Double-target chimeric antigen receptor, immune effector cell, and use

Abstract

The present invention provides a double-target chimeric antigen receptor (CAR), an immune effector cell, and a use. Disclosed in the present invention are a CAR targeting an IL13Ra2 binding protein and an EGFRvIII binding protein, an immune effector cell modified by the CAR, a preparation method therefor, and a use thereof in inhibition of tumors.

Description

Dual-target chimeric antigen receptor, immune effector cell and application

[0001] The present application claims priority to the patent application filed on May 20, 2024, with the application number CN 202410625952.6; the entire content of which is incorporated herein. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine; more particularly, the present application relates to a dual-target chimeric antigen receptor, immune effector cell and application. BACKGROUND

[0003] Glioma is the most common primary tumor of the central nervous system, accounting for more than 40% of intracranial tumors. In children under the age of 15 and adult cancer death patients aged 15-34, brain glioma is the second largest cause. Among them, glioblastoma (GBM) is the most malignant type. Studies have shown that GBM is a highly heterogeneous disease, and this heterogeneity is not only manifested between tumors, but also within tumors, resulting in a complex cellular biological heterogeneity environment for each tumor. The current treatment method for GMB is mainly surgery, supplemented by comprehensive treatment such as radiotherapy and drug chemotherapy, but the effect is not satisfactory, and recurrence often occurs after surgery. Therefore, there is an urgent need to explore new effective medical means as a supplement.

[0004] In recent years, with the development of tumor immunotherapy and the progress of clinical research, chimeric antigen receptor T cell (CAR-T) immunotherapy has been developed and applied. CAR-T cells are connected by the single-chain variable region of a monoclonal antibody and the T cell signal transduction region, and the antibody can bind to the corresponding tumor antigen to activate the T cell in a major histocompatibility complex in a non-restricted manner, thereby exerting an anti-tumor effect. The structure of CAR can include: an extracellular antigen binding region, a hinge region, a transmembrane domain and an intracellular signal transduction domain.

[0005] When developing CAR-modified immune effector cells, the targeted antigen is actually a relatively critical choice. The selection of the target antigen is a key determinant for the specificity, effectiveness of CAR and the safety of the genetically modified T cells.

[0006] However, given the complexity of gene expression in vivo and various uncontrollable factors, it is very difficult to successfully select a suitable gene for CAR-based treatment. In a large amount of research work in the field, many tumor-specific antigens have been found to be unsuitable for CAR-modified targeted therapy. In addition, although CAR-T therapy has had some successful cases, it is challenged by the complexity of its production and adverse events related to cell activity, such as cytokine storm (CRS), etc. At present, there is no report on CAR T cell treatment for many tumors

[0007] In summary, the field is in urgent need of finding a more ideal antigen molecule for glioma disease to achieve better therapeutic effect. SUMMARY

[0008] The present application aims to provide a dual-targeting chimeric antigen receptor, immune effector cell and application.

[0009] In a first aspect of the present application, a chimeric antigen receptor (CAR) is provided, comprising, in sequence: an extracellular binding region, a transmembrane region and an intracellular signaling region; wherein the extracellular binding region comprises an IL13Ra2 binding protein and / or an EGFRvIII binding protein.

[0010] In one or more embodiments, the IL13Ra2 binding protein is an antibody; preferably, the antibody is an anti-IL13Ra2 single-domain antibody, the complementarity determining region CDR of which comprises CDR1-CDR3 with the amino acid sequences as shown below: CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, CDR3 as shown in SEQ ID NO: 3.

[0011] In one or more embodiments, the EGFRvIII binding protein is an antibody; preferably, the antibody is an anti-EGFRvIII single-domain antibody, the complementarity determining region CDR of which comprises CDR1-CDR3 with the amino acid sequences as shown below: CDR1 as shown in SEQ ID NO: 12, CDR2 as shown in SEQ ID NO: 13, CDR3 as shown in SEQ ID NO: 14.

[0012] In one or more embodiments, the anti-IL13Ra2 single-domain antibody further comprises a framework region (FR) comprising FR1-FR4 with the amino acid sequence as set forth in: FR1 of SEQ ID NO: 8, FR2 of SEQ ID NO: 9, FR3 of SEQ ID NO: 10, FR4 of SEQ ID NO: 11 (humanized structure); or FR1 of SEQ ID NO: 4, FR2 of SEQ ID NO: 5, FR3 of SEQ ID NO: 6, FR4 of SEQ ID NO: 7.

[0013] In one or more embodiments, the anti-EGFRvIII single-domain antibody further comprises a framework region (FR) comprising FR1-FR4 with the amino acid sequence as set forth in: FR1 of SEQ ID NO: 19, FR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 20, FR4 of SEQ ID NO: 21 (humanized structure); or FR1 of SEQ ID NO: 15, FR2 of SEQ ID NO: 16, FR3 of SEQ ID NO: 17, FR4 of SEQ ID NO: 18.

[0014] In one or more embodiments, the single-domain antibody is a VHH of llama origin or a humanized VHH.

[0015] In one or more embodiments, the anti-IL13Ra2 single-domain antibody comprises: (a) a single-domain antibody with the amino acid sequence as set forth in SEQ ID NO: 23 (humanized structure) or SEQ ID NO: 22; or (b) a single-domain antibody with an amino acid sequence having 80% or more (such as 85%, 90%, 93%, 95%, 97%, or 99% or more) identity to the sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 22, and having the function of the single-domain antibody of (a).

[0016] In one or more embodiments, the anti-EGFRvIII single-domain antibody comprises: (a) a single-domain antibody with the amino acid sequence as set forth in SEQ ID NO: 25 (humanized structure) or SEQ ID NO: 24; or (b) a single-domain antibody with an amino acid sequence having 80% or more (such as 85%, 90%, 93%, 95%, 97%, or 99% or more) identity to the sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 24, and having the function of the single-domain antibody of (a).

[0017] In one or more embodiments, the transmembrane region is a sequence comprising a CD8 transmembrane region or a CD28 transmembrane region; preferably, the transmembrane region is a sequence comprising a CD8 transmembrane region.

[0018] In one or more embodiments, the intracellular signaling region comprises an intracellular signaling region sequence selected from the group consisting of 4-1BB, CD3 zeta, Fc epsilon RI gamma, CD27, CD28, CD134, ICOS, GITR, or a combination thereof; preferably, the intracellular signaling region comprises 4-1BB and CD3 zeta.

[0019] In one or more embodiments, the transmembrane region is a protein having an amino acid sequence as set forth in SEQ ID NO: 26, or a protein having an amino acid sequence that is 80% or more (such as 85%, 90%, 93%, 95%, 97%, or 99% or more) identical to the sequence as set forth in SEQ ID NO: 26, and that has the function of the protein of the sequence as set forth in SEQ ID NO: 26.

[0020] In one or more embodiments, the 4-1BB is a protein having an amino acid sequence as set forth in SEQ ID NO: 27, or a protein having an amino acid sequence that is 80% or more (such as 85%, 90%, 93%, 95%, 97%, or 99% or more) identical to the sequence as set forth in SEQ ID NO: 27, and that has the function of the protein of the sequence as set forth in SEQ ID NO: 27.

[0021] In one or more embodiments, the CD3 zeta is a protein having an amino acid sequence as set forth in SEQ ID NO: 28, or a protein having an amino acid sequence that is 80% or more (such as 85%, 90%, 93%, 95%, 97%, or 99% or more) identical to the sequence as set forth in SEQ ID NO: 28, and that has the function of the protein of the sequence as set forth in SEQ ID NO: 28.

[0022] In one or more embodiments, in the chimeric antigen receptor, the extracellular binding region comprises an IL13Ra2 binding protein and an EGFRvIII binding protein; preferably, the chimeric antigen receptor comprises, in order (extracellular region to intracellular region): EGFRvIII binding protein-IL13Ra2 binding protein-transmembrane region-intracellular signaling region; or, IL13Ra2 binding protein-EGFRvIII binding protein-transmembrane region-intracellular signaling region.

[0023] In one or more embodiments, the chimeric antigen receptor is a protein having an amino acid sequence as set forth in SEQ ID NO: 31 (B010-C-03 CAR), SEQ ID NO: 32 (B010-C-04 CAR), SEQ ID NO: 29 (EGFRvIII CAR), or SEQ ID NO: 30 (IL13Ra2 CAR), or a protein having an amino acid sequence that is 80% or more (such as 85%, 90%, 93%, 95%, 97%, or 99% or more) identical to the sequence as set forth in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 29, or SEQ ID NO: 30.

[0024] In another aspect of the present application, there is provided a nucleic acid, an expression vector containing the nucleic acid, or a virus (obtained from packaging of the expression vector and a helper plasmid) containing the expression vector; wherein the nucleic acid encodes the chimeric antigen receptor.

[0025] In another aspect of the present application, there is provided a use of the aforementioned chimeric antigen receptor, the nucleic acid, the expression vector, or the virus for preparing an immune effector cell having a targeted killing effect on a cell expressing IL13Ra2 and / or EGFRvIII.

[0026] In another aspect of the present application, there is provided an IL13Ra2 binding protein, which is an anti-IL13Ra2 single-domain antibody, the complementarity determining regions (CDRs) of which include CDR1, CDR2, and CDR3 having the amino acid sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0027] In one or more embodiments, the anti-IL13Ra2 single-domain antibody in the IL13Ra2 binding protein includes: (a) a single-domain antibody having an amino acid sequence as set forth in SEQ ID NO: 23 (humanized structure) or SEQ ID NO: 22; or (b) a single-domain antibody having an amino acid sequence that is 80% or more (such as 85%, 90%, 93%, 95%, 97%, or 99% or more) identical to the sequence as set forth in SEQ ID NO: 23 or SEQ ID NO: 22, and having the function of the single-domain antibody of (a).

[0028] In another aspect of the present application, there is provided an EGFRvIII binding protein, which is an anti-EGFRvIII single-domain antibody, the complementarity determining regions (CDRs) of which include CDR1, CDR2, and CDR3 having the amino acid sequences as set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.

[0029] In one or more embodiments, the EGFRvIII binding protein, the anti-EGFRvIII single domain antibody comprises: (a) a single domain antibody having an amino acid sequence as set forth in SEQ ID NO: 25 (humanized structure) or SEQ ID NO: 24; or (b) a single domain antibody having an amino acid sequence with 80% or more (such as 85%, 90%, 93%, 95%, 97% or 99% or more) identity to the sequence as set forth in SEQ ID NO: 25 or SEQ ID NO: 24, and having the function of the single domain antibody of (a).

[0030] In another aspect of the present application, there is provided an isolated nucleic acid, a construct or an expression system comprising the nucleic acid; wherein the nucleic acid encodes the binding protein of any of the preceding aspects; wherein the expression system comprises the construct or the nucleic acid integrated into the genome of the expression system; preferably the expression system is a cellular expression system.

[0031] In one or more embodiments, the expression system is used to express the antibody under conditions suitable for expression of the antibody, thereby expressing the binding protein (single domain antibody); preferably, the antibody is further isolated and purified.

[0032] In another aspect of the present application, there is provided a modified immune effector cell transduced with the nucleic acid, the construct or the expression vector or the virus.

[0033] In one or more embodiments, the immune effector cell expresses on its surface the chimeric antigen receptor of any of the preceding aspects; or the binding protein of any of the preceding aspects.

[0034] In one or more embodiments, the immune effector cell comprises a cell selected from the group consisting of: a T lymphocyte, an NK cell or an NKT cell; or a combination thereof; preferably, the immune effector cell is a T lymphocyte.

[0035] In one or more embodiments, the immune effector cell further expresses a cytokine having a characteristic selected from the group consisting of: having an immunomodulatory activity or an anti-tumor activity, having an enhanced function of the immune effector cell; preferably, the cytokine comprises (but is not limited to): IL-12, IL-15, IL-21, IL-2, IL-4, IL-7, IL-9, IL-17, IL-18, IL-23.

[0036] In one or more embodiments, the immune effector cell further expresses a chemokine receptor (which blocks metastasis of the tumor); preferably, the chemokine receptor comprises: CCR2 or CCR7.

[0037] In one or more embodiments, the immune effector cell further expresses an inhibitory molecule (such as siRNA) that reduces PD-1 expression or a protein that blocks PD-L1.

[0038] In one or more embodiments, the immune effector cell further expresses a dominant negative receptor for TGFb, such as TGFbRII with a deletion of the intracellular signaling domain.

[0039] In one or more embodiments, the immune effector cell further expresses a safety switch; preferably, the safety switch comprises iCaspase-9, Truancated EGFR or RQR8.

[0040] In another aspect of the present application, there is provided a use of the modified immune effector cell of any one of the preceding aspects for the manufacture of a pharmaceutical composition for inhibiting a tumor; wherein the tumor is a tumor that expresses IL13Ra2 and / or EGFRvIII.

[0041] In one or more embodiments, the tumor comprises glioma, astrocytoma, high-grade astrocytoma, glioblastoma.

[0042] In another aspect of the present application, there is provided a pharmaceutical composition for inhibiting a tumor, comprising the modified immune effector cell, and a pharmaceutically acceptable pharmaceutical carrier or excipient.

[0043] In another aspect of the present application, there is provided a kit for inhibiting a tumor, comprising: a container, and the pharmaceutical composition in the container; or comprising: a container, and the modified immune effector cell in the container.

[0044] In another aspect of the present application, there is provided a method for inhibiting a tumor, comprising administering the modified immune effector cell or the pharmaceutical composition to a subject in need thereof.

[0045] Other aspects of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1, flow cytometry detection of humanized binding protein binding to protein expressing cell lines.

[0047] Figure 2, flow cytometry detection of humanized EGFRvIII binding protein binding to human / monkey EGFRvIII T cells. A, EGFRvIII antigen binding protein binding to human EGFRvIII expressed on cell surface; B, EGFRvIII antigen binding protein binding to monkey EGFRvIII expressed on cell surface.

[0048] Figure 3, schematic diagram of CAR structure.

[0049] Figure 4, flow cytometry-based determination of the binding rate of the antigen binding protein prepared in the application to the single target CAR-T and proximal protein. A, binding rate of the prepared EGFRvIII CAR-T to EGFR protein and average fluorescence intensity; B, binding rate of the prepared IL13Ra2 CAR-T to IL13Ra2 protein and average fluorescence intensity.

[0050] Figure 5, flow cytometry-based determination of the binding rate of the antigen binding protein prepared in the application to the CAR-T and EGFRvIII protein.

[0051] Figure 6, ONE-Glo luciferase detection system for determination of the killing effect of single and double target CAR-T on U87 MG related cell lines.

[0052] Figure 7, determination of the killing effect of CAR-T on U87 MG constructed cell lines based on live cell imaging system.

[0053] Figure 8, flow cytometry-based detection of cytokine secretion after co-incubation of CAR-T and U87 MG constructed cells for 24 hours.

[0054] Figure 9, CAR-T tumor inhibition effect in U87MG-EGFRvIII(+) cell mouse model.

[0055] Figure 10, CAR-T tumor inhibition effect in U87MG mix cell mouse model.

[0056] Figure 11, CAR-T tumor inhibition effect in U373-EGFRvIII(+) cell mouse model.

[0057] Figure 12, flow cytometry-based detection of CD8 T cell CD107a expression after co-culture of PDC and CAR T in vitro.

[0058] Figure 13, flow cytometry-based detection of the apoptosis rate of PDC after co-culture of PDC and CAR T in vitro. DETAILED DESCRIPTION

[0059] The present inventors have made intensive studies and disclosed a chimeric antigen receptor (CAR) targeting IL13Ra2 binding protein and EGFRvIII binding protein, immune effector cells modified by the CAR, a preparation method thereof and its application in inhibiting tumors (particularly glioma).

[0060] Terminology

[0061] As used herein, a "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct comprising an extracellular domain capable of binding an antigen, a transmembrane domain, and to a cytoplasmic signaling domain (also referred to as "intracellular signaling region"), wherein the intracellular signaling region comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. For example, the stimulatory molecule can be the zeta chain associated with the T cell receptor complex, in particular, it can be 4-1BB (CD137) and / or CD18.

[0062] As used herein, the term "immune cell" and "immune effector cell" are used interchangeably and include T lymphocytes, NK cells or NKT cells, etc.; preferably T lymphocytes.

[0063] The term "single (structural) domain antibody (VHH)" refers to a variable region of a heavy chain of an antibody, which is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment having a complete function. Generally, after obtaining an antibody naturally lacking a light chain and a heavy chain constant region 1 (CH1) from a llama immune serum, the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0064] The term "single (structural) domain antibody (VHH)" refers to an immunoglobulin domain comprising four "framework regions" referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, which are separated by three "complementarity determining regions" or "CDRs" referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of a single domain antibody (VHH) can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The single domain antibody (VHH) confers specificity of an antibody to an antigen due to the antigen binding site.

[0065] The terms "single domain antibody", "VHH domain", "VHH", "VHH antibody fragment", "VHH antibody", and "nanobody" are used interchangeably.

[0066] As used herein, an "intracellular signaling domain" or "intracellular signaling region" or "intracellular signaling activation region" refers to the intracellular portion of a CAR molecule. The intracellular signaling domain generates a signal that can promote an immunologic effector function of a CAR immune effector cell, e.g., a CAR T cell. Examples of immunologic effector functions, e.g., in a CAR T cell, include cytolytic activity and helper activity, including cytokine secretion.

[0067] As used herein, "specifically binds" means that the extracellular binding domain (e.g., a single domain antibody) does not cross-react, or does not substantially cross-react, with any polypeptide other than the target antigen. The degree of specificity can be judged by immunological techniques, including but not limited to immunoblotting, immunoaffinity chromatography, flow cytometry, and the like. In the present application, specific recognition is preferably determined by flow cytometry, and the criteria for specific recognition can be judged by one of ordinary skill in the art based on his common general knowledge in the art.

[0068] As used herein, "operatively linked" or "operably linked" refers to the functional placement of two or more nucleic acid / protein regions or nucleic acid / protein sequences in a spatial relationship.

[0069] As used herein, "construct" refers to a single- or double-stranded DNA molecule that has been modified by the hand of man, to contain segments of DNA combined and arranged in a sequence not existing in nature. The "construct" includes an expression vector; alternatively, the "construct" is contained in, or is part of, an expression vector.

[0070] The "IMGT Numbering System" is an integrated information system for immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complex (MHC) of human and other vertebrates, i.e., THE INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). The IMGT® database (http: / / www.imgt.org / IMGT_vquest) analyzes antibody light and heavy chain genes to determine the framework regions (FR) and complementarity determining regions (CDR) of the variable region. CDRs are "located" within the structure of immunoglobulin variable domains and exist in structures called loops, so CDR and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and substitute CDR residues from one species of immunoglobulin into an acceptor framework, often from a human antibody. Unless otherwise indicated, in the specification, claims and drawings, the single domain antibodies are numbered according to the IMGT Numbering System method to determine CDR and FR regions.

[0071] Target molecule

[0072] For such refractory tumors as glioma, the present inventors previously investigated a variety of tumor-related genes and found that a considerable part of such genes is also expressed in normal cells of some tissues, which are difficult to be applied to CAR modified immune effector cell technology; some other tumor-specific genes have good tumor-specific expression characteristics, but the CAR modified immune effector cells based on the design thereof have no or very low tumor cell killing activity, which may be because the target can trigger tumor cells to secrete factors that inhibit immune effector cells. After repeated investigation and screening of a variety of genes, the present inventors determined IL13Ra2 and EGFRvIII as target genes for designing CAR. The amino acid sequence of IL13Ra2 is disclosed in GenBank accession number NP_000631.1. The amino acid sequence of EGFRvIII is disclosed in GenBank accession number NP_001333870.1.

[0073] Compared with wild-type EGFR, EGFRvIII has a 2-7 exon in-frame deletion in the extracellular domain, resulting in the deletion of amino acids 6-273, so it does not have the ability to bind to epidermal growth factor (EGF), while the domain EGFRvIII at the C-terminus is completely the same as wild-type EGFR and can mediate intracellular signal transduction. EGFRvIII establishes a signal pathway regulation network through non-receptor-dependent autophosphorylation and tyrosine kinase activity, regulates the cell proliferation ability and invasion ability of glioblastoma through EGFRvIII-PI3K-AKT, EGFRvIII-Ras-Raf-MEK ERK / MAPK and EGFRvIII-JAK-STAT signaling pathways, and improves its resistance to chemotherapeutic drugs.

[0074] As an IL13 high-affinity induced receptor, IL13Ra2 is used as a negative prognostic marker for infiltrating glioma. So far, the downstream signal pathway of IL13Ra2 has not been found.

[0075] At present, although CAR T cells have become a potential treatment, the selection of its therapeutic targets has always been a difficulty, and many tumors have not been reported for CAR T cell treatment. In the early stage of research, it is not known whether targeting IL13Ra2 and EGFRvIII can be successfully used for glioma. After in-depth research, the present inventors determined the relatively applicable single domain antibody. The research of the present inventors showed that the CAR T cells composed of the two single domain antibodies described in the present application retained high efficiency and selective killing effect on target cells.

[0076] Glioma (especially as glioblastoma) has strong tumor heterogeneity, and single-target CAR-T treatment can cause tumor cell escape and drug resistance and recurrence, and double-target CAR-T performs better. In view of the relationship between EGFRvIII and IL13Ra2 targets and glioma development and specific high expression, the development of EGFRvIII-IL13Ra2 double-target CAR-T has great clinical significance.

[0077] Antigen binding protein

[0078] The application also provides preferred antigen binding proteins against the target molecules, which can be used as part of the extracellular binding region of the CAR.

[0079] On the basis of extensive screening, the application provides preferred anti-IL13Ra2 single-domain antibodies, the complementarity determining regions CDR of which include CDR1-CDR3 with the amino acid sequences as shown below: CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3. On the basis of extensive screening, the application also provides preferred anti-EGFRvIII single-domain antibodies, the complementarity determining regions CDR of which include CDR1-CDR3 with the amino acid sequences as shown below: CDR1 as shown in SEQ ID NO: 12, CDR2 as shown in SEQ ID NO: 13, and CDR3 as shown in SEQ ID NO: 14.

[0080] The antigen binding property of an antibody is usually determined by three complementarity determining regions CDR, which are arranged in order with FR regions, and the FR regions are not directly involved in the binding reaction. These CDRs form a loop structure, and the beta sheets formed by the FRs therebetween are close to each other in the spatial structure, and constitute the antigen binding site of the antibody. The CDR region is the sequence of the protein of immunological interest, and the CDR region of the antibody of the application is brand new.

[0081] The single-domain antibody provided by the application can specifically bind to the target antigen, increase the expression of IFN-γ, TNF-α and / or IL-2 in T lymphocytes, and can inhibit tumor growth.

[0082] As a preferred mode of the application, the single-domain antibody provided by the application is a humanized antibody.

[0083] The application also provides an isolated polynucleotide encoding the single-domain antibody. Methods for providing the isolated polynucleotide should be known to those skilled in the art, for example, can be prepared by automatic DNA synthesis and / or recombinant DNA technology, and can also be isolated from suitable natural sources.

[0084] The polynucleotide can be contained in a construct or expression system to express the single domain antibody.

[0085] Chimeric antigen receptors and nucleic acids encoding the same

[0086] The present application provides a CAR expressed on the surface of immune effector cells, which comprises, in sequence: an extracellular binding region, a transmembrane region and an intracellular signaling region. The CAR of the present application combines a specific extracellular binding region and an intracellular signaling region. The expression of the CAR on the surface of immune effector cells can make the immune effector cells have a highly specific cytotoxic effect on tumors expressing IL13Ra2 and / or EGFRvIII.

[0087] The extracellular binding region comprises a protein that specifically recognizes IL13Ra2 and / or EGFRvIII. The expression of the CAR on the surface of immune effector cells can make the immune effector cells have a highly specific cytotoxic effect on tumor cells highly expressing IL13Ra2 and / or EGFRvIII. As a preferred mode of the present application, the extracellular binding region is a single domain antibody.

[0088] In the chimeric antigen receptor of the present application, the single domain antibody can be operatively linked to a hinge region sequence (such as a CD8 hinge region), a transmembrane region sequence (such as a CD8 transmembrane region), and then operatively linked to an intracellular signaling region.

[0089] The transmembrane region of the CAR can be selected from the transmembrane region of proteins such as CD8 or CD28. Human CD8 protein is a heterodimer composed of αβ or γδ two chains. In a preferred embodiment of the present application, the transmembrane region is selected from the transmembrane region of CD8 (CD8a) or CD28.

[0090] The intracellular signaling region can be selected from the intracellular signaling region of CD3ζ, FcεRIγ, CD27, CD28, 4-1BB, CD134, ICOS, GITR protein, and combinations thereof. The CD3 molecule is composed of five subunits, of which the CD3ζ subunit (also known as CD3 zeta, abbreviated as Z) contains 3 ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex. FcεRIγ is mainly distributed on the surface of mast cells and basophils, and contains an ITAM motif, which is similar to CD3ζ in structure, distribution and function. In addition, CD28, 4-1BB, CD134 are costimulatory signaling molecules, and the costimulatory effect produced by the intracellular signaling segment after binding to the respective ligands causes the sustained proliferation of immune effector cells (mainly T lymphocytes), and can increase the level of cytokines such as IL-2 and IFN-γ secreted by immune effector cells, and at the same time improve the survival period of CAR immune effector cells in vivo and the anti-tumor effect.

[0091] In embodiments of the application, the preferred CARs (EGFRvIII binding protein-IL13Ra2 binding protein-transmembrane region-intracellular signaling region; IL13Ra2 binding protein-EGFRvIII binding protein-transmembrane region-intracellular signaling region) of the inventors are provided. It is also understood that CARs that have been altered or modified from the optimized CARs of the application can also be encompassed by the application.

[0092] As one way of the application, the CAR can further comprise a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain during cellular processing and localization of the CAR to the cell membrane.

[0093] The application also includes nucleic acids encoding the CARs. The nucleic acid sequences of the application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand. The nucleic acid codons of the application that encode the amino acid sequences of the CAR proteins can be degenerate, i.e., a plurality of degenerate nucleic acid sequences that encode the same amino acid sequence are encompassed by the application. Degenerate nucleic acid codons that correspond to an amino acid are well known in the art.

[0094] The application also includes variants of the above polynucleotides that encode polypeptides or fragments, analogs, and derivatives of the polypeptides having the same amino acid sequences of the application. The variants of the polynucleotides can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which can result from a natural process such as mutation or genetic recombination. The allelic variant can be a substitution, deletion, or insertion variant, but does not substantially alter the functional properties of the encoded polypeptide.

[0095] The current CAR-T technology has limited targets for treating glioma, which can be related to the difficulty in determining suitable target antigens. The application provides a new dual-target combination for targeting tumors by CAR-T technology, which has a suitable expression density on tumor cells. The CAR molecules of the application can be modified T cells, and the specific CAR-T cells can efficiently and specifically treat tumors, especially glioma.

[0096] Expression constructs and immune effector cells

[0097] The application also provides modified immune effector cells that are transduced with the nucleic acids of the application or are transduced with the above-mentioned recombinant plasmids containing the nucleic acids or viruses containing the plasmids of the application. The cells are cells or cell populations containing the cells, preferably T cells or T cell-containing cell populations.

[0098] Conventional nucleic acid transduction methods, including non-viral and viral transduction methods, can be used in the present application. Non-viral based transduction methods include electroporation and transposon methods.

[0099] The present application also provides an expression construct (vector) comprising the above-mentioned nucleic acid encoding a chimeric antigen receptor protein expressed on the surface of an immune effector cell, including a viral vector or a non-viral vector.

[0100] The non-viral vector system, such as the Sleeping Beauty system or PiggyBac transposon, etc., has a higher transduction efficiency than ordinary electroporation. The combination of nucleofector transfection instrument and Sleeping Beauty system has been reported [Davies JK., et al. Combining CD19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies. Cancer Res, 2010, 70(10): OF1-10.], which has both high transduction efficiency and the ability to achieve site-specific integration of the target gene. In addition, mRNA transfection technology can also be applied.

[0101] A variety of vectors for viral packaging can be used in the present application, including lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, etc., and also including viral vectors formed by further modification based on these viral vectors. In a specific embodiment of the present application, the backbone vector used in the present application is a lentiviral plasmid vector. The CAR lentiviral vector plasmid, in the presence of a helper packaging plasmid, is co-transfected into virus production cells, which can be packaged into a lentivirus with CAR molecules.

[0102] The present application also includes viruses packaged by viral vectors. The viruses can be lentiviruses, adenoviruses, adeno-associated viruses, etc., and also include viruses formed by further modification based on these viruses. The viruses of the present application include packaged viruses with infectivity, and also include viruses to be packaged containing the necessary components for packaging viruses with infectivity. It should be understood that although lentiviruses are preferred in the present application, other viruses known in the art that can be used to transduce exogenous genes into immune effector cells and their corresponding plasmid vectors can also be used in the present application.

[0103] In one embodiment of the present application, the method for transducing the chimeric antigen receptor modified immune effector cell is based on viral transduction method, such as lentivirus. This method has the advantages of high transduction efficiency, stable expression of exogenous genes, and shortening the time for in vitro culture of immune effector cells to reach the clinical level. In the surface of the transgenic immune effector cell, the transduced nucleic acid is expressed on its surface by transcription and translation. Through in vitro cytotoxicity experiments on various cultured tumor cells, the modified immune effector cells of the present application have a high specific tumor cell killing effect (also known as cytotoxicity). Therefore, the nucleic acid encoding the chimeric antigen receptor protein of the present application, the plasmid containing the nucleic acid, the virus containing the plasmid, and the transgenic immune effector cell transduced with the above nucleic acid, plasmid or virus can be effectively used for immunotherapy of tumors, especially glioma.

[0104] The immune cells described in the present application can also carry the coding sequence of an exogenous cytokine; the cytokine includes but is not limited to IL-12, IL-15 or IL-21, etc. These cytokines have immunoregulatory or anti-tumor activity, and can enhance the function of effector T cells and activated NK cells, or directly play an anti-tumor role. Therefore, those skilled in the art can understand that the use of these cytokines helps the immune cells to better perform their functions.

[0105] The immune cells described in the present application can also express another chimeric antigen receptor in addition to the above-mentioned chimeric antigen receptor, which can not contain CD3ζ, but contains the intracellular signaling domain of CD28, the intracellular signaling domain of CD137, or a combination of the two.

[0106] The immune cells described in the present application can also express a chemokine receptor; the chemokine receptor includes but is not limited to CCR2. Those skilled in the art can understand that the CCR2 chemokine receptor can compete with CCR2 in vivo, which is beneficial for blocking tumor metastasis.

[0107] The immune cells described in the present application can also express a safety switch; preferably, the safety switch includes iCaspase-9, Truancated EGFR or RQR8. As described previously, the current CA therapy is also challenged by the complexity of its production and adverse events related to cell activity, such as cytokine storm (CRS), etc. Therefore, it is more preferred to have a drug or therapy that can effectively regulate CAR-T, such as setting a safety switch.

[0108] Pharmaceutical composition

[0109] The modified immune effector cells of the present application can be used in the preparation of a composition, particularly a pharmaceutical composition. The composition can comprise, in addition to an effective amount of the immune effector cells, a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that the molecular entity and the composition, when properly administered to an animal or human, do not produce adverse, allergic, or other untoward reactions.

[0110] Specific examples of some substances which can serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; powdered tragacanth; 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 oil of theobroma; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as the wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline; phosphate buffer solutions and the like.

[0111] The compositions of the present application can be formulated into various dosage forms as desired and can be administered to patients by a physician in a dose which is beneficial to the patient, taking into account the patient's age, body weight, and general condition, the mode of administration, and the like. The mode of administration can be, for example, injection or other therapeutic means.

[0112] The immune effector cells of the present application or the compositions containing the cells can also be placed in an appropriate kit for ease of use by a clinician. Preferably, the kit can also contain instructions for using the compositions of the present application.

[0113] The present application is further illustrated by the following examples. It is to be understood that these examples are merely for the purpose of illustration and are not to be construed as limiting the scope of the present application. The experimental procedures in the following examples, unless otherwise indicated, were carried out under conventional conditions as described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, or as recommended by the manufacturer.

[0114] Example 1, Immunization of Llama

[0115] A healthy female adult alpaca was immunized with recombinant human EGFRvIII protein (Cat. No. EG8-HM154, Kaybio) or human IL13Ra2 protein (Cat. No. 10350-H03H, Beijing Yiqiao Shenzhou Technology Co., Ltd.). The first immunization was performed by injecting 500 μg of recombinant human EGFRvIII or human IL13Ra2 protein emulsified with an equal volume of Freund's complete adjuvant into the left and right sides near the cervical lymph nodes; the booster immunization was performed by injecting 500 μg of recombinant human EGFRvIII or human IL13Ra2 protein emulsified with an equal volume of Freund's complete or incomplete adjuvant into the left and right sides near the cervical lymph nodes, and several booster immunizations were performed. One week after the last immunization, blood was collected to monitor the titer of the antisera.

[0116] Example 2, Construction of Alpaca Immunized Library and Screening of Antigen Binding Protein

[0117] After the immunization was completed, the peripheral blood of the alpaca was collected, and the lymphocytes were separated using a lymphocyte separation medium; total RNA was extracted using TRIzol reagent; and cDNA was reversely transcribed using a PrimeScript TM The VHH gene was amplified by nested PCR, and the VHH gene fragment was recovered using a gel purification kit and then digested by restriction enzyme Bgl 1. Subsequently, the VHH gene fragment was cloned into the phagemid vector pADL-10b, and the constructed cloning product was transformed into E. coli TG1 electrocompetent cells to construct a VHH gene library. The library capacity was determined to be 2.3 x 10 9 pfu (EGFRvIII phage library) and 8.2 x 10 8 pfu (IL13Ra2 phage library) by a plate gradient dilution method. The colony PCR results showed that the library insertion rate was greater than 95%. The live cells of 10-100 times the library capacity were inoculated and cultured, and the M13K07 phage was rescued in the logarithmic phase. After rescue culture, the phage was collected by centrifugation, and the phage was purified using PEG-NaCl, thereby obtaining the phage display library, which can be directly used for subsequent screening. After phage panning and Phage ELISA verification, the positive clones were selected for sequencing to obtain the single-domain antibody sequence.

[0118] By the above method, the single-domain antibody (sdAb) SPH005-02 targeting human IL3Ra2 with high affinity was obtained. At the same time, the single-domain antibody (sdAb) SPH006-16 targeting human EGFRvIII with high affinity was also obtained.

[0119] Example 3, Design of Humanization of Antibody

[0120] In order to reduce the immunogenicity of the camelid antibodies, the biologically active antibodies screened can optionally be humanized. Humanization of the camelid monoclonal antibodies is performed according to the methods published in many literatures in the art. Briefly, the human antibody constant domains can be used to replace the parent (camelid antibody) constant domains, the human germline antibody sequences are selected according to the homology of the camelid antibody and the human antibody, and CDR grafting is performed. Then, based on the three-dimensional structure of the camelid antibody, the constant region of the camelid antibody is replaced by the human constant region by back-mutation of the amino acid residues of VH to obtain the final single-domain antibody (sdAb) SPH005-02 humanized binding protein SPH005-02-3 targeting human IL3Ra2, and the single-domain antibody (sdAb) SPH006-16 humanized binding protein SPH006-16-3 targeting human EGFRvIII.

[0121] Table 1 and Table 2 are the CDRs, heavy chain variable regions VH, FRs of the antigen binding proteins of the present application.

[0122] Table 1

[0123] Table 2

[0124] Example 4, binding affinity of the antigen binding proteins

[0125] The binding affinity of different EGFRvIII antigen binding proteins to antigen (EGFRvIII protein, source: Beijing Yiqiao God Science and Technology Co., Ltd., item number 11958-H08H1-B) and IL13Ra2 antigen binding proteins to antigen (IL13Ra2 protein, item number 10350-H03H, Beijing Yiqiao God Science and Technology Co., Ltd.) was detected by Biacore.

[0126] Mix 100 mL of 10x HBS-EP+buffer and 900 mL of Milli-Q water to obtain 1 L of 1x HBS-EP+buffer.

[0127] The surface of the CM5 chip 1-8 channels was activated with 1:1 mixed 50 mM NHS and 200 mM EDC (NHS and EDC from Amino Coupling Kit) at a flow rate of 10 μL / min for 420 seconds. Anti-hFc or anti-mouse Fc antibody (diluted in sodium acetate solution at pH 4.5, concentration 20 μg / mL) was injected at a flow rate of 10 μL / min for 200 seconds, and finally 1 M ethanolamine hydrochloride (pH 8.5) was used to block the excess active carboxyl groups on the chip. The chip surface was washed with 1x HBS-EP+at a flow rate of 10 μL / min for 2 hours to stabilize the baseline, and the instrument was set to a temperature of 25°C.

[0128] The initial cycle consists of two steps: sampling and regeneration. It is repeated three times before measurement to stabilize the baseline.

[0129] Sample testing: Inject 1×HBS-EP+ buffer into channels 1-8 at a flow rate of 30 μL / min for 120 seconds, followed by dissociation for 60 seconds.

[0130] Regeneration: Inject 10 mM glycine (pH 1.5) into channels 1-8 at a rate of 30 μL / min for 30 seconds, followed by stabilization for 30 seconds.

[0131] Experimental procedures for determining kinetic parameters: The running buffer for kinetic measurements was 1×HBS-EP+ (pH 7.4) solution. Capture: Different antibodies were injected into test channels 1-8 of the Anti-hFc or Anti-mouse Fc chip at a flow rate of 10 μL / min for 60 s. Antigen IL13Ra2 protein (catalog number 10350-H03H, Beijing Yiqiao Shenzhou Technology Co., Ltd.) was diluted to 100 nM with 1×HBS-EP+ (pH 7.4). Antigen EGFRvIII protein (catalog number 29662-H08H, Beijing Yiqiao Shenzhou Technology Co., Ltd.) was diluted to 200 nM. Sample testing: One 0 concentration sample was injected into channels 1-8 at a flow rate of 30 μL / min to remove background signal; the binding and dissociation times of the antigen and antibody were 180 and 400 seconds, respectively. Regeneration: Inject 10 mM glycine (pH 1.5) into channels 1-8 at a flow rate of 30 μL / min for 30 seconds, then stabilize for 60 seconds. The equilibrium dissociation constant (K0) of each antigen-binding protein of this invention was calculated using Biacore8K analysis software. D (Value). The reference channel (FC1) is used for background subtraction.

[0132] The results of the Biacore assay for the binding affinity of different antigen-binding proteins to antigen proteins are shown in Table 3. This experiment demonstrates that the IL13Ra2 antigen-binding protein and the EGFRvIII antigen-binding protein of this invention both possess good binding affinity to their respective antigens.

[0133] Table 3

[0134] Example 5: Binding of IL13Ra2 antigen-binding protein to human IL13Ra2 expressed on the cell surface

[0135] The binding affinity of the IL13Ra2 antigen-binding protein (SPH005-02-3) to human IL13Ra2 expressed on the surface of CHOK1 cells was determined by flow cytometry. The binding affinity was assessed by comparing the binding curves of different IL13Ra2 antigen-binding proteins of this invention with human IL13Ra2 expressed on the surface of CHOK1 cells.

[0136] 1) In order to screen human IL13Ra2 binding proteins, CHOK1 cells were reconstituted to overexpress human IL13Ra2, and the cells were named CHOK1-hIL13Ra2, respectively. CHOK1-hIL13Ra2 cells were digested and plated in 96-well plates.

[0137] 2) hlgG (Nearshore Technology, Cat. No. NC002) and the antigen binding protein antibody of the present application were prepared in PBS containing 2% FBS, respectively, at a maximum concentration of 15 μg / ml, 3-fold dilution, 8 points, and the diluted samples were added to the 96-well plates and incubated at 4°C for 1 hour.

[0138] 3) The plates were washed 3 times with PBS containing 2% FBS.

[0139] 4) PE-labeled human IgG (Biolegend, Cat. No. 410708) was diluted with PBS containing 2% FBS according to the product instructions, and the diluted sample was added to the 96-well plates and incubated at 4°C for 0.5 hours.

[0140] 5) The plates were washed 2 times with PBS containing 2% FBS.

[0141] 6) The cells were resuspended with PBS containing 2% FBS, and the median fluorescence value (MFI) of the PE channel was measured using a flow cytometer.

[0142] The binding curve of the antigen binding protein to CHOK1 cells overexpressing human IL13Ra2 was determined based on flow cytometry. The results are shown in Figure 1, indicating that the antigen binding protein has binding activity to CHOK1 cells overexpressing human IL13Ra2.

[0143] Example 6, Binding of EGFRvIII antigen binding protein to human / monkey EGFRvIII expressed on the surface of cells

[0144] The binding ability of EGFRvIII antigen binding protein (SPH006-16-3) to human / monkey EGFRvIII expressed on the surface of CHOK1 cells was determined based on flow cytometry. The binding ability of different EGFRvIII antigen binding proteins of the present application to human / monkey EGFRvIII expressed on the surface of CHOK1 cells was determined by comparing the binding curves.

[0145] 1) To screen human / monkey EGFRvIII binding proteins, CHOK1 cells were genetically engineered to overexpress human / monkey EGFRvIII, which were named CHOK1-hEGFRvIII and CHOK1-cynoEGFRvIII cells, respectively. CHOK1-hEGFRvIII and CHOK1-cynoEGFRvIII cells were digested and plated in 96-well plates.

[0146] 2) The negative control antibody hIgG (Nearshore Biotech, Cat. No. NC002) and the antigen binding protein antibody of the present application were prepared in PBS containing 2% FBS at a maximum concentration of 15 μg / ml, 3-fold dilution, 8 points, and the diluted samples were added to the 96-well plates and incubated at 4°C for 1 hour.

[0147] 3) The plates were washed 3 times with PBS containing 2% FBS.

[0148] 4) PE-labeled hIgG (Biolegend, Cat. No. 410708) was diluted with PBS containing 2% FBS according to the product instructions, and the diluted sample was added to the 96-well plates and incubated at 4°C for 0.5 hours.

[0149] 5) The plates were washed 2 times with PBS containing 2% FBS.

[0150] 6) The cells were resuspended with 2% FBS in PBS, and the median fluorescence value (MFI) of the PE channel was measured using a flow cytometer.

[0151] The binding curve of the antigen binding protein to CHOK1 cells overexpressing human EGFRvIII and CHOK1 cells overexpressing monkey EGFRvIII was determined based on flow cytometry, as shown in Figure 2. The above test shows that the antigen binding protein has binding activity to CHOK1 cells overexpressing human EGFRvIII and monkey EGFRvIII, and is particularly excellent for human EGFRvIII.

[0152] In subsequent examples, the antigen binding protein is the humanized protein.

[0153] Example 7, EGFRvIII / IL13Ra2 bispecific CAR molecule and its corresponding single-target CAR molecule EGFRvIII CAR, IL13Ra2 CAR plasmid construction and lentivirus packaging

[0154] The gene sequence of the genetically synthesized humanized EGFRvIII / IL13Ra2 targeted chimeric antigen receptor molecule specifically includes: a human EGFRvIII targeting single domain antibody VHH (SPH006-16-3) sequence, a Linker1, a human IL13Ra2 targeting antibody VHH (SPH005-02-3) sequence, a membrane domain (CD8 transmembrane domain), a 4-1BB costimulatory signaling domain (CD137 costimulatory domain), and a CD3 zeta intracellular signaling domain, which are connected in the order of tandem connection. The obtained chimeric antigen receptor molecule (CAR molecule) is named B010-C-03CAR (the amino acid sequence is shown as SEQ ID NO: 31), and the chimeric antigen receptor molecule (CAR molecule) obtained by position transposition of the human EGFRvIII targeting single domain antibody VHH sequence and the human IL13Ra2 targeting antibody VHH sequence is named B010-C-04CAR (the amino acid sequence is shown as SEQ ID NO: 32).

[0155] The gene sequence of the genetically synthesized humanized IL13Ra2 targeted chimeric antigen receptor molecule specifically includes: a human IL13Ra2 targeting single domain antibody VHH sequence, a membrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta intracellular signaling domain, which are connected in the order of tandem connection, and the obtained chimeric antigen receptor molecule (CAR molecule) is named IL13Ra2 CAR (the amino acid sequence is shown as SEQ ID NO: 30).

[0156] The gene sequence of the genetically synthesized humanized EGFRvIII targeted chimeric antigen receptor molecule specifically includes: a human EGFRvIII targeting antibody VHH sequence, a membrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta intracellular signaling domain, which are connected in the order of tandem connection, and the obtained chimeric antigen receptor molecule (CAR molecule) is named EGFRvIII CAR (the amino acid sequence is shown as SEQ ID NO: 29).

[0157] The 806-hu08 CAR (the amino acid sequence is shown as SEQ ID NO: 33) sequence is derived from the patent WO2021041725A1, and after the synthesis of the chimeric antigen receptor gene sequence, it is cloned into the pLVX vector between the NheI and SalI enzyme cutting sites.

[0158] The schematic diagram of the CAR structure is shown in FIG. 3. The sequence of each domain and the CAR sequence are shown in Table 4.

[0159] Table 4

[0160] Establish lentivirus core plasmid: pLvx-EGFRVIIICAR, pLvx-IL13Ra2 CAR, pLvx-B010-C-03CAR, pLvx-B010-C-04CAR. The constructed lentivirus core plasmid and auxiliary plasmid pCMV-VSV-G, pMDLg_pRRE, pRSV-Rev were transfected into 293T cells with PEI transfection reagent, and the virus was collected after a certain period of culture and concentrated for use.

[0161] Example 8, preparation of CAR-T cells

[0162] Human peripheral blood T lymphocytes were collected, transfected with lentivirus prepared in Example 7, and CAR-T cells were prepared, which were named IL13Ra2 CAR-T, EGFRvIIICAR-T, B010-C-03CAR-T, and B010-C-04CAR-T, respectively.

[0163] 1) Take a 12-well plate, prepare an antibody mixture containing 5ug / ml anti-CD3, 2.5ug / ml anti-CD28 and 20ug / ml Retronectin antibody, coat the antibody mixture at 500ul / well, and incubate at room temperature for 2h or more.

[0164] 2) Thaw one vial of frozen PBMC cells. Wash once with 5ml PBS, and perform the sorting step according to the kit (MojoSort TM Human CD3 T cell isolation kit, biolegend, 480022) to obtain CD3+T cells.

[0165] 3) Take the incubated 12-well plate, wash once with 500ul / well PBS solution, and add PBMC cell solution to the well plate at 1E6 / well. Supplement T cell culture medium to 1ml / well, and place in a 37℃, 5% CO2 incubator for activation for 24 hours.

[0166] 4) Take a 12-well plate, add virus, and place in a 37℃, 5% CO2 incubator for culture for 24h. Supplement T cell culture medium to 1ml / well, and place in a 37℃, 5% CO2 incubator for culture for 48 hours or more. Transfer the CAR T cell suspension to a T25 culture flask, supplement T cell culture medium, and then expand the culture to obtain the required CAR-T cells.

[0167] Example 9, detection of the binding of CAR-T cells to EGFRvIII and IL13Ra2 proteins

[0168] The binding ability of the prepared CAR-T to EGFRvIII and IL13Ra2 proteins was determined based on flow cytometry assay.

[0169] 1) Take an appropriate amount of CAR-T cells and place them in a 96-well plate, add 10 ug / ml of EGFRvIII protein solution and IL13Ra2 protein solution 100ul respectively, and incubate at 4°C for 30 minutes.

[0170] 2) Wash the plate with PBS containing 2% FBS for 3 times.

[0171] 3) Dilute PE-labeled-streptin (Biolegend, item number 554061) with 2% FBS PBS according to the product instructions, and add the diluted sample to the 96-well plate, and incubate at 4°C for 30 minutes.

[0172] 4) Wash the plate with PBS containing 2% FBS for 2 times.

[0173] 5) Resuspend the cells with 2% FBS PBS, and use flow cytometry to determine the positive rate and median fluorescence value (MFI) of PE channel.

[0174] The results of the binding rate and average fluorescence intensity of the single-target CAR-T prepared by the antigen binding protein of the application based on flow cytometry are shown in Figure 4, A is the binding rate and average fluorescence intensity of the prepared EGFRvIII CAR-T and EGFR protein; B is the binding rate and average fluorescence intensity of the prepared IL13Ra2 CAR-T and IL13Ra2 protein.

[0175] The results of the binding rate and average fluorescence intensity of the CAR-T prepared by the antigen binding protein of the application based on flow cytometry are shown in Figure 5.

[0176] The above results show that the CAR-T prepared by the anti-IL13Ra2 antigen binding protein specifically binds to IL13Ra2 protein, the CAR-T prepared by the anti-EGFRvIII antigen binding protein specifically binds to EGFRvIII protein, and the double-target CAR-T containing anti-IL13Ra2 antigen binding protein and anti-EGFRvIII antigen binding protein simultaneously binds to IL13Ra2 protein and EGFRvIII protein, while the T cells not transfected with virus (NC group) do not bind to EGFRvIII protein.

[0177] Example 10, Comparison of the killing effect of CAR-T cells on target cells

[0178] To verify the killing function of the prepared CAR-T to the cells expressing EGFRvIII and IL13Ra2, human glioma U87 MG cells were genetically modified to overexpress human EGFRvIII, and the cells were named U87MG-EGFRvIII(+) cells. U87MG cells were also genetically edited to knock out EGFR and IL13Ra2 and then overexpress EGFRvIII, and the cells were named U87 MG-EGFR(-)-IL13Ra2(-)-EGFRvIII(+) cells. In addition, the cells were genetically edited to knock out EGFR and only overexpress IL13Ra2, and the cells were named U87MG-EGFR(-)-IL13Ra2(+) cells. The cells modified based on U87 MG simultaneously expressed luciferase and green fluorescent protein (GFP). The killing effect of CAR-T cells on EGFRvIII / IL3Ra2 cell lines at different effector-to-target ratios was compared by luciferase reporter system.

[0179] 1) Digest U87 MG-EGFR(-)-IL13Ra2(-)-EGFRvIII(+) cell lines and U87 MG-EGFR(-)-IL13Ra2(+) cell lines respectively, mix and plate in 96-well plates at a ratio of 1:1 after counting, and the mixed target cells are labeled as U87 MG mix; at the same time, digest U87 MG-EGFRvIII(+) cell lines.

[0180] 2) Count CAR-T cells, and add CAR-T cells to U87 MG-EGFRvIII(+) / U87 MG mix well plates at different ratios of CAR-T:U87 MG-EGFRvIII(+) / U87 MG mix = 5:1, 2.5:1, 1.25:1, and 0.625:1, respectively, for incubation, and this group is the test group; at the same time, use T lymphocytes transfected with virus packaged by irrelevant sequence preparation vector as the control group NC.

[0181] 3) After 20 hours of incubation, add ONE-Glo luciferase detection system (Promega, item number E6120) solution, and incubate in the dark for 10 minutes, then measure the luminescence value by enzyme label instrument to calculate the CAR-T in vitro killing effect result.

[0182] The killing effect of CAR-T on U87 MG related cell lines based on ONE-Glo luciferase detection system is shown in Figure 6.

[0183] The above experiments show that EGFRvIII CAR-T, IL13Ra2 CAR-T, B010-C-03 CAR-T, and B010-C-04 CAR-T all exhibit cytotoxic effects on U87 MG-EGFRvIII(+) / U87 MG mix cells, and the killing effect is enhanced with the increase of the effector target. EGFRvIII CAR-T does not exhibit killing effect on cell lines that do not express EGFRvIII, and IL13Ra2 CAR-T does not exhibit killing effect on cell lines that do not express IL13Ra2, indicating that the target specificity of CAR-T killing effect is excellent. For the IL13Ra2 and EGFRvIII co-expressed cell line U87 MG-EGFRvIII(+), the cytotoxic effect of double-target CAR-T is comparable to that of single-target CAR-T; for the IL13Ra2 single-positive and EGFRvIII single-positive mixed cell line U87 MG mix, the cytotoxic effect of double-target CAR-T is significantly better than that of single-target CAR-T, showing a strong killing advantage.

[0184] Example 11, Detection of Long-term Killing Effect of CAR-T Cells on Target Cells

[0185] To verify the killing function of the further prepared CAR-T cells on cells expressing IL13Ra2 and EGFRvIII, long-term killing ability monitoring of CAR-T cells was performed, and a live cell imager was used to monitor the killing effect of different CAR-T cells on U87 MG constructed cell lines at the same effector target ratio for a long time.

[0186] 1) Digest U87 MG-EGFR(-)-IL13 Ra2(-)-EGFRvIII(+) cell line and U87 MG-EGFR(-)-IL13Ra2(+) cell line respectively, count and mix at a ratio of 1:1, then plate in a 96-well plate. The mixed target cells are labeled as U87 MG mix.

[0187] 2) Count the prepared CAR-T cells and U87 MG mix, add cells to the 96-well plate according to the ratio of CAR-T:U87 MG mix=1:5, and incubate according to the group. This group is used as the test group; at the same time, T lymphocytes transfected with virus packaged by irrelevant sequence preparation vector are used as the control group NC. Add 2ul APC labeled Annexin V dye to each well for labeling dead cells.

[0188] 3) Place the well plate in a live cell imager for continuous monitoring for 120h, and process the images after the monitoring is completed to obtain a long-term killing effect diagram of CAR-T cells.

[0189] The killing effect of CAR-T on U87 MG constructed cell lines was determined based on live cell imaging system as shown in Figure 7.

[0190] The above experiments showed that both double-target B010-C-03 CAR-T and B010-C-04 CAR-T exhibited cytotoxic effects on U87 MG-EGFRvIII(+) / U87 MG mix cell lines, and T cells transfected with virus (NC group) could not kill the cell lines. EGFRvIII CAR-T did not exhibit killing effect on cell lines not expressing EGFRvIII, and IL13Ra2 CAR-T did not exhibit killing effect on cell lines not expressing IL13Ra2, indicating that CAR-T killing has target specificity. For IL13Ra2 and EGFRvIII co-expressed cell line U87 MG-EGFRvIII(+), the cytotoxic effect of double-target CAR-T was comparable to that of single-target CAR-T; for IL13Ra2 single-positive and EGFRvIII single-positive mixed cell line U87 MG mix (i.e. Tumor only group), the cytotoxic effect of double-target CAR-T was superior to that of single-target CAR-T, showing a killing advantage.

[0191] Example 12, Detection of cytokine secretion during the action of CAR-T cells and target cells

[0192] Flow cytometry was used to detect the secretion of cytokines during the action of CAR-T cells and U87 MG constructed cell lines expressing EGFRvIII and IL13Ra2, in order to compare the cytokine secretion ability of different CAR-T under the action of target cells.

[0193] 1) U87 MG-EGFR(-)-IL13 Ra2(-)-EGFRvIII(+) cell lines and U87 MG-EGFR(-)-IL13Ra2(+) cell lines were digested respectively, counted and mixed at a ratio of 1:1, then plated in a 96-well plate, and the mixed target cells were labeled as U87 MG mix. U87 MG-EGFRvIII(+) cell lines were digested.

[0194] 2) The prepared CAR-T cells and U87 MG-EGFRvIII(+) / U87 MG mix were counted respectively, and the cells were added to the 96-well plate according to the ratio of CAR-T:U87 MG-EGFRvIII(+) / U87 MG mix=1:1 for incubation, and this group was used as the test group; at the same time, T lymphocytes transfected with virus packaged by irrelevant sequence preparation vector were used as the control group NC group.

[0195] 3) After 24 hours of incubation, take the supernatant, follow the operation of Human Th1 / Th2 Cytokine Cytometric Bead Array (CBA) Kit II (BD, item number 551809) to detect the cytokines using flow cytometry.

[0196] The results of the analysis of cytokine secretion after 24 hours of co-incubation of CAR-T with U87 MG-EGFRvIII(+)-LUC cells and U87 MG-EGFR(-)-EGFRvIII(+)-LUC cells based on flow cytometry are shown in Figure 8 (where C is the group without target cells).

[0197] The above experiments show that EGFRvIII CAR-T, IL13Ra2 CAR-T, B010-C-03 CAR-T, B010-C-04 CAR-T all secrete IL-2, TNF-a, IFN-g after co-incubation with U87 MG-EGFRvIII(+) / U87 MG mix cell lines. EGFRvIII CAR-T does not detect significant cytokine secretion in the absence of EGFRvIII-expressing cell lines, and IL13Ra2 CAR-T does not detect significant cytokine secretion in the absence of IL13Ra2-expressing cell lines, indicating that the CAR-T cell activation has target specificity. Co-incubation of T cells without virus transfection (NC group) cannot secrete cytokines.

[0198] Example 13, in vivo tumor inhibition experiment of CAR-T cell mouse model

[0199] To verify the tumor inhibition effect of the constructed CAR-T in the mouse model, three types of constructed mouse models were used. The first used the constructed target tumor cell U87MG-EGFRvIII(+)-LUC overexpressing EGFRvIII protein and luciferase, the second used U87 MG mix model, and the third constructed target tumor cell U373-EGFRvIII(+)-LUC cells overexpressing EGFRvIII protein and luciferase (replacing U87MG in U87MG-EGFRvIII(+)-LUC with human glioma U373). After successful construction of the tumor model, the tumor inhibition effect of CAR-T in the mouse model was compared.

[0200] 1) The model mouse used NOG mice (purchased from Beijing Vantoll Life Science and Technology Co., Ltd. Shanghai Branch), and the brain was injected in situ to form a tumor. 2.5 x 10 5 The above target cells were resuspended in 4 ul of serum-free culture medium containing 20% Matrigel, and the mouse brain was injected in situ.

[0201] 2) After the model tumor formation, the mice were injected with luciferase substrate D-Luciferin intraperitoneally, and then the mice were anesthetized and placed in a small animal live imaging instrument for imaging. The CAR-T cells were washed with PBS and resuspended with serum-free medium, and the cell density was adjusted. Each mouse was injected with 4x10 6

[0202] 3) After injection of CAR-T cells, bioluminescence was measured twice a week, and tumor elimination effect was collected.

[0203] The tumor inhibition effect of CAR-T cells in U87 MG-EGFRvIII(+)-LUC cell mouse model was detected based on a live imaging instrument, as shown in Figure 9.

[0204] The tumor inhibition effect of CAR-T cells in U87 MG mix cell mouse model was detected based on a live imaging instrument, as shown in Figure 10.

[0205] The tumor inhibition effect of CAR-T cells in U373-EGFRvIII(+)-LUC cell mouse model was detected based on a live imaging instrument, as shown in Figure 11.

[0206] The above experiments show that the tumor inhibition effect of double-target CAR-T in U87 MG-EGFRvIII(+)-LUC cell mouse model is better than that of single-target EGFRvIII CAR-T, IL13Ra2 CAR-T or comparable to that of single-target CAR-T, and single-target CAR-T cannot completely inhibit tumor in U87 MG mix cell mouse model, while double-target CAR-T can inhibit the growth of U87 MG mix cells and even eliminate tumors. In the U373-EGFRvIII(+)-LUC cell mouse model, B010-C-03 CAR-T showed a tumor inhibition advantage compared with the University of Pennsylvania CAR-T (806-hu08 CAR). The untransfected virus T cells (NT-T) group in the three models grew rapidly without showing tumor inhibition.

[0207] Example 14, PDC and CAR T in vitro co-culture cytotoxicity experiment

[0208] ​To further verify the cytotoxicity of the constructed CAR-T to the patient glioma cells, two patient-derived cell (PDC) HN-13-0059 and HN-13-0063 (provided by WuXi) were selected. After counting the CAR-T and target cells, the cells were plated in the well plate at the ratio of E:T = 16:1, 4:1, and 1:1, and incubated for 4 h. Then, the expression rate of CD8 T cell CD107a was detected by flow cytometry. In addition, the cells were plated in the well plate at the ratio of E:T = 4:1, 1:1, and 0.25:1, and incubated overnight. Then, the apoptosis rate of PDC was detected by flow cytometry, wherein the cells were labeled with AnnexinV / PI.

[0209] The results of detecting the expression of CD8 T cell CD107a after co-culturing PDC with CAR T in vitro by flow cytometry are shown in FIG. 12. The results of detecting the apoptosis rate of PDC after co-culturing PDC with CAR T in vitro by flow cytometry are shown in FIG. 13.

[0210] The above experiments show that for the two PDC target cells, B010-C-03 CAR-T shows significantly higher CD107a level compared with the NT-T group, proving that CAR-T can kill both target cells. Compared with HN-13-0059 target cells, CAR-T shows higher CD107a level when co-cultured with HN-13-0063, proving that CAR-T can better kill target cell HN-13-0063. This result is consistent with the apoptosis rate of PDC. Meanwhile, as the ratio of effector to target increases, the apoptosis rate of target cells increases. Compared with HN-13-0059, HN-13-0063 is more sensitive to the cytotoxicity of B010-C-03 CAR-T, while NT-T has little killing effect on target cells.

[0211] The foregoing detailed description has been presented for purposes of illustration and description. Various changes in the described embodiments can be made without departing from the scope of the following claims and their equivalents.

Claims

1. A chimeric antigen receptor comprising, in sequence: an extracellular binding region, a transmembrane region, and an intracellular signaling region; wherein the extracellular binding region comprises an IL13Ra2 binding protein and / or an EGFRvIII binding protein.

2. The chimeric antigen receptor of claim 1, wherein, the IL13Ra2 binding protein is an antibody; preferably, the antibody is an anti-IL13Ra2 single domain antibody, the complementarity determining regions (CDRs) of which comprise CDR1-CDR3 having the amino acid sequences of CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2, and CDR3 of SEQ ID NO: 3; or the EGFRvIII binding protein is an antibody; preferably, the antibody is an anti-EGFRvIII single domain antibody, the complementarity determining regions (CDRs) of which comprise CDR1-CDR3 having the amino acid sequences of CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 13, and CDR3 of SEQ ID NO:

14.

3. The chimeric antigen receptor of claim 2, wherein, the anti-IL13Ra2 single domain antibody comprises: (a) a single domain antibody having the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 22; or (b) a single domain antibody having an amino acid sequence that is more than 80% identical to the sequence of SEQ ID NO: 23 or SEQ ID NO: 22 and that has the function of the single domain antibody of (a); or the anti-EGFRvIII single domain antibody comprises: (a) a single domain antibody having the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 24; or (b) a single domain antibody having an amino acid sequence that is more than 80% identical to the sequence of SEQ ID NO: 25 or SEQ ID NO: 24 and that has the function of the single domain antibody of (a).

4. The chimeric antigen receptor of claim 1, wherein, the transmembrane region comprises a sequence comprising a CD8 transmembrane region or a CD28 transmembrane region; preferably, the transmembrane region comprises a sequence comprising a CD8 transmembrane region; or the intracellular signaling region comprises an intracellular signaling region sequence selected from the group consisting of 4-1BB, CD3 zeta, Fc epsilon RI gamma, CD27, CD28, CD134, ICOS, GITR, or a combination thereof; preferably, the intracellular signaling region comprises 4-1BB and CD3 zeta.

5. The chimeric antigen receptor of claim 1, wherein, in the chimeric antigen receptor, the extracellular binding region comprises an IL13Ra2 binding protein and an EGFRvIII binding protein; preferably, the chimeric antigen receptor comprises, in sequence: an EGFRvIII binding protein-IL13Ra2 binding protein-transmembrane region-intracellular signaling region; or an IL13Ra2 binding protein-EGFRvIII binding protein-transmembrane region-intracellular signaling region.

6. A nucleic acid, an expression vector comprising the nucleic acid, or a virus comprising the expression vector; wherein the nucleic acid encodes the chimeric antigen receptor of any one of claims 1-5.

7. Use of the chimeric antigen receptor of any one of claims 1 to 5, the nucleic acid, expression vector or virus of claim 6, for the manufacture of an immune effector cell having a targeted killing effect on a cell expressing IL13Ra2 and / or EGFRvIII.

8. An IL13Ra2 binding protein which is an anti-IL13Ra2 single domain antibody, the complementarity determining regions (CDRs) of which comprise CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

9. The IL13Ra2 binding protein of claim 8, wherein, The anti-IL13Ra2 single domain antibody comprises: (a) a single domain antibody having the amino acid sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 22; or (b) a single domain antibody having an amino acid sequence which is 80% or more identical to the sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 22 and which has the function of the single domain antibody of (a).

10. An EGFRvIII binding protein which is an anti-EGFRvIII single domain antibody, the complementarity determining regions (CDRs) of which comprise CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

11. The EGFRvIII binding protein of claim 10, wherein, The anti-EGFRvIII single domain antibody comprises: (a) a single domain antibody having the amino acid sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 24; or (b) a single domain antibody having an amino acid sequence which is 80% or more identical to the sequence set forth in SEQ ID NO: 25 or SEQ ID NO: 24 and which has the function of the single domain antibody of (a).

12. An isolated nucleic acid, a construct or an expression system comprising the nucleic acid; wherein the nucleic acid encodes the binding protein of any one of claims 8 to 11; wherein the expression system comprises the construct or the nucleic acid integrated into the genome of the expression system; preferably the expression system is a cellular expression system.

13. A modified immune effector cell transduced with the nucleic acid, construct or expression vector or virus of claim 6 or claim 12.

14. The modified immune effector cell of claim 13, wherein the modified immune effector cell is a T cell. The immune effector cell expresses on its surface the chimeric antigen receptor of any one of claims 1 to 5; or the binding protein of any one of claims 8 to 11.

15. The immune effector cells as described in claim 13 or 14, characterized in that, The immune effector cell comprises a cell selected from the group consisting of a T lymphocyte, an NK cell or an NKT cell; preferably the immune effector cell is a T lymphocyte.

16. Use of the modified immune effector cell of any one of claims 13 to 15 for the manufacture of a pharmaceutical composition for inhibiting a tumor; wherein the tumor is a tumor expressing IL13Ra2 and / or EGFRvIII.

17. The use according to claim 16, characterized in that, The tumor comprises a glioma, an astrocytoma, a high-grade astrocytoma, a glioblastoma.

18. A pharmaceutical composition for inhibiting a tumor, comprising the modified immune effector cell of any one of claims 13-15, and a pharmaceutically acceptable pharmaceutical carrier or excipient.

19. A kit for inhibiting a tumor, comprising: a container, and the pharmaceutical composition of claim 18 in the container; or wherein comprising: a container, and the modified immune effector cell of any one of claims 13-15 in the container.

Images