WO2018149358A1 - 靶向il-13ra2的抗体及其应用 - Google Patents

靶向il-13ra2的抗体及其应用 Download PDF

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Publication number
WO2018149358A1
WO2018149358A1 PCT/CN2018/075859 CN2018075859W WO2018149358A1 WO 2018149358 A1 WO2018149358 A1 WO 2018149358A1 CN 2018075859 W CN2018075859 W CN 2018075859W WO 2018149358 A1 WO2018149358 A1 WO 2018149358A1
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seq
antibody
sequence
chain variable
variable region
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PCT/CN2018/075859
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English (en)
French (fr)
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王鹏
王华茂
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科济生物医药（上海）有限公司
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Priority claimed from CN201810079015.XA external-priority patent/CN108456250A/zh
Priority to KR1020197027172A priority Critical patent/KR20190127740A/ko
Priority to EP18753921.8A priority patent/EP3594241A4/en
Priority to AU2018221110A priority patent/AU2018221110B9/en
Priority to NZ756954A priority patent/NZ756954A/en
Priority to JP2019544896A priority patent/JP7064663B2/ja
Application filed by 科济生物医药（上海）有限公司 filed Critical 科济生物医药（上海）有限公司
Priority to US16/486,481 priority patent/US11530270B2/en
Priority to CA3053592A priority patent/CA3053592A1/en
Priority to RU2019128921A priority patent/RU2756623C2/ru
Priority to SG11201907528TA priority patent/SG11201907528TA/en
Publication of WO2018149358A1 publication Critical patent/WO2018149358A1/zh
Priority to IL26868719A priority patent/IL268687A/en

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    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464419—Receptors for interleukins [IL]
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present invention relates to the field of tumor immunotherapy or diagnosis, and more particularly to antibodies that specifically recognize IL-13RA2 and uses thereof.
  • MG Malignant gliomas
  • MG Malignant gliomas
  • pleomorphic glioblastoma and glioblastoma have 20,000 new cases each year in the United States.
  • MG is a rare disease, its malignancy and mortality are very high.
  • the current standard of treatment has very limited results, and the five-year survival rate after surgery and radiotherapy is also very low.
  • new treatments are urgent needs of the majority of patients.
  • Interleukin-13 Receptor subunit alpha 2 (IL-13RA2) is a tumor-specific marker that is highly expressed on the surface of malignant tumor cells such as human glioma (Dehinski et al., (1995) Clin. Cancer Res. 1, 1253-1258).
  • Human IL-13RA2 has been the target of treatment for human gliomas. It has attracted the attention of the US FDA since 1988.
  • the organization has prepared the drug IL-13-PE38 and the human IL- for human IL-13RA2 therapeutic targets. Single-chain antibody scFv-PE fusion molecule of 13RA2.
  • IL-13-PE38 has achieved efficacy in the treatment of malignant tumors such as glioma, head and neck tumors, ovarian cancer and kidney cancer and has been approved by the US FDA for clinical treatment, due to the treatment process, IL-13-PE38 not only binds to human IL-13RA2 specifically expressed on the surface of tumor cells, but also binds to IL13-RA1 expressed on the surface of normal tissue cells, damaging normal tissues and cells. Further application of IL-13-PE38 is limited due to the lack of stringent targeting.
  • the present invention aims to find antibodies specific for IL-13RA2 and to develop immune effector cells targeting IL-13RA2.
  • the present invention provides an antibody that specifically recognizes IL-13RA2, which binds to U251 cells endogenously expressing IL-13RA2 with a relative affinity EC50 of not more than 100 nM, preferably no more than 10 nM, More preferably, it is 0.01-10 nM.
  • the processing of the relative affinity data uses GraphPad Prism 5 software (GraphPad Software, Inc).
  • the antibody is selected from any of the following:
  • an antibody comprising a heavy chain variable region comprising the HCDR1 set forth in SEQ ID NO: 9, 45, 46, 47, 48, 49, 50, 51, 63, or 64, And/or comprising HCDR2 as set forth in SEQ ID NO: 10, 52, 53, 54, 55, 56, 57, 58, 65, or 66, and/or comprising any of SEQ ID NO: 11 or SEQ ID NO: HCDR3 as shown;
  • an antibody comprising a light chain variable region comprising the LCDR1 set forth in SEQ ID NO: 13, and/or comprising the LCDR2 set forth in SEQ ID NO: 14, and/or comprising the SEQ ID NO: 15 or LCDR3 shown in any one of SEQ ID NO: 16;
  • an antibody comprising (1) a heavy chain variable region of said antibody and (2) a light chain variable region of said antibody;
  • the antibody is selected from any of the following:
  • An antibody comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 4 and SEQ ID NO a sequence of variants of 8;
  • an antibody comprising a heavy chain variable region having the SEQ ID NO: 2, 6, 29, 31, 33, 35, 37, 39, 41, 43, 59 or 61 a sequence, or a variant of the above sequence;
  • An antibody comprising (1) a heavy chain variable region of the antibody and (2) a light chain variable region of the antibody.
  • the light chain variable region of the antibody comprises LCDR1 of SEQ ID NO: 13, LCDR2 and SEQ ID NO: 15 or SEQ ID NO: 16 of SEQ ID NO: LCDR3 shown.
  • the light chain variable region of the antibody has the sequence set forth in SEQ ID NO: 4 or 8, or has at least 80%, eg, 85%, 90%, 91 of any of the above sequences. Sequences of %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity.
  • the heavy chain variable region of the antibody comprises HCDR1 set forth in SEQ ID NO: 9, 45, 46, 47, 48, 49, 50, 51, 63 or 64, SEQ ID NO: 10.
  • the heavy chain variable region of the antibody has the heavy chain variable region of the antibody having SEQ ID NOs: 2, 6, 29, 31, 33, 35, 37, 39, 41, 43 a sequence of 59 or 61, or having at least 80%, more preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 of any of the above sequences %, 99% similarity sequence.
  • the CDR regions of the light chain variable region and the CDR regions of the heavy chain variable region have the following optional sequences or variants thereof:
  • LCDR1 shown in SEQ ID NO: 13 LCDR2 shown in SEQ ID NO: 14 and LCDR3 shown in SEQ ID NO: 15;
  • LCDR1 shown in SEQ ID NO: 13 LCDR2 shown in SEQ ID NO: 14 and LCDR3 shown in SEQ ID NO: 15;
  • LCDR1 shown in SEQ ID NO: 13 LCDR2 shown in SEQ ID NO: 14 and LCDR3 shown in SEQ ID NO: 15;
  • LCDR1 shown in SEQ ID NO: 13 LCDR2 shown in SEQ ID NO: 14 and LCDR3 shown in SEQ ID NO: 15; HCDR1 shown in SEQ ID NO: 49, SEQ ID NO: 58 HCDR2 and HCDR3 as shown in SEQ ID NO:11.
  • the light chain variable region has the sequence of the sequence of SEQ ID NO: 4 or a variant thereof, the heavy chain variable region having the sequence of SEQ ID NO: 2 or a variant thereof ;
  • the light chain variable region has the sequence of SEQ ID NO: 8 or a variant thereof, and the heavy chain variable region has the sequence of SEQ ID NO: 6 or a variant thereof ;
  • the light chain variable region has the sequence of the sequence of SEQ ID NO: 8 or a variant thereof, the heavy chain variable region having the sequence of SEQ ID NO: 61 or a variant thereof ;
  • the light chain variable region has the sequence of the sequence of SEQ ID NO: 4 or a variant thereof, and the heavy chain variable region has the sequence of SEQ ID NO: 29 or a variant thereof ;
  • the light chain variable region has the sequence of the sequence of SEQ ID NO: 8 or a variant thereof, the heavy chain variable region having the sequence of SEQ ID NO: 59 or a variant thereof ;
  • the light chain variable region has the sequence of the sequence of SEQ ID NO: 4 or a variant thereof, the heavy chain variable region having the sequence of SEQ ID NO: 39 or a variant thereof ;
  • the light chain variable region has the sequence of the sequence of SEQ ID NO: 4 or a variant thereof, the heavy chain variable region having the sequence of SEQ ID NO: 31 or a variant thereof ;
  • the light chain variable region has the sequence of the sequence of SEQ ID NO: 4 or a variant thereof, the heavy chain variable region having the sequence of SEQ ID NO: 35 or a variant thereof ;
  • the light chain variable region has the sequence of the sequence of SEQ ID NO: 4 or a variant thereof, the heavy chain variable region having the sequence of SEQ ID NO: 33 or a variant thereof ;
  • the light chain variable region has the sequence of the sequence of SEQ ID NO: 4 or a variant thereof, the heavy chain variable region having the sequence of SEQ ID NO: 37 or a variant thereof ;
  • the light chain variable region has the sequence of the sequence of SEQ ID NO: 4 or a variant thereof, the heavy chain variable region having the sequence of SEQ ID NO: 41 or a variant thereof ;
  • the light chain variable region has the sequence of the sequence of SEQ ID NO: 4 or a variant thereof, the heavy chain variable region having the sequence of SEQ ID NO: 43 or a variant thereof .
  • the invention provides an antibody that specifically recognizes IL-13RA2, which antibody recognizes the same epitope as the antibody of the first aspect.
  • the invention provides an antibody that specifically recognizes IL-13RA2, which competitively binds IL-13RA2 to an antibody of the first aspect.
  • the invention provides a nucleic acid encoding the antibody of the first to third aspects.
  • the present invention provides an expression vector comprising the nucleic acid of the fourth aspect.
  • the present invention provides a host cell comprising the expression vector of the fifth aspect or the nucleic acid according to the fourth aspect integrated in the genome.
  • the present invention provides a multifunctional immunoconjugate comprising:
  • a functional molecule linked thereto said functional molecule being selected from the group consisting of a molecule that targets a tumor surface marker, a molecule that inhibits tumors, a molecule that targets a surface marker of an immune cell, or a detectable label.
  • the molecule that targets a tumor surface marker is an antibody or ligand that binds to other tumor surface markers other than IL-13RA2;
  • the tumor suppressing molecule is an antitumor cytokine or an antitumor toxin; preferably, the cytokine is selected from the group consisting of IL-12, IL-15, type I interferon, and TNF-alpha.
  • the molecule that targets a surface marker of an immune cell is an antibody that binds to a surface marker of an immune cell.
  • the binding immunocyte surface marker is selected from the group consisting of: CD3, CD16, CD28 More preferably, the antibody that binds to an immunocyte surface marker is an anti-CD3 antibody.
  • the molecule that targets a surface marker of an immune cell is an antibody that binds to a T cell surface marker, and the antibody of any of the first aspect to the third aspect forms a T cell involved.
  • Bifunctional antibody is an antibody that binds to a T cell surface marker, and the antibody of any of the first aspect to the third aspect forms a T cell involved.
  • the multifunctional immunoconjugate is a fusion polypeptide, further comprising a linker peptide between the antibody of any of the first aspect to the third aspect, and the functional molecule linked thereto.
  • the invention provides a nucleic acid encoding the multifunctional immunoconjugate of the seventh aspect.
  • the present invention provides the chimeric antigen receptor of the antibody of the first to third aspect, wherein the chimeric antigen receptor comprises a sequence of linkages: the first to the third aspect Antibodies, transmembrane regions, and intracellular signaling regions.
  • the intracellular signal region is selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FcR ⁇ (FCER1G), FcR ⁇ (Fc ⁇ R1b), CD79a, CD79b, Fc ⁇ RIIa, DAP10, and DAP12. Domain, or a combination thereof.
  • the intracellular signal region further has a costimulatory signaling domain comprising a functional signaling domain selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11
  • the chimeric antigen receptor comprises the following sequentially linked antibodies, transmembrane regions, and intracellular signal regions:
  • the antibody of the first to third aspects the transmembrane region of the CD28 molecule, the intracellular signal region of the CD28 molecule, and CD3 ⁇ ; or
  • the antibody of the first to third aspects the transmembrane region of the CD28 molecule, the intracellular signal region of the CD28 molecule, CD137 and CD3 ⁇ .
  • the invention provides a nucleic acid encoding the chimeric antigen receptor of the ninth aspect.
  • the present invention provides an expression vector, the nucleic acid of the tenth aspect.
  • the present invention provides a virus comprising the vector of the eleventh aspect.
  • the present invention provides a chimeric antigen receptor-modified immune cell, which is transduced with the nucleic acid of the tenth aspect, or the expression vector of the eleventh aspect or the twelfth aspect a virus; or a surface thereof expressing the chimeric antigen receptor of the ninth aspect;
  • the immune cells are: T lymphocytes, NK cells or NKT lymphocytes.
  • the immune cell further carries a coding sequence for a foreign cytokine;
  • chemokine receptor preferably, the chemokine receptor comprises: CCR;
  • siRNA that reduces PD-1 expression or a protein that blocks PD-L1; or endogenous PD-1 in its cells is knocked out by gene editing techniques; or
  • the present invention provides a pharmaceutical composition comprising:
  • the antibody of the first or third aspect or the nucleic acid encoding the antibody or
  • a chimeric antigen receptor according to the ninth aspect, or a nucleic acid encoding the chimeric antigen receptor;
  • the chimeric antigen receptor-modified immune cell of the thirteenth aspect is a chimeric antigen receptor-modified immune cell of the thirteenth aspect.
  • the present invention provides a kit comprising:
  • a container or an antibody according to the first to third aspect, or a nucleic acid encoding the antibody; or the immunoconjugate of the seventh aspect or the nucleic acid encoding the same; or the ninth aspect A chimeric antigen receptor or a nucleic acid encoding the chimeric antigen receptor; or a chimeric antigen receptor modified immune cell according to the thirteenth aspect.
  • the invention provides the antibody of the first or third aspect, or the nucleic acid encoding the antibody; or the immunoconjugate of the seventh aspect or the nucleic acid encoding the same; or the ninth aspect
  • the tumor expressing IL-13RA2 is brain cancer, pancreatic cancer, ovarian cancer, kidney cancer, bladder cancer, pancreatic cancer, gastric cancer, intestinal cancer, head and neck cancer, thyroid cancer, prostate cancer, Kaposi's sarcoma.
  • the brain cancer is selected from the group consisting of astrocytoma, meningioma, oligodendroglioma, and glioma.
  • Figure 1 shows an SDS electropherogram (reduction conditions) of IL-13RA2_huFc, IL13RA1_huFc;
  • Figure 2 shows the binding of 31C2, 32H4 to IL-13RA2 and IL13Ra1 by ELISA
  • Figure 3 shows the binding of antibody 31C2, 32H4 to murine IL-13RA2 by ELISA
  • FIG. 4 shows the binding of FACs detection antibody 31C2, 32H4 to U251 (IL-13RA2 positive) and 293T (IL-13RA2 negative) cells;
  • FIG. 5 shows the affinity of Biacore assay antibody 31C2, 32H4 (scFv_Fc);
  • Figure 6 shows the EC50 of FACs detecting antibody 31C2, 32H4 binding to U215 cells
  • Figure 7 shows primer information for affinity maturation
  • Figure 8 shows the dissociation constant Kd of 10 clones screened after affinity maturation
  • Figure 9A shows the affinity matured clone heavy chain sequence alignment of 31C2
  • Figure 9B shows the sequence of HCDR1 and HCDR2 of the 31C2 affinity matured clone
  • Figure 9C shows the 32H4 affinity matured clone heavy chain sequence alignment
  • Figure 9D shows Affinity of the 32H4 mature mature cloned sequences of HCDR1 and HCDR2;
  • Figure 10A shows the affinity binding antibody binding dissociation constant
  • Figure 10B shows the specific identification results of the antibodies 5D7, 2C7, 5G3, 2D4, 2D3, 1B11;
  • Figure 11A shows the results of assay of the yield of the scFv_Fc form of the antibody in the 30 ml expression system and the degree of aggregation of the purified product after affinity maturation;
  • Figure 11B-G shows the affinity of the scFv_Fc form of the antibody;
  • Figure 11H shows the result of antibody binding dissociation constant ;
  • Figure 12 shows the EC50 binding to U251 cells of the scFv_Fc form of the antibodies 5D7, 2C7, 5G3, 2D4, 2D3, 1B11;
  • Figure 13 shows the in vitro killing activity of different CAR-T cells.
  • the inventors have obtained intensive research and screening to obtain antibodies that specifically recognize IL-13RA2, including single-chain antibodies and humanized antibodies.
  • the antibody of the present invention can be applied to the preparation of various targeted antitumor drugs as well as drugs for diagnosing tumors.
  • IL-13RA2 also referred to herein as CD213A2
  • CD213A2 is a subunit of the interleukin-13 receptor complex. Transmembrane protein consisting of 380 amino acid residues (NCBI Reference Sequence: NP_000631.1). It is similar to IL-13RA1 (NCBI Reference Sequence: NP_001551.1) and binds strongly to IL-13 but has no intracellular signal domain.
  • antibody refers to an antigen binding protein of the immune system, including intact full length antibodies having an antigen binding region, and also includes fragments having an "antigen binding portion” or “antigen binding region”, or a single strand thereof such as a single Chain variable fragments (scFv), also including variants of the antibodies provided herein.
  • scFv single Chain variable fragments
  • Antibody fragments include, but are not limited to, (i) Fab fragments consisting of VL, VH, CL, and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments consisting of VH and CH1 domains, (iii) An Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a single variable region (Ward et al., 1989, Nature 341: 544-546); (v) a F(ab')2 fragment a bivalent fragment comprising two linked Fab fragments; (vi) a single-chain Fv molecule antigen binding site; (vii) a bispecific single chain Fv dimer (PCT/US92/09965); (viii) "two a "poly” or “trisomy", a multivalent or multispecific fragment constructed by genetic fusion; and (ix) a scFv genetically fused to the same or a different antibody.
  • Fc or "Fc region” as used herein includes a polypeptide comprising an antibody constant region other than the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinges at the N-terminus of these domains.
  • IgA and IgM Fc can include a J chain.
  • Fc includes the immunoglobulin domains C ⁇ 2 and C ⁇ 3 and the hinge between C ⁇ 1 and C ⁇ 2.
  • the human IgG heavy chain Fc region is generally defined as comprising residues C226 or P230 at its carboxy terminus, where numbering is according to the EU index of Kabat.
  • Fc is defined herein to include residue P232 to its carboxy terminus, where numbering is based on the EU index in Kabat.
  • Fc may refer to this region of isolation, or to that region of an Fc polypeptide, such as an antibody, environment.
  • the above “hinge” includes a flexible polypeptide comprising an amino acid between the first and second constant domains of the antibody.
  • the IgG CH1 domain ends at position EU220 and the IgG CH2 domain begins at residue EU237.
  • the antibody hinge herein is defined to include 221 (D221 of IgG1) to 231 (A231 of IgG1), where the numbering is according to the EU index of Kabat.
  • variant refers to one or more active polypeptides having substantially the same amino acid sequence as the sequences of the antibodies provided herein or encoded by substantially the same nucleotide sequence.
  • the variant has the same or similar activity as the antibodies provided in the examples of the present application.
  • a variant has at least one amino acid modification compared to a parent antibody.
  • the variant sequences herein preferably have at least about 80%, most preferably at least about 90%, more preferably at least about 95%, more preferably at least about 98%, and most preferably at least about the parent antibody sequence. 99% amino acid sequence identity.
  • a variant may refer to the antibody itself, and may also refer to a composition comprising a parent antibody.
  • the term "amino acid modification” includes amino acid substitutions, additions and/or deletions, "amino acid substitution” means replacement of an amino acid at a particular position in the parent polypeptide sequence with another amino acid, and "amino acid insertion” means a specificity in the parent polypeptide sequence. Addition of an amino acid at a position, "amino acid deletion” or “deletion” means removal of an amino acid at a particular position in the parent polypeptide sequence.
  • amino acid modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are substitutions in which amino acid residues are replaced with amino acid residues having similar side chains.
  • a family of amino acid residues having similar side chains has been defined in the art.
  • amino acids containing basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged acute side chains eg, , glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains eg, alanine, valine, leucine, isoleucine
  • Acid, proline, phenylalanine, methionine ⁇ -branched side chains (eg, threonine, valine, isoleucine)
  • aromatic side chains eg, tyrosine, benzene
  • parent antibody refers to an antibody obtained by the present invention or an antibody obtained by mutation or affinity maturation according to the antibody provided herein, and preferably refers to the antibody shown in the examples.
  • the parent antibody can be a naturally occurring antibody, or a variant or modified version of a naturally occurring antibody.
  • a parent antibody can refer to the antibody itself, a composition comprising the parent antibody, or a nucleic acid sequence encoding the same.
  • antigenic epitope as used herein, also referred to as an epitope, may consist of a contiguous sequence of IL-13RA2 protein sequences or a discontinuous three-dimensional structure of the IL-13RA2 protein sequence.
  • antigen binding proteins having an antigen-binding region based on scFv including antibodies, are described.
  • the scFv was selected from the human scFv phage display library using recombinant IL-13RA2. These molecules display fine specificity.
  • the antibody only recognizes IL-13RA2 and does not recognize IL-13RA1.
  • IL-13RA2 refers to human IL-13RA2 unless otherwise specified.
  • the invention encompasses an antibody having a scFv sequence fused to one or more heavy chain constant regions to form an antibody having a human immunoglobulin Fc region to produce a bivalent protein, thereby increasing antibody Overall affinity and stability.
  • the Fc portion allows for direct conjugation of other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to, for example, antibodies used in antigen quantification studies in order to immobilize antibodies for affinity measurement, for targeted delivery therapy. Drugs, use of immune effector cells to test Fc-mediated cytotoxicity and many other applications.
  • the molecules of the invention are based on the use of phage display to identify and select single-chain variable fragments (scFv), the amino acid sequence of which confers specificity for the molecule against IL-13RA2 and forms the basis of all antigen binding proteins of the present disclosure.
  • scFv single-chain variable fragments
  • the scFv can be used to design a range of different "antibody” molecules including, for example, full length antibodies, fragments thereof such as Fab and F(ab')2, fusion proteins (including scFv_Fc), multivalent antibodies, ie, having More than one specific antibody of the same antigen or different antigens, for example, bispecific T cell binding antibody (BiTE), triabodies, etc.
  • BiTE bispecific T cell binding antibody
  • the heavy and light chains of the antibodies of the invention may be full length (eg, the antibody may comprise at least one and preferably two intact heavy chains, and at least one and preferably The two intact light chains) may alternatively comprise an antigen binding moiety (Fab, F(ab')2, Fv or scFv).
  • the antibody heavy chain constant region is selected, for example, from IgGl, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. The choice of antibody type will depend on the immune effector function that the designed antibody is intended to elicit. Suitable amino acid sequences for the constant regions of various immunoglobulin isotypes and methods for producing a wide variety of antibodies are known to those skilled in the art in the construction of recombinant immunoglobulins.
  • the invention provides an antibody that specifically recognizes IL-13RA2, which binds to U251 cells stably transfected with human IL-13RA2 with a relative affinity EC50 of less than 100 nM, preferably less than 10 nM, more preferably 0.1-1 nM, most It is preferably 0.3-0.6 nM.
  • the antibody of IL-13RA2 provided by the present invention comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, and/or a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and / Or a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or 12.
  • the antibody that binds IL-13RA2 provided by the present invention comprises: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14. And/or the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15 or 16.
  • an antibody that binds to IL-13RA2 comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, and/or a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: And/or a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11 or 12, and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, and/or a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: And/or the light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15 or 16.
  • the antibody that binds IL-13RA2 comprises: HCDR1 represented by SEQ ID NO: 9, HCDR2 represented by SEQ ID NO: 10, HCDR3 represented by SEQ ID NO: 11, and SEQ ID NO: 13. LCDR1 shown in LCDR1, SEQ ID NO: 14 or LCDR3 shown in SEQ ID NO: 15; or HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10, SEQ ID NO: HCDR3 shown in FIG. 12, and LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, and LCDR3 shown in SEQ ID NO: 16.
  • the antibody that binds to IL-13RA2 comprises HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10, HCDR3 shown in SEQ ID NO: 12, and SEQ ID NO: 13 LCDR1 shown in LCDR1, SEQ ID NO: 14, and LCDR3 shown in SEQ ID NO: 16.
  • the invention provides an antibody that binds to IL-13RA2, the heavy chain variable region thereof being selected from the sequence of SEQ ID NO: 2 or SEQ ID NO: 6, or a variant of both.
  • the invention provides an antibody or fragment thereof that binds IL-13RA2 comprising a light chain variable region sequence selected from the group consisting of SEQ ID NO: 4 or SEQ ID NO: 8.
  • heavy and light chain variable region sequences can each bind IL-13RA2
  • the heavy and light chain variable region sequences can be "mixed and matched" to produce the anti-IL-13RA2 binding molecules of the invention.
  • the invention provides a variant of an antibody or fragment thereof that binds IL-13RA2.
  • the invention provides an antibody or fragment thereof having a heavy chain and/or light chain variable region that is at least 80% identical to the variable region sequence of a heavy or light chain.
  • the amino acid sequence identity of the heavy and/or light chain variable regions is at least 85%, more preferably at least 90%, most preferably at least 95%, especially 96%, more particularly 97%, even more particularly 98%.
  • the most special 99% including, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
  • the variant can be obtained by using the antibody described in the present application as a parent antibody, by yeast library screening, phage library screening, point mutation or the like.
  • the invention provides an antibody that recognizes the same epitope as the anti-IL-13RA2 antibody described above.
  • Standard assays for assessing the binding ability of antibodies are known in the art and include, for example, ELISA, biacore, Western blot, and flow cytometry analysis. Suitable assays are described in detail in the examples.
  • the invention also provides nucleic acids, vectors, and host cells comprising the nucleic acids or vectors, which are isolated from antibodies and fragments thereof that bind to IL-13RA2.
  • the nucleic acid can be located in intact cells, in cell lysates, or in partially purified or substantially purified form.
  • the nucleic acids of the invention can be obtained using standard molecular biology techniques, for example, by standard PCR amplification or cDNA cloning techniques, obtaining light and heavy chains encoding antibodies or cDNA encoding VH and VL segments.
  • standard molecular biology techniques for example, by standard PCR amplification or cDNA cloning techniques, obtaining light and heavy chains encoding antibodies or cDNA encoding VH and VL segments.
  • antibodies obtained from immunoglobulin gene libraries eg, using phage display technology
  • one or more nucleic acids encoding the antibodies can be recovered from the library. Methods for introducing foreign nucleic acids into host cells are generally known in the art and can vary with the host cell used.
  • a preferred nucleic acid molecule of the invention is a heavy chain variable region encoding a light chain variable region selected from SEQ ID NO: 3 or SEQ ID NO: 7, and/or selected from SEQ ID NO: 1 or SEQ ID NO: Those. More preferred is a nucleic acid molecule comprising the heavy chain sequence of SEQ ID NO: 1, and a light chain sequence comprising SEQ ID NO: 3 or a heavy chain sequence comprising SEQ ID NO: 5, and comprising SEQ ID NO: The light chain sequence of 7.
  • a nucleic acid encoding an antibody of the invention can be integrated into an expression vector.
  • expression vectors are available for protein expression.
  • Expression vectors can include self-replicating extra-chromosomal vectors, or vectors integrated into the host genome.
  • Expression vectors for use in the present invention include, but are not limited to, those which enable expression of proteins in mammalian cells, bacteria, insect cells, yeast, and in vitro systems. As is known in the art, a variety of expression vectors are commercially available or otherwise available. It can be used in the present invention to express antibodies.
  • the invention also provides a multifunctional immunoconjugate comprising an antibody described herein and further comprising at least one other type of functional molecule.
  • the functional molecule is selected from, but not limited to, a molecule that targets a tumor surface marker, a molecule that inhibits tumors, a molecule that targets a surface marker of an immune cell, or a detectable label.
  • the antibody and the functional molecule may constitute a composition by covalent attachment, coupling, attachment, crosslinking, or the like.
  • the immunoconjugate may comprise: an antibody of the invention and at least one molecule that targets a tumor surface marker or a tumor suppressor molecule.
  • the tumor suppressing molecule may be an anti-tumor cytokine or an anti-tumor toxin; preferably, the cytokine includes, but is not limited to, IL-2, IL-7, IL-12, IL. -15, type I IFN, TNF-alpha.
  • the molecule that targets a tumor surface marker is a molecule that targets a surface marker of the same tumor to which the antibody of the invention is targeted.
  • the molecule that targets a tumor surface marker can be an antibody or ligand that binds to a tumor surface marker, for example, can cooperate with the antibody of the invention to more precisely target tumor cells.
  • the tumor suppressing molecule may be an anti-tumor cytokine or an anti-tumor toxin; preferably, the cytokine includes, but is not limited to, IL-2, IL-7, IL
  • the immunoconjugate can comprise: an antibody of the invention and a detectable label.
  • the detectable label includes, but is not limited to, a fluorescent label, a chromogenic label; eg, an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, a radioactive material, a positron emitting metal, and a non-radioactive paramagnetic Metal ion. More than one marker may also be included.
  • the label used to label the antibody for detection and/or analysis and/or diagnostic purposes depends on the particular detection/analysis/diagnostic technique and/or method used, such as immunohistochemical staining (tissue) samples, flow cytometry, and the like. Suitable labels for detection/analysis/diagnostic techniques and/or methods known in the art are well known to those skilled in the art.
  • the immunoconjugate may comprise: an antibody of the invention and a molecule that targets a surface marker of an immune cell.
  • the molecule targeting the surface marker of the immune cell may be an antibody or a ligand that binds to an immunocyte surface marker, and is capable of recognizing an immune cell, which carries the antibody of the present invention to the immune cell, and the antibody of the present invention can immunize the cell Targeting tumor cells, thereby inducing immune cells to specifically kill tumors.
  • the immune cell surface marker may be selected from the group consisting of CD3, CD16, CD28, and more preferably, the antibody that binds to the immune cell surface marker is an anti-CD3 antibody.
  • the immune cells can be selected from the group consisting of T cells, NK cells, and NKT cells.
  • the immunoconjugate can be produced as a fusion protein comprising an antibody of the invention and a suitable other protein.
  • the fusion protein can be produced by methods known in the art, for example by constructing a nucleic acid molecule comprising a nucleotide sequence encoding the antibody and encoding a suitable labeled nucleoside, and subsequently expressing the nucleic acid molecule. Acid sequence.
  • Another aspect of the invention provides a nucleic acid molecule encoding at least one antibody, functional variant or immunoconjugate thereof of the invention.
  • the invention provides a chimeric antigen receptor comprising an extracellular binding domain, a transmembrane domain, and an intracellular domain.
  • Chimeric Antigen Receptor refers to a tumor antigen binding domain fused to an intracellular signal transduction domain that activates T cells.
  • the extracellular binding domain of CAR is derived from a mouse or humanized or human monoclonal antibody.
  • the extracellular binding domain is an antibody of the invention, non-limiting examples comprising a single-chain variable fragment (scFv) derived from an antibody, a fragment antigen binding region (Fab) selected from a library, a single domain fragment or ligated A natural ligand for its cognate receptor.
  • the extracellular antigen binding region can comprise an scFv, Fab, or natural ligand, as well as any derivatives thereof.
  • An extracellular antigen binding region can refer to a molecule other than an intact antibody, which can comprise a portion of an intact antibody and can bind to an antigen to which the intact antibody binds.
  • antibody fragments can include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bifunctional antibodies, linear antibodies; single-chain antibody molecules (eg, scFv); and formed from antibody fragments Multispecific antibodies.
  • An extracellular antigen binding region such as a scFv, Fab or natural ligand, can be part of a CAR that determines antigen specificity.
  • the extracellular antigen binding region can bind to any complementary target.
  • the extracellular antigen binding region can be derived from an antibody of known variable region sequence.
  • the extracellular antigen binding region can be obtained from antibody sequences obtained from available mouse hybridomas.
  • extracellular antigen binding regions can be obtained from whole-out cleavage of tumor cells or primary cells, such as tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the binding specificity of the extracellular antigen binding region can be determined by a complementarity determining region or CDR, such as a light chain CDR or a heavy chain CDR.
  • CDR complementarity determining region
  • binding specificity can be determined by light chain CDRs and heavy chain CDRs.
  • a given combination of heavy chain CDRs and light chain CDRs can provide a given binding pocket that can confer greater affinity and/or specificity to the antigen than other reference antigens.
  • the extracellular antigen binding region can comprise a light chain CDR specific for the antigen.
  • the light chain CDR can be a complementarity determining region of an scFv light chain of an antibody, such as a CAR.
  • the light chain CDRs may comprise contiguous amino acid residue sequences, or two or more contiguous sequence of amino acid residues separated by non-complementarity determining regions (eg, framework regions).
  • a light chain CDR can comprise two or more light chain CDRs, which can be referred to as a light chain CDR-1, CDR-2, and the like.
  • the light chain CDRs can comprise three light chain CDRs, which can be referred to as light chain CDR-1, light chain CDR-2 and light chain CDR-3, respectively.
  • a set of CDRs present on a common light chain can be collectively referred to as a light chain CDR.
  • the extracellular antigen binding region can comprise a heavy chain CDR that is specific for the antigen.
  • the heavy chain CDR can be the heavy chain complementarity determining region of an antibody, such as an scFv.
  • the heavy chain CDRs may comprise a contiguous sequence of amino acid residues, or a contiguous sequence of two or more amino acid residues separated by a non-complementarity determining region (eg, a framework region).
  • the heavy chain CDRs can comprise two or more heavy chain CDRs, which can be referred to as heavy chain CDR-1, CDR-2, and the like.
  • the heavy chain CDRs can comprise three heavy chain CDRs, which can be referred to as heavy chain CDR-1, heavy chain CDR-2 and heavy chain CDR-3, respectively.
  • a set of CDRs present on a common heavy chain can be collectively referred to as a heavy chain CDR.
  • the extracellular antigen binding region can be modified in various ways by using genetic engineering.
  • the extracellular antigen binding region can be mutated such that the extracellular antigen binding region can be selected to have a higher affinity for its target.
  • the affinity of the extracellular antigen binding region for its target can be optimized for targets that can be expressed at low levels on normal tissues. This optimization can be done to minimize potential toxicity.
  • clones of the extracellular antigen binding region with a higher affinity for the membrane-bound form of the target may be preferred over the counterpart of its soluble form. This modification can be made because different levels of soluble forms of the target can also be detected and their targeting can cause undesirable toxicity.
  • the extracellular antigen binding region comprises a hinge or spacer.
  • the terms hinge and spacer are used interchangeably.
  • the hinge can be considered as part of a CAR for providing flexibility to the extracellular antigen binding region.
  • the hinge can be used to detect CAR on the cell surface of a cell, particularly when detecting antibodies to the extracellular antigen binding region are ineffective or available.
  • the length of the hinge derived from an immunoglobulin may need to be optimized, depending on the location of the extracellular antigen binding region that targets the epitope on the target.
  • the hinge may not belong to an immunoglobulin, but to another molecule, such as the native hinge of a CD8 alpha molecule.
  • the CD8 alpha hinge may contain cysteine and proline residues known to play a role in the interaction of the CD8 co-receptor and the MHC molecule. The cysteine and proline residues can affect the performance of the CAR.
  • the CAR hinge can be adjustable in size. This morphology of the immunological synapse between the immune response cell and the target cell also defines the distance that cannot be functionally bridged by the CAR due to the distal membrane epitope on the cell surface target molecule, ie, the use of a short hinge CAR does not The synaptic distance reaches an approximation of the signal's ability to conduct. Similarly, the membrane proximal CAR target epitope was only observed for signal output in the context of a long hinged CAR.
  • the hinge can be adjusted depending on the extracellular antigen binding region used. The hinge can be of any length.
  • the transmembrane domain can anchor the CAR to the plasma membrane of the cell.
  • the natural transmembrane portion of CD28 can be used for CAR.
  • the natural transmembrane portion of CD8 ⁇ can also be used in the CAR.
  • CD8 may be a protein having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to the NCBI reference number: NP_001759 or a fragment thereof having stimulatory activity.
  • a “CD8 nucleic acid molecule” may be a polynucleotide encoding a CD8 polypeptide, and in some cases, the transmembrane region may be a natural transmembrane portion of CD28, and “CD28” may refer to NCBI reference number: NP_006130 or its stimulating activity.
  • a fragment has a protein of at least 85, 90, 95, 96, 97, 98, 99 or 100% identity.
  • a "CD28 nucleic acid molecule” can be a polynucleotide encoding a CD28 polypeptide.
  • the transmembrane portion can comprise a CD8 alpha region.
  • the (fine) intracellular signaling region of CAR may be responsible for activating at least one of the effector functions of the immune response cells into which the CAR has been placed.
  • CAR can induce effector functions of T cells, for example, the effector function is cytolytic activity or helper activity, including secretion of cytokines.
  • intracellular signaling region refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specific function. Although the entire intracellular signaling region can generally be used, in many cases it is not necessary to use the entire chain of the signal domain. In some embodiments, a truncated portion of an intracellular signaling region is used. In some embodiments, the term intracellular signaling region is therefore intended to include any truncated portion of an intracellular signaling region sufficient to transduce an effector function signal.
  • Preferred examples of signal domains for use in CAR may include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction after target-receptor binding, as well as any derivatives thereof or Variant sequences and any synthetic sequences of these sequences that have the same functionality.
  • TCRs T cell receptors
  • co-receptors that act synergistically to initiate signal transduction after target-receptor binding
  • the intracellular signaling region can contain a known signal motif for an immunoreceptor tyrosine activation motif (ITAM).
  • ITAMs containing cytoplasmic signaling sequences include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d.
  • the intracellular signal domain is derived from a CD3 ⁇ chain.
  • T cell signaling domain containing one or more ITAM motifs is the CD3 ⁇ domain, also known as the T cell receptor T3 ⁇ chain or CD247.
  • This domain is part of the T cell receptor-CD3 complex and plays an important role in binding antigen recognition of several intracellular signal transduction pathways to the main effector activation of T cells.
  • CD3 ⁇ primarily refers to human CD3 ⁇ and its isoforms, as known from the Swissprot entry P20963, including proteins having substantially the same sequence.
  • the full T cell receptor T3 ⁇ chain is not required and that any derivative of the signal domain comprising the T cell receptor T3 ⁇ chain is suitable, including any functional equivalent thereof. .
  • the intracellular signaling domain can be selected from any one of the domains of Table 1.
  • the domain can be modified such that identity to the reference domain can range from about 50% to about 100%.
  • Any of the domains of Table 1 can be modified such that the modified form can comprise about 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or up to about 100% identity.
  • the intracellular signaling region of CAR may further comprise one or more costimulatory domains.
  • the intracellular signaling region may comprise a single costimulatory domain, such as an ⁇ chain (first generation CAR) or it is with CD28 or 4-1BB (second generation CAR).
  • the intracellular signaling region can comprise two costimulatory domains, such as CD28/OX40 or CD28/4-1BB (third generation).
  • CD28 phosphatidylinositol-4,5-diphosphate 3-kinase
  • 4-1BB/OX40 TNF-receptor-associated factor adapter protein
  • signals generated by the CAR may be combined with an auxiliary or costimulatory signal.
  • costimulatory signaling domains chimeric antigen receptor-like complexes can be designed to contain several possible costimulatory signal domains.
  • costimulatory signaling domains chimeric antigen receptor-like complexes can be designed to contain several possible costimulatory signal domains.
  • the individual ligation of T cell receptors is not sufficient to induce complete activation of T cells into cytotoxic T cells.
  • a second co-stimulatory signal is required for complete productive T cell activation.
  • receptors have been reported to provide co-stimulation for T cell activation including, but not limited to, CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4- 1BB.
  • the signaling pathways used by these costimulatory molecules work synergistically with the primary T cell receptor activation signal.
  • the signals provided by these costimulatory signaling regions can act synergistically with primary effect activation signals derived from one or more ITAM motifs (eg, the CD3zeta signal transduction domain) and can fulfill the requirements for T cell activation.
  • the addition of a costimulatory domain to a chimeric antigen receptor-like complex can enhance the efficacy and durability of engineered cells.
  • the T cell signal domain and the costimulatory domain are fused to each other to form a signaling region.
  • the chimeric antigen receptor binds to the target antigen.
  • the target antigen can be obtained or isolated from various sources.
  • a target antigen as used herein is an antigenic epitope on an antigen or antigen that is critical in mammals for immune recognition and ultimately elimination or control of pathogenic factors or disease states.
  • the immune recognition can be a cell and/or a body fluid. In the case of intracellular pathogens and cancer, the immune recognition can be, for example, a T lymphocyte reaction.
  • the target antigen comprises an antigen associated with a pre-cancerous or proliferative state.
  • Target antigens may also be associated with or caused by cancer.
  • a chimeric antigen receptor of the invention recognizes and binds to a tumor antigen comprising IL-13RA2 as described hereinbefore.
  • a chimeric antigen receptor herein is present on the plasma membrane of a cell, and when bound to its target and activated, the cell expressing the chimeric antigen receptor can produce a cell carrying the target Cytotoxicity.
  • the chimeric antigen receptor is present on a cytotoxic cell, such as an NK cell or a cytotoxic T cell, and, when activated by a target, increases the toxicity of the cytotoxic cell to the target cell .
  • a chimeric antigen receptor herein can increase the effect of immunoreactive cells on cells expressing IL-13RA2, such as tumor cells.
  • a cell expressing a chimeric antigen receptor described herein increases the cytotoxic effect on cells expressing IL-13RA2 by at least 10%, at least 15 compared to a cell that does not express a chimeric antigen receptor herein.
  • a transgene encoding a receptor or a CAR that binds to an antigen can be incorporated into the cell.
  • a transgene can be incorporated into an immune response cell, such as a T cell.
  • the transgene can be a complementary DNA (cDNA) fragment that is a copy of messenger RNA (mRNA); or the gene itself (with or without introns) located in the original region of its genomic DNA.
  • cDNA complementary DNA
  • mRNA messenger RNA
  • a nucleic acid encoding a transgene sequence, such as DNA can be randomly inserted into the chromosome of the cell. Random integration can be produced by any method that introduces a nucleic acid, such as DNA, into a cell.
  • the method can include, but is not limited to, electroporation, ultrasound, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viruses including adenovirus, AAV, and retroviral vectors.
  • Vector, and/or type II ribozyme can be produced by any method that introduces a nucleic acid, such as DNA, into a cell.
  • the method can include, but is not limited to, electroporation, ultrasound, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viruses including adenovirus, AAV, and retroviral vectors.
  • Vector, and/or type II ribozyme
  • the DNA encoding the transgene can also be designed to include a reporter gene such that the presence of the transgene or its expression product can be detected by activation of the reporter gene. Any reporter gene can be used, such as those described above.
  • the cells containing the transgene can be selected by selecting cells in the cell culture in which the reporter gene has been activated.
  • Expression of CAR can be verified by expression assays such as qPCR or by measuring the level of RNA.
  • the level of expression can also indicate the number of copies. For example, if the level of expression is very high, this may indicate that more than one copy of the CAR is integrated into the genome. Alternatively, high expression may indicate that the transgene is integrated in a high transcribed region, such as near a highly expressed promoter. Expression can also be verified by measuring protein levels, for example by Western blotting.
  • an immune response cell of the invention may comprise one or more transgenes.
  • the one or more transgenes can express a CAR protein that recognizes and binds to at least one epitope on the antigen or binds to a mutant epitope on the antigen.
  • CAR can be a functional CAR.
  • the immune response cells of the invention may comprise one or more CARs, or they may comprise a single CAR and a secondary engineered receptor.
  • the transgene can encode a suicide gene.
  • CAR immune response cells cause tumor regression but can be associated with toxicity.
  • the target antigen when the target antigen is shared in normal tissues and tumor cells, the CAR immune response cells may not be able to distinguish between tumors and normal tissues ("target/off-target toxicity").
  • a systemic disturbance of the immune system called cytokine release syndrome (CRS)
  • CRS may comprise a systemic inflammatory response syndrome or a cytokine storm, which may be a consequence of rapid expansion of the CAR immune response cells in vivo.
  • CRS is a condition characterized by fever and hypotension, which can lead to multiple organ failure.
  • the toxicity is associated with in vivo expansion of infused CAR immune response cells, which can cause an overall disturbance of the immune system, as well as release high levels of pro-inflammatory cytokines such as TNF[alpha] and IL-6.
  • Suicide genes can induce the elimination of CAR immunoreactive cells.
  • the suicide gene may be any gene that induces apoptosis in the CAR immunoreactive cells.
  • a suicide gene can be encoded in the viral vector together with the antigen-binding receptor. The coding of the suicide gene allows for the mitigation or complete abortion of the toxicity caused by in vivo expansion of the infused CAR immune response cells under specific conditions.
  • CAR immunoreactive cells that are present in antigens of normal tissues can be produced such that they transiently express CAR, eg, after electroporating the mRNA encoding the receptor.
  • a major effort to further strengthen CAR immunoreactive cells by including a safety switch can substantially eliminate CAR immunoreactive cells in the case of severe target toxicity.
  • the CAR-encoding vector can be associated with, for example, an inducible caspase-9 gene (activated by a dimeric chemical inducer) or a truncated form of EGF receptor R (by the monoclonal antibody Cetuximab) Monoclonal activation) or RQR8 safety switch combination.
  • transgenes used herein may be from different species.
  • one or more of the transgenes can comprise a human gene, a mouse gene, a rat gene, a porcine gene, a bovine gene, a dog gene, a cat gene, a monkey gene, a chimpanzee gene, or any combination thereof.
  • a transgene can be from a human having a human genetic sequence.
  • One or more transgenes may comprise a human gene. In some cases, one or more of the transgenes are not adenoviral genes.
  • the transgene can be inserted into the genome of the immunoreactive cell in a random or site-specific manner.
  • a transgene can be inserted into a random site in the genome of an immune cell.
  • These transgenes can be functional, for example, fully functional when inserted into any part of the genome.
  • a transgene can encode its own promoter or can be inserted into a position controlled by its internal promoter.
  • the transgene can be inserted into a gene, such as an intron of a gene or an exon, promoter or non-coding region of a gene.
  • a transgene can be inserted to insert a disruptive gene, such as an endogenous immune checkpoint.
  • more than one copy of the transgene can be inserted into multiple random sites within the genome. For example, multiple copies can be inserted into random sites in the genome. This may result in an increase in overall expression compared to random insertion of the transgene once.
  • a copy of the transgene can be inserted into the gene and another copy of the transgene can be inserted into a different gene.
  • the transgene can be targeted such that it can be inserted into a specific site in the genome of the immunoreactive cell.
  • a polynucleic acid comprising a receptor sequence encoding an antigen binding agent can take the form of a plasmid vector.
  • the plasmid vector may comprise a promoter. In some cases, the promoter can be constitutive. In some embodiments, the promoter is inducible. The promoter may be or may be derived from CMV, U6, MND or EF1a. In some embodiments, the promoter can be adjacent to the CAR sequence. In some embodiments, the plasmid vector further comprises a splice acceptor. In some embodiments, the splice acceptor can be adjacent to the CAR sequence.
  • the promoter sequence can be a PKG or MND promoter.
  • the MND promoter may be a synthetic promoter of the U3 region of the MoMuLV LTR modified with myeloproliferative sarcoma virus enhancer.
  • a polynucleic acid encoding a receptor of interest can be designed to be delivered to a cell by non-viral techniques.
  • the polynucleic acid can be a Good Manufacturing Practice (GMP) compatible reagent.
  • GMP Good Manufacturing Practice
  • Promoters can be ubiquitous, constitutive (unrestricted promoters, allowing for continuous transcription of related genes), tissue-specific promoters or inducible promoters. Expression of a transgene inserted adjacent to or proximate to the promoter can be modulated. For example, a transgene can be inserted near or beside a ubiquitous promoter.
  • Some ubiquitous promoters may be the CAGGS promoter, the hCMV promoter, the PGK promoter, the SV40 promoter or the ROSA26 promoter.
  • Promoters can be endogenous or exogenous.
  • one or more transgenes can be inserted adjacent to or proximate to the endogenous or exogenous ROSA26 promoter.
  • the promoter may be specific for immunoreactive cells.
  • one or more transgenes can be inserted adjacent to or proximate to the porcine ROSA26 promoter.
  • Tissue-specific promoters or cell-specific promoters can be used to control the location of expression.
  • one or more transgenes can be inserted into proximity or proximity of a tissue-specific promoter.
  • Tissue-specific promoters may be FABP promoter, Lck promoter, CamKII promoter, CD19 promoter, keratin promoter, albumin promoter, aP2 promoter, insulin promoter, MCK promoter, MyHC promoter, WAP Promoter, or Col2A promoter.
  • Inducible promoters can also be used. These inducible promoters can be turned on and off by adding or removing an inducer if necessary.
  • the inducible promoter is contemplated to be, but not limited to, Lac, tac, trc, trp, araBAD, phoA, recA, proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx, and/or Trex.
  • inducible promoter is a controlled promoter which does not express or underexpress a gene operably linked thereto before the desired condition is reached, and is achieved under the expected conditions. A gene that is operably linked to it is expressed or expressed at a high level.
  • the transgenic sequences may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
  • transcriptional or translational regulatory sequences such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
  • the transgene encodes a receptor or CAR that binds to the antigen, wherein the transgene is inserted into a safe harbor such that the antigen-binding receptor is expressed.
  • the transgene is inserted into the PD1 and/or CTLA-4 locus.
  • the transgene is delivered as a lentivirus to the cells for random insertion, while a PD1- or CTLA-4 specific nuclease can be provided as mRNA.
  • the transgene is delivered by a viral vector system such as retrovirus, AAV or adenovirus, and mRNA encoding a nuclease specific for safe harbor (eg, AAVS1, CCR5, albumin, or HPRT). Cells can also be treated with mRNA encoding PD1 and/or CTLA-4 specific nucleases.
  • the polynucleotide encoding the CAR is provided by a viral delivery system with an mRNA encoding a HPRT-specific nuclease and a PD1- or CTLA-4 specific nuclease. CARs that can be used with the methods and compositions disclosed herein can include all types of these chimeric proteins.
  • a transgene can be introduced into an immunoreactive cell using a retroviral vector (gamma-retroviral or lentiviral vector).
  • a transgene encoding a CAR or any receptor that binds an antigen, or a variant or fragment thereof can be cloned into a retroviral vector and can be derived from an endogenous promoter, a retroviral long terminal repeat, or a target Cell type-specific promoter drive.
  • Non-viral vectors can also be used.
  • Non-viral vector delivery systems can include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from retroviruses such as murine leukemia virus because they can transduce non-proliferating cells. They also have the added advantage of low immunogenicity.
  • An advantage of adenoviral vectors is that they do not fuse into the genome of the target cell, thereby bypassing negative integration-related events.
  • the cells can be transfected with a transgene encoding the antigen-binding receptor.
  • the transgenic concentration can range from about 100 picograms to about 50 micrograms.
  • the amount of nucleic acid (eg, ssDNA, dsDNA, or RNA) introduced into the cell can be altered to optimize transfection efficiency and/or cell viability. For example, 1 microgram of dsDNA can be added to each cell sample for electroporation.
  • the amount of nucleic acid (eg, double stranded DNA) required for optimal transfection efficiency and/or cell viability varies depending on the cell type.
  • the amount of nucleic acid (eg, dsDNA) used for each sample can directly correspond to transfection efficiency and/or cell viability. For example, a range of transfection concentrations.
  • the transgene encoded by the vector can be integrated into the genome of the cell. In some embodiments, the transgene encoded by the vector is forward integrated. In other cases, the reverse integration of the transgene encoded by the vector.
  • the vector by administration to an individual patient is typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or topical application, as described below.
  • the vector can be delivered ex vivo to the cells, such as cells removed from an individual patient (eg, lymphocytes, T cells, bone marrow aspirate, tissue biopsy), and then typically after re-selecting the cells into which the vector is incorporated Implanted in a patient. Cells can be expanded before or after selection.
  • Suitable immunoreactive cells for expression of a receptor that binds to an antigen may be cells that are autologous or non-autologous to the individual in need thereof.
  • T cells can be obtained.
  • the T cells can be obtained from a number of sources, including PBMC, bone marrow, lymph node tissue, cord blood, thymus tissue, and tissues from infected sites, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells can be obtained from blood collected from the individual using any number of techniques known to those skilled in the art, such as FicollTM separation.
  • cells from circulating blood of an individual are obtained by apheresis.
  • Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • lymphocytes including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • cells collected by apheresis collection can be washed to remove plasma fractions and placed in a suitable buffer or medium for subsequent processing steps.
  • cells can be derived from a healthy donor, from a patient diagnosed with cancer, or a patient diagnosed with an infection.
  • the cells can be part of a mixed cell population with different phenotypic characteristics.
  • Cell lines can also be obtained from transformed T cells according to the methods previously described.
  • Cells can also be obtained from a cell therapy library.
  • Modified cells that are resistant to immunosuppressive therapy can be obtained by any of the methods described herein. It is also possible to select a suitable cell population prior to modification.
  • the engineered cell population can also be selected after modification.
  • Engineered cells can be used for autologous transplantation.
  • the cells can be used for allogeneic transplantation.
  • the cells are administered to a sample for identification of the same patient of a cancer associated target sequence. In other instances, the cells are administered to a patient different from the patient whose sample is used to identify the cancer-related target sequence.
  • suitable primary cells include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), and other blood cell subpopulations such as, but not limited to, T cells, natural killer cells, monocytes, Natural killer T cells, monocyte precursor cells, hematopoietic stem cells or non-pluripotent stem cells.
  • the cell can be any immune cell, including any T cell such as a tumor infiltrating cell (TIL), such as a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, or any other type of T cell.
  • T cells can also include memory T cells, memory stem T cells, or effector T cells.
  • T cells can also be expanded from a large population.
  • T cells may also be inclined to specific populations and phenotypes.
  • a T cell can be tilted to a phenotype comprising CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), and/or IL-7R ⁇ (+).
  • Suitable cells may be selected from one or more of the following list: CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and/or IL-7R ⁇ (+).
  • Suitable cells also include stem cells such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells.
  • stem cells such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells.
  • Suitable cells can comprise any number of primary cells, such as human cells, non-human cells, and/or mouse cells.
  • Suitable cells can be progenitor cells.
  • Suitable cells can be derived from a subject (eg, a patient) to be treated.
  • the amount of therapeutically effective cells required in a patient can vary depending on the viability of the cells and the efficiency with which the cells are genetically modified (eg, the efficiency with which the transgene is integrated into one or more cells, or the level of expression of the protein encoded by the transgene) ).
  • the product (eg, doubling) of the cell viability after genetic modification and the efficiency of transgene integration can correspond to a therapeutic amount of cells available for administration to a subject.
  • an increase in cell viability after genetic modification may correspond to a reduction in the amount of essential cells effective to administer the treatment to the patient.
  • an increase in the efficiency of integration of the transgene into one or more cells can correspond to a reduction in the number of cells necessary to administer a therapeutically effective in a patient.
  • determining the amount of therapeutically effective cells required can include determining a function associated with changes in cells over time.
  • determining the amount of cells that are required to be therapeutically effective can include determining a function corresponding to a change in efficiency of integrating the transgene into one or more cells according to a time-dependent variable (eg, cell culture time, electroporation time, Cell stimulation time).
  • the therapeutically effective cell can be a population of cells comprising about 30% to about 100% of the expression of a receptor that binds to the antigen on the surface of the cell.
  • the therapeutically effective cells can express about 30%, 35%, 40%, 45%, 50%, 55%, 60 of the antigen-binding receptor on the cell surface as measured by flow cytometry. %, 65%, 70%, 75% 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9 % or more than about 99.9%.
  • the invention also encompasses a nucleic acid encoding the antigen-binding receptor.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • the present invention also provides a vector comprising the above nucleic acid encoding a receptor protein that binds to an antigen expressed on the surface of an immune response cell.
  • the invention also includes viruses comprising the vectors described above.
  • the virus of the present invention includes a packaged infectious virus, and also includes a virus to be packaged containing components necessary for packaging as an infectious virus.
  • Other viruses known in the art that can be used to transduce foreign genes into immune response cells and their corresponding plasmid vectors can also be used in the present invention.
  • a host cell comprising an antibody or chimeric antigen receptor as described herein, and optionally a Type I interferon.
  • a host cell comprising a nucleic acid encoding an antibody or chimeric antigen receptor described herein, and optionally a type I interferon.
  • the host cell is an immune response cell.
  • the immune response cell is a T cell, a natural killer cell, a cytotoxic T lymphocyte, a natural killer T cell, a DNT cell, and/or a regulatory T cell.
  • the host cell is an NK92 cell.
  • the immune response cell of the present invention may further carry a coding sequence of a foreign cytokine; the cytokine includes, but not limited to, IL-12, IL-15 or IL-21 and the like.
  • cytokine includes, but not limited to, IL-12, IL-15 or IL-21 and the like.
  • These cytokines have further immunomodulatory or anti-tumor activity, enhance the function of effector T cells and activated NK cells, or directly exert anti-tumor effects.
  • cytokines will help the immune response cells to function better.
  • the immune response cell of the present invention may also express another antigen-binding receptor other than the antigen-binding receptor described above.
  • the immune response cells of the invention may also express a chemokine receptor; the chemokine receptors include, but are not limited to, CCR2. Those skilled in the art will appreciate that the CCR2 chemokine receptors may allow CCR2 binding in vivo to compete with it, which is advantageous for blocking tumor metastasis.
  • the immune response cells of the present invention can also express siRNA that reduces PD-1 expression or a protein that blocks PD-L1.
  • siRNA that reduces PD-1 expression
  • the immune response cells of the present invention may also express a safety switch; preferably, the safety switch comprises: iCaspase-9, Truncated EGFR or RQR8.
  • the immune response cells of the invention do not express a costimulatory ligand such as 4-1BBL.
  • a method of producing an antibody or chimeric antigen receptor described herein or a composition comprising the same comprising culturing a host cell described herein under suitable conditions.
  • the method comprises isolating and obtaining an expression product of the host cell.
  • composition comprising an antibody, chimeric antigen receptor, or nucleic acid described herein.
  • the composition is a pharmaceutical composition comprising the antibody, chimeric antigen receptor or nucleic acid.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • composition comprising a host cell as described herein and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that when the molecular body and composition are suitably administered to an animal or a human, they do not produce an adverse, allergic or other untoward reaction.
  • the composition comprises another therapeutic agent.
  • the additional therapeutic agent is a chemotherapeutic agent, such as those described in US20140271820 and/or a pharmaceutically acceptable salt or analog thereof.
  • the therapeutic agent includes, but is not limited to, a mitotic inhibitor (vinca alkaloid), including vincristine, vinblastine, vindesine, and novibin (TM) (vinorelbine, 5' - dehydrohydrogen sulfide); topoisomerase I inhibitors, such as camptothecin compounds, including CamptosarTM (irinotecan HCL), HycamtinTM (topotecan HCL), and others derived from camptothecin and the like a compound; a podophyllotoxin derivative such as etoposide, teniposide and midozozod; an alkylating agent cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine
  • TM novibin
  • the additional therapeutic agent is selected from one or more of epirubicin, oxaliplatin, and 5-fluorouracil.
  • the additional therapeutic agent includes, but is not limited to, an anti-angiogenic agent, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides), and other blood vessels Inhibitors such as angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta) interleukin 12, tissue inhibitors of retinoic acid and metalloproteinases-1 and -2, and the like occur.
  • anti-VEGF antibodies including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides
  • Inhibitors such as angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta) interleukin 12, tissue inhibitors of retinoic acid and metalloproteinases-1 and -2, and the like
  • sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof such as carboxymethyl fibers Sodium, ethyl cellulose and methyl cellulose; western yellow gum powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; Coloring agents; flavoring agents; compressed tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate
  • compositions described herein may comprise one or more pharmaceutically acceptable salts.
  • “Pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not produce any adverse toxicological effects (see, for example, Berge, SM et al., 1977, J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, and derived from non-toxic organic acids such as aliphatic monocarboxylic acids and A salt of a dicarboxylic acid, a phenyl-substituted alkanoic acid, a hydroxyalkanoic acid, an aromatic acid, an aliphatic or an aromatic sulfonic acid.
  • non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like
  • non-toxic organic acids such as aliphatic monocarboxylic acids and A salt of a dicarboxylic acid, a phenyl-substituted alkanoic acid, a hydroxyalkanoic acid, an aromatic
  • Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as salts derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucosamine. Glucosamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.
  • antioxidants include, but are not limited to, water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, Phosphoric acid, etc.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • composition of the present invention can be formulated into various dosage forms as needed, and can be administered by a physician in accordance with factors such as patient type, age, body weight, and general disease condition, mode of administration, and the like.
  • the mode of administration can be, for example, parenteral administration (e.g., injection) or other treatment.
  • parenteral administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection or infusion techniques.
  • Formulations comprising an immunoreactive cell population administered to an individual comprise a plurality of immunoreactive cells effective to treat and/or prevent a particular indication or disease.
  • a therapeutically effective population of immunoreactive cells can be administered to an individual.
  • administration contain from about 1 ⁇ 10 4 to about 1 ⁇ 10 10 cells reactive immunization formulation.
  • the formulation will comprise from about 1 x 10 5 to about 1 x 10 9 immunoreactive cells, from about 5 x 10 5 to about 5 x 10 8 immunoreactive cells, or about 1 x 10 6 to About 1 ⁇ 10 7 immunoreactive cells.
  • the number of CAR immunoreactive cells administered to the individual will vary from wide range. The doctor will finalize the appropriate dose to use.
  • a chimeric antigen receptor is used to stimulate an immune cell mediated immune response.
  • a T cell mediated immune response is an immune response involving T cell activation.
  • Activated antigen-specific cytotoxic T cells are capable of inducing apoptosis in target cells that exhibit a foreign antigenic epitope on the surface, such as cancer cells that display tumor antigens.
  • the chimeric antigen receptor is used to provide anti-tumor immunity in a mammal. Subjects will develop anti-tumor immunity due to T cell-mediated immune responses.
  • a method of treating a subject having cancer can involve administering one or more immune response cells of the invention to a subject in need of treatment.
  • the immune response cell binds to a tumor target molecule and induces cancer cell death.
  • the invention also provides a method of treating a pathogen infection in an individual comprising administering to the individual a therapeutically effective amount of an immune response cell of the invention.
  • the frequency of administration of the immunoreactive cells of the present invention will depend on factors including the disease being treated, the elements of the particular immunoreactive cells, and the mode of administration. For example, it can be administered 4 times, 3 times, 2 times a day, once a day, every other day, every three days, every four days, every five days, every six days, once a week, once every eight days, every time. Dosing once every nine days, every ten days, once a week, or twice a month.
  • the immune response cells of the present application have improved viability, they can be administered not only in a therapeutically effective amount that is lower than an immune response cell that is similar but does not express exogenous type I interferon, and can Administration at a lower frequency to achieve at least a similar, and preferably more pronounced, effect.
  • the compositions may be isotonic, ie they may have the same osmotic pressure as blood and tears.
  • the desired isotonicity of the compositions of the present invention can be achieved using sodium chloride or other pharmaceutically acceptable agents such as glucose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent.
  • Suitable thickeners include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The preferred concentration of thickener will depend on the reagent selected. It will be apparent that the choice of suitable carrier and other additives will depend on the exact route of administration and the nature of the particular formulation, such as a liquid dosage form.
  • kits comprising the antibodies, chimeric antigen receptors, nucleic acids or immune response cells described herein.
  • a kit can include a therapeutic or prophylactic composition comprising an effective amount of an antibody, chimeric antigen receptor, nucleic acid, or immune response cell described herein comprising one or more unit dosage forms.
  • the kit comprises a sterile container that can contain a therapeutic or prophylactic composition; such a container can be a cartridge, ampule, bottle, vial, tube, bag, blister pack, or other suitable as is known in the art.
  • Such containers may be made of plastic, glass, laminated paper, metal foil or other materials suitable for holding the drug.
  • the kit comprises an antibody, chimeric antigen receptor, nucleic acid or immune response cell as described herein, and the antibody, chimeric antigen receptor, nucleic acid or immune response cell described herein is administered to the individual Instructions. Methods for treating or preventing cancer or tumors using the antibodies, chimeric antigen receptors, nucleic acids or immune response cells described herein are typically included in the instructions.
  • the kits comprise a host cell as described herein and can include from about 1 x 10 4 cells to about 1 x 10 6 cells.
  • the kit can include at least about 1 x 10 5 cells, at least about 1 x 10 6 cells, at least about 1 x 10 7 cells, at least about 4 x 10 7 cells, at least about 5 x 10 7 cells, at least about 6 ⁇ 10 7 cells, at least about 6 ⁇ 10 7 cells, 8 ⁇ 10 7 cells, at least about 9 ⁇ 10 7 cells, at least about 1 ⁇ 10 8 cells, at least about 2 ⁇ 10 8 cells, at least about 3 ⁇ 10 8 cells, at least about 4 ⁇ 10 8 cells, at least about 5 ⁇ 10 8 cells, at least about 6 ⁇ 10 8 cells, at least about 6 ⁇ 10 8 cells , at least about 8 ⁇ 10 8 cells, at least about 9 ⁇ 10 8 cells, at least about 1 ⁇ 10 9 cells, at least about 2 ⁇ 10 9 cells, at least about 3 ⁇ 10 9 cells, at least about 4 ⁇ 10 9 cells, at least about 5 ⁇ 10 9 cells, at least about 6 ⁇ 10 9 cells, at least about 8 ⁇ 10 9 cells, at least about 9 ⁇ 10 9 cells, at least about 9 ⁇ 10 9
  • the kit can include allogeneic cells.
  • a kit can include cells that can include genomic modifications.
  • the kit can comprise "off the shelf" cells.
  • the kit can include cells that can be expanded for clinical use. In some cases, the kit may contain content for research purposes.
  • the instructions include at least one of: a description of a therapeutic agent; a dosage regimen and administration for treating or preventing a tumor or a symptom thereof; preventive measures, warnings, contraindications, excessive information, adverse reactions, animals Pharmacology, clinical studies, and/or citations. Instructions can be printed directly on the container (if any), or as a label on the container, or as a separate paper, booklet, card or folder in the container or in the container. In some embodiments, the instructions provide methods of administering an immune response cell of the invention for treating or preventing a tumor. In some cases, the instructions provide methods of administering an immunoreactive cell of the invention before, after or simultaneously with the administration of a chemotherapeutic agent.
  • a method of inducing cell death comprising IL-13RA2, the method comprising: ligating the cell with an antibody described herein, a chimeric antigen receptor described herein, described herein
  • the composition, or the host cell described herein is contacted.
  • the contacting is in vitro contact. In some embodiments, the contacting is in vivo contact.
  • the cell is a tumor cell.
  • the cell is a brain tumor, and more specifically, may be astrocytoma, meningioma, glioma.
  • provided herein is a method of treating a tumor in an individual in need thereof, the method comprising administering to the individual an effective amount of an antibody, chimeric antigen receptor, composition, vector or host described herein. cell.
  • the subject can administer immunoreactive cells, wherein the immunoreactive cells that can be administered can be from about 1 to about 35 days of age.
  • the cells administered may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or up to about 40 days.
  • the age of CAR immunoreactive cells can be calculated from the time of stimulation.
  • the age of the immunoreactive cells can be calculated from the time of blood collection.
  • the age of the immunoreactive cells can be calculated from the time of transduction.
  • the immunoreactive cells that can be administered to the subject are from about 10 to about 14 or about 20 days of age.
  • the "age" of an immunoreactive cell can be determined by telomere length.
  • a "young" immune response cell can have a longer telomere length than "depleted” or "old” immunoreactive cells.
  • immunoreactive cells lose an estimated telomere length of about 0.8 kb per week in culture, and young immunoreactive cell cultures can have longer than about 44 days of immunoreactive cells.
  • About 1.4 kb of telomeres it is believed that a longer telomere length can be associated with a positive objective clinical response in a patient and persistence of cells in vivo.
  • Cells can be functional before, after, and/or during transplantation.
  • the transplanted cells may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 after transplantation. 20, 21, 22, 23, 24, 25, 6, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 days.
  • the transplanted cells can function at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after transplantation.
  • the transplanted cells can function at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 years after transplantation.
  • the transplanted cells can function during the life of the recipient.
  • transplanted cells can function at 100% of their normal expected function.
  • the transplanted cells can also perform their normal expected functions of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 , 96, 97, 98, or up to about 100% of the functionality.
  • Transplanted cells can also perform more than 100% of their normal intended function.
  • the transplanted cells can function as approximately 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 as normal expected functions. Or up to about 5,000% of the functionality.
  • Porting can be done by any type of transplant.
  • Topography may include, but is not limited to, subhepatic sac space, subsplenic sac space, subcapsular space, omentum, gastric or intestinal submucosa, small intestinal vascular segment, venous sac, testis, brain, spleen, or cornea.
  • the transplant can be a subcapsular transplant.
  • Transplantation can also be intramuscular transplantation.
  • the transplant can be a portal vein transplant.
  • transplant rejection can be improved after treatment with the immune response cells of the present invention as compared to when one or more wild type cells are transplanted to the recipient.
  • transplant rejection can be a hyperacute rejection.
  • Transplant rejection can also be an acute rejection.
  • Other types of rejection may include chronic rejection.
  • Transplant rejection can also be cell-mediated rejection or T cell-mediated rejection.
  • Transplant rejection can also be a natural killer cell mediated rejection.
  • Improving transplantation may mean alleviating hyperacute rejection, which may include reducing, alleviating or reducing adverse effects or symptoms.
  • Transplantation can refer to adoptive transplantation of cellular products.
  • Another indication of successful transplantation may be the number of days the recipient does not need immunosuppressive therapy.
  • the recipient may not require at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days of immunosuppressive therapy. This can indicate that the transplant was successful. This can also indicate that the transplanted cells, tissues and/or organs are not repelled.
  • an antibody, chimeric antigen receptor, composition, vector or host cell described herein can be administered in combination with another therapeutic agent.
  • the additional therapeutic agent is a chemotherapeutic agent, such as those described in US20140271820.
  • Chemotherapeutic agents that can be used in conjunction with the immune response cells of the invention include, but are not limited to, mitotic inhibitors (vinca alkaloids), including vincristine, vinblastine, vindesine, and novibin (TM) (vinorelbine) , 5'-dehydrohydrogen sulfide); topoisomerase I inhibitors, such as camptothecin compounds, including CamptosarTM (irinotecan HCL), HycamtinTM (topotecan HCL), and derived from camptothecin and the like Other compounds; podophyllotoxin derivatives such as etoposide, teniposide and midozozoz; alkylating agents cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, card Mustastine, busulfan, chlorambucil, briquetazine, uracil mustard, cloprofen and dacarbazine; antimetabolites, including
  • chemotherapeutic agents that can be used in conjunction with the immune response cells of the invention include, but are not limited to, anti-angiogenic agents, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligos) Nucleotide) and other angiogenesis inhibitors such as angiostatin, endostatin, interferon, interleukin-1 (including alpha and beta) interleukin 12, retinoic acid and metalloproteinase-1 and -2 tissue inhibition Agent.
  • anti-angiogenic agents including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligos) Nucleotide) and other angiogenesis inhibitors such as angiostatin, endostatin, interferon, interleukin-1 (including alpha and beta) interleukin 12, retinoic acid and metalloproteinase-1 and -2 tissue inhibition Agent.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • the extracellular domain Asp27-Arg343 (SEQ ID NO: 18) gene (SEQ ID NO: 17) of human IL-13RA2 was synthesized in vitro, and the gene was inserted into the eukaryotic expression of the Fc fragment Asp104-Lys330 containing the human IgG1 heavy chain constant region.
  • the middle is ligated with "GS” to form a fusion expression protein IL-13RA2_huFc (SEQ ID NO: 22), and the corresponding gene sequence is shown in SEQ ID NO: 11.
  • the IL-13RA1 extracellular domain gene (SEQ ID NO: 19) was inserted into the eukaryotic expression plasmid containing the Fc fragment Asp104-Lys330 of the human IgG1 heavy chain constant region, and ligated in the middle to form a fusion expression protein IL.
  • -13RA1_huFc (SEQ ID NO: 24), the corresponding gene sequence is shown in SEQ ID NO: 23.
  • Example 2 Screening of scFv specific for IL-13RA2 using a fully human phage display library
  • the phage display library used in the present invention is a whole human natural scFv phage library constructed by the company, and has a storage capacity of 1E+11.
  • a highly specific scFv fragment directed against IL-13RA2 is obtained using screening methods known to those skilled in the art. Briefly, 10 ug/ml of antigen IL-13RA2_huFc and IL-13RA1_huFc were coated in the immunotube, respectively. To screen for antibodies that specifically bind to IL-13RA2, the phage library was added to an immunotube coated with IL-13RA1_huFc for 1 hr.
  • the supernatant was added to the immunotube containing IL-13RA2_huFc for 1.5 hours, then the non-specific phage was washed away, the bound phage was eluted and infected with E. coli TG1 in logarithmic growth phase.
  • the phage eluted by expansion was expanded and the expanded phage library was purified using PEG/NaCl precipitation for the next round of screening. Panning was performed for 3-4 cycles to enrich for scFv phage clones that specifically bind to IL-13RA2. Positive clones were determined by standard ELISA methods against IL-13RA2_huFc.
  • the ELISA used IL-13RA1_huFc as an unrelated antigen to verify the specificity of the antibody.
  • a total of 3,420 clones were screened, of which 44 cloned ELISA assays specifically bound IL-13RA2_huFc and did not bind IL-13RA1_huFc.
  • After sequencing, 5 single sequences were obtained. These 5 clones were expressed and purified, and only 2 of them specifically bound U251 cells expressing IL13RA2 (purchased from the Chinese Academy of Sciences cell bank) (Fig. 2, 4), and the clone names were 31C2, 32H4.
  • amino acid sequence of the heavy chain variable region of 31C2 is set forth in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 4
  • amino acid sequence of the heavy chain variable region of 32H4 is SEQ ID NO:
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8.
  • the amino acid sequence of HDCR1 of 31C2 is shown in SEQ ID NO: 9, the amino acid sequence of HDCR2 is shown in SEQ ID NO: 10, the amino acid sequence of HDCR3 is shown in SEQ ID NO: 11, and the amino acid sequence of LDCR1 is SEQ ID NO:
  • the amino acid sequence of LDCR2 is shown in SEQ ID NO: 14
  • the amino acid sequence of LDCR3 is shown in SEQ ID NO: 15
  • the amino acid sequence of HDCR1 of 32H4 is shown in SEQ ID NO: 9
  • the amino acid sequence of HDCR2 is as SEQ ID NO: 10
  • the amino acid sequence of HDCR3 is shown in SEQ ID NO: 12
  • the amino acid sequence of LDCR1 is shown in SEQ ID NO: 13
  • the amino acid sequence of LDCR2 is shown in SEQ ID NO: 14
  • the sequence is shown in SEQ ID NO: 16.
  • the species specificity of antibody 31C2, 32H4 was determined by standard ELISA.
  • Mouse IL-13RA2 was purchased from Sino Biological Inc. Coated 5 ug/ml, 100 ul/well of murine IL-13RA2 and ELISA plate, 4 degrees overnight. Wash the ELISA plate three times with PBS. 200 ul/well of 2% skim milk powder in PBS was added for 1 hr at room temperature. Wash three times with PBS. Gradiently diluted antibody was added at a starting concentration of 10 ug/ml, diluted 3 fold, and incubated for 1 hr at room temperature. Wash three times with PBST and wash three times with PBS.
  • Example 4 Construction of anti-IL-13RA2scFv_Fc fusion antibody and its transient expression purification and activity identification in eukaryotic cells
  • Primers were designed for the VH and VL fragments of 31C2 and 32H4, respectively, and a linker consisting of 15 flexible amino acids (GGGGSGGGGSGGGGS) was introduced to form a scFv; a suitable restriction site and a protective base were introduced upstream of the VH, and a suitable downstream was introduced in the VL.
  • the enzyme cleaves the site and protects the base.
  • the PCR product was analyzed by 1% agarose gel electrophoresis and purified and recovered. After digestion, it is ligated into a suitable eukaryotic expression vector.
  • the binding of the antibody to U251 cells endogenously expressing IL-13RA2 was tested by flow cytometry, and 293T cells were used as negative cell controls.
  • the specific method for FACs detection is as follows: cells are harvested, cells are washed once with growth medium, resuspended in PBS, and the cell concentration is adjusted to 4E+5 cells/ml.
  • the diluted scFv_Fc fusion antibody was incubated with the cells for 30 minutes on ice with an antibody concentration of 111 nM. Thereafter, it was incubated with a FITC-labeled anti-human IgG secondary antibody. After two washing steps, it was tested using a Guava easyCyteTM HT System instrument.
  • Figure 4 shows the binding of the antibody 31C2, 32H4scFv_Fc fusion form to U251 and 293T cells. Both of these antibodies specifically bind to U251 cells endogenously expressing IL-13RA2 and do not bind to negative cell 293T.
  • IL-13RA2_huFc was coated on the CM5 chip by amino coupling, coated to about 500 RU, and the gradient-diluted antibody was passed through the antigen-coated channel at a flow rate of 30 ul/min as a mobile phase.
  • the running buffer was HBS-N and the temperature was 25 degrees.
  • the experimental data was analyzed by BIAevaluation 3.2 and the kinetic curves were fitted using a 1:1 langmuir model.
  • the KD of 31C2 (scFv_Fc) was 1.79 nM
  • the KD of 32H4 (scFv_Fc) was 3.76 nM (see Figure 5).
  • the cells were harvested, washed once with growth medium, resuspended in PBS, and adjusted to a cell concentration of 4E+5 cells/ml.
  • the gradient diluted scFv_Fc fusion antibody was incubated with the cells for 30 minutes on ice with an initial concentration of 500 nM, 5 fold dilutions, 8 gradients. Thereafter, it was incubated with a FITC-labeled anti-human IgG secondary antibody. After two washing steps, it was tested using a Guava easyCyteTM HT System instrument.
  • Affinity maturation is performed using phage display technology.
  • 31C2 and 32H4 as parent antibodies, two phage libraries were constructed, one CDR1 and CDR2 of the randomized light chain, and the other CDR2 and CDR2 of the heavy chain.
  • the antigen is then panned, and high affinity antibodies, i.e., variants of 31C2 and 32H4, are screened by SPR techniques and the like. Primer information is shown in Figure 7.
  • the template plasmid was first constructed based on the antibody 31C2 (scFv) (amino acid sequence SEQ ID NO: 25, nucleic acid sequence SEQ ID NO: 26).
  • scFv amino acid sequence SEQ ID NO: 25, nucleic acid sequence SEQ ID NO: 26.
  • primer 1 was amplified by PCR using primers LMF and IL1R
  • fragment 2 was amplified by PCR using primers IL2F and FdR
  • fragment 1 and fragment 2 were ligated by bridge PCR to obtain randomization
  • the full length of the sequence of scFv was then digested with NcoI and NotI, and ligated into the same digested template plasmid by T4 ligase and electroporated into TG1 competent cells with a storage capacity of 1.68E+9.
  • primer 3 was amplified by PCR using primers LMF and BH1R; fragment 4 was amplified by PCR using primers BH2F and FdR; then fragment 3 and fragment 4 were ligated by bridge PCR to obtain randomization
  • the full length of the sequence of scFv was then digested with NcoI and NotI, and ligated into the same digested template plasmid by T4 ligase and electroporated into TG1 competent cells with a storage capacity of 1.75E+9.
  • a template plasmid was constructed based on antibody 32H4 (scFv) (amino acid sequence SEQ ID NO: 26, nucleic acid sequence SEQ ID NO: 27).
  • the CDR1 and CDR2 of the light chain were randomized using the same primers as 31C2, and the resulting phage library library capacity was 2.1E+9.
  • the CDR1 and CDR2 of the heavy chain were similarly randomized using the same primers as 31C2, and the resulting phage library library capacity was 1.5E+9.
  • Example 2 of this patent The initial concentration of the antigen IL13RA2_huFc was 50 nM and a 2-fold gradient dilution was performed for the next round of screening. Panning was performed for 2-3 cycles to enrich for scFv phage clones that specifically bind to IL13RA2_huFc. Positive clones were determined by standard ELISA methods against IL13RA2_huFc. The ELISA used the human IL13RA1_huFc fragment as an unrelated antigen to verify the specificity of the antibody. A total of 111 ELISA-positive clones were picked and the dissociation constant Kd of the induced supernatant was determined by biacore after re-induction. Among them, 10 clones had a dissociation constant Kd more than 10 times lower than that of the female clone, as shown in Fig. 8.
  • Figure 9A compares clone 2C7 (amino acid sequence SEQ ID NO: 29, nucleic acid sequence SEQ ID NO: 30), 2D3 (amino acid sequence SEQ ID NO: 31, nucleic acid sequence SEQ ID NO: 32), 1D11 (amino acid sequence SEQ ID NO: :33, nucleic acid sequence SEQ ID NO: 34), 1B11 (amino acid sequence SEQ ID NO: 35, nucleic acid sequence SEQ ID NO: 36), 2A5 (amino acid sequence SEQ ID NO: 37, nucleic acid sequence SEQ ID NO: 38), 2D4 (amino acid sequence SEQ ID NO: 39, nucleic acid sequence SEQ ID NO: 40), 1H7 (amino acid sequence SEQ ID NO: 41, nucleic acid sequence SEQ ID NO: 42), 1D8 (amino acid sequence SEQ ID NO: 43, nucleic acid sequence The heavy chain amino acid sequences of SEQ ID NOS: 44) and 31C2 (amino acid sequence SEQ
  • the sequences of the HCDR1 of the affinity matured clone of 31C2 are shown in SEQ ID NOs: 45-51, respectively, and the sequences of HCDR2 are shown in SEQ ID NOs: 52-58, respectively, as shown in Figure 9B.
  • 2C7 has 4 mutations, the similarity is 96.7%; 2D3 has 5 mutations, the similarity is 95.8%; 1D11 has 6 mutations, similarity 95%; 1B11 has 5 locus mutations, the similarity is 95.8%; 2A5 has 4 locus mutations, the similarity is 96.7%; 2D4 has 5 locus mutations, the similarity is 95.8%; 1H7 There were 4 loci mutations with a similarity of 96.7%; 1D8 had 4 locus mutations with a similarity of 96.7%.
  • the sequences of the HCDR1 of the affinity matured clone of 32H4 are shown in SEQ ID NO: 63, 64, respectively, and the sequences of HCDR2 are shown in SEQ ID NO: 65, 66, respectively, as shown in Figure 9D.
  • 5G3 has 5 mutations, the similarity is 95.7%; 2D3 has 5 mutations, the similarity is 95.8%; 5D7 has 8 mutations, similarity It was 95%; 1B11 had mutations at 5 sites with a similarity of 93.2%.
  • the cells were collected by centrifugation, suspended in 30 mM Tris HCl, 20% sucrose, 1 mM EDTA pH 8.0 (80 ml per gram of cells), and then ice-bathed, 4 degrees, centrifuged at 8000 g, and the supernatant A was taken. The precipitate was suspended in 8 ml of 5 mM MgSO 4 . In an ice bath, gently shake for 10 minutes, 4 degrees, centrifuge at 8000 g, and take supernatant B. Combine supernatant A and supernatant B, centrifuge at 12000g for 15 minutes, and take the supernatant as cold osmotic shock fluid.
  • Affinity purification was performed using a nickel column, and affinity was measured using biacore T200, and the antibody-binding dissociation constant of the affinity matured is shown in Fig. 10A.
  • the specificity of the antibodies 5D7, 2C7, 5G3, 2D4, 2D3, 1B11 was determined by standard ELISA following the method of Example 3. The result is shown in Fig. 10B.
  • clones 1B11, 2C7, 2D3, 2D4 from the parent antibody 31C2 specifically bind to human IL13RA2, do not bind to human IL13RA1, and cross-react with murine IL13RA2.
  • the clone from the parental antibody 32H4, 5D7, 5G3 specifically binds to human IL13RA2, does not bind to human IL13RA1, and does not bind to murine IL13RA2.
  • the six antibodies 5D7, 2C7, 5G3, 2D4, 2D3, 1B11 with high affinity were picked for construction of the scFv_Fc fusion form.
  • cleavage sites and protecting bases were introduced upstream of the VH, and appropriate cleavage sites and protecting bases were introduced downstream of the VL.
  • the PCR product was analyzed by 1% agarose gel electrophoresis and purified and recovered. After digestion, it was ligated into the eukaryotic expression vector V152 containing human Fc fragment (purchased from Shanghai Ruijin Biotechnology Co., Ltd.). Transiently transfected into 30 ml of 293F cells by 293Fectin and expressed. The culture supernatant was collected for 5-7 days after transfection and subjected to affinity purification by Protein A. The aggregation of the antibodies was analyzed by SEC. The result is shown in FIG.
  • Affinity was determined using the method of Example 5 using biacore T200, and the results are shown in Figures 11B-11G.
  • the affinity of the antibody after affinity maturation is 3 to 10 times higher than that of the parent antibody.
  • the antibody binding dissociation constant is shown in Figure 11F.
  • Example 11 Determination of EC50 binding to U251 cells in the scFv_Fc form of the antibody
  • the cells were harvested according to the method of Example 6, and the cells were washed once with growth medium, resuspended in PBS, and adjusted to a cell concentration of 4E+5 cells/ml.
  • the gradient diluted scFv_Fc fusion antibody was incubated with the cells for 30 minutes on ice with an initial concentration of antibody of 2000 nM, 5 fold dilution, 11 gradients. Thereafter, it was incubated with a FITC-labeled anti-human IgG secondary antibody. After two washing steps, it was tested using a Guava easyCyteTM HT System instrument. Results As shown in Fig.
  • the EC50 binding EC50 of the scFv_Fc form of the antibodies 5D7, 2C7, 5G3, 2D4, 2D3, 1B11 was 0.56 nM, 0.57 nM, 0.53 nM, 0.37 nM, 0.33 nM, 0.47 nM, respectively. There is also a 2 to 8 fold increase compared to the parent antibody.
  • 2D4 and 5G3 were selected for CAR-T cell preparation and antitumor activity studies.
  • PRRLSIN-cPPT.EF-1 ⁇ a lentiviral plasmid expressing the chimeric antigen receptors of antibodies 2D4 and 5G3 was constructed, including PRRLSIN-cPPT.EF-1 ⁇ -2D4-28Z, PRRLSIN-cPPT.EF-1 ⁇ - 2D4-BBZ, PRRLSIN-cPPT.EF-1 ⁇ -2D4-28BBZ and PRRLSIN-cPPT.EF-1 ⁇ -5G3-28Z, PRRLSIN-cPPT.EF-1 ⁇ -5G3-BBZ, PRRLSIN-cPPT.EF-1 ⁇ -5G3- 28BBZ.
  • the 2D4-28Z sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 68), 2D4scFv (SEQ ID NO: 67), CD8hinge (SEQ ID NO: 69), CD28 transmembrane region (SEQ ID NO: 70), and intracellular signaling
  • the domain (SEQ ID NO: 71) and the intracellular domain CD3 (CD ID NO: 72) of CD3 consist.
  • the 2D4-BBZ sequence consists of the CD8 ⁇ signal peptide (SEQ ID NO: 68), 2D4 scFv (SEQ ID NO: 67), CD8hinge (SEQ ID NO: 69), CD8 transmembrane region (SEQ ID NO: 73), intracellular CD137
  • the signaling domain (SEQ ID NO: 74) and the intracellular domain CD3 (CD ID NO: 72) of CD3 consist.
  • the 2D4-28BBZ sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 68), 2D4scFv (SEQ ID NO: 67), CD8hinge (SEQ ID NO: 69), CD28 transmembrane region (SEQ ID NO: 70), and intracellular signaling
  • the domain SEQ ID NO: 71
  • the intracellular signaling domain of CD137 SEQ ID NO: 74
  • the intracellular domain CD3 CD3
  • the 5G3-28Z sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 68), 5G3 scFv (SEQ ID NO: 75), CD8hinge (SEQ ID NO: 69), CD28 transmembrane region (SEQ ID NO: 70), and intracellular signaling.
  • the domain (SEQ ID NO: 71) and the intracellular domain CD3 (CD ID NO: 72) of CD3 consist.
  • the 5G3-BBZ sequence consists of the CD8 ⁇ signal peptide (SEQ ID NO: 68), 5G3 scFv (SEQ ID NO: 75), CD8hinge (SEQ ID NO: 69), CD8 transmembrane region (SEQ ID NO: 73), intracellular CD137
  • the signaling domain (SEQ ID NO: 74) and the intracellular domain CD3 (CD ID NO: 72) of CD3 consist.
  • the 5G3-28BBZ sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 68), 5G3 scFv (SEQ ID NO: 75), CD8hinge (SEQ ID NO: 69), CD28 transmembrane region (SEQ ID NO: 70), and intracellular signaling.
  • 293T cells were seeded at a density of 1.7 x 107 in a 15 cm dish in DMEM containing 10% fetal bovine serum (BioWest).
  • 158.4 ⁇ g of PEI (1 ⁇ g/ ⁇ l) was dissolved in 2048 ⁇ l of serum-free DMEM medium and incubated at room temperature.
  • the plasmid mixture was added to the PEI mixture and incubated for 20 min at room temperature.
  • 4.096 ml of the transfection complex was added dropwise to a 15 cm culture dish containing 20 ml of DMEM medium. After 4-5 hours, the transfected 293T cells were exchanged with DMEM medium of 10% FBS, and incubated at 37 ° C for 72 h to collect the virus. The supernatant was concentrated and concentrated to determine the virus titer.
  • the concentrated virus titer was:
  • T lymphocytes cultured in a lymphocyte culture medium at a density of about 5 ⁇ 10 5 /mL, and magnetic beads (Invitrogen) coated with anti-CD3 and CD28 antibodies simultaneously at a magnetic bead:cell ratio of 2:1 Recombinant human IL-2 (Shanghai Huaxin Biotech Co., Ltd.) with a concentration of 500 U/mL was stimulated for 24-48 h;
  • Retronectin coated 24-well plates 380 ⁇ l of 5 ⁇ g/ml retronectin solution (PBS) was added to each well and incubated overnight at 4 °C. Discard the retronectin solution (PBS) in a 24-well plate, wash twice with 1 ml PBS, wash once with the wells (well kept moist); inoculate the cells in a 24-well plate coated with retronectin, the number of cells per well 5 ⁇ 105
  • T lymphocytes On day 7 of culture, 1 ⁇ 10 6 T cells were taken, aliquoted at 4 degrees, 5000 rpm, centrifuged for 5 min, the supernatant was discarded, and PBS was washed twice. The control cells were incubated with 50 ⁇ l of PE-SA (1:200 dilution) antibody for 45 min on ice, washed twice with PBS (2% NBS), and resuspended as a control.
  • the cells in the test group were +50 ⁇ l 1:50 diluted biotin-Goat anti Human IgG, F(ab')2 antibody, incubated on ice for 45 min; wash twice with PBS (2% NBS); add 50 ⁇ l of PE-SA (1:200 dilution) antibody for 45 min on ice; add 2 ml PBS (2% NBS) Resuspend the cells, discard the supernatant at 4 ° C, 5000 rpm / min, centrifugation for 5 minutes; repeat twice; flow cytometry to detect the proportion of CAR-positive T cells.
  • Target cells Inoculate 50 ul of 1 ⁇ 104/mL U251 cells into E-Plate 16 plates, let stand for 30-40 min, and set them on the real-time monitor to start monitoring;
  • Effector cells after 18 hours, add UTD and CAR T cells expressing different chimeric antigen receptors at a target ratio of 3:1, 1:1 or 1:3;
  • Each group is provided with two duplicate wells, and the average of two replicate wells is taken.
  • the detection time is 38h.
  • Each experimental group each target cell + CAR T expressing different chimeric antigen receptors
  • Control group 1 target cells
  • Control group 2 blank medium
  • % cytotoxicity [(experimental group - effector cell spontaneous group - target cell spontaneous group) / (target cell max - target cell spontaneous)] * 100

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Abstract

提供了特异性识别IL-13RA2的抗体，其可以用于制备靶向性抗肿瘤药物以及诊断肿瘤的药物。

Description

靶向IL-13RA2的抗体及其应用 技术领域
本发明涉及肿瘤免疫治疗或诊断领域，更具体地，涉及特异识别IL-13RA2的抗体及其应用。
背景技术
恶性神经胶质瘤(malignant gliomas,MG),包括多形性成胶质细胞瘤和胶质母细胞瘤,在美国每年的新增病例有20000例。根据美国脑部肿瘤协会的统计，截至2010年，美国有140000人患有恶性的脑部肿瘤。尽管MG是一种罕见的疾病，但是它的恶性程度以及致死率是非常高的。现有的标准治疗手段，效果非常有限，外科手术以及放疗后的五年生存率也非常低。对于手术后复发的病人，新的治疗选择也非常少。因此，开发新的靶点，新的治疗手段是广大病人的迫切需求。
白介素13受体a2(Interleukin-13 Receptor subunit alpha 2，IL-13RA2)是特异性高表达于人神经胶质瘤等恶性肿瘤细胞表面的肿瘤特异性标志物(Dehinski等，(1995)Clin.Cancer Res.1,1253-1258)。人IL-13RA2做为人神经胶质瘤的治疗靶点早在1988年已引起美国FDA的注意，该组织先后制备了针对人IL-13RA2治疗靶点的药物IL-13-PE38和针对人IL-13RA2的单链抗体scFv-PE融合分子。尽管IL-13-PE38已在神经胶质瘤、头颈部肿瘤、卵巢癌和肾癌等恶性肿瘤的治疗中取得了疗效并已被美国FDA批准进入临床期治疗，但由于在治疗过程中，IL-13-PE38不仅与肿瘤细胞表面特异性表达的人IL-13RA2结合，也可以与表达于正常组织细胞表面的IL13-RA1结合，损伤正常组织和细胞。由于缺乏严格的靶向性，限制了IL-13-PE38的进一步应用。
本发明目的在于发现特异性针对IL-13RA2的抗体以及开发靶向IL-13RA2的免疫效应细胞。
发明内容
本发明的目的在于提供抗IL-13RA2的抗体及其应用。
在第一方面，本发明提供了一种特异性识别IL-13RA2的抗体，所述抗体与内源性表达IL-13RA2的U251细胞的结合相对亲和力EC50不高于100nM，优选不高于10nM，更优选0.01-10nM。
在一优选例中，所述相对亲和力数据的处理使用GraphPad Prism 5软件(GraphPad Software,Inc)。
在具体的实施方式中，所述抗体选自以下的任一种:
(1)抗体，其包含重链可变区，所述重链可变区包含SEQ ID NO:9、45、46、47、48、49、50、51、63、或64所示的HCDR1，和/或包含SEQ ID NO:10、52、53、54、 55、56、57、58、65、或66所示的HCDR2，和/或包含SEQ ID NO:11或SEQ ID NO:12任一所示的HCDR3；
(2)抗体，其包含轻链可变区，所述轻链可变区包含SEQ ID NO:13所示的LCDR1，和/或包含SEQ ID NO:14所示的LCDR2，和/或包含SEQ ID NO:15或SEQ ID NO:16任一所示的LCDR3；
(3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区；
(4)抗体，(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
在具体的实施方式中，所述抗体选自以下的任一种:
(1)抗体，包含轻链可变区，该轻链可变区包含SEQ ID NO:4所示的氨基酸序列、SEQ ID NO:8所示的氨基酸序列或SEQ ID NO:4和SEQ ID NO:8的变体的序列；
(2)抗体，包含重链可变区，所述重链可变区具有SEQ ID NO:2、6、29、31、33、35、37、39、41、43、59或61所示的序列、或上述序列的变体；
(3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区。
在具体的实施方式中，所述的抗体的轻链可变区包含SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15或SEQ ID NO:16所示的LCDR3。
在具体的实施方式中，所述的抗体的轻链可变区具有SEQ ID NO:4或8所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
在具体的实施方式中，所述抗体的重链可变区包含SEQ ID NO:9、45、46、47、48、49、50、51、63或64所示的HCDR1，SEQ ID NO:10、52、53、54、55、56、57、58、65或66所示的HCDR2，和SEQ ID NO:11或SEQ ID NO:12所示的HCDR3。
在具体的实施方式中，所述抗体的重链可变区具有所述抗体的重链可变区具有SEQ ID NO:2、6、29、31、33、35、37、39、41、43、59或61所示的序列，或具有与上述任一序列至少80％，更优选85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
在具体的实施方式中，所述轻链可变区的CDR区和所述重链可变区的CDR区具有下述任选的序列或其变体：
(1)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:9所示的HCDR1，SEQ ID NO:10所示的HCDR2和SEQ ID NO:11所示的HCDR3；
(2)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:16所示的LCDR3；SEQ ID NO:9所示的HCDR1，SEQ ID NO:10所示的HCDR2和SEQ ID NO:12所示的HCDR3；
(3)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:16 所示的LCDR3；SEQ ID NO:64所示的HCDR1，SEQ ID NO:66所示的HCDR2和SEQ ID NO:12所示的HCDR3；
(4)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:45所示的HCDR1，SEQ ID NO:52所示的HCDR2和SEQ ID NO:11所示的HCDR3；
(5)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:16所示的LCDR3；SEQ ID NO:63所示的HCDR1，SEQ ID NO:65所示的HCDR2和SEQ ID NO:12所示的HCDR3；
(6)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:50所示的HCDR1，SEQ ID NO:56所示的HCDR2和SEQ ID NO:11所示的HCDR3；
(7)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:46所示的HCDR1，SEQ ID NO:52所示的HCDR2和SEQ ID NO:11所示的HCDR3；
(8)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:48所示的HCDR1，SEQ ID NO:54所示的HCDR2和SEQ ID NO:11所示的HCDR3；
(9)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:47所示的HCDR1，SEQ ID NO:53所示的HCDR2和SEQ ID NO:11所示的HCDR3；
(10)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:49所示的HCDR1，SEQ ID NO:55所示的HCDR2和SEQ ID NO:11所示的HCDR3；
(11)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:51所示的HCDR1，SEQ ID NO:57所示的HCDR2和SEQ ID NO:11所示的HCDR3；
(12)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:49所示的HCDR1，SEQ ID NO:58所示的HCDR2和SEQ ID NO:11所示的HCDR3。
在具体的实施方式中，
(1)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:2所示的序列或其变体的序列；
(2)所述轻链可变区具有SEQ ID NO:8所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:6所示的序列或其变体的序列；
(3)所述轻链可变区具有SEQ ID NO:8所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:61所示的序列或其变体的序列；
(4)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:29所示的序列或其变体的序列；
(5)所述轻链可变区具有SEQ ID NO:8所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:59所示的序列或其变体的序列；
(6)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:39所示的序列或其变体的序列；
(7)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:31所示的序列或其变体的序列；
(8)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:35所示的序列或其变体的序列；
(9)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:33所示的序列或其变体的序列；
(10)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:37所示的序列或其变体的序列；
(11)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:41所示的序列或其变体的序列；
(12)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:43所示的序列或其变体的序列。
在第二方面，本发明提供一种特异性识别IL-13RA2的抗体，该抗体与第一方面所述的抗体识别相同的抗原决定部位。
在第三方面，本发明提供一种特异性识别IL-13RA2的抗体，该抗体与第一方面所述的抗体竞争性结合IL-13RA2。
在第四方面，本发明提供编码第一-第三方面所述的抗体的核酸。
在第五方面，本发明提供一种表达载体，其包含第四方面所述的核酸。
在第六方面，本发明提供一种宿主细胞，其包含第五方面所述的表达载体或基因组中整合有第四方面所述的核酸。
在第七方面，本发明提供一种多功能免疫辍合物，所述的多功能免疫辍合物包括:
第一-第三方面所述的抗体；以及
与之连接的功能性分子；所述的功能性分子选自:靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。
在具体的实施方式中，所述的靶向肿瘤表面标志物的分子是结合除IL-13RA2外的其他肿瘤表面标志物的抗体或配体；或
所述的抑制肿瘤的分子是抗肿瘤的细胞因子或抗肿瘤的毒素；较佳地，所述的细胞因子选自:IL-12、IL-15、I型干扰素、TNF-alpha。
在具体的实施方式中，所述的靶向免疫细胞的表面标志物的分子是结合免疫细胞表面标志物的抗体，优选的，所述的结合免疫细胞表面标志物选自:CD3，CD16，CD28，更佳的，所述的结合免疫细胞表面标志物的抗体是抗CD3抗体。
在具体的实施方式中，所述的靶向免疫细胞的表面标志物的分子是结合T细胞表面标志物的抗体，其与第一方面-第三方面任一所述的抗体形成T细胞参与的双功能抗体，
在具体的实施方式中，所述多功能免疫辍合物是融合多肽，在第一方面-第三方面任一所述的抗体以及与之连接的功能性分子之间，还包括连接肽。
在第八方面，本发明提供编码第七方面所述的多功能免疫辍合物的核酸。
在第九方面，本发明提供第一-第三方面所述的抗体的嵌合抗原受体，其特征在于，所述的嵌合抗原受体包含顺序连接的：第一-第三方面所述的抗体、跨膜区和胞内信号区。
在具体的实施方式中，所述的胞内信号区选自：CD3ζ、CD3γ、CD3δ、CD3ε、FcRγ(FCER1G)、FcRβ(FcεR1b)、CD79a、CD79b、FcγRIIa、DAP10和DAP12的蛋白质的功能信号传导结构域，或其组合。
在具体的实施方式中，所述的胞内信号区还具有共刺激信号传导结构域，所述共刺激信号传导结构域包含选自以下蛋白的功能信号传导结构域：CD27、CD28、4-1BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关的抗原-1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3、特异性结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244,2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A，Ly108)、SLAM(SLAMF1，CD150，IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46和NKG2D，或其组合。
在具体的实施方式中，所述的嵌合抗原受体包括如下的顺序连接的抗体、跨膜区和胞内信号区：
第一-第三方面所述的抗体、CD8和CD3ζ；
第一-第三方面所述的抗体、CD8、CD137和CD3ζ；或
第一-第三方面所述的抗体、CD28分子的跨膜区、CD28分子的胞内信号区和CD3ζ；或
第一-第三方面所述的抗体、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
在第十方面，本发明提供编码第九方面述的嵌合抗原受体的核酸。
在第十一方面，本发明提供一种表达载体，其第十方面所述的核酸。
在第十二方面，本发明提供一种病毒，所述病毒包含第十一方面所述的载体。
在第十三方面，本发明提供一种嵌合抗原受体修饰的免疫细胞，其转导有第十方面所述的核酸，或第十一方面所述的表达载体或第十二方面所述的病毒；或其表面表达第九方面所述的嵌合抗原受体；
较佳地，所述的免疫细胞为：T淋巴细胞、NK细胞或者NKT淋巴细胞。
在具体的实施方式中，所述的免疫细胞还携带外源的细胞因子的编码序列；或
其还表达另一种嵌合抗原受体，该受体不含有CD3ζ；或
其还表达趋化因子受体；较佳地，所述的趋化因子受体包括：CCR；或
其还表达能降低PD-1表达的siRNA或者阻断PD-L1的蛋白；或其细胞中内源性的PD-1被基因编辑技术敲除；或
其还表达安全开关。
在第十四方面，本发明提供一种药物组合物，其包括：
第一-第三方面所述的抗体或编码该抗体的核酸；或
第七方面所述的免疫辍合物或编码该辍合物的核酸；或
第九方面所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或
第十三方面所述的嵌合抗原受体修饰的免疫细胞；
以及药学上可接受的载体或赋形剂。
在第十五方面，本发明提供一种试剂盒，其包括：
容器，以及位于容器中的第十四方面所述的药物组合物；或
容器，以及位于容器中的第一-第三方面所述的抗体或编码该抗体的核酸；或第七方面所述的免疫辍合物或编码该辍合物的核酸；或第九方面所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或第十三方面所述的嵌合抗原受体修饰的免疫细胞。
在第十六方面，本发明提供第一-第三方面所述的抗体或编码该抗体的核酸；或第 七方面所述的免疫辍合物或编码该辍合物的核酸；或第九方面所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或第十三方面所述的嵌合抗原受体修饰的免疫细胞的用途，用于治疗表达IL-13RA2的肿瘤，
较佳的，所述的表达IL-13RA2的肿瘤为脑癌、胰腺癌、卵巢癌、肾癌、膀胱癌、胰腺癌、胃癌、肠癌、头颈癌、甲状腺癌、前列腺癌、卡波氏肉瘤。更佳的，所述的脑癌选自星形细胞瘤、脑膜瘤、少突神经胶质瘤、神经胶质瘤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了IL-13RA2_huFc、IL13RA1_huFc的SDS电泳图(还原条件)；
图2显示了ELISA检测31C2，32H4与IL-13RA2以及IL13Ra1的结合；
图3显示了ELISA检测抗体31C2，32H4与鼠的IL-13RA2的结合；
图4显示了FACs检测抗体31C2，32H4与U251(IL-13RA2阳性)以及293T(IL-13RA2阴性)细胞的结合；
图5显示了Biacore测定抗体31C2，32H4(scFv_Fc)的亲和力；
图6显示了FACs检测抗体31C2，32H4结合U215细胞的EC50；
图7显示了亲和力成熟的引物信息；
图8显示了亲和力成熟后筛选的10个克隆的解离常数Kd；
图9A显示了31C2的亲和力成熟克隆重链序列比对，图9B显示了31C2的亲和力成熟克隆的HCDR1和HCDR2的序列，图9C显示了32H4的亲和力成熟克隆重链序列比对，图9D显示了32H4的亲和力成熟克隆的HCDR1和HCDR2的序列；
图10A显示了亲和力成熟的抗体结合解离常数；图10B显示了抗体5D7、2C7、5G3、2D4、2D3、1B11的特异性鉴定结果；
图11A显示了亲和力成熟后，抗体的scFv_Fc形式在30ml表达体系的产出以及纯化产物的聚集程度测定结果；图11B-G显示了抗体的scFv_Fc形式的亲和力；图11H为抗体结合解离常数结果；
图12显示了抗体5D7、2C7、5G3、2D4、2D3、1B11的scFv_Fc形式的与U251细胞结合的EC50；
图13显示了不同CAR-T细胞的体外杀伤活性。
具体实施方式
本发明人经过深入的研究筛选，获得了特异性识别IL-13RA2的抗体，包括单链抗体和人源化抗体。本发明的抗体可以被应用于制备各种靶向性抗肿瘤药物以及诊断肿瘤 的药物。
为了更易于理解本发明，首先定义一些术语。
本文中的术语“IL-13RA2”，也是CD213A2，是白介素-13受体复合物的一个亚单位。由380个氨基酸残基组成的跨膜蛋白(NCBI Reference Sequence:NP_000631.1)。它与IL-13RA1(NCBI Reference Sequence:NP_001551.1)相类似，与IL-13结合很强，但是没有胞内信号域。
本文中的术语“抗体”指免疫系统的抗原结合蛋白，包括具有抗原结合区域的完整的全长抗体，还包括具有“抗原结合部分”或“抗原结合区域”的片段、或其单链例如单链可变片段(scFv)，还包括本文提供的抗体的变体。抗体片断包括但不限于:(i)由VL、VH、CL和CH1结构域组成的Fab片段，包括Fab’和Fab’-SH，(ii)VH和CH1结构域组成的Fd片段，(iii)由单个抗体的VL和VH结构域组成的Fv片段；(iv)由单个可变区组成的dAb片段(Ward等，1989，Nature 341:544-546)；(v)F(ab’)2片段，包含2个连接的Fab片段的二价片段；(vi)单链Fv分子抗原结合位点；(vii)双特异性单链Fv二聚体(PCT/US92/09965)；(viii)“二体”或“三体”，通过基因融合构建的多价或多特异性片段；和(ix)与相同或不同抗体遗传融合的scFv。
本文中的术语“Fc”或“Fc区”包括包含除第一恒定区免疫球蛋白结构域以外的抗体恒定区的多肽。因而，Fc指IgA、IgD和IgG的最后两个恒定区免疫球蛋白结构域，和IgE和IgM的最后三个恒定区免疫球蛋白结构域，和这些结构域N端的柔性铰链。对于IgA和IgM，Fc可包括J链。对于IgG，Fc包括免疫球蛋白结构域Cγ2和Cγ3和在Cγ1和Cγ2之间的铰链。虽然Fc区的边界可以改变，但人IgG重链Fc区通常定义为在其羧基端包含残基C226或P230，其中编号是根据Kabat的EU索引。对于人IgG1，Fc在本文定义为包含残基P232至其羧基端，其中编号是根据Kabat中的EU索引。Fc可以指分离的该区域，或者位于Fc多肽，例如抗体，环境中的该区域。上述“铰链”包括包含在抗体的第一和第二恒定结构域之间的氨基酸的柔性多肽。结构上，IgG CH1结构域中止于EU220位，IgG CH2结构域始于残基EU237位。因而，对于IgG，本文中抗体铰链定义为包括221(IgG1的D221)至231(IgG1的A231)位，其中编号是根据Kabat的EU索引。
术语“变体”指与本申请所提供的抗体的序列具有基本上相同氨基酸序列或由基本上相同的核苷酸序列编码的一种或多种活性的多肽。所述变体与本申请实施例中所提供的抗体具有相同或相似的活性。
变体相比亲本抗体，具有至少一个氨基酸修饰。在具体的实施方式中，本文中的变体序列优选的具有与亲本抗体序列至少约80％，最优选至少约90％，更优选至少约95％，更优选至少约98％、最优选至少约99％的氨基酸序列同一性。变体可以指抗体本身，也可以指包含亲本抗体的组合物。术语“氨基酸修饰”包括氨基酸取代、添加和/或缺失，“氨基酸取代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸，“氨基酸插入”意指在亲本多肽序列中的特定位置添加氨基酸，“氨基酸缺失”或“缺失”意指去除亲本多 肽序列中特定位置上的氨基酸。
“氨基酸修饰”可通过本领域已知的标准技术将修饰导入本发明的抗体中，例如定点诱变和PCR介导的诱变。保守的氨基酸取代是用具有相似侧链的氨基酸残基替换氨基酸残基的取代。本领域已经定义了具有相似侧链的氨基酸残基家族。这些家族包括含碱性侧链的氨基酸(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电的急性侧链(例如，甘氨酸、天冬酰胺、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、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因而，可以用其他相同侧链家族的氨基酸残基替换本发明抗体的CDR区中或框架区中的一个或多个氨基酸残基，并可以测试所改变的抗体(变体抗体)保留的功能。
上述术语“亲本抗体”指本申请所提供的抗体或依据本申请所提供的抗体进行突变、或亲和力成熟等处理后后所得的抗体，优选的，指实施例所示的抗体。所述亲本抗体可以是天然存在的抗体，或者天然存在的抗体的变体或改造版本。亲本抗体可以指抗体本身，包含所述亲本抗体的组合物，或其编码氨基酸序列。
本文中使用的术语“抗原决定部位”又称抗原表位，可以由IL-13RA2蛋白序列的连续序列组成，也可以由IL-13RA2蛋白序列不连续的三维结构组成。
抗IL-13RA2的抗体
在本公开中，描述了具有基于scFv的抗原结合区域的抗原结合蛋白，包括抗体。其中使用重组IL-13RA2，从人scFv噬菌体展示文库选择scFv。这些分子展示精细的特异性。例如，该抗体仅识别IL-13RA2,不识别IL-13RA1。本发明中如果没有特别说明，IL-13RA2指人的IL-13RA2。
在一些实施方案中，本发明包括具有scFv序列的抗体，所述scFv序列与一个或多个重链恒定区域融合以形成具有人免疫球蛋白Fc区的抗体以产生双价蛋白，从而增加抗体的总体亲和力和稳定性。此外，Fc部分允许将其他分子(包括但不限于荧光染料、细胞毒素、放射性同位素等)与例如用于抗原定量研究中的抗体直接缀合，以便固定抗体用于亲和力测量、用于定向递送治疗药、使用免疫效应细胞测试Fc介导的细胞毒性和许多其它应用。
本文提供的结果突出本发明抗体在靶向IL-13RA2时的特异性、灵敏性和效用。
本发明的分子基于使用噬菌体展示鉴定和选择单链可变片段(scFv)，所述单链可变片段的氨基酸序列赋予分子针对IL-13RA2的特异性并且形成本公开的全部抗原结合蛋白的基础。因此，所述scFv可以用来设计一系列不同“抗体”分子，包括例如全长抗体、其片段如Fab和F(ab’)2、融合蛋白(包括scFv_Fc)、多价抗体、即，具有针对相同抗原或不同抗原的多于一种特异性的抗体，例如，双特异性T细胞结合抗体(BiTE)、三抗体等(见Cuesta等人，Multivalent antibodies:when design surpasses evolution,Trends in  Biotechnology 28:355-362,2010)。
在抗原结合蛋白是全长抗体的一个实施方案中，本发明抗体的重链和轻链可以是全长(例如，抗体可以包括至少一条并优选地两条完整重链，和至少一条并优选地两条完整轻链)或可以包括抗原结合部分(Fab、F(ab’)2、Fv或scFv)。在其他实施方案中，抗体重链恒定区选自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE。抗体类型的选择将取决于所设计的抗体欲引发的免疫效应子功能。在构建重组免疫球蛋白时，各种免疫球蛋白同种型的恒定区的适宜氨基酸序列和用于产生广泛种类抗体的方法是本领域技术人员已知的。
在第一方面，本发明提供了特异性识别IL-13RA2的抗体，其与稳定转染了人IL-13RA2的U251细胞的结合相对亲和力EC50小于100nM，优选小于10nM，更优选0.1-1nM，最优选0.3-0.6nM。
在一优选方案中，本发明提供的IL-13RA2的抗体包括:包含SEQ ID NO:9的氨基酸序列的重链CDR1，和/或包含SEQ ID NO:10的氨基酸序列的重链CDR2，和/或包含SEQ ID NO:11或者12的氨基酸序列的重链CDR3。在另一优选方案中，本发明提供的结合IL-13RA2的抗体包括:包含SEQ ID NO:13的氨基酸序列的轻链CDR1，和/或包含SEQ ID NO:14的氨基酸序列的轻链CDR2，和/或包含SEQ ID NO:15或者16的氨基酸序列的轻链CDR3。在另一优选方案中，本发明提供了结合IL-13RA2的抗体包括:包含SEQ ID NO:9的氨基酸序列的重链CDR1，和/或包含SEQ ID NO:10的氨基酸序列的重链CDR2，和/或包含SEQ ID NO:11或者12的氨基酸序列的重链CDR3，以及包含SEQ ID NO:13的氨基酸序列的轻链CDR1，和/或包含SEQ ID NO:14的氨基酸序列的轻链CDR2，和/或包含SEQ ID NO:15或者16的氨基酸序列的轻链CDR3。优选的，所述结合IL-13RA2的抗体包括:包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:10所示的HCDR2、SEQ ID NO:11所示的HCDR3，以及SEQ ID NO:13所示的LCDR1、SEQ ID NO:14所示的LCDR2、SEQ ID NO:15所示的LCDR3；或者包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:10所示的HCDR2、SEQ ID NO:12所示的HCDR3，以及SEQ ID NO:13所示的LCDR1、SEQ ID NO:14所示的LCDR2、SEQ ID NO:16所示的LCDR3。
更优选的，所述结合IL-13RA2的抗体包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:10所示的HCDR2、SEQ ID NO:12所示的HCDR3，以及SEQ ID NO:13所示的LCDR1、SEQ ID NO:14所示的LCDR2、SEQ ID NO:16所示的LCDR3。
在另一方面，本发明提供了结合IL-13RA2的抗体，其重链可变区选自SEQ ID NO:2或者SEQ ID NO:6的序列，或者二者的变体的序列。
在另一方面，本发明提供了结合IL-13RA2的抗体或其片段，其包括选自SEQ ID NO:4或者SEQ ID NO:8的轻链可变区序列。
考虑到这些重链和轻链可变区序列各自可以结合IL-13RA2，可以“混合和匹配”重链和轻链可变区序列来产生本发明的抗IL-13RA2的结合分子。
在另一个方面，本发明提供了结合IL-13RA2的抗体或其片段的变体。因而本发明 提供了抗体或其片段，具有与重链或轻链的可变区序列至少80％相同的重链和/或轻链可变区。优选的，重链和/或轻链可变区的氨基酸序列同一性是至少85％，更优选至少90％，最优选至少95％，特别是96％，更特别97％，甚至更特别98％，最特别99％，包括例如80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％和100％。变体可以以本申请所述的抗体为母本抗体，通过酵母库筛选、噬菌体库筛选、点突变等方法得到。
在另一个方面，本发明提供了与前述的抗IL-13RA2的抗体识别相同的抗原决定部位的抗体。
抗IL-13RA2的抗体特性
评估抗体例如抗IL-13RA2的抗体的结合能力的标准测定是本领域已知的，包括例如ELISA、biacore、Western印迹和流式细胞仪分析。合适的测定详细描述在实施例中。
核酸、载体和宿主细胞
本发明还提供了编码结合IL-13RA2的抗体和其片段分离的核酸、载体以及包含所述核酸或载体的宿主细胞。核酸可位于完整细胞中、细胞裂解液中或者以部分纯化的或基本纯化的形式。
可以使用标准的分子生物学技术获得本发明的核酸，例如可以通过标准的PCR扩增或cDNA克隆技术，获得编码抗体的轻链和重链或者编码VH和VL区段的cDNA。对于从免疫球蛋白基因文库获得的抗体(例如，使用噬菌体展示技术)，可以从文库回收编码抗体的一种或多种核酸。向宿主细胞中导入外源核酸的方法是本领域普遍已知的，并可随所使用的宿主细胞而变化。
优选的本发明核酸分子是编码选自SEQ ID NO:3或者SEQ ID NO:7的轻链可变区，和/或选自SEQ ID NO:1或者SEQ ID NO:5的重链可变区的那些。更优选的是这样的核酸分子，所述核酸分子包含SEQ ID NO:1的重链序列，和包含SEQ ID NO:3的轻链序列或者包含SEQ ID NO:5的重链序列，和包含SEQ ID NO:7的轻链序列。
为了表达蛋白质，可以将编码本发明抗体的核酸整合到表达载体中。多种表达载体可用于蛋白质表达。表达载体可包括自我复制的染色体外载体，或整合到宿主基因组中的载体。用于本发明的表达载体包括但不限于使蛋白质能够在哺乳动物细胞、细菌、昆虫细胞、酵母和体外系统中表达的那些。如本领域已知的，多种表达载体是可商业或其他方式获得的。可用于本发明中来表达抗体。
免疫辍合物
本发明还提供了多功能免疫缀合物，其包含本文所述抗体以及进一步包含至少一种其它类型的功能性分子。所述的功能性分子选自但不限于:靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。所述抗体与所述 功能性分子可以通过共价连接、偶联、附着、交联等方式构成辍合物。
作为一种优选方式，所述免疫缀合物可包含:本发明的抗体以及至少一种靶向肿瘤表面标志物的分子或抑制肿瘤的分子。所述的抑制肿瘤的分子可以是抗肿瘤的细胞因子，或抗肿瘤的毒素；较佳地，所述的细胞因子包括(但不限于):IL-2、IL-7、IL-12、IL-15、I型IFN、TNF-alpha。在具体的实施方式中，所述的靶向肿瘤表面标志物的分子是靶向本发明抗体所靶向的相同肿瘤的表面标志物的分子。例如，所述的靶向肿瘤表面标志物的分子可以是结合肿瘤表面标志物的抗体或配体，例如可以与本发明的抗体协同作用，更精准地靶向肿瘤细胞。可选的，
作为一种优选方式，所述免疫缀合物可包含:本发明的抗体以及可检测标记物。所述的可检测标记物包括但不限于:荧光标记物、显色标记物；如:酶、辅基、荧光材料、发光材料，生物发光材料、放射性材料、正电子发射金属以及非放射性顺磁性金属离子。也可包含一个以上的标记物。为了检测和/或分析和/或诊断目的用于标记抗体的标记依赖于使用的特定检测/分析/诊断技术和/或方法例如免疫组织化学染色(组织)样品、流式细胞计量术等。对于本领域已知的检测/分析/诊断技术和/或方法合适的标记为本领域技术人员所熟知。
作为一种优选方式，所述免疫缀合物可包含:本发明的抗体以及靶向免疫细胞的表面标志物的分子。所述靶向免疫细胞的表面标志物的分子可以是结合免疫细胞表面标志物的抗体或配体，能够识别免疫细胞，其携带本发明的抗体达到免疫细胞，同时本发明的抗体可将免疫细胞靶向于肿瘤细胞，从而引发免疫细胞特异性地杀伤肿瘤。所述的免疫细胞表面标志物可以选自CD3，CD16，CD28，更佳的，结合免疫细胞表面标志物的抗体是抗CD3抗体。免疫细胞可选自T细胞、NK细胞、NKT细胞。
作为通过直接或间接(例如通过接头)缀合而化学产生免疫缀合物的一种方式，所述免疫缀合物可以作为融合蛋白而产生，所述融合蛋白包含本发明的抗体及合适的其它蛋白。融合蛋白可以通过本领域已知方法产生，例如通过构建核酸分子以及随后表达所述核酸分子而重组产生，所述核酸分子包含符合读框的编码抗体的核苷酸序列以及编码合适标记的核苷酸序列。
本发明另一方面提供了编码本发明的至少一种抗体、其功能变体或者免疫缀合物的核酸分子。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
在另一个方面，本发明提供了嵌合抗原受体，其包含胞外结合结构域、跨膜域和胞内域。本文所用的术语“嵌合抗原受体(Chimeric Antigen Receptor，CAR)”指一种融合到细胞内信号转导域的肿瘤抗原结合结构域，能激活T细胞。常见地，CAR的胞外结合结构域来源于小鼠或人源化或人的单克隆抗体。
胞外结合结构域为本发明所述的抗体，非限制性实例包括衍生自抗体的单链可变片段(scFv)、选自文库的片段抗原结合区(Fab)、单结构域片段或与接合其同源受体的自然 配体。在一些实施方案中，胞外抗原结合区域可以包含scFv、Fab或天然配体，以及它们的任何衍生物。胞外抗原结合区可以指除完整抗体之外的分子，其可以包含完整抗体的一部分并且可以与完整抗体所结合的抗原结合。抗体片段的实例可以包括但不限于Fv、Fab、Fab'、Fab'-SH、F(ab')2；双功能抗体、线性抗体；单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。
胞外抗原结合区，例如scFv、Fab或天然配体，可以是确定抗原特异性的CAR的一部分。胞外抗原结合区可以结合任何互补靶。胞外抗原结合区可以衍生自已知可变区序列的抗体。胞外抗原结合区可以从获自可获得的小鼠杂交瘤的抗体序列中得到。或者，可以从肿瘤细胞或原代细胞例如肿瘤浸润淋巴细胞(TIL)的全外切割测序获得胞外抗原结合区。
在一些实施方案中，胞外抗原结合区的结合特异性可以通过互补决定区或CDR，如轻链CDR或重链CDR来确定。在许多情况下，结合特异性可以通过轻链CDR和重链CDR来确定。与其他参考抗原相比，给定的重链CDR和轻链CDR的组合可以提供给定的结合袋，其可以赋予抗原更大的亲和力和/或特异性。
在本文公开的任何实施方案的某些方面，胞外抗原结合区，例如scFv可以包含对抗原特异性的轻链CDR。轻链CDR可以是抗体例如CAR的scFv轻链的互补决定区。轻链CDR可以包含连续的氨基酸残基序列，或由非互补决定区(例如框架区)隔开的两个或更多个连续的氨基酸残基序列，。在一些实施方案中，轻链CDR可以包含两个或更多个轻链CDR，其可以被称为轻链CDR-1，CDR-2等。在一些实施方案中，轻链CDR可以包含三个轻链CDR，其可分别称为轻链CDR-1，轻链CDR-2和轻链CDR-3。在一些实例中，存在于普通轻链上的一组CDR可统称为轻链CDR。
在本文公开的任何实施方案的某些方面，胞外抗原结合区，例如scFv可以包含对抗原特异的重链CDR。重链CDR可以是抗体例如scFv的重链互补决定区。重链CDR可以包含氨基酸残基的连续序列，或由非互补决定区(例如框架区)隔开的两个或更多个氨基酸残基的连续序列。在一些实施方案中，重链CDR可以包含两个或更多个重链CDR，其可以称为重链CDR-1，CDR-2等。在一些实施方案中，重链CDR可以包含三个重链CDR，其可分别称为重链CDR-1，重链CDR-2和重链CDR-3。在一些实施方案中，存在于共同重链上的一组CDR可统称为重链CDR。
通过使用基因工程，可以以各种方式修饰胞外抗原结合区。在一些实施方案中，可以突变胞外抗原结合区域，从而可以选择胞外抗原结合区域以对其靶标具有更高的亲和力。在一些实施方案中，胞外抗原结合区域对其靶标的亲和力可针对可在正常组织上以低水平表达的靶标进行优化。可以进行此优化，以尽量减少潜在的毒性。在其他情况下，对靶标的膜结合形式具有更高亲和力的胞外抗原结合区域的克隆可以优于其可溶形式的对应物。可以进行这种修饰，因为也可以检测到不同水平的可溶形式的靶标，并且它们的靶向可引起不期望的毒性。
在一些实施方案中，胞外抗原结合区域包括铰链或间隔区。术语铰链和间隔区可以 互换使用。铰链可以被认为是用于向胞外抗原结合区提供柔性的CAR的一部分。在一些实施方案中，铰链可用于检测细胞的细胞表面上的CAR，特别是当检测胞外抗原结合区的抗体不起作用或可用时。例如，衍生自免疫球蛋白的铰链的长度可能需要优化，这取决于胞外抗原结合区域靶向靶上的表位的位置。
在一些实施方案中，铰链可能不属于免疫球蛋白，而是属于另一种分子，如CD8α分子的天然铰链。CD8α铰链可以含有已知在CD8辅助受体和MHC分子的相互作用中起作用的半胱氨酸和脯氨酸残基。所述半胱氨酸和脯氨酸残基可影响所述CAR的性能。
CAR铰链可以是尺寸可调的。免疫应答细胞和靶细胞之间的免疫突触的这种形貌还限定了由于细胞表面靶分子上的膜远端表位而不能由CAR进行功能桥接的距离，即使用短铰链CAR也不能使突触距离达到信号能够传导的近似值。同样，膜近端CAR靶抗原表位仅在长铰链CAR的背景下观察到信号输出。可以根据所使用的胞外抗原结合区域来调节铰链。铰链可以是任何长度的。
跨膜结构域可以将CAR锚定在细胞的质膜上。CD28的天然跨膜部分可用于CAR。在其他情况下，也可以在CAR中使用CD8α的天然跨膜部分。“CD8”可以是与NCBI参考号:NP_001759或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD8核酸分子”可以是编码CD8多肽的多核苷酸，在某些情况下，跨膜区可以是CD28的天然跨膜部分，“CD28”可以指与NCBI参考号:NP_006130或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD28核酸分子”可以是编码CD28多肽的多核苷酸。在一些实施方案中，跨膜部分可以包含CD8α区域。
CAR的(细)胞内信号传导区域可以负责活化CAR已经置于其中的免疫应答细胞的效应子功能中的至少一种。CAR可以诱导T细胞的效应子功能，例如，所述效应子功能是细胞溶解活性或辅助活性，包括细胞因子的分泌。因此，术语细胞内信号传导区域是指转导效应子功能信号并引导细胞进行特异功能的蛋白质部分。虽然通常可以使用整个细胞内信号传导区域，但是在许多情况下，不必使用信号结构域的整个链。在一些实施方案中，使用细胞内信号传导区的截短部分。在一些实施方案中，术语细胞内信号传导区域因此意在包括足以转导效应子功能信号的细胞内信号传导区的任何截短部分。
在CAR中使用的信号结构域的优选实例可以包括T细胞受体(TCR)的细胞质序列和协同作用以在靶-受体结合之后启动信号转导的共同受体，以及它们的任何衍生物或变体序列和这些序列的具有相同功能性的任何合成序列。
在一些实施方案中，所述细胞内信号传导区域可以含有已知的免疫受体酪氨酸激活基序(ITAM)的信号基序。含有细胞质信号传导序列的ITAM的实例包括衍生自TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。然而，在优选的实施方案中，细胞内信号结构域衍生自CD3ζ链。
含有一个或多个ITAM基序的T细胞信号结构域的实例是CD3ζ结构域，也称为T细胞受体T3ζ链或CD247。该结构域是T细胞受体-CD3复合物的一部分，并且在将几 种细胞内信号转导途径的抗原识别与T细胞的主效应激活相结合方面起重要作用。如本文所用，CD3ζ主要是指人类CD3ζ及其同种型，如从Swissprot条目P20963所知的，包括具有基本相同序列的蛋白质。作为嵌合抗原受体的一部分，再次重申，不需要全T细胞受体T3ζ链，并且其包含T细胞受体T3ζ链的信号结构域的任何衍生物都是合适的，包括其任何功能等同物。
细胞内信号传导结构域可以选自表1的任何一个结构域。在一些实施方案中，可以修饰结构域，使得与参考结构域的同一性可以为约50％至约100％。可以修饰表1的任何一个结构域，使得修饰形式可以包含约50、60、70、80、90、95、96、97、98、99或至多约100％的同一性。
CAR的细胞内信号传导区可以进一步包含一个或多个共刺激结构域。细胞内信号传导区可以包含单个共刺激结构域，例如ζ链(第一代CAR)或其与CD28或4-1BB(第二代CAR)。在其他实例中，细胞内信号传导区可以包含两个共刺激结构域，例如CD28/OX40或CD28/4-1BB(第三代)。
与细胞内信号结构域如CD8一起，这些共刺激结构域可以产生激酶途径的下游激活，从而支持基因转录和功能性细胞反应。CAR的共刺激结构域可以激活与CD28(磷脂酰肌醇-4,5-二磷酸3-激酶)或4-1BB/OX40(TNF-受体相关因子衔接蛋白)途径以及MAPK和Akt激活相关的近端信号蛋白。
在某些情况下，通过CAR产生的信号可能与辅助或共刺激信号相结合。对于共刺激信号结构域，嵌合抗原受体样复合物可被设计成包含若干可能的共刺激信号结构域。如本领域众所周知的，在幼稚T细胞中，T细胞受体的单独接合不足以诱导T细胞的完全活化为细胞毒性T细胞。完整的生产性T细胞激活需要第二共刺激信号。已经报道对T细胞活化提供共刺激的几种受体，包括但不限于CD28、OX40、CD27、CD2、CD5、ICAM-1、LFA-1(CD11a/CD18)、4-1BBL、MyD88和4-1BB。这些共刺激分子使用的信号传导途径均能与主T细胞受体激活信号协同作用。这些共刺激信号传导区域提供的信号可以与源自一个或多个ITAM基序(例如CD3zeta信号转导域)的主效应激活信号协同作用，并且可以完成T细胞激活的要求。
在一些实施方案中，向嵌合抗原受体样复合物添加共刺激结构域可以增强工程细胞的功效和耐久性。在另一些实施方案中，T细胞信号结构域和共刺激结构域彼此融合从而构成信号传导区。
表4.共刺激结构域
Figure PCTCN2018075859-appb-000001
Figure PCTCN2018075859-appb-000002
嵌合抗原受体结合靶抗原。当在体外或离体测定T细胞活化时，可以从各种来源获得或分离靶抗原。本文使用的靶抗原是抗原或抗原上的免疫表位，其在哺乳动物中对于免疫识别和最终消除或控制致病因素或疾病状态中至关重要。免疫识别可以是细胞和/或体液。在细胞内病原体和癌症的情况下，免疫识别可以是，例如T淋巴细胞反应。
在一些实施方案中，靶抗原包括与癌前或增生状态相关的抗原。靶抗原也可能与癌症相关或起因于癌症。例如，在一些实施方案中，本发明的嵌合抗原受体识别并结合包括本文前述的IL-13RA2的肿瘤抗原。
在一些实施方案中，本文的嵌合抗原受体当存在于细胞的质膜上，并且当与其靶标结合并激活时，表达所述嵌合抗原受体的细胞可对携带所述靶标的细胞产生细胞毒性。例如，在一些实施方案中，所述嵌合抗原受体存在于细胞毒性细胞上，如NK细胞或者细胞毒性T细胞，并且在被靶标激活时，可以增加所述细胞毒性细胞对靶细胞的毒性。在一些实施方案中，本文的嵌合抗原受体可增加免疫反应性细胞对表达IL-13RA2的细胞如肿瘤细胞的作用。在一些实施方案中，与不表达本文的嵌合抗原受体的细胞相比，表达本文所述嵌合抗原受体的细胞对表达IL-13RA2的细胞的细胞毒性作用提高至少10％、至少15％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少1倍、至少1.5倍、至少2倍、至少2.5倍、至少3倍、至少3.5倍、至少4倍、至少4.5倍、至少5倍、至少6倍、至少7倍、至少8倍、至少9倍、或者至少10倍。
可以将编码目标结合抗原的受体或CAR的转基因并入细胞中。例如，可将转基因 并入免疫应答细胞，例如T细胞。当插入细胞时，转基因可以是互补DNA(cDNA)片段，其是信使RNA(mRNA)的拷贝；或者位于其基因组DNA原始区域的基因本身(含或不含内含子)。
编码转基因序列的核酸如DNA可以随机插入细胞的染色体。随机整合可以由将核酸(例如DNA)引入细胞的任何方法产生。例如，该方法可以包括但不限于电穿孔、超声、使用基因枪、脂转染、磷酸钙转染、使用树枝状大分子、显微注射和使用包括腺病毒、AAV和逆转录病毒载体的病毒载体、和/或II型核酶。
编码转基因的DNA也可以设计成包括报告基因，从而可以通过报告基因的活化检测转基因或其表达产物的存在。可以使用任何报道基因，例如上述那些。通过在细胞培养物中选择其中报道基因已经被活化的细胞，可以选择含有转基因的细胞。
CAR的表达可以通过表达测定法，例如qPCR或通过测量RNA的水平来验证。表达水平也可以指示拷贝数。例如，如果表达水平非常高，这可以表明CAR的多于一个拷贝被整合到基因组中。或者，高表达可以指示转基因整合在高转录区域中，例如高度表达的启动子附近。也可以通过测量蛋白质水平来验证表达，例如通过Western印迹。
在一些实施方案中，本发明的免疫应答细胞可以包含一种或多种转基因。所述一种或多种转基因可以表达CAR蛋白，而该CAR蛋白识别并结合抗原上的至少一个表位或结合抗原上的突变表位。CAR可以是功能性CAR。在一些实施方案中本发明的免疫应答细胞可以包含一种或多种CAR，或者其可以包含单一CAR和二次工程化受体。
在一些实施方案中，转基因可编码自杀基因。如癌症患者的许多有效治疗所证明的，CAR免疫应答细胞使得肿瘤消退但可伴随毒性。在一些实施方案中，当靶标抗原在正常组织和肿瘤细胞中共享时，CAR免疫应答细胞可能不能区分肿瘤和正常组织(“在靶/脱靶毒性”)。在另一些情况下，可以发生免疫系统的全身扰动，称为细胞因子释放综合征(CRS)。所述CRS可以包含全身炎症反应综合征或细胞因子风暴，这可能是CAR免疫应答细胞体内快速膨胀的后果。CRS是以发热和低血压为特征的病症，严重者可导致多器官功能衰竭。在大多数情况下，所述毒性与输注的CAR免疫应答细胞的体内扩增相关，其可引起免疫系统的整体扰动，以及释放高水平的促炎细胞因子，例如TNFα和IL-6。自杀基因可以诱导消除CAR免疫反应性细胞。自杀基因可以是在所述CAR免疫反应性细胞中诱导细胞凋亡的任何基因。自杀基因可以与所述结合抗原的受体一起编码在病毒载体内。编码自杀基因使得在特定的情况下可以缓解或者彻底中止由输注的CAR免疫应答细胞在体内扩增引起的毒性。
在一些实施方案中，可以产生存在于正常组织的抗原的CAR免疫反应性细胞，使得它们瞬时表达CAR，例如在电穿孔编码受体的mRNA之后。此外，通过包括安全开关来进一步加强CAR免疫反应性细胞的重大努力，在严重的在靶毒性的情况下，可以大大消除CAR免疫反应性细胞。
在一些实施方案中，编码CAR的载体可以与诸如可诱导的半胱天冬酶-9基因(由二聚化学诱导剂激活)或截短形式的EGF受体R(由单克隆抗体西妥昔单抗激活)或RQR8 的安全开关组合。
本文所用的一个或多个转基因可以来自不同的物种。例如，一个或多个转基因可以包含人基因、小鼠基因、大鼠基因、猪基因、牛基因、狗基因、猫基因、猴基因、黑猩猩基因或其任何组合。例如，转基因可以来自具有人类遗传序列的人。一个或多个转基因可以包含人基因。在某些情况下，一个或多个转基因不是腺病毒基因。
如上所述，转基因可以以随机或位点特异性方式插入免疫反应性细胞的基因组中。例如，可以将转基因插入到免疫细胞的基因组中的随机位点。这些转基因可以是功能性的，例如，在插入到基因组中的任何地方中时是完全功能性的。例如，转基因可以编码其自身的启动子，或者可以插入其内部启动子控制的位置。或者，可以将转基因插入基因，例如基因的内含子或基因的外显子、启动子或非编码区。可以插入转基因使得插入破坏基因，例如内源性免疫检查点。
在一些实施方案中，一个以上拷贝的转基因可以插入到基因组内的多个随机位点。例如，可将多个拷贝插入基因组中的随机位点。与转基因随机插入一次相比，这可能导致整体表达增加。或者，转基因的拷贝可以插入到基因中，转基因的另一拷贝可以插入到不同的基因中。可以靶向转基因，使其可以插入到免疫反应性细胞的基因组中的特定位点。
在一些实施方案中，包含编码结合抗原的受体序列的多核酸可以采取质粒载体的形式。质粒载体可以包含启动子。在某些情况下，启动子可以是组成型的。在一些实施方案中，启动子是可诱导的。启动子可以是或可以衍生自CMV、U6、MND或EF1a。在一些实施方案中，启动子可以与CAR序列相邻。在一些实施方案中，质粒载体还包含剪接受体。在一些实施方案中，剪接受体可以与CAR序列相邻。启动子序列可以是PKG或MND启动子。MND启动子可以是含有骨髓增生性肉瘤病毒增强子修饰的MoMuLV LTR的U3区域的合成启动子。
在一些实施方案中，可以设计编码目标受体的多核酸，以通过非病毒技术递送至细胞。在某些情况下，多核酸可以是良好的制造规范(GMP)兼容试剂。
编码目标结合抗原的受体或CAR的多核酸的表达可以由一种或多种启动子控制。启动子可以是普遍存在的，组成型(不受限制的启动子，允许相关基因的连续转录)，组织特异性启动子或诱导型启动子。可以调节插入邻近或接近启动子的转基因的表达。例如，转基因可插入到普遍存在的启动子附近或旁边。一些普遍存在的启动子可以是CAGGS启动子、hCMV启动子、PGK启动子、SV40启动子或ROSA26启动子。
启动子可以是内源的或外源的。例如，可以将一个或多个转基因插入到内源或外源ROSA26启动子的邻近或接近处。此外，启动子可以是免疫反应性细胞的特异性。例如，一个或多个转基因可以插入猪ROSA26启动子的邻近或接近。
组织特异性启动子或细胞特异性启动子可用于控制表达的位置。例如，可以将一个或多个转基因插入组织特异性启动子的邻近或接近。组织特异性启动子可以是FABP启动子、Lck启动子、CamKII启动子、CD19启动子、角蛋白启动子、白蛋白启动子、aP2 启动子、胰岛素启动子、MCK启动子、MyHC启动子、WAP启动子、或Col2A启动子。
也可以使用诱导型启动子。如果需要，可以通过添加或除去诱导剂来开启和关闭这些诱导型启动子。预期诱导型启动子可以是但不限于Lac、tac、trc、trp、araBAD、phoA、recA、proU、cst-1、tetA、cadA、nar、PL、cspA、T7、VHB、Mx和/或Trex。
本文所用的术语“诱导型启动子”是一种受控的启动子，其在所期待的条件未达成前不表达或者低表达与其可操作地连接的基因，而在所期待的条件达成的情况下表达或者高水平表达与其可操作地连接的基因。
此外，尽管不是表达必需的，但转基因序列还可以包括转录或翻译调控序列，例如启动子、增强子、绝缘体、内部核糖体进入位点、编码2A肽和/或多腺苷酸化信号的序列。
在一些实施方案中，转基因编码目标结合抗原的受体或CAR，其中将转基因插入安全港，使得表达所述结合抗原的受体。在一些实施方案中，将转基因插入PD1和/或CTLA-4基因座。在其他情况下，将转基因以慢病毒递送至细胞随机插入，而PD1-或CTLA-4特异性核酸酶可作为mRNA提供。在一些实施方案中，转基因通过病毒载体系统如逆转录病毒、AAV或腺病毒以及编码对于安全港特异的核酸酶(例如AAVS1、CCR5、白蛋白或HPRT)的mRNA递送。也可以用编码PD1和/或CTLA-4特异性核酸酶的mRNA处理细胞。在一些实施方案中，编码CAR的多核苷酸通过病毒递送系统与编码HPRT特异性核酸酶和PD1-或CTLA-4特异性核酸酶的mRNA一起提供。可以与本文公开的方法和组合物一起使用的CAR可以包括所有类型的这些嵌合蛋白。
在一些实施方案中，可以使用逆转录病毒载体(γ-逆转录病毒或慢病毒载体)将转基因导入免疫反应性细胞。例如，编码CAR的转基因或结合抗原的任何受体或其变体或片段可被克隆到逆转录病毒载体中，并且可以由其内源性启动子、逆转录病毒长末端重复序列、或对靶细胞类型特异性的启动子驱动。也可以使用非病毒载体。非病毒载体递送系统可以包括DNA质粒、裸核酸和与递送载体如脂质体或泊洛沙姆复合的核酸。
已经开发了许多基于病毒的系统用于将基因转移到哺乳动物细胞中。例如，逆转录病毒为基因递送系统提供了便利的平台。可以使用本领域已知的技术将所选择的基因插入载体并包装在逆转录病毒颗粒中。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及其在子细胞中的繁殖。慢病毒载体与衍生自逆转录病毒如鼠类白血病病毒的载体相比具有附加优点，因为它们可以转导非增殖细胞。它们还具有低免疫原性的附加优点。腺病毒载体的优点是它们不融合到靶细胞的基因组中，从而绕过负面的整合相关事件。
可以用编码所述结合抗原的受体的转基因转染细胞。转基因浓度可以为约100皮克至约50微克。在一些实施方案中，可以改变引入细胞的核酸(例如，ssDNA、dsDNA或RNA)的量以优化转染效率和/或细胞活力。例如，可以向每个细胞样品中加入1微克dsDNA用于电穿孔。在一些实施方案中，最佳转染效率和/或细胞活力所需的核酸(例如，双链DNA)的量根据细胞类型而不同。在一些实施方案中，用于每个样品的核酸(例如， dsDNA)的量可以直接对应于转染效率和/或细胞活力。例如，一系列转染浓度。由载体编码的转基因可以整合到细胞基因组中。在一些实施方案中，由载体编码的转基因前向整合。在其他情况下，由载体编码的转基因的反向整合。
通常通过全身给药(例如静脉内、腹膜内、肌内、皮下或颅内输注)或局部应用，通过给予个体患者体内递送载体，如下所述。或者，载体可以离体递送到细胞，例如从个体患者(例如，淋巴细胞、T细胞、骨髓抽吸物、组织活检)移出的细胞，然后通常在选择并入了该载体的细胞后将细胞再植入患者体内。在选择之前或之后，可以扩增细胞。
用于表达结合抗原的受体的合适的免疫反应性细胞可以是对于有需要的个体是自体的或非自体的细胞。
可以从个体获得合适的免疫应答细胞的来源。在某些情况下，可以获得T细胞。所述T细胞可以从许多来源获得，包括PBMC、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织和肿瘤的组织。在某些情况下，可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离，从自所述个体收集的血液获得T细胞。在一些实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一些实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆级分并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。
或者，可以从健康供体，来自诊断患有癌症的患者或诊断为感染的患者衍生细胞。在一些实施方案中，细胞可以是具有不同表型特征的混合细胞群体的一部分。还可以根据前述方法从转化的T细胞获得细胞系。还可以从细胞治疗库获得细胞。可以通过本文所述的任何方法获得对免疫抑制治疗有抗性的修饰细胞。还可以在修饰前选择合适的细胞群。修饰后也可以选择工程细胞群。工程细胞可用于自体移植。或者，细胞可用于同种异体移植。在一些实施方案中，将细胞施用于样品用于鉴定癌症相关靶序列的同一患者。在其他情况下，将细胞施用于不同于其样本用于鉴定癌症相关靶序列的患者的患者。
在一些实施方案中，合适的原代细胞包括外周血单核细胞(PBMC)、外周血淋巴细胞(PBL)和其它血液细胞亚群，例如但不限于T细胞、天然杀伤细胞、单核细胞、天然杀伤剂T细胞、单核细胞前体细胞、造血干细胞或非多能干细胞。在一些实施方案中，细胞可以是任何免疫细胞，包括任何T细胞如肿瘤浸润细胞(TIL)，如CD3+T细胞、CD4+T细胞、CD8+T细胞或任何其他类型的T细胞。T细胞还可以包括记忆T细胞、记忆干T细胞或效应T细胞。也可以从大量群体中选择T细胞，例如从全血中选择T细胞。T细胞也可以从大量群体中扩增。T细胞也可能倾向于特定种群和表型。例如，T细胞可以倾斜于表型包含CD45RO(-)、CCR7(+)、CD45RA(+)、CD62L(+)、CD27(+)、CD28(+)和/或IL-7Rα(+)。合适的细胞可以选自以下列表中的一种或多种标志物:CD45RO(-)、CCR7(+)、CD45RA(+)、CD62L(+)、CD27(+)、CD28(+)和/或IL-7Rα(+)。合适的细胞还包括干细胞，例如，例如胚胎干细胞、诱导的多能干细胞、造血干细胞、神经元干细胞和间充质干细胞。合适的细胞可以包含任何数量的原代细胞，例如人细胞、 非人细胞和/或小鼠细胞。合适的细胞可以是祖细胞。合适的细胞可以衍生自要治疗的受试者(例如，患者)。
患者中需要的治疗有效的细胞的量可以根据细胞的存活力和细胞被遗传修饰的效率而变化(例如，转基因被整合到一个或多个细胞中的效率，或者由转基因编码的蛋白质的表达水平)。在一些实施方案中，遗传修饰后细胞存活力的产物(例如，倍增)和转基因整合的效率可以对应于可用于给予受试者的细胞的治疗量。在一些实施方案中，遗传修饰后细胞存活力的增加可能对应于给予治疗对患者有效的必需细胞量的减少。在一些实施方案中，转基因整合到一个或多个细胞中的效率的增加可以对应于给予在患者中治疗有效的必需的细胞数量的减少。在一些实施方案中，确定所需的治疗有效的细胞的量可以包括确定与细胞随时间变化相关的功能。在一些实施方案中，确定需要治疗有效的细胞的量可以包括确定与根据时间相关变量将转基因整合到一个或多个细胞中的效率变化相对应的功能(例如，细胞培养时间、电穿孔时间、细胞刺激时间)。在一些实施方案中，治疗有效的细胞可以是细胞群，其包含在细胞表面上约30％至约100％的结合抗原的受体的表达。在一些实施方案中，通过流式细胞术测量，治疗有效的细胞可以在细胞表面上表达所述结合抗原的受体约30％、35％、40％、45％、50％、55％、60％、65％、70％、75％80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％或超过约99.9％。
根据本发明的一个方面，本发明也包括编码所述结合抗原的受体的核酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。
本发明还提供了包含上述编码表达于免疫应答细胞表面的结合抗原的受体蛋白的核酸的载体。
本发明还包括包含上述载体的病毒。本发明的病毒包括包装后的具有感染力的病毒，也包括包含包装为具有感染力的病毒所必需成分的待包装的病毒。本领域内已知的其它可用于将外源基因转导入免疫应答细胞的病毒及其对应的质粒载体也可用于本发明。
在另一个方面，本文提供了宿主细胞，其包含本文所述的抗体或嵌合抗原受体、以及任选的I型干扰素。在另一个方面，本文提供了宿主细胞，其包含编码本文所述的抗体或嵌合抗原受体、以及任选的I型干扰素的核酸。
在一些实施方案中，所述宿主细胞是免疫应答细胞。在一些实施方案中，所述免疫应答细胞是T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、和/或调节性T细胞。在一些实施方案中，所述宿主细胞是NK92细胞。
本发明所述的免疫应答细胞还可以进一步携带外源的细胞因子的编码序列；所述的细胞因子包括但不限于:IL-12，IL-15或IL-21等。这些细胞因子具有进一步的免疫调节或抗肿瘤的活性，能增强效应T细胞及活化的NK细胞的功能，或直接发挥抗肿瘤作用。因此，本领域技术人员可以理解，这些细胞因子的运用有助于所述的免疫应答细胞更好 地发挥作用。
本发明所述的免疫应答细胞还可以表达除了上述结合抗原的受体以外的另一种结合抗原的受体。
本发明所述的免疫应答细胞还可以表达趋化因子受体；所述的趋化因子受体包括但不限于CCR2。本领域技术人员可以理解，所述的CCR2趋化因子受体可以使得体内的CCR2与之竞争性结合，对于阻断肿瘤的转移是有利的。
本发明所述的免疫应答细胞还可以表达能降低PD-1表达的siRNA或者阻断PD-L1的蛋白。本领域技术人员可以理解，竞争性阻断PD-L1与其受体PD-1的相互作用，有利于恢复抗肿瘤T细胞反应，从而抑制肿瘤生长。
本发明所述的免疫应答细胞还可以表达安全开关；较佳地，所述的安全开关包括:iCaspase-9，Truancated EGFR或RQR8。
在一些实施方案中，本发明的免疫应答细胞不表达诸如4-1BBL这类的共刺激配体。
因此，在另一方面，本文还提供了一种生成本文所述的抗体或嵌合抗原受体或者包含它们的组合物的方法，其包括在适合的条件下培养本文所述的宿主细胞。在一些实施方案中，所述方法包括分离并获得所述宿主细胞的表达产物。
在另一方面，本文提供了一种组合物，其包含本文所述的抗体、嵌合抗原受体、或核酸。在一些实施方案中，所述组合物是包含所述抗体、嵌合抗原受体或核酸的药物组合物。在一些实施方案中，所述药物组合物还包含药学可接受的载体。
在另一方面，本文提供了一种药物组合物，其包含本文所述的宿主细胞和药学可接受的载体。
术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。
在一些实施方案中，所述组合物包含另一治疗剂。在一些实施方案中，所述另一治疗剂是化疗剂，如US20140271820中记载的那些和/或其药学上可接受的盐或类似物。在一些实施方案中，所述治疗剂包括但不限于有丝分裂抑制剂(长春花生物碱)，包括长春新碱、长春花碱、长春地辛和诺维宾(TM)(长春瑞滨，5'-去氢硫化氢)；拓扑异构酶I抑制剂，例如喜树碱化合物，包括CamptosarTM(伊立替康HCL)、HycamtinTM(托泊替康HCL)和衍生自喜树碱及其类似物的其它化合物；鬼臼毒素衍生物，例如依托泊苷、替尼泊苷和米多昔佐兹；烷基化剂顺铂、环磷酰胺、氮芥、三亚甲基硫代磷酰胺、卡莫司汀、白消安、苯丁酸氮芥、布列喹嗪、尿嘧啶芥末、氯洛芬和达卡巴嗪；抗代谢物，包括阿糖胞苷、5-氟尿嘧啶、甲氨蝶呤、巯嘌呤、硫唑嘌呤和丙卡巴肼；抗生素，包括但不限于多柔比星、博来霉素、更生霉素、柔红霉素、霉素霉素、丝裂霉素、肉瘤霉素C和道诺霉素；以及其它化疗药物，包括但不限于抗肿瘤抗体、达卡巴嗪、氮胞苷、阿姆沙康、美法仑、异环磷酰胺和米托蒽醌。在一些实施方案中，所述另外的治疗剂选自表柔比星、奥沙利铂和5-氟尿嘧啶中的一种或多种。在一些实施方案中，所述另外的治疗剂包括但不限于抗血管生成剂，包括抗VEGF抗体(包括人源化和嵌合抗体、抗VEGF 适体和反义寡核苷酸)以及其他血管发生抑制剂，例如血管抑素、内皮抑制素、干扰素、白细胞介素1(包括α和β)白介素12、视黄酸和金属蛋白酶-1和-2的组织抑制剂等。
可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。
本文所述的药物组合物可包含一种或多种药学可接受的盐。“药学可接受的盐”指这样一种盐，其保留亲本化合物的期望生物学活性且不产生任何不利的毒物学效果(参见例如，Berge,S.M等人.,1977,J.Pharm.Sci.66:1-19)。此类盐的例子包括酸加成盐和碱加成盐。
酸加成盐包括衍生自无毒无机酸，诸如盐酸、硝酸、磷酸、硫酸、氢溴酸、氢碘酸、亚磷酸等的盐，以及衍生自无毒有机酸，诸如脂肪族单羧酸和二羧酸、苯基取代的链烷酸、羟基链烷酸、芳香族酸、脂肪族和芳香族磺酸等的盐。碱加成盐包括衍生自碱土金属(诸如钠、钾、镁、钙等)的盐，以及衍生自无毒有机胺的盐，诸如N,N'-二苄乙二胺、N-甲基葡糖胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺、普鲁卡因等。
本文所述的药物组合物还可包含抗氧化剂。抗氧化剂的实例包括但不限于:水溶性抗氧化剂，诸如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠、亚硫酸钠等；油溶性抗氧化剂，诸如抗坏血酸棕榈酸酯、丁基化羟基茴香醚(BHA)、丁基化羟基甲苯(BHT)，卵磷脂、没食子酸丙酯、α-生育酚等；和金属螯合剂，诸如柠檬酸、乙二胺四乙酸(EDTA)、山梨醇、酒石酸、磷酸等。
本发明的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用肠胃外给药(如注射)或其它治疗方式。
免疫原性组合物的“肠胃外”施用包括例如皮下(s.c.)、静脉内(i.v.)、肌内(i.m.)或胸骨内注射或输注技术。
给予个体的包含免疫反应性细胞群体的制剂包含有效治疗和/或预防特定适应症或疾病的多个免疫反应性细胞。因此，可以向个体施用免疫反应性细胞的治疗有效群体。通常，施用包含约1×10 4至约1×10 10个免疫反应性细胞的制剂。在大多数情况下，制剂将包含约1×10 5至约1×10 9个免疫反应性细胞、约5×10 5至约5×10 8个免疫反应性细胞、或约1×10 6至约1×10 7个免疫反应性细胞。然而，根据癌症的位置、来源、身份、程度和严重程度、待治疗的个体的年龄和身体状况等，对个体施用的CAR免疫反应性细胞的数量将在宽的范围之间变化。医生将最终确定要使用的适当剂量。
在一些实施方案中，使用嵌合抗原受体来刺激免疫细胞介导的免疫应答。例如，T 细胞介导的免疫应答是涉及T细胞活化的免疫应答。活化的抗原特异性细胞毒性T细胞能够在表面上显示外源抗原表位的靶细胞中诱导细胞凋亡，例如显示肿瘤抗原的癌细胞。在另一些实施方案中，使用嵌合抗原受体在哺乳动物中提供抗肿瘤免疫。由于T细胞介导的免疫应答，受试者将产生抗肿瘤免疫。
在某些情况下，治疗患有癌症的受试者的方法可以涉及向需要治疗的受试者施用一种或多种本发明所述的免疫应答细胞。所述免疫应答细胞可结合肿瘤靶分子并诱导癌细胞死亡。如前文所述，本发明还提供治疗个体中的病原体感染的方法，包括向所述个体施用治疗有效量的本发明的免疫应答细胞。
本发明的免疫反应性细胞的给药频率将根据包括所治疗疾病的因素、特定免疫反应性细胞的元件和给药方式。例如可以每日给药4次、3次、2次或每日一次、每隔一天、每三天、每四天、每五天、每六天一次、每周一次、每八天一次、每九天一次、每十天、每周一次、或者每月两次给药。如本文所述，由于本申请的免疫应答细胞具有改善的活力，从而可以不仅以与类似的但不表达外源性I型干扰素的免疫应答细胞更低的治疗有效的量给药，并且可以以更低的频率给药，以获得至少类似、并且优选更加显著的疗效。
在一些实施方案中，组合物可以是等渗的，即它们可以具有与血液和泪液相同的渗透压。本发明组合物的期望等渗性可以使用氯化钠或其它药学上可接受的试剂如葡萄糖、硼酸、酒石酸钠、丙二醇或其它无机或有机溶质来实现。如果需要，组合物的粘度可以使用药学上可接受的增稠剂维持在选定的水平。合适的增稠剂包括，例如，甲基纤维素、黄原胶、羧甲基纤维素、羟丙基纤维素、卡波姆等。增稠剂的优选浓度将取决于所选择的试剂。显然，合适的载体和其它添加剂的选择将取决于确切的给药途径和特定剂型的性质，例如液体剂型。
本发明还提供了包含本文所述的抗体、嵌合抗原受体、核酸或免疫应答细胞的试剂盒。在一些实施方案中，试剂盒可以包括含有有效量的包含一种或多种单位剂型的本文所述的抗体、嵌合抗原受体、核酸或免疫应答细胞的治疗或预防组合物。在一些实施方案中，试剂盒包含可含有治疗或预防性组合物的无菌容器；这样的容器可以是盒、安瓿、瓶、小瓶、管、袋、泡罩包装或本领域已知的其它合适的容器形式。这种容器可以由塑料、玻璃、层压纸、金属箔或其他适合于保持药物的材料制成。在一些实施方案中，所述试剂盒包含本文所述的抗体、嵌合抗原受体、核酸或免疫应答细胞，以及将本文所述的抗体、嵌合抗原受体、核酸或免疫应答细胞给予个体的说明书。说明书中通常包含使用本文所述的抗体、嵌合抗原受体、核酸或免疫应答细胞来治疗或预防癌症或肿瘤的方法。在一些实施方案中，试剂盒包含本文所述的宿主细胞，并且可以包括约1×10 4个细胞至约1×10 6个细胞。在一些实施方案中，试剂盒可以包括至少约1×10 5个细胞，至少约1×10 6个细胞，至少约1×10 7个细胞，至少约4×10 7个细胞，至少约5×10 7个细胞，至少约6×10 7个细胞，至少约6×10 7个细胞，8×10 7个细胞，至少约9×10 7个细胞，至少约1×10 8个细胞，至少约2×10 8个细胞，至少约3×10 8个细胞，至少约4×10 8个细胞，至少约5×10 8个细胞，至少约6×10 8个细胞，至少约6×10 8细胞，至少约 8×10 8个细胞，至少约9×10 8细胞，至少约1×10 9个细胞，至少约2×10 9个细胞，至少约3×10 9个细胞，至少约4×10 9个细胞，至少约5×10 9个细胞，至少约6×10 9个细胞，至少约8×10 9个细胞，至少约9×10 9个细胞，至少约1×10 10个细胞，至少约2×10 10个细胞，至少约3×10 10个细胞，至少约4×10 10个细胞，至少约5×10 10个细胞，至少约6×10 10个细胞，至少约7×10 10个细胞、至少约8×10 10个细胞、至少约9×10 10个细胞，至少约1×10 11个细胞，至少约2×10 11个细胞，至少约3×10 11个细胞，至少约4×10 11个细胞，至少约5×10 11个细胞，至少约8×10 11个细胞，至少约9×10 11个细胞，或至少约1×10 12个细胞。例如，可以在试剂盒中包括大约5×10 10个细胞。在另一个实例中，试剂盒可以包括3×10 6个细胞；细胞可以扩增至约5×10 10个细胞并施用于受试者。
在一些实施方案中，试剂盒可以包括同种异体细胞。在一些实施方案中，试剂盒可以包括可以包含基因组修饰的细胞。在一些实施方案中，试剂盒可以包含“现成的”细胞。在一些实施方案中，试剂盒可以包括可以扩展用于临床使用的细胞。在某些情况下，试剂盒可能包含用于研究目的的内容物。
在一些实施方案中，说明书包括以下中的至少一个:治疗剂的描述；用于治疗或预防肿瘤或其症状的剂量方案和给药；预防措施、警示、禁忌症、过量信息、不良反应、动物药理学、临床研究、和/或引用文献。说明书可以直接打印在容器上(如果有的话)，或作为容器上的标签，或作为容器内或容器中提供的单独的纸张、小册子、卡片或文件夹打印。在一些实施方案中，说明书提供施用本发明所述的免疫应答细胞用于治疗或预防肿瘤的方法。在某些情况下，说明书提供了施用化学治疗剂之前、之后或同时给予本发明的免疫反应性细胞的方法。
在另一方面，本文还提供了一种诱导包含IL-13RA2的细胞死亡的方法，所述方法包括使所述细胞与本文所述的抗体、本文所述的嵌合抗原受体、本文所述的组合物、或本文所述的宿主细胞接触。在一些实施方案中，所述接触是在体外接触。在一些实施方案中，所述接触是在体内接触。
在一些实施方案中，所述细胞是肿瘤细胞。在一些实施方案中，所述细胞是脑瘤，更具体的，可以是星形细胞瘤、脑膜瘤、神经胶质瘤。
在另一方面，本文提供了一种治疗有需要的个体的肿瘤的方法，所述方法包括向所述个体给予有效量的本文所述的抗体、嵌合抗原受体、组合物、载体或者宿主细胞。
在一些实施方案中，受试者可以给予免疫反应性细胞，其中可以施用的免疫反应性细胞可以是约1至约35天龄。例如，所施用的细胞可以是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或最多约40天。可以从刺激时起计算CAR免疫反应性细胞的年龄。可以从血液采集的时间开始计算免疫反应性细胞的年龄。可以从转导时起计算免疫反应性细胞的年龄。在一些实施方案中，可以给予受试者的免疫反应性细胞为约10至约14或约20天龄。在一些实施方案中，免疫反应性细胞的“年龄”可以通过端粒长 度来确定。例如，“年轻”的免疫反应细胞可以具有比“耗尽”或“老”的免疫反应性细胞更长的端粒长度。不受特定理论的束缚，可以认为免疫反应性细胞在培养物中每周丢失约0.8kb的估计端粒长度，并且年轻的免疫反应性细胞培养物可以具有比约44天的免疫反应性细胞长约1.4kb的端粒。不受特定理论的束缚，人们认为更长的端粒长度可以与患者中的阳性客观临床反应和体内细胞的持久性相关联。
在移植之前、之后和/或期间，细胞(例如，工程细胞或工程化的原代T细胞)可以是功能性的。例如，移植的细胞可以是在移植后至少约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18，19、20、21、22、23、24、25、6、27、28、29、30、40、50、60、70、80、90或100天起作用。移植细胞可以在移植后至少约1、2、3、4、5、6、7、8、9、10、11或12个月起作用。移植细胞可以在移植后至少约1、2、3、4、5、6、7、8、9、10、15、20、25或30年起作用。在一些实施方案中，移植细胞可以在接受者的寿命期间起作用。
此外，移植细胞可以以其正常预期功能的100％起作用。移植细胞还可以发挥其正常预期功能约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、或高达约100％的功能。
移植细胞也可以发挥其正常预期功能的超过100％的作用。例如，移植的细胞可以起到正常预期功能的约110、120、130、140、150、160、170、180、190、200、250、300、400、500、600、700、800、900、1000或至多约5000％的功能。
移植可以通过任何类型的移植。局部可以包括但不限于肝下囊空间、脾囊下空间、肾囊下空间、网膜、胃或肠粘膜下层、小肠血管节段、静脉囊、睾丸、脑、脾或角膜。例如，移植可以是囊下移植。移植也可以是肌内移植。移植可以是门静脉移植。
与当一个或多个野生型细胞被移植到接受者之时相比，采用本发明的免疫应答细胞治疗之后可以改善移植排斥反应。例如，移植排斥可以是超急性排斥反应。移植排斥也可以是急性排斥反应。其他类型的排斥可能包括慢性排斥反应。移植排斥也可以是细胞介导的排斥反应或T细胞介导的排斥反应。移植排斥也可以是自然杀伤细胞介导的排斥反应。
改善移植可能意味着减轻超急性排斥反应，其可以包括减少、减轻或降低不良作用或症状。移植可以指细胞产品的过继性移植。
移植成功的另一个迹象可以是接受者不需要免疫抑制治疗的天数。例如，在提供本发明的免疫应答细胞之后，接受者可以不要求至少约1、2、3、4、5、6、7、8、9、10或更多天的免疫抑制治疗。这可以表明移植成功。这也可以表明移植的细胞，组织和/或器官没有排斥。
在一些实施方案中，本文所述的抗体、嵌合抗原受体、组合物、载体或者宿主细胞可以与另一治疗剂联合给药。在一些实施方案中，所述另一治疗剂是化疗剂，如US20140271820中记载的那些。可以与本发明的免疫应答细胞联合应用的化疗药物包括但不限于有丝分裂抑制剂(长春花生物碱)，包括长春新碱、长春花碱、长春地辛和诺维宾(TM)(长春瑞滨，5'-去氢硫化氢)；拓扑异构酶I抑制剂，例如喜树碱化合物，包括CamptosarTM(伊立替康HCL)、HycamtinTM(托泊替康HCL)和衍生自喜树碱及其类似物的其它化合物；鬼臼毒素衍生物，例如依托泊苷、替尼泊苷和米多昔佐兹；烷基化剂顺铂、环磷酰胺、氮芥、三亚甲基硫代磷酰胺、卡莫司汀、白消安、苯丁酸氮芥、布列喹嗪、尿嘧啶芥末、氯洛芬和达卡巴嗪；抗代谢物，包括阿糖胞苷、5-氟尿嘧啶、甲氨蝶呤、巯嘌呤、硫唑嘌呤和丙卡巴肼；抗生素，包括但不限于多柔比星、博来霉素、更生霉素、柔红霉素、霉素霉素、丝裂霉素、肉瘤霉素C和道诺霉素；以及其它化疗药物，包括但不限于抗肿瘤抗体、达卡巴嗪、氮胞苷、阿姆沙康、美法仑、异环磷酰胺和米托蒽醌。在一些实施方案中，所述额外的治疗剂选自表柔比星、奥沙利铂和5-氟尿嘧啶中的一种或多种。
在一些实施方案中，可以与本发明的免疫应答细胞联合应用的化疗药物包括但不限于抗血管生成剂，包括抗VEGF抗体(包括人源化和嵌合抗体、抗VEGF适体和反义寡核苷酸)以及其他血管发生抑制剂，例如血管抑素、内皮抑制素、干扰素、白细胞介素1(包括α和β)白介素12、视黄酸和金属蛋白酶-1和-2的组织抑制剂。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1.IL-13RA2和IL-13RA1重组蛋白的制备
a.IL-13RA2_huFc，IL-13RA1_huFc表达质粒的构建
体外合成人IL-13RA2的胞外段Asp27-Arg343(SEQ ID NO:18)基因(SEQ ID NO:17)，将该基因插入含有人IgG1重链恒定区的Fc段Asp104-Lys330的真核表达质粒中，中间以“GS”相连接，形成融合表达蛋白IL-13RA2_huFc(SEQ ID NO:22)，相对应的基因序列如SEQ ID NO:11所示。或者将IL-13RA1胞外段基因(SEQ ID NO:19)插入含有人IgG1重链恒定区的Fc段Asp104-Lys330的真核表达质粒中，中间以“GS”相连接，形成融合表达蛋白IL-13RA1_huFc(SEQ ID NO:24)，相对应的基因序列如SEQ ID NO: 23所示。
b.瞬时转染表达IL-13RA2_huFc、IL-13RA1_huFc
1)转染前一天，接种6～7×105/ml 293F细胞于125ml培养瓶中
2)转染当天，调整3×107细胞于28ml FreeStyleTM 293expression medium
3)按照下面的操作步骤制备lipid-DNA复合物:
用Opti-MEM I稀释30ug DNA，终体积1ml,充分混匀
用Opti-MEM I稀释60ul 293fectinTM,终体积1ml，充分混匀
室温孵育5分钟，
4)将稀释好的DNA与293fectinTM混合，温孵育20分钟
5)将2ml DNA-293fectin复合物加入28ml细胞中，37度，8％CO2，125rpm培养3～4天，收取上清
c.IL-13RA2_huFc、IL-13RA1_huFc的纯化
1)13000rpm离心培养上清15min
2)采用protein A填料进行亲和纯化，具体具体操作步骤如下:
平衡:10个柱体积的平衡buffer平衡protein A填料。
上样:0.45μm滤膜处理好的样品全部上样。
洗杂:20个柱体积平衡buffer洗杂，直至流穿无物质流出。
洗脱:加入10个柱体积的洗脱buffer洗脱目的蛋白(收集管中事先加入6％的中和buffer)。
溶液配方
平衡buffer:PBS pH 7.4
洗脱buffer:0.1M 甘氨酸pH 2.6
中和buffer:1M Tris
3)洗脱用0.22um的膜过滤后，使用截流量为10KD的millipore超滤管进行浓缩，浓缩至体积1ml以内，使用PD-Midi脱盐柱脱盐，收集1.5ml样品。通过OD280/1.47测蛋白浓度
取2ug跑 SDS-PAGE，结果如图1所示。
实施例2.使用全人噬菌体展示文库筛选针对IL-13RA2特异的scFv
本发明使用的噬菌体展示文库为本公司构建的全人的天然的scFv噬菌体文库,库容为1E+11。利用本领域技术人员已知的筛选方法得到针对IL-13RA2高度特异的scFv片段。简言之，分别包被10ug/ml抗原IL-13RA2_huFc和IL-13RA1_huFc于免疫管。为了筛选到特异性结合IL-13RA2的抗体，将噬菌体文库加入包被了IL-13RA1_huFc的免疫管中结合1hr。取上清加入包被IL-13RA2_huFc的免疫管中结合1.5小时，随后将非特异性的噬菌体洗掉，将结合的噬菌体洗脱下来并感染对数生长期的大肠杆菌TG1。扩大培养洗脱下来得噬菌体并使用PEG/NaCl沉淀纯化扩大后的噬菌体文库用于下一轮的筛 选。将淘选进行3-4个循环以富集与IL-13RA2特异性结合的scFv噬菌体克隆。通过针对IL-13RA2_huFc的标准ELISA方法确定阳性克隆。ELISA使用IL-13RA1_huFc做为无关抗原来验证抗体的特异性。一共筛选了3420个克隆，其中44个克隆ELISA实验特异性的结合IL-13RA2_huFc，不结合IL-13RA1_huFc。经测序，得到5个单一的序列。表达纯化这5个克隆，其中只有2个特异性的结合表达IL13RA2的U251细胞(购自中国科学院细胞库)(图2、4)，克隆名称为31C2、32H4。
31C2的重链可变区的氨基酸序列如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:4所示；32H4的重链可变区的氨基酸序列如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示。31C2的HDCR1的氨基酸序列如SEQ ID NO:9所示、HDCR2的氨基酸序列如SEQ ID NO:10所示、HDCR3的氨基酸序列如SEQ ID NO:11所示、LDCR1的氨基酸序列如SEQ ID NO:13所示、LDCR2的氨基酸序列如SEQ ID NO:14所示、LDCR3的氨基酸序列如SEQ ID NO:15所示；32H4的HDCR1的氨基酸序列如SEQ ID NO:9所示、HDCR2的氨基酸序列如SEQ ID NO:10所示、HDCR3的氨基酸序列如SEQ ID NO:12所示、LDCR1的氨基酸序列如SEQ ID NO:13所示、LDCR2的氨基酸序列如SEQ ID NO:14所示、LDCR3的氨基酸序列如SEQ ID NO:16所示。
实施例3.ELISA结合试验
通过标准的ELISA检测抗体31C2，32H4的种属特异性。鼠IL-13RA2购自Sino Biological Inc公司。包被5ug/ml,100ul/孔鼠IL-13RA2与ELISA板，4度过夜。用PBS洗三遍包被好的ELISA板。加入200ul/孔的含2％脱脂奶粉的PBS室温封闭1hr。用PBS洗三遍。加入梯度稀释的抗体，起始浓度10ug/ml，3倍稀释，室温孵育1hr。PBST洗三遍，PBS洗三遍。加入HRP标记的羊抗人Fc，室温孵育1hr。PBST洗三遍，PBS洗三遍。加入TMB显色15分钟后，加入硫酸终止反应并在酶标仪上读数。结果如图3所示。抗体31C2能结合鼠IL-13RA2，32H4不结合鼠IL-13RA2。
实施例4.抗IL-13RA2scFv_Fc融合抗体的构建及其在真核细胞中的瞬转表达纯化、活性鉴定
分别针对31C2，32H4的VH和VL片段设计引物，引入由15个柔性氨基酸(GGGGSGGGGSGGGGS)组成的linker连接组成scFv；在VH上游引入合适的酶切位点和保护碱基，在VL的下游引入合适的酶切位点和保护碱基。1％琼脂糖凝胶电泳分析PCR产物并纯化回收。酶切后连接入合适的真核表达载体。采用293fectin TMTransfection reagent(Invitrogen，12347-019)或者聚乙烯亚胺(PEI)(Sigma-Aldrich，408727)瞬时转染对数生长期的293F细胞。转染5-7天后收集培养上清通过Protein A进行亲和纯化。
通过流式细胞术测试抗体与内源性表达IL-13RA2的U251细胞的结合，293T细胞作为阴性细胞对照。FACs检测的具体方法如下:收获细胞，用生长培养基洗涤细胞一次， 重悬于PBS中，调整细胞浓度为4E+5细胞/ml。在冰上将稀释的scFv_Fc融合抗体与细胞孵育30分钟，抗体的浓度为111nM。其后与FITC标记的抗人IgG第二抗体孵育。两次洗涤步骤之后，使用Guava easyCyteTM HT System仪器检测。图4显示了抗体31C2，32H4scFv_Fc融合形式和U251以及293T细胞结合的情况。这两个抗体都特异性的结合内源性表达IL-13RA2的U251细胞，不结合阴性细胞293T。
实施例5.利用表面等离子共振技术(SPR)测定抗体的亲和力
不同抗体针对IL-13RA2的亲和力是使用biacoreT200测定的。具体做法如下:
将IL-13RA2_huFc通过氨基偶联的方式包被在CM5芯片上，包被至500RU左右，梯度稀释的抗体作为流动相以30ul/min的流速通过包被抗原的通道。运行缓冲液为HBS-N，温度为25度。实验数据通过BIAevaluation3.2进行分析，动力学曲线使用1:1的langmuir模型进行拟合。其中31C2(scFv_Fc)的KD为1.79nM，32H4(scFv_Fc)的KD为3.76nM(见图5)。
实施例6.利用FACs测定抗体与U251细胞的结合EC50
收获细胞，用生长培养基洗涤细胞一次，重悬于PBS中，调整细胞浓度为4E+5细胞/ml。在冰上将梯度稀释的scFv_Fc融合抗体与细胞温育30分钟，抗体的起始浓度为500nM，5倍稀释，8个梯度。其后与FITC标记的抗人IgG第二抗体孵育。两次洗涤步骤之后，使用Guava easyCyteTM HT System仪器检测。结果如图6所示，其中两个抗体都和U251细胞有浓度梯度依赖的结合，31C2(ScFv_Fc)的EC50为2.8nM，32H4(ScFv_Fc)的EC50为1nM。
实施例7.抗体的亲和力成熟
利用噬菌体展示技术进行亲和力成熟。
以31C2和32H4为亲本抗体，分别构建两个噬菌体文库，一个随机化轻链的CDR1以及CDR2，另一个随机化重链的CDR2以及CDR2。然后针对抗原进行淘选，通过SPR技术等筛选出高亲和力的抗体，即31C2和32H4的变体。引物信息如图7所示。
首先基于抗体31C2(scFv)(氨基酸序列SEQ ID NO:25，核酸序列SEQ ID NO:26)构建模板质粒。对于轻链CDR1和CDR2随机化的噬菌体文库，使用引物LMF和IL1R，PCR扩增片段1；使用引物IL2F和FdR，PCR扩增片段2；然后通过搭桥PCR连接片段1和片段2得到含有随机化序列的scFv全长，然后用NcoI和NotI酶切全长片段，通过T4连接酶连接入同样酶切的模板质粒中，并电转化至TG1感受态细胞中，库容为1.68E+9。对于重链CDR1和CDR2随机化的噬菌体文库，使用引物LMF和BH1R，PCR扩增片段3；使用引物BH2F和FdR，PCR扩增片段4；然后通过搭桥PCR连接片段3和片段4得到含有随机化序列的scFv全长，然后用NcoI和NotI酶切全长片段，通过T4连接酶连接入同样酶切的模板质粒中，并电转化至TG1感受态细胞中，库容为 1.75E+9。
抗体32H4亲和力成熟文库的构建与31C2类似，基于抗体32H4(scFv)(氨基酸序列SEQ ID NO:26，核酸序列SEQ ID NO:27)构建模板质粒。使用与31C2相同的引物随机化轻链的CDR1和CDR2，得到的噬菌体文库库容为2.1E+9。类似地使用与31C2相同的引物随机化重链的CDR1和CDR2，得到的噬菌体文库库容为1.5E+9。
实施例8.噬菌体文库的筛选
参照本专利实施例2中方法。抗原IL13RA2_huFc的起始浓度为50nM，2倍梯度稀释进行下一轮筛选。将淘选进行2-3个循环以富集与IL13RA2_huFc特异性结合的scFv噬菌体克隆。通过针对IL13RA2_huFc的标准ELISA方法确定阳性克隆。ELISA使用人IL13RA1_huFc段作为无关抗原来验证抗体的特异性。一共挑取111个ELISA阳性的克隆，重新诱导以后通过biacore测定诱导上清的解离常数Kd。其中有10个克隆的解离常数Kd比母本克隆低10倍以上，如图8所示。
经测序，克隆2C7、2D3、1D11、1B11、2A5、2D4、1H7、1D8的轻链与31C2一样(氨基酸序列SEQ ID NO:4，核酸序列SEQ ID NO:3)。图9A比较了克隆2C7(氨基酸序列SEQ ID NO:29，核酸序列SEQ ID NO:30)，2D3(氨基酸序列SEQ ID NO:31，核酸序列SEQ ID NO:32)，1D11(氨基酸序列SEQ ID NO:33，核酸序列SEQ ID NO:34)、1B11(氨基酸序列SEQ ID NO:35，核酸序列SEQ ID NO:36)，2A5(氨基酸序列SEQ ID NO:37，核酸序列SEQ ID NO:38)，2D4(氨基酸序列SEQ ID NO:39，核酸序列SEQ ID NO:40)，1H7(氨基酸序列SEQ ID NO:41，核酸序列SEQ ID NO:42)，1D8(氨基酸序列SEQ ID NO:43，核酸序列SEQ ID NO:44)及31C2(氨基酸序列SEQ ID NO:2，核酸序列SEQ ID NO:1)的重链氨基酸序列。
其中，31C2的亲和力成熟克隆的HCDR1的序列分别如SEQ ID NO:45-51所示，HCDR2的序列分别如SEQ ID NO:52-58所示，具体见图9B。
与亲本抗体31C2的VH相比，2C7有4个位点的突变，相似性为96.7％；2D3有5个位点的突变，相似性为95.8％；1D11有6个位点的突变，相似性为95％；1B11有5个位点的突变，相似性为95.8％；2A5有4个位点的突变，相似性为96.7％；2D4有5个位点的突变，相似性为95.8％；1H7有4个位点的突变，相似性为96.7％；1D8有4个位点的突变，相似性为96.7％。
经测序，克隆5G3，5D7的轻链与32H4一样(氨基酸序列SEQ ID NO:8，核酸序列SEQ ID NO:7)。图9C比较了克隆5G3(氨基酸序列SEQ ID NO:59，核酸序列SEQ ID NO:60)，5D7(氨基酸序列SEQ ID NO:61，核酸序列SEQ ID NO:62)及32H4(氨基酸序列SEQ ID NO:6，核酸序列SEQ ID NO:5)的重链氨基酸序列。
其中，32H4的亲和力成熟克隆的HCDR1的序列分别如SEQ ID NO:63、64所示，HCDR2的序列分别如SEQ ID NO:65、66所示，具体见图9D。
与亲本抗体32H4的VH相比，5G3有5个位点的突变，相似性为95.7％；2D3有5 个位点的突变，相似性为95.8％；5D7有8个位点的突变，相似性为95％；1B11有5个位点的突变，相似性为93.2％。
实施例9.scFv的表达纯化
将含有抗体基因的TG1划线培养，以挑取单个克隆接种于2xTY-Amp-5％Glucose培养基中，37℃，220rpm培养至OD600nM＝0.8～0.9，加入终浓度为1mM IPTG，25度220rpm过夜培养，诱导scFv的表达。
离心收集菌体，用30mM Tris HCl、20％蔗糖、1mM EDTA pH 8.0(按每克菌体80ml计)悬浮后冰浴，4度，8000g离心，取上清A，沉淀用5mM MgSO4 8ml悬浮后冰浴，轻轻振荡10分钟，4度，8000g离心，取上清B。合并上清A和上清B，12000g离心15分钟，取上清即为cold osmotic shock fluid
使用镍柱进行亲和纯化，采用biacoreT200测定亲和力，亲和力成熟的抗体结合解离常数如图10A所示。
参照实施例3的方法，通过标准的ELISA检测抗体5D7、2C7、5G3、2D4、2D3、1B11的特异性。结果如图10B所示。其中来自母本抗体31C2的克隆1B11、2C7、2D3、2D4特异性的结合人IL13RA2，不结合人IL13RA1，并且和鼠IL13RA2有交叉反应。来自母本抗体32H4的克隆5D7、5G3特异性的结合人IL13RA2，不结合人IL13RA1，不结合鼠IL13RA2。
实施例10.抗体scFv_Fc形式的表达及亲和力测定
挑取其中亲和力较高的六个抗体5D7、2C7、5G3、2D4、2D3、1B11进行scFv_Fc融合形式的构建。
参照实施例4所示，在VH上游引入合适的酶切位点和保护碱基，在VL的下游引入合适的酶切位点和保护碱基。1％琼脂糖凝胶电泳分析PCR产物并纯化回收。酶切后连接入含有人Fc段的真核表达载体V152中(购自上海锐劲生物技术有限公司)。通过293Fectin瞬时转染至30ml 293F细胞中并表达。转染5-7天后收集培养上清通过Protein A进行亲和纯化。通过SEC分析抗体的聚集情况。结果如图11所示。
参照实施例5的方法使用biacoreT200测定亲和力，结果如图11B-11G所示。经过亲和力成熟以后的抗体，亲和力与母本抗体相比有3～10倍的提高。抗体结合解离常数如图11F所示。
实施例11.抗体的scFv_Fc形式的与U251细胞结合EC50的测定
参照实施例6的方法，收获细胞，用生长培养基洗涤细胞一次，重悬于PBS中，调整细胞浓度为4E+5细胞/ml。在冰上将梯度稀释的scFv_Fc融合抗体与细胞育30分钟,抗体的起始浓度为2000nM，5倍稀释，11个梯度。其后与FITC标记的抗人IgG第二抗体孵育。两次洗涤步骤之后，使用Guava easyCyteTM HT System仪器检测。结果 如图12所示:抗体5D7、2C7、5G3、2D4、2D3、1B11的scFv_Fc形式的与U251细胞结合EC50分别为0.56nM、0.57nM、0.53nM、0.37nM、0.33nM、0.47nM。与母本抗体相比也有2～8倍的提高。
实施例12.CAR-T细胞的制备
选择2D4和5G3进行CAR-T细胞制备和抗肿瘤活性研究。
1.慢病毒包装质粒pRRL-hu8E3-28Z的构建
以PRRLSIN-cPPT.EF-1α为载体，构建了表达抗体2D4和5G3的嵌合抗原受体的慢病毒质粒，包括PRRLSIN-cPPT.EF-1α-2D4-28Z、PRRLSIN-cPPT.EF-1α-2D4-BBZ、PRRLSIN-cPPT.EF-1α-2D4-28BBZ以及PRRLSIN-cPPT.EF-1α-5G3-28Z、PRRLSIN-cPPT.EF-1α-5G3-BBZ、PRRLSIN-cPPT.EF-1α-5G3-28BBZ。
2D4-28Z序列由CD8α信号肽(SEQ ID NO:68)、2D4scFv(SEQ ID NO:67)、CD8hinge(SEQ ID NO:69)、CD28跨膜区(SEQ ID NO:70)和胞内信号传导结构域(SEQ ID NO:71)以及CD3的胞内段CD3ξ(SEQ ID NO:72)组成。
2D4-BBZ序列由CD8α信号肽(SEQ ID NO:68)、2D4scFv(SEQ ID NO:67)、CD8hinge(SEQ ID NO:69)、CD8跨膜区(SEQ ID NO:73)、CD137的胞内信号传导结构域(SEQ ID NO:74)以及CD3的胞内段CD3ξ(SEQ ID NO:72)组成。
2D4-28BBZ序列由CD8α信号肽(SEQ ID NO:68)、2D4scFv(SEQ ID NO:67)、CD8hinge(SEQ ID NO:69)、CD28跨膜区(SEQ ID NO:70)和胞内信号传导结构域(SEQ ID NO:71)、CD137的胞内信号传导结构域(SEQ ID NO:74)以及CD3的胞内段CD3ξ(SEQ ID NO:72)组成。
5G3-28Z序列由CD8α信号肽(SEQ ID NO:68)、5G3scFv(SEQ ID NO:75)、CD8hinge(SEQ ID NO:69)、CD28跨膜区(SEQ ID NO:70)和胞内信号传导结构域(SEQ ID NO:71)以及CD3的胞内段CD3ξ(SEQ ID NO:72)组成。
5G3-BBZ序列由CD8α信号肽(SEQ ID NO:68)、5G3scFv(SEQ ID NO:75)、CD8hinge(SEQ ID NO:69)、CD8跨膜区(SEQ ID NO:73)、CD137的胞内信号传导结构域(SEQ ID NO:74)以及CD3的胞内段CD3ξ(SEQ ID NO:72)组成。
5G3-28BBZ序列由CD8α信号肽(SEQ ID NO:68)、5G3scFv(SEQ ID NO:75)、CD8hinge(SEQ ID NO:69)、CD28跨膜区(SEQ ID NO:70)和胞内信号传导结构域(SEQ ID NO:71)、CD137的胞内信号传导结构域(SEQ ID NO:74)以及CD3的胞内段CD3ξ(SEQ ID NO:72)组成。
2.靶向IL13Ra2CAR慢病毒载体的慢病毒包装、病毒浓缩及滴度测定
以1.7×107的密度接种293T细胞于15cm培养皿中，培养基为含10％胎牛血清(BioWest)的DMEM。分别将目的基因质粒PRRLSIN-2D4-28Z、PRRLSIN-2D4-BBZ、PRRLSIN-2D4-28BBZ、PRRLSIN-5G3-28Z、PRRLSIN-5G3-BBZ、PRRLSIN-5G3-28BBZ质粒13.73μg与包装质粒pRsv-REV 16.4μg、RRE-PMDLg 16.4μg、Vsvg 6.4μg溶入2048 μL空白DMEM培养液，混匀。
将158.4μg PEI(1μg/μl)溶解于2048μl的无血清DMEM培养液中，混匀室温孵育。将质粒混合液加入PEI混合液中，混匀室温下孵育20min。将转染复合物4.096ml滴加入含20ml DMEM培养基的15cm培养皿中，4-5h小时后，用10％FBS的DMEM培基给转染的293T细胞换液，37℃孵育72h，收集病毒液上清并浓缩，测定病毒滴度，浓缩后的病毒滴度分别为：
2D4-28Z:3.89E×108U/ml
2D4-BBZ:3.08E×108U/ml
2D4-28BBZ:2.72E×108U/ml
5G3-28Z:3.7E×108U/ml
5G3-BBZ:1.88E×108U/ml
5G3-28BBZ:3.11E×108U/ml
3.慢病毒转导T淋巴细胞------CAR阳性的T淋巴细胞的制备
T淋巴细胞活化：以约5×105/mL密度加入淋巴细胞培养基液培养，并按照磁珠:细胞比例为2:1加入同时包被有抗CD3和CD28抗体的磁珠(Invitrogen)和终浓度500U/mL的重组人IL-2(上海华新生物高技术有限公司)刺激培养24-48h；
Retronectin包被24孔板：每孔加入380μl 5μg/ml的retronectin溶液(PBS)，4℃孵育过夜。弃去24孔板中的retronectin溶液(PBS)，1ml PBS洗两次，培基洗一次(孔保持湿润)；将细胞接种于包被了retronectin的24孔板内，每孔细胞数目5×105，培养液体积500μl；按照MOI＝15在PBMC细胞中加入浓缩后的慢病毒，32℃，1200g，离心60min后，转移至细胞培养箱中，感染病毒24h后，低速离心换液(300rpm,10min,大型离心机)。感染后3～4天可以去磁珠。
4.T淋巴细胞嵌合抗原受体表达
慢病毒感染的T淋巴细胞在培养第7天，取1×106的T细胞，二等分，4度、5000rpm，离心5min，弃上清，PBS洗两次。对照组细胞加入50μl PE-SA(1:200稀释)抗体冰上孵育45min，PBS(2％NBS)洗两次，重悬后作为对照；检测组细胞+50μl 1:50稀释的biotin-Goat anti human IgG，F(ab’)2抗体，冰上孵育45min；PBS(2％NBS)洗两次；加入50μl PE-SA(1:200稀释)抗体冰上孵育45min；加入2ml PBS(2％NBS)重悬细胞，4℃，5000rpm/min，离心5分钟弃上清；重复两次；流式细胞仪检测CAR阳性的T细胞比例。
5.靶向IL13Ra2CAR T细胞的细胞毒性测定
在比较UTD、2D4-28Z、2D4-BBZ、2D4-28BBZ、5G3-28Z、5G3-BBZ、5G3-28BBZ CAR T细胞的体外杀伤活性时，六种CAR T细胞的感染阳性率分别为66.0％、26.3％、34.8％、59.9％、35.5％以及23.8％。
三块E-Plate 16中分别加入50ul RMPI+10％胎牛血清(Gibco)+双抗，置于实时监测仪上调整基线。
靶细胞:分别接种50ul 1×104/mL的U251细胞于E-Plate 16板，静置30-40min， 置于实时监测仪开始监测；
效应细胞:18小时后，按效靶比3:1、1:1或1:3加UTD及表达不同嵌合抗原受体的CAR T细胞；
各组均设2个复孔，取2个复孔的平均值。检测时间为第38h。
其中各实验组和各对照组如下:
各实验组：各靶细胞+表达不同嵌合抗原受体的CAR T；
对照组1：靶细胞
对照组2：空白培基；
细胞毒性计算公式为：％细胞毒性＝[(实验组-效应细胞自发组-靶细胞自发组)/(靶细胞最大-靶细胞自发)]*100
实验结果如图13所示，各表达不同嵌合抗原受体的CAR T细胞对IL13Ra2阳性的细胞均有显著的体外杀伤活性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (32)

  1. 一种特异性识别IL-13RA2的抗体，其特征在于，所述抗体与内源性表达IL-13RA2的U251细胞的结合相对亲和力EC50不高于100nM，优选不高于10nM，更优选0.01-10nM。
  2. 如权利要求1所述的抗体，其特征在于，所述抗体选自以下的任一种：
    (1)抗体，其包含重链可变区，所述重链可变区包含SEQ ID NO:9、45、46、47、48、49、50、51、63、或64所示的HCDR1，和/或包含SEQ ID NO:10、52、53、54、55、56、57、58、65、或66所示的HCDR2，和/或包含SEQ ID NO:11或SEQ ID NO:12任一所示的HCDR3；
    (2)抗体，其包含轻链可变区，所述轻链可变区包含SEQ ID NO:13所示的LCDR1，和/或包含SEQ ID NO:14所示的LCDR2，和/或包含SEQ ID NO:15或SEQ ID NO:16任一所示的LCDR3；
    (3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区；
    (4)抗体，(1)～(3)中任一项所述的抗体的变体，且具备与(1)～(3)中任一项所述的抗体相同或相似的活性。
  3. 如权利要求2所述的抗体，其特征在于，所述抗体选自以下的任一种：
    (1)抗体，包含轻链可变区，该轻链可变区包含SEQ ID NO:4所示的氨基酸序列、SEQ ID NO:8所示的氨基酸序列或SEQ ID NO:4和SEQ ID NO:8的变体的序列；
    (2)抗体，包含重链可变区，所述重链可变区具有SEQ ID NO:2、6、29、31、33、35、37、39、41、43、59或61所示的序列、或上述序列的变体；
    (3)抗体，包含(1)所述抗体的重链可变区及(2)所述抗体的轻链可变区。
  4. 如权利要求1所述的抗体，其特征在于，所述的抗体的轻链可变区包含SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15或SEQ ID NO:16所示的LCDR3。
  5. 根据权利要求4所述的抗体，其特征在于，所述的抗体的轻链可变区具有SEQ ID NO:4或8所示的序列，或具有与上述任一序列至少80％，例如，85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
  6. 根据权利要求4或5所述的抗体，其特征在于，所述抗体的重链可变区包含SEQ ID NO:9、45、46、47、48、49、50、51、63或64所示的HCDR1，SEQ ID NO:10、52、53、54、55、56、57、58、65或66所示的HCDR2，和SEQ ID NO:11或SEQ ID NO:12所示的HCDR3。
  7. 根据权利要求6所述的抗体，其特征在于，所述抗体的重链可变区具有所述抗体的重链可变区具有SEQ ID NO:2、6、29、31、33、35、37、39、41、43、59或61所示的序列，或具有与上述任一序列至少80％，更优选85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％相似性的序列。
  8. 根据权利要求6所述的抗体，其特征在于，所述轻链可变区的CDR区和所述重链可变区的CDR区具有下述任选的序列或其变体：
    (1)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:9所示的HCDR1，SEQ ID NO:10所示的HCDR2和SEQ ID NO:11所示的HCDR3；
    (2)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:16所示的LCDR3；SEQ ID NO:9所示的HCDR1，SEQ ID NO:10所示的HCDR2和SEQ ID NO:12所示的HCDR3；
    (3)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:16所示的LCDR3；SEQ ID NO:64所示的HCDR1，SEQ ID NO:66所示的HCDR2和SEQ ID NO:12所示的HCDR3；
    (4)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:45所示的HCDR1，SEQ ID NO:52所示的HCDR2和SEQ ID NO:11所示的HCDR3；
    (5)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:16所示的LCDR3；SEQ ID NO:63所示的HCDR1，SEQ ID NO:65所示的HCDR2和SEQ ID NO:12所示的HCDR3；
    (6)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:50所示的HCDR1，SEQ ID NO:56所示的HCDR2和SEQ ID NO:11所示的HCDR3；
    (7)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:46所示的HCDR1，SEQ ID NO:52所示的HCDR2和SEQ ID NO:11所示的HCDR3；
    (8)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:48所示的HCDR1，SEQ ID NO:54所示的HCDR2和SEQ ID NO:11所示的HCDR3；
    (9)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:47所示的HCDR1，SEQ ID NO:53所示的HCDR2和SEQ ID NO:11所示的HCDR3；
    (10)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:49所示的HCDR1，SEQ ID NO:55所示的HCDR2和SEQ ID NO:11所示的HCDR3；
    (11)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO:15所示的LCDR3；SEQ ID NO:51所示的HCDR1，SEQ ID NO:57所示的HCDR2和SEQ ID NO:11所示的HCDR3；
    (12)SEQ ID NO:13所示的LCDR1，SEQ ID NO:14所示的LCDR2和SEQ ID NO: 15所示的LCDR3；SEQ ID NO:49所示的HCDR1，SEQ ID NO:58所示的HCDR2和SEQ ID NO:11所示的HCDR3。
  9. 如权利要求7所述的抗体，其特征在于，
    (1)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:2所示的序列或其变体的序列；
    (2)所述轻链可变区具有SEQ ID NO:8所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:6所示的序列或其变体的序列；
    (3)所述轻链可变区具有SEQ ID NO:8所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:61所示的序列或其变体的序列；
    (4)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:29所示的序列或其变体的序列；
    (5)所述轻链可变区具有SEQ ID NO:8所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:59所示的序列或其变体的序列；
    (6)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:39所示的序列或其变体的序列；
    (7)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:31所示的序列或其变体的序列；
    (8)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:35所示的序列或其变体的序列；
    (9)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:33所示的序列或其变体的序列；
    (10)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:37所示的序列或其变体的序列；
    (11)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:41所示的序列或其变体的序列；
    (12)所述轻链可变区具有SEQ ID NO:4所示的序列或其变体的序列，所述重链可变区具有SEQ ID NO:43所示的序列或其变体的序列。
  10. 一种特异性识别IL-13RA2的抗体，其特征在于，该抗体与权利要求1-9任一所述的抗体识别相同的抗原决定部位。
  11. 一种特异性识别IL-13RA2的抗体，其特征在于，该抗体与权利要求1-9任一所述的抗体竞争性结合IL-13RA2。
  12. 编码权利要求1-11任一所述的抗体的核酸。
  13. 一种表达载体，其包含权利要求12所述的核酸。
  14. 一种宿主细胞，其包含权利要求13所述的表达载体或基因组中整合有权利要求12所述的核酸。
  15. 一种多功能免疫辍合物，其特征在于，所述的多功能免疫辍合物包括：
    权利要求1-11任一所述的抗体；以及
    与之连接的功能性分子；所述的功能性分子选自：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。
  16. 如权利要求15所述的多功能免疫辍合物，其特征在于，所述的靶向肿瘤表面标志物的分子是结合除IL-13RA2外的其他肿瘤表面标志物的抗体或配体；或
    所述的抑制肿瘤的分子是抗肿瘤的细胞因子或抗肿瘤的毒素；较佳地，所述的细胞因子选自：IL-12、IL-15、I型干扰素、TNF-alpha。
  17. 如权利要求15所述的多功能免疫辍合物，其特征在于，所述的靶向免疫细胞的表面标志物的分子是结合免疫细胞表面标志物的抗体，优选的，所述的结合免疫细胞表面标志物选自：CD3，CD16，CD28，更佳的，所述的结合免疫细胞表面标志物的抗体是抗CD3抗体。
  18. 如权利要求17所述的多功能免疫辍合物，其特征在于，所述的靶向免疫细胞的表面标志物的分子是结合T细胞表面标志物的抗体，其与权利要求1-11中任一所述的抗体形成T细胞参与的双功能抗体。
  19. 如权利要求17所述的多功能免疫辍合物，其特征在于，其是融合多肽，在权利要求1-11中任一所述的抗体以及与之连接的功能性分子之间，还包括连接肽。
  20. 编码权利要求15-19中任一所述的多功能免疫辍合物的核酸。
  21. 包含权利要求1-11任一所述的抗体的嵌合抗原受体，其特征在于，所述的嵌合抗原受体包含顺序连接的：权利要求1-11任一所述的抗体、跨膜区和胞内信号区。
  22. 如权利要求21所述的嵌合抗原受体，其特征在于，所述的胞内信号区选自：CD3ζ、CD3γ、CD3δ、CD3ε、FcRγ(FCER1G)、FcRβ(FcεR1b)、CD79a、CD79b、FcγRIIa、DAP10和DAP12的蛋白质的功能信号传导结构域，或其组合。
  23. 如权利要求22所述的嵌合抗原受体，其特征在于，所述的胞内信号区还具有共刺激信号传导结构域，所述共刺激信号传导结构域包含选自以下蛋白的功能信号传导结构域：CD27、CD28、4-1BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关的抗原-1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3、特异性结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244,2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A，Ly108)、SLAM(SLAMF1，CD150，IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46和NKG2D，或其组合。
  24. 如权利要求21所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体包括 如下的顺序连接的抗体、跨膜区和胞内信号区：
    权利要求1-11任一所述的抗体、CD8和CD3ζ；
    权利要求1-11任一所述的抗体、CD8、CD137和CD3ζ；或
    权利要求1-11任一所述的抗体、CD28分子的跨膜区、CD28分子的胞内信号区和CD3ζ；或
    权利要求1-11任一所述的抗体、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
  25. 编码权利要求21-24中任一所述的嵌合抗原受体的核酸。
  26. 一种表达载体，其特征在于，其包含权利要求25所述的核酸。
  27. 一种病毒，其特征在于，所述的病毒包含权利要求26所述载体。
  28. 一种嵌合抗原受体修饰的免疫细胞，其特征在于，其转导有权利要求27所述的核酸，或权利要求26所述的表达载体或权利要求27所述的病毒；或其表面表达权利要求21-24任一所述的嵌合抗原受体；
    较佳地，所述的免疫细胞为：T淋巴细胞、NK细胞或者NKT淋巴细胞。
  29. 如权利要求28所述的免疫细胞，其特征在于，其还携带外源的细胞因子的编码序列；或
    其还表达另一种嵌合抗原受体，该受体不含有CD3ζ；或
    其还表达趋化因子受体；较佳地，所述的趋化因子受体包括:CCR；或
    其还表达能降低PD-1表达的siRNA或者阻断PD-L1的蛋白；或其细胞中内源性的PD-1被基因编辑技术敲除；或
    其还表达安全开关。
  30. 一种药物组合物，其特征在于，其包括：
    权利要求1-11任一所述的抗体或编码该抗体的核酸；或
    权利要求15-19任一所述的免疫辍合物或编码该辍合物的核酸；或
    权利要求21-24任一所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或
    权利要求28或29所述的嵌合抗原受体修饰的免疫细胞；
    以及药学上可接受的载体或赋形剂。
  31. 一种试剂盒，其特征在于，其包括：
    容器，以及位于容器中的权利要求30所述的药物组合物；或
    容器，以及位于容器中的权利要求1-11任一所述的抗体或编码该抗体的核酸；或权利要求15-19任一所述的免疫辍合物或编码该辍合物的核酸；或权利要求21-24任一所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或权利要求28或29所述的嵌合抗原受体修饰的免疫细胞。
  32. 权利要求1-11任一所述的抗体或编码该抗体的核酸；或权利要求15-19任一所述的免疫辍合物或编码该辍合物的核酸；或权利要求21-24任一所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或权利要求28或29所述的嵌合抗原受体修饰的免疫细胞 的用途，用于治疗表达IL-13RA2的肿瘤，
    较佳的，所述的表达IL-13RA2的肿瘤为脑癌、胰腺癌、卵巢癌、肾癌、膀胱癌、胰腺癌、胃癌、肠癌、头颈癌、甲状腺癌、前列腺癌、卡波氏肉瘤。更佳的，所述的脑癌选自星形细胞瘤、脑膜瘤、少突神经胶质瘤、神经胶质瘤。
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