WO2023130462A1 - 靶向IL13Rα2的嵌合抗原受体及其用途 - Google Patents

靶向IL13Rα2的嵌合抗原受体及其用途 Download PDF

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WO2023130462A1
WO2023130462A1 PCT/CN2022/071078 CN2022071078W WO2023130462A1 WO 2023130462 A1 WO2023130462 A1 WO 2023130462A1 CN 2022071078 W CN2022071078 W CN 2022071078W WO 2023130462 A1 WO2023130462 A1 WO 2023130462A1
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cells
car
amino acid
cell
domain
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French (fr)
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钟晓松
白玥
续畅
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卡瑞济(北京)生命科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates generally to chimeric antigen receptors targeting IL13R ⁇ 2, immune effector cells (e.g., T cells, NK cells) engineered to express chimeric antigen receptors of the invention, and such engineered immune Use of effector cells for the treatment of diseases associated with the expression of IL13R ⁇ 2.
  • immune effector cells e.g., T cells, NK cells
  • GBM Glioblastoma
  • GBM is one of the most common and aggressive primary malignant brain tumors worldwide, and most cases are highly malignant. Due to the high morbidity, high mortality and low cure rate, GBM has caused a huge social and medical burden worldwide, and the current conventional treatment is almost impossible to cure glioblastoma. Even with the best multimodal treatments, the median patient survival rate is only about 21 months, and overall survival for glioblastoma patients has not improved substantially in half a century. For most patients, the main cause of treatment failure is the relapse of aggressive, drug-resistant malignant cells. Studies have shown that the continued growth and recurrence of tumors is due to the failure to mount an effective immune response.
  • a chimeric antigen receptor is a synthetic molecule that guides immune effector cells (such as T cells, T cells, NK cells) clear tumors (Sampson JH, Choi BD, Sanchez-Perez L et al., EGFRvIIImCAR-modified T-cell therapy cures mice with established intracerebralglioma and generates host immunity against tumor-antigen loss. Clinical cancer research: an off icial journal of the American Association for Cancer Research. 2014;20(4):972-984).
  • chimeric antigen receptor T cells directly target the surface antigens of tumor cells through chimeric antigen receptor (CAR) molecules on T cells, so as to achieve the purpose of recognizing and killing tumors.
  • the N-terminus of the chimeric antigen receptor contains an extracellular domain that recognizes an antigen.
  • the CAR-T cells can recognize and kill these antigen-positive cells.
  • CD19-CAR T cells can reduce the incidence of brain metastatic leukemia (Abramson JS, McGree B, Noyes S et al., Anti-CD19 CAR T Cells in CNS Diffuse Large-B-Cell Lymphoma. The New England journal of medicine. 2017; 377(8):783-784).
  • CAR T cells can migrate into the brain parenchyma through chemokines, target and kill tumor cells (Hong JJ, Rosenberg SA, Dudley ME et al, Successful treatment of melanoma brain metastases with adoptive cell therapy. Clinical cancer research: an official journal of the American Association for Cancer Research. 2010; 16(19):4892-4898).
  • the application of CAR T cells in the treatment of brain tumors is still in its early stages.
  • the therapeutic effect of CAR T cells is not ideal, the reasons are lack of tumor-specific antigens, insufficient survival and expansion of T cells, low migration efficiency of T cells to tumor tissues, and immunosuppressive tumor microenvironment and other factors related.
  • IL13R ⁇ 2 is the receptor polypeptide of IL-13, which is almost only expressed in cancer cells and not expressed in normal tissue cells (except testis). In GBM patients, more than 50% of patients express IL13R ⁇ 2, making it a CAR-T An effective target for the treatment of GBM (Sharma P, Debinski W. Receptor-Targeted Glial Brain Tumor Therapies. International journal of molecular sciences. 2018; 19(11)).
  • IL13R ⁇ 2 vaccine for GBM (Iwami K, Shimato S, Ohno M et al., Peptide-pulsed dendritic cell vaccination targeting interleukin-13 receptor alpha2chain in recurrent malignant glioma patients with HLA-A*24/A*02 allele .Cytotherapy .2012; 14(6):733-742) and first-generation IL13 ⁇ CAR T cells and second-generation IL13BB ⁇ CAR T cells in the treatment of recurrent glioblastoma, making IL13R ⁇ 2 an attractive target for immunotherapy (Brown CE, Aguilar B, Starr R et al., Optimization of IL13Ralpha2-Targeted Chimeric Antigen Receptor T Cells for Improved Anti-tumor Efficacy against Glioblastoma. Molecular therapy: the journal of the American Society of Gene The rapy.2018;26(1):31 -44).
  • CAR-T cell therapy breaks through the restriction that antigens need to be presented by MHC molecules, and can overcome the barriers of the blood-brain barrier, it has been proved to be a breakthrough therapy with long-lasting and effective anti-tumor activity. Side effects and inefficiencies have gradually become a challenge.
  • Chimeric antigen receptor (CAR)-modified T (CAR-T) cell therapy has been proven to be an effective anticancer approach, but its side effects and low efficacy in solid tumors have gradually become a challenge.
  • the present invention provides a variety of third-generation CAR T cells targeting IL13R ⁇ 2, which respectively contain different transmembrane regions from CD4, CD8 or CD28.
  • the transmembrane domain (TMD) of CAR is used to improve the anti-tumor effect of CAR-T.
  • the present invention provides various third-generation IL13R ⁇ -specific CAR polypeptides with different transmembrane domains, comprising
  • transmembrane domain selected from the group consisting of: CD4 transmembrane domain or variants thereof with 1-10 amino acid modifications, CD8 transmembrane domain or variants thereof with 1-10 amino acid modifications, CD28 transmembrane domain The membrane domain or a variant thereof having 1-10 amino acid modifications;
  • costimulatory domains selected from the group consisting of: (a) CD28 costimulatory domains or variants thereof with 1-10 amino acid modifications, (b) 4-1BB costimulatory domains or variants thereof with 1-10 amino acid modifications Amino acid modified variants, (c) OX40 co-stimulatory domains or variants thereof with 1-10 amino acid modifications, and any combination of (a), (b) and/or (c) described; and
  • CD3 ⁇ signaling domain or variants thereof having 1-10 amino acid modifications CD3 ⁇ signaling domain or variants thereof having 1-10 amino acid modifications
  • amino acid modification is addition, deletion or substitution of amino acid.
  • the chimeric antigen receptor polypeptide of the present invention further comprises a spacer between said (ii) and said (iii), for example, said spacer is an IgG hinge region or has a 1- A variant with 2 amino acid modifications, e.g., an IgG4 hinge region or a variant thereof having 1-2 amino acid modifications, e.g., the IgG4 hinge region shown in ESKYGPPCPSCP (SEQ ID NO: 1), wherein the amino acid modification is an amino acid addition, deletion or substitution.
  • said spacer is an IgG hinge region or has a 1- A variant with 2 amino acid modifications, e.g., an IgG4 hinge region or a variant thereof having 1-2 amino acid modifications, e.g., the IgG4 hinge region shown in ESKYGPPCPSCP (SEQ ID NO: 1), wherein the amino acid modification is an amino acid addition, deletion or substitution.
  • (i) in the chimeric antigen receptor polypeptide of the invention is human IL-13 or a variant thereof having 1-2 amino acid modifications, for example, the amino acid at position 13 of human IL-13
  • the amino acid at position 13 of human IL-13 is Y, also known as IL-13(E13Y) or IL-13E13Y
  • said (i) is SPGPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRF N (SEQ ID NO: 2).
  • (ii) in the chimeric antigen receptor polypeptide of the invention is a CD4 transmembrane domain or a variant thereof having 1-5 amino acid modifications, a CD8 transmembrane domain or a variant thereof having 1-5 amino acid modifications
  • the CD4 transmembrane domain is The sequence shown in MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO:3)
  • the CD8 transmembrane domain is the sequence shown in IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:4)
  • the CD28 transmembrane domain is VWWWLVESWLVIACYYQWPLLFSG (SEQ ID NO:5) sequence shown.
  • (iii) in a chimeric antigen receptor polypeptide of the invention is (a) a CD28 costimulatory domain or a variant thereof having 1-5 amino acid modifications, (b) a 4-1BB costimulatory Domain or its variant with 1-5 amino acid modifications, (c) OX40 co-stimulatory domain or its variant with 1-5 amino acid modifications, and said (a), (b) and/or ( Any combination of c), wherein the amino acid modification is an addition, deletion or substitution of an amino acid; for example, said (iii) is two different co-stimulatory domains, for example, (a) a CD28 co-stimulatory domain or its A combination of a 1-5 amino acid modified variant and (b) a 4-1BB costimulatory domain or a variant thereof with 1-5 amino acid modifications, for example, the CD28 costimulatory domain is SKRSRLMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO : the sequence shown in 6), the 4-1BB co
  • (iv) in the chimeric antigen receptor polypeptide of the present invention is a CD3 ⁇ signaling domain or a variant thereof having 1-5 amino acid modifications, wherein the amino acid modification is an addition or deletion of an amino acid or replace;
  • the CD3 ⁇ signaling domain is the sequence shown in RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAKDTYDALHMQALPPRYQPLE (SEQ ID NO: 8).
  • a chimeric antigen receptor polypeptide of the invention comprises:
  • transmembrane domain which is a CD4 transmembrane domain or a variant thereof with 1-2 amino acid modifications, for example, the CD4 transmembrane domain shown in SEQ ID NO:3; the CD8 transmembrane domain or It has a variant of 1-2 amino acid modifications, for example, the CD8 transmembrane domain shown in SEQ ID NO: 4; or a CD28 transmembrane domain or a variant thereof with 1-2 amino acid modifications, for example, SEQ ID NO:4 CD28 transmembrane domain shown in ID NO:5;
  • costimulatory domains which are two different costimulatory domains, for example, (a) CD28 costimulatory domain or variants thereof with 1-2 amino acid modifications and (b) 4-1BB costimulatory A combination of domains or variants thereof with 1-2 amino acid modifications, for example, a combination of a CD28 costimulatory domain set forth in SEQ ID NO:6 and a 4-1BB costimulatory domain set forth in SEQ ID NO:7 ;and
  • CD3 ⁇ signaling domain or a variant thereof with 1-2 amino acid modifications for example, the CD3 ⁇ signaling domain shown in SEQ ID NO:8.
  • amino acid modification is addition, deletion or substitution of amino acid.
  • the invention provides a nucleic acid encoding a chimeric antigen receptor (CAR polypeptide) as described herein, a vector comprising a nucleic acid encoding a CAR polypeptide as described herein, and a nucleic acid molecule comprising a CAR nucleic acid molecule or vector as described herein.
  • a cell, or a cell expressing a CAR polypeptide described herein, preferably, the cell is an autologous T cell or an allogeneic T cell.
  • the present invention uses human PBMCs to prepare primary CAR-T cells.
  • the CAR-T cell transduced with the CAR molecule of the present invention has an in vitro effector function, and has the activity of continuously killing target cells in vitro.
  • the CAR-T cells transduced with the CAR molecules of the present invention also have the function of killing tumor cells in vivo.
  • the CAR-T cells of the present invention have enhanced anti-tumor activity.
  • the present invention provides a method of producing a cell, such as an immune effector cell, comprising introducing a nucleic acid molecule (for example, an RNA molecule, such as an mRNA molecule) encoding a CAR polypeptide described herein, or comprising a nucleic acid molecule encoding a CAR polypeptide described herein.
  • a nucleic acid molecule for example, an RNA molecule, such as an mRNA molecule
  • the vector of the nucleic acid molecule of the CAR polypeptide is introduced (eg, transduced) into immune effector cells.
  • the immune effector cells are T cells, NK cells, for example, the T cells are autologous T cells or allogeneic T cells, for example, the immune effector cells are T cells isolated from human PBMC, prepared after NK cells.
  • the three CARs exemplified in the present invention namely IL13-CD4-28BB ⁇ , IL13-CD8-28BB ⁇ , or IL13-CD28-BB ⁇ , are introduced into primary T cells with retroviruses to obtain the CARs of the present invention.
  • CAR-T cells are introduced into primary T cells with retroviruses to obtain the CARs of the present invention.
  • the in vitro anti-glioma effect of the CAR-T cells of the present invention was detected by flow cytometry and real-time cell analysis (RTCA), and the U373 cell xenograft mouse model with higher expression of IL13R ⁇ 2 and the mouse model with low expression of IL13R ⁇ 2 were respectively tested.
  • RTCA real-time cell analysis
  • the in vivo anti-glioma efficacy of the CAR-T cells of the present invention was verified on the U251 cell xenograft mouse model.
  • the present invention shows that the T cells transduced by the three CARs exemplified by the present invention have similar anti-tumor activity when co-cultured with U373 cells expressing higher IL13R ⁇ 2, but the T cells transduced by the three CARs have similar anti-tumor activity when co-cultured with U373 cells expressing higher IL13R ⁇ 2.
  • U251 cells were co-cultured, their antitumor activities were different.
  • All three CAR-T cells of the present invention can be activated by U373 cells that highly express IL13R ⁇ 2, but only IL13-CD28BB ⁇ CAR-T cells can be activated by U251 cells, and after co-culture with U251 cells, the expression of IFN- ⁇ increases significantly.
  • IL13-CD28BB ⁇ -type CAR-T cells can infiltrate tumors and exhibit the best anti-tumor activity in xenograft mouse models.
  • the present invention screens out differentially expressed genes related to different anti-GBM activities.
  • the superior antitumor effect of IL13-CD28BB ⁇ CAR-T cells is partly due to the differential expression of extracellular assembly, extracellular matrix, cell migration, and adhesion-related genes that contribute to lower IL13R ⁇ 2 thresholds required for their activity, increased CAR-T cell proliferation and improve the migration ability of CAR-T cells.
  • the present invention provides the use of the immune effector cells expressing the CAR polypeptide of the present invention, for the preparation of drugs for preventing or treating tumors (such as cancer) or providing anti-tumor immunity in subjects, preferably, the The tumor is a glioma, more preferably, the tumor is a glioblastoma.
  • the present invention provides the use of the CAR polypeptide-expressing immune effector cells of the present invention for treating diseases related to the expression of IL13R ⁇ 2 in a subject, comprising administering to the subject a therapeutically effective amount of the CAR polypeptide-expressing The immune effector cells, wherein the disease associated with the expression of IL13R ⁇ 2 is, for example, glioma, preferably glioblastoma.
  • the present invention provides a method for treating a mammal suffering from a disease related to the expression of IL13R ⁇ 2, comprising administering to the mammal an effective amount of an immune effector cell expressing a CAR polypeptide of the present invention, for example, wherein the disease related to the expression of IL13R ⁇ 2 is glial
  • the tumor preferably, is a glioblastoma.
  • the present invention firstly confirmed that the immune effector cells expressing the CAR polypeptide of the present invention can regulate the function of CAR-T cells in vitro and in vivo; it confirmed that the immune effector cells expressing the CAR polypeptide of the present invention are suitable for targeting the therapeutic target IL13R ⁇ 2 Treatment of glioma, in particular treatment of glioblastoma.
  • FIG. 1 Schematic diagram showing the structure of three CAR constructs, SD in the figure represents the splice donor (Splice donor); SA represents the splice acceptor (splice acceptor); LTR represents the long terminal repeat sequence (long terminal repeat); "TM " or "TMD” means a transmembrane domain.
  • the extracellular domain comprises IL13 or a variant thereof capable of specifically binding IL13R ⁇ 2.
  • FIG. 2 Flowchart illustrating the preparation and evaluation of CAR-T cells, in which mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors at day 0.
  • PBMCs mononuclear cells
  • T cells in PBMCs were stimulated with the anti-CD3/CD28 T cell activator Dynabeads (Invitrogen).
  • T cells in PBMCs were stimulated with the anti-CD3/CD28 T cell activator Dynabeads (Invitrogen).
  • T cells were transfected with retroviral supernatant containing the nucleotide sequence encoding CAR.
  • the positive rate of CAR expression in transfected T cells was detected by flow cytometry.
  • days 9-14 cells were collected for in vitro experiments.
  • Figure 3 It shows that after culturing CAR-T cells in vitro in X-VIVO-15 medium containing 5% human AB serum, 100U/ml IL-2, 100U/ml penicillin and 100 ⁇ g/ml streptomycin, the CAR- The proliferative capacity of T cells.
  • Figure 4 Shows the proliferation of CAR-T cells when co-cultured with U251 cells in vitro.
  • Figure 5 Shows the expression of intracellular IFN- ⁇ of CAR-T cells when co-cultured with U251 cells or U373 cells in vitro.
  • Figure 6 Shows the detection of human interferon- Expression of gamma (IFN- ⁇ ), tumor necrosis factor-alpha (TNF- ⁇ ), IL17, IL4, IL6 and IL10.
  • IFN- ⁇ interferon- Expression of gamma
  • TNF- ⁇ tumor necrosis factor-alpha
  • IL17 IL17
  • IL4 IL6
  • IL10 interferon- Expression of gamma
  • Figure 6 Shows the detection of human interferon- Expression of gamma (IFN- ⁇ ), tumor necrosis factor-alpha (TNF- ⁇ ), IL17, IL4, IL6 and IL10.
  • Figure 7 Shows that CAR-T cells were co-cultured with U373-GL cells and U251-GL cells at different E:T ratios, luciferase substrate was added after 24 hours, and the survival of target cells in vitro was assessed by an optical imaging system .
  • FIG. 8 Shows the cell growth results of tumor cells detected by real-time cell analysis (RTCA), and evaluates the anti-tumor ability of CAR-T cells in vitro.
  • RTCA real-time cell analysis
  • Figure 9 shows the expression percentage of IL13Ra2 on the surface of U251 cell line and U373 cell line, in the figure, U373-GL represents U373 cell line; U251-GL represents U251 cell line.
  • Figure 10 shows the protocol for generating xenograft model mice using the glioma cell line U373, injection of CAR-T cells and detection. Among them, 2 ⁇ 10 5 U373 cells were injected into the striatum of the right brain of female NOD-SCID mice on day 1 (also abbreviated as D1). On day 6 (D6), 3 ⁇ 10 7 CAR-T cells were injected through the tail vein.
  • Figure 11 and Figure 12 shows the inhibition of tumor growth in U373-GL model mice after injection of CAR-T cells.
  • Figure 13 shows the survival curve of U373-GL model mice after injection of CAR-T cells.
  • Figure 14 Shows the infiltration of CAR-T cells into tumor tissue in U373-GL model mice after injection of CAR-T cells.
  • Figure 15 shows the protocol for generating xenograft model mice using the glioma cell line U251, injection of CAR-T cells and detection. Among them, 2 ⁇ 10 5 U251 cells were injected into the striatum of the right brain of female NOD-SCID mice on day 1 (also abbreviated as D1). On day 6 (D6), 3 ⁇ 10 7 CAR-T cells were injected through the tail vein.
  • Figure 16 and Figure 17 shows the inhibition of tumor growth in U251-GL model mice after injection of CAR-T cells.
  • Figure 18 shows the survival curve of U251-GL model mice after injection of CAR-T cells.
  • Figure 19 shows the infiltration of CAR-T cells into tumor tissue in U251-GL model mice after injection of CAR-T cells.
  • Figure 20- Figure 22 Shows the comparison of differentially expressed genes of three CAR-T cells.
  • Figure 23 shows the results of GO analysis of differentially expressed genes.
  • Figure 24 shows a protein-protein interaction network diagram.
  • Figure 25 shows differentially expressed genes verified by RT-qPCR.
  • Figure 26 shows the percentage of memory T cells and the percentage of effector T cells after CAR-T cells were co-cultured with U251 and U373 cells.
  • Figure 27 A flowchart showing the ability of CAR-T cells to recognize tumor cells in vitro by cell binding assays.
  • Figure 28 shows the fluorescence imaging results of the cell adhesion experiment, and the statistics of the number of adhered cells.
  • Figure 29 shows that CD28-TMD on the CAR forms heterodimers with naturally occurring CD28 molecules on T cells.
  • chimeric receptor chimeric antigen receptor
  • CAR CAR
  • the term "stimulatory molecule” refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that modulates TCR complex activation in a stimulatory manner in at least some aspect of the T cell signaling pathway.
  • primary activation In one embodiment, primary signals are elicited, eg, by binding of the TCR/CD3 complex to peptide-loaded MHC molecules and result in mediation of T cell responses including, but not limited to, proliferation, activation, differentiation, and the like.
  • the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence, eg, the primary signaling sequence of CD3 ⁇ .
  • CD3 ⁇ is defined as the protein given by GenBan Accession No. BAG36664.1 or its equivalent
  • CD3 ⁇ stimulatory signaling domain is defined as amino acid residues from the cytoplasmic domain of the CD3 ⁇ chain sufficient for functional Transmits the initial signal necessary for T cell activation.
  • the cytoplasmic domain of CD3 ⁇ comprises residues 52 to 164 of GenBank accession number BAG36664.1 or as a functional ortholog thereof from a non-human species (e.g., mouse, rodent, equivalent residues for monkeys, apes, etc.).
  • the "CD3 ⁇ stimulatory signaling domain” is the sequence provided in SEQ ID NO: 8 or a variant thereof.
  • costimulatory molecule refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand to mediate a costimulatory response (eg, but not limited to, proliferation) of the cell.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response.
  • Costimulatory molecules include but are not limited to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, OX40 , CD40, GITR, 4-1BB (ie CD137), CD27 and CD28.
  • the "co-stimulatory molecule” is CD28, 4-1BB (ie CD137).
  • Costimulatory signaling domain refers to the intracellular portion of a costimulatory molecule.
  • 4-1BB refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank Accession No. AAA62478.2 or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) and "4-1BB co-stimulatory signaling domain” is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2 or equivalent residues from a non-human species (eg, mouse, rodent, monkey, ape, etc.) .
  • the "4-1BB co-stimulatory domain” is the sequence provided as SEQ ID NO: 7 or equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).
  • signaling pathway refers to the biochemical relationship between various signaling molecules that play a role in propagating a signal from one part of a cell to another.
  • cytokine is a general term for proteins released by one cell population to act as intercellular mediators on another cell.
  • cytokines are lymphokines, monokines, interleukins (IL), such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7.
  • amino acid change and “amino acid modification” are used interchangeably to refer to additions, deletions, substitutions and other modifications of amino acids. Any combination of amino acid additions, deletions, substitutions and other modifications can be made, provided that the final polypeptide sequence possesses the desired properties.
  • the amino acid substitutions are non-conservative amino acid substitutions, ie, the substitution of one amino acid with another amino acid having different structural and/or chemical properties.
  • Amino acid substitutions include non-naturally occurring amino acids or naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxy lysine) substitution.
  • Amino acid changes can be made using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. A method of changing amino acid side chain groups by methods other than genetic engineering, such as chemical modification, may be useful. Various names may be used herein to refer to the same amino acid change. For example, a glutamic acid to tyrosine substitution at position 13 of IL-13 can be represented as 13Y, E13Y.
  • conservative sequence modification and “conservative sequence change” refer to amino acid modifications or changes that do not significantly affect or change the characteristics of the CAR containing the amino acid sequence or its constituent elements. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the CAR of the invention or its constituent elements by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), ⁇ -side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenyl Alanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • autologous refers to any substance that is derived from the same individual into whom the substance is later reintroduced.
  • allogeneic refers to any substance derived from a different animal of the same species as the individual into whom it is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently dissimilar genetically to interact antigenically.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • apheresis refers to an art-recognized extracorporeal method by which blood from a donor or patient is removed from a donor or patient and passed through a device that separates selected specific components and return the remainder to the donor or patient's circulation, for example, by retransfusion.
  • single sample refers to a sample obtained using apheresis.
  • immune effector cell refers to a cell that participates in an immune response, eg, participates in promoting an immune effector response.
  • immune effector cells include T cells, eg, ⁇ / ⁇ T cells and ⁇ / ⁇ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • immune effector function refers to, for example, the enhancement of immune effector cells or the function or response of immune attack target cells.
  • immune effector function or response refers to T cell or NK cell properties that promote killing of target cells or inhibit growth or proliferation of target cells.
  • primary stimulation and co-stimulation are examples of immune effector functions or responses.
  • effector function refers to a specialized function of a cell.
  • the effector function of T cells may be, for example, cytolytic activity or helper activity, including secretion of cytokines.
  • T cell activation or “T cell activation” are used interchangeably and refer to one or more cellular responses of T lymphocytes, especially cytotoxic T lymphocytes, selected from: proliferation, differentiation, cytokine secretion , release of cytotoxic effector molecules, expression of cytotoxic activity and activation markers.
  • cytotoxic T lymphocytes selected from: proliferation, differentiation, cytokine secretion , release of cytotoxic effector molecules, expression of cytotoxic activity and activation markers.
  • the chimeric antigen receptors of the invention are capable of inducing T cell activation. Suitable assays for measuring T cell activation are described in the Examples and are known in the art.
  • lentivirus refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information to host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentiviral genome, including inter alia self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009).
  • Other examples of lentiviral vectors that may be used clinically include, but are not limited to, the Lentiviral vector from Oxford BioMedica Gene delivery technology, LENTIMAX TM vector system from Lentigen, etc.
  • Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.
  • disease associated with expression of IL13R ⁇ 2 refers to any condition caused by, aggravated by, or otherwise associated with increased expression or activity of IL13R ⁇ 2.
  • mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rodents). mouse).
  • domesticated animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rodents.
  • rodents e.g., mice and rodents.
  • an individual or subject is a human.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • Tumor immune evasion refers to the process by which tumors evade immune recognition and clearance.
  • tumor immunity is "cured” when such evasions are weakened, and the tumor is recognized and attacked by the immune system.
  • Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.
  • treating means slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
  • Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, reduction of the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • the CAR immune effector cells of the invention are used to delay the development of a disease or to slow down the progression of a disease.
  • an effective amount refers to such an amount or dose of the CAR immune effector cells of the present invention that, after being administered to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.
  • An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering various factors such as: the species of mammal; body weight, age and general health; the particular disease involved; the extent or severity of the disease; the individual The patient's response; the specific CAR immune effector cells administered; the mode of administration; the bioavailability characteristics of the formulation administered; the selected dosing regimen; and the use of any concomitant therapy.
  • a “therapeutically effective amount” refers to an amount effective, at dosages required, and for periods of time required, to achieve the desired therapeutic result.
  • a therapeutically effective amount of CAR immune effector cells can vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the CAR immune effector cells to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the CAR immune effector cells are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 50%, relative to an untreated subject. 60% or 70% and still more preferably at least about 80% or 90%.
  • the ability of CAR immune effector cells to suppress a measurable parameter eg, cancer
  • vector refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • Subject/patient sample refers to a collection of cells, tissues or body fluids obtained from a patient or subject.
  • the source of the tissue or cell sample can be solid tissue like from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood components; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid ), peritoneal fluid (ascites), or interstitial fluid; cells from any time during pregnancy or development of a subject.
  • Tissue samples may contain compounds that are not naturally intermingled with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • tumor samples herein include, but are not limited to, tumor biopsy, fine needle aspirate, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimen, circulating tumor cells, serum, plasma, circulating Plasma proteins in ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples such as formalin-fixed, paraffin-embedded tumor samples, or frozen tumors sample.
  • Glioma is a type of tumor with the highest incidence rate in the brain. It has high malignancy, poor curative effect, and easy recurrence. Its pathogenesis, pathogenesis and effective treatment methods have always been the difficulties of research.
  • the prior art has found that glioma is closely related to many cytokines, among which IL13 is particularly closely related.
  • IL13 is a multifunctional cytokine, which has important biological functions in the intricate cytokine network in vivo. A large number of studies have shown that IL13 plays a very important role in the process of immune response and inflammatory response.
  • IL13R ⁇ 2 IL-13 receptor ⁇ Chain isomer 2
  • the present invention relates to a chimeric antigen receptor (CAR) polypeptide capable of specifically binding IL13R ⁇ 2.
  • a chimeric antigen receptor (CAR) is a recombinant polypeptide that at least includes an extracellular recognition domain, a transmembrane region and an intracellular signaling domain.
  • Extracellular recognition domains also referred to simply as "ectodomains” specifically recognize and bind molecules present on the cell surface of target cells. Since the CAR can both bind antigen and induce T cell activation, and the T cell activation is independent of MHC restriction, CAR can be used to treat antigen-positive tumor patients regardless of the HLA genotype of the tumor patient .
  • Adoptive immunotherapy using T lymphocytes expressing tumor-specific CARs can be a powerful therapeutic strategy for treating cancer.
  • the present invention constructs a variety of CAR polypeptides containing different transmembrane domains that specifically bind IL13R ⁇ 2, and confirms their anti-tumor effects, providing an alternative treatment plan for the immunotherapy of GBM patients.
  • the chimeric antigen receptor of the present invention comprises
  • transmembrane domain selected from the group consisting of: CD4 transmembrane domain or variants thereof with 1-10 amino acid modifications, CD8 transmembrane domain or variants thereof with 1-10 amino acid modifications, CD28 transmembrane domain The membrane domain or a variant thereof having 1-10 amino acid modifications;
  • costimulatory domains selected from the group consisting of: (a) CD28 costimulatory domains or variants thereof with 1-10 amino acid modifications, (b) 4-1BB costimulatory domains or variants thereof with 1-10 amino acid modifications Amino acid modified variants, (c) OX40 co-stimulatory domains or variants thereof with 1-10 amino acid modifications, and any combination of (a), (b) and/or (c) described; and
  • CD3 ⁇ signaling domain or variants thereof having 1-10 amino acid modifications CD3 ⁇ signaling domain or variants thereof having 1-10 amino acid modifications
  • amino acid modification is addition, deletion or substitution of amino acid.
  • the extracellular domain of a chimeric antigen receptor of the invention is human IL-13 or a variant thereof having 1-10 amino acid modifications, e.g., it has 1-2 amino acid modifications, that specifically binds IL13R ⁇ 2 variant of .
  • the CAR of the present invention includes an amino acid modification at the 13th amino acid of the IL13R ⁇ 2 binding part of the CAR, that is, IL13, for example, the amino acid at position 13 of human IL-13 is not E, for example, E becomes Y, E becomes some other amino acid like K or R or L or V.
  • the natural glutamic acid residue at position 13 of ⁇ -helix A of human IL-13 contained in the ectodomain of the CAR of the present invention is replaced with tyrosine to obtain an IL13 (E13Y) mutant.
  • the IL13(E13Y) mutant selectively reduces the affinity for the IL13R ⁇ 1 receptor, while the binding of the IL13(E13Y) mutant to IL13R ⁇ 2 is increased relative to the binding of wild-type IL13 to IL13R ⁇ 2.
  • the IL13R ⁇ 2-binding portion of the CAR of the present invention contains the E13Y mutation, which increases the binding specificity of IL13 to IL13R ⁇ 2 and reduces the binding to IL13R ⁇ 1, thereby reducing the targeting of healthy tissues.
  • the IL13 (E13Y) mutant has the amino acid sequence shown in SPGPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN (SEQ ID NO: 2) or a variant thereof.
  • the extracellular domain of the CAR of the invention comprises native sequence IL13.
  • CAR immune effector cells e.g., T cells, NK cells
  • the extracellular domain of the CAR i.e., IL13 or variants thereof
  • target immune effector cells e.g., T cells, NK cells
  • the cells expressing IL13R ⁇ 2 are tumor cells (including glioma cells).
  • the transmembrane domain comprised in the chimeric antigen receptors of the invention is an anchored transmembrane domain, which is an integral part of a polypeptide chain capable of integrating in a cell membrane.
  • Transmembrane domains can be fused to other extracellular and/or intracellular polypeptide domains whereby these extracellular and/or intracellular polypeptide domains will also be confined to the cell membrane.
  • the transmembrane domain confers membrane attachment to the CAR polypeptide of the invention.
  • the CAR polypeptide of the present invention comprises at least one transmembrane domain, which may be derived from a natural source or a recombinant source, comprising predominantly hydrophobic residues such as leucine and valine.
  • the transmembrane domain may be derived from the transmembrane domain of a membrane-bound or transmembrane protein such as CD4, CD28, CD8 (eg, CD8 ⁇ , CD8 ⁇ ).
  • the transmembrane domain in the chimeric antigen receptor of the invention is a CD4 transmembrane domain or a variant thereof having 1-10 amino acid modifications, for example, it has 1-5 amino acid modifications Variants.
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the CD4 transmembrane domain is the sequence shown in MALIVLGGVAGLLLFIGLGIFF (SEQ ID NO: 3).
  • the transmembrane domain in the chimeric antigen receptor of the invention is a CD8 transmembrane domain or a variant thereof having 1-10 amino acid modifications, for example, it has 1-5 amino acid modifications Variants.
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the CD8 transmembrane domain is the sequence shown in IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 4).
  • the transmembrane domain in the chimeric antigen receptor of the invention is a CD28 transmembrane domain or a variant thereof having 1-10 amino acid modifications, for example, it has 1-5 amino acid modifications Variants.
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the CD28 transmembrane domain is the sequence shown in VWWWLVESWLVIACYYQWPLLFSG (SEQ ID NO: 5).
  • IL13R ⁇ 2-specific IL13R ⁇ 2-containing CAR-T cells comprising the CD28 transmembrane domain have excellent anti-GBM activity in vivo.
  • the transmembrane domain in the CAR of the invention can be linked to the extracellular region of the CAR (ie, IL-13 or a variant thereof) via a hinge/spacer.
  • Glycine-serine doublets provide a particularly suitable linker as hinge/spacer.
  • the linker comprises the amino acid sequence of GGGGS.
  • the cytoplasmic domain comprised in the CAR of the present invention comprises an intracellular signaling domain.
  • the intracellular signaling domain is capable of activating at least one effector function of the immune cell into which the CAR of the present invention has been introduced.
  • the CAR of the present invention further comprises a spacer between the transmembrane domain and the intracellular signaling domain, for example, the spacer is an IgG hinge region or it has 1-2 amino acid modifications
  • the spacer is an IgG hinge region or it has 1-2 amino acid modifications
  • intracellular signaling domains for use in the CARs of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act cooperatively to initiate signal transduction following binding of the extracellular domain to IL13R ⁇ 2, and these Any derivative or variant of the sequence and any recombinant sequence having the same functional capacity.
  • TCR T cell receptor
  • co-receptors that act cooperatively to initiate signal transduction following binding of the extracellular domain to IL13R ⁇ 2
  • the CAR of the present invention is also designed with a co-stimulatory signal domain (CSD) that can generate co-stimulatory signals.
  • CSD co-stimulatory signal domain
  • Activation of T cells is mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domain) and those that act in an antigen-independent manner to provide co-stimulatory signals Those sequences of (secondary cytoplasmic domain, eg, co-stimulatory domain).
  • the CAR of the present invention comprises a primary intracellular signaling domain, e.g., a primary signaling domain of CD3 ⁇ , e.g., a CD3 ⁇ signaling domain as shown in SEQ ID NO: 8 or it has 1-10 amino acids Modified variants, eg, variants having 1-5 amino acid modifications, wherein the amino acid modifications are additions, deletions or substitutions of amino acids.
  • the intracellular signaling domain in the CAR of the present invention also includes a secondary signaling domain (ie, co-stimulatory signaling domain).
  • a co-stimulatory signaling domain refers to the portion of the CAR comprising the intracellular domain of a co-stimulatory molecule.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for immune effector cells to mount an effective response to an antigen.
  • co-stimulatory molecules include, but are not limited to, CD28, 4-1BB (CD137), OX40, which cause co-stimulation that enhances the proliferation, effector function, and survival of human CART cells in vitro and enhances human T cell antitumor activity.
  • the co-stimulatory signaling domain in the chimeric antigen receptor of the invention is a CD28 co-stimulatory domain or a variant thereof having 1-10 amino acid modifications, for example, it has 1-5 amino acid modifications variant of .
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the co-stimulatory signaling domain in the chimeric antigen receptor of the invention is a 4-1BB costimulatory domain or a variant thereof having 1-10 amino acid modifications, for example, it has 1-5 Amino acid modified variants.
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the intracellular region of the CAR of the present invention comprises one or more co-stimulatory domains in tandem with CD3 ⁇ , such as the co-stimulatory domain of 4-1BB (also known as CD137).
  • CD3 ⁇ such as the co-stimulatory domain of 4-1BB (also known as CD137).
  • 4-1BB also known as CD137.
  • the CAR When expressed on the surface of cells (eg, T cells, NK cells), the CAR enables the T cells to receive co-stimulatory signals.
  • the co-stimulatory signaling domain in the chimeric antigen receptor of the invention is an OX40 co-stimulatory domain or a variant thereof having 1-10 amino acid modifications, for example, it has 1-5 amino acid modifications variant of .
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the intracellular signaling sequences of the CAR of the present invention can be connected to each other in a random order or in a specified order.
  • short oligopeptide linkers or polypeptide linkers can form linkages between intracellular signaling sequences.
  • a glycine-serine doublet can be used as a suitable linker.
  • single amino acids, eg, alanine, glycine can be used as suitable linkers.
  • the intracellular signaling domain of the CAR of the present invention is designed to include a co-stimulatory signaling domain of CD28 and a stimulating signaling domain of CD3 ⁇ .
  • the intracellular signaling domain is designed to comprise the co-stimulatory signaling domain of 4-1BB and the stimulatory signaling domain of CD3 ⁇ .
  • the intracellular signaling domain of the CAR of the present invention is designed to include a co-stimulatory signaling domain of CD28, a co-stimulatory signaling domain of 4-1BB and a stimulating signaling domain of CD3 ⁇ .
  • CAR polypeptides of the present invention may also be modified so as to vary in amino acid sequence but not in desired activity.
  • additional nucleotide substitutions that result in amino acid substitutions at "non-essential" amino acid residues can be made to the CAR polypeptide.
  • a non-essential amino acid residue in a molecule can be replaced with another amino acid residue from the same side chain family.
  • an amino acid stretch can be substituted for a structurally similar stretch that differs in the order and composition of side chain family members, for example, conservative substitutions can be made in which the amino acid residue Substitute with an amino acid residue with a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, , glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains (e.g., threonine, valine, iso leucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • the present invention also contemplates the generation of functionally equivalent CAR polypeptide molecules.
  • the invention provides nucleic acid molecules encoding the CAR constructs described herein.
  • the nucleic acid molecule is provided as a DNA construct.
  • Constructs encoding the CARs of the invention can be obtained using recombinant methods well known in the art.
  • the nucleic acid of interest may be produced synthetically rather than by genetic recombination methods.
  • the present invention also provides a vector inserted with the CAR construct of the present invention.
  • Expression of a natural or synthetic nucleic acid encoding a CAR is achieved by operably linking the nucleic acid encoding the CAR polypeptide to a promoter and incorporating the construct into an expression vector.
  • the vector may be suitable for replication and integration in eukaryotes. Common cloning vectors contain transcriptional and translational terminators, initiation sequences and promoters for regulating the expression of the desired nucleic acid sequence.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the CAR constructs of the invention can be inserted into vectors and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject in vivo or ex vivo.
  • Numerous retroviral systems are known in the art.
  • lentiviral vectors are used.
  • the nucleic acid sequence of the CAR construct of the present invention is cloned into a lentiviral vector to generate a full-length CAR construct in a single coding frame, and the EF1 ⁇ promoter is used for expression.
  • Retroviruses such as lentiviruses
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses, such as murine leukemia virus, because they can transduce non-proliferative cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • a retroviral vector may also be, for example, a gamma retroviral vector.
  • a gamma retroviral vector may, for example, comprise a promoter, a packaging signal ( ⁇ ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest, e.g., encoding a CAR gene.
  • Gamma retroviral vectors may lack viral structural genes such as gag, pol, and env.
  • a promoter capable of expressing a CAR transgene in mammalian T cells is the EF1a promoter.
  • the native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome.
  • the EF1a promoter has been used extensively in mammalian expression plasmids and has been shown to efficiently drive CAR expression from transgenes cloned into lentiviral vectors. See, eg, Milone et al., Mol. Ther. 17(8):1453-1464 (2009).
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a constitutively strong promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked thereto.
  • other constitutive promoter sequences can also be used, including but not limited to Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) Long terminal repeat (LTR) promoters, MoMuLV promoters, avian leukemia virus promoters, Epstein-Barr virus immediate early promoters, Rous sarcoma virus promoters, and human gene promoters such as but not limited to the actin promoter , myosin promoter, elongation factor-1 ⁇ promoter, hemoglobin promoter and creatine kinase promoter. Additionally, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of
  • the invention provides methods of expressing a CAR construct of the invention in mammalian immune effector cells (eg, mammalian T cells or mammalian NK cells) and immune effector cells produced thereby.
  • mammalian immune effector cells eg, mammalian T cells or mammalian NK cells
  • a source of cells eg, immune effector cells, eg, T cells or NK cells
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from blood components collected from a subject using any technique known to those of skill in the art, such as Ficoll (TM ) separation.
  • the cells from the circulating blood of the individual are obtained by apheresis.
  • Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • T cell subsets such as CD3+, CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells can be further isolated by positive or negative selection techniques.
  • anti-CD3/anti-CD28 conjugated beads such as M-450CD3/CD28T
  • the period of time is between about 30 minutes and 36 hours or longer. Longer incubation times can be used to isolate T cells wherever small numbers of T cells are present, such as for isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals.
  • TILs tumor infiltrating lymphocytes
  • Enrichment of T cell populations can be accomplished through the process of negative selection using a combination of antibodies directed against surface markers unique to the negatively selected cells.
  • One method is the sorting and/or selection of cells by means of negative magnetic immunoadhesion or flow cytometry using the presence of cells on negatively selected cells Monoclonal antibody cocktail for surface markers.
  • the immune effector cells can be allogeneic immune effector cells, eg, T cells or NK cells.
  • the cell can be an allogeneic T cell, e.g., one lacking expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA) (e.g., HLA class I and/or HLA class II) T cells.
  • TCR T cell receptor
  • HLA human leukocyte antigen
  • a T cell lacking a functional TCR can, for example, be engineered such that it does not express any functional TCR on its surface; engineered such that it does not express one or more subunits that make up a functional TCR (e.g. engineered so that it does not express or exhibit reduced expression of TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ and/or TCR ⁇ ); or engineered so that it produces very few functional TCRs on its surface.
  • a T cell as described herein can, for example, be engineered such that it does not express functional HLA on its surface.
  • T cells described herein can be engineered such that cell surface expression of HLA (e.g., HLA class I and/or HLA class II) is downregulated.
  • HLA e.g., HLA class I and/or HLA class II
  • downregulation of HLA can be achieved by reducing or eliminating beta-2 microglobulin (B2M) expression.
  • the T cells may lack a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II.
  • the cells transduced with the nucleic acid encoding the CAR of the present invention are proliferated, for example, the cells are proliferated in culture for 2 hours to about 14 days.
  • the immune effector cells expressing CAR obtained after in vitro proliferation can be tested for effector function as described in the Examples.
  • expressing the CAR polypeptide with CD28-TMD against IL13R ⁇ 2 of the present invention can significantly improve the proliferation, migration, and expression of protein kinase B activation and protein kinase C signaling of its transduced CAR-T cells, suggesting CD28-TMD-induced isomerization makes T cells easier to activate.
  • CD28-TMD-induced isomerization makes T cells easier to activate.
  • the T cell co-stimulatory molecule CD28 can provide signals that help CAR-T cell survival.
  • the CD28-TMD on the transfected CAR can form heterodimers with the naturally occurring CD28 molecule on T cells, which may also be related to the enhanced survival and expansion of CAR-T cells.
  • the CAR-T cells of the present invention can maintain the killing activity against tumors expressing IL13R ⁇ 2.
  • T cells transduced with the IL13-CD428BB ⁇ or IL13-CD828BB ⁇ CAR of the present invention are co-cultured with U373 cells, the intracellular IFN- ⁇ level is significantly increased, and after co-cultured with U251 cells, intracellular IFN - Gamma levels were not elevated. This may be due to the relatively low level of IL13R ⁇ 2 in U251 cells. Therefore, CAR-T cells containing CD4 or CD8 transmembrane regions may require relatively high levels of IL13R ⁇ 2 on tumor cells to be active, also referred to herein as requiring a higher activation threshold.
  • IL13-CD28BB ⁇ CAR-T cells expressed similar IFN- ⁇ before co-culture with GBM cells, and exhibited similar antitumor activity to IL13-CD428BB ⁇ or IL13-CD828BB ⁇ CAR-T cells in vitro.
  • IL13-CD28BB ⁇ CAR-T cells showed excellent anti-tumor activity in vivo, which may be due to the fact that CARs containing the CD28 transmembrane domain induced more stable and efficient immune interactions and lowered the activation threshold.
  • the ability of the CAR-T cells of the present invention to recognize tumor cells is detected by a cell adhesion detection method.
  • Various CAR-T cells of the present invention have similar interactions with U373 cells, but have different interactions with U251 cells, indicating that the transmembrane domain (TMD) contained in CAR changes the combination of CAR-T cells and tumor cells ability, which was demonstrated when co-cultured with tumor cells expressing less IL13 receptor IL13R ⁇ 2.
  • the CAR-T cells of the present invention are co-cultured with tumor cells
  • the CAR-T cells are separated using DynabeadsTM CD4 Positive Isolation Kit and DynabeadsTM CD8 Positive Isolation Kit (Invitrogen, Thermo Fisher Science).
  • DynabeadsTM CD4 Positive Isolation Kit and DynabeadsTM CD8 Positive Isolation Kit Invitrogen, Thermo Fisher Science.
  • high-throughput sequencing is then performed to detect the differentially expressed genes of the CAR-T cells of the present invention.
  • RNA extraction, cDNA library construction, and sequencing were all performed in strict accordance with transcriptome sequencing standards.
  • Gene Ontology (GO) analysis was performed on genes differentially expressed in CAR-T cells of the present invention using the online bioinformatics tool DAVID bioinformatics Resources 6.8.
  • the upregulation of chemokines such as CXCL5, cytoskeleton genes such as ACTN2, and the reduction of inhibitory cytokines such as IL-10 in the CAR-T cells of the present invention may facilitate the migration of CAR-T cells to tumor sites more easily in vivo.
  • PKC is a family of serine/threonine kinases widely expressed in mammalian cells, with multiple isoforms identified, mainly serving as key regulators of cytoskeletal remodeling, cell polarity and T cell migration.
  • Real et al proved that PKC ⁇ and PKC ⁇ affect T cell motility and scanning behavior of chemokine receptor molecules on DC cell surface (Real E, Faure S, Donnadieu E, Delon J. Cutting edge: Atypical PKCs regulate T lymphocyte polarity and scanning behavior . Journal of Immunology. 2007;179(9):5649-5652).
  • PKC ⁇ was up-regulated more than 2.9-fold in IL13-CD28BB ⁇ CD4+ CAR-T cells. Upregulation of PKC ⁇ may be an upstream signal of overexpression of downstream genes, contributing to altered extracellular matrix assembly and cell adhesion assembly.
  • increased T cell infiltration in tumors may also be a result of activation of PKC signaling.
  • PKB/Akt serine/threonine kinase protein kinase B ⁇
  • PKB/Akt serine/threonine kinase protein kinase B ⁇
  • FGF1, ANG and PINK1 were significantly upregulated in IL13-CD28BB ⁇ CD8+ CAR-T cells, and these genes are functionally related to the activation of PKB.
  • the CD28 transmembrane domain-containing third-generation IL13BB ⁇ CAR-T cells of the present invention have excellent in vivo anti-GBM activity due to their lower activity threshold and altered transcriptome.
  • T-cell therapy was first applied in the treatment of hematological B-cell malignancies and showed effective and encouraging results.
  • the antitumor activity of CAR-T cells in the treatment of solid tumors is limited and still in the experimental stage.
  • the occurrence of side effects and inefficiency in solid tumors have also become a challenge.
  • immune effector cells expressing the CAR polypeptide of the present invention are obtained, which are used to treat diseases related to the expression of IL13R ⁇ 2 in subjects.
  • T cells eg, patient-specific autologous T cells
  • ACT adoptive cell therapy
  • T cells eg, patient-specific autologous T cells
  • ACT adoptive cell therapy
  • T cell subsets can be used to express the CAR polypeptides of the invention.
  • the immune effector cell when treating a patient with an immune effector cell expressing a CAR polypeptide of the present invention, may be an autologous T cell or an allogeneic T cell.
  • the T cells used are CD4+ and CD8+ central memory T cells (T CM ), which are CD45RO+CD62L+, and the use of such cells improves adoptive T cells compared to other types of patient-specific T cells. Long-term survival of cells after sexual transfer.
  • other immune cells eg, NK cells
  • NK cells are engineered to express a CAR polypeptide of the invention. After expanding the engineered other immune cells (eg, NK cells), they are used for adoptive cell therapy (ACT).
  • ACT adoptive cell therapy
  • an immune effector cell expressing a CAR polypeptide of the invention is used to treat a cancer expressing or overexpressing IL13R ⁇ 2 in a subject and is capable of reducing the severity of at least one symptom or indication of cancer or inhibiting cancer cells grow.
  • the present invention provides a method for treating a disease associated with the expression of IL13R ⁇ 2 (e.g., a cancer that expresses or overexpresses IL13R ⁇ 2) in a subject, comprising administering to an individual in need thereof a therapeutically effective amount of an immune effector expressing a CAR polypeptide of the present invention cell.
  • a disease associated with the expression of IL13R ⁇ 2 e.g., a cancer that expresses or overexpresses IL13R ⁇ 2
  • administering to an individual in need thereof a therapeutically effective amount of an immune effector expressing a CAR polypeptide of the present invention cell.
  • the present invention provides the use of the aforementioned immune effector cells expressing the CAR polypeptide of the present invention in the preparation of medicines for treating diseases related to the expression of IL13R ⁇ 2 (for example, cancers expressing or overexpressing IL13R ⁇ 2).
  • Immune effector cells expressing a CAR polypeptide of the invention can also be administered to individuals whose cancer has been treated with one or more prior therapies but has subsequently relapsed or metastasized.
  • immune effector cells e.g., T cells, NK cells
  • a CAR polypeptide of the invention are used for parenteral, transdermal, intracavity, intraarterial, intravenous, intrathecal administration, or direct injection into tissue or tumors.
  • the immune effector cells expressing the CAR polypeptide of the present invention can be administered to a subject at an appropriate dose.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dose for any one patient depends on many factors, including the patient's weight, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, and concomitant other medications administered.
  • the immune effector cells for example, T cells, NK cells
  • the immune effector cells expressing the CAR polypeptide of the present invention are 1 ⁇ 10 6 -1 ⁇ 10 12 immune effector cells, preferably 1 ⁇ 10 7 -1 ⁇ 10 10 immune effector cells, for example Doses of 5x107 , 1x108 , 5x108, 1x109 , 5x109 immune effector cells are administered parenterally, preferably intravenously, in single or multiple doses.
  • administering results in complete disappearance of the tumor. In some embodiments, administration of immune effector cells expressing a CAR polypeptide of the invention to an individual with cancer results in at least an 85% or greater reduction in tumor cells or tumor size.
  • Tumor reduction can be measured by any method known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetics analyze.
  • Human GBM cell lines U251, U373 and retroviral packaging cell lines PG13 and Phoenix ECO were purchased from American Tissue Culture Collection (ATCC).
  • U251 cells and U373 cells expressed ffLuc reporter gene and GFP gene by retroviral transduction.
  • the GBM cell line grows well in DMEM medium (Lonza) containing 10% fetal bovine serum (FBS, Biosera), 100 U/mL penicillin and 100 ⁇ g/mL streptomycin (Eall Bio Life Sciences).
  • Retroviral producing cell lines were cultured in DMEM containing 10% FBS without penicillin and streptomycin.
  • BD FacsCanto II Plus instrument (BD Biosciences) was used for flow cytometry detection, and FlowJo v.10 software (Tree star, Inc. Ashland, OR) was used to analyze the flow cytometry results.
  • the antibodies used are as follows: anti-human CD3-APC-R700 antibody (BD Bioscience), anti-human-CD4-V450 (BD Bioscience), anti-human-CD8-PE-Cy7 (BD Bioscience), anti-human IL13Ra- APC (BD Bioscience) and goat anti-mouse IgG-APC (Sigma).
  • anti-human CD107a-APC BD-Biosciences
  • blocking agent eBioscience TM Protein Transport Inhibitor Cocktail, Invitrogen
  • 1 ⁇ blocking agent eBioscience TM Protein Transport Inhibitor Cocktail, Invitrogen
  • IFN- ⁇ interferon- ⁇
  • TNF- ⁇ tumor necrosis factor- ⁇
  • RTCA Real-Time Cell Analysis
  • the proliferation/cytotoxicity of CAR T cells was assessed using the xCELLigence RTCA system (Roche Applied Sciences, Basel, Switzerland). The system is based on a gold plate sensor electrode for electrical impedance reading, located at the bottom of a cytotoxicity plate (E-16 plate).
  • E-16 plate cytotoxicity plate
  • the target cells U251-GL and U373-GL cells were seeded in E-16 plates at 1 ⁇ 104 cells per well. After 24 hours, 1 ⁇ 10 5 CAR-T cells were added to the E-16 plate, incubated with human GBM cells, monitored every 15 minutes, and the cell index was obtained for 48 hours. Each independent experiment was performed in triplicate. Using RTCA software to automatically calculate the slope of the interval, and evaluate the rate of change of the cell index. To demonstrate the effect of treatment, cell indices were normalized to equal values at normalized time points.
  • CAR-T cells were co-cultured with U251 cells at an effect-to-target ratio of 10:1.
  • CD4 and CD8 CAR T cells were isolated 4 hours later using DynabeadsTM CD4 Positive Isolation Kit and DynabeadsTM CD8 Positive Isolation Kit (Invitrogen, Thermo Fisher Science).
  • the co-cultured mixed cells, sorted CD4-CAR-T cells, CD8-CAR-T cells and U251 cells were sent to Annoroad Gene Technology Co., Ltd. (Beijing, China) for RNA sequencing.
  • RNA extraction, cDNA library construction, and sequencing were all performed in strict accordance with transcriptome sequencing standards.
  • the gene ontology (GO) analysis of differentially expressed genes among various CAR-T cells was performed using the online bioinformatics tool DAVID bioinformatics Resources 6.8. Data visualization and analysis were handled by custom R studio scripts following packages (ggplot2 and Tree map). Gene enrichment analysis was performed using Fisher's exact test.
  • Example 1 Construction of IL13R ⁇ 2-specific CAR retroviral vector and preparation of retroviral supernatant Three different IL13R ⁇ 2-targeted CARs shown in Figure 1 were constructed using retroviruses.
  • IL13CD428BB ⁇ CAR contains from the N-terminus to the C-terminus: a human IL13 domain (SEQ ID NO: 2) with a mutation at the E13Y site on the cell membrane surface, a CD4 transmembrane domain (SEQ ID NO: 3), A human CD28 and 4-1BB co-stimulatory domain (SEQ ID NO:6 and SEQ ID NO:7), a human CD3 ⁇ cytoplasmic domain (SEQ ID NO:8);
  • IL13CD828BB ⁇ CAR contains from N-terminus to C-terminus: a human IL13 domain (SEQ ID NO:2) with E13Y site mutation located on the cell membrane surface, a CD8 transmembrane domain (SEQ ID NO:4) and an IgG4 hinge domain (SEQ ID NO:1), a human CD28 and 4-1BB co-stimulatory domain (SEQ ID NO:6 and SEQ ID NO:7), a human CD3 ⁇ cytoplasmic domain (SEQ ID NO:8);
  • IL13CD28BB ⁇ CAR contains from N-terminus to C-terminus: a human IL13 domain (SEQ ID NO: 2) with E13Y site mutation located on the cell membrane surface, a CD28 transmembrane domain (SEQ ID NO: 5), a human CD28 and 4 - 1BB co-stimulatory domain (SEQ ID NO:6 and SEQ ID NO:7), a human CD3 ⁇ cytoplasmic domain (SEQ ID NO:8).
  • CARs share a human IL13 domain with E13Y site mutation located on the cell membrane surface, a human CD28 and 4-1BB co-stimulatory domain, and a human CD3 ⁇ cytoplasmic domain in structure (see Figure 1). All three CARs recognize IL13R ⁇ 2 through a single-site (E13Y) modified membrane-tethered IL-13 as a ligand, and have the intracellular signaling domain of CD28 and 4-1BB as co-stimulatory sequences to promote CAR-T Cellular persistence and protection against anergy, the intracellular segments of these three CARs also possess CD3 ⁇ .
  • E13Y single-site
  • IL13CD428BB ⁇ connects a CD4 transmembrane domain after the IL13(E13Y) sequence
  • IL13CD828BB ⁇ connects a CD8 transmembrane domain and IgG4 (hinge) region after the IL13(E13Y) sequence
  • IL13CD28BB ⁇ connects after the IL13(E13Y) sequence A transmembrane domain of CD28.
  • the coding sequence of IL13 (E13Y) sequence was synthesized, primers were designed, and it was connected with the other domain coding sequences of the above three different IL13R ⁇ 2-targeted CARs by means of homologous recombination to form a complete Finally, the above three CAR sequences were subcloned into the transcription virus vector SFG by homologous recombination to obtain three target plasmids.
  • the three target plasmids encoding CAR retroviral vector were co-transduced with RD114 packaging plasmid into 293GP packaging cell line, and the retroviral supernatant was harvested after 48 hours for transduction of T cells .
  • the retroviral supernatant harvested in Example 1 was used to transduce T cells, thereby preparing CAR-T cells.
  • PBMC mononuclear cells
  • GE-healthcare lymphocyte separation medium
  • T cells in PBMCs were stimulated with the anti-CD3/CD28 T cell activator Dynabeads (Invitrogen). After 48 hours of stimulation, T cells were transfected with the retroviral supernatant of Example 1.
  • the retrovirus transfection experiment was carried out according to the instructions of the calcium phosphate transfection kit (Sigma). On day 7, the positive rate of CAR expression in transfected T cells was detected by flow cytometry. On days 9-14, cells were collected for experiments.
  • the specific CAR-T cell preparation and evaluation process is shown in Figure 2.
  • CAR-T cells were cultured in X-VIVO-15 medium containing 5% human AB serum (SIGMA), 100 U/ml IL-2, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin (EallBio Life Sciences). This study was approved by the Institutional Review Board of Beijing Shijitan Hospital, and informed consent was obtained from all participants.
  • SIGMA human AB serum
  • IL-2 100 U/ml IL-2
  • penicillin 100 ⁇ g/ml streptomycin
  • Example 1 The results showed that the three CARs constructed in Example 1 were successfully introduced into T cells respectively. As shown in Figure 3, the obtained three CAR-T cells all showed good viability and proliferation ability in vitro, among which IL13CD428BB ⁇ CAR-T cells had the strongest proliferation ability, followed by IL13CD28BB ⁇ CAR-T proliferation ability.
  • Example 3 In vitro proliferation of CAR-T cells stimulated by IL13R ⁇ 2+ tumor cells
  • Example 2 The three kinds of CAR-T cells prepared in Example 2 were co-cultured with IL13R ⁇ 2+ tumor cells in vitro, and the cell growth and cytokine release of CAR-T cells were observed.
  • Figure 4 shows the proliferation of CAR-T cells co-cultured with U251 cells. The results showed that when the three CAR-T cells prepared in Example 2 were co-cultured with the glioma cell U251 cells with low expression of IL13R ⁇ 2, the CAR-T cells could be effectively expanded, and the IL13CD28BB ⁇ CAR-T cells had the best proliferative ability (Figure 4).
  • CBA Cytometric Bead Array
  • CAR-T cells In order to further evaluate the killing ability of CAR-T cells on tumor cells, three kinds of CAR-T cells were combined with glioma cells U251-GL or U373-GL cells according to the ratio of effector cells to target cells (E:T) 0.5: After 1, 1:1, 2.5:1, 5:1 and 10:1 co-culture for 24 hours, add luciferase substrate, and evaluate the survival of target cells in vitro by optical imaging system, thus, by means of the measured luminescence The signal evaluates the in vitro killing ability of CAR-T cells on tumor cells.
  • a negative control (NT) group using peripheral blood mononuclear cells (PBMC) as effector cells was set up.
  • Figure 7 shows the results of in vitro survival of target cells after CAR-T cells were co-cultured with U373-GL and U251-GL cells at different E:T ratios for 24 hours.
  • the three groups of CAR-T cells of the present invention significantly inhibited the luminescent signal, and the inhibitory effect of IL13CD28BB ⁇ CAR-T cells was the strongest, especially at low E:T ratios (0.5:1 and 1:1), compared with the other two groups of CAR-T cells, IL13CD28BB ⁇ CAR-T cells had a stronger inhibitory effect on glioma cells in vitro.
  • the cell growth analysis of tumor cells was carried out by RTCA real-time cell analyzer.
  • the bottom of the detection plate E-Plate 16 is integrated with a micro-gold electronic sensor chip.
  • the change in the impedance value directly reflects the growth of the cells.
  • Figure 8 shows the cell growth results of tumor cells detected by RTCA method, and the anti-tumor ability of CAR-T cells was evaluated in vitro. The results showed that all three groups of CAR-T cells significantly inhibited the growth of U373-GL and U251-GL cells, and had obvious anti-tumor activity, and there was no significant difference among the three groups (Figure 8).
  • an orthotopic xenograft mouse model was constructed by intracranial injection of glioma cells.
  • Glioma cell lines U373 and U251 expressed IL13R ⁇ 2 on the surface.
  • the expression levels of IL13R ⁇ 2 on these two glioma cells were detected by flow cytometry.
  • An orthotopic xenograft mouse model was constructed using glioma cell lines U373 and U251 cells. Specifically, the mice used in the experiment were six to eight-week-old NOD-SCID mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. 2 ⁇ 10 5 U251-GL cells or U373-GL cells were respectively injected into the striatum of the right brain of female NOD-SCID mice (denoted as the first day, also abbreviated as D1). On the sixth day (ie, D6), 3 ⁇ 10 7 CAR-T cells prepared in Example 2 were injected through the tail vein ( FIG. 10 , FIG. 15 ). A blank control group was set up in the experiment, and the CAR-T cells of the present invention were not administered to the orthotopic xenograft mouse model on D6, but PBS was administered.
  • the development of the tumor was monitored by observing the tumor growth by detecting the fluorescent signal using a small animal imaging instrument (IVIS, Xenogen, Alameda, CA, USA), and the mice were sacrificed when the tumor diameter reached 20 mm. Animal experiments were approved by the Ethics Committee of Beijing Shijitan Hospital.
  • mice generated by U373-GL were generated by U373-GL.
  • Figure 10 shows the protocol for generating xenograft model mice using the glioma cell line U373 (also referred to herein as U373-GL; or U373), injection of three CAR-T cells of the present invention, and detection.
  • U373 also referred to herein as U373-GL; or U373
  • Figure 15 shows the protocol for generating xenograft model mice using the glioma cell line U251 (also referred to herein as U251-GL; or U251), injection of three CAR-T cells of the present invention, and detection.
  • U251 also referred to herein as U251-GL; or U251
  • the CAR-T cells were divided into CD4+T cells and CD8+T cells, and then high-throughput sequencing was performed to detect the three CAR-T cells differentially expressed genes.
  • Figure 20- Figure 22 shows the comparison of differentially expressed genes of three CAR-T cells;
  • Figure 23 shows the results of GO analysis of differentially expressed genes.
  • FIG 20- Figure 22 compared with CD4+ T cells transfected by the other two CARs of the present invention, there are 413 up-regulated genes in the CD4+ T cells transfected by the IL13-CD28BB ⁇ CAR; compared with the other two CARs of the present invention Compared with CAR-transfected CD8+ T cells, there were 509 up-regulated genes in IL13-CD28BB ⁇ CAR-transfected CD8+ T cells.
  • FIG. 24 shows a protein-protein interaction network diagram.
  • RNA-seq results CAR-T cells co-cultured with U251 or U373 cells for 4 hours were collected, and then RT-qPCR was used to detect the expression of candidate genes CYBB, IL-10, CXCL5, TCF7 and ACTN2 in CAR-T cells. expression in .
  • the method was as follows: Total RNA was extracted from cells using TRIzol reagent (Invitrogen) following the manufacturer's instructions. RNA quantity and purity were measured using a Nanodrop One spectrophotometer (Thermo Fisher Scientific).
  • cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), and then amplified using SYBR Green PCR Master Mix (Thermo Fisher Scientific) and gene-specific primers. GAPDH was used as an internal control. Relative expression of genes was calculated using the 2- ⁇ Ct method.
  • FIG. 25 shows the differentially expressed genes verified by RT-qPCR. The results showed that IL13-CD28BB ⁇ CAR-T cells had the lowest IL10 level and the highest Tcf7 expression ( FIG. 25 ). The Tcf7 encodes TCF1 protein, which is highly expressed in memory T cells.
  • Example 7 The ability of CAR-T cells to recognize tumor cells in vitro
  • GBM cells were first seeded on a 24-well plate; after they adhered to the wall, T cells were stained with CSFE; then T cells were co-cultured with tumor cells, shaken for 5 minutes and then photographed (Pic1); washed 3 times, after removing the cells that did not adhere to the 24-well plate, take a second photoshoot (Pic2).
  • Figure 28 shows the fluorescence imaging results of the cell adhesion experiment and the statistics of the number of adhered cells. As shown in Figure 28, more IL13-CD28BB ⁇ CAR-T cells combined with U251 cells after incubation for 5 min, but the three groups of CAR-T cells combined with U373 cells in similar numbers.
  • Figure 29 shows that on IL13-CD28BB ⁇ CAR-T cells, the CD28 transmembrane domain on the CAR forms a heterodimer with the naturally occurring CD28 molecule on T cells, which may enhance the survival and expansion of CAR-T cells .

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Abstract

本发明涉及靶向IL13Rα2的嵌合抗原受体、编码所述嵌合抗原受体的核酸分子、包含所述核酸分子的载体、以及经工程化以表达所述嵌合抗原受体的免疫效应细胞(例如，T细胞、NK细胞)。本发明还涉及所述经工程化的免疫效应细胞的用途，用于治疗与表达IL13Rα2相关的疾病，例如，胶质瘤。

Description

靶向IL13Rα2的嵌合抗原受体及其用途 技术领域
本发明总体上涉及靶向IL13Rα2的嵌合抗原受体、经工程化以表达本发明的嵌合抗原受体的免疫效应细胞(例如，T细胞、NK细胞)、以及所述经工程化的免疫效应细胞的用途，用于治疗与表达IL13Rα2相关的疾病。
背景技术
胶质母细胞瘤(Glioblastoma，GBM)是世界范围内最常见、最具侵袭性的原发性恶性脑肿瘤之一，大多数病例恶性程度比较高。由于高发病率、高死亡率和低治愈率，GBM在世界范围内造成了巨大的社会和医疗负担，而且目前常规的治疗手段几乎无法治愈胶质母细胞瘤。即使采用最好的多模式治疗，患者的中位生存率也只有21个月左右，半个世纪以来，胶质母细胞瘤患者的总体生存率没有实质性的提高。对于大多数患者来说，治疗失败的主要原因是侵袭性耐药恶性细胞的复发。研究表明，肿瘤的持续生长和复发是由于未能产生有效的免疫反应。
因此，有必要开发在GBM患者体内能够产生有效的免疫反应的新疗法，以改善GBM患者的预后。
嵌合抗原受体(Chimeric antigen receptor，CAR)是一种人工合成的分子，其通过特异性识别肿瘤细胞表面表达的抗原来引导经基因工程化以表达CAR的免疫效应细胞(例如，T细胞、NK细胞)清除肿瘤(Sampson JH,Choi BD,Sanchez-Perez L等人,EGFRvIIImCAR-modified T-cell therapy cures mice with established intracerebralglioma and generates host immunity against tumor-antigen loss.Clinical cancer research:an official journal of the American Association for Cancer Research.2014；20(4):972-984)。例如，嵌合抗原受体T细胞(CAR-T)是通过T细胞上的嵌合抗原受体(CAR)分子直接靶向肿瘤细胞的表面抗原，从而达到识别和杀伤肿瘤的目的，其中所述嵌合抗原受体的N端包含识别抗原的胞外域。当所述CAR-T细胞针对的抗原阳性细胞存在时，CAR-T细胞能识别并杀伤这些抗原阳性细胞。
CAR T细胞介导的CD19 +恶性血液病的临床成功为CAR T细胞过继免疫治疗的发展提供了支持(Batlevi CL,Matsuki E,Brentjens RJ,Younes A.Novel immunotherapies in lymphoid malignancies.Nature reviews.Clinical oncology.2016；13(1):25-40)。研究发现，CD19-CAR T细胞可以降低脑转移性白血病的发病率(Abramson JS,McGree B,Noyes S等人,Anti-CD19 CAR T Cells in CNS Diffuse Large-B-Cell Lymphoma.The New England journal of medicine.2017； 377(8):783-784)。CAR T细胞可以通过趋化因子迁移到脑实质中，靶向并杀死肿瘤细胞(Hong JJ,Rosenberg SA,Dudley ME等人,Successful treatment of melanoma brain metastases with adoptive cell therapy.Clinical cancer research:an official journal of the American Association for Cancer Research.2010；16(19):4892-4898)。然而，CAR T细胞在脑肿瘤治疗中的应用尚处于早期阶段。在实体瘤领域，CAR T细胞治疗效果并不理想，其原因与缺乏肿瘤特异性抗原、T细胞的存活及扩增不足、T细胞向肿瘤组织的迁移效率低和免疫抑制的肿瘤微环境等因素有关。
IL13Rα2是IL-13的受体多肽，几乎只在癌细胞中表达，在正常组织细胞中不表达(睾丸除外)，在GBM患者中，有50％以上的患者表达IL13Rα2，使其成为CAR-T治疗GBM的有效靶点(Sharma P,Debinski W.Receptor-Targeted Glial Brain Tumor Therapies.International journal of molecular sciences.2018；19(11))。早期临床试验包括IL13Rα2疫苗治疗GBM(Iwami K,Shimato S,Ohno M等人,Peptide-pulsed dendritic cell vaccination targeting interleukin-13 receptor alpha2chain in recurrent malignant glioma patients with HLA-A*24/A*02 allele.Cytotherapy.2012；14(6):733-742)和第一代IL13ζCAR T细胞和第二代IL13BBζCAR T细胞治疗复发性胶质母细胞瘤，使IL13Rα2成为免疫治疗的一个有吸引力的靶点(Brown CE,Aguilar B,Starr R等人,Optimization of IL13Ralpha2-Targeted Chimeric Antigen Receptor T Cells for Improved Anti-tumor Efficacy against Glioblastoma.Molecular therapy:the journal of the American Society of Gene Therapy.2018；26(1):31-44)。
尽管CAR-T细胞治疗突破了抗原需要被MHC分子呈递的限制，且能够克服血脑屏障的障碍，已被证明是一种具有持久有效抗肿瘤活性的突破性治疗方法，但其在实体瘤中的副作用和低效性已逐渐成为一个挑战。
本领域仍然需要结构得到进一步优化的靶向IL13Rα2的CAR，以降低CAR-T细胞治疗胶质母细胞瘤的副作用并提高其疗效。
发明内容
嵌合抗原受体(CAR)修饰T(CAR-T)细胞治疗已被证明是一种有效的抗癌手段，但其副作用的发生，以及在实体瘤中的低效性逐渐成为一个挑战。为了提高CAR-T治疗的抗胶质母细胞瘤效果，本发明提供了多种针对IL13Rα2的第三代CAR T细胞，它们分别包含来自CD4、CD8或CD28的不同跨膜区，旨在通过优化CAR的跨膜结构域(TMD)来提高CAR-T的抗肿瘤效果。
因此，在第一方面，本发明提供了跨膜结构域不同的多种第三代IL13Rα特异性CAR多肽，其包含
(i)人IL-13或其具有1-10个氨基酸修饰的变体；
(ii)跨膜结构域，其选自：CD4跨膜结构域或其具有1-10个氨基酸修饰的变体、CD8跨膜结构域或其具有1-10个氨基酸修饰的变体、CD28跨膜结构域或其具有1-10个氨基酸修饰的变体；
(iii)共刺激结构域，其选自：(a)CD28共刺激结构域或其具有1-10个氨基酸修饰的变体、(b)4-1BB共刺激结构域或其具有1-10个氨基酸修饰的变体、(c)OX40共刺激结构域或其具有1-10个氨基酸修饰的变体、和所述(a)、(b)和/或(c)的任意组合；和
(iv)CD3ζ信号传导结构域或其具有1-10个氨基酸修饰的变体，
其中所述氨基酸修饰是氨基酸的添加、缺失或取代。
在一些实施方案中，本发明的嵌合抗原受体多肽还包含在所述(ii)和所述(iii)之间的间隔区，例如，所述间隔区是IgG铰链区或其具有1-2个氨基酸修饰的变体，例如，IgG4铰链区或其具有1-2个氨基酸修饰的变体，例如，ESKYGPPCPSCP(SEQ ID NO:1)所示的IgG4铰链区，其中所述氨基酸修饰是氨基酸的添加、缺失或取代。
在一些实施方案中，本发明的嵌合抗原受体多肽中的(i)是人IL-13或其具有1-2个氨基酸修饰的变体，例如，人IL-13的位置13上的氨基酸不是E，例如，人IL-13的位置13上的氨基酸是Y，也称为IL-13(E13Y)或IL-13E13Y，例如，所述(i)是SPGPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN(SEQ ID NO:2)。
在一些实施方案中，本发明的嵌合抗原受体多肽中的(ii)是CD4跨膜结构域或其具有1-5个氨基酸修饰的变体、CD8跨膜结构域或其具有1-5个氨基酸修饰的变体、或CD28跨膜结构域或其具有1-5个氨基酸修饰的变体，其中所述氨基酸修饰是氨基酸的添加、缺失或取代；例如，所述CD4跨膜结构域是MALIVLGGVAGLLLFIGLGIFF(SEQ ID NO:3)所示的序列；所述CD8跨膜结构域是IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:4)所示的序列；所述CD28跨膜结构域是VWWWLVESWLVIACYYQWPLLFSG(SEQ ID NO:5)所示的序列。
在一些实施方案中，本发明的嵌合抗原受体多肽中的(iii)是(a)CD28共刺激结构域或其 具有1-5个氨基酸修饰的变体、(b)4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体、(c)OX40共刺激结构域或其具有1-5个氨基酸修饰的变体、和所述(a)、(b)和/或(c)的任意组合，其中所述氨基酸修饰是氨基酸的添加、缺失或取代；例如，所述(iii)是两个不同的共刺激结构域，例如，(a)CD28共刺激结构域或其具有1-5个氨基酸修饰的变体和(b)4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体的组合，例如，所述CD28共刺激结构域是SKRSRLLMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:6)所示的序列，所述4-1BB共刺激结构域是AERNSCIYSNNHLCDQYKLLKRKMAVAADFQKKKKEDV(SEQ ID NO:7)所示的序列。
在一些实施方案中，本发明的嵌合抗原受体多肽中的(iv)是CD3ζ信号传导结构域或其具有1-5个氨基酸修饰的变体，其中所述氨基酸修饰是氨基酸的添加、缺失或取代；例如，所述CD3ζ信号传导结构域是RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAKDTYDALHMQALPPRYQPLE(SEQ ID NO:8)所示的序列。
在一些实施方案中，本发明的嵌合抗原受体多肽包含：
(i)人IL-13或其具有1-2个氨基酸修饰的变体，例如，IL-13(E13Y)；
(ii)跨膜结构域，其是CD4跨膜结构域或其具有1-2个氨基酸修饰的变体，例如，SEQ ID NO:3所示的CD4跨膜结构域；CD8跨膜结构域或其具有1-2个氨基酸修饰的变体，例如，SEQ ID NO:4所示的CD8跨膜结构域；或者CD28跨膜结构域或其具有1-2个氨基酸修饰的变体，例如，SEQ ID NO:5所示的CD28跨膜结构域；
(iii)共刺激结构域，其是两个不同的共刺激结构域，例如，(a)CD28共刺激结构域或其具有1-2个氨基酸修饰的变体和(b)4-1BB共刺激结构域或其具有1-2个氨基酸修饰的变体的组合，例如，SEQ ID NO:6所示的CD28共刺激结构域和SEQ ID NO:7所示的4-1BB共刺激结构域的组合；和
(iv)CD3ζ信号传导结构域或其具有1-2个氨基酸修饰的变体，例如，SEQ ID NO:8所示的CD3ζ信号传导结构域。
任选地，包含在所述(ii)和所述(iii)之间的间隔区，例如，IgG4铰链区或其具有1-2个氨基酸修饰的变体，例如，SEQ ID NO:1所示的IgG4铰链区，
其中所述氨基酸修饰是氨基酸的添加、缺失或取代。
在第二方面，本发明提供了编码如本文所述的嵌合抗原受体(CAR多肽)的核酸、包含编码本文所述CAR多肽的核酸的载体、和包含本文所述CAR核酸分子或载体的细胞、或表达本文所述CAR多肽的细胞，优选地，所述细胞是自体T细胞或同种异体T细胞。
在一个实施方案中，本发明采用人PBMC制备了原代CAR-T细胞。用本发明的CAR分子转导的CAR-T细胞具有体外效应功能，在体外具有持续杀伤靶细胞的活性。
用本发明的CAR分子转导的CAR-T细胞还具有体内杀伤肿瘤细胞的功能。在肿瘤细胞异种移植小鼠模型中，本发明的CAR-T细胞具有提高的抗肿瘤活性。
在第三方面，本发明提供了一种产生细胞、例如免疫效应细胞的方法，所述方法包括将编码本文所述CAR多肽的核酸分子(例如，RNA分子，例如mRNA分子)，或包含编码本文所述CAR多肽的核酸分子的载体引入(例如转导)免疫效应细胞。
在一些实施方案中，所述免疫效应细胞是T细胞、NK细胞，例如，所述T细胞是自体T细胞或同种异体T细胞，例如，所述免疫效应细胞是自人PBMC分离T细胞、NK细胞后制备的。
在一些实施方案中，用逆转录病毒将本发明例示的三种CAR，即IL13-CD4-28BBζ、IL13-CD8-28BBζ或IL13-CD28-BBζ分别导入原代T细胞中，获得了本发明的CAR-T细胞。
用流式细胞术和实时细胞分析(RTCA)检测了本发明的CAR-T细胞的体外抗胶质瘤作用，并分别在表达较高IL13Rα2的U373细胞异种移植小鼠模型和在低表达IL13Rα2的U251细胞异种移植小鼠模型上验证了本发明的CAR-T细胞的体内抗胶质瘤功效。
本发明显示，用本发明例示的三种CAR转导的T细胞与表达较高IL13Rα2的U373细胞共培养时具有相似的抗肿瘤活性，但所述三种CAR转导的T细胞与低表达IL13Rα2的U251细胞共培养时，它们的抗肿瘤活性不同。本发明的三种CAR-T细胞均能被高表达IL13Rα2的U373细胞激活，但只有IL13-CD28BBζCAR-T细胞能被U251细胞激活，且在与U251细胞共培养后，IFN-γ表达显著增加。另外，IL13-CD28BBζ型CAR-T细胞在异种移植小鼠模型中，能浸润肿瘤且表现出最好的抗肿瘤活性。
通过高通量测序，本发明筛选出与不同抗GBM活性相关的差异表达基因。IL13-CD28BBζCAR-T细胞的优越抗肿瘤作用的部分原因是细胞外组装、细胞外基质、细胞迁移和粘附相关基因的差异表达，这些基因有助于降低其发挥活性所需的IL13Rα2阈值、增加CAR-T细胞增殖和提高CAR-T细胞的迁移能力。
在第四方面，本发明提供了本发明的表达CAR多肽的免疫效应细胞的用途，用于制 备在受试者中预防或治疗肿瘤(例如癌症)或提供抗肿瘤免疫的药物，优选地，所述肿瘤为胶质瘤，更优选地，所述肿瘤为胶质母细胞瘤。
在第五方面，本发明提供了本发明的表达CAR多肽的免疫效应细胞的用途，用于在受试者中治疗与表达IL13Rα2相关的疾病，包括向受试者施用治疗有效量的表达CAR多肽的免疫效应细胞，其中所述与表达IL13Rα2相关的疾病是例如胶质瘤，优选地胶质母细胞瘤。
本发明提供了治疗患有与表达IL13Rα2相关的疾病的哺乳动物的方法，包括向哺乳动物施用有效量的本发明的表达CAR多肽的免疫效应细胞，例如，其中与表达IL13Rα2相关的疾病是胶质瘤，优选地，是胶质母细胞瘤。
由此，本发明首次证实了表达本发明CAR多肽的免疫效应细胞能够在体内外调节CAR-T细胞功能；证实了本发明的表达CAR多肽的免疫效应细胞适用于靶向治疗性靶点IL13Rα2来治疗胶质瘤，特别地治疗胶质母细胞瘤。
附图说明
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1：显示了三种CAR构建体的结构示意图，图中SD表示剪接供体(Splice donor)；SA表示剪接受体(splice acceptor)；LTR表示长末端重复序列(long terminal repeat)；“TM”或“TMD”表示跨膜结构域(transmembrane domain)。在所述构建体中，胞外域包含能够特异性结合IL13Rα2的IL13或其变体。
图2：例示了CAR-T细胞的制备和评价流程图，其中，在第0天分离健康捐赠者外周血中的单个核细胞(PBMC)。用抗CD3/CD28T细胞激活剂Dynabeads(Invitrogen)刺激PBMC中的T细胞。在第2天(即，刺激48小时后)，用包含编码CAR的核苷酸序列的逆转录病毒上清液转染T细胞。在第7天，使用流式细胞术检测转染的T细胞中CAR表达阳性率。在第9-14天，收集细胞进行体外实验。
图3：显示了体外在含有5％人AB血清、100U/ml IL-2、100U/ml青霉素和100μg/ml链霉素的X-VIVO-15培养基中培养CAR-T细胞后，CAR-T细胞的增殖能力。
图4：显示了体外与U251细胞共培养时，CAR-T细胞的增殖。
图5：显示了体外与U251细胞或U373细胞共培养时，CAR-T细胞的胞内IFN-γ的表达。
图6：显示了体外CAR-T细胞与U251细胞或U373细胞共培养时，通过流式细胞仪珠阵列(CBA)试剂盒(BD Biosciences)检测来自共培养细胞的上清液中人干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)、IL17、IL4、IL6和IL10的表达。
图7：显示了CAR-T细胞与U373-GL细胞和U251-GL细胞以不同的E:T比例共培养，24小时后加入荧光素酶底物，通过光学成像系统评估靶细胞在体外的存活。
图8：显示了实时细胞分析(RTCA)法检测的肿瘤细胞的细胞生长结果，体外评价了CAR-T细胞的抗肿瘤能力。
图9：显示了IL13Ra2在U251细胞系和U373细胞系表面的表达百分数，图中，U373-GL表示U373细胞系；U251-GL表示U251细胞系。
图10：显示了使用胶质瘤细胞系U373产生异种移植模型小鼠、以及注射CAR-T细胞和检测的方案。其中，在第1天(也简写为D1)向雌性NOD-SCID小鼠右脑纹状体注射2×10 5个U373细胞。在第6天(D6)通过尾静脉注射3×10 7个CAR-T细胞。
图11和图12：显示了注射CAR-T细胞后对U373-GL模型小鼠的肿瘤生长的抑制。
图13：显示了注射CAR-T细胞后U373-GL模型小鼠生存曲线。
图14：显示了注射CAR-T细胞后U373-GL模型小鼠中，CAR-T细胞对肿瘤组织的浸润。
图15：显示了使用胶质瘤细胞系U251产生异种移植模型小鼠、以及注射CAR-T细胞和检测的方案。其中，在第1天(也简写为D1)向雌性NOD-SCID小鼠右脑纹状体注射2×10 5个U251细胞。在第6天(D6)通过尾静脉注射3×10 7个CAR-T细胞。
图16和图17：显示了注射CAR-T细胞后对U251-GL模型小鼠的肿瘤生长的抑制。
图18：显示了注射CAR-T细胞后U251-GL模型小鼠生存曲线。
图19：显示了注射CAR-T细胞后U251-GL模型小鼠中，CAR-T细胞对肿瘤组织的浸润。
图20-图22：显示了三种CAR-T细胞的差异表达基因的比较。
图23：显示了差异表达基因的GO分析结果。
图24：显示了蛋白质-蛋白质相互作用网络图。
图25：显示了RT-qPCR验证的差异表达基因。
图26：显示了CAR-T细胞在与U251和U373细胞共培养后，记忆性T细胞的百分数和效应T细胞的百分数。
图27：显示了通过细胞结合试验检测CAR-T细胞在体外识别肿瘤细胞的能力的流程图。
图28：显示了细胞粘附实验的荧光成像结果，及粘附细胞数的统计。
图29：显示了CAR上的CD28-TMD与T细胞上天然存在的CD28分子形成异源二聚体。
具体实施方式
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。本文所提及的全部出版物、专利申请、专利和其他参考文献通过引用的方式完整地并入。此外，本文中所述的材料、方法和例子仅是说明性的并且不意在是限制性的。本发明的其他特征、目的和优点将从本说明书及附图并且从后附的权利要求书中显而易见。
定义
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。
在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“嵌合受体”、“嵌合抗原受体”或“CAR”在本文中可互换使用，是指至少包含胞外抗原结合结构域、跨膜结构域及胞内信号结构域的重组多肽。
术语“刺激分子”指由提供初级胞质信号传导序列的T细胞表达的分子，所述的初级胞质信号传导序列在T细胞信号传导途径的至少某个方面以刺激性方式调节TCR复合体的初级活化。在一个实施方案中，初级信号例如通过TCR/CD3复合体与载有肽的MHC分子的结合引发并且导致介导T细胞反应，包括但不限于增殖、活化、分化等。在本发明的具体CAR中，本发明的任一种或多种CAR中的胞内信号结构域包含胞内信号传导序列，例如，CD3ζ的初级信号传导序列。
术语“CD3ζ”定义为GenBan登录号BAG36664.1提供的蛋白质或其等同物，并且“CD3ζ刺激信号结构域”定义为来自CD3ζ链胞质结构域的氨基酸残基，所述氨基酸残基足以在功能上传播T细胞活化必需的初始信号。在一个实施方案中，CD3ζ的胞质结构域包含GenBank登录号BAG36664.1的残基52至残基164或作为其功能直向同源物的来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。在一个实施方案中，“CD3ζ刺激信号结构域”是在SEQ ID NO:8中提供的序列或其变体。
术语“共刺激分子”是指细胞上的与共刺激配体特异性结合从而介导细胞的共刺激反应(例如但不限于增殖)的相应结合配偶体。共刺激分子是除抗原受体或其配体之外的有助于有效免疫应答的细胞表面分子。共刺激分子包括但不限于MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号传导淋巴细胞活化分子(SLAM蛋白)、激活NK细胞受体、OX40、CD40、GITR、4-1BB(即CD137)、CD27和CD28。在一些实施方案中，“共刺激分子”是CD28、4-1BB(即CD137)。共刺激信号结构域是指共刺激分子的胞内部分。
术语“4-1BB”指TNFR超家族成员，所述成员具有作为GenBank登录号AAA62478.2提供的氨基酸序列或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基；并且“4-1BB共刺激信号结构域”定义为GenBank登录号AAA62478.2的氨基酸残基214-255或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。在一个实施方案中，“4-1BB共刺激结构域”是作为SEQ ID NO:7提供的序列或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。
术语“信号传导途径”指在从细胞一个部分传播信号至细胞的另一个部分中发挥作用的多种信号传导分子之间的生物化学关系。
术语“细胞因子”是由一种细胞群释放，作为细胞间介质作用于另一细胞的蛋白质的通称。此类细胞因子的例子有淋巴因子、单核因子、白介素(IL)，诸如IL-1，IL-1α，IL-2，IL-3，IL-4，IL-5，IL-6，IL-7，IL-8，IL-9，IL-11，IL-12，IL-13，IL-15；肿瘤坏死因子，诸如TNF-α或TNF-β；及其它多肽因子，包括γ-干扰素。
术语“氨基酸变化”和“氨基酸修饰”可互换地使用，是指氨基酸的添加、缺失、取代和其他修饰。可以进行氨基酸的添加、缺失、取代和其他修饰的任意组合，条件是最终的多肽序列具有所需的特性。在一些实施方案中，氨基酸的取代是非保守氨基酸取代，即用具有不同结构和/或化学性质的另一种氨基酸取代一种氨基酸。氨基酸取代包括用非天然存在的 氨基酸或二十种标准氨基酸的天然存在的氨基酸衍生物(例如、4-羟基脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟基赖氨酸)的取代。可以使用本领域公知的遗传或化学方法产生氨基酸变化。遗传方法可包括定点诱变、PCR、基因合成等。通过除基因工程化之外的方法(如化学修饰)改变氨基酸侧链基团的方法可能是有用的。本文可使用多种名称来表示相同的氨基酸变化。例如，IL-13第13位的谷氨酸到酪氨酸的取代可以表示为13Y、E13Y。
术语“保守序列修饰”、“保守序列变化”指未显著影响或改变含有氨基酸序列的CAR或其组成元件的特征的氨基酸修饰或变化。这类保守修饰包括氨基酸取代、添加和缺失。可以通过本领域已知的标准技术，如位点定向诱变和PCR介导的诱变向本发明的CAR或其组成元件中引入修饰。保守性取代是氨基酸残基由具有相似侧链的氨基酸残基替换的氨基酸取代。已经在本领域中定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电荷极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸)、β-侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因而，可以将本发明CAR内部的一个或多个氨基酸残基替换为来自相同侧链家族的其他氨基酸残基，并且可以使用本文所述的功能测定法测试改变的CAR的功能。
术语“自体的”指这样的任何物质，所述物质从稍后将向个体再次引入所述物质的相同个体衍生。
术语“同种异体的”指这样的任何物质，所述物质从与引入所述物质的个体相同的物种的不同动物衍生。当一个或多个基因座处的基因不相同时，两位或更多位个体据称彼此是同种异体的。在一些方面，来自相同物种的个体的同种异体物质可以在遗传上足够地不相似以发生抗原性相互作用。
术语“异种的”指从不同物种的动物衍生的移植物。
如本文所用的术语“单采血液成分术”指本领域认可的体外方法，借助所述方法，供体或患者的血液从供体或患者取出并且穿过这样的装置，所述装置分离选择的特定组分并将剩余部分返回供体或患者的循环，例如，通过再输血。因此，在“单采样品”的语境中，指使用单采血液成分术获得的样品。
术语“免疫效应细胞”指参与免疫应答，例如参与促进免疫效应反应的细胞。免疫效应细胞的例子包括T细胞，例如，α/βT细胞和γ/δT细胞、B细胞、天然杀伤(NK)细胞、 天然杀伤T(NKT)细胞、肥大细胞、和髓细胞衍生的吞噬细胞。
“免疫效应功能”、“免疫效应应答”或“免疫效应反应”指例如免疫效应细胞的增强或促进免疫攻击靶细胞的功能或应答。例如，免疫效应功能或应答指促进杀伤靶细胞或抑制靶细胞生长或增殖的T细胞或NK细胞特性。在T细胞的情况下，初级刺激和共刺激是免疫效应功能或应答的例子。
术语“效应功能”指细胞的特化功能。T细胞的效应功能例如可以是溶细胞活性或辅助活性，包括分泌细胞因子。
术语“T细胞激活”或“T细胞活化”可互换地使用，是指T淋巴细胞，特别是细胞毒性T淋巴细胞的一种或多种细胞应答，选自：增殖、分化、细胞因子分泌、细胞毒性效应分子释放、细胞毒活性和活化标志物的表达。本发明的嵌合抗原受体能够诱导T细胞激活。用于测量T细胞激活的合适测定法在实施例中描述，并是本领域中已知的。
术语“慢病毒”指逆转录病毒科(Retroviridae)的一个属。慢病毒在逆转录病毒当中的独特之处在于能够感染非分裂性细胞；它们可以递送显著量的遗传信息至宿主细胞，从而它们是基因递送载体的最高效方法之一。HIV、SIV和FIV均是慢病毒的例子。
术语“慢病毒载体”指从慢病毒基因组的至少一部分衍生的载体，尤其包括如Milone等人,Mol.Ther.17(8):1453–1464(2009)中提供的自我失活慢病毒载体。可以在临床使用的慢病毒载体的其他例子例如包括但不限于来自Oxford BioMedica的
Figure PCTCN2022071078-appb-000001
基因递送技术、来自Lentigen的LENTIMAX TM载体系统等。非临床类型的慢病毒载体也是可获得的并且是本领域技术人员已知的。
术语“与表达IL13Rα2相关的疾病”是指由IL13Rα2增加的表达或活性引起、加重或以其它方式与其相关的任何病症。
术语“个体”或“受试者”可互换地使用，包括哺乳动物。哺乳动物包括但不限于驯化动物(例如，牛、羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，个体或受试者是人。
术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。
“肿瘤免疫逃逸”指肿瘤逃避免疫识别和清除的过程。如此，作为治疗概念，肿瘤免疫在此类逃避减弱时得到“治疗”，并且肿瘤被免疫系统识别并攻击。肿瘤识别的例子包括肿瘤结合，肿瘤收缩和肿瘤清除。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症 状、病症、病况或疾病的进展或严重性。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明的CAR免疫效应细胞用来延缓疾病发展或用来减慢疾病的进展。
术语“有效量”指本发明的CAR免疫效应细胞的这样的量或剂量，其以单一或多次剂量施用患者后，在需要治疗或预防的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如哺乳动物的物种；体重、年龄和一般健康状况；涉及的具体疾病；疾病的程度或严重性；个体患者的应答；施用的具体CAR免疫效应细胞；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。CAR免疫效应细胞的治疗有效量可以根据多种因素如疾病状态、个体的年龄、性别和重量和CAR免疫效应细胞在个体中激发所需反应的能力而变动。治疗有效量也是这样的一个量，其中CAR免疫效应细胞的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率、肿瘤体积等)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％和仍更优选地至少约80％或90％。可以在预示人肿瘤中的功效的动物模型系统中评价CAR免疫效应细胞抑制可度量参数(例如，癌症)的能力。
在本文中当谈及核酸时使用的术语“载体(vector)”是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其有效相连的核酸的表达。这样的载体在本文中被称为“表达载体”。
“受试者/患者样品”指从患者或受试者得到的细胞、组织或体液的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。肿瘤样品的例子在本文中包括但不限于肿瘤活检、细针吸出物、支气管灌洗液、胸膜液(胸水)、痰液、尿液、手术标本、循环中的肿瘤细胞、血清、血浆、循环中的血浆蛋白质、腹水、衍 生自肿瘤或展现出肿瘤样特性的原代细胞培养物或细胞系，以及保存的肿瘤样品，诸如福尔马林固定的、石蜡包埋的肿瘤样品或冷冻的肿瘤样品。
本发明的嵌合抗原受体(CAR)
脑胶质瘤是在颅内发病率最高的一类肿瘤，其恶性程度高，疗效差，且容易复发，其发病诱因、发病机制及有效治疗方法一直是研究的难点。现有技术发现脑胶质瘤与许多细胞因子存在密切的关系，其中与IL13的关系尤为密切。IL13是一种多功能的细胞因子，其在体内错综复杂的细胞因子网络中有着重要的生物学功能。大量研究表明IL13在免疫应答和炎症反应过程中起着相当重要的作用。利用放射标记的IL13的放射自显影技术已经证明在所研究的几乎所有脑胶质瘤组织上存在大量的IL13结合，这是因为脑胶质瘤中IL13R表达异常，其中，IL-13受体α链异构体2(IL13Rα2)在脑胶质瘤细胞表面存在高表达，并在脑胶质瘤恶性增殖的过程中起着重要的作用。
本发明涉及能够特异性结合IL13Rα2的嵌合抗原受体(CAR)多肽。嵌合抗原受体(CAR)是一种重组多肽，其至少包含胞外识别域、跨膜区和胞内信号传导域。胞外识别域(也简称为“胞外域”)特异性识别并结合靶细胞的细胞表面上存在的分子。由于所述CAR可以既结合抗原又转导T细胞激活，且所述T细胞激活是不依赖于MHC限制的，因此，CAR能够用于治疗抗原阳性的肿瘤患者，不论该肿瘤患者HLA基因型如何。使用表达肿瘤特异性CAR的T淋巴细胞的过继性免疫疗法可以是用于治疗癌症的强大治疗策略。
本发明构建了特异性结合IL13Rα2的多种包含不同跨膜结构域的CAR多肽，并证实了它们的抗肿瘤效果，为GBM患者的免疫治疗提供了备选的治疗方案。本发明的嵌合抗原受体包含
(i)人IL-13或其具有1-10个氨基酸修饰的变体；
(ii)跨膜结构域，其选自：CD4跨膜结构域或其具有1-10个氨基酸修饰的变体、CD8跨膜结构域或其具有1-10个氨基酸修饰的变体、CD28跨膜结构域或其具有1-10个氨基酸修饰的变体；
(iii)共刺激结构域，其选自：(a)CD28共刺激结构域或其具有1-10个氨基酸修饰的变体、(b)4-1BB共刺激结构域或其具有1-10个氨基酸修饰的变体、(c)OX40共刺激结构域或其具有1-10个氨基酸修饰的变体、和所述(a)、(b)和/或(c)的任意组合；和
(iv)CD3ζ信号传导结构域或其具有1-10个氨基酸修饰的变体，
其中所述氨基酸修饰是氨基酸的添加、缺失或取代。
在一些实施方案中，本发明的嵌合抗原受体的胞外域是特异性结合IL13Rα2的人IL-13或其具有1-10个氨基酸修饰的变体，例如，其具有1-2个氨基酸修饰的变体。
在一个实施方案中，本发明的CAR包括在CAR的IL13Rα2结合部分即IL13的第13位氨基酸处具有氨基酸修饰，例如，人IL-13的位置13上的氨基酸不是E，例如E变成Y、E变成一些其他氨基酸如K或R或L或V。
在一个实施方案中，将本发明CAR的胞外域包含的人IL-13上α螺旋A位置13处的天然谷氨酸残基用酪氨酸取代，获得IL13(E13Y)突变体。IL13(E13Y)突变体选择性地降低对IL13Rα1受体的亲和力，同时，IL13(E13Y)突变体与IL13Rα2的结合相对于野生型IL13与IL13Rα2的结合是增加的。由此，本发明的CAR的IL13Rα2结合部分包含E13Y突变，该突变增加IL13对IL13Rα2的结合特异性，并减少与IL13Rα1的结合，从而减少对健康组织的靶向性。在一个实施方案中，所述IL13(E13Y)突变体具有SPGPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN(SEQ ID NO:2)所示的氨基酸序列或其变体。
在一个实施方案中，本发明CAR的胞外域包含天然序列的IL13。在通过局部施用直接将CAR免疫效应细胞(如，T细胞、NK细胞)注射到肿瘤块中的情形，特别地可以使用CAR的胞外域包含天然序列的IL13。
当本发明的CAR在免疫效应细胞(如，T细胞、NK细胞)表面上表达时，CAR的胞外域(即，IL13或其变体)使得其能够将免疫效应细胞(如，T细胞、NK细胞)特异性导向表达IL13Rα2的那些细胞，例如，所述表达IL13Rα2的细胞是肿瘤细胞(包括神经胶质瘤细胞)。
在一些实施方案中，本发明的嵌合抗原受体中包含的跨膜结构域是锚定的跨膜结构域，其是能够整合在细胞膜中的多肽链的组成部分。跨膜结构域可以与其他胞外和/或胞内多肽结构域融合，由此，这些胞外和/或胞内多肽结构域也将被限制在细胞膜上。在本发明的嵌合抗原受体(CAR)多肽中，跨膜结构域赋予本发明的CAR多肽的膜附着。本发明的CAR多肽包含至少一个跨膜结构域，其可以衍生自天然来源或重组来源，包含优势疏水的残基如亮氨酸和缬氨酸。在天然来源的情况下，跨膜结构域可以衍生自膜结合蛋白或跨膜蛋白例如CD4、CD28、CD8(例如，CD8α，CD8β)的跨膜结构域。
在一个实施方案中，本发明的嵌合抗原受体中的跨膜结构域是CD4跨膜结构域或其具 有1-10个氨基酸修饰的变体，例如，其具有1-5个氨基酸修饰的变体。所述氨基酸修饰是氨基酸的添加、缺失或取代。例如，所述CD4跨膜结构域是MALIVLGGVAGLLLFIGLGIFF(SEQ ID NO:3)所示的序列。
在一个实施方案中，本发明的嵌合抗原受体中的跨膜结构域是CD8跨膜结构域或其具有1-10个氨基酸修饰的变体，例如，其具有1-5个氨基酸修饰的变体。所述氨基酸修饰是氨基酸的添加、缺失或取代。例如，所述CD8跨膜结构域是IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:4)所示的序列。
在一个实施方案中，本发明的嵌合抗原受体中的跨膜结构域是CD28跨膜结构域或其具有1-10个氨基酸修饰的变体，例如，其具有1-5个氨基酸修饰的变体。所述氨基酸修饰是氨基酸的添加、缺失或取代。例如，所述CD28跨膜结构域是VWWWLVESWLVIACYYQWPLLFSG(SEQ ID NO:5)所示的序列。
在一个实施方案中，包含CD28跨膜结构域的特异性结合IL13Rα2的CAR-T细胞具有极好的体内抗GBM活性。
在一些实施方案中，本发明的CAR中的跨膜结构域可以借助铰链区/间隔区与CAR的胞外区(即，IL-13或其变体)连接。甘氨酸-丝氨酸双联体提供了特别合适的接头作为铰链区/间隔区。例如，在一个实施方案中，接头包含GGGGS的氨基酸序列。
本发明的CAR中包含的胞质结构域包含胞内信号结构域。胞内信号结构域能够活化引入了本发明CAR的免疫细胞的至少一个效应功能。
在一些实施方案中，本发明的CAR还包含在跨膜结构域和所述胞内信号结构域之间的间隔区，例如，所述间隔区是IgG铰链区或其具有1-2个氨基酸修饰的变体，例如，IgG4铰链区或其具有1-2个氨基酸修饰的变体，例如，ESKYGPPCPSCP(SEQ ID NO:1)所示的IgG4铰链区。
用于本发明CAR中的胞内信号结构域的例子包括协同发挥作用以在胞外结构域结合IL13Rα2后启动信号转导的T细胞受体(TCR)和共受体的胞质序列，以及这些序列的任何衍生物或变体和具有相同功能性能力的任何重组序列。
考虑到仅通过TCR生成的信号尚不足以完全活化T细胞，因此本发明的CAR还设计了能够产生共刺激信号的共刺激信号结构域(CSD)。T细胞的活化由两类不同的胞质信号传导序列介导：通过TCR启动抗原依赖性初级活化的那些序列(初级胞内信号结构域)和以抗原非依赖性方式发挥作用以提供共刺激信号的那些序列(次级胞质结构域，例如，共刺激结构域)。
在一个实施方案中，本发明的CAR包含初级胞内信号结构域，例如，CD3ζ的初级信号结构域，例如，SEQ ID NO:8所示的CD3ζ信号传导结构域或其具有1-10个氨基酸修饰的变体，例如，其具有1-5个氨基酸修饰的变体，其中所述氨基酸修饰是氨基酸的添加、缺失或取代。
本发明CAR中的胞内信号结构域还包次级信号结构域(即，共刺激信号结构域)。共刺激信号结构域指包含共刺激分子的胞内结构域的CAR部分。共刺激分子是除抗原受体或其配体之外免疫效应细胞对抗原作出有效反应所需要的细胞表面分子。在一些实施方案中，共刺激分子包括但不限于CD28、4-1BB(CD137)、OX40，其引起的共刺激作用在体外增强人CART细胞的增殖、效应功能和存活并且在体内增进人T细胞的抗肿瘤活性。
在一个实施方案中，本发明的嵌合抗原受体中的共刺激信号结构域是CD28共刺激结构域或其具有1-10个氨基酸修饰的变体，例如，其具有1-5个氨基酸修饰的变体。所述氨基酸修饰是氨基酸的添加、缺失或取代。
在一个实施方案中，本发明的嵌合抗原受体中的共刺激信号结构域是4-1BB共刺激结构域或其具有1-10个氨基酸修饰的变体，例如，其具有1-5个氨基酸修饰的变体。所述氨基酸修饰是氨基酸的添加、缺失或取代。
在一个实施方案中，本发明CAR的胞内区包含与CD3ζ串联的一个或多个共刺激结构域，如4-1BB(也称为CD137)的共刺激结构域，当所述CAR在免疫效应细胞(如，T细胞、NK细胞)表面上表达时，所述CAR使得T细胞能够接受共刺激信号。
在一个实施方案中，本发明的嵌合抗原受体中的共刺激信号结构域是OX40共刺激结构域或其具有1-10个氨基酸修饰的变体，例如，其具有1-5个氨基酸修饰的变体。所述氨基酸修饰是氨基酸的添加、缺失或取代。
在本发明CAR的胞内信号传导序列可以彼此按随机顺序或按指定的顺序连接。任选地，短寡肽接头或多肽接头可以在胞内信号传导序列之间形成键接。在一个实施方案中，甘氨酸-丝氨酸双联体可以用作合适的接头。在一个实施方案中，单个氨基酸，例如，丙氨酸、甘氨酸，可以用作合适的接头。
在一个实施方案中，本发明CAR的胞内信号结构域设计成包含CD28的共刺激信号结构域和CD3ζ的刺激信号结构域。又在一个实施方案中，胞内信号结构域设计成包含4-1BB的共刺激信号结构域和CD3ζ的刺激信号结构域。
在一个优选的实施方案中，本发明CAR的胞内信号结构域设计成包含CD28的共刺激 信号结构域、4-1BB的共刺激信号结构域和CD3ζ的刺激信号结构域。
本领域普通技术人员将理解，本发明的CAR多肽还可以进行修饰，从而在氨基酸序列上变动，但是在所需的活性方面不变动。例如，可以对CAR多肽进行导致“非必需”氨基酸残基处氨基酸置换的额外核苷酸置换。例如，可以将分子中的非必需氨基酸残基替换为来自相同侧链家族的另一个氨基酸残基。在另一个实施方案中，可以将一个氨基酸片段替换为结构上相似的片段，所述的片段在侧链家族成员的顺序和组成方面中不同，例如，可以进行保守性置换，其中将氨基酸残基替换为具有相似侧链的氨基酸残基。
本领域中已经定义了具有相似侧链的氨基酸残基家族，所述侧链包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电荷极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸)、β分枝侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
在一些实施方案中，本发明也构思了产生功能上等同的CAR多肽分子。
编码本发明的CAR的核酸分子、载体和表达本发明CAR的细胞
本发明提供了编码本文所述的CAR构建体的核酸分子。在一个实施方案中，核酸分子作为DNA构建体提供。
可以使用本领域公知的重组方法获得编码本发明CAR的构建体。备选地，可以合成地产生目的核酸，而非通过基因重组方法产生目的核酸。
本发明还提供了插入有本发明CAR构建体的载体。通过将编码CAR多肽的核酸有效连接至启动子并将构建体并入表达载体中，实现编码CAR的天然或合成的核酸的表达。载体可以适合在真核生物中复制和整合。常见的克隆载体含有用于调节所需核酸序列的表达的转录和翻译终止子、起始序列和启动子。
已经开发了众多基于病毒的系统用于转移基因至哺乳动物细胞中。例如，逆转录病毒提供了用于基因递送系统的便利平台。可以使用本领域已知的技术，将本发明CAR构建体插入载体并且包装在逆转录病毒粒子中。随后可以分离重组病毒并将其在体内或离体递送至受试者的细胞。众多逆转录病毒系统是本领域已知的。在一些实施方案中，使用慢病毒载体。例如，将本发明CAR构建体的核酸序列克隆至慢病毒载体中，以在单个编码框中产生全长CAR构建体，并用EF1α启动子用于表达。
衍生自逆转录病毒(如慢病毒)的载体是实现长期基因转移的合适工具，因为它们允许 转基因的长期、稳定整合和其在子代细胞中增殖。慢病毒载体具有胜过衍生自癌-逆转录病毒(如鼠白血病病毒)的载体的额外优点，因为它们可以转导非增殖性细胞，如肝细胞。它们还具有额外的低免疫原性优点。逆转录病毒载体也可以例如是γ逆转录病毒载体。γ逆转录病毒载体可以例如包含启动子、包装信号(ψ)、引物结合位点(PBS)、一个或多个(例如，两个)长末端重复序列(LTR)和目的转基因，例如，编码CAR的基因。γ逆转录病毒载体可以缺少病毒结构性基因如gag、pol和env。
能够在哺乳动物T细胞中表达CAR转基因的启动子的例子是EF1a启动子。天然EF1a启动子驱动延伸因子-1复合体的α亚基表达，所述α亚基负责酶促递送氨酰基tRNA至核糖体。已经在哺乳动物表达质粒中广泛使用了EF1a启动子并且已经显示有效驱动从克隆至慢病毒载体中的转基因表达CAR。参见，例如，Milone等人,Mol.Ther.17(8):1453–1464(2009)。
启动子的另一个例子是立即早期巨细胞病毒(CMV)启动子序列。这个启动子序列是能够驱动与之有效连接的任何多核苷酸序列高水平表达的组成型强启动子序列。但是，也可以使用其他组成型启动子序列，所述其他组成型启动子序列包括但不限于猴病毒40(SV40)早期启动子、小鼠乳腺瘤病毒(MMTV)、人类免疫缺陷病毒(HIV)长末端重复序列(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、埃巴病毒立即早期启动子、劳斯肉瘤病毒启动子以及人基因启动子，如但不限于肌动蛋白启动子、肌球蛋白启动子、延伸因子-1α启动子、血红蛋白启动子和肌酸激酶启动子。另外，本发明不应当限于使用组成型启动子。还构思了诱导型启动子作为本发明的部分。
在一些实施方案中，本发明提供了在哺乳动物免疫效应细胞(例如哺乳动物T细胞或哺乳动物NK细胞)中表达本发明的CAR构建体的方法和由此产生的免疫效应细胞。
从受试者获得细胞来源(例如，免疫效应细胞，例如，T细胞或NK细胞)。术语“受试者”意在包括可以激发免疫应答的活生物(例如，哺乳动物)。可以从众多来源获得T细胞，包括外周血单个核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织和肿瘤。
可以使用本领域技术人员已知的任何技术(如Ficoll TM分离法)，从采集自受试者的血液成分中获得T细胞。在一个优选的方面，通过单采血液成分术获得来自个体循环血液的细胞。单采产物一般含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核的白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采血液成分术采集的细胞，以除去血浆级分并且以在用于后续加工步骤的适宜缓冲液或培养基中放置细胞。在本发明的一个方面， 用磷酸盐缓冲盐水(PBS)洗涤细胞。
可以通过正向或负向选择技术进一步分离特定的T细胞亚群，如CD3+、CD28+、CD4+、CD8+、CD45RA+和CD45RO+T细胞。例如，在一个实施方案中，通过与抗CD3/抗CD28缀合的珠(如
Figure PCTCN2022071078-appb-000002
M-450CD3/CD28T)温育一段足够正向选择所需T细胞的时间，分离T细胞。在一些实施方案中，该时间段是约30分钟至36小时之间或更长时间。较长的温育时间可以用来在存在少量T细胞的任何情况下分离T细胞，如用于从肿瘤组织或从免疫受损个体分离肿瘤浸润型淋巴细胞(TIL)。另外，使用较长的温育时间可以增加捕获CD8+T细胞的效率。因此，通过简单地缩短或延长该时间，允许T细胞与CD3/CD28珠结合和/或通过增加或减少珠对T细胞的比率，可以在培养伊始或在培养过程期间的其他时间点偏好地选择T细胞亚群。
可以用抗体的组合，通过负选择过程完成T细胞群体的富集，其中所述抗体针对负向选择的细胞独有的表面标志物。一种方法是借助负向磁力免疫粘附法或流式细胞术分选和/或选择细胞，所述负向磁力免疫粘附法或流式细胞术使用针对负向选择的细胞上存在的细胞表面标志物的单克隆抗体混合物。
在一些实施方案中，免疫效应细胞可以是同种异体免疫效应细胞，例如，T细胞或NK细胞。例如，细胞可以是同种异体T细胞，例如，缺少功能性T细胞受体(TCR)和/或人白细胞抗原(HLA)(例如，HLA I类和/或HLA II类)表达的同种异体T细胞。
缺少功能性TCR的T细胞可以例如经工程化，从而它在其表面上不表达任何功能性TCR；经工程化，从而它不表达构成功能性TCR的一个或多个亚基(例如，经工程化，从而它不表达或显示出减少表达的TCRα、TCRβ、TCRγ、TCRδ、TCRε和/或TCRζ)；或经工程化，从而它在其表面上产生非常少的功能性TCR。
本文所述的T细胞例如可以如此工程化，从而它不在其表面上表达功能性HLA。例如，本文所述的T细胞可以如此工程化，从而HLA(例如，HLA I类和/或HLA II类)的细胞表面表达下调。在一些方面，可以通过减少或消除β-2微球蛋白(B2M)表达实现HLA的下调。
在一些实施方案中，T细胞可以缺少功能性TCR和功能性HLA，例如，HLA I类和/或HLA II类。
在一个实施方案中，对编码本发明所述的CAR的核酸转导的细胞进行增殖，例如，将细胞在培养下增殖2小时至约14天。
对经体外增殖后获得的表达CAR的免疫效应细胞可以如实施例中所述进行效应功能 的检测。
在一些实施方案中，表达本发明的针对IL13Rα2的具有CD28-TMD的CAR多肽可显著地提高其转导的CAR-T细胞增殖、迁移、促进蛋白激酶B活化和蛋白激酶C信号的表达，提示CD28-TMD诱导的异构化使T细胞更容易被激活。同时，在T细胞活化过程中，认为T细胞共刺激分子CD28能够提供有助于CAR-T细胞存活的信号。另外，所转染的CAR上具有的CD28-TMD与T细胞上天然存在的CD28分子能够形成异源二聚体，这也可能与增强的CAR-T细胞存活和扩增有关。
在一些实施方案中，即使肿瘤细胞上的IL13Rα2肿瘤抗原密度非常低，本发明的CAR-T细胞也能维持对表达IL13Rα2的肿瘤的杀伤活性。
在一些实施方案中，用本发明的IL13-CD428BBζ或IL13-CD828BBζCAR转导的T细胞与U373细胞共培养后，细胞内IFN-γ水平显著升高，而与U251细胞共培养后，细胞内IFN-γ水平没有升高。这可能是由于U251细胞IL13Rα2水平相对较低所致。因此，包含CD4或CD8跨膜区的CAR-T细胞可能需要肿瘤细胞上的IL13Rα2水平相对较高才能发挥活性，在本文中也称为需要更高的激活阈值。另一方面，IL13-CD28BBζCAR-T细胞在与GBM细胞共培养前表达相似的IFN-γ，在体外表现出与IL13-CD428BBζ或IL13-CD828BBζCAR-T细胞相似的抗肿瘤活性。IL13-CD28BBζCAR-T细胞在体内显示出极佳的抗肿瘤活性，这可能是由于包含CD28跨膜结构域的CAR诱导了更稳定有效的免疫相互作用，降低了激活阈值。
在一个实施方案中，通过细胞粘附检测方法检测了本发明的CAR-T细胞对肿瘤细胞的识别能力。本发明的多种CAR-T细胞与U373细胞的相互作用相似，但与U251细胞的相互作用存在不同，表明CAR中包含的跨膜结构域(TMD)改变了CAR-T细胞与肿瘤细胞的结合能力，这在与表达较少IL13受体IL13Rα2的肿瘤细胞共培养时表现出来。
在一些实施方案中，将本发明的CAR-T细胞与肿瘤细胞共培养后，用Dynabeads TM CD4阳性分离试剂盒和Dynabeads TM CD8阳性分离试剂盒(Invitrogen，Thermo Fisher Science)将CAR-T细胞分为CD4+T细胞和CD8+T细胞，然后进行高通量测序，检测到本发明的CAR-T细胞的差异表达基因。RNA提取、cDNA文库构建和测序均严格按照转录组测序标准进行。使用在线生物信息学工具DAVID bioinformatics Resources 6.8对本发明的CAR-T细胞差异表达的基因进行基因本体(GO)分析。数据可视化和分析由定制的R studio脚本按照包(ggplot2和Tree map)进行处理。基因富集分析采用Fisher精确检验。GO分析结果表明，IL13-CD28BB ζCAR-T细胞中的大多数差异表达基因定位于细胞外区域，在功能上与细胞外基质的组装和解离、粘着斑组装有关。本发明的CAR-T细胞增殖、迁移、蛋白激酶B活性的激活、蛋白激酶C(PKC)信号转导和转录激活活性的增强可能诱导CAR-T细胞与肿瘤细胞发生较强的相互作用，从而有助于提高体内抗肿瘤的疗效。同时，本发明的CAR-T细胞中的趋化因子如CXCL5、细胞骨架基因如ACTN2的上调以及抑制性细胞因子如IL-10的减少可能促进CAR-T细胞在体内更容易移向肿瘤部位。
PKC是一个在哺乳动物细胞中广泛表达的丝氨酸/苏氨酸激酶家族，已鉴定出多种亚型，主要作为细胞骨架重塑、细胞极性和T细胞迁移的关键调节因子。Real等人证明PKCζ和PKCι影响T细胞运动和对DC细胞表面趋化因子受体分子的扫描行为(Real E,Faure S,Donnadieu E,Delon J.Cutting edge:Atypical PKCs regulate T lymphocyte polarity and scanning behavior.Journal of immunology.2007；179(9):5649-5652)。在本发明中，IL13-CD28BBζCD4+CAR-T细胞中PKCζ的上调超过2.9倍。PKCζ的上调可能是下游基因过度表达的上游信号，有助于改变细胞外基质组装和细胞粘附组装。此外，肿瘤中T细胞浸润的增加也可能是PKC信号激活的结果。
丝氨酸/苏氨酸激酶蛋白激酶Bα(PKB/Akt)是PI3K活性的下游靶点，在多种模型中作为一个关键分子参与调节细胞存活。活化的PKB已被证实在生长因子和细胞因子介导的生存中发挥作用，并保护细胞免受各种刺激诱导的凋亡。在本发明中，IL13-CD28BBζCD8+CAR-T细胞中FGF1、ANG和PINK1显著上调，这些基因在功能上与PKB的激活有关。
在一些实施方案中，本发明的含有CD28跨膜结构域的第三代IL13BBζCAR-T细胞由于其较低的活性阈值和改变的转录组而具有极佳的体内抗GBM活性。
表达本发明CAR多肽的免疫效应细胞的用途和使用表达本发明CAR多肽的免疫效应细胞的治疗方法
T细胞治疗首次应用于血液学B细胞恶性肿瘤的治疗，并显示出有效和令人鼓舞的结果。然而，CAR-T细胞治疗实体瘤的抗肿瘤活性有限，尚处于实验阶段。同时，在实体瘤中副作用的发生和低效性也逐渐成为一个挑战。本发明通过优化CAR构建体的结构，获得了表达本发明CAR多肽的免疫效应细胞，其用于在受试者中治疗与表达IL13Rα2相关的疾病。
在一些实施方案中，工程化改造T细胞(例如患者特异的自体T细胞)以表达本发明的CAR多肽。通过扩增所述工程化改造的T细胞后，将其用于过继细胞治疗(ACT)。可以使用多种T细胞亚群表达本发明的CAR多肽。
在一些实施方案中，在利用表达本发明的CAR多肽的免疫效应细胞治疗患者时，所述免疫效应细胞可以是自体T细胞或同种异体T细胞。在一些实施方案中，所用的T细胞是CD4+和CD8+中央记忆型T细胞(T CM)，其是CD45RO+CD62L+，并且使用此类细胞相比于使用其他类型的患者特异性T细胞可以提高过继性转移后细胞的长期存活。
在一些实施方案中，工程化改造其他免疫细胞(例如，NK细胞)以表达本发明的CAR多肽。通过扩增所述工程化改造的其他免疫细胞(例如，NK细胞)后，将其用于过继细胞治疗(ACT)。
在一个实施方案中，表达本发明CAR多肽的免疫效应细胞用于在受试者中治疗表达或过表达IL13Rα2的癌症，并且能够减轻癌症的至少一种症状或指征的严重性或抑制癌细胞生长。
本发明提供了在受试者中治疗与表达IL13Rα2相关的疾病(例如，表达或过表达IL13Rα2的癌症)的方法，其包括向有需要的个体施用治疗有效量的表达本发明CAR多肽的免疫效应细胞。
本发明提供了前述表达本发明CAR多肽的免疫效应细胞在制备用于治疗与表达IL13Rα2相关的疾病(例如，表达或过表达IL13Rα2的癌症)的药物中的用途。
表达本发明CAR多肽的免疫效应细胞也可以施用于已经用一种或多种先前疗法治疗癌症但随后复发或转移的个体。
在一些实施方案中，表达本发明CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)用于胃肠外、经皮、腔内、动脉内、静脉内、鞘内施用，或直接注入组织或肿瘤中。
表达本发明CAR多肽的免疫效应细胞可以以合适的剂量施用于受试者。剂量方案将由主治医生和临床因素决定。如医学领域中公知的，用于任何一名患者的剂量取决于许多因素，包括患者的体重、身体表面积、年龄、待施用的特定化合物、性别、施用时间和途径、一般健康状况、和待并行施用的其他药物。
在一些实施方案中，表达本发明CAR多肽的免疫效应细胞(例如，T细胞、NK细胞)以1x10 6个－1x10 12个免疫效应细胞、优选地1x10 7个－1x10 10个免疫效应细胞、例如5x10 7个、1x10 8个、5x10 8个、1x10 9个、5x10 9个免疫效应细胞的剂量以单次或多次肠胃外施用，优选为静脉内施用。
在一些实施方案中，向患有癌症的个体施用表达本发明CAR多肽的免疫效应细胞导致肿瘤的完全消失。在一些实施方案中，向患有癌症的个体施用表达本发明CAR多肽的免疫 效应细胞导致肿瘤细胞或肿瘤大小减少至少85％或更多。可以通过本领域已知的任何方法测量肿瘤的减少，例如X-线、正电子发射断层扫描(PET)、计算机断层扫描(CT)、磁共振成像(MRI)、细胞学、组织学或分子遗传分析。
本发明所述的各个实施方案/技术方案以及各个实施方案/技术方案中的特征应当被理解为可以任意进行相互组合，这些相互组合得到的各个方案均包括在本发明的范围内，就如同在本文中具体地且逐一地列出了这些相互组合而得到的方案一样，除非上下文清楚地显示并非如此。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成对本发明的保护范围的限制。
实施例
一般方法：
除非另外说明，本发明的实施将利用本领域技术人员已知并可获得的细胞生物学、细胞培养、分子生物学(包括重组技术)、微生物学、生物化学、动物学、病毒学和免疫学的常规技术。这类技术在如下文献中描述：Molecular Cloning:A laboratory Manual,第3版(Sambrook等人,2001)Cold Spring Harbor Press；Oligonucleotide Synthesis(P.Herdewijn编著,2004)；Animal Cell Culture(R.I.Freshney编著,1987)；Methods in Enzymology(Academic Press,Inc.)；Current Protocols in Molecular Biology(F.M.Ausubel等人编著,1987)；PCR:The Polymerase Chain Reaction(Mullis等人编著,1994)；Current Protocols in Immunology(J.E.Coligan等人编著,1991)；和Short Protols in Molecular Biology(Wiley and Sons,1999)。除非另外规定，本文中所用的全部术语及科学术语具有与本发明所属领域的技术人员通常理解的相同含义。
细胞系
人GBM细胞系U251、U373及逆转录病毒包装细胞系PG13和Phoenix ECO购自美国组织培养收藏中心(ATCC)。U251细胞和U373细胞通过逆转录病毒转导表达ffLuc报告基因和GFP基因。GBM细胞系在含有10％胎牛血清(FBS，Biosera)、100U/mL青霉素和100μg/mL链霉素(EallBio Life Sciences)的DMEM培养基(Lonza)中良好培养。逆转录病毒产生细胞系在不含青霉素和链霉素的含10％FBS的DMEM培养基中培养。
流式细胞术分析
使用BD FacsCanto II Plus仪器(BD Biosciences)进行流式细胞术检测，利用FlowJo v.10软 件(Tree star，Inc.Ashland，OR)对流式结果进行分析。所用抗体具体如下：anti-human CD3-APC-R700抗体(BD Bioscience)，anti-human-CD4-V450(BD Bioscience)，anti-human-CD8-PE-Cy7(BD Bioscience)，anti-human IL13Ra-APC(BD Bioscience)和羊抗鼠IgG-APC(Sigma)。
脱颗粒实验
CAR-T细胞与靶细胞按E:T＝10:1进行共培养，加入适量的anti-human CD107a-APC(BD-Biosciences)和封闭剂(eBioscience TM Protein Transport Inhibitor Cocktail,Invitrogen)，共培养6h后，收集细胞，制备单细胞悬液，加入anti-human-CD3-APC-R700抗体，避光孵育，用PBS洗涤细胞，然后用含有1％FBS的PBS重悬细胞，用流式细胞仪检测T细胞的脱颗粒情况。
胞内IFN-γ实验
CAR-T细胞与靶细胞按E:T＝10:1进行共培养，加入2μl 1×封闭剂(eBioscience TM Protein Transport Inhibitor Cocktail,Invitrogen)，共培养6H后，收集细胞，制备单细胞悬液，加anti-human-CD3-APC-R700抗体，避光孵育，用PBS洗涤细胞，然后用含有1％FBS的PBS重悬细胞，用流式细胞仪检测胞内IFN-γ的释放情况。
增殖实验
先用CSFE染料标记CAR-T细胞，然后再与靶细胞U251按E:T＝5:1进行共培养，5天后，收集细胞，加适量anti-human CD3-APC-R700抗体孵育，PBS洗去多余的抗体，加含有1％FBS的PBS重悬细胞，用流式细胞仪检测T细胞增殖。并使用Flow Jo v.10软件(Tree star，Inc.Ashland，OR)分析细胞增殖情况。
功能检测
CAR-T细胞与靶细胞(U251-GL和U373-GL细胞系)按不同的效靶比(E:T＝10:1；5:1；2.5:1；1:1)共培养24H后，采用Perkinelmer光化学成像系统采集信号，加入底物D-荧光素钾盐工作液后，混匀拍照，Living Imaging分析系统分析荧光信号的强度，评估CAR-T细胞对靶细胞的杀伤能力。
细胞因子产生的测定
CAR-T细胞与人GBM细胞系以E:T＝10:1的比例共培养24小时。使用商业化的流式细胞仪珠阵列(CBA)试剂盒(BD Biosciences)检测来自共培养细胞的上清液中人干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)、IL17A、IL4、IL6和IL10的表达情况，具体操作步骤 根据试剂盒的说明书进行。
实时细胞分析(RTCA)
使用xCELLigence RTCA系统(瑞士巴塞尔罗氏应用科学公司)评估CAR T细胞的增殖/细胞毒性。本系统基于一个电阻抗读数的金板传感器电极，位于细胞毒性板(E-16板)的底部。首先，将靶细胞U251-GL和U373-GL细胞按每孔1×10 4个细胞接种于E-16板中。24H后，在E-16平板上加入1×10 5个CAR-T细胞，与人GBM细胞共同孵育，每15min监测一次，得到48小时的细胞指数。每一个独立的实验分三次进行。利用RTCA软件自动计算区间斜率，评价细胞指数的变化率。为了证明处理的效果，在标准化时间点将细胞指数标准化为相等的值。
转录组测序
CAR-T细胞与U251细胞以10:1的效靶比共培养。4小时后用Dynabeads TM CD4阳性分离试剂盒和Dynabeads TM CD8阳性分离试剂盒(Invitrogen，Thermo Fisher Science)分离CD4和CD8CAR T细胞。将共培养的混合细胞、分选的CD4-CAR-T细胞、CD8-CAR-T细胞和U251细胞送至安诺优达基因科技有限公司(中国北京)进行RNA测序。RNA提取、cDNA文库构建和测序均严格按照转录组测序标准进行。
使用在线生物信息学工具DAVID bioinformatics Resources 6.8对各种CAR-T细胞之间差异表达的基因进行基因本体(GO)分析。数据可视化和分析由定制的R studio脚本按照包(ggplot2和Tree map)进行处理。基因富集分析采用Fisher精确检验。
统计分析
使用GraphPad Prism 7软件(GraphPad软件，加利福尼亚州圣地亚哥)进行数据分析。数据以平均值±SEM表示，采用非配对t检验来评估差异。采用Kaplan-Meier法测定GBM异种移植小鼠的总存活率，并采用Cox比例风险回归分析进行组间比较。P<0.05为有统计学意义。
实施例1.IL13Rα2特异性CAR逆转录病毒载体的构建和逆转录病毒上清液的制备使用逆转录病毒构建了图1所示的三种不同的IL13Rα2靶向的CAR。
由图1可见，IL13CD428BBζCAR从N端至C端包含：一个位于细胞膜表面的E13Y位点突变的人IL13结构域(SEQ ID NO:2)、一个CD4跨膜结构域(SEQ ID NO:3)、一个人CD28和4-1BB共刺激结构域(SEQ ID NO:6和SEQ ID NO:7)、一个人CD3ζ胞浆结构域(SEQ ID NO:8)；
IL13CD828BBζCAR从N端至C端包含：一个位于细胞膜表面的E13Y位点突变的人IL13 结构域(SEQ ID NO:2)、一个CD8跨膜结构域(SEQ ID NO:4)和一个IgG4铰链结构域(SEQ ID NO:1)、一个人CD28和4-1BB共刺激结构域(SEQ ID NO:6和SEQ ID NO:7)、一个人CD3ζ胞浆结构域(SEQ ID NO:8)；
IL13CD28BBζCAR从N端至C端包含：一个位于细胞膜表面的E13Y位点突变的人IL13结构域(SEQ ID NO:2)、一个CD28跨膜结构域(SEQ ID NO:5)、一个人CD28和4-1BB共刺激结构域(SEQ ID NO:6和SEQ ID NO:7)、一个人CD3ζ胞浆结构域(SEQ ID NO:8)。
这三个CAR在结构上均共有一个位于细胞膜表面的E13Y位点突变的人IL13结构域、一个人CD28和4-1BB共刺激结构域和一个人CD3ζ胞浆结构域(参见图1)。这三个CAR均通过单一位点(E13Y)修饰的膜栓系IL-13作为配体来识别IL13Rα2，并具有CD28和4-1BB的细胞内信号结构域作为共刺激序列，以促进CAR-T细胞的持久性和防止无能，这三个CAR的胞内段还具有CD3ζ。
这三个CAR唯一的区别在于跨膜结构域的不同。IL13CD428BBζ在IL13(E13Y)序列后连接一个CD4的跨膜结构域，IL13CD828BBζ在IL13(E13Y)序列后连接一个CD8的跨膜结构域和IgG4(铰链)区域，而IL13CD28BBζ在IL13(E13Y)序列后连接一个CD28的跨膜结构域。
采用Thermo公司的GeneArt基因合成技术，合成IL13(E13Y)序列的编码序列，设计引物，通过同源重组的方式将其与上述三种不同的IL13Rα2靶向的CAR的其他结构域编码序列连接形成完整的IL13Rα2特异的CAR序列，最后再通过同源重组的方式将上述三种CAR序列分别亚克隆至转录病毒载体SFG上，获得三种目的质粒。
采用脂质体转导的方法将编码CAR逆转录病毒载体的三种目的质粒分别与RD114包装质粒共同转导293GP包装细胞系，48小时后收获逆转录病毒上清液，用于转导T细胞。
实施例2.CAR-T细胞的产生
在本实施例中，使用实施例1收获的逆转录病毒上清液转导T细胞，由此制备CAR-T细胞。
首先，用淋巴细胞分离液(GE-healthcare)通过梯度离心法分离健康捐赠者外周血中的单个核细胞(PBMC)。用抗CD3/CD28T细胞激活剂Dynabeads(Invitrogen)刺激PBMC中的T细胞。刺激48小时后，用实施例1的逆转录病毒上清液转染T细胞。逆转录病毒转染实验参照磷酸钙转染试剂盒(Sigma)的说明书进行。在第7天，使用流式细胞术检测转染的T细胞中CAR表达阳性率。在第9-14天，收集细胞，进行实验。具体的CAR-T细胞制备和评价流程详见图2。
在含有5％人AB血清(SIGMA)、100U/ml IL-2、100U/ml青霉素和100μg/ml链霉素(EallBio Life Sciences)的X-VIVO-15培养基中培养CAR-T细胞。本研究由北京世纪坛医院机构评审委员会批准，并获得所有参与者的知情同意。
结果表明，实施例1中构建的三种CAR分别被成功地导入T细胞。如图3所示，所获得的三种CAR-T细胞在体外都表现出良好的生存能力和增殖能力，其中，IL13CD428BBζCAR-T细胞增殖能力最强，IL13CD28BBζCAR-T增殖能力次之。
实施例3.CAR-T细胞在IL13Rα2+肿瘤细胞刺激下的体外增殖
将实施例2制备的三种CAR-T细胞分别与IL13Rα2+肿瘤细胞进行体外共培养，观察了CAR-T细胞的细胞生长及细胞因子释放情况。
图4显示了CAR-T细胞与U251细胞共培养时的增殖情况。结果表明，当实施例2制备的三种CAR-T细胞与低表达IL13Rα2的胶质瘤细胞U251细胞共培养时，所述CAR-T细胞均能够有效扩增，其中IL13CD28BBζCAR-T细胞具有最佳的增殖能力(图4)。
当实施例2制备的三种CAR-T细胞与高表达IL13Rα2的胶质瘤细胞U373细胞共培养6小时后，检测到能够发生胞内IFN-γ释放的IFN-γ阳性细胞比例均增加(图5)。另外，当实施例2制备的三种CAR-T细胞与U251细胞共培养6小时后，只有IL13CD28BBζCAR-T细胞IFN-γ表达显著增加(图5)。
CAR-T细胞与U251细胞、CAR-T细胞与U373细胞共培养24小时后，收集细胞上清液，采用细胞因子微球检测技术(Cytometric Bead Array(CBA))基于流式细胞检测系统进行多种细胞因子水平的定量检测。结果表明，三种CAR-T细胞与U373细胞共培养后，培养液中IFN-γ、TNF-α、IL-17和IL-4水平升高；而与U251细胞共培养后，只有IL13CD28BBζCAR-T细胞的IFN-γ、TNF-α、IL-17和IL-4水平显著升高(图6)。这些结果表明，IL13CD28BBζCAR-T细胞具有较低的活性阈值。
实施例4.CAR-T细胞对肿瘤细胞的体外杀伤
为了进一步评价CAR-T细胞对肿瘤细胞的杀伤能力，将三种CAR-T细胞分别与胶质瘤细胞U251-GL或U373-GL细胞按效应细胞与靶细胞之比(E:T)0.5:1、1:1、2.5:1、5:1和10:1共培养24小时后，加入荧光素酶底物，通过光学成像系统评估靶细胞在体外的存活情况，由此，借助测定的发光信号评价CAR-T细胞对肿瘤细胞的体外杀伤能力。设置了阴性对照(NT)组，其使用外周血单个核细胞(PBMC)作为效应细胞。
图7显示了CAR-T细胞与U373-GL和U251-GL细胞以不同的E:T比例共培养24小 时后，靶细胞在体外存活的结果。如图7所示，与阴性对照(NT)组相比，本发明的三组CAR-T细胞均显著抑制发光信号，其中IL13CD28BBζCAR-T细胞的抑制作用最强，尤其是在低E:T比值(0.5:1和1:1)时，与其他两组CAR-T细胞比较，IL13CD28BBζCAR-T细胞对胶质瘤细胞的体外抑制作用更强。
采用RTCA实时细胞分析仪(real-time cell analyzer)进行肿瘤细胞的细胞生长分析。检测板E-Plate 16的底部整合有微金电子传感器芯片，当生长在微电极表面的肿瘤细胞引起电极界面阻抗的改变时，该阻抗值的变化直接反映细胞的生长。
图8显示了RTCA法检测的肿瘤细胞的细胞生长结果，体外评价了CAR-T细胞的抗肿瘤能力。结果显示，三组CAR-T细胞均显著抑制U373-GL和U251-GL细胞生长，具有明显的抗肿瘤活性，且三组间无显著性差异(图8)。
实施例5.CAR-T细胞的小鼠体内抗肿瘤作用
首先，通过颅内注射胶质瘤细胞构建了原位异种移植小鼠模型。
胶质瘤细胞系U373和U251细胞表面表达IL13Rα2。采用流式细胞术检测了这两种胶质瘤细胞上IL13Rα2的表达水平。
结果如图9所示，IL13Ra2在U251和U373细胞系表面的表达分别为36.1％和90.9％。
使用胶质瘤细胞系U373和U251细胞构建了原位异种移植小鼠模型。具体而言，实验所用小鼠为六到八周龄的NOD-SCID小鼠，购自北京维通利华实验动物技术有限公司。向雌性NOD-SCID小鼠右脑纹状体分别注射2×10 5个U251-GL细胞或U373-GL细胞(记为第一天，也简写为D1)。在第六天(即，D6)通过尾静脉注射3×10 7个实施例2制备的CAR-T细胞(图10、图15)。实验设置了空白对照组，对于原位异种移植小鼠模型在D6不施用本发明的CAR-T细胞，而是施用PBS。
使用小动物成像仪器(IVIS，Xenogen，Alameda，CA，USA)通过检测荧光信号观察肿瘤生长情况，由此监测肿瘤的发展，当肿瘤直径达到20mm时处死小鼠。动物实验经北京世纪坛医院伦理委员会批准通过。
i)对于U373-GL生成的异种移植模型小鼠
图10显示了使用胶质瘤细胞系U373(本文中也称为U373-GL；或U373)产生异种移植模型小鼠、以及注射本发明的三种CAR-T细胞和检测的方案。
如图11和图12所示，注射本发明的三种CAR-T细胞均能够抑制U373-GL模型小鼠的肿瘤生长，其中，IL13-CD28BBζCAR-T细胞对U373-GL模型小鼠的控制效果更好。图 13显示，IL13-CD28BBζCAR-T细胞使得U373-GL模型小鼠生存曲线显著延长，达90天以上。
免疫组化结果显示，本发明的三种CAR-T细胞均可以浸润到肿瘤组织中，而对照组则无T细胞浸润到肿瘤组织中(图14)。
ii)对于U251-GL生成的异种移植模型小鼠
图15显示了使用胶质瘤细胞系U251(本文中也称为U251-GL；或U251)产生异种移植模型小鼠、以及注射本发明的三种CAR-T细胞和检测的方案。
如图16和图17所示，注射本发明的三种CAR-T细胞均能够抑制U251-GL模型小鼠的肿瘤生长，其中，IL13-CD28BBζCAR-T细胞对U251-GL模型小鼠的控制效果更好。图18显示，IL13-CD28BBζCAR-T细胞使得U251-GL模型小鼠生存曲线显著延长，小鼠的存活率最高。
免疫组化结果显示，本发明的三种CAR-T细胞均可以浸润到脑组织中，并有效的杀伤U251细胞，而对照组则无T细胞浸润到肿瘤组织中(图19)。
实施例6.高通量RNA测序分析CAR-T细胞的差异表达基因
分别将本发明的三种CAR-T细胞与U251细胞共培养4h后，将CAR-T细胞分为CD4+T细胞和CD8+T细胞，然后进行高通量测序，检测三种CAR-T细胞之间的差异表达基因。
图20-图22显示了三种CAR-T细胞的差异表达基因比较；图23显示了差异表达基因的GO分析结果。如图20-图22所示，与其他两种本发明的CAR转染的CD4+T细胞比较，在IL13-CD28BBζCAR转染的CD4+T细胞中有413个上调基因；与其他两种本发明的CAR转染的CD8+T细胞比较，在IL13-CD28BBζCAR转染的CD8+T细胞中有509个上调基因。
GO分析结果表明，IL13-CD28BBζCAR-T细胞中的大多数上调基因定位于细胞外区域，在功能上与细胞外基质的组装和解离有关(图23)。
经过蛋白质-蛋白质相互作用(PPI)分析，观察到CYBB、IL-10、CXCL5、TCF7和ACTN2位于差异表达基因的网络中心。图24显示了蛋白质-蛋白质相互作用网络图。
为了验证RNA-seq结果，收集了与U251或U373细胞共培养4小时后的CAR-T细胞，然后用RT-qPCR检测了候选基因CYBB、IL-10、CXCL5、TCF7和ACTN2在CAR-T细胞中的表达情况。方法如下：使用TRIzol试剂(Invitrogen)按照制造商的说明从细胞中提取总RNA。使用Nanodrop One分光光度计(Thermo Fisher Scientific)测量RNA的数量和纯度。使用高容量cDNA反转录试剂盒(Thermo Fisher Scientific)合成cDNA，然后使用SYBR Green  PCR Master Mix(Thermo Fisher Scientific)和基因特异引物进行扩增。GAPDH被用来作为内部对照。使用2-ΔΔCt方法计算基因的相对表达。
图25显示了RT-qPCR验证的差异表达基因。结果显示，IL13-CD28BBζCAR-T细胞具有最低的IL10水平和最高的Tcf7表达(图25)。所述Tcf7编码TCF1蛋白，该蛋白在记忆性T细胞中高表达。
本发明的CAR-T细胞在与U251和U373细胞共培养后，发现记忆性T细胞在IL13-CD28BBζCAR-T细胞与U251细胞共培养中所占比例较高，IL13-CD28BBζCAR-T细胞与U373细胞共培养7d后记忆性T细胞减少，而效应T细胞的比例正好相反(图26)。
实施例7.CAR-T细胞在体外识别肿瘤细胞的能力
在本实施例中，通过使用细胞结合试验来检测CAR-T细胞在体外识别肿瘤细胞的能力(图27)。
如图27所示，首先将GBM细胞接种于24孔板上；待其贴壁后，对T细胞进行CSFE染色；后将T细胞与肿瘤细胞共培养，振荡5min后进行拍照(Pic1)；洗涤3次，将未粘附在24孔板的细胞清除后，进行第二次拍照(Pic2)。
图28显示了细胞粘附实验荧光成像结果，及粘附细胞数的统计。如图28所示，更多的IL13-CD28BBζCAR-T细胞在孵育5min后与U251细胞结合，但这三组CAR-T细胞与U373细胞结合的数量相似。
图29显示了在IL13-CD28BBζCAR-T细胞上，CAR上的CD28跨膜结构域与T细胞上天然存在的CD28分子形成异源二聚体，由此可能增强CAR-T细胞的存活和扩增。
以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。

Claims (19)

  1. 靶向IL13Rα2的嵌合抗原受体多肽，其特征在于，包含
    (i)人IL-13或其具有1-2个氨基酸修饰的变体，
    (ii)跨膜结构域，其选自：VWWWLVESWLVIACYYQWPLLFSG的SEQ ID NO:5所示CD28跨膜结构域或其具有1-2个氨基酸修饰的变体；
    (iii)共刺激结构域，其选自：(a)CD28共刺激结构域或其具有1-5个氨基酸修饰的变体、(b)4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体、(c)OX40共刺激结构域或其具有1-5个氨基酸修饰的变体、和所述(a)、(b)和/或(c)的任意组合；和
    (iv)CD3ζ信号传导结构域或其具有1-5个氨基酸修饰的变体。
  2. 根据权利要求1所述的嵌合抗原受体多肽，其中，所述(i)是人IL-13的位置13上的氨基酸是Y，也称为IL-13 E13Y。
  3. 根据权利要求2所述的嵌合抗原受体多肽，其中，所述(i)是
    SPGPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN的SEQ ID NO:2。
  4. 根据权利要求1所述的嵌合抗原受体多肽，其中，所述(iii)是两个不同的共刺激结构域的组合。
  5. 根据权利要求4所述的嵌合抗原受体多肽，其中，所述(iii)是(a)CD28共刺激结构域或其具有1-5个氨基酸修饰的变体和(b)4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体的组合。
  6. 根据权利要求5所述的嵌合抗原受体多肽，其中，所述CD28共刺激结构域是SKRSRLLMTPRRPGPTRKHYQPYAPPRDFAAYRS的SEQ ID NO:6所示序列，所述4-1BB共刺激结构域是AERNSCIYSNNHLCDQYKLLKRKMAVAADFQKKKKEDV的SEQ ID NO:7所示序列。
  7. 根据权利要求1所述的嵌合抗原受体多肽，其中，所述(iv)是RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAKDTYDALHMQALPPRYQPLE的SEQ ID NO:8所示CD3ζ信号传导结构域。
  8. 根据权利要求1所述的嵌合抗原受体多肽，其中，所述嵌合抗原受体多肽包含：
    (i)人IL-13或IL-13 E13Y；
    (ii)跨膜结构域，其是SEQ ID NO:5所示的CD28跨膜结构域或其具有1-2个氨基酸修饰的变体；
    (iii)共刺激结构域，其是(a)CD28共刺激结构域或其具有1-2个氨基酸修饰的变体和(b)4-1BB共刺激结构域或其具有1-2个氨基酸修饰的变体的组合；和
    (iv)SEQ ID NO:8所示的CD3ζ信号传导结构域或其具有1-2个氨基酸修饰的变体。
  9. 根据权利要求8所述的嵌合抗原受体多肽，其中，所述嵌合抗原受体多肽包含：
    (i)IL-13 E13Y；
    (ii)跨膜结构域，其是SEQ ID NO:5所示的CD28跨膜结构域；
    (iii)共刺激结构域，其是SEQ ID NO:6所示的CD28共刺激结构域和SEQ ID NO:7所示的4-1BB共刺激结构域的组合；和
    (iv)SEQ ID NO:8所示的CD3ζ信号传导结构域或其具有1-2个氨基酸修饰的变体。
  10. 核酸分子，其特征在于，编码权利要求1-9中任一项所述的嵌合抗原受体多肽。
  11. 载体，其特征在于，包含权利要求10所述的核酸分子。
  12. 细胞，其特征在于，包含权利要求1-9中任一项所述的嵌合抗原受体多肽、权利要求10所述的核酸分子、或权利要求11所述的载体。
  13. 根据权利要求12所述的细胞，其中，所述细胞是免疫效应细胞。
  14. 根据权利要求13所述的细胞，其中，所述免疫效应细胞是T细胞、NK细胞。
  15. 根据权利要求14所述的细胞，其中，所述T细胞是自体T细胞或同种异体T细胞。
  16. 制备权利要求12-15中任一项所述的细胞的方法，其特征在于，包括用权利要求11所述的载体转导细胞。
  17. 权利要求12-15中任一项所述的细胞的用途，其特征在于，用于制备在受试者中预防或治疗肿瘤或提供抗肿瘤免疫的药物组合物。
  18. 根据权利要求17所述的用途，其中，所述肿瘤为胶质瘤。
  19. 根据权利要求17所述的用途，其中，所述肿瘤为胶质母细胞瘤。
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WO2021183960A1 (en) * 2020-03-12 2021-09-16 City Of Hope TARGETED CHIMERIC ANTIGEN RECEPTOR MODIFIED T CELLS FOR TREATMENT OF IL13RαA2 POSITIVE MALIGNANCIES

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Publication number Priority date Publication date Assignee Title
US20030031666A1 (en) * 1998-04-03 2003-02-13 Waldemar Debinski Antibodies that bind IL-13 mutants
CN105142677A (zh) * 2013-02-15 2015-12-09 加利福尼亚大学董事会 嵌合抗原受体及其使用方法
CN107002084A (zh) * 2014-09-19 2017-08-01 希望之城公司 靶向IL13Rα2的共刺激嵌合抗原受体T细胞
WO2021183960A1 (en) * 2020-03-12 2021-09-16 City Of Hope TARGETED CHIMERIC ANTIGEN RECEPTOR MODIFIED T CELLS FOR TREATMENT OF IL13RαA2 POSITIVE MALIGNANCIES
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