymphocytes are capable of selectively targeting T lymphocytes to tumor cells and specifically killing tumors in a non-limiting manner in a major Histocompatibility Complex (MHC). CAR T lymphocytes are a new immunotherapeutic 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 natural killer cells. Cytotherapy. 2014 Dec 18.pii: S1465-3249 (14) 00791-9) or NKT cells also showed good results in preclinical studies. Antitumor activity (Heczey A1, Liu D1, Tian G2, Courtney AN1, Wei J1, Marinova E1, Gao X1, Guo L1, Yvon E3, Hicks J2, Liu H4, Dotti G5, Metelitsa LS6. Invariant NKT cells with chimeric antigen Receptor provides a novel platform for safe and effective cancer immunotherapy. Blood. 2014; 124(18): 2824-33).

Chimeric antigen receptors include extracellular binding regions, transmembrane regions, and intracellular signaling regions. Usually, the extracellular region contains a scFv capable of recognizing a tumor-associated antigen, a transmembrane region adopts a transmembrane region of a molecule such as CD8, CD28, and the intracellular signal region adopts an immunoreceptor tyrosine activation motif (ITAM) CD3ζ or FcεRIγ and co-stimulation. The intracellular signal region of the signaling molecules CD28, CD27, CD137, CD134, and the like.

The intracellular signal domain contains only ITAM as the first generation of CAR T lymphocytes, wherein the chimeric antigen receptor portions are linked as follows: scFv-TM-ITAM. This kind of CAR T can stimulate the anti-tumor cytotoxic effect, but the cytokine secretion is relatively small, and can not stimulate the 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.].

Subsequent development of second-generation CAR T lymphocytes was added to the intracellular signaling region of CD28 or CD137 (aka 4-1BB), in which the chimeric antigen receptor moieties were joined as follows: scFv-TM-CD28-ITAM or scFv -TM-/CD137-ITAM. Co-stimulation of B7/CD28 or 4-1BBL/CD137 in the intracellular signal domain causes sustained proliferation of T lymphocytes, and can increase the secretion of cytokines such as IL-2 and IFN-γ by T lymphocytes, and increase CAR T Survival cycle and anti-tumor effect in vivo [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.].

The third generation of CAR T lymphocytes developed in recent years, in which the chimeric antigen receptor parts are linked as follows: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, further enhancing CAR 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, specific antigens recognized by low expression of CAR in certain/normal tissues may cause damage to normal tissues expressing antigens by CART lymphocytes. For example, the antigen carbonic anhydrase IX (CAIX) expressed on tumor cells of patients with renal cell carcinoma is the first case for the clinical treatment of CAR T lymphocytes. It is also the first case to report the off-target effect of CAR cells. . Patients developed grade 2-4 hepatotoxicity after multiple injections of CAR T lymphocytes. The reason for the analysis is that the liver bile duct epithelial cells have low expression of CAIX, and the original clinical trial was interrupted while excluding any evaluation of the patient's therapeutic effect [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 adoptive immunotherapy of cancer. Human Molecular Genetics, 2011, R1-R7]. In addition, excessive costimulatory signals in CAR reduce the threshold required for effector cell activation, allowing genetically modified T lymphocytes to be activated under conditions of low or no antigen triggering, resulting in the release of large amounts of cytokines. As a result, a so-called "cytokine storm" may be triggered. This signal leakage can lead to off-target cytotoxicity, resulting in non-specific tissue damage. For example, in the clinical treatment of a late-stage colon cancer patient with liver and lung metastases with a third-generation CAR for Her2, the so-called "cytokine storm" caused by the low expression of Her2 in normal lung tissue caused sudden death [Morgan] RA., et al. Report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing Erbb 2. 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 more accurately on tumors and avoid the higher risks of tumor immunotherapy.

Summary of the invention

It is an object of the present invention to provide an activatable chimeric receptor, a process for its preparation and its use.

In a first aspect of the invention, an 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) capable of inhibiting binding of a chimeric receptor to an antigen highly expressed by the pathological tissue;

A cleavable element (CE) located between the chimeric receptor and the blocking element.

In a preferred embodiment, the chimeric receptor comprises a sequence-ligated: 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, intracellular signal 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 by the pathological tissue.

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

In another preferred embodiment, the chimeric receptor is capable of efficiently recognizing the antigen highly expressed in the pathological tissue in the absence of the blocking element, and the blocking element can interfere with or compete for high expression of the chimeric receptor and the pathological tissue. The combination of antigens.

In another preferred embodiment, the activatable chimeric receptor comprises, in order from the amino terminus to the carboxy terminus, a blocking element, a cleavable element, and a chimeric receptor.

In another preferred embodiment, the chimeric receptor, the blocking element, and the cleavable element further comprise a linker peptide.

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

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

In another preferred embodiment, the blocking element is 2 to 100 aa (e.g., 10 aa, 20 aa, 30 aa, 40 aa, 50 aa, 60 aa, 70 aa, 80 aa); preferably a polypeptide of 2 to 40 aa.

In another preferred embodiment, the cleavable element is an element that is cleaved, reduced or degraded by a protease that is specifically expressed or highly expressed by a pathological tissue, and the protease is highly expressed with the pathological tissue. The antigen is co-localized in the same pathological tissue.

In another preferred embodiment, the pathological tissue includes, but is not limited to, a tumor, an autoimmune disease tissue, a tissue infected with a virus such as HIV virus.

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

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

In another preferred embodiment, the antigenically expressed antigen of the pathological tissue includes but is not limited to: GPC3, EGFR, HER2, EphA2, Claudin 18.1, Claudin 18.2, Claudin 6, GD2, EpCAM, mesothelin, CD19, CD20 Or ASGPR1.

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

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

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

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

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

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

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

In another aspect of the invention, a chimeric receptor immune effector cell transduced with a nucleic acid encoding an activatable chimeric receptor of any of the foregoing, or an expression vector as described above or The virus; or its surface expresses an activatable chimeric receptor as described above.

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

In another aspect of the invention, there is provided the use of said chimeric receptor immune effector cell for the preparation of a medicament for targeting a pathological tissue, said pathological tissue highly expressing an antigen to which said chimeric receptor is capable of binding.

In a preferred embodiment, the pathological tissue is a tumor, and the drug targeting the pathological tissue is inhibited. Tumor-causing drugs.

In another aspect of the invention, there is provided a pharmaceutical composition comprising: the chimeric receptor immune effector cell as described above.

In another aspect of the invention, an activatable chimeric receptor is provided, comprising:

a chimeric receptor capable of targeting an antigen expressed in a pathological tissue in an activated state;

a blocking element capable of inhibiting binding of a chimeric receptor to an antigen expressed by said pathological tissue;

A shearable element positioned between the chimeric receptor and the closure element.

In a preferred embodiment, the chimeric receptor is capable of targeting an antigen that is highly expressed or specifically expressed in a pathological tissue in an activated state.

In a preferred embodiment, the extracellular antigen binding region is an antibody that specifically binds to an antigen expressed by the pathological tissue.

In a preferred embodiment, the antibody that specifically binds to the antigen expressed by the pathological tissue is selected from the group consisting of a single chain antibody or a single domain antibody.

In a preferred embodiment, the activatable chimeric receptor comprises, in order from the amino terminus to the carboxy terminus, a blocking element, a cleavable element, and a chimeric receptor.

In a preferred embodiment, the chimeric receptor, the blocking element, and the cleavable element further comprise a linker peptide.

In a preferred embodiment, the closure element is selected from the group consisting of:

a polypeptide that binds directly to the chimeric receptor; or

A polypeptide that sterically hinders binding of the chimeric receptor to an antigen.

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

In a preferred embodiment, the pathological tissue comprises: a tumor, an autoimmune disease tissue, a tissue infected by a virus. In a preferred embodiment, the pathological tissue is a tumor, and the pathological tissue-specifically expressed or highly expressed protease comprises: a urokinase-type plasminogen activator, a legumain protease or a matriptase.

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

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

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

DRAWINGS

Figure 1. Schematic diagram of the structure of an activating chimeric receptor ACR.

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

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

According to the direction from the N-terminus to the C-terminus, each module in the figure is CD8 secretion signal, BE (closed element), LP1, CE (shearable element, UP1 substrate), LP2, CR (GC33-28BBZ), F2A, eGFP.

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

The positive rate by GFP was used as the GC33-28BBZ-ACR positive rate indicating T cells after lentivirus infection.

Figure 4. In vitro toxicity test. The hepatoma cell line Huh-7 was used as the target cell, and the effector cells were T cells expressing GC33-28BBZ-ACR for 11 days in vitro, and the target ratios were 3:1, 1:1 and 1:3, respectively. It is 10000 cells/well, corresponding to effector cells according to different target-to-target ratios. UPA and MTSP1 were added separately, one low dose group, one high dose group and one blank group were set. Five replicate wells were set for each group, and the average of five replicate wells was taken. The detection time is 18h.

detailed description

The inventors have extensively studied and revealed an activatable chimeric receptor based on chimeric antigen receptor (CAR) technology, which is only expressed in pathological tissue-specific or high expression. In the presence of proteases, the corresponding antigen can be targeted to achieve the killing effect of pathological tissues or cells; but it does not function in the absence of protease. The present invention provides a viable solution to avoid on-target-off-tumor.

As used herein, the "chimeric receptor" and "chimeric antigen receptor" are used interchangeably.

In the present invention, the high expression or specific expression means that the ratio of the expression level of an antigen or protease in a pathological tissue to the expression level of the antigen or protease in a non-pathological tissue such as a paracancerous tissue or a normal tissue is greater than 1.5, or greater than 2, greater than 3, greater than 4, more preferably greater than 5 or greater than 6.

As used herein, "a pathologically highly expressed antigen" refers to an antigen targeted by an activated chimeric receptor that is highly expressed in a pathological tissue or cell. In the present invention, the "antigen which is 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 of pathological tissue" is a tumor associated antigen, for example selected from, but not limited to: GPC3, EGFR, HER2, EphA2, Claudin 18.1, Claudin 18.2, Claudin 6, GD2, EpCAM , mesothelin, CD19, CD20 or ASGPR1.

As used herein, "pathological tissue-specifically expressed or highly expressed protease" refers to a protein (hydrolase) enzyme that is expressed only in pathological tissues or cells, or is highly expressed in pathological tissues or cells, It is capable of hydrolyzing its specific substrate.

As used herein, a "blocking element" refers to a polypeptide that is capable of blocking the binding of the chimeric receptor to its corresponding antigen by directly binding to or sterically hindering the chimeric receptor. The chimeric receptor binds to the antigen to exert a function of blocking the binding of the chimeric receptor to the target site.

As used herein, a "cleavable element" is a polypeptide between a chimeric receptor and a blocking element that is a substrate for said "pathologically tissue-specifically expressed or highly expressed protease". When the protease is present, the "cleavable element" can be cleaved, reduced or decomposed such that the blocking element no longer blocks binding of the chimeric receptor to its corresponding antigen.

As used herein, "pathological tissue" includes, but is not limited to, tumors, autoimmune disease tissues, tissues infected with viruses such as HIV, and the like.

In the present invention, the pathological tissue may be various harmful tissues or lesions that are not healthy for the body, and it is necessary to remove it from the body. The pathological target tissue includes a tumor. Any tumor known in the art can be included in the present invention as long as the tumor is capable of expressing a tumor-associated antigen that is lowly expressed in normal tissues. For example, the tumor includes, but is 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, Renal tumor, adrenal tumor, bladder tumor, testicular tumor, malignant lymphoma, multiple myeloma, nervous system tumor, esophageal cancer, thymic mesothelioma, pancreatic cancer, leukemia, head and neck cancer, cervical cancer, skin cancer, melanin 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, Claudin 18.1, Claudin 18.2, 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 effector cells" is well known in the art and is an immunopotentiating cell that expresses an antigen (e.g., tumor antigen)-specific chimeric receptor by genetic engineering, and can exert a targeted killing effect. The immune effector cells include, for example, T cells, NK cells, NKT cells, Regulatory cells (Tregs for short). Conventional methods of preparing "chimeric receptor immune effector cells" are known to those skilled in the art, including allowing them to express intracellular co-stimulatory cellular intracellular domains, such as CD28 (preferably including CD28a, CD28b), CD137, CD27, CD3ζ (preferably CD3ζ intracellular domain), CD8, CD19, CD134, One or more of CD20, FcεRIγ. By binding to the corresponding ligands, the second signal of the immune effector cells is activated, the proliferation ability of the immune cells and the secretory function of the cytokines are enhanced, and the survival time of the activated immune cells is prolonged.

In view of the fact that tumors have absolutely few specific targets, most of the CAR-modified immune cells (such as CAR T cells) target antigens (such as CD19, CD20, Her2, EGFR, EpCAM, etc.) more or less normal. Expression in tissues, so it is difficult to avoid the side effects of on-target-off-tumor. How to reduce or reduce the role of this On-target-off-tumor becomes very important. The present inventors have extensively compared the microenvironment of tumor tissue with the microenvironment of normal tissues, and for the first time, based on some proteolytic enzymes such as uPA, MT-sp1, Legumamain protease, etc., high expression in tumor tissues is not expressed or low in normal tissues. The characteristics of expression, using the specificity of these hydrolases, modified the chimeric receptor-modified immune cells, and the obtained immune cells can exert anti-tumor function more effectively only after the action of these hydrolases, thereby effectively improving immunity. Cell safety.

Accordingly, the present invention provides an Activatable Chimeric Receptor (ACR) comprising a Chimeric Receptor (CR), a Cleavable element (CE), and a blocking element ( Blocking element, BE) linkage, the three can be linked by a linker polypeptide. Where the CE antigen is cleaved, reduced, optically resolved or otherwise modified, the ACRs can exhibit an activated conformation such that CR can more readily bind to the target.

As a preferred mode of the present invention, a schematic structural view of the activatable chimeric receptor ACR of the present invention is shown in FIG. A schematic representation of the chimeric receptor ACR can be activated by aligning from left to right, a transmembrane receptor consisting of the N-terminus to the C-terminus of the protein. In the unactivated state, BE binds to the Ab region or spatially blocks the binding of the Ab to the target antigen; and once CE1 is cleaved by a protease or the like, the Ab can bind to the target antigen.

The blocking element may be any which 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 peptide. The blocking element can be selected based on 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 a binding polypeptide of the antibody. In a specific embodiment of the invention, the antigen binding region of the chimeric receptor is the anti-GPC3 antibody GC33 and the binding polypeptide (NSQQATPKDNEISTFH) is used as the blocking element.

The cleavable element is a substrate that can be cleaved, reduced or decomposed by a protease that is specifically expressed or highly expressed by a pathological tissue. The protease in which the expression is specifically expressed or highly expressed can be selected according to the corresponding indication, and the substrate can be used as a cleavable element. The higher the expression specificity of the protease, the more preferable. In a preferred mode of the present invention, the indication is a tumor, and the microenvironment of the tumor tissue is different from the microenvironment of the normal tissue, wherein the proteolytic enzymes such as uPA, MT-sp1, and Legumain protease are highly expressed in the tumor tissue, and Normal tissues do not express or underexpress, so the inventors used the specificity of these hydrolases to engineer chimeric receptor-modified immune cells, and the immune cells can exert anti-tumor functions more effectively only after the action of these hydrolases. Thereby effectively improving the safety of CAR-modified immune cells. In a specific embodiment of the invention, the substrate polypeptide LSGRSDNH of uPA and MT-SP1 protease is employed as a cleavable element.

Between the chimeric receptor, the blocking element, and the cleavable element, a linker peptide can also be included. The linker peptide is not particularly limited and may be any polypeptide capable of providing a flexible linkage between the chimeric receptor, the blocking member, and the cleavable member without affecting the function of each member. Preferably, the linker comprises 2-40 amino acids; preferably 3-30 amino acids, such as 5, 8, 10, 15, 20, 25 amino acids.

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

The invention also provides a vector comprising a nucleic acid of the above-described activating chimeric receptor. In a specific embodiment, the vector used in the invention is a lentiviral plasmid vector pWPT. It should be understood that other expression vectors are also available.

The invention also includes viruses comprising the vectors described above. The virus of the present invention includes a packaged infectious virus, and also includes a virus to be packaged containing components necessary for packaging as an infectious virus. Other viruses known in the art that can be used to transduce foreign genes into immune effector cells and their corresponding plasmid vectors 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-described recombinant plasmid comprising the nucleic acid, or comprising the plasmid Virus. Conventional nucleic acid transduction methods, 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 nuclear transfection device developed by Amaxa can directly introduce foreign genes into the nucleus to obtain efficient transduction of the target gene. In addition, the transduction efficiency of the transposon system based on Sleeping Beauty system or PiggyBac transposon is much higher than that of ordinary electroporation, and the nucleofector transfection apparatus is combined with the Sleeping Beauty transposon system. 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.], The method has high transduction efficiency and can achieve targeted integration of the target gene. In one embodiment of the invention, the transduction method for effecting a genetically modified cell of a chimeric receptor (in this invention, an activatable chimeric receptor) is based on the transduction of a virus such as a retrovirus or a lentivirus. method. The method has the advantages of high transduction efficiency, stable expression of the exogenous gene, and shortening the time for the cultured immune effector cells to reach the clinical level in vitro. On the surface of the transgenic immune effector cells, the transduced nucleic acid is expressed on its surface by transcription and translation. The in vitro cytotoxicity assay of various cultured tumor cells demonstrates 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 of the present invention, the plasmid containing the nucleic acid, the virus comprising the plasmid, and the transgenic immune effector cell transduced with the above nucleic acid, plasmid or virus can be effectively used for immunotherapy of tumors.

The immune cells of the present invention may also carry a coding sequence of a foreign cytokine; 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 activity, enhance the function of effector T cells and activated NK cells, or directly exert anti-tumor effects. Thus, those skilled in the art will appreciate that the use of these cytokines will help the immune cells to function better. use.

The immune cells of the present invention may also express another chimeric receptor other than the chimeric receptor described above, which does not contain CD3ζ, but contains the intracellular signal domain of CD28, the intracellular signal domain of CD137. Or a combination of the two.

The immune cells of the invention may also express a chemokine receptor; the chemokine receptors include, but are not limited to, CCR2. Those skilled in the art will appreciate that the CCR2 chemokine receptors may allow CCR2 binding in vivo to compete with it, which is advantageous for blocking tumor metastasis.

The immune cells of the present invention can also express siRNA that reduces PD-1 expression or a protein that blocks PD-L1. Those skilled in the art will appreciate that competitively blocking the interaction of PD-L1 with its receptor PD-1 facilitates the recovery of anti-tumor T cell responses, thereby inhibiting tumor growth.

The immune cells of the present invention may also express a safety switch; preferably, the safety switch comprises: iCaspase-9, Truncated EGFR or RQR8.

The chimeric receptor immune effector cells of the invention can be used to prepare pharmaceutical compositions or diagnostic reagents. The composition may comprise a pharmaceutically acceptable carrier in addition to an effective amount of the immune cells. The term "pharmaceutically acceptable" means that when the molecular body and composition are suitably administered to an animal or a human, they do not produce an adverse, allergic or other untoward reaction.

Specific examples of some substances which 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 derivatives thereof such as carboxymethyl fibers Sodium, ethyl cellulose and methyl cellulose; western yellow gum 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; compressed tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions and phosphate buffers.

The composition of the present invention can be formulated into various dosage forms as needed, and can be administered by a physician in accordance with factors such as patient type, age, body weight, and general disease condition, mode of administration, and the like. The mode of administration can be, for example, injection or other treatment.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the manufacturer. The suggested conditions.

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

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

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

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

Primers as shown in Table 1 were designed.

Table 1

The fragment of Sp+BE+LP1+CE+LP2 was obtained by Overlap PCR using primers as shown in Table 1, and the amplification conditions were:

Pre-denaturation: 94 ° C, 4 min;

The following 25 cycles: Denaturation: 94 ° C, 30 s,

Annealing: 50 ° C, 30 s,

Extension: 68 ° C, 20 s;

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

Subsequently, the PCR amplification product was subjected to gel recovery using a conventional method to obtain a DNA fragment in which Sp+BE+LP1+CE+LP2 was ligated to each other.

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

Design primers such as 2.

Table 2

Take GC33-28BBZ plasmid (this plasmid see CN201310164725.X patent) 1 microliter (100ng) as template, GPC3scfv-F, 3z-F2A-R as the upstream and downstream primers, 50 microliter system, PCR amplification The PCR product was subjected to gelatinization recovery to obtain a LP2-GC33-28BBZ fragment.

(3) Acquisition of F2A-EGFP

Design primers such as 3.

table 3

One microliter (100 ng) of GC33-28BBZ plasmid was used as a template, and F2A-EGFP-F and pwpt-EGFP-R were used as upstream and downstream primers for PCR amplification, and the obtained PCR product was subjected to gel extraction to obtain F2A- EGFP.

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

The three-stage fragment obtained by the above (1) to (3) is subjected to overlap PCR, specifically, three-stage fragment, mixed according to an equimolar ratio, and overlap PCR is carried out as described in the above step (1), and the upstream and downstream primers are CD8sp. -F and EGFP-R. The obtained PCR product was subjected to gelatinization recovery.

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

The obtained recombinant plasmid was transformed into E. coli Top10 competent cells, and cultured for about 12 hours for colony verification. Clones containing the recombinant plasmid were selected for sequencing. Select the correct clone for the virus preparation and packaging.

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

Example 3, Preparation of CAR-T

1, the packaging of the slow virus of GC33-28BBZ-ACR

HEK-293T cells (ATCC: CRL-11268) cultured to the 6th to 10th passages were inoculated at a density of 6 × 10 ⁶ in a 10 cm culture dish, and cultured overnight at 37 ° C, 5% CO ₂ for transfection. The medium was DMEM (purchased from Gibco) containing 10% fetal bovine serum (available from Gibco).

The steps for transfection are as follows:

(1) 10 μg of the target gene plasmid pWPT-GC33-28BBZ-ACR, and 6.5 μg of the packaging plasmid PAX2: and 3.5 μg of the envelope plasmid pMD2.G, respectively, were dissolved in 800 μL of serum-free DMEM culture medium, and mixed.

(2) 60 μl of PEI (polyethyleneimine, purchased from Polysciences, formulated into a working solution of 1 μg / μL concentration), added to the above-mentioned plasmid-containing 800 μL of serum-free DMEM culture medium, vortexed and mixed, and allowed to stand at room temperature. Incubate for 25 min.

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

(4) Transfection efficiency (i.e., the proportion of cells showing green fluorescence) was observed about 24 hours after transfection. After 72 h of transfection, the virus was collected by filtration using a 0.45 μm filter (purchased from Millipore), ultracentrifuged (28,000 rpm at 28,000 rpm in a Beckman Optima L-100XP ultracentrifuge), and the virus was concentrated. The pellet obtained by centrifugation was resuspended in a 1/30 stock volume of AIM-V medium (purchased from Gibco), and stored at -80 ° C in 100 μL/tube for infection of 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. Peripheral blood mononuclear cells were obtained by CD4+/CD8+ cell magnetic beads (purchased from Stem Cell Technologies) negative sorting method to obtain CD4+ and CD8+ positive primary human T lymphocytes. The sorted T cells were tested for purity by flow cytometry, and the positive rate of the target cells was ≥95%. AIM-V lymphocyte medium (purchased from Gibco, containing 2% human AB serum) was added at a density of 1 × 10 ⁶ /mL and added with 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.) at a final concentration of 300 U/mL were stimulated for 24 h. Before infection, CD4+CD8+ T lymphocytes were mixed in a ratio of 1:1, and then the above virus concentrate was mixed with the T cells to be infected with MOI≈5-10, and a polybrene having a final concentration of 6 μg/mL was added. The infected cells are changed every other day and replaced with a virus-free medium. Passage was carried out every other day at a density of 5 × 10 ⁵ /mL, and a recombinant human IL-2 having a final concentration of 300 U/mL was added to the lymphocyte culture solution.

Primary T cells after infection were tested for expression of chimeric antigen receptor GC33-28BBZ-ACR by flow cytometry on days 9-11 (ie at the end of the first round of amplification), due to eGFP and GC33-28BBZ - ACR co-expression, positive cells detecting eGFP are positive cells expressing chimeric antigen receptor, and uninfected T lymphocytes are used as a negative control.

As a result, as shown in Fig. 3, the positive rate of GFP was used as the positive rate of GC33-28BBZ-ACR indicating T cells after lentivirus infection, and the positive rate was 61.0%.

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

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

The hepatoma cell line Huh-7 was used as the target cell, and the effector cells were T cells expressing GC33-28BBZ-ACR for 11 days in vitro (positive rate 61%), and the target ratios were 3:1, 1:1 and 1 respectively. : 3, the number of target cells is 10,000 / hole, corresponding to effector cells according to different target ratios. UPA and MTSP1 were added separately, one low dose group (uPA: 0.08 μg/mL; MT-SP1: 0.04 μg/mL) and one high dose group (uPA: 0.4 μg/mL; MT-SP1: 0.2 μg/mL) and blank group (uPA: 0 μg/mL; MT-SP1: 0 μg/mL). Five replicate wells were set for each group, and the average of five replicate wells was taken. The detection time is 18h.

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: The maximum release of LDH+ different concentrations of enzyme from target cells,

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

Control group 3: Effector cells spontaneously released LDH+ different concentrations of enzyme.

Detection method: Detection was carried out using a CytoTox 96 non-radioactive cytotoxicity test kit (Promega).

The cytotoxicity calculation formula is:

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

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

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

An activatable chimeric receptor comprising:

a chimeric receptor capable of targeting an antigen highly expressed by a pathological tissue in an activated state;

a blocking element capable of inhibiting binding of a chimeric receptor to an antigen highly expressed by said pathological tissue;

A shearable element positioned between the chimeric receptor and the closure element.
The activatable chimeric receptor according to claim 1, wherein said chimeric receptor comprises a sequence-ligated: an extracellular antigen binding region, a transmembrane region, and an intracellular signal region; The intracellular signal region is selected from: CD3
, FcεRIγ, CD27, CD28, CD137, CD134, the intracellular signal sequence of CD40 or Myd88, or a combination thereof.
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 by the pathological tissue.
The activatable chimeric receptor according to claim 3, wherein the antibody that specifically binds to the antigen highly expressed by the pathological tissue is selected from the group consisting of a single chain antibody or a single domain antibody.
The activatable chimeric receptor according to claim 1, wherein said activatable chimeric receptor comprises, in order from amino terminus to carboxy terminus, a blocking element, a cleavable element, and an inlay. Receptor.
The activatable chimeric receptor of claim 1 wherein said chimeric receptor, blocking element, and cleavable element further comprise a linker peptide.
The activatable chimeric receptor of claim 1 wherein said blocking member is selected from the group consisting of:

a polypeptide that binds directly to the chimeric receptor; or

A polypeptide that sterically hinders binding of the chimeric receptor to an antigen.
The activatable chimeric receptor according to claim 1, wherein said cleavable element is an element that can be cleaved, reduced or decomposed by a protease that is specifically expressed or highly expressed by a pathological tissue. The protease colocalizes with the antigen highly expressed by the pathological tissue in the same pathological tissue.
The activatable chimeric receptor according to claim 8, wherein said pathological tissue comprises: a tumor, an autoimmune disease tissue, a tissue infected with a virus.
The activatable chimeric receptor according to claim 9, wherein the pathological tissue is a tumor, and the pathological tissue-specifically expressed or highly expressed protease comprises: urokinase-type plasmin The original activator, legumain protease or matriptase.
The activatable chimeric receptor according to claim 10, wherein said pathological tissue-specifically expressed or highly expressed protease is urokinase-type plasminogen activator or matriptase, said The splicing element is a polypeptide of the amino acid sequence shown in SEQ ID NO: 2.
The activatable chimeric receptor according to claim 9, wherein the antigen highly expressed by the pathological tissue comprises: GPC3, EGFR, HER2, EphA2, Claudin 18.1, Claudin 18.2, Claudin 6, GD2. , EpCAM, mesothelin, CD19, CD20 or ASGPR1.
The activatable chimeric receptor according to claim 12, wherein said pathological tissue The highly expressed antigen is GPC3, and the blocking element is a binding polypeptide of a GC33 antibody; preferably, the blocking element is a polypeptide of the amino acid sequence shown in SEQ ID NO: 1.
A polynucleotide encoding the activatable chimeric receptor of any of claims 1-13.
An expression vector comprising a nucleic acid encoding the activatable chimeric receptor of claim 14.
A virus, characterized in that the virus comprises the vector of claim 15.
Use of an activatable chimeric receptor according to any one of claims 1 to 13, or a nucleic acid encoding the same, or an expression vector or virus comprising the nucleic acid, for preparing chimeric receptor immunity against pathological tissues Effector cells.
A chimeric receptor immune effector cell transduced with a nucleic acid encoding the activatable chimeric receptor of any of claims 1-13, or the expression vector of claim 15 or the method of claim 16. And a surface thereof expressing the activatable chimeric receptor of any of claims 1-13.
The chimeric receptor immune effector cell according to claim 18, wherein the immune effector cells comprise: T lymphocytes, NK cells or NKT cells, and Treg cells.
Use of the chimeric receptor immune effector cell of claim 18 or 19 for the preparation of a medicament for targeting a pathological tissue which highly expresses an antigen to which the chimeric receptor binds.
The use according to claim 20, wherein said pathological tissue is a tumor, and said drug targeted to pathological tissue is a drug for inhibiting tumor.
A pharmaceutical composition comprising: the chimeric receptor immune effector cell of claim 18 or 19.
An activatable chimeric receptor comprising:

a chimeric receptor capable of targeting an antigen expressed in a pathological tissue in an activated state;

a blocking element capable of inhibiting binding of a chimeric receptor to an antigen expressed by said pathological tissue;

A shearable element positioned between the chimeric receptor and the closure element.
The activatable chimeric receptor according to claim 23, wherein the chimeric receptor is capable of targeting an antigen that is highly expressed or specifically expressed in a pathological tissue in an activated state.