WO2024066026A1 - 靶向IL13Rα2的经优化的嵌合抗原受体及其用途 - Google Patents

靶向IL13Rα2的经优化的嵌合抗原受体及其用途 Download PDF

Info

Publication number
WO2024066026A1
WO2024066026A1 PCT/CN2022/136205 CN2022136205W WO2024066026A1 WO 2024066026 A1 WO2024066026 A1 WO 2024066026A1 CN 2022136205 W CN2022136205 W CN 2022136205W WO 2024066026 A1 WO2024066026 A1 WO 2024066026A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
sequence
variant
cells
Prior art date
Application number
PCT/CN2022/136205
Other languages
English (en)
French (fr)
Inventor
钟晓松
白玥
Original Assignee
卡瑞济(北京)生命科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 卡瑞济(北京)生命科技有限公司 filed Critical 卡瑞济(北京)生命科技有限公司
Publication of WO2024066026A1 publication Critical patent/WO2024066026A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • 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
    • C12N15/11—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
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • 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 to the field of genetic engineering technology. Specifically, the present invention relates to an optimized chimeric antigen receptor targeting IL13R ⁇ 2, an immune effector cell (e.g., T cell, NK cell) engineered to express the optimized chimeric antigen receptor of the present invention, and the use of the engineered immune effector cell for treating diseases associated with the expression of IL13R ⁇ 2.
  • an immune effector cell e.g., T cell, NK cell
  • Glioblastoma is the most malignant primary brain tumor in adults. Current standard treatment includes surgical resection, radiotherapy and chemotherapy (eg, using temozolomide), however, the five-year overall survival rate is less than 10%.
  • Immunotherapy is a very attractive treatment method that can improve the prognosis of GBM patients without the cytotoxic reactions caused by chemotherapy or radiotherapy.
  • Adoptive immunotherapy using antibodies or T cells is currently the most effective immunotherapy in clinical and experimental settings.
  • immunotherapy based on dendritic cell vaccines has shown encouraging results in the clinical treatment of relapsed and newly diagnosed GBM patients, stabilizing the disease and increasing the survival of patients, these clinical results need to be confirmed by further randomized clinical trials, and published research articles have also shown that it is difficult to induce glioma-specific T cells in patients. Therefore, there is an urgent need to study and develop new immunotherapy methods.
  • Chimeric antigen receptor T cell (CAR-T) immunotherapy uses transgenic technology to express the single-chain variable region (scFv) of monoclonal antibodies that can specifically recognize target antigens on the surface of T cells, while activating the intracellular activation and proliferation signaling domains of T cells (CD3 ⁇ chain and co-stimulatory molecules CD28/4-1BB), thereby enabling T cells to produce highly efficient and specific anti-tumor responses.
  • scFv single-chain variable region
  • CAR-T is currently the most popular cell immunotherapy.
  • Five drugs have been successfully launched in the United States (Novartis' Kymriah, Kite's Yescarta and Tecartus, BMS' Breyanzi and Abecma). Its earliest subject, Emily Whitedhead (an acute lymphoblastic leukemia patient), has been tumor-free for 8 years, which has rekindled the hope of life for countless cancer patients.
  • the high-affinity interleukin-13 receptor ⁇ 2 (IL13R ⁇ 2) is selectively expressed at high frequency by glioblastoma (GBM) as well as several other tumor types.
  • GBM glioblastoma
  • One approach to targeting this tumor-specific receptor is to utilize the corresponding ligand IL-13 conjugated to a cytotoxic molecule.
  • this approach lacks specificity because the lower affinity receptor IL13R ⁇ 1 that binds IL-13 is widely expressed by normal tissues.
  • the object of the present invention is to provide an optimized third-generation chimeric antigen receptor that specifically targets IL13R ⁇ 2 and immune effector cells (e.g., T cells, NK cells) engineered to express the chimeric antigen receptor, for use in treating diseases associated with the expression of IL13R ⁇ 2, such as glioblastoma (GBM), so as to solve the problems existing in the above-mentioned prior art.
  • IL13R ⁇ 2 e.g., T cells, NK cells
  • GBM glioblastoma
  • the present invention provides an optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2, comprising:
  • An optimized anti-IL13R ⁇ 2 scFv sequence wherein the scFv sequence specifically binds to IL13R ⁇ 2 and comprises:
  • a heavy chain complementary determining region CDR H1 represented by the amino acid sequence KYGVH (SEQ ID NO: 15), or a variant of said CDR H1 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR H2 represented by the amino acid sequence VKWAGGSTDTDSALMS (SEQ ID NO:16), or a variant of said CDR H2 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR L light chain complementary determining region 1 represented by the amino acid sequence TASLSVSSTYLH (SEQ ID NO:18), or a variant of said CDR L1 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR L3 represented by the amino acid sequence HQYHRSPLT (SEQ ID NO: 20), or a variant of said CDR L3 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • amino acid change is an addition, deletion or substitution of an amino acid
  • CD8 hinge region (SEQ ID NO 8), or a CD8 hinge region having at least 80% sequence identity thereto.
  • TM transmembrane region
  • a CD28 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO:9 or a variant thereof having 1-2 amino acid modifications;
  • a CD4 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 10 or a variant thereof having 1-2 amino acid modifications;
  • a CD8 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 11 or a variant thereof having 1-2 amino acid modifications;
  • a CD28 co-stimulatory domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO:12 or a variant thereof having 1-2 amino acid modifications;
  • a 4-1BB co-stimulatory domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 13 or a variant thereof having 1-2 amino acid modifications;
  • Stimulatory signal domain which is the CD3 ⁇ signal transduction domain or a variant thereof having 1-10 amino acid modifications, for example, the sequence shown in SEQ ID NO:14 or a variant thereof having 1-10 or 1-5 amino acid modifications.
  • the optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2 of the present invention comprises:
  • An optimized anti-IL13R ⁇ 2 scFv sequence wherein the scFv sequence specifically binds to IL13R ⁇ 2 and comprises:
  • a heavy chain variable region comprising the sequence of SEQ ID NO:2, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and
  • a light chain variable region comprising the sequence of SEQ ID NO:4, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • an IgG4 hinge region (SEQ ID NO 7), or an IgG4 hinge region having at least 90% or at least 95% sequence identity thereto;
  • CD8 hinge region (SEQ ID NO 8), or a CD8 hinge region having at least 90% or at least 95% sequence identity thereto.
  • TM transmembrane region
  • a stimulatory signaling domain which is the CD3 ⁇ signaling domain shown in SEQ ID NO: 14 or a variant thereof having one amino acid modification;
  • the amino acid modification is the addition, deletion or substitution of amino acids.
  • the optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2 of the present invention comprises:
  • An optimized anti-IL13R ⁇ 2 scFv sequence wherein the scFv sequence specifically binds to IL13R ⁇ 2 and comprises:
  • TM transmembrane region
  • the optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2 of the present invention further comprises a signal peptide sequence at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO: 21,
  • the optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2 of the present invention has the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence that has at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto.
  • the present 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 cell comprising a CAR nucleic acid molecule or vector as described herein, or a cell expressing a CAR polypeptide as described herein, preferably, the cell is an autologous T cell or an allogeneic T cell.
  • CAR polypeptide chimeric antigen receptor
  • the present invention provides a method for producing cells, such as immune effector cells, comprising introducing (e.g., transducing) a nucleic acid molecule (e.g., an RNA molecule, such as an mRNA molecule) encoding a CAR polypeptide described herein, or a vector comprising a nucleic acid molecule encoding a CAR polypeptide described herein into an immune effector cell.
  • a nucleic acid molecule e.g., an RNA molecule, such as an mRNA molecule
  • a vector comprising a nucleic acid molecule encoding a CAR polypeptide described herein into an immune effector cell.
  • 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 prepared by isolating T cells, NK cells from human PBMCs.
  • a nucleic acid molecule encoding a CAR polypeptide described herein is introduced into primary T cells using a retrovirus to obtain a CAR-T cell of the present invention.
  • the CAR-T cells of the present invention have significant killing activity against glioma U373 cells that highly express IL13R ⁇ 2 and glioma U251 cells that lowly express IL13R ⁇ 2 in vitro, and produce cytokines such as IFN- ⁇ and TNF- ⁇ .
  • the CAR-T cells of the present invention also have the function of killing tumor cells in vivo.
  • the CAR-T cells of the present invention have improved anti-tumor activity.
  • the present invention provides the use of the immune effector cells expressing the CAR polypeptide of the present invention for preparing a medicament for preventing or treating a tumor (e.g., cancer) or providing anti-tumor immunity in a subject, preferably, the tumor is a glioma, more preferably, the tumor is a glioblastoma.
  • a tumor e.g., cancer
  • the tumor is a glioma
  • the tumor is a glioblastoma.
  • the present invention provides the use of the immune effector cells expressing the CAR polypeptide of the present invention for treating a disease associated with the expression of IL13R ⁇ 2 in a subject, comprising administering a therapeutically effective amount of immune effector cells expressing the CAR polypeptide to the subject, 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 associated with 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 associated with the expression of IL13R ⁇ 2 is glioma, preferably, glioblastoma.
  • the present invention uses the anti-IL13R ⁇ 2 scFv sequence for the first time to construct the third-generation CAR-T cells, which proves that it can treat GBM by specifically targeting IL13R ⁇ 2, thereby improving the safety and effectiveness of CAR-T cell treatment of GBM, avoiding rejection reactions or off-target phenomena, and being more conducive to the promotion and clinical application of CAR-T.
  • FIG1 is a schematic diagram of the optimized CAR structure constructed by the present invention, in which SD represents a splice donor, SA represents a splice acceptor, and LTR represents a long terminal repeat.
  • the extracellular domain comprises an optimized scFv sequence against IL13R ⁇ 2 and a short IgG4 hinge region.
  • Figure 2 shows the results of CD107a staining and detection of CD107a expression rate by flow cytometry when the optimized CAR-T cells targeting IL13R ⁇ 2 and containing the anti-IL13R ⁇ 2 scFv sequence were cultured alone.
  • Figure 3 shows the results of CD107a staining of CAR-T cells when the optimized CAR-T cells targeting IL13R ⁇ 2 and containing the anti-IL13R ⁇ 2 scFv sequence were co-cultured with the U373 cell line that highly expresses IL13R ⁇ 2, and the expression rate of CD107a was detected by flow cytometry.
  • Figure 4 shows the results of CD107a staining of CAR-T cells containing anti-IL13R ⁇ 2 scFv sequence targeting IL13R ⁇ 2 when they were co-cultured with U251 cell line with low expression of IL13R ⁇ 2, and the expression rate of CD107a was detected by flow cytometry.
  • Figure 5 shows the analysis results of the difference in CD107a expression rate after co-culture of optimized CAR-T cells targeting IL13R ⁇ 2 containing anti-IL13R ⁇ 2 scFv sequence with U373 cell line or U251 cell line.
  • Figure 6 shows the experimental results of the optimized CAR-T cells targeting IL13R ⁇ 2 and containing the anti-IL13R ⁇ 2 scFv sequence killing the target cells U373 cell line or U251 cell line.
  • Figure 7 shows the results of differential analysis of the killing of target cells U373 cell line or U251 cell line by optimized CAR-T cells targeting IL13R ⁇ 2 and containing anti-IL13R ⁇ 2 scFv sequences.
  • Figure 8 shows the content of cytokine IFN- ⁇ in the supernatant after co-culture of optimized CAR-T cells targeting IL13R ⁇ 2 containing anti-IL13R ⁇ 2 scFv sequence (abbreviated as "IL13R ⁇ 2-CAR-T” in the figure) with IL13R ⁇ 2-positive human GBM cell lines (U373, U251, U87).
  • Figure 9 shows the content of cytokine TNF- ⁇ in the supernatant after co-culture of optimized CAR-T cells targeting IL13R ⁇ 2 containing anti-IL13R ⁇ 2 scFv sequence (abbreviated as "IL13R ⁇ 2-CAR-T” in the figure) with IL13R ⁇ 2-positive human GBM cell lines (U373, U251, U87).
  • Figure 10 shows the RTCA detection results after co-culture of optimized CAR-T cells targeting IL13R ⁇ 2 containing anti-IL13R ⁇ 2 scFv sequence with U373 cell line.
  • FIG. 11 shows the RTCA detection results after co-culture of CAR-T cells targeting IL13R ⁇ 2 and containing an optimized scFv sequence against IL13R ⁇ 2 with the U251 cell line.
  • Figure 12 is the in vivo imaging results of the treatment of the U251 orthotopic animal model with optimized CAR-T cells containing anti-IL13R ⁇ 2 scFv sequences targeting IL13R ⁇ 2 (abbreviated as "IL13R ⁇ 2-CAR-T” in the figure) or CAR-T cells containing IL13 shown in SEQ ID NO: 24 targeting IL13R ⁇ 2 (abbreviated as "IL13-CAR-T” in the figure), wherein 2 ⁇ 10 5 U251 cells were injected into the right brain striatum of female NOD-SCID mice on day 1 (also abbreviated as D1). 3 ⁇ 10 7 CAR-T cells were injected through the tail vein on day 6 (D6).
  • IL13R ⁇ 2-CAR-T CAR-T cells containing IL13 shown in SEQ ID NO: 24 targeting IL13R ⁇ 2
  • IL13-CAR-T 2 ⁇ 10 5 U251 cells were injected into the right brain striatum of female NOD-SC
  • FIG. 13 shows the survival period of the U251 orthotopic animal model treated with optimized CAR-T cells containing anti-IL13R ⁇ 2 scFv sequence targeting IL13R ⁇ 2 (abbreviated as "IL13R ⁇ 2-CAR-T” in the figure) or CAR-T cells containing IL13 shown in SEQ ID NO:24 targeting IL13R ⁇ 2 (abbreviated as "IL13-CAR-T” in the figure).
  • the experiment is the same as FIG. 12.
  • Figure 14 is the in vivo imaging results of the treatment of U373 orthotopic animal model with optimized CAR-T cells containing anti-IL13R ⁇ 2 scFv sequences targeting IL13R ⁇ 2 (abbreviated as "IL13R ⁇ 2-CAR-T” in the figure) or CAR-T cells containing IL13 shown in SEQ ID NO: 24 targeting IL13R ⁇ 2 (abbreviated as "IL13-CAR-T” in the figure), wherein 2 ⁇ 10 5 U373 cells were injected into the right striatum of female NOD-SCID mice on day 1 (also abbreviated as D1). 3 ⁇ 10 7 CAR-T cells were injected through the tail vein on day 6 (D6).
  • IL13R ⁇ 2-CAR-T CAR-T cells containing IL13 shown in SEQ ID NO: 24 targeting IL13R ⁇ 2
  • IL13-CAR-T 2 ⁇ 10 5 U373 cells were injected into the right striatum of female NOD-SCID mice on
  • FIG. 15 shows the survival period of the U373 orthotopic animal model treated with optimized CAR-T cells containing anti-IL13R ⁇ 2 scFv sequence targeting IL13R ⁇ 2 (abbreviated as "IL13R ⁇ 2-CAR-T” in the figure) or CAR-T cells containing IL13 shown in SEQ ID NO:24 targeting IL13R ⁇ 2 (abbreviated as "IL13-CAR-T” in the figure).
  • the experiment is the same as FIG. 14.
  • chimeric receptor refers to a recombinant polypeptide comprising at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
  • anti-IL13R ⁇ 2 antibody As used herein, the terms “anti-IL13R ⁇ 2 antibody”, “antibody against IL13R ⁇ 2”, “antibody that specifically binds to IL13R ⁇ 2”, “antibody that specifically targets IL13R ⁇ 2”, “antibody that specifically recognizes IL13R ⁇ 2” are used interchangeably and refer to antibodies that can specifically bind to IL13R ⁇ 2. In particular, in some specific embodiments, it refers to an antibody that specifically binds to human IL13R ⁇ 2, in particular, an antibody that specifically binds to human IL13R ⁇ 2 but does not specifically bind to human IL13R ⁇ 1.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are optionally connected continuously by means of a flexible short polypeptide linker and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived.
  • scFv can have a VL variable region and a VH variable region in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv can comprise VL-linker-VH or can comprise VH-linker-VL.
  • Each heavy chain variable region (VH) and light chain variable region (VL) are respectively composed of four conserved framework regions (FR) and three complementary determining regions (CDR), arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the scFv sequence for IL13R ⁇ 2 contained in the CAR of the present invention is an optimized sequence, for example, a sequence in which the framework region is optimized.
  • the amino acid sequence of the framework region is optimized, thereby retaining the improved binding affinity of the optimized scFv sequence for IL13R ⁇ 2 to IL13R ⁇ 2.
  • CDR region or “CDR” or “hypervariable region” is a region of an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and/or contains antigen contact residues ("antigen contact points").
  • the CDRs are primarily responsible for binding to antigen epitopes.
  • the CDRs of the heavy and light chains are usually referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus.
  • the CDRs located within the antibody heavy chain variable domain are referred to as CDR H1, CDR H2 and CDR H3, while the CDRs located within the antibody light chain variable domain are referred to as CDR L1, CDR L2 and CDR L3.
  • each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al.
  • CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the CDRs exemplified in the present invention).
  • a reference CDR sequence e.g., any of the CDRs exemplified in the present invention.
  • the present invention when referring to antibody variable regions and specific CDR sequences (including heavy chain variable region residues), it refers to the numbering position according to the Kabat numbering system.
  • CDR is different from antibody to antibody, only a limited number of amino acid positions in CDR are directly involved in antigen binding.
  • the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding.
  • the minimum binding unit can be a sub-portion of the CDR.
  • the residues of the rest of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR given herein.
  • the amino acid residues of the minimum binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia or AbM can be replaced by conservative amino acid residues.
  • variable region refers to the domain of an antibody heavy chain or light chain that is involved in binding of the antibody to an antigen.
  • the variable domains of the heavy and light chains of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementary determining regions
  • binding means that the binding effect is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm interferometry or other conventional binding assays known in the art.
  • the term "stimulatory molecule” refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway.
  • the primary signal is initiated, for example, by the binding of the TCR/CD3 complex to the MHC molecule loaded with a peptide and leads to mediating a T cell response, including but not limited to proliferation, activation, differentiation, etc.
  • the intracellular signaling domain in any one or more CARs of the present invention comprises an intracellular signaling sequence, for example, a primary signaling sequence of CD3 ⁇ .
  • CD3 ⁇ is defined as a protein provided by GenBank 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 that are sufficient to functionally propagate the initial signal necessary for T cell activation.
  • the cytoplasmic domain of CD3 ⁇ comprises residues 52 to residue 164 of GenBank Accession No. BAG36664.1 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.) that are functional homologs thereof.
  • the "CD3 ⁇ stimulatory signaling domain” is the sequence provided in SEQ ID NO:14 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 of the cell (e.g., but not limited to, proliferation).
  • 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 (i.e., CD137), CD27, and CD28.
  • costimulatory molecules are CD28, 4-1BB (i.e., CD137).
  • the costimulatory signaling domain refers to the intracellular portion of a costimulatory molecule.
  • the nucleic acid sequence encoding the CAR of the present invention can be introduced into cells by "transfection”, “conversion” or “transduction”.
  • transfection refers to the introduction of one or more exogenous polynucleotides into host cells by physical or chemical methods.
  • Many transfection techniques are known in the art, and include, for example, calcium phosphate DNA coprecipitation (see, for example, Murray E.J. (ed.), Methods in Molecular Biology, Vol.
  • signaling pathway refers to the biochemical relationships between multiple signaling molecules that play a role in propagating a signal from one part of a cell to another part of the cell.
  • cytokine is a generic term for proteins released by one cell population that act as intercellular mediators on another cell.
  • cytokines include lymphokines, monokines, interleukins (ILs), such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15; tumor necrosis factors, such as TNF- ⁇ or TNF- ⁇ ; and other polypeptide factors, including gamma-interferon.
  • ILs interleukins
  • activation or “activation” of immune cells refers to the ability of immune cells to respond and exhibit the immune function of the corresponding cells known to those skilled in the art at a measurable level. Methods for measuring immune cell activity are also known to those skilled in the art.
  • amino acid change and “amino acid modification” are used interchangeably and refer to the addition, deletion, substitution and other modifications of amino acids. Any combination of the addition, deletion, substitution and other modifications of amino acids can be performed, provided that the final polypeptide sequence has the desired characteristics.
  • the substitution of amino acids is a non-conservative amino acid substitution, i.e., replacing one amino acid with another amino acid having different structures and/or chemical properties.
  • Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine).
  • Amino acid changes can be produced using genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods for changing amino acid side chain groups by methods other than genetic engineering (such as chemical modification) may be useful.
  • 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 or its constituent elements containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the CAR of the present invention or its constituent elements by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are amino acid substitutions in which amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids 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
  • non-polar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • ⁇ -side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • one or more amino acid residues within the CAR of the present invention can be replaced with other amino acid residues from the same side chain family, and the function of the altered CAR can be tested using the functional assays described here
  • autologous refers to any substance that is derived from the same individual into which the substance is later reintroduced.
  • allogeneic refers to any material that is derived from a different animal of the same species as the individual into which the material 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 can be genetically dissimilar enough to interact antigenically.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • apheresis refers to an art-recognized in vitro method by which a donor's or patient's blood is removed from the donor or patient and passed through a device that separates selected specific components and returns the remainder to the donor's or patient's circulation, e.g., by retransfusion.
  • apheresis sample a sample obtained using apheresis is referred to.
  • immune effector cell refers to a cell that participates in an immune response, e.g., participates in promoting an immune effector reaction.
  • immune effector cells include T cells, e.g., ⁇ / ⁇ T cells and ⁇ / ⁇ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid derived phagocytes.
  • Immuno effector function refers to, for example, a function or response of an immune effector cell that enhances or promotes immune attack on a target cell.
  • an immune effector function or response refers to a T cell or NK cell property that promotes killing of target cells or inhibits 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 a T cell may be, for example, cytolytic activity or helper activity, including the secretion of cytokines.
  • T cell activation or “T cell activation” are used interchangeably and refer to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.
  • the chimeric antigen receptors of the present 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 Retroviridae family. 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 all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentiviral genome, and particularly includes self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8): 1453–1464 (2009).
  • Other examples of lentiviral vectors that can be used clinically include, but are not limited to, lentiviral vectors 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, exacerbated 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 rats).
  • 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 rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • tumor and cancer are used interchangeably herein and encompass both solid and liquid tumors.
  • Tumor immune escape refers to the process by which a tumor evades immune recognition and clearance.
  • tumor immunity is “treated” when such escape is weakened and the tumor is recognized and attacked by the immune system.
  • tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.
  • treatment refers to slowing down, interrupting, blocking, alleviating, stopping, reducing, or reversing the progress or severity of existing symptoms, illnesses, conditions, or diseases.
  • the desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis.
  • the CAR immune effector cells of the present invention are used to delay disease development or to slow down the progression of the disease.
  • the term "effective amount” refers to such an amount or dosage of the CAR immune effector cells of the present invention, which is administered to the patient in a single or multiple doses, and produces the desired effect in the patient in need of treatment or prevention.
  • the effective amount can be easily determined by the attending physician as a technician in the field by considering the following factors: such as mammalian species; weight, age and general health; the specific disease involved; the extent or severity of the disease; the response of individual patients; the specific CAR immune effector cells administered; the mode of administration; the bioavailability characteristics of the administered preparation; the selected dosage regimen; and the use of any concomitant therapy.
  • “Therapeutically effective amount” refers to the amount of the desired therapeutic result effectively achieved at the required dosage and for the required time period.
  • the therapeutically effective amount of CAR immune effector cells can be changed according to various factors such as disease state, individual age, sex and weight and the ability of CAR immune effector cells to stimulate the desired response in individuals.
  • the therapeutically effective amount is also such an amount, in which any toxic or harmful effects of CAR immune effector cells are less than the therapeutic beneficial effects.
  • "therapeutically effective amount” preferably suppresses measurable parameters (such as tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60% or 70% and still more preferably at least about 80% or 90%.
  • the ability of CAR immune effector cells to suppress measurable parameters e.g., cancer
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively attached. 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, such as from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject.
  • Tissue samples may contain compounds that are naturally not mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.
  • tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, 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 tumor samples.
  • IL-13 receptor ⁇ chain isoform 2 (IL13R ⁇ 2) is a receptor polypeptide for IL-13. It is almost exclusively expressed in cancer cells and not in normal tissue cells (except testes). It is highly expressed on the surface of glioma cells and plays an important role in the malignant proliferation of gliomas.
  • IL13R ⁇ 2 IL-13 receptor ⁇ chain isoform 2
  • GBM patients more than 50% of patients express IL13R ⁇ 2, making it an effective target for CAR-T therapy of GBM (Sharma P, Debinski W. Receptor-Targeted Glial Brain Tumor Therapies. International journal of molecular sciences. 2018; 19(11)).
  • the present invention relates to an optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2.
  • a chimeric antigen receptor (CAR) is a recombinant polypeptide comprising at least an extracellular recognition domain, a transmembrane region, and an intracellular signaling domain.
  • the extracellular recognition domain (also referred to as "extracellular domain") of the CAR polypeptide of the present invention is an optimized anti-IL13R ⁇ 2 scFv sequence that specifically recognizes and binds to IL13R ⁇ 2 on the surface of target cells.
  • CAR can both bind to antigens and transduce 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 of lymphocytes using CAR can be a powerful therapeutic strategy for treating cancer.
  • the optimized chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2 of the present invention comprises:
  • An optimized anti-IL13R ⁇ 2 scFv sequence wherein the scFv sequence specifically binds to IL13R ⁇ 2 and comprises:
  • a heavy chain complementary determining region CDR H1 represented by the amino acid sequence KYGVH (SEQ ID NO: 15), or a variant of said CDR H1 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR H2 represented by the amino acid sequence VKWAGGSTDTDSALMS (SEQ ID NO:16), or a variant of said CDR H2 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR L light chain complementary determining region 1 represented by the amino acid sequence TASLSVSSTYLH (SEQ ID NO:18), or a variant of said CDR L1 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • CDR L3 represented by the amino acid sequence HQYHRSPLT (SEQ ID NO: 20), or a variant of said CDR L3 having no more than 2 amino acid changes or no more than 1 amino acid change;
  • amino acid change is an addition, deletion or substitution of an amino acid
  • CD8 hinge region (SEQ ID NO 8), or a CD8 hinge region having at least 80% sequence identity thereto.
  • TM transmembrane region
  • a CD28 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO:9 or a variant thereof having 1-2 amino acid modifications;
  • a CD4 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 10 or a variant thereof having 1-2 amino acid modifications;
  • a CD8 transmembrane domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 11 or a variant thereof having 1-2 amino acid modifications;
  • a CD28 co-stimulatory domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 12 or a variant thereof having 1-2 amino acid modifications;
  • a 4-1BB co-stimulatory domain or a variant thereof having 1-5 amino acid modifications for example, the sequence shown in SEQ ID NO: 13 or a variant thereof having 1-2 amino acid modifications;
  • a stimulatory signaling domain which is a CD3 ⁇ signaling domain or a variant thereof having 1-10 amino acid modifications, for example, a sequence as shown in SEQ ID NO: 14 or a variant thereof having 1-10 or 1-5 amino acid modifications;
  • amino acid modification is the addition, deletion or substitution of amino acids
  • the extracellular domain of the chimeric antigen receptor of the present invention is an optimized anti-IL13R ⁇ 2 scFv sequence that specifically binds to IL13R ⁇ 2, wherein the scFv sequence specifically binds to IL13R ⁇ 2 and comprises:
  • a heavy chain variable region comprising the sequence of SEQ ID NO:2, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and
  • a light chain variable region comprising the sequence of SEQ ID NO:4 or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the extracellular domain of CAR i.e., the optimized anti-IL13R ⁇ 2 scFv sequence
  • immune effector cells e.g., T cells, NK cells
  • the cells expressing IL13R ⁇ 2 are tumor cells (including glioma cells).
  • the transmembrane domain included in the chimeric antigen receptor of the present invention is an anchored transmembrane domain, which is a component of a polypeptide chain that can be integrated into a cell membrane.
  • the transmembrane domain can be fused with other extracellular and/or intracellular polypeptide domains, whereby these extracellular and/or intracellular polypeptide domains will also be restricted to the cell membrane.
  • the transmembrane domain confers membrane attachment to the CAR polypeptide of the present invention.
  • the CAR polypeptide of the present invention includes at least one transmembrane domain, which may be derived from a natural source or a recombinant source, comprising dominant hydrophobic residues such as leucine and valine.
  • the transmembrane domain may be derived from a membrane-bound protein or a transmembrane protein such as CD4, CD28, CD8 (e.g., CD8 ⁇ , CD8 ⁇ ) transmembrane domain.
  • the transmembrane domain in the chimeric antigen receptor of the present invention is a CD4 transmembrane domain or a variant thereof having 1-10 amino acid modifications, for example, a variant thereof having 1-5 amino acid modifications.
  • the amino acid modification is an addition, deletion or substitution of an amino acid.
  • the CD4 transmembrane domain is the sequence shown in SEQ ID NO: 10.
  • the transmembrane domain in the chimeric antigen receptor of the present invention is a CD8 transmembrane domain or a variant thereof having 1-10 amino acid modifications, for example, a variant thereof having 1-5 amino acid modifications.
  • the amino acid modification is an addition, deletion or substitution of an amino acid.
  • the CD8 transmembrane domain is the sequence shown in SEQ ID NO: 11.
  • the transmembrane domain in the chimeric antigen receptor of the present invention is a CD28 transmembrane domain or a variant thereof having 1-10 amino acid modifications, for example, a variant thereof having 1-5 amino acid modifications.
  • the amino acid modification is an addition, deletion or substitution of an amino acid.
  • the CD28 transmembrane domain is the sequence shown in SEQ ID NO:9.
  • a CAR-T cell comprising a CD28 transmembrane domain that specifically binds to IL13R ⁇ 2 has excellent anti-GBM activity in vivo.
  • the transmembrane domain in the CAR of the present invention is connected to the extracellular region of the CAR (ie, the anti-IL13R ⁇ 2 scFv sequence) by means of a hinge region/spacer.
  • a hinge region/spacer comprises the entire or part of an immunoglobulin (eg, IgG1, IgG2, IgG3, IgG4) hinge region, that is, a sequence falling between the CH1 and CH2 domains of an immunoglobulin, for example, an IgG4Fc hinge or a CD8 hinge.
  • Some hinge regions/spacers comprise an immunoglobulin CH3 domain or both a CH3 domain and a CH2 structure.
  • the sequence derived from an immunoglobulin may comprise one or more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, for example, substitutions that reduce off-target binding.
  • the hinge region/spacer is an IgG hinge region or a variant thereof having 1-2 amino acid modifications, e.g., an IgG4 hinge region or a variant thereof having 1-2 amino acid modifications.
  • the length of the hinge region/spacer region is optimized, and it is found that for IL13R ⁇ 2, a short hinge region/spacer region (for example, the IgG4 hinge region shown in SEQ ID NO:7) is easier to activate CAR-T than a long hinge region/spacer region.
  • the IgG4 hinge region shown in SEQ ID NO:7 does not retain the heavy chain constant region 2 of IgG4 and therefore does not have the ability to bind to Fc receptors (e.g., Fc ⁇ R), thereby avoiding off-target activation of CAR-T cells.
  • Fc receptors e.g., Fc ⁇ R
  • the cytoplasmic domain included in the CAR of the present invention includes an intracellular signaling domain.
  • the intracellular signaling domain can activate at least one effector function of the immune cell introduced with the CAR of the present invention.
  • intracellular signaling domains used in the CAR of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction after the extracellular domain binds to IL13R ⁇ 2, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capacity.
  • TCRs T cell receptors
  • co-receptors that act synergistically to initiate signal transduction after the extracellular domain binds to IL13R ⁇ 2, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capacity.
  • the CAR of the present invention is also designed to generate a costimulatory signal domain (CSD).
  • CSD costimulatory signal domain
  • the activation of T cells is mediated by two different cytoplasmic signaling sequences: those sequences (primary intracellular signaling domains) that initiate antigen-dependent primary activation through TCR and those sequences (secondary cytoplasmic domains, e.g., costimulatory domains) that act in an antigen-independent manner to provide costimulatory signals.
  • the CAR of the present invention comprises a primary intracellular signaling domain, for example, a primary signaling domain of CD3 ⁇ , for example, a CD3 ⁇ signaling domain shown in SEQ ID NO: 14 or a variant thereof having 1-10 amino acid modifications, for example, a variant thereof having 1-5 amino acid modifications, wherein the amino acid modification is an addition, deletion or substitution of an amino acid.
  • the intracellular signaling domain in the CAR of the present invention also includes a secondary signaling domain (i.e., a co-stimulatory signaling domain).
  • the co-stimulatory signaling domain refers to the CAR portion that includes the intracellular domain of a co-stimulatory molecule.
  • Co-stimulatory molecules are cell surface molecules required for immune effector cells to effectively respond to antigens in addition to antigen receptors or their ligands.
  • co-stimulatory molecules include but are not limited to CD28, 4-1BB (CD137), and OX40, the co-stimulatory effects of which enhance the proliferation, effector function, and survival of human CAR T cells in vitro and enhance the anti-tumor activity of human T cells in vivo.
  • the costimulatory signaling domain in the chimeric antigen receptor of the present invention is a CD28 costimulatory domain or a variant thereof having 1-10 amino acid modifications, for example, a variant thereof having 1-5 amino acid modifications.
  • the amino acid modification is the addition, deletion or substitution of an amino acid.
  • the costimulatory signaling domain in the chimeric antigen receptor of the present invention is a 4-1BB costimulatory domain or a variant thereof with 1-10 amino acid modifications, for example, a variant with 1-5 amino acid modifications.
  • the amino acid modification is the addition, deletion or substitution of an amino acid.
  • the intracellular region of the CAR of the present invention comprises multiple co-stimulatory domains in series with CD3 ⁇ , such as a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain, and when the CAR is expressed on the surface of an immune effector cell (e.g., a T cell, a NK cell), the CAR enables the T cell to receive a co-stimulatory signal.
  • an immune effector cell e.g., a T cell, a NK cell
  • 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 bonds between intracellular signaling sequences.
  • a glycine-serine doublet can be used as a suitable linker.
  • a single amino acid, for example, alanine, glycine can be used as a suitable linker.
  • the intracellular signaling domain of the CAR of the present invention is designed to include the costimulatory signaling domain of CD28, the costimulatory signaling domain of 4-1BB, and the stimulatory signaling domain of CD3 ⁇ .
  • the CAR polypeptides of the present invention can also be modified so that the amino acid sequence is changed, but the desired activity is not changed.
  • the CAR polypeptide can be subjected to additional amino acid substitutions that result in amino acid substitutions at "non-essential" amino acid residues.
  • 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 fragment can be replaced with a structurally similar fragment that differs in the order and composition of the side chain family members, for example, a conservative substitution can be performed in which an amino acid residue is replaced with an amino acid residue having 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, isoleucine) and aromatic side chains (e.g., 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 present invention provides nucleic acid molecules encoding CAR constructs described herein.
  • the nucleic acid molecule is provided as a DNA construct.
  • the construct encoding the CAR of the present invention can be obtained using recombinant methods known in the art.
  • the target nucleic acid can be produced synthetically rather than by genetic recombination methods.
  • the present invention also provides a vector inserted with a CAR construct of the present invention.
  • a vector By effectively connecting a nucleic acid encoding a CAR polypeptide to a promoter and incorporating the construct into an expression vector, expression of a natural or synthetic nucleic acid encoding CAR is achieved.
  • the vector may be suitable for replication and integration in eukaryotic organisms. Common cloning vectors contain transcription and translation 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 construct of the present invention can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro.
  • Numerous retroviral systems are known in the art.
  • a lentiviral vector is used.
  • the nucleic acid sequence of the CAR construct of the present invention is cloned into a lentiviral vector to produce a full-length CAR construct in a single coding frame, and the EF1 ⁇ promoter is used for expression.
  • Retroviral vectors can also be, for example, ⁇ retroviral vectors.
  • ⁇ retroviral vectors can, for example, include promoters, packaging signals ( ⁇ ), primer binding sites (PBS), one or more (e.g., two) long terminal repeats (LTR) and target transgenics, for example, genes encoding CAR.
  • ⁇ retroviral vectors can 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 natural EF1a promoter drives the expression of the ⁇ subunit of the elongation factor-1 complex, which is responsible for enzymatic delivery of aminoacyl tRNA to the ribosome.
  • the EF1a promoter has been widely used in mammalian expression plasmids and has been shown to effectively drive the expression of CAR from a transgene cloned into a lentiviral vector. See, for example, Milone et al., Mol. Ther. 17 (8): 1453–1464 (2009).
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a constitutive strong promoter sequence that can drive any polynucleotide sequence effectively connected thereto to express at a high level.
  • other constitutive promoter sequences may 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) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter and human gene promoter, such as but not limited to actin promoter, myosin promoter, elongation factor-1 ⁇ promoter, hemoglobin promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention.
  • the present invention provides methods for expressing a CAR construct of the present invention in a mammalian immune effector cell (e.g., a mammalian T cell or a mammalian NK cell) and the immune effector cells produced thereby.
  • a mammalian immune effector cell e.g., a mammalian T cell or a mammalian NK cell
  • a cell source e.g., immune effector cells, e.g., T cells or NK cells
  • T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells from blood components collected from a subject can be obtained by apheresis.
  • Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets.
  • the cells collected by apheresis can be washed to remove the plasma fraction and to place cells in a suitable buffer or culture medium for subsequent processing steps.
  • cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Specific T cell subsets such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques.
  • the time period is about 30 minutes to 36 hours or longer.
  • Longer incubation time can be used to separate T cells in any case where there is a small amount of T cells, such as for separating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals.
  • TIL tumor infiltrating lymphocytes
  • the efficiency of capturing CD8+T cells can be increased using longer incubation time.
  • T cell subsets can be preferentially selected at the beginning of culture or at other time points during the culture process.
  • Enrichment of T cell populations can be accomplished by a negative selection process using a combination of antibodies directed against surface markers unique to the negatively selected cells.
  • One approach is to sort and/or select cells using negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells.
  • the immune effector cell can be an allogeneic immune effector cell, e.g., a T cell or a NK cell.
  • the cell can be an allogeneic T cell, e.g., an allogeneic T cell 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).
  • TCR functional T cell receptor
  • HLA human leukocyte antigen
  • a T cell lacking a functional TCR can, for example, be engineered so that it does not express any functional TCR on its surface; engineered so 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 shows reduced expression of TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ and/or TCR ⁇ ); or engineered so that it produces very few functional TCRs on its surface.
  • the T cells described herein can be engineered, for example, so that it does not express functional HLA on its surface.
  • the T cells described herein can be engineered so 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 cell may lack a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II.
  • cells transduced with a nucleic acid encoding a CAR described herein are proliferated, for example, the cells are proliferated in culture for 2 hours to about 14 days.
  • the CAR-expressing immune effector cells obtained after in vitro proliferation can be tested for effector function as described in the examples.
  • the CAR-T cells of the present invention can maintain the killing activity against tumors expressing IL13R ⁇ 2.
  • the optimized third-generation chimeric antigen receptor (CAR) polypeptide targeting IL13R ⁇ 2 of the present invention has excellent safety and in vivo anti-GBM activity due to its high selectivity and specific targeting of IL13R ⁇ 2.
  • the present invention obtains immune effector cells expressing the CAR polypeptide of the present invention by optimizing the structure of the CAR construct, which is used to treat solid tumors associated with the expression of IL13R ⁇ 2, such as gliomas, in subjects.
  • engineered T cells e.g., patient-specific autologous T cells
  • ACT adoptive cell therapy
  • a variety of T cell subsets can be used to express the CAR polypeptides of the present invention.
  • the immune effector cells when using immune effector cells expressing the CAR polypeptides of the present invention to treat patients, can be autologous T cells or allogeneic T cells.
  • the T cells used are CD4+ and CD8+ central memory T cells (T CM ), which are CD45RO+CD62L+, and the use of such cells can improve the long-term survival of cells after adoptive transfer compared to the use of other types of patient-specific T cells.
  • NK cells are engineered to express the CAR polypeptides of the present invention. After amplifying the other immune cells (e.g., NK cells) engineered, they are used for adoptive cell therapy (ACT).
  • ACT adoptive cell therapy
  • the immune effector cells expressing the CAR polypeptide of the present invention are used to treat cancer that expresses or overexpresses IL13R ⁇ 2 in a subject, and are capable of reducing the severity of at least one symptom or indication of cancer or inhibiting cancer cell growth.
  • 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 immune effector cells expressing a CAR polypeptide of the present invention.
  • 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 immune effector cells expressing a CAR polypeptide of the present invention.
  • the present invention provides the use of the aforementioned immune effector cells expressing the CAR polypeptide of the present invention in the preparation of a medicament for treating a disease associated with the expression of IL13R ⁇ 2 (eg, a cancer expressing or overexpressing IL13R ⁇ 2).
  • a disease associated with the expression of IL13R ⁇ 2 eg, a cancer 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, intracavitary, intraarterial, intravenous, intrathecal administration, or directly injected into a tissue or tumor.
  • the immune effector cells expressing the CAR polypeptides of the present invention can be administered to the subject at a suitable dose.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's weight, body surface area, age, specific compound to be administered, sex, administration time and route, general health status, and other drugs to be administered in parallel.
  • immune effector cells e.g., T cells, NK cells
  • a CAR polypeptide of the invention are administered parenterally, preferably intravenously, in a single or multiple doses of 1 ⁇ 10 6 to 1 ⁇ 10 12 immune effector cells, preferably 1 ⁇ 10 7 to 1 ⁇ 10 10 immune effector cells, for example 5 ⁇ 10 7 , 1 ⁇ 10 8 , 5 ⁇ 10 8 , 1 ⁇ 10 9 , 5 ⁇ 10 9 immune effector cells.
  • immune effector cells expressing the CAR polypeptides of the present invention are administered to individuals with cancer, resulting in the complete disappearance of the tumor. In some embodiments, immune effector cells expressing the CAR polypeptides of the present invention are administered to individuals with cancer, resulting in a reduction of tumor cells or tumor size by at least 85% or more.
  • the reduction of tumors can be measured by any method known in the art, such as X-rays, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analysis.
  • GBM cell lines U251, U373, U87 and retroviral packaging cell lines PG13 and Phoenix ECO were purchased from the American Type Culture Collection (ATCC).
  • U251 cells and U373 cells were transduced with retrovirus to express the green fluorescent protein (GFP) gene and the firefly luciferase (LUC) reporter gene (sometimes abbreviated as "GL”).
  • GBM cell lines grow well in DMEM medium (Lonza) containing 10% fetal bovine serum (FBS, Biosera), 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin (EallBio Life Sciences).
  • Retroviral production cell lines were cultured in DMEM medium containing 10% FBS without penicillin and streptomycin.
  • Flow cytometry was performed using a BD FacsCanto II Plus instrument (BD Biosciences), and the results were analyzed using FlowJo v.10 software (Tree star, Inc. Ashland, OR).
  • the antibodies used were 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).
  • CAR-T cells and target cells were co-cultured at an E:T ratio of 10:1 for 24 h, and the killing of target cells was detected by flow cytometry.
  • CAR-T cells were co-cultured with human GBM cell lines (U373, U251, and U87) at an E:T ratio of 10:1 for 24 hours.
  • the expression of human interferon- ⁇ (IFN- ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ), IL17A, IL4, IL6, and IL10 in the supernatant of co-cultured cells was detected using a commercial flow cytometry bead array (CBA) kit (BD Biosciences). The specific operation steps were performed according to the instructions of the kit.
  • CBA flow cytometry bead array
  • RTCA Real-time cell analysis
  • the proliferation/cytotoxicity of CAR T cells was evaluated using the xCELLigence RTCA system (Roche Applied Science, Basel, Switzerland). The system is based on a gold plate sensor electrode with electrical impedance reading, located at the bottom of the cytotoxicity plate (E-16 plate).
  • E-16 plate a gold plate sensor electrode with electrical impedance reading, located at the bottom of the cytotoxicity plate (E-16 plate).
  • target cells U251-GL or target cells U373-GL were seeded in E-16 plates at 1 ⁇ 104 cells per well.
  • 1 ⁇ 105 CAR-T cells were added to the E-16 plate and incubated with human GBM cells and monitored every 15 min to obtain the cell index for 48 h. Each independent experiment was performed in triplicate. The interval slope was automatically calculated using the RTCA software to evaluate the rate of change of the cell index.
  • the cell index was normalized to equal values at standardized time points.
  • mice used in the experiment were 6-8 week old NOD-SCID mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. 2 ⁇ 10 5 U251-GL cells and U373-GL cells were injected into the right striatum of female NOD-SCID mice to construct an orthotopic xenograft mouse model, which was recorded as day 1. On day 6, 3 ⁇ 10 7 CAR-T cells were injected through the tail vein. The development of the tumor was monitored using a small animal imaging instrument (IVIS, Xenogen, Alameda, CA, USA), and the mice were killed when the tumor diameter reached 20 mm. The animal experiment was approved by the Ethics Committee of Beijing Century Altar Hospital.
  • IVIS small animal imaging instrument
  • the CAR targeting IL13R ⁇ 2 shown in FIG. 1 and comprising an optimized scFv sequence for IL13R ⁇ 2 was constructed using a retroviral vector.
  • the CAR targeting IL13R ⁇ 2 contains an optimized anti-IL13R ⁇ 2 scFv sequence located in the extracellular domain, an optimized IgG4 hinge region, a CD28 transmembrane region, a CD28 co-stimulatory domain located in the intracellular part, a 4-1BB co-stimulatory domain, and a CD3 ⁇ signaling domain.
  • Thermo Company's GeneArt gene synthesis technology was used to synthesize a nucleotide sequence encoding the anti-IL13R ⁇ 2 scFv sequence, which, from N-terminus to C-terminus, consists of the antibody heavy chain variable region VH nucleotide sequence shown in SEQ ID NO:1, the nucleotide sequence encoding the linker shown in SEQ ID NO:5, and the antibody light chain variable region VL nucleotide sequence shown in SEQ ID NO:3.
  • nucleotide sequences encoding the IgG4 hinge region shown in SEQ ID NO:7, the CD28 transmembrane domain shown in SEQ ID NO:9, the CD28 co-stimulatory domain shown in SEQ ID NO:12, the 4-1BB co-stimulatory domain shown in SEQ ID NO:13, and the CD3 ⁇ signaling domain shown in SEQ ID NO:14 were ligated into the retroviral vector SFG (Addgene, also known as "vector pMSGV1" to obtain the vector retro-SFG-IgG4.
  • the constructed IL13R ⁇ 2-specific CAR retroviral vector was used for virus packaging. Specifically, the retroviral vector retro-SFG-IL13R ⁇ 2 scFv-IgG4-CD28-4-1BB-CD3 ⁇ was co-transfected into PG13 cells using a calcium phosphate reagent. The culture medium was replaced 48 hours after transfection, and the culture supernatant was collected. The culture supernatant was collected again 72 hours later and filtered through 0.45 ⁇ m. Syringe filter to obtain the retroviral stock solution.
  • the virus stock solution was ultracentrifuged at 4°C and 32,000 r/min for 2 hours, and the retrovirus precipitate was dissolved in X-VIVO culture medium to obtain the retrovirus concentrate, which was aliquoted and stored at -80°C.
  • the titer of the retroviral concentrate was tested. 0.5 ⁇ 10 6 Jurkat cells were plated in each well of a 96-well pointed bottom plate, 200 ⁇ L/well, the retroviral concentrate was diluted 100 times, and 400 ⁇ L, 40 ⁇ L and 10 ⁇ L were added to each well at a ratio of 1:50, 1:500 and 1:2000. Centrifuge at 32°C and 1200 ⁇ g for 90 min. After 4 hours, the cells were washed once with DPBS and plated on a 12-well plate, and detected by flow cytometry 48 hours after transduction. The results showed that the retroviral concentrate prepared in this example was able to infect Jurkat cells and express CAR after being diluted 100 times. Therefore, the production of CAR-T cells was implemented using the retroviral concentrate.
  • Example 1 the concentrated retrovirus solution harvested in Example 1 was used to transduce T cells, thereby preparing CAR-T cells.
  • PBMC mononuclear cells
  • GE-healthcare lymphocyte separation fluid
  • T cells in PBMC were stimulated with anti-CD3/CD28 T cell activator Dynabeads (Invitrogen). After 48 hours of stimulation, T cells were transfected with the retroviral concentrate of Example 1.
  • the retroviral transfection experiment was performed with reference to the instructions of the calcium phosphate transfection kit (Sigma). On day 7, flow cytometry was used to detect the positive rate of CAR expression in transfected T cells.
  • CAR-T cells were cultured in X-VIVO-15 medium containing 5% human AB serum (SIGMA), 100U/ml IL-2, 100U/ml penicillin, and 100 ⁇ g/ml streptomycin (EallBio Life Sciences). This study was approved by the Institutional Review Board of Beijing Century Altar Hospital, and informed consent was obtained from all participants.
  • SIGMA human AB serum
  • 100U/ml IL-2 100U/ml
  • penicillin 100U/ml
  • streptomycin EallBio Life Sciences
  • T cells from healthy donors and activated by CD3/CD28 antibodies were transduced with the retrovirus prepared in Example 1, and the gene-modified T cells were detected by FACS analysis 7 days later.
  • the CAR containing the anti-IL13R ⁇ 2 scFv sequence was stably expressed on the surface of T cells.
  • the killing effect of CAR-T cells on tumor cells can be detected by the degranulation ability of CAR-T cells, that is, the expression of CD107a.
  • the CAR-T cells containing the anti-IL13R ⁇ 2 scFv sequence produced in Example 2 contain high concentrations of cytotoxic granules in the form of vesicles, such as perforin, granzyme, etc.
  • Lysosomal associated membrane protein-1 (LAMP-1) also known as CD107a, is a marker of cell degranulation. Under the stimulation of tumor cells, the expression of CD107a is associated with the lysis of target cells mediated by the CAR-T cells produced in Example 2.
  • the CD107a expression rate was positively correlated with the killing activity of the CAR-T cells.
  • Example 5 Determination of cytokines produced by CAR-T cells containing anti-IL13R ⁇ 2 scFv sequences
  • CAR-T cells containing anti-IL13R ⁇ 2 scFv sequence and IL13R ⁇ 2-positive human GBM cell lines (U373, U251, and U87 cells) were co-cultured at an E:T ratio of 10:1 for 24 hours, interferon- ⁇ (IFN- ⁇ ) and tumor necrosis factor- ⁇ (TNF- ⁇ ) in the culture supernatant were detected by CBA method, wherein IFN- ⁇ and TNF- ⁇ were positively correlated with the killing ability of CAR-T cells on target cells.
  • IFN- ⁇ interferon- ⁇
  • TNF- ⁇ tumor necrosis factor- ⁇
  • the cell growth analysis of tumor cells was performed using a real-time cell analyzer (RTCA).
  • RTCA real-time cell analyzer
  • a micro-gold electronic sensor chip is integrated at the bottom of the detection plate E-Plate 16.
  • Figures 10 and 11 show the cell growth results of tumor cells detected by the RTCA method, and the anti-tumor ability of CAR-T cells containing anti-IL13R ⁇ 2 scFv sequences was evaluated in vitro.
  • the number of tumor cells in the U373 tumor cell control group increased significantly with the extension of time; the number of tumor cells in the co-culture group of CAR-T cells containing anti-IL13R ⁇ 2 scFv sequence and U373 tumor cells ( ⁇ line) decreased significantly after 20 hours of co-culture, and the CAR-T cells significantly killed the tumor cells.
  • the number of tumor cells in the U251 tumor cell control group increased significantly with the extension of time; after 20 hours of co-culture with U251 tumor cells in the CAR-T cells containing the anti-IL13R ⁇ 2 scFv sequence ( ⁇ line), the number of tumor cells decreased significantly, and the CAR-T cells had a significant killing effect on tumor cells.
  • the CAR-T cells of the present invention containing the anti-IL13R ⁇ 2 scFv sequence significantly inhibited the growth of U373 and U251 tumor cells and had obvious anti-tumor activity.
  • an orthotopic xenograft mouse model was established by intracranial injection of glioma cells.
  • Glioma cell lines U373 and U251 express IL13R ⁇ 2 on their cell surface.
  • 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 injected into the right striatum of female NOD-SCID mice (recorded as the first day, also abbreviated as D1).
  • a positive control group was set up in the experiment, using IL13-CAR-T.
  • the preparation of the positive control IL13-CAR-T cells was similar to that of Example 1 and Example 2, except that the anti-IL13R ⁇ 2 scFv sequence was replaced by the IL13 sequence shown in SEQ ID NO:24.
  • Tumor growth was observed by detecting fluorescence signals using a small animal imaging instrument (IVIS, Xenogen, Alameda, CA, USA) to monitor tumor development, and mice were killed when the tumor diameter reached 20 mm. Animal experiments were approved by the Ethics Committee of Beijing Shijitan Hospital.
  • IVIS small animal imaging instrument
  • the longest survival period of the U251 in situ animal model treated with IL13-CAR-T in the positive control group was 34 days, while the longest survival period of mice treated with IL13R ⁇ 2-CAR-T of the present invention was 50 days.
  • the longest survival period of the U373 in situ animal model treated with IL13-CAR-T in the positive control group was 60 days, while the longest survival period of mice treated with IL13R ⁇ 2-CAR-T of the present invention was 64 days.

Abstract

提供了一种靶向IL13Rɑ2的嵌合抗原受体，其包含:(1)经优化的抗IL13Rɑ2 scFv序列；(2)经优化的铰链区/间隔区；(3)跨膜区(TM); (4)CD28共刺激结构域和4-1BB共刺激结构域；和(5)CD3ζ信号传导结构域。还提供了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、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)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IL13Ro2相关的疾病，例如，胶质瘤。

Description

靶向IL13Rα2的经优化的嵌合抗原受体及其用途 技术领域
本发明涉及基因工程技术领域。具体地，本发明涉及经优化的靶向IL13Rα2的嵌合抗原受体、经工程化以表达本发明的所述经优化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
神经胶质母细胞瘤(Glioblastoma，GBM)是成年人中恶性程度最高的原发性脑肿瘤。目前的标准治疗包括手术切除、放疗和化疗(例如，使用替莫唑胺(temozolomide))，但是，五年总体生存率小于10％。
免疫治疗是一种非常有吸引力的治疗方法，它能改善GBM患者的预后，而且没有化疗或放疗辐射产生的细胞毒性反应。使用抗体或T细胞的过继免疫疗法是目前临床中及实验中最有效的免疫疗法。虽然以树突状细胞疫苗为主的免疫治疗在临床治疗复发和治疗新诊断的GBM患者中已显示出令人鼓舞的结果，能让患者疾病稳定并且增加患者的生存期，但这些临床结果有待进一步的随机临床试验证实，而且已发表的研究文章也表明，它很难在患者体内诱导胶质瘤特异性T细胞。因此迫切需要研究和发展新的免疫治疗方法。
大多数的人类癌症缺乏可预测的、并且充当有效靶标以被T细胞根除的特异性抗原。每一癌细胞类型具有产生不同肿瘤特异性抗原的独特的突变集合。尽管抗原鉴定技术取得了巨大进展，但仍难以鉴别有效独特的抗原，也难以分离合适的TCR用于自体T细胞的转导而实施过继免疫疗法。
嵌合抗原受体T细胞(chimeric antigen receptor-T cell,CAR-T)免疫疗法是通过转基因技术，使能够特异性识别靶抗原的单克隆抗体单链可变区(scFv)等表达在T细胞表面，同时激活T细胞的胞内活化增殖信号域(CD3ζ链和共刺激分子CD28/4-1BB)，从而使T细胞产生高效的特异性抗肿瘤反应。
CAR-T作为目前最为瞩目的细胞免疫疗法，在美国已有5款药物成功上市(诺华公司的Kymriah、Kite公司的Yescarta和Tecartus、BMS公司的Breyanzi和Abecma)，其最早的受试者Emily Whitedhead(急性淋巴细胞白血病患者)已无瘤生存8年，这重燃起无数肿瘤患者对生的希望。
当前，随着CAR-T技术的快速发展，CAR分子结构已从第一代发展到了第三代，但CAR分子中所包含的特异性识别靶抗原的单克隆抗体单链可变区(scFv)大部分均为鼠源的scFv序列，尤其在治疗GBM方面，尚未出现有效的第三代CAR-T细胞，这使在临床治疗过程中，CAR-T细胞存在发生排斥反应或脱靶现象。另外目前CAR-T研究 多以治疗血液系统疾病为主，对于实体瘤的研究较少。
高亲和力白介素-13受体α2(IL13Rα2)由胶质母细胞瘤(GBM)以及若干其它肿瘤类型以高频率选择性地表达。靶向这种肿瘤特异性受体的一种方法是利用缀合到细胞毒性分子的相应配体IL-13。然而，这种方法缺乏特异性，因为结合IL-13的较低亲和力受体IL13Rα1由正常组织广泛地表达。
因此，本领域仍然需要高选择性地特异性靶向IL13Rα2的抗IL13Rα2CAR，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。
发明概述
本发明的目的是提供一种特异性靶向IL13Rα2的经优化第三代嵌合抗原受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)，用于治疗与表达IL13Rα2相关的疾病，例如，神经胶质母细胞瘤(GBM)以解决上述现有技术存在的问题。
因此，在第一方面，本发明提供了一种靶向IL13Rα2的经优化嵌合抗原受体(CAR)多肽，其包含：
(1)经优化的抗IL13Rα2 scFv序列，其中所述scFv序列特异性结合IL13Rα2且包含：
(i)重链可变区，其包含根据Kabat编号的
(a)氨基酸序列KYGVH(SEQ ID NO:15)所示的重链互补决定区CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(b)氨基酸序列VKWAGGSTDTDSALMS(SEQ ID NO:16)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
(c)氨基酸序列DHRDAMDY(SEQ ID NO:17)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
(ii)轻链可变区，其包含根据Kabat编号的
(d)氨基酸序列TASLSVSSTYLH(SEQ ID NO:18)所示的轻链互补决定区(CDR L)1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(e)氨基酸序列STSNLAS(SEQ ID NO:19)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
(f)氨基酸序列HQYHRSPLT(SEQ ID NO:20)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
其中所述氨基酸变化是氨基酸的添加、缺失或取代；
(2)经优化的铰链区/间隔区，其选自
(i)IgG4铰链区(SEQ ID NO 7)，或其具有至少80％的序列同一性的IgG4铰链区；
(ii)CD8铰链区(SEQ ID NO 8)，或其具有至少80％的序列同一性的CD8铰链区。
(3)跨膜区(TM)，其选自
(i)CD28跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:9所示的序列或其具有1-2个氨基酸修饰的变体；
(ii)CD4跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:10所示的序列或其具有1-2个氨基酸修饰的变体；
(iii)CD8跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:11所示的序列或其具有1-2个氨基酸修饰的变体；
(4)共刺激信号结构域(CSD)，其是：
(i)CD28共刺激结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:12所示的序列或其具有1-2个氨基酸修饰的变体；和
(ii)4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:13所示的序列或其具有1-2个氨基酸修饰的变体；
(5)刺激信号结构域(SSD)，为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，SEQ ID NO:14所示的序列或其具有1-10个、1-5个氨基酸修饰的变体。
在一些实施方案中，本发明的靶向IL13Rα2的经优化的嵌合抗原受体(CAR)多肽包含：
(1)经优化的抗IL13Rα2 scFv序列，其中所述scFv序列特异性结合IL13Rα2且包含：
(i)重链可变区，其包含SEQ ID NO:2的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，和
(ii)轻链可变区，其包含SEQ ID NO:4的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
例如，(i)重链可变区，其包含SEQ ID NO:2的序列，和
(ii)轻链可变区，其包含SEQ ID NO:4的序列；
(2)经优化的铰链区/间隔区，其选自
(i)IgG4铰链区(SEQ ID NO 7)，或其具有至少90％、至少95％的序列同一性的IgG4铰链区；
(ii)CD8铰链区(SEQ ID NO 8)，或其具有至少90％、至少95％的序列同一性的CD8铰链区。
(3)跨膜区(TM)，其选自
(i)SEQ ID NO:9所示的CD28跨膜结构域或其具有1个氨基酸修饰的变体；
(ii)SEQ ID NO:10所示的CD4跨膜结构域或其具有1个氨基酸修饰的变体；
(iii)SEQ ID NO:11所示的CD8跨膜结构域或其具有1个氨基酸修饰的变体；
(4)共刺激信号结构域(CSD)，其是：
(i)SEQ ID NO:12所示的CD28共刺激结构域或其具有1个氨基酸修饰的变体；和
(ii)SEQ ID NO:13所示的4-1BB共刺激结构域或其具有1个氨基酸修饰的变体；
(5)刺激信号结构域(SSD)，为SEQ ID NO:14所示的CD3ζ信号传导结构域或其具有1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，本发明的靶向IL13Rα2的经优化的嵌合抗原受体(CAR)多肽包含：
(1)经优化的抗IL13Rα2 scFv序列，其中所述scFv序列特异性结合IL13Rα2且包含：
(i)SEQ ID NO:2所示的重链可变区，和
(ii)SEQ ID NO:4所示的轻链可变区；
(2)SEQ ID NO 7所示的经优化的铰链区；
(3)跨膜区(TM)，其是SEQ ID NO:9所示的CD28跨膜结构域；
(4)共刺激信号结构域(CSD)，其是：
(i)SEQ ID NO:12所示的CD28共刺激结构域；和
(ii)SEQ ID NO:13所示的4-1BB共刺激结构域；
(5)SEQ ID NO:14所示的CD3ζ信号传导结构域。
在一些实施方案中，本发明的靶向IL13Rα2的经优化的嵌合抗原受体(CAR)多肽还包含位于N端的信号肽序列，例如，SEQ ID NO:21所示的信号肽序列，
在一些实施方案中，本发明的靶向IL13Rα2的经优化的嵌合抗原受体(CAR)多肽具有SEQ ID NO:22所示的氨基酸序列或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上的同一性的氨基酸序列。
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，用逆转录病毒将编码本文所述CAR多肽的核酸分子导入原代 T细胞中，获得了本发明的CAR-T细胞。
本发明的CAR-T细胞在体外对高表达IL13Rα2的胶质瘤U373细胞和对低表达IL13Rα2的胶质瘤U251细胞均具有显著的杀伤活性且产生IFN-γ、TNF-α等细胞因子。
本发明的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，本发明首次采用抗IL13Rα2 scFv序列构建第三代CAR-T细胞，证实了其能够通过特异性靶向IL13Rα2来治疗GBM，提高了CAR-T细胞治疗GBM的安全性与有效性，避免了发生排斥反应或脱靶现象，更利于CAR-T的推广与临床应用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些优选的实施方案，对于本领域普通技术人员而言，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的技术方案。
图1为本发明构建的经优化的CAR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)。在所述构建体中，胞外域包含抗IL13Rα2的经优化的scFv序列和短的IgG4铰链区。
图2为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞单独培养时，对CD107a染色，通过流式细胞术检测CD107a的表达率结果。
图3为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞与高表达IL13Rα2的U373细胞株共培养时，对CAR-T细胞实施CD107a染色，通过流式细胞术检测CD107a的表达率结果。
图4为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞与低表 达IL13Rα2的U251细胞株共培养时，对CAR-T细胞实施CD107a染色，通过流式细胞术检测CD107a的表达率结果。
图5为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞与U373细胞株或U251细胞株共培养后，对CD107a表达率差异的分析结果。
图6为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞杀伤靶细胞U373细胞株或U251细胞株的实验结果。
图7为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞杀伤靶细胞U373细胞株或U251细胞株的差异分析结果。
图8为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞(图中缩写为“IL13Rα2-CAR-T”)与IL13Rα2阳性的人GBM细胞株(U373、U251、U87)共培养后，上清液中细胞因子IFN-γ的含量。
图9为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞(图中缩写为“IL13Rα2-CAR-T”)与IL13Rα2阳性的人GBM细胞株(U373、U251、U87)共培养后，上清液中细胞因子TNF-α的含量。
图10为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞与U373细胞系共培养后，RTCA检测结果。
图11为靶向IL13Rα2的包含抗IL13Rα2的经优化的scFv序列的CAR-T细胞与U251细胞系共培养后，RTCA检测结果。
图12为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞(图中缩写为“IL13Rα2-CAR-T”)或靶向IL13Rα2的包含SEQ ID NO:24所示IL13的CAR-T细胞(图中缩写为“IL13-CAR-T”)治疗U251原位动物模型的活体成像结果，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13为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞(图中缩写为“IL13Rα2-CAR-T”)或靶向IL13Rα2的包含SEQ ID NO:24所示IL13的CAR-T细胞(图中缩写为“IL13-CAR-T”)治疗U251原位动物模型的生存周期。实验同图12。
图14为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞(图中缩写为“IL13Rα2-CAR-T”)或靶向IL13Rα2的包含SEQ ID NO:24所示IL13的CAR-T细胞(图中缩写为“IL13-CAR-T”)治疗U373原位动物模型的活体成像结果，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15为经优化的靶向IL13Rα2的包含抗IL13Rα2 scFv序列的CAR-T细胞(图中缩写为“IL13Rα2-CAR-T”)或靶向IL13Rα2的包含SEQ ID NO:24所示IL13的CAR-T细胞(图中缩写为“IL13-CAR-T”)治疗U373原位动物模型的生存周期。实验同图14。
发明详述
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。
I.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。
如本文所用的术语“抗IL13Rα2抗体”、“针对IL13Rα2的抗体”、“特异性结合IL13Rα2的抗体”、“特异性靶向IL13Rα2的抗体”、“特异性识别IL13Rα2的抗体”可互换地使用，意指能够与IL13Rα2特异性结合的抗体。特别地，在一些具体实施方案中，意指与人IL13Rα2特异性结合的抗体，特别是与人IL13Rα2特异性结合而不与人IL13Rα1特异性结合的抗体。
术语“scFv”指一种融合蛋白，其包含至少一个包含轻链可变区的抗体片段和至少一个包含重链可变区的抗体片段，其中轻链可变区和重链可变区任选地借助柔性短多肽接头连续地连接，并且能够表达为单链多肽，并且其中scFv保留衍生它的完整抗体的特异性。除非另外指出，否则如本文所用，scFv可以具有按任何顺序(例如，相对于多肽的N末端和C末端)的VL可变区和VH可变区，scFv可以包含VL-接头-VH或可以包含VH-接头-VL。每个重链可变区(VH)和轻链可变区(VL)分别由四个保守的构架区(FR)和三个互补决定区(CDR)组成，按以下顺序从氨基端到羧基端排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。在一些实施方案中，本发明的CAR所包含的针对IL13Rα2的scFv序列是经优化的序列，例如，是对构架区进行了优化的序列。在一些具体实施方案中，对构架区的氨基酸序列进行了优化，从而保留优化后的针对 IL13Rα2的scFv序列对IL13Rα2的结合亲和力提高。
“互补决定区”或“CDR区”或“CDR”或“高变区”是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作CDR H1、CDR H2和CDR H3，而位于抗体轻链可变结构域内的CDR被称作CDR L1、CDR L2和CDR L3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
CDR也可以基于与参考CDR序列(例如本发明示例的CDR之任一)具有相同的Kabat编号位置而确定。在本发明中，当提及抗体可变区和具体CDR序列(包括重链可变区残基)时，是指根据Kabat编号系统的编号位置。
尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia,AbM和Contact方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia或AbM定义的其余CDR残基可以被保守氨基酸残基替代。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的构架区(FR)和三个互补决定区(CDR)。(参见例如，Kindt等人，Kuby Immunology，6 th ed.，W.H.Freeman and Co.91页(2007))。
如本文所用，术语“结合”或“特异性结合”意指结合作用对抗原是选择性的并且可以与不想要的或非特异的相互作用区别。抗体与特定抗原结合的能力可以通过酶联免疫 吸附测定法(ELISA)、SPR或生物膜层干涉技术或本领域已知的其他常规结合测定法测定。
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:14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。
可以通过“转染”、“转化”或“转导”将编码本发明的CAR的核酸序列引入细胞。本文使用的术语“转染”、“转化”或“转导”指通过利用物理或化学方法将一种或多种外源多核苷酸引入宿主细胞。许多转染技术在本领域是已知的，并且其包括，例如磷酸钙DNA共沉淀(参见，例如Murray E.J.(编著)，Methods in Molecular Biology，第7卷，Gene Transfer and Expression Protocols,Humana Press(1991))；DEAE-葡聚糖；电穿孔；阳离子脂质体介导的转染；钨颗粒促进的微粒轰击(Johnston,Nature,346:776-777(1990))；以及磷酸锶DNA共沉淀(Brash等人，Mol.Cell Biol.,7:2031-2034(1987))。在感染性颗粒于合适的包装细胞(它们中的许多可商购获得)中生长之后，可将噬菌体载体或病毒载体引入宿主细胞。
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。
如本文中所用，免疫细胞的“活化”或“激活”是指免疫细胞响应并在可测量的水平上表现出本领域技术人员已知的相应细胞的免疫功能的能力。测量免疫细胞活性的方法也是本领域技术人员已知的。
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。
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 PCTCN2022136205-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。
II.本发明的经优化的嵌合抗原受体(CAR)
脑胶质瘤是在颅内发病率最高的一类实体肿瘤，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。IL-13受体α链异构体2(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。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))。
本发明涉及靶向IL13Rα2的经优化的嵌合抗原受体(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。本发明CAR多肽的胞外识别域(也简称为“胞外域”)是特异性识别并结合靶细胞表面上的IL13Rα2的经优化的抗IL13Rα2 scFv序列。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CAR的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。
本发明靶向IL13Rα2的经优化的嵌合抗原受体(CAR)多肽包含：
(1)经优化的抗IL13Rα2 scFv序列，其中所述scFv序列特异性结合IL13Rα2且包含：
(i)重链可变区，其包含根据Kabat编号的
(a)氨基酸序列KYGVH(SEQ ID NO:15)所示的重链互补决定区CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(b)氨基酸序列VKWAGGSTDTDSALMS(SEQ ID NO:16)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
(c)氨基酸序列DHRDAMDY(SEQ ID NO:17)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
(ii)轻链可变区，其包含根据Kabat编号的
(d)氨基酸序列TASLSVSSTYLH(SEQ ID NO:18)所示的轻链互补决定区(CDR L)1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
(e)氨基酸序列STSNLAS(SEQ ID NO:19)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
(f)氨基酸序列HQYHRSPLT(SEQ ID NO:20)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
其中所述氨基酸变化是氨基酸的添加、缺失或取代；
(2)经优化的铰链区/间隔区，其选自
(i)IgG4铰链区(SEQ ID NO 7)，或其具有至少80％的序列同一性的IgG4铰链区；
(ii)CD8铰链区(SEQ ID NO 8)，或其具有至少80％的序列同一性的CD8铰链区。
(3)跨膜区(TM)，其选自
(i)CD28跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:9所示的序列或其具有1-2个氨基酸修饰的变体；
(ii)CD4跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:10所示的序列或其具有1-2个氨基酸修饰的变体；
(iii)CD8跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:11所示的序列或其具有1-2个氨基酸修饰的变体；
(4)共刺激信号结构域(CSD)，其是：
(i)CD28共刺激结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:12所示的序列或其具有1-2个氨基酸修饰的变体；和
(ii)4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:13所示的序列或其具有1-2个氨基酸修饰的变体；
(5)刺激信号结构域(SSD)，为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，SEQ ID NO:14所示的序列或其具有1-10个、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；
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的经优化的抗IL13Rα2 scFv序列，其中所述scFv序列特异性结合IL13Rα2且包含：
(i)重链可变区，其包含SEQ ID NO:2的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，和
(ii)轻链可变区，其包含SEQ ID NO:4的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
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，经优化的抗IL13Rα2 scFv序列)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SEQ ID NO:10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SEQ ID NO:11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SEQ ID NO:9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中的跨膜结构域借助铰链区/间隔区与CAR的胞外区(即，抗IL13Rα2 scFv序列)连接。可以使用多种不同的铰链区/间隔区。在一些实施方案中，铰链区/间隔区包含整个或部分的免疫球蛋白(例如，IgG1、IgG2、IgG3、IgG4)铰链区，即，落在免疫球蛋白的CH1和CH2结构域之间的序列，例如，IgG4Fc铰链或CD8铰链。一些铰链区/间隔区包含免疫球蛋白CH3结构域或CH3结构域和CH2结构两者。免疫球蛋白来源的序列可以包含一个或多个氨基酸修饰，例如，1、2、3、4或5个取代，例如，减少脱靶结合的取代。
在一个实施方案中，所述铰链区/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。
在一些优选的实施方案中，为了使本发明的CAR-T细胞与表达IL13Rα2的靶细胞处于最佳的细胞间距离，从而有利于免疫突触形成，且增强CAR信号，对所述铰链区/间隔区的长度进行了优化，发现针对IL13Rα2而言，短的铰链区/间隔区(例如，SEQ ID NO:7所示的IgG4铰链区)比长的铰链区/间隔区更容易激活CAR-T。
另外，SEQ ID NO:7所示的IgG4铰链区没有保留IgG4的重链恒定区2，因此，不具有结合Fc受体(例如，FcγR)的能力，从而避免了CAR-T细胞的脱靶激活。
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。
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:14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含次级信号结构域(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CAR T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串联的多个共刺激结构域，如CD28共刺激结构域和4-1BB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氨基酸置换。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。
III.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 PCTCN2022136205-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，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，本发明的靶向IL13Rα2的经优化的第三代嵌合抗原受体(CAR)多肽由于其高选择性地特异性靶向IL13Rα2而具有极佳的安全性和体内抗GBM活性。
IV.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相关的实体瘤，例如，胶质瘤。
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1×10 6个－1×10 12个免疫效应细胞、优选地1×10 7个－1×10 10个免疫效应细胞、例如5×10 7个、1×10 8个、5×10 8个、1×10 9个、5×10 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、U87及逆转录病毒包装细胞系PG13和Phoenix ECO购自美国典型培养物保藏中心(ATCC)。U251细胞和U373细胞通过逆转录病毒转导表达绿色荧光蛋白(GFP)基因和萤火虫荧光素酶(Fireflyluciferase，LUC)报告基因(有时也简写为“GL”))。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。
细胞杀伤实验
CAR-T细胞与靶细胞(例如，U251细胞系或U373细胞系)按E:T＝10:1共培养24h，流式细胞术检测对靶细胞的杀伤情况。
细胞因子产生的测定
CAR-T细胞与人GBM细胞系(U373、U251、U87)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或靶细胞U373-GL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。
通过颅内注射胶质瘤细胞构建原位异种移植小鼠模型
实验所用小鼠为6-8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细胞和U373-GL细胞，构建原位异种移植小鼠模型，记为第1天。在第6天，通过尾静脉注射3×10 7个CAR-T细胞。使用小动物成像仪器(IVIS，Xenogen，Alameda，CA，USA)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。
统计分析
使用GraphPadPrism7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
使用逆转录病毒载体构建了图1所示的靶向IL13Rα2的包含针对IL13Rα2的经优 化的scFv序列的CAR。
靶向IL13Rα2的CAR包含位于胞外结构域的经优化的抗IL13Rα2 scFv序列、经优化的IgG4铰链区、CD28跨膜区、位于胞内部分的CD28共刺激结构域、4-1BB共刺激结构域和CD3ζ信号传导结构域。
采用Thermo公司的GeneArt基因合成技术，合成编码抗IL13Rα2 scFv序列的核苷酸序列，其从N端至C端为SEQ ID NO:1所示的抗体重链可变区VH核苷酸序列、编码SEQ ID NO:5所示接头的核苷酸序列和SEQ ID NO:3所示的抗体轻链可变区VL核苷酸序列。
将编码SEQ ID NO:7所示的IgG4铰链区、SEQ ID NO:9所示的CD28跨膜结构域、SEQ ID NO:12所示的CD28共刺激结构域、SEQ ID NO:13所示的4-1BB共刺激结构域和SEQ ID NO:14所示的CD3ζ信号传导结构域的核苷酸序列连接入逆转录病毒载体SFG(Addgene公司，也称为“载体pMSGV1”)，获得载体retro-SFG-IgG4-CD28-4-1BB-CD3ζ，用Sma Ⅰ/Mlu Ⅰ双酶切，然后利用克隆技术将编码抗IL13Rα2 scFv序列的核苷酸序列连接至该载体，37℃连接1h后，转化涂于氨苄抗性LB平板上，通过挑选亚克隆、抽提质粒、酶切验证后测序鉴定重组载体，构建出第三代CAR-T的逆转录病毒载体retro-SFG-IL13Rα2 scFv-IgG4-CD28-4-1BB-CD3ζ(如图1所示)。
使用所构建的IL13Rα2特异性CAR逆转录病毒载体，进行病毒包装。具体而言，将逆转录病毒载体retro-SFG-IL13Rα2 scFv-IgG4-CD28-4-1BB-CD3ζ通过磷酸钙试剂共转染至PG13细胞，转染后48h换液，收取培养上清，72h后再次收取培养上清，过0.45μm
Figure PCTCN2022136205-appb-000003
针式滤器，获得逆转录病毒原液。
将病毒原液4℃、32000r/min超速离心2h，将逆转录病毒沉淀溶解在X-VIVO培养液中。获得逆转录病毒浓缩液，分装后-80℃冻存。
对逆转录病毒浓缩液进行了滴度检测。96孔尖底板每孔铺0.5×10 6个Jurkat细胞，200μL/孔，将逆转录病毒浓缩液稀释100倍，按1:50、1:500和1:2000的比例加入各孔400μL、40μL和10μL。32℃、1200×g离心90min。4h后用DPBS将细胞洗一次后铺于12孔板，转导48h后通过流式细胞术检测。结果表明，本实施例制备的逆转录病毒浓缩液稀释100倍后能够感染Jurkat细胞并表达CAR，因此，使用所述逆转录病毒浓缩液实施了CAR-T细胞的产生。
实施例2.包含抗IL13Rα2 scFv序列的CAR-T细胞的产生
在本实施例中，使用实施例1收获的逆转录病毒浓缩液转导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的逆转录病毒浓缩液转染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，用实施例1制备的逆转录病毒转导来自健康供体的并经CD3/CD28抗体活化的T细胞，7天后用FACS分析检测基因修饰的T细胞，T细胞表面稳定表达包含抗IL13Rα2 scFv序列的CAR。
实施例3.包含抗IL13Rα2 scFv序列的CAR-T细胞的脱颗粒能力检测
CAR-T细胞对肿瘤细胞的杀伤效果可以通过CAR-T细胞的脱颗粒能力，即，CD107a的表达来检测。
实施例2产生的包含抗IL13Rα2 scFv序列的CAR-T细胞胞浆内含有高浓度以囊泡形式存在的细胞毒性颗粒，如穿孔素、颗粒酶等。溶酶体相关膜蛋白-1(LAMP-1)也称为CD107a是细胞脱颗粒的标志物，在肿瘤细胞的刺激下，CD107a的表达与实施例2产生的CAR-T细胞介导的靶细胞裂解相关。
将实施例2产生的包含抗IL13Rα2 scFv序列的CAR-T细胞与U373及U251两种神经胶质母细胞瘤(GBM)细胞株按E:T＝10:1进行共培养6h后，用流式细胞仪检测所述CAR-T细胞的CD107a的表达率，所述CD107a表达率与CAR-T细胞的杀伤活性呈正相关。
结果显示，包含抗IL13Rα2 scFv序列的CAR-T细胞单独培养时CD107a的表达率为0％(图2)；包含抗IL13Rα2 scFv序列的CAR-T细胞与U373细胞株共培养时CD107a表达率为3.7％(图3)；包含抗IL13Rα2 scFv序列的CAR-T细胞与U251细胞株共培养时CD107a表达率为2％(图4)；所述CAR-T细胞与U373细胞株或U251细胞株共培养后CD107a的表达率均显著高于单独培养的所述CAR-T细胞，且所述CAR-T细胞与U373细胞株共培养组(图5中表示为“CAR-T+U373”)和所述CAR-T细胞与U251细胞株共培养组(图5中表示为“CAR-T+U251”)之间不具有显著性差异(图5中，“NS”表示“无显著性差异”；＊表示P<0.05，有显著性差异)。
实施例4.包含抗IL13Rα2 scFv序列的CAR-T细胞对肿瘤细胞的杀伤效果
将实施例2的包含抗IL13Rα2 scFv序列的CAR-T细胞与U373、U251细胞株按E:T＝10:1共培养24h后，流式细胞术检测所述CAR-T细胞对U373细胞株、U251细胞 株的杀伤情况。
结果如图6所示，与所述CAR-T细胞共培养24h后，U373细胞的细胞数由91.4％降到了5.1％，杀伤效率为94.42％；U251细胞的细胞数由48.3％降到了1.7％，杀伤效率为96.48％。
另外，由图7可见，所述CAR-T细胞对U373细胞株的杀伤效率与所述CAR-T细胞对U251细胞株的杀伤效率之间无显著性差异，杀伤效率均在90％以上(图7中，“NS”表示“无显著性差异”)。
实施例5.测定包含抗IL13Rα2 scFv序列的CAR-T细胞产生的细胞因子
包含抗IL13Rα2 scFv序列的CAR-T细胞与IL13Rα2阳性的人GBM细胞株(U373、U251、U87细胞)以E:T＝10:1的比例分别共培养24小时后，采用CBA法对培养上清液中的干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)进行检测，其中所述IFN-γ、TNF-α与CAR-T细胞对靶细胞的杀伤能力呈正相关。
结果分别如图8和图9所示，表明了包含抗IL13Rα2 scFv序列的CAR-T细胞(图中缩写为“IL13Rα2-CAR-T”)与U373、U251、U87细胞株共培养后，上清液中IFN-γ、TNF-α均显著升高，且与对照组CD19-CAR-T细胞具有统计学显著性差异(P＜0.05)，其中所述对照CD19-CAR-T细胞的制备类似于实施例1和实施例2，不同之处仅在于用SEQ ID NO:23所示的抗CD19scFv序列替换掉抗IL13Rα2 scFv序列。
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10和图11显示了RTCA法检测的肿瘤细胞的细胞生长结果，体外评价了包含抗IL13Rα2 scFv序列的CAR-T细胞的抗肿瘤能力。
如图10所示，U373肿瘤细胞对照组(△线)随着时间的延长，肿瘤细胞数显著升高；包含抗IL13Rα2 scFv序列的CAR-T细胞与U373肿瘤细胞共培养组(○线)在共培养20h后，肿瘤细胞数显著下降，所述CAR-T细胞对肿瘤细胞的杀伤显著。
如图11所示，U251肿瘤细胞对照组(△线)随着时间的延长，肿瘤细胞数显著升高；包含抗IL13Rα2 scFv序列的CAR-T细胞与U251肿瘤细胞共培养组(○线)在共培养20h后，肿瘤其细胞数显著下降，所述CAR-T细胞对肿瘤细胞的杀伤现象显著。
因此，本发明的包含抗IL13Rα2 scFv序列的CAR-T细胞均显著抑制U373和U251肿瘤细胞生长，具有明显的抗肿瘤活性。
实施例6.包含抗IL13Rα2 scFv序列的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。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制备的包含抗IL13Rα2 scFv序列的CAR-T细胞(图中缩写为“IL13Rα2-CAR-T”)。
实验设置了阳性对照组，使用的是IL13-CAR-T。所述阳性对照IL13-CAR-T细胞的制备类似于实施例1和实施例2，不同之处仅在于用SEQ ID NO:24所示的IL13序列替换掉抗IL13Rα2 scFv序列。
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U251-GL生成的异种移植模型小鼠
如图12和图13所示，阳性对照组IL13-CAR-T治疗U251原位动物模型的最长生存周期为34天，而经本发明的IL13Rα2-CAR-T治疗的小鼠最长生存周期为50天。
ii)对于U373-GL生成的异种移植模型小鼠
如图14和图15所示，阳性对照组IL13-CAR-T治疗U373原位动物模型的最长生存周期为60天，而经本发明的IL13Rα2-CAR-T治疗的小鼠最长生存周期为64天。
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。
序列表
Figure PCTCN2022136205-appb-000004
Figure PCTCN2022136205-appb-000005
Figure PCTCN2022136205-appb-000006

Claims (11)

  1. 一种靶向IL13Rα2的嵌合抗原受体(CAR)多肽，其包含：
    (1)抗IL13Rα2 scFv序列，其中所述scFv序列特异性结合IL13Rα2且包含：
    (i)重链可变区，其包含根据Kabat编号的
    (a)氨基酸序列KYGVH(SEQ ID NO:15)所示的重链互补决定区CDR H1、或所述CDR H1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
    (b)氨基酸序列VKWAGGSTDTDSALMS(SEQ ID NO:16)所示的CDR H2、或所述CDR H2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
    (c)氨基酸序列DHRDAMDY(SEQ ID NO:17)所示的CDR H3、或所述CDR H3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
    (ii)轻链可变区，其包含根据Kabat编号的
    (d)氨基酸序列TASLSVSSTYLH(SEQ ID NO:18)所示的轻链互补决定区(CDR L)1、或所述CDR L1的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
    (e)氨基酸序列STSNLAS(SEQ ID NO:19)所示的CDR L2、或所述CDR L2的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；和
    (f)氨基酸序列HQYHRSPLT(SEQ ID NO:20)所示的CDR L3、或所述CDR L3的不超过2个氨基酸变化或不超过1个氨基酸变化的变体；
    其中所述氨基酸变化是氨基酸的添加、缺失或取代；
    (2)铰链区/间隔区，其选自
    (i)IgG4铰链区(SEQ ID NO 7)，或其具有至少80％的序列同一性的IgG4铰链区；
    (ii)CD8铰链区(SEQ ID NO 8)，或其具有至少80％的序列同一性的CD8铰链区；
    (3)跨膜区(TM)，其选自
    (i)CD28跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:9所示的序列或其具有1-2个氨基酸修饰的变体；
    (ii)CD4跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:10所示的序列或其具有1-2个氨基酸修饰的变体；
    (iii)CD8跨膜结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:11所示的序列或其具有1-2个氨基酸修饰的变体；
    (4)共刺激信号结构域(CSD)，其是：
    (i)CD28共刺激结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:12所示的序列或其具有1-2个氨基酸修饰的变体；和
    (ii)4-1BB共刺激结构域或其具有1-5个氨基酸修饰的变体，例如，SEQ ID NO:13所示的序列或其具有1-2个氨基酸修饰的变体；
    (5)刺激信号结构域(SSD)，为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，SEQ ID NO:14所示的序列或其具有1-10个、1-5个氨基酸修饰的变体。
  2. 根据权利要求1所述的CAR多肽，其从N端到C端包含：
    (1)抗IL13Rα2 scFv序列，其中所述scFv序列特异性结合IL13Rα2且包含：
    (i)重链可变区，其包含根据Kabat编号的
    (a)氨基酸序列KYGVH(SEQ ID NO:15)所示的CDR H1；
    (b)氨基酸序列VKWAGGSTDTDSALMS(SEQ ID NO:16)所示的CDR H2；和
    (c)氨基酸序列DHRDAMDY(SEQ ID NO:17)所示的CDR H3；和
    (ii)轻链可变区，其包含根据Kabat编号的
    (d)氨基酸序列TASLSVSSTYLH(SEQ ID NO:18)所示的CDR L1；
    (e)氨基酸序列STSNLAS(SEQ ID NO:19)所示的CDR L2；和
    (f)氨基酸序列HQYHRSPLT(SEQ ID NO:20)所示的CDR L3；
    (2)铰链区/间隔区，其选自
    (i)IgG4铰链区(SEQ ID NO 7)，或其具有至少90％、至少95％的序列同一性的IgG4铰链区；
    (ii)CD8铰链区(SEQ ID NO 8)，或其具有至少90％、至少95％的序列同一性的CD8铰链区；
    (3)跨膜区(TM)，其选自
    (i)SEQ ID NO:9所示的CD28跨膜结构域或其具有1个氨基酸修饰的变体；
    (ii)SEQ ID NO:10所示的CD4跨膜结构域或其具有1个氨基酸修饰的变体；
    (iii)SEQ ID NO:11所示的CD8跨膜结构域或其具有1个氨基酸修饰的变体；
    (4)共刺激信号结构域(CSD)，其是：
    (i)SEQ ID NO:12所示的CD28共刺激结构域或其具有1个氨基酸修饰的变体；和
    (ii)SEQ ID NO:13所示的4-1BB共刺激结构域或其具有1个氨基酸修饰的变体；
    (5)刺激信号结构域(SSD)，为SEQ ID NO:14所示的CD3ζ信号传导结构域或其具有1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。
  3. 根据权利要求2所述的CAR多肽，其包含：
    (1)抗IL13Rα2 scFv序列，其中所述scFv序列特异性结合IL13Rα2且包含：
    (i)重链可变区，其包含SEQ ID NO:2的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，和
    (ii)轻链可变区，其包含SEQ ID NO:4的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
    例如，(i)重链可变区，其包含SEQ ID NO:2的序列，和
    (ii)轻链可变区，其包含SEQ ID NO:4的序列；
    (2)铰链区/间隔区，其选自
    (i)IgG4铰链区(SEQ ID NO 7)，或其具有至少90％、至少95％的序列同一性的IgG4铰链区；
    (ii)CD8铰链区(SEQ ID NO 8)，或其具有至少90％、至少95％的序列同一性的CD8铰链区；
    (3)跨膜区(TM)，其选自
    (i)SEQ ID NO:9所示的CD28跨膜结构域或其具有1个氨基酸修饰的变体；
    (ii)SEQ ID NO:10所示的CD4跨膜结构域或其具有1个氨基酸修饰的变体；
    (iii)SEQ ID NO:11所示的CD8跨膜结构域或其具有1个氨基酸修饰的变体；
    (4)共刺激信号结构域(CSD)，其是：
    (i)SEQ ID NO:12所示的CD28共刺激结构域或其具有1个氨基酸修饰的变体；和
    (ii)SEQ ID NO:13所示的4-1BB共刺激结构域或其具有1个氨基酸修饰的变体；
    (5)刺激信号结构域(SSD)，为SEQ ID NO:14所示的CD3ζ信号传导结构域或其具有1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。
  4. 根据权利要求3所述的CAR多肽，其包含：
    (1)抗IL13Rα2 scFv序列，其中所述scFv序列特异性结合IL13Rα2且包含：
    (i)SEQ ID NO:2所示的重链可变区，和
    (ii)SEQ ID NO:4所示的轻链可变区；
    (2)SEQ ID NO 7所示的铰链区；
    (3)跨膜区(TM)，其是SEQ ID NO:9所示的CD28跨膜结构域；
    (4)共刺激信号结构域(CSD)，其是：
    (i)SEQ ID NO:12所示的CD28共刺激结构域；和
    (ii)SEQ ID NO:13所示的4-1BB共刺激结构域；
    (5)SEQ ID NO:14所示的CD3ζ信号传导结构域。
  5. 根据权利要求1-4中任一项所述的CAR多肽，其还包含位于N端的信号肽序列，例如，SEQ ID NO:21所示的信号肽序列，
    优选地，所述CAR多肽具有SEQ ID NO:22所示的氨基酸序列或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上的同一性的氨基酸序列。
  6. 编码权利要求1-5中任一项所述的CAR多肽的核酸分子。
  7. 载体，其包含权利要求6所述的核酸分子，例如，所述载体选自DNA载体、RNA载体、质粒、慢病毒载体、腺病毒载体或逆转录病毒载体。
  8. 细胞，其包含权利要求1-5中任一项所述的CAR多肽、权利要求6所述的核酸、或权利要求7所述的载体，所述细胞是例如免疫效应细胞，例如，所述免疫效应细胞是T细胞、NK细胞，例如，所述T细胞、NK细胞是自体T细胞、NK细胞或同种异体T细胞、NK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。
  9. 制备权利要求8的细胞的方法，包括用权利要求7所述的载体导入所述细胞，例如，自人PBMC分离T细胞、NK细胞，用权利要求7所述的载体转导所述分离的T细胞、NK细胞。
  10. 权利要求8所述的细胞的用途，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组合物，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。
  11. 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权利要求8所述的细胞，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。
PCT/CN2022/136205 2022-09-29 2022-12-02 靶向IL13Rα2的经优化的嵌合抗原受体及其用途 WO2024066026A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211201075.7A CN115960257B (zh) 2022-09-29 2022-09-29 靶向IL13Rα2的经优化的嵌合抗原受体及其用途
CN202211201075.7 2022-09-29

Publications (1)

Publication Number Publication Date
WO2024066026A1 true WO2024066026A1 (zh) 2024-04-04

Family

ID=87357564

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/136205 WO2024066026A1 (zh) 2022-09-29 2022-12-02 靶向IL13Rα2的经优化的嵌合抗原受体及其用途

Country Status (2)

Country Link
CN (1) CN115960257B (zh)
WO (1) WO2024066026A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104936621A (zh) * 2012-11-07 2015-09-23 辉瑞公司 抗IL-13受体α2抗体和抗体-药物缀合物
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
CN112236151A (zh) * 2018-03-14 2021-01-15 西雅图儿童医院（Dba西雅图儿童研究所） 用于肿瘤特异性T细胞免疫疗法的IL-13受体α2(IL13RA2)嵌合抗原受体
WO2021154543A2 (en) * 2020-01-31 2021-08-05 City Of Hope TARGETED CHIMERIC ANTIGEN RECEPTOR MODIFIED T CELLS FOR TREATMENT OF IL13Rα2 POSITIVE MALIGNANCIES
CN114014941A (zh) * 2022-01-10 2022-02-08 卡瑞济(北京)生命科技有限公司 靶向IL13Rα2的嵌合抗原受体及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104936621A (zh) * 2012-11-07 2015-09-23 辉瑞公司 抗IL-13受体α2抗体和抗体-药物缀合物
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
CN112236151A (zh) * 2018-03-14 2021-01-15 西雅图儿童医院（Dba西雅图儿童研究所） 用于肿瘤特异性T细胞免疫疗法的IL-13受体α2(IL13RA2)嵌合抗原受体
WO2021154543A2 (en) * 2020-01-31 2021-08-05 City Of Hope TARGETED CHIMERIC ANTIGEN RECEPTOR MODIFIED T CELLS FOR TREATMENT OF IL13Rα2 POSITIVE MALIGNANCIES
CN114014941A (zh) * 2022-01-10 2022-02-08 卡瑞济(北京)生命科技有限公司 靶向IL13Rα2的嵌合抗原受体及其用途

Also Published As

Publication number Publication date
CN115960257B (zh) 2023-10-27
CN115960257A (zh) 2023-04-14

Similar Documents

Publication Publication Date Title
US11085021B2 (en) Compositions and methods for TCR reprogramming using fusion proteins
US11242376B2 (en) Compositions and methods for TCR reprogramming using fusion proteins
US20210079057A1 (en) Compositions and methods for tcr reprogramming using fusion proteins
CN107074929B (zh) 嵌合自身抗体受体t细胞的组合物和方法
JP7475088B2 (ja) ヒトメソセリンを特異的に認識する細胞表面分子、ｉｌ－７、及びｃｃｌ１９を発現する免疫担当細胞
EP3805270A1 (en) Improved anti-cd19 car-t cell
CN114014941B (zh) 靶向IL13Rα2的嵌合抗原受体及其用途
JP2019513394A (ja) キメラアロ抗原受容体ｔ細胞の組成物および方法
CN115175695A (zh) 制备表达嵌合抗原受体的细胞的方法
JP2021536245A (ja) 血液中または濃縮ｐｂｍｃ中のリンパ球を遺伝子改変するための方法および組成物
WO2021133959A2 (en) Compositions and methods for gamma delta tcr reprogramming using fusion proteins
WO2024055339A1 (zh) 用于制备和扩增通用型人源化抗cd19 car-nk细胞的方法及其用途
JP7057975B2 (ja) 殺細胞効果を有するキメラ抗原受容体遺伝子改変リンパ球
WO2024066026A1 (zh) 靶向IL13Rα2的经优化的嵌合抗原受体及其用途
WO2021259334A1 (zh) 自我调节型嵌合抗原受体及其在肿瘤免疫中的应用
JP6842688B2 (ja) キメラ抗原受容体
CN115807020A (zh) 白介素15受体α装甲CAR-T细胞在降低白介素15诱导的细胞毒性中的用途
EP4199959A1 (en) A chimeric antigen receptor construct encoding a checkpoint inhibitory molecule and an immune stimulatory cytokine and car-expressing cells recognizing cd44v6
JP2015092865A (ja) ヒト化抗ｃｄ２０キメラ抗原レセプター
WO2023130462A1 (zh) 靶向IL13Rα2的嵌合抗原受体及其用途
CN115925985B (zh) Car-t细胞及其在非小细胞肺癌治疗中的应用
JP2019517518A (ja) 早期治療選択肢としての養子細胞療法