WO2022148332A1 - 改造的免疫效应细胞及其用途 - Google Patents

改造的免疫效应细胞及其用途 Download PDF

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WO2022148332A1
WO2022148332A1 PCT/CN2022/070012 CN2022070012W WO2022148332A1 WO 2022148332 A1 WO2022148332 A1 WO 2022148332A1 CN 2022070012 W CN2022070012 W CN 2022070012W WO 2022148332 A1 WO2022148332 A1 WO 2022148332A1
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antigen
amino acid
acid sequence
immune effector
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French (fr)
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杨选明
王海勇
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Shanghai Jiao Tong University
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Shanghai Jiao Tong University
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Definitions

  • the present application relates to the field of biomedicine, in particular to a modified CAR-T cell and its use.
  • CLDN18 is a member of the Claudins protein family, and CLDN18.1 and CLDN18.2 are alternative splices of CLDN18.
  • CLDN18.1 was mainly expressed in the lungs, and CLDN18.2 was only expressed in gastric mucosal epithelial cells.
  • CLDN18.2 is expressed in a variety of tumor tissues, such as gastric cancer, pancreatic cancer, esophageal cancer, ovarian cancer, lung cancer, etc., and is an ideal tumor CAR-T therapeutic target.
  • CD20 is specifically expressed in B cells or B cell-derived leukemias or lymphomas.
  • Rituxan an antibody drug targeting CD20, has been marketed, but there are drug resistance and recurrence in the treatment.
  • Glypican-3 (GPC-3) is not expressed in normal liver tissue, but is highly expressed in fetal liver and liver cancer tissue. It is a specific marker of primary hepatocellular carcinoma and is involved in the occurrence and development of liver cancer. The target of small molecule targeted therapy is also the recognition target of immunotherapy.
  • CAR-T cells chimeric antigen receptor T cells
  • T cells are artificially modified tumor killer cells, which combine the target recognition function of antibodies and the tumor killing function of T cells.
  • solid tumors such as gastric cancer and pancreatic cancer
  • new CAR-T therapy is the key to the treatment of solid tumors.
  • the present application provides an immune effector cell comprising and/or expressing a chimeric antigen receptor (CAR), and a Bcl-2 protein or a functionally active fragment thereof.
  • the immune effector cells have one or more of the following properties: 1) able to specifically bind to antigens; 2) have strong in vitro expansion ability; 3) have strong in vivo anti-tumor ability; 4) have Strong anti-tumor ability in vitro.
  • the application provides an immune effector cell comprising and/or expressing a chimeric antigen receptor (CAR), and a Bcl-2 protein or a functionally active fragment thereof.
  • CAR chimeric antigen receptor
  • the CAR comprises an antigen-binding domain
  • the antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds CD20.
  • the antibody or antigen-binding fragment thereof comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:1.
  • the antibody or antigen-binding fragment thereof comprises HCDR2, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2.
  • the antibody or antigen-binding fragment thereof comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:3.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:4.
  • the antibody or antigen-binding fragment thereof comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5.
  • the antibody or antigen-binding fragment thereof comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:6.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:7.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence set forth in SEQ ID NO:8.
  • the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:10.
  • the CAR comprises an antigen-binding domain
  • the antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds CLDN18.2.
  • the antibody or antigen-binding fragment thereof comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:11.
  • the antibody or antigen-binding fragment thereof comprises HCDR2, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:12.
  • the antibody or antigen-binding fragment thereof comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:13.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:14.
  • the antibody or antigen-binding fragment thereof comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:15.
  • the antibody or antigen-binding fragment thereof comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:16.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:17.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence set forth in SEQ ID NO:18.
  • the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:19.
  • the CAR comprises an antigen-binding domain
  • the antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds GPC-3.
  • the antibody or antigen-binding fragment thereof comprises a HCDR3, and the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:20, SEQ ID NO:29, and SEQ ID NO:38.
  • the antibody or antigen-binding fragment thereof comprises HCDR2, and the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:21, SEQ ID NO:30, and SEQ ID NO:39.
  • the antibody or antigen-binding fragment thereof comprises HCDR1
  • the HCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:22, SEQ ID NO:31, and SEQ ID NO:40.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:23, SEQ ID NO:32, and SEQ ID NO:41.
  • the antibody or antigen-binding fragment thereof comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:24, SEQ ID NO:33, and SEQ ID NO:42.
  • the antibody or antigen-binding fragment thereof comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:25, SEQ ID NO:34, and SEQ ID NO:43.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in any one of SEQ ID NO:26, SEQ ID NO:35, and SEQ ID NO:44.
  • the antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence set forth in any one of SEQ ID NO:27, SEQ ID NO:36, and SEQ ID NO:45.
  • the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in any one of SEQ ID NO:28, SEQ ID NO:37, and SEQ ID NO:46.
  • the antibody comprises a single chain antibody.
  • the CAR comprises a transmembrane domain comprising a transmembrane domain derived from a protein selected from the group consisting of CD28, CD3e, CD45, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:47.
  • the CAR comprises a costimulatory domain
  • the costimulatory domain comprises one or more costimulatory domains selected from the group consisting of CD28, 4-1BB, CD40L, TIM1, CD226, DR3 , SLAM, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD27, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, and costimulatory signaling domains in DAP12.
  • the costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:48.
  • the CAR comprises an intracellular signaling domain
  • the intracellular signaling domain comprises an intracellular signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO:49.
  • the CAR comprises a hinge region located between the antigen binding domain and the transmembrane domain.
  • the hinge region comprises at least one of the hinge regions of CD8, CD28, 4-1BB, CD4, CD27, CD7, and PD-1.
  • the hinge region comprises the amino acid sequence set forth in SEQ ID NO:50.
  • the CAR comprises the amino acid sequence set forth in any one of SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, and SEQ ID NO:61 .
  • the Bcl-2 protein or functionally active fragment thereof comprises an exogenous Bcl-2 protein or functionally active fragment thereof.
  • the Bcl-2 protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:52.
  • the immune effector cells comprise T cells.
  • the present application also provides nucleic acid molecules encoding the CAR and the Bcl-2 protein or functionally active fragments thereof.
  • the nucleic acid molecule comprises a sequence encoding a self-cleaving peptide located between the sequence encoding the CAR and the sequence encoding the Bcl-2 protein.
  • the self-cleaving peptide comprises a 2A peptide.
  • the 2A peptide is one or more selected from the group consisting of P2A, T2A, E2A and F2A.
  • the 2A peptide comprises the amino acid sequence set forth in SEQ ID NO:51.
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the vector is selected from one or more of plasmids, retroviral vectors, and lentiviral vectors.
  • the present application also provides immune effector cells, which comprise the nucleic acid molecule or the vector.
  • the present application also provides a method for preparing the immune effector cells, the method comprising culturing the immune effector cells under conditions such that the chimeric antigen receptor is expressed.
  • the present application also provides a composition comprising the immune effector cells.
  • the present application also provides the use of the immune effector cell, the nucleic acid molecule, the carrier or the composition for preparing a medicament for preventing and/or treating a disease and/or disease.
  • the present application also provides the immune effector cell, the nucleic acid molecule, the vector or the composition for preventing and/or treating a disease and/or disorder.
  • the present application also provides a method for preventing and/or treating a disease and/or disorder, the method comprising administering the immune effector cell, the nucleic acid molecule, the vector described in the present application to a subject in need thereof or the composition.
  • the disease and/or disorder is associated with CD20 expression.
  • the disease and/or disorder is associated with CLDN18.2 expression.
  • the disease and/or disorder is associated with GPC-3 expression.
  • the disease and/or disorder comprises a tumor.
  • the tumor comprises a solid tumor and/or a hematological tumor.
  • the tumor comprises a CD20 positive tumor.
  • the tumor comprises a CLDN18.2 positive tumor.
  • the tumor comprises a GPC-3 positive tumor.
  • the tumor comprises lymphoma.
  • the tumor comprises pancreatic cancer.
  • Figure 1 shows a schematic diagram of the construction of the chimeric antigen receptor described in this application.
  • FIGS 2A-2B show the expression levels of the CARs described in this application.
  • FIG. 3 shows the expression of the CAR of the immune effector cells described in this application.
  • Figure 4 shows the expansion capacity of anti-CD20 Bcl-2 CAR-T cells.
  • Figure 5 shows the expansion capacity of anti-CLDN18.2 Bcl-2 CAR-T cells.
  • Figure 6 shows the expansion capacity of anti-GPC-3 Bcl-2 CAR-T cells.
  • Figure 7 shows the in vitro tumor killing ability of the immune effector cells described in the present application.
  • Figures 8A-8B show the in vivo tumor killing capacity of the immune effector cells described in the present application.
  • Figure 9 shows the effect of anti-CLDN18.2 Bcl-2 CAR-T cells on tumor volume.
  • Figure 10 shows the in vivo tumor killing ability of anti-GPC-3 Bcl-2 CAR-T cells.
  • immune effector cells generally refers to immune cells that participate in immune responses and perform effector functions. For example, performing effector functions may include removing foreign antigens or promoting immune effector responses.
  • Immune effector cells can include plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.
  • the immune effector cells described herein can include engineered immune effector cells.
  • the immune effector cells described in the present application may contain chimeric antigen receptors, and at the same time may express Bcl-2 protein or a functionally active fragment thereof.
  • the Bcl-2 protein is an exogenous Bcl-2 protein or a functionally active fragment thereof.
  • chimeric antigen receptor or “CAR” is also referred to as “chimeric receptor", “T body”, chimeric immunoreceptor, and generally refers to comprising at least one extracellular antigen binding domain, Recombinant polypeptide constructs of transmembrane and cytoplasmic signaling domains (also referred to as “intracellular signaling domains").
  • the chimeric antigen receptor can include a targeting moiety (eg, a moiety that binds a tumor-associated antigen), a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.
  • Bcl-2 and Bcl-2 protein are used interchangeably and generally refer to the encoded product of the bcl-2 proto-oncogene.
  • the amino acid sequence of the Bcl-2 protein can be set forth in SEQ ID NO:52.
  • the term encompasses full-length Bcl-2 protein and homologues, analogs, truncations, mutants and functionally active fragments thereof.
  • the term covers exogenous Bcl-2 protein or a functionally active fragment thereof.
  • an antigen binding domain generally refers to a domain capable of binding a target antigen.
  • An antigen-binding domain may comprise a chimeric antigen receptor and fragments thereof, antibodies or antigen-binding fragments thereof capable of (specifically) binding an antigen.
  • the antigen binding domain can be of natural, synthetic, semi-synthetic or recombinant origin.
  • the antigen-binding domain may comprise an antibody or antigen-binding fragment thereof.
  • the antigen binding domain may comprise a single chain antibody.
  • the term "specific binding” or “specific” generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that can be found in a heterogeneous population of molecules, including biomolecules
  • the presence may determine the presence of the target.
  • an antibody that specifically binds a target (which can be an epitope) can be an antibody that binds to that target with greater affinity, avidity, easier, and/or for a longer duration than it binds to other targets .
  • the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species.
  • specific binding may include, but does not require, exclusive binding.
  • an antibody generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a particular antigen.
  • an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof.
  • the term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (eg, dAbs), single chain antibodies (eg, scFvs), As well as antigen-binding antibody fragments (eg, Fab, Fab' and (Fab) 2 fragments).
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • the VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL can consist of three CDRs and four FR regions, which can be arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • CDRs also referred to as “complementarity determining regions” generally refers to regions within the variable domains of antibodies, the sequences of which are highly variable and/or form structurally defined loops.
  • an antibody typically includes six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3).
  • naturally occurring camelid antibodies consisting only of heavy chains are also functional and stable in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc. in combination. These coding systems are known in the art, see eg http://www.bioinf.org.uk/abs/index.html#kabatnum. For example, the amino acid sequence of the antibody can be numbered according to the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system@imgt.cines.fr; http://imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27: 209-212; Ruiz et al., 2000 Nucleic Acids Res.
  • IMGT the international ImMunoGeneTics information system@imgt.cines.fr
  • Lefranc et al. 1999, Nucleic Acids Res. 27: 209-212; Ruiz et al., 2000 Nucleic
  • the CDRs of the antibody can be determined according to the Kabat numbering system (see e.g. Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
  • variable domains of native heavy and light chains each comprise four FR regions, namely four in VH (H-FR1, H-FR2, H-FR3 and H-FR4), and four in VL (L-FR1, L-FR2, L-FR3 and L-FR4).
  • variable domain and “variable region” are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain.
  • the variable domains of heavy and light chains may be referred to as “ VH “ and “ VL “, respectively (or “VH” and “VL”, respectively). These domains are usually the most variable part of the antibody (relative to other antibodies of the same type) and contain the antigen binding site.
  • single-chain antibody generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain. wherein the light and heavy chain variable regions are contiguous (eg, via a synthetic linker, eg, a short flexible polypeptide linker).
  • the scFv can be expressed as a single-chain polypeptide and can also retain the specificity of the intact antibody from which it is derived.
  • the scFv may have the VL and VH in any order (eg, relative to the N- and C-terminus of the polypeptide), the scFv may also include a VL-linker-VH or may include a VH-linker- VL.
  • transmembrane domain generally refers to a domain capable of spanning the plasma membrane of a cell.
  • the transmembrane domain may generally comprise three domains: an N-terminal extracellular domain, an intermediate transmembrane stretch domain, and a C-terminal cytoplasmic domain.
  • the transmembrane domain may also contain an intracellular or cytoplasmic domain.
  • costimulatory domain generally refers to an intracellular domain that can provide an immune costimulatory molecule.
  • the costimulatory molecule may be a cell surface molecule required for an effective lymphocyte response to an antigen.
  • the costimulatory domain may be the intracellular portion of a costimulatory molecule or a truncated form thereof.
  • intracellular signaling domain generally refers to a domain located inside a cell capable of transducing a signal.
  • the intracellular signaling domain can transmit a signal into a cell.
  • the term encompasses intracellular signaling domains and any truncations thereof capable of inducing effector function signals.
  • 4-1BB also referred to as “CD137” generally refers to a member of the tumor necrosis factor (TNF) receptor family, encoded by the tumor necrosis factor receptor superfamily member 9 (TNFRSF9) gene.
  • TNF tumor necrosis factor
  • TNFRSF9 tumor necrosis factor receptor superfamily member 9
  • the "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host for transfer of the inserted nucleic acid molecule into and/or between host cells.
  • the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce the desired expression product by culturing a suitable host cell containing the vector.
  • Plasmid generally refers to DNA molecules other than chromosomes or nucleoids in organisms such as bacteria and yeast, which exist in the cytoplasm and have the ability to autonomously replicate, enabling them to maintain a constant copy in daughter cells number and express the genetic information it carries. Plasmids are used as carriers of genes in genetic engineering research.
  • retroviral vector generally refers to a virus particle that can controllably and express foreign genes, but cannot self-package into proliferative virus particles. Most of these viruses have reverse transcriptase. Retroviruses contain at least three genes: gag, the genes that contain the proteins that make up the center and structure of the virus; pol, the genes that make up the reverse transcriptase, and env, the genes that make up the viral coat. Through retroviral transfection, the retroviral vector can randomly and stably integrate its own genome and the foreign genes it carries into the host cell genome, for example, CAR molecules can be integrated into the host cell.
  • lentiviral vector generally refers to a diploid RNA viral vector that is a retrovirus.
  • the lentiviral vector is based on the genome of the lentivirus, and many sequence structures related to the virus activity are removed to make it biologically safe, and then the sequence of the target gene required for the experiment is introduced into the genome backbone. and expression constructs, and prepared into vectors.
  • retroviral vectors can randomly and stably integrate their own genomes and the foreign genes they carry into the host cell genome, for example, CAR molecules can be integrated into host cells.
  • tumor generally refers to any new pathological tissue growth. Tumor cells can spread to other parts of the body locally or through the bloodstream and lymphatic system.
  • the tumors may include benign tumors and malignant tumors.
  • the tumor may include solid tumors and/or hematological tumors.
  • the tumor may include cancer.
  • examples of such tumors include, but are not limited to, glioma, breast cancer, melanoma, non-small cell lung cancer, bladder cancer, ovarian cancer, and colorectal cancer.
  • the protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues with the same or similar functions as the protein or polypeptide.
  • the variant may be, for example, one or more substitutions, deletions, or additions to the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds the CD73 protein).
  • amino acid protein or polypeptide may comprise at least 1, such as 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, or 5 amino acid substitutions that have been made , a protein or polypeptide with amino acid changes, deletions and/or insertions.
  • the functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (eg, substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding capacity) of the protein or polypeptide prior to alteration.
  • the substitutions can be conservative substitutions.
  • the homologue may be at least about 85% (eg, having at least about 85%) the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds the CD73 protein). 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology Sexual proteins or polypeptides.
  • the homology generally refers to the similarity, similarity or relatedness between two or more sequences. "Percent sequence homology" can be calculated by comparing the two sequences to be aligned in a comparison window to determine the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences.
  • the same amino acid residue eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met
  • Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • FASTA and BLAST A description of the FASTA algorithm can be found in W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D.J. Lipman and W.R. Pearson, "Fast and Sensitive Protein Similarity Search", Science, 227: 1435-1441, 1989.
  • a description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, "A Basic Local Alignment Search Tool", J. Molecular Biology, 215: 403-410 , 1990.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides an immune effector cell, which can comprise and/or express a chimeric antigen receptor (CAR), and the immune effector cell can also comprise and/or express a Bcl-2 protein or a functionally active fragment thereof.
  • the Bcl-2 protein or functionally active fragment thereof may be exogenous (eg, artificially synthesized sequences of: hinge region, transmembrane domain, costimulatory domain, intracellular signaling domain, cleavage peptide and Bcl-2 or its Functionally active fragments, scFv added after ligation of each part, molecular cloning, production of viral vectors, infecting immune effector cells for expression) are introduced into the immune effector cells.
  • the immune effector cell can be an engineered or modified immune effector cell (eg, a T cell), wherein the engineering can include sending the immune effector cell (eg, derived from a recipient other naturally derived immune effector cells of the subject) (e.g., by constructing cells encoding antigen-binding proteins, hinge regions, transmembrane domains, costimulatory domains, intracellular signaling domains, cleavage peptides, Bcl-2 Nucleic acid sequence, expressed by viral vector infection of immune effector cells) the CAR and/or the Bcl-2 protein, and/or the nucleic acid molecule expressing the CAR and/or the Bcl-2 protein.
  • the immune effector cell eg, derived from a recipient other naturally derived immune effector cells of the subject
  • the engineering can include sending the immune effector cell (eg, derived from a recipient other naturally derived immune effector cells of the subject) (e.g., by constructing cells encoding antigen-binding proteins,
  • the CAR comprises an antigen binding domain, a transmembrane domain, a costimulatory domain, an intracellular signaling domain and/or a hinge region.
  • the immune effector cells overexpress Bcl-2 protein or a functionally active fragment thereof.
  • the CAR comprises an antigen-binding domain
  • the antigen-binding domain comprises an antibody or antigen-binding fragment thereof that targets CD20.
  • the CD20-targeting antibody or antigen-binding fragment thereof comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 1; in certain embodiments, the antibody or its The antigen-binding fragment comprises HCDR2, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2; in certain embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, and the HCDR1 comprises SEQ ID NO:3 amino acid sequence shown.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 4; in certain embodiments, the antibody or antigen-binding fragment thereof comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:5; in certain embodiments, the antibody or antigen-binding fragment thereof comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:6 sequence.
  • the antibody or antigen-binding fragment thereof comprises HCDR3, HCDR2, and HCDR1, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 1, and the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2 sequence, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, LCDR2, and LCDR1, the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:4, and the LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5 sequence, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:6.
  • the antibody or antigen-binding fragment thereof comprises HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1,
  • the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 1
  • the HCDR2 comprises SEQ ID NO : the amino acid sequence shown in 2
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5 amino acid sequence
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:6.
  • the antibody or antigen-binding fragment thereof comprises a VH, which may comprise the amino acid sequence set forth in SEQ ID NO:7. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VL, which may comprise the amino acid sequence set forth in SEQ ID NO:8.
  • the antibody or antigen-binding fragment thereof comprises VH and VL, the VH comprising the amino acid sequence set forth in SEQ ID NO:7 and the VL comprising the amino acid sequence set forth in SEQ ID NO:8 sequence.
  • the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:10.
  • the CAR comprises an antigen binding domain
  • the antigen binding domain comprises an antibody or antigen binding fragment thereof targeting CLDN18.2.
  • the antibody or antigen-binding fragment thereof targeting CLDN18.2 comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11; in certain embodiments, the antibody or an antigen-binding fragment thereof comprising HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 12; in certain embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, and the HCDR1 comprises SEQ ID NO : the amino acid sequence shown in 13.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 14; in certain embodiments, the antibody or antigen-binding fragment thereof comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15; in certain embodiments, the antibody or antigen-binding fragment thereof comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 16 sequence.
  • the antibody or antigen-binding fragment thereof comprises HCDR3, HCDR2, and HCDR1, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, and the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12 sequence, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:13.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, LCDR2, and LCDR1, the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 14, and the LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15 sequence, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof comprises HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1,
  • the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11
  • the HCDR2 comprises SEQ ID NO The amino acid sequence shown in: 12
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 13
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 15 amino acid sequence
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof comprises a VH, which may comprise the amino acid sequence set forth in SEQ ID NO:17. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence set forth in SEQ ID NO:18.
  • the antibody or antigen-binding fragment thereof comprises VH and VL, the VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and the VL comprising the amino acid sequence set forth in SEQ ID NO: 18 sequence.
  • the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:19.
  • the CAR comprises an antigen-binding domain
  • the antigen-binding domain comprises an antibody or antigen-binding fragment thereof targeting GPC-3.
  • the antibody or antigen-binding fragment thereof targeting GPC-3 comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20; in certain embodiments, the antibody or an antigen-binding fragment thereof comprising HCDR2, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 21; in certain embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, and the HCDR1 comprises SEQ ID NO : the amino acid sequence shown in 22.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 23; in certain embodiments, the antibody or antigen-binding fragment thereof comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:24; in certain embodiments, the antibody or antigen-binding fragment thereof comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:25 sequence.
  • the antibody or antigen-binding fragment thereof comprises HCDR3, HCDR2, and HCDR1, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:20, and the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:21 sequence, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:22.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, LCDR2, and LCDR1, the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:23, and the LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:24 sequence, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:25.
  • the antibody or antigen-binding fragment thereof comprises HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1,
  • the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20
  • the HCDR2 comprises SEQ ID NO The amino acid sequence shown in: 21
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 23
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 24 amino acid sequence
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:25.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:26. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence set forth in SEQ ID NO:27.
  • the antibody or antigen-binding fragment thereof comprises VH and VL, the VH comprising the amino acid sequence set forth in SEQ ID NO:26 and the VL comprising the amino acid sequence set forth in SEQ ID NO:27 sequence.
  • the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:28.
  • the antibody or antigen-binding fragment thereof targeting GPC-3 comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29; in certain embodiments, the antibody or an antigen-binding fragment thereof comprising HCDR2, and said HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 30; in certain embodiments, said antibody or antigen-binding fragment thereof comprising HCDR1, and said HCDR1 comprising SEQ ID NO : amino acid sequence shown in 31.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 32; in certain embodiments, the antibody or antigen-binding fragment thereof comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:33; in certain embodiments, the antibody or antigen-binding fragment thereof comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:34 sequence.
  • the antibody or antigen-binding fragment thereof comprises HCDR3, HCDR2, and HCDR1, the HCDR3 comprising the amino acid sequence set forth in SEQ ID NO:29, and the HCDR2 comprising the amino acid sequence set forth in SEQ ID NO:30 sequence, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:31.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, LCDR2 and LCDR1
  • the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:32
  • the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:33 amino acid sequence
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:34.
  • the antibody or antigen-binding fragment thereof comprises HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1,
  • the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29
  • the HCDR2 comprises SEQ ID NO The amino acid sequence shown in: 30
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 31
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 32
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 33 amino acid sequence
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:34.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:35. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence set forth in SEQ ID NO:36.
  • the antibody or antigen-binding fragment thereof comprises VH and VL, the VH comprising the amino acid sequence set forth in SEQ ID NO:35 and the VL comprising the amino acid sequence set forth in SEQ ID NO:36 sequence.
  • the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:37.
  • the antibody or antigen-binding fragment thereof targeting GPC-3 may comprise HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 38; in certain embodiments, the The antibody or antigen-binding fragment thereof comprises HCDR2, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 39; in certain embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, and the HCDR1 comprises SEQ ID The amino acid sequence shown in NO:40.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 41; in certain embodiments, the antibody or antigen-binding fragment thereof comprises LCDR2, and the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:42; in certain embodiments, the antibody or antigen-binding fragment thereof comprises LCDR1, and the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:43 sequence.
  • the antibody or antigen-binding fragment thereof comprises HCDR3, HCDR2 and HCDR1
  • the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:38
  • the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:39 amino acid sequence
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:40.
  • the antibody or antigen-binding fragment thereof comprises LCDR3, LCDR2, and LCDR1, the LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:41, and the LCDR2 comprising the amino acid sequence set forth in SEQ ID NO:42 sequence, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:43.
  • the antibody or antigen-binding fragment thereof comprises HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1,
  • the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 38
  • the HCDR2 comprises SEQ ID NO The amino acid sequence shown in: 39
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 40
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 41
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 42 amino acid sequence
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:43.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:44. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VL comprising the amino acid sequence set forth in SEQ ID NO:45.
  • the antibody or antigen-binding fragment thereof comprises VH and VL, the VH comprising the amino acid sequence set forth in SEQ ID NO:44 and the VL comprising the amino acid sequence set forth in SEQ ID NO:45 sequence.
  • the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:46.
  • the antigen-binding domain of the CAR may include an antibody or antigen-binding fragment thereof that specifically binds to CD20, CLDN18.2 or GPC-3.
  • the antibodies or antigen-binding fragments thereof described herein may include, but are not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, diabodies, tribodies Antibodies, tetrabodies, Fv fragments, scFv fragments, Fab fragments, Fab' fragments, F(ab') 2 fragments.
  • the antigen-binding fragment may include Fab, Fab', F(ab')2, F(ab) 2 , Fv fragment, scFv, di-scFv and/or dAb.
  • the antigen binding domain may be a single chain antibody.
  • the CD20 binding domain is an scFv.
  • the scFv may comprise the sequence set forth in SEQ ID NO:10.
  • the CD20 binding domain may comprise the VH, VL and linker peptide of the antibody.
  • the linker peptide comprises the amino acid sequence shown in SEQ ID NO:9.
  • the CLDN18.2 binding domain is an scFv.
  • the scFv may comprise the sequence shown in SEQ ID NO:19.
  • the CLDN18.2 binding domain may comprise the VH, VL and linker peptide of the antibody.
  • the linker peptide comprises the amino acid sequence shown in SEQ ID NO:9.
  • the GPC-3 binding domain is an scFv.
  • the scFv may comprise the sequence set forth in SEQ ID NO:28.
  • the GPC-3 binding domain may comprise the VH, VL and linker peptide of an antibody.
  • the linker peptide comprises the amino acid sequence shown in SEQ ID NO:9.
  • the GPC-3 binding domain is an scFv.
  • the scFv may comprise the sequence shown in SEQ ID NO:37.
  • the GPC-3 binding domain may comprise the VH, VL and linker peptide of an antibody.
  • the linker peptide comprises the amino acid sequence shown in SEQ ID NO:9.
  • the GPC-3 binding domain is an scFv.
  • the scFv may comprise the sequence set forth in SEQ ID NO:46.
  • the GPC-3 binding domain may comprise the VH, VL and linker peptide of an antibody.
  • the linker peptide comprises the amino acid sequence shown in SEQ ID NO:9.
  • the CAR may comprise a transmembrane domain
  • the transmembrane domain may comprise a transmembrane domain selected from the following proteins: CD28, CD3e, CD45, CD5, CD8, CD9, CD16, CD22, CD33 , CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:47.
  • the CAR may comprise a costimulatory domain, which may comprise a costimulatory domain selected from the following proteins: CD28, 4-1BB, CD40L, TIM1, CD226, DR3, SLAM, ICOS, OX40, Costimulatory signaling regions in NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD27, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT and DAP12.
  • the costimulatory domain comprises the costimulatory domain of 4-1BB.
  • the costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:48.
  • the CAR may comprise an intracellular signaling domain, which may comprise a CD3 ⁇ -derived signaling domain.
  • the intracellular signaling domain may comprise the amino acid sequence set forth in SEQ ID NO:49.
  • the CAR may include an antigen binding domain, a transmembrane domain, a costimulatory domain and an intracellular signaling domain in order from the N-terminal to the C-terminal.
  • the antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:37 or SEQ ID NO:46
  • the transmembrane structure The domain may comprise the amino acid sequence shown in SEQ ID NO:47
  • the costimulatory domain may comprise the amino acid sequence shown in SEQ ID NO:48
  • the intracellular signaling domain may comprise the amino acid shown in SEQ ID NO:49 sequence.
  • the CAR may further comprise a hinge region connecting the antigen binding domain and the transmembrane domain.
  • the hinge region is derived from the IgG family.
  • the hinge region is derived from IgG1.
  • the hinge region is derived from IgG4.
  • the hinge region is derived from IgD.
  • the hinge region is derived from CD8.
  • the hinge region may comprise the amino acid sequence set forth in SEQ ID NO:50.
  • the CAR can also be linked to a signal peptide, for example, the signal peptide can be derived from CD8.
  • the signal peptide can be CD8 ⁇ .
  • the signal peptide is linked to the N-terminus of the antigen binding domain.
  • the immune effector cells may further comprise and/or express Bcl-2 protein or a functionally active fragment thereof.
  • the Bcl-2 protein or functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO:52.
  • the Bcl-2 protein is introduced exogenously, eg, by adding a cleavage peptide to the nucleotide sequence encoding the chimeric antigen receptor and encoding the Bcl-2 protein or its functional activity The sequence of the fragment such that the Bcl-2 protein or its functionally active fragment is expressed in immune effector cells.
  • the engineered immune effector cell wherein the engineering of the immune effector cell comprises causing the immune effector cell to contain and/or express a chimeric antigen receptor, and introducing exogenous Bcl into the immune effector cell -2 protein or a functionally active fragment thereof.
  • the functionally active fragment of Bcl-2 includes a fragment that enables Bcl-2 protein to exert anti-apoptotic function, for example, the functionally active fragment of Bcl-2 protein includes its BH4 domain.
  • the BBZ comprises a hinge region, a transmembrane domain, a costimulatory domain, an intracellular signaling domain.
  • the 20BBZ comprises the sequence of a CAR targeting CD20.
  • the 20BBZ comprises the amino acid sequence shown in SEQ ID NO:54.
  • the 20BBZ-Bcl-2 comprises sequences capable of expressing CD20-targeting CAR and Bcl-2 proteins or functionally active fragments thereof.
  • the 20BBZ-Bcl-2 comprises the amino acid sequence shown in SEQ ID NO:53.
  • the Ab10BBZ comprises the sequence of a CAR targeting CLDN18.2.
  • the Ab10BBZ comprises the amino acid sequence shown in SEQ ID NO:56.
  • the Ab10BBZ-Bcl-2 comprises sequences capable of expressing a CAR targeting CLDN18.2 and a Bcl-2 protein or a functionally active fragment thereof.
  • the Ab10BBZ-Bcl-2 comprises the amino acid sequence shown in SEQ ID NO:55.
  • the GC33BBZ comprises the sequence of a CAR targeting GPC-3.
  • the GC33BBZ comprises the amino acid sequence shown in SEQ ID NO:58.
  • the GC33BBZ-Bcl-2 comprises sequences capable of expressing GPC-3 targeting CAR and Bcl-2 proteins or functionally active fragments thereof. Described GC33BBZ-Bcl-2 comprises the amino acid sequence shown in SEQ ID NO:57.
  • the GC179BBZ comprises the sequence of a CAR targeting GPC-3.
  • the GC179BBZ comprises the amino acid sequence shown in SEQ ID NO:60.
  • the GC179BBZ-Bcl-2 comprises sequences capable of expressing CAR and Bcl-2 proteins targeting GPC-3 or functionally active fragments thereof.
  • the GC179BBZ-Bcl-2 comprises the amino acid sequence shown in SEQ ID NO:59.
  • the M3C11BBZ comprises the sequence of a CAR targeting GPC-3.
  • the M3C11BBZ comprises the amino acid sequence shown in SEQ ID NO:62.
  • the M3C11BBZ-Bcl-2 comprises sequences capable of expressing GPC-3 targeting CAR and Bcl-2 proteins or functionally active fragments thereof.
  • the M3C11BBZ-Bcl-2 comprises the amino acid sequence shown in SEQ ID NO:61.
  • the CAR comprises the amino acid sequence of any one of SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59 and SEQ ID NO:61.
  • nucleic acid molecule Nucleic acid molecule, vector, cell, preparation method, composition
  • the application also provides isolated one or more nucleic acid molecules that can encode the CARs described herein.
  • the isolated nucleic acid molecule(s) described herein may be nucleotides, deoxyribonucleotides or ribonucleotides in isolated form of any length, or analogs isolated from their natural environment or synthetically synthesized , but can encode the CAR described in this application.
  • the nucleic acid molecule encodes the chimeric antigen receptor and the Bcl-2 protein or a functionally active fragment thereof.
  • the nucleic acid molecule further comprises a nucleotide sequence encoding a cleavage peptide.
  • the cleavage peptide can be the 2A peptide.
  • the cleavage peptide may be selected from one or more of P2A, T2A, E2A and F2A.
  • the cleavage peptide may comprise the amino acid sequence shown in SEQ ID NO:51.
  • the nucleic acid molecule comprises, in order from 5' end to 3' end: a sequence encoding an antigen binding domain, a sequence encoding a transmembrane domain, a sequence encoding a costimulatory domain, a sequence encoding an intracellular signaling domain
  • a sequence encoding an antigen binding domain a sequence encoding a transmembrane domain
  • a sequence encoding a costimulatory domain a sequence encoding an intracellular signaling domain
  • the nucleic acid molecule may comprise nucleotide sequences encoding scFv targeting CD20, 4-1BB, CD3 ⁇ , 2A, Bcl-2 in order from the 5' end to the 3' end.
  • the nucleic acid sequences of the various moieties may be linked directly or indirectly.
  • the indirect connection may be via a linker.
  • the nucleic acid molecule may comprise nucleotide sequences encoding scFv, 4-1BB, CD3 ⁇ , 2A, Bcl-2 targeting CLDN18.2 in order from the 5' end to the 3' end.
  • the nucleic acid sequences of the various moieties may be linked directly or indirectly.
  • the indirect connection may be via a linker.
  • the nucleic acid molecule may comprise, in order from the 5' end to the 3' end, nucleotide sequences encoding scFv, 4-1BB, CD3 ⁇ , 2A, Bcl-2 targeting GPC-3.
  • the nucleotide sequences of the various moieties may be linked directly or indirectly. The indirect connection may be via a linker.
  • the nucleotide sequence encoding the scFv targeting GPC-3 may be the nucleotide sequence encoding GC33 scFv, GC179 scFv or M3C11 scFv provided in the Examples.
  • the present application also provides vectors, which may comprise the nucleic acid molecules described herein.
  • the vector can be transformed, transduced or transfected into a host cell so that the genetic material elements it carries can be expressed in the host cell.
  • vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes of P1 origin (PAC); bacteriophages such as lambda phage or M13 phage and Animal viruses, etc.
  • Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Viruses (eg, SV40).
  • the vector may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication.
  • the carrier may also include components to assist its entry into cells, such as viral particles, liposomes or protein coats, but not only these materials.
  • the vectors described herein may be selected from one or more of plasmids, retroviral vectors and lentiviral vectors.
  • the vectors described herein can be lentiviral vectors.
  • the present application also provides immune effector cells, which may comprise the nucleic acid molecule described herein or the vector described herein.
  • the cells may include progeny of a single cell. Progeny may not necessarily be identical (in morphology or in genome) to the original parent cell due to natural, accidental or intentional mutation.
  • the cells can also be mammalian cells.
  • the immune effector cells also include T lymphocytes, such as alpha/beta T lymphocytes and gamma/delta T lymphocytes.
  • the immune effector cells can be derived from human PBMCs.
  • the T lymphocytes can be CD4+ T cells or CD8+ T cells.
  • the immune effector cells include natural killer cells, natural killer T cells, mast cells, and bone marrow-derived phagocytic cells.
  • the present application also provides a method for preparing the immune effector cells described in the present application, the method may include introducing the isolated nucleic acid molecule described in the present application or the vector described in the present application into the immune effector cells.
  • compositions which may comprise the immune effector cells described herein.
  • the composition further includes, optionally, a pharmaceutically acceptable carrier.
  • compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.
  • the pharmaceutically acceptable adjuvant includes any and all solvents, dispersion media, isotonic and absorption delaying agents that are compatible with the immune effector cells, are generally safe, nontoxic, and Neither biologically nor otherwise undesirable.
  • the composition comprises parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the composition can be administered to a patient or subject by infusion or injection.
  • the pharmaceutical composition is administered by various means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the composition is administered without interruption.
  • the uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the influx of the therapeutic agent into the patient, as described in WO2015/036583.
  • the present application also provides the use of the immune effector cell, the nucleic acid molecule, the carrier, and the composition in the preparation of a medicament that can be used to prevent and/or treat diseases and/or or disease.
  • the present application also provides methods of preventing and/or treating diseases and/or disorders, which methods may comprise administering to a subject in need thereof the immune effector cells or compositions described herein.
  • the subject may include humans and non-human animals.
  • the subject can include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • the immune effector cells described in this application, the nucleic acid molecules described in this application, the vectors described in this application and/or the compositions described in this application can be used for preventing, relieving or treating tumors.
  • the disease and/or disorder is associated with CD20 expression.
  • the disease and/or disorder is associated with CLDN18.2 expression.
  • the disease and/or disorder is associated with GPC-3 expression.
  • the disease and/or disorder comprises a tumor.
  • the tumor comprises a solid tumor and/or a hematological tumor.
  • the tumor comprises a CD20 positive tumor.
  • the tumor comprises a CLDN18.2 positive tumor.
  • the tumor comprises a GPC-3 positive tumor.
  • the tumor comprises lymphoma.
  • the tumor comprises pancreatic cancer.
  • a CAR targeting CD20 (20BBZ), a CAR targeting CLDN18.2 (Ab10BBZ) and a CAR targeting GPC-3 (GC33BBZ, GC179BBZ and M3C11BBZ) were prepared, and the structures of the CARs are shown in Figure 1.
  • the following sequences were artificially synthesized: scFv 20, scFv Ab10, scFv GC33, scFv GC179, scFv M3C11, hinge region, transmembrane domain, 4-1BB costimulatory domain, CD3 ⁇ intracellular signaling domain.
  • the hinge region, transmembrane domain, 4-1BB costimulatory domain and CD3 ⁇ intracellular signaling domain are linked end to end to obtain BBZ.
  • the scFv20 that can specifically bind to CD20, the scFv Ab10 that specifically binds to CLDN18.2, and the scFv GC33, scFv GC179, scFv GC179, scFv M3C11 and BBZ that specifically bind to GPC-3 are subjected to overlap PCR, and XbaI and BamHI restriction sites are added at both ends.
  • the pCDH-MSCVEF vector was cloned.
  • Viruses containing CAR against CD20 (20BBZ virus), CAR against CLDN18.2 (Ab10BBZ virus) and CAR against GPC-3 (GC33BBZ virus, GC179BBZ virus and M3C11BBZ virus) were prepared.
  • the correctly sequenced clones were extracted with endotoxin-free NucleoBond Xtra Midi Plus EF kit, and co-transfected with lentiviral packaging plasmids (VSV-g, pMD Gag/Pol or RSV-REV) in 293 cells at 37°C, 5% CO 2 After 48 hours of culture, collect the supernatant, filter it at 0.45 ⁇ M, use a Beckman ultracentrifuge and a SW28 rotor, and centrifuge at 25,000 RPM for 2 hours to concentrate the virus, which is the virus containing pCDH-MSCVEF-20BBZ, containing pCDH- Viruses containing MSCVEF-Ab10BBZ, viruses containing pCDH-
  • FIG. 293 cells were infected with the obtained virus, and the virus titer was measured by flow cytometry using an anti-mouse Fab antibody (Jackson ImmunoResearch #115-605-006).
  • Figure 2 shows the flow detection results when 1 ⁇ L, 3 ⁇ L, and 9 ⁇ L of the virus were added, and no virus was added as a blank control. The results showed that with the increase of the added virus dose, the CAR expression levels of the CARs: 20BBZ, Ab10BBZ, GC33BBZ, GC179BBZ and M3C11BBZ also increased.
  • anti-CD20 CAR-T cells (20BBZ CAR-T cells), anti-CLDIN18.2 CAR-T cells (Ab10BBZ CAR-T cells) and anti-GPC-3 CAR-T cells (GC33BBZ CAR-T cells, GC179BBZ CAR-T cells and M3C11BBZ CAR-T cells).
  • Human PBMCs were purified by Stemcell T cell isolation kit (purchased from stem cell Catlog#19671), then inoculated into anti-hCD3 (purchased from Bioxcell#BE0001-2) and anti-hCD28 (purchased from Bioxcell#BE0248) coated cells.
  • the medium is RPMI complete medium containing 10% FBS, IL2 (50IU/ml), IL21 (4ng/ml), and artificial antigen-presenting cells (X-ray 100Gray irradiated) are used every 6 days.
  • the obtained cells are 20BBZ CAR-T cells, Ab10BBZ CAR-T cells , GC33BBZ CAR-T cells, GC179BBZ CAR-T cells and M3C11BBZ CAR-T cells, using Alexa 647 AffiniPure F(ab') 2 Fragment Goat Anti-Mouse IgG, Fab fragment specific antibody staining and flow analysis, the results are shown in Figure 3, the results show that the obtained cells are all CAR positive.
  • a CAR targeting CD20 expressing Bcl-2 (20BBZ-Bcl-2, the structure is shown in Figure 1)
  • a CAR of CLDN18.2 (Ab10BBZ-Bcl-2, the structure is shown in Figure 1) was prepared )
  • GPC-3 CARs (GC33BBZ-Bcl-2, GC179BBZ-Bcl-2 and M3C11BBZ-Bcl-2, the structure is shown in Figure 1)
  • Bcl-2-expressing anti-CD20 CAR-T virus (20BBZ- Bcl-2 virus), CAR-T virus of CLDN18.2 (Ab10BBZ-Bcl-2 virus), CAR-T virus of GPC-3 (GC33BBZ-Bcl-2 virus, GC179BBZ-Bcl-2 virus and M3C11BBZ-Bcl-2 virus) 2 virus).
  • the virus titer was measured by the method of flow detection in Example 1.
  • Figure 2 shows the flow detection results when 1 ⁇ L, 3 ⁇ L, and 9 ⁇ L of the virus were added, and no virus was added as a blank control.
  • the results showed that with the increase of the added virus dose, the CAR expression levels of the CARs: 20BBZ-Bcl-2, Ab10BBZ-Bcl-2, GC33BBZ-Bcl-2, GC179BBZ-Bcl-2 and M3C11BBZ-Bcl-2 also increased. increased accordingly.
  • Bcl-2-expressing anti-CD20 CAR-T cells (20BBZ-Bcl-2 CAR-T cells), anti-CLDIN18.2 CAR-T cells (Ab10BBZ-Bcl-2 CAR-T cells) and anti-GPC-3 CAR-T cells cells (GC33BBZ-Bcl-2 CAR-T cells, GC179BBZ-Bcl-2 CAR-T cells and M3C11BBZ-Bcl-2 CAR-T cells).
  • Human PBMC-derived T cells were purified, activated, and infected and expanded using 20BBZ-Bcl-2 virus, Ab10BBZ-Bcl-2 virus, GC33BBZ-Bcl-2 virus, GC179BBZ-Bcl-2 virus, and M3C11BBZ-Bcl-2 virus.
  • the CD20BBZ CAR-T cells prepared in Example 1 and the CD20BBZ-Bcl-2 CAR-T cells prepared in Example 2 were continuously cultured, stimulated with artificial antigen-presenting cells every 6 days, and the cells were counted. The results are shown in the figure 4 shown. It can be seen from Figure 4 that CD20BBZ-Bcl-2 CAR-T cells have stronger expansion ability than CD20BBZ CAR-T cells.
  • the Ab10BBZ CAR-T cells prepared in Example 1 and the Ab10BBZ-Bcl-2 CAR-T cells prepared in Example 2 were continuously cultured and stimulated once every 6 days with artificial antigen-presenting cells, and the cells were counted, and the results were as follows: shown in Figure 5. It can be seen from Figure 5 that Ab10BBBZ-Bcl-2 CAR-T cells have stronger in vitro expansion ability than Ab10BBZ CAR-T cells.
  • GC33BBZ CAR-T cells prepared in Example 1 and the GC33BBZ-Bcl-2 CAR-T cells prepared in Example 2 were continuously cultured and stimulated with artificial antigen-presenting cells every 6 days. The cells were counted, and the results were as follows: shown in Figure 6. It can be seen from Figure 6 that GC33BBZ-Bcl-2 CAR-T cells have stronger in vitro expansion ability than GC33BBZ CAR-T cells.
  • the 20BBZ CAR-T cells prepared in Example 1 and the 20BBZ-Bcl-2 CAR-T cells prepared in Example 2 were inoculated into a 96-well plate, according to the CAR-T:tumor cell ratio of 1:1, 0.5:1 CD20-positive tumor cells (Raji) were added at 0.25:1, and the survival of Raji was detected by flow cytometry after 24 hours.
  • 20BBZ-Bcl-2 CAR-T cells had similar in vitro tumor killing ability than 20BBZ CAR-T cells.
  • B-NDG mice were inoculated with 3x10 6 Raji cells intravenously, and 6 days later, they were treated with 10 7 CD20BBZ CAR-T cells or CD20BBZ-Bcl-2 CAR-T cells, and PBS was used as a blank control to measure T in the bone marrow of mice
  • the ratio of cells and Raji cells the results are shown in Figure 8A, respectively, the ratio of CD20BBZ-Bcl-2 CAR-T cells and CD20BBZ CAR-T cells in the bone marrow was statistically significantly different (P ⁇ 0.01).
  • CD20BBZ-Bcl-2 CAR-T cell treatment significantly reduced the proportion of Raji cells in bone marrow, and there was a statistically significant difference (P ⁇ 0.001).
  • B-NDG mice were inoculated with 3x10 6 Raji cells intravenously, and 6 days later, they were treated with 10 7 CD20BBZ CAR-T cells or CD20BBZ-Bcl-2 CAR-T cells, and PBS was given as a blank control to draw the survival curve of the mice.
  • the results are shown in Figure 8B, compared with the control group CD20BBZ CAR-T cell treatment, the mice treated with CD20BBZ-Bcl-2 CAR-T cells survived longer, up to 48 days, demonstrating that CD20BBZ-Bcl -2 CAR-T cells have significant antitumor ability in vitro.
  • Example 8 In vivo tumor killing ability of anti-CLDN18.2 Bcl-2 CAR-T cells
  • B-NDG mice were subcutaneously inoculated with 3x10 6 CFPAC-1 tumor cells, and then treated with 10 7 Ab10BBZ CAR-T cells or Ab10BBZ-Bcl-2 CAR-T cells 6 days later, and the tumor burden of the mice was measured. The results are shown in the figure As shown in 9, Ab10BBZ-Bcl-2 CAR-T cells can better control tumor burden than Ab10BBZ CAR-T cells.
  • Huh-7 tumor cells were subcutaneously inoculated into B-NDG mice, and 7 days later, 10 7 GC33BBZ CAR-T cells or GC33BBZ-Bcl-2 CAR-T cells were treated, and the tumor burden of the mice was measured.
  • the results are shown in the figure As shown in Figure 10, GC33BBZ-Bcl-2 CAR-T cells can better control tumor burden and inhibit tumor growth than GC33BBZ CAR-T cells, which proves that GC33BBZ-Bcl-2 CAR-T cells have obvious anti-tumor ability in vivo.

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Abstract

一种经改造的免疫效应细胞，该免疫效应细胞可以包含和/或表达嵌合抗原受体，以及Bcl-2蛋白或其功能活性片段，还提供了该免疫效应细胞的应用。

Description

改造的免疫效应细胞及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种经改造的CAR-T细胞及其用途。
背景技术
CLDN18属于Claudins蛋白家族成员，CLDN18.1和CLDN18.2是CLDN18的可变剪切体。在正常组织中，CLDN18.1主要表达在肺部，CLDN18.2只表达于胃粘膜上皮细胞。但是CLDN18.2在多种肿瘤组织表达，譬如胃癌，胰腺癌，食管癌，卵巢癌，肺癌等，是理想的肿瘤CAR-T治疗靶点。
CD20特异表达于B细胞或B细胞来源的白血病或淋巴瘤，已有靶向CD20的抗体药物美罗华上市，但治疗中存在耐药和复发现象。
Glypican-3(GPC-3)在正常肝组织中无表达,而在胎肝和肝癌组织高表达，是原发性肝细胞性肝癌特异标志物，并参与到肝癌的发生和发展进程，是肝癌小分子靶向治疗的靶点，也是免疫治疗的识别靶点。
过继性细胞疗法中如嵌合抗原受体T细胞(CAR-T细胞)是一种经人工修饰的肿瘤杀伤细胞，其结合了抗体的靶向识别功能和T细胞的肿瘤杀伤功能，是肿瘤免疫治疗领域的一大突破。然而，CAR-T对胃癌、胰腺癌等实体瘤的疗效尚不理想，新型CAR-T疗法治疗实体瘤的关键。
发明内容
本申请提供了一种免疫效应细胞，其包含和/或表达嵌合抗原受体(CAR)，以及Bcl-2蛋白或其功能活性片段。所述免疫效应细胞具有下述性质中的一种或多种：1)能够特异性结合抗原；2)具有较强的体外扩增能力；3)具有较强的体内抗肿瘤能力；4)具有较强的体外抗肿瘤能力。
一方面，本申请提供了一种免疫效应细胞，其包含和/或表达嵌合抗原受体(CAR)，以及Bcl-2蛋白或其功能活性片段。
在某些实施方式中，所述CAR包含抗原结合结构域，且所述抗原结合结构域包含特异性结合CD20的抗体或其抗原结合片段。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，且所述HCDR3包含SEQ  ID NO:1所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR2，且所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR1，且所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，且所述LCDR3包含SEQ ID NO:4所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR2，且所述LCDR2包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR1，且所述LCDR1包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH，所述VH包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VL，所述VL包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述CAR包含抗原结合结构域，且所述抗原结合结构域包含特异性结合CLDN18.2的抗体或其抗原结合片段。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，且所述HCDR3包含SEQ ID NO:11所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR2，且所述HCDR2包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR1，且所述HCDR1包含SEQ ID NO:13所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，且所述LCDR3包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR2，且所述LCDR2包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR1，且所述LCDR1包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH，所述VH包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VL，所述VL包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述CAR包含抗原结合结构域，且所述抗原结合结构域包含特异性结合GPC-3的抗体或其抗原结合片段。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，且所述HCDR3包含SEQ ID NO:20、SEQ ID NO:29和SEQ ID NO:38中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR2，且所述HCDR2包含SEQ ID NO:21、SEQ ID NO:30和SEQ ID NO:39中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR1，且所述HCDR1包含SEQ ID NO:22、SEQ ID NO:31和SEQ ID NO:40中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，且所述LCDR3包含SEQ ID NO:23、SEQ ID NO:32和SEQ ID NO:41中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR2，且所述LCDR2包含SEQ ID NO:24、SEQ ID NO:33和SEQ ID NO:42中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR1，且所述LCDR1包含SEQ ID NO:25、SEQ ID NO:34和SEQ ID NO:43中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH，所述VH包含SEQ ID NO:26、SEQ ID NO:35和SEQ ID NO:44中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VL，所述VL包含SEQ ID NO:27、SEQ ID NO:36和SEQ ID NO:45中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO:28、SEQ ID NO:37和SEQ ID NO:46中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体包含单链抗体。
在某些实施方式中，所述CAR包含跨膜结构域，所述跨膜结构域包含源自选自下述蛋白的跨膜结构域：CD28、CD3e、CD45、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
在某些实施方式中，所述跨膜结构域包含如SEQ ID NO:47所示的氨基酸序列。
在某些实施方式中，所述CAR包含共刺激域，且所述共刺激域包含一种或多种选自下述蛋白的共刺激域：CD28、4-1BB、CD40L、TIM1、CD226、DR3、SLAM、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD27、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT和DAP12中的共刺激信号传导区。
在某些实施方式中，所述共刺激域包含如SEQ ID NO:48所示的氨基酸序列。
在某些实施方式中，所述CAR包含胞内信号传导域，且所述胞内信号传导域包含源自CD3ζ的胞内信号传导域。
在某些实施方式中，所述胞内信号传导域包含如SEQ ID NO:49所示的氨基酸序列。
在某些实施方式中，所述CAR包含铰链区，所述铰链区位于所述抗原结合结构域和所述跨膜结构域之间。
在某些实施方式中，所述铰链区包含以下至少一项：CD8、CD28、4-1BB、CD4、CD27、CD7和PD-1的铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:50所示的氨基酸序列。
在某些实施方式中，所述CAR包含如SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59和SEQ ID NO:61中任一项所示的氨基酸序列。
在某些实施方式中，所述Bcl-2蛋白或其功能活性片段包含外源Bcl-2蛋白或其功能活性片段。
在某些实施方式中，所述Bcl-2蛋白或其功能活性片段包含SEQ ID NO:52所示的氨基酸序列。
在某些实施方式中，所述免疫效应细胞包括T细胞。
另一方面，本申请还提供了核酸分子，所述核酸分子编码所述的CAR和所述的Bcl-2蛋白或其功能活性片段。
在某些实施方式中，所述核酸分子包含编码自剪切肽的序列，所述编码自剪切肽的序列位于编码所述CAR的序列和编码所述Bcl-2蛋白的序列之间。
在某些实施方式中，所述自剪切肽包含2A肽。
在某些实施方式中，所述2A肽为选自下述的一种或多种：P2A，T2A，E2A和F2A。
在某些实施方式中，所述2A肽包含如SEQ ID NO:51所示的氨基酸序列。
另一方面，本申请还提供了载体，其包含所述的核酸分子。
在某些实施方式中，所述载体选自质粒、逆转录病毒载体和慢病毒载体中的一种或多种。
另一方面，本申请还提供了免疫效应细胞，其包含所述的核酸分子或所述的载体。
另一方面，本申请还提供了制备所述免疫效应细胞的方法，所述方法包括在使得所述嵌合抗原受体表达的条件下，培养所述免疫效应细胞。
另一方面，本申请还提供了组合物，其包含所述免疫效应细胞。
另一方面，本申请还提供了所述免疫效应细胞、所述核酸分子、所述载体或所述组合物用于制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
另一方面，本申请还提供了所述免疫效应细胞、所述核酸分子、所述载体或所述组合物，其用于预防和/或治疗疾病和/或病症。
另一方面，本申请还提供了预防和/或治疗疾病和/或病症的方法，所述方法包括向有需要的受试者施用本申请所述免疫效应细胞、所述核酸分子、所述载体或所述组合物。
在某些实施方式中，所述疾病和/或病症与CD20表达相关。
在某些实施方式中，所述疾病和/或病症与CLDN18.2表达相关。
在某些实施方式中，所述疾病和/或病症与GPC-3表达相关。
在某些实施方式中，所述疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或血液瘤。
在某些实施方式中，所述肿瘤包括CD20阳性肿瘤。
在某些实施方式中，所述肿瘤包括CLDN18.2阳性肿瘤。
在某些实施方式中，所述肿瘤包括GPC-3阳性肿瘤。
在某些实施方式中，所述肿瘤包括淋巴瘤。
在某些实施方式中，所述肿瘤包括胰腺癌。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是本申请所述嵌合抗原受体的构建示意图。
图2A-2B显示的是本申请所述CAR的表达量情况。
图3显示的是本申请所述免疫效应细胞的CAR的表达情况。
图4显示的是抗CD20 Bcl-2 CAR-T细胞的扩增能力。
图5显示的是抗CLDN18.2 Bcl-2 CAR-T细胞的扩增能力。
图6显示的是抗GPC-3 Bcl-2 CAR-T细胞的扩增能力。
图7显示的是本申请所述免疫效应细胞的体外肿瘤杀伤能力。
图8A-8B显示的是本申请所述免疫效应细胞的体内肿瘤杀伤能力。
图9显示的是抗CLDN18.2 Bcl-2 CAR-T细胞对肿瘤体积的影响。
图10显示的是抗GPC-3 Bcl-2 CAR-T细胞的体内肿瘤杀伤能力。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“免疫效应细胞”通常是指参与免疫应答，行使效应功能的免疫细胞。例如所述行使效应功能可以包括清除异物抗原或促进免疫效应子应答等。免疫效应细胞可以包括浆细胞、T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和骨髓源性吞噬细胞。本申请所述免疫效应细胞可以包括经工程化修饰的免疫效应细胞。例如，本申请所述免疫效应细胞可以包含嵌合抗原受体，同时可以表达Bcl-2蛋白或其功能活性片段。例如，所述Bcl-2蛋白为外源性Bcl-2蛋白或其功能活性片段。
在本申请中，术语“嵌合抗原受体”或“CAR”也称“嵌合受体”、“T体”、嵌合免疫受体，通常是指包含至少一个胞外抗原结合结构域，跨膜结构域和细胞质信号传导结构域(也称“胞内信号传导结构域”)的重组多肽构建体。例如，所述嵌合抗原受体可以包括靶向部分(例如，结合肿瘤相关抗原的部分)、铰链区、跨膜结构域、共刺激域和胞内信号传导域。
术语“Bcl-2”与“Bcl-2蛋白”可以互换使用，通常指bcl-2原癌基因的编码产物。在某些实施方式中，Bcl-2蛋白的氨基酸序列可以如SEQ ID NO:52所示。在本申请中，该术语涵盖全长Bcl-2蛋白及其同源物、类似物、截短体、突变体及功能活性片段。在本申请中，该术语涵盖外源Bcl-2蛋白或其功能活性片段。
在本申请中，术语“抗原结合结构域”通常指能够结合靶抗原的结构域。抗原结合结构域可以包含能(特异性)结合抗原的嵌合抗原受体及其片段、抗体或其抗原结合片段。所述抗原结合结构域可以为天然来源、合成来源、半合成来源或重组来源。在某些实施方式中， 所述抗原结合结构域可以包含抗体或其抗原结合片段。例如，所述抗原结合结构域可以包含单链抗体。
在本申请中，术语“特异性结合”或“特异性的”通常指可测量的和可再现的相互作用，例如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况可决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体可以是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更长的持续时间结合该靶标的抗体。在某些实施方案中，抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在某些实施方案中，特异性结合可以包括但不要求排他性地结合。
在本申请中，术语“抗体”通常指能够特异性识别和/或中和特定抗原的多肽分子。例如，抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链组成的免疫球蛋白，并且包括任何包含其抗原结合部分的分子。术语“抗体”包括单克隆抗体、抗体片段或抗体衍生物，包括但不限于人抗体、人源化抗体、嵌合抗体、单域抗体(例如，dAb)，单链抗体(例如，scFv)，以及与抗原结合的抗体片段(例如，Fab、Fab’和(Fab) 2片段)。术语“抗体”还包括抗体的所有重组体形式，例如在原核细胞中表达的抗体、未糖基化的抗体以及本申请所述的任何与抗原结合的抗体片段及其衍生物。每条重链可由重链可变区(VH)和重链恒定区构成。每条轻链可由轻链可变区(VL)和轻链恒定区构成。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为构架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。
在本申请中，术语“CDR”也称“互补决定区”，通常指抗体可变结构域中的区域，其序列是高度可变的和/或形成结构定义环。通常，抗体包括六个CDR；在VH中三个(HCDR1、HCDR2、HCDR3)，和在VL中三个(LCDR1、LCDR2、LCDR3)。在某些实施方案中，仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况下，其功能也能够正常且稳定。参见，例如，Hamers-Casterman et al.,Nature 363:446-448(1993)；Sheriff et al,Nature Struct.Biol.3:733-736(1996)。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，http://www.bioinf.org.uk/abs/index.html#kabatnum。例如，所述抗体的氨基酸序列编号可以按照IMGT编号方案(IMGT,the international ImMunoGeneTics information system@imgt.cines.fr；http：//imgt.cines.fr；Lefranc等,1999,Nucleic Acids Res.27:209-212；Ruiz等,2000Nucleic Acids  Res.28:219-221；Lefranc等,2001,Nucleic Acids Res.29:207-209；Lefranc等,2003,Nucleic Acids Res.31:307-310；Lefranc等,2005,DevComp Immunol 29:185-203)。例如，所述抗体的CDR可以根据Kabat编号系统确定(参见例如Kabat EA&Wu TT(1971)Ann NY AcadSci 190:382-391和Kabat EAet al.,(1991)Sequences of Proteins of Immunological Interest,FifthEdition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)。
在本申请中，术语“FR”通常指抗体可变结构域的更高度保守的部分，其被称为框架区。通常，天然重链和轻链的可变结构域各自包含四个FR区，即在VH中四个(H-FR1，H-FR2，H-FR3和H-FR4)，和在VL中四个(L-FR1，L-FR2，L-FR3和L-FR4)。
在本申请中，术语“可变结构域”与“可变区”可以互换使用，通常指抗体重链和/或轻链的一部分。重链和轻链的可变结构域可以分别称为“V H”和“V L”(或者分别称为“VH”和“VL”)。这些结构域通常是抗体的变化最大的部分(相对于相同类型的其它抗体)，且包含抗原结合位点。
在本申请中，术语“单链抗体(scFv)”通常指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白。其中，所述轻链可变区和重链可变区是邻接的(例如经由合成接头，例如短的柔性多肽接头)。所述scFv能够以单链多肽形式表达，也可以保留其所来源的完整抗体的特异性。在本申请中，所述scFv可以以任何顺序(例如相对于多肽的N末端和C末端)具有所述的VL和VH，所述scFv也可以包括VL-接头-VH或可以包括VH-接头-VL。
在本申请中，术语“跨膜结构域”通常指能够跨越细胞质膜的结构域。所述跨膜结构域一般可以包含三个结构区：N末端胞外区、中间的跨膜伸展区以及C末端胞质区。跨膜结构域可能还包含胞内区或胞质区。
在本申请中，术语“共刺激结构域”通常是指可以提供免疫共刺激分子的胞内结构域。其中所述共刺激分子可以为淋巴细胞对抗原的有效应答所需要的细胞表面分子。在某些实施方式中，所述共刺激域可以是共刺激分子的胞内部分或其截短形式。
在本申请中，术语“胞内信号传导域”通常指位于细胞内部能够传导信号的结构域。在本申请中，所述胞内信号传导结构域可以将信号传导至细胞内。该术语涵盖能够诱导效应子功能信号的胞内信号传导域及其任何截短部分。
在本申请中，术语“4-1BB”也称“CD137”，通常指一种肿瘤坏死因子(TNF)受体家族的成员，由肿瘤坏死因子受体超家族成员9(TNFRSF9)基因编码。人4-1BB的氨基酸序列可见于GenBank登录号No.AAA62478.2下。本申请所述4-1BB涵盖其同源物、类似物或突 变体。
在本申请中，所述“载体”通常是指能够在合适的宿主中自我复制的核酸分子，用以将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“质粒”通常是指细菌、酵母菌等生物中染色体或拟核以外的DNA分子，存在于细胞质中，具有自主复制能力，使其能够在子代细胞中保持恒定的拷贝数，并表达所携带的遗传信息。质粒在遗传工程研究中被用作基因的载体。
在本申请中，术语“逆转录病毒载体”通常是指可以可控并表达外源基因，但不能自我包装成有增殖能力的病毒颗粒。此类病毒多具有反转录酶。反转录病毒至少含有三种基因：gag，包含组成病毒中心和结构的蛋白质的基因；pol，包含反转录酶的基因和env，包含组成病毒外壳的基因。通过逆转录病毒转染，逆转录病毒载体可将自身基因组及其携带的外源基因随机、稳定地整合入宿主细胞基因组中，例如，可将CAR分子整合进宿主细胞中。
在本申请中，术语“慢病毒载体”通常是指属于逆转录病毒的一种二倍体RNA病毒载体。慢病毒载体是以慢病毒的基因组为基础，将其中多个和病毒活性相关的序列结构去除，使其具有生物学的安全性，然后再在这个基因组骨架中引入实验所需要的目标基因的序列和表达结构，并将之制备成载体。通过慢病毒载体转染，逆转录病毒载体可将自身基因组及其携带的外源基因随机、稳定地整合入宿主细胞基因组中，例如，可将CAR分子整合进宿主细胞中。
术语“肿瘤”通常指任何新的病理性的组织增生。肿瘤细胞可以局部地或通过血流和淋巴系统扩散到身体其他部分。在本申请中，所述肿瘤可以包括良性肿瘤和恶性肿瘤。在本申请中，所述肿瘤可以包括实体瘤和/或血液瘤。在本申请中，所述肿瘤可以包括癌症。在本申请中，所述肿瘤的实例包括但不限于：脑胶质瘤、乳腺癌、黑色素瘤、非小细胞肺癌、膀胱癌、卵巢癌和结直肠癌。
在本申请中，涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。
在本申请中，所述变体可以为，例如在所述蛋白质和/或所述多肽(例如，特异性结合CD73蛋白的抗体或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1- 10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述同源物可以为与所述蛋白质和/或所述多肽(例如，特异性结合CD73蛋白的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
免疫效应细胞
一方面，本申请提供一种免疫效应细胞，其可以包含和/或表达嵌合抗原受体(CAR)，所述免疫效应细胞还可以包含和/或表达Bcl-2蛋白或其功能活性片段。所述Bcl-2蛋白或其功能活性片段可以是外源(例如，人工合成以下序列：铰链区，跨膜结构域，共刺激域，胞内信号传导域，剪切肽和Bcl-2或其功能活性片段，将各部分连接后加入scFv，分子克隆，生产病毒载体，感染免疫效应细胞进行表达)引入所述免疫效应细胞中的。例如，所述免疫效应细胞(例如T细胞)可以是经工程化改造或经修饰的免疫效应细胞(例如T细胞)，其中所述工程化改造可以包括向该免疫效应细胞(例如，源自受试者的其他天然来源的免疫效应细胞)中引入(例如，通过构建具有编码抗原结合蛋白、铰链区、跨膜结构域、共刺激域、胞内信号传导域、剪切肽、Bcl-2的核酸序列，通过病毒载体感染免疫效应细胞表达)所述CAR和/或所述Bcl-2蛋白，和/或表达所述CAR和/或所述Bcl-2蛋白的核酸分子。在某些实施方式中，所述CAR包含抗原结合结构域、跨膜结构域、共刺激域、胞内信号传导域和/或铰链区。在某些实施方式中，所述免疫效应细胞过表达Bcl-2蛋白或其功能活性片段。
在某些实施方式中，所述CAR包含抗原结合结构域，且该抗原结合结构域包含靶向CD20的抗体或其抗原结合片段。
在某些实施方式中，所述靶向CD20的抗体或其抗原结合片段包含HCDR3，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含HCDR2，且所述HCDR2包含SEQ ID NO:2所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含HCDR1，且所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，且所述LCDR3包含SEQ ID NO:4所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含LCDR2，且所述LCDR2包含SEQ ID NO:5所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含LCDR1，且所述LCDR1包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，HCDR2和HCDR1，所述HCDR3包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，且所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，LCDR2和LCDR1，所述LCDR3包含SEQ ID NO:4所示的氨基酸序列，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列，且所述LCDR1包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，HCDR2，HCDR1，LCDR3，LCDR2和LCDR1，所述HCDR3包含SEQ ID NO:1所示的氨基酸序列，所述HCDR2包含SEQ ID NO:2所示的氨基酸序列，所述HCDR1包含SEQ ID NO:3所示的氨基酸序列，所述LCDR3包含SEQ ID NO:4所示的氨基酸序列，所述LCDR2包含SEQ ID NO:5所示的氨基酸序列，且所述LCDR1包含SEQ ID NO:6所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH，所述VH可包含SEQ ID NO:7所示的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含VL，所述VL可包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH和VL，所述VH包含如SEQ ID NO:7所示的氨基酸序列且所述VL包含如SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述CAR包含抗原结合结构域，且该抗原结合结构域包含靶向CLDN18.2的抗体或其抗原结合片段。
在某些实施方式中，所述靶向CLDN18.2的抗体或其抗原结合片段包含HCDR3，且所述HCDR3包含SEQ ID NO:11所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含HCDR2，且所述HCDR2包含SEQ ID NO:12所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含HCDR1，且所述HCDR1包含SEQ ID NO:13所示的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，且所述LCDR3包含SEQ ID NO:14所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含LCDR2，且所述LCDR2包含SEQ ID NO:15所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含LCDR1，且所述LCDR1包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，HCDR2和HCDR1，所述HCDR3包含SEQ ID NO:11所示的氨基酸序列，所述HCDR2包含SEQ ID NO:12所示的氨基酸序列，且所述HCDR1包含SEQ ID NO:13所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，LCDR2和LCDR1，所述LCDR3包含SEQ ID NO:14所示的氨基酸序列，所述LCDR2包含SEQ ID NO:15所示的氨基酸序列，且所述LCDR1包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，HCDR2，HCDR1，LCDR3，LCDR2和LCDR1，所述HCDR3包含SEQ ID NO:11所示的氨基酸序列，所述HCDR2包含SEQ ID NO:12所示的氨基酸序列，所述HCDR1包含SEQ ID NO:13所示的氨基酸序列，所 述LCDR3包含SEQ ID NO:14所示的氨基酸序列，所述LCDR2包含SEQ ID NO:15所示的氨基酸序列，且所述LCDR1包含SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH，所述VH可包含SEQ ID NO:17所示的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含VL，所述VL包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH和VL，所述VH包含如SEQ ID NO:17所示的氨基酸序列且所述VL包含如SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述CAR包含抗原结合结构域，且该抗原结合结构域包含靶向GPC-3的抗体或其抗原结合片段。
在某些实施方式中，所述靶向GPC-3的抗体或其抗原结合片段包含HCDR3，且所述HCDR3包含SEQ ID NO:20所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含HCDR2，且所述HCDR2包含SEQ ID NO:21所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含HCDR1，且所述HCDR1包含SEQ ID NO:22所示的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，且所述LCDR3包含SEQ ID NO:23所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含LCDR2，且所述LCDR2包含SEQ ID NO:24所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含LCDR1，且所述LCDR1包含SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，HCDR2和HCDR1，所述HCDR3包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR1包含SEQ ID NO:22所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，LCDR2和LCDR1，所述LCDR3包含SEQ ID NO:23所示的氨基酸序列，所述LCDR2包含SEQ ID NO:24所示的氨基酸序列，且所述LCDR1包含SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，HCDR2，HCDR1，LCDR3，LCDR2和LCDR1，所述HCDR3包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，所述HCDR1包含SEQ ID NO:22所示的氨基酸序列，所述LCDR3包含SEQ ID NO:23所示的氨基酸序列，所述LCDR2包含SEQ ID NO:24所示的氨基酸序列，且所述LCDR1包含SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH，所述VH包含SEQ ID NO:26 所示的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含VL，所述VL包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH和VL，所述VH包含如SEQ ID NO:26所示的氨基酸序列且所述VL包含如SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO:28所示的氨基酸序列。
在某些实施方式中，所述靶向GPC-3的抗体或其抗原结合片段包含HCDR3，且所述HCDR3包含SEQ ID NO:29所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含HCDR2，且所述HCDR2包含SEQ ID NO:30所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含HCDR1，且所述HCDR1包含SEQ ID NO:31所示的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，且所述LCDR3包含SEQ ID NO:32所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含LCDR2，且所述LCDR2包含SEQ ID NO:33所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含LCDR1，且所述LCDR1包含SEQ ID NO:34所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，HCDR2和HCDR1，所述HCDR3包含SEQ ID NO:29所示的氨基酸序列，所述HCDR2包含SEQ ID NO:30所示的氨基酸序列，且所述HCDR1包含SEQ ID NO:31所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，LCDR2和LCDR1，所述LCDR3可以包含SEQ ID NO:32所示的氨基酸序列，所述LCDR2包含SEQ ID NO:33所示的氨基酸序列，且所述LCDR1包含SEQ ID NO:34所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，HCDR2，HCDR1，LCDR3，LCDR2和LCDR1，所述HCDR3包含SEQ ID NO:29所示的氨基酸序列，所述HCDR2包含SEQ ID NO:30所示的氨基酸序列，所述HCDR1包含SEQ ID NO:31所示的氨基酸序列，所述LCDR3包含SEQ ID NO:32所示的氨基酸序列，所述LCDR2包含SEQ ID NO:33所示的氨基酸序列，且所述LCDR1包含SEQ ID NO:34所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH，所述VH包含SEQ ID NO:35所示的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含VL，所述VL包含SEQ ID NO:36所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH和VL，所述VH包含如SEQ ID NO:35所示的氨基酸序列且所述VL包含如SEQ ID NO:36所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO:37所示的氨基酸序列。
在某些实施方式中，所述靶向GPC-3的抗体或其抗原结合片段可以包含HCDR3，且所述HCDR3包含SEQ ID NO:38所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含HCDR2，且所述HCDR2包含SEQ ID NO:39所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含HCDR1，且所述HCDR1包含SEQ ID NO:40所示的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，且所述LCDR3包含SEQ ID NO:41所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含LCDR2，且所述LCDR2包含SEQ ID NO:42所示的氨基酸序列；在某些实施方式中，所述抗体或其抗原结合片段包含LCDR1，且所述LCDR1包含SEQ ID NO:43所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，HCDR2和HCDR1，所述HCDR3可以包含SEQ ID NO:38所示的氨基酸序列，所述HCDR2包含SEQ ID NO:39所示的氨基酸序列，且所述HCDR1包含SEQ ID NO:40所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含LCDR3，LCDR2和LCDR1，所述LCDR3包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR1包含SEQ ID NO:43所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含HCDR3，HCDR2，HCDR1，LCDR3，LCDR2和LCDR1，所述HCDR3包含SEQ ID NO:38所示的氨基酸序列，所述HCDR2包含SEQ ID NO:39所示的氨基酸序列，所述HCDR1包含SEQ ID NO:40所示的氨基酸序列，所述LCDR3包含SEQ ID NO:41所示的氨基酸序列，所述LCDR2包含SEQ ID NO:42所示的氨基酸序列，且所述LCDR1包含SEQ ID NO:43所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH，所述VH包含SEQ ID NO:44所示的氨基酸序列。在某些实施方式中，所述抗体或其抗原结合片段包含VL，所述VL包含SEQ ID NO:45所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含VH和VL，所述VH包含如SEQ ID NO:44所示的氨基酸序列且所述VL包含如SEQ ID NO:45所示的氨基酸序列。
在某些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO:46所示的氨基酸序列。
在本申请中，所述CAR的抗原结合结构域可以包括特异性结合CD20、CLDN18.2或GPC-3的抗体或其抗原结合片段。例如，本申请所述的抗体或其抗原结合片段可以包括但不限于重组抗体、单克隆抗体、人抗体、人源化抗体、嵌合抗体、双特异性抗体、单链抗体、双抗体、三抗体、四抗体、Fv片段，scFv片段、Fab片段、Fab’片段、F(ab’) 2片段。
在本申请中，所述抗原结合片段可以包括Fab、Fab’、F(ab’)2、F(ab) 2、Fv片段、scFv、di-scFv和/或dAb。
在本申请中，所述抗原结合结构域可以是单链抗体。例如，所述CD20结合结构域为scFv。scFv可包含SEQ ID NO:10所示的序列。例如，CD20结合结构域可包含抗体的VH，VL和连接肽。例如，所述连接肽包含SEQ ID NO:9所示的氨基酸序列。
例如，所述CLDN18.2结合结构域为scFv。scFv可包含SEQ ID NO:19所示的序列。例如，CLDN18.2结合结构域可包含抗体的VH，VL和连接肽。例如，所述连接肽包含SEQ ID NO:9所示的氨基酸序列。
例如，所述GPC-3结合结构域为scFv。scFv可包含SEQ ID NO:28所示的序列。例如，GPC-3结合结构域可包含抗体的VH，VL和连接肽。例如，所述连接肽包含SEQ ID NO:9所示的氨基酸序列。
例如，所述GPC-3结合结构域为scFv。scFv可包含SEQ ID NO:37所示的序列。例如，GPC-3结合结构域可包含抗体的VH，VL和连接肽。例如，所述连接肽包含SEQ ID NO:9所示的氨基酸序列。
例如，所述GPC-3结合结构域为scFv。scFv可包含SEQ ID NO:46所示的序列。例如，GPC-3结合结构域可包含抗体的VH，VL和连接肽。例如，所述连接肽包含SEQ ID NO:9所示的氨基酸序列。
在本申请中，所述CAR可以包含跨膜结构域，该跨膜结构域可以包含选自下述蛋白的跨膜结构域：CD28、CD3e、CD45、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。在某些实施方式中，所述跨膜结构域包含SEQ ID NO:47所示的氨基酸序列。
在本申请中，所述CAR可以包含共刺激域，该共刺激域可以包含选自下述蛋白的共刺激域：CD28、4-1BB、CD40L、TIM1、CD226、DR3、SLAM、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD27、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT和DAP12中的共刺激信号传导区。在某些实施方式中，所述共刺激域包含4-1BB的共刺激域。在某些实施方式中，所述共刺激域包含SEQ ID NO:48所示的氨基酸序列。
在本申请中，所述CAR可以包含胞内信号传导域，该胞内信号传导域可以包含源自CD3ζ的信号传导域。例如，所述胞内信号传导域可以包含SEQ ID NO:49所示的氨基酸序列。
在本申请中，所述CAR可以从N端到C端依次包含抗原结合结构域、跨膜结构域、共刺激域以及胞内信号传导域。例如，所述抗原结合结构域可以包含SEQ ID NO:10、SEQ ID  NO:19、SEQ ID NO:28、SEQ ID NO:37或SEQ ID NO:46所示的氨基酸序列、所述跨膜结构域可以包含SEQ ID NO:47所示的氨基酸序列、所述共刺激域可以包含SEQ ID NO:48所示的氨基酸序列、所述胞内信号传导域可以包含SEQ ID NO:49所示的氨基酸序列。
在本申请中，所述CAR还可以包含铰链区，所述铰链区连接所述抗原结合结构域和所述跨膜结构域。例如，所述铰链区来源于IgG家族。例如，所述铰链区来源于IgG1。例如，所述铰链区来源于IgG4。例如，所述铰链区来源于IgD。例如，所述铰链区来源于CD8。例如，所述铰链区可以包含SEQ ID NO:50所示的氨基酸序列。
在本申请中，所述CAR还可以连接信号肽，例如，所述信号肽可以来源于CD8。例如，所述信号肽可以为CD8α。所述信号肽连接于所述抗原结合结构域的N端。
在本申请中，所述免疫效应细胞还可包含和/或表达Bcl-2蛋白或其功能活性片段。在某些实施方式中，所述Bcl-2蛋白或其功能活性片段包含SEQ ID NO:52所示的氨基酸序列。
在某些实施方式中，所述Bcl-2蛋白是外源引进的，例如，在编码所述嵌合抗原受体的核苷酸序列中加入剪切肽和编码Bcl-2蛋白或其功能活性片段的序列，使得Bcl-2蛋白或其功能活性片段在免疫效应细胞中表达。
在某些实施方式中，所述经改造的免疫效应细胞，其中对免疫效应细胞的改造包括使免疫效应细胞包含和/或表达嵌合抗原受体，以及，向免疫效应细胞中引入外源Bcl-2蛋白或其功能活性片段。
在本申请中，所述Bcl-2的功能活性片段包含能够使Bcl-2蛋白发挥抗凋亡功能的片段，例如，所述Bcl-2蛋白的功能活性片段包含其BH4结构域。
在某些实施方式中，所述BBZ包含铰链区、跨膜结构域、共刺激域、胞内信号传导域。
在某些实施方式中，所述20BBZ包含靶向CD20的CAR的序列。所述20BBZ包含SEQ ID NO:54所示的氨基酸序列。所述20BBZ-Bcl-2包含能够表达靶向CD20的CAR和Bcl-2蛋白或其功能活性片段的序列。所述20BBZ-Bcl-2包含SEQ ID NO:53所示的氨基酸序列。
在某些实施方式中，所述Ab10BBZ包含靶向CLDN18.2的CAR的序列。所述Ab10BBZ包含SEQ ID NO:56所示的氨基酸序列。所述Ab10BBZ-Bcl-2包含能够表达靶向CLDN18.2的CAR和Bcl-2蛋白或其功能活性片段的序列。所述Ab10BBZ-Bcl-2包含SEQ ID NO:55所示的氨基酸序列。
在某些实施方式中，所述GC33BBZ包含靶向GPC-3的CAR的序列。所述GC33BBZ包含SEQ ID NO:58所示的氨基酸序列。所述GC33BBZ-Bcl-2包含能够表达靶向GPC-3的CAR和Bcl-2蛋白或其功能活性片段的序列。所述GC33BBZ-Bcl-2包含SEQ ID NO:57所示的氨 基酸序列。
在某些实施方式中，所述GC179BBZ包含靶向GPC-3的CAR的序列。所述GC179BBZ包含SEQ ID NO:60所示的氨基酸序列。所述GC179BBZ-Bcl-2包含能够表达靶向GPC-3的CAR和Bcl-2蛋白或其功能活性片段的序列。所述GC179BBZ-Bcl-2包含SEQ ID NO:59所示的氨基酸序列。
在某些实施方式中，所述M3C11BBZ包含靶向GPC-3的CAR的序列。所述M3C11BBZ包含SEQ ID NO:62所示的氨基酸序列。所述M3C11BBZ-Bcl-2包含能够表达靶向GPC-3的CAR和Bcl-2蛋白或其功能活性片段的序列。所述M3C11BBZ-Bcl-2包含SEQ ID NO:61所示的氨基酸序列。
在本申请中，所述CAR包含SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59和SEQ ID NO:61中任一项所述的氨基酸序列。
核酸分子、载体、细胞、制备方法、组合物
另一方面，本申请还提供了分离的一种或多种核酸分子，其可以编码本申请所述的CAR。本申请所述的分离的一种或多种核酸分子可以为任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物，但可以编码本申请所述的CAR。
在本申请中，所述核酸分子编码所述嵌合抗原受体和所述Bcl-2蛋白或其功能活性片段。
在本申请中，所述核酸分子还包含编码剪切肽的核苷酸序列。例如，所述剪切肽可以是2A肽。例如，所述剪切肽可以选自P2A，T2A，E2A和F2A中的一种或多种。例如，所述剪切肽可以包含SEQ ID NO:51所示的氨基酸序列。
在某些实施方式中，所述核酸分子从5’端到3’端依次包含：编码抗原结合结构域的序列、编码跨膜结构域的序列、编码共刺激域的序列、编码胞内信号传导域的序列、编码2A肽的序列和编码Bcl-2蛋白或其功能活性片段的序列。
例如，所述核酸分子从5’端到3’端可依次包含编码靶向CD20的scFv、4-1BB、CD3ζ、2A、Bcl-2的核苷酸序列。所述各个部分的核酸序列可以直接或间接连接。所述间接连接可以通过接头连接。
例如，所述核酸分子从5’端到3’端可依次包含编码靶向CLDN18.2的scFv、4-1BB、CD3ζ、2A、Bcl-2的核苷酸序列。所述各个部分的核酸序列可以直接或间接连接。所述间接连接可以通过接头连接。
例如，所述核酸分子从5’端到3’端可依次包含编码靶向GPC-3的scFv、4-1BB、CD3ζ、 2A、Bcl-2的核苷酸序列。所述各个部分的核苷酸序列可以直接或间接连接。所述间接连接可以通过接头连接。例如，所述编码所述靶向GPC-3的scFv的核苷酸序列可以为实施例中提供的编码GC33scFv、GC179scFv或M3C11scFv的核苷酸序列。
另一方面，本申请还提供了载体，其可以包含本申请所述的核酸分子。所述载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内得以表达。例如，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40).又例如，所述载体可以含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，所述载体还可以含有复制起始位点。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。例如，本申请所述载体可以选自质粒、逆转录病毒载体和慢病毒载体中的一种或多种。例如，本申请所述载体可以为慢病毒载体。
另一方面，本申请还提供了免疫效应细胞，其可以包含本申请所述的核酸分子或本申请所述的载体。所述细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。例如，所述细胞还可以为哺乳动物细胞。在某些实施方式中，所述免疫效应细胞还包括T淋巴细胞，例如α/βT淋巴细胞和γ/δT淋巴细胞。例如所述免疫效应细胞可以为人PBMC来源。例如，所述T淋巴细胞可以为CD4+T细胞或CD8+T细胞。在某些实施方式中，所述免疫效应细胞包括自然杀伤细胞、自然杀伤T细胞、肥大细胞和骨髓源性吞噬细胞。
另一方面，本申请还提供了制备本申请所述的免疫效应细胞的方法，所述方法可以包括向免疫效应细胞中引入本申请所述的分离的核酸分子或本申请所述的载体。
另一方面，本申请还提供了组合物，其可以包含本申请所述的免疫效应细胞。在某些实施方式中，所述组合物还包括任选地药学上可接受的载剂。
在某些实施方式中，所述组合物的可接受成分在所用剂量和浓度下对接受者无毒。本发明的药物组合物包括但不限于液体、冷冻和冻干组合物。
在某些实施方式中，所述药学上可接受的佐剂包括与所述免疫效应细胞相容的任何和所有的溶剂、分散介质、等渗剂和吸收延迟剂，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。
在某些实施方式中，所述组合物包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述组合物可以通过输注或注射施用于患者或者受试者。在某些实施方式中，所述药物组合物的施用通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。在某些实施方式中，所述组合物不间断施用。所述不间断(或连续)施用可以通过患者佩戴的小泵系统来实现，以测量流入患者体内的治疗剂，如WO2015/036583所述。
用途和应用
另一方面，本申请还提供了所述免疫效应细胞、所述核酸分子、所述载体、所述组合物在制备药物中的用提，所述药物可以用于预防和/或治疗疾病和/或病症。
另一方面，本申请还提供了预防和/或治疗疾病和/或病症的方法，所述方法可以包括向有需要的受试者施用本申请所述的免疫效应细胞或组合物。
在本申请中，所述受试者可以包括人类和非人类动物。例如，所述受试者可以包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
另一方面，本申请所述的免疫效应细胞、本申请所述的核酸分子、本申请所述载体和/或本申请所述的组合物，其可以用于预防、缓解或治疗肿瘤。
在某些实施方式中，所述疾病和/或病症与CD20表达相关。
在某些实施方式中，所述疾病和/或病症与CLDN18.2表达相关。
在某些实施方式中，所述疾病和/或病症与GPC-3表达相关。
在某些实施方式中，所述疾病和/或病症包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或血液瘤。
在某些实施方式中，所述肿瘤包括CD20阳性肿瘤。
在某些实施方式中，所述肿瘤包括CLDN18.2阳性肿瘤。
在某些实施方式中，所述肿瘤包括GPC-3阳性肿瘤。
在某些实施方式中，所述肿瘤包括淋巴瘤。
在某些实施方式中，所述肿瘤包括胰腺癌。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的免疫效应细胞、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1抗CD20CAR-T细胞、抗CLDN18.2 CAR-T细胞和抗GPC-3 CAR-T细胞的制备
制备靶向CD20的CAR(20BBZ)，靶向CLDN18.2的CAR(Ab10BBZ)和靶向GPC-3的CAR(GC33BBZ，GC179BBZ和M3C11BBZ)，所述CAR的结构如图1所示。人工合成以下序列：scFv 20，scFv Ab10，scFv GC33，scFv GC179，scFv M3C11，铰链区，跨膜结构域，4-1BB共刺激域，CD3ζ胞内信号传导结构域。其中，将铰链区，跨膜结构域，4-1BB共刺激域和CD3ζ胞内信号传导结构域首尾相连可获得BBZ。将可特异性结合CD20的scFv20，特异性结合CLDN18.2的scFv Ab10，特异性结合GPC-3的scFv GC33、scFv GC179和scFv M3C11和BBZ通过overlap PCR，两端加入XbaI和BamHI酶切位点克隆pCDH-MSCVEF载体。进行PCR扩增，并用延伸PCR在5’端依次带上XbaI酶切位点(含保护碱基)、scFv20/scFv Ab10/scFv GC33/scFv scFv GC179/scFv M3C11、铰链区、跨膜结构域、4-1BB共刺激域、CD3ζ胞内信号传导结构域、BamHI酶切位点，PCR扩增得到所述CAR：20BBZ，Ab10BBZ，GC33BBZ，GC179BBZ和M3C11BBZ。
制备含有抗CD20的CAR的病毒(20BBZ病毒)，含有抗CLDN18.2的CAR的病毒(Ab10BBZ病毒)和含有抗GPC-3的CAR的病毒(GC33BBZ病毒，GC179BBZ病毒和M3C11BBZ病毒)。将测序正确的克隆用NucleoBond Xtra Midi Plus EF试剂盒无内毒素大提，和慢病毒包装质粒(VSV-g，pMD Gag/Pol或RSV-REV)共同转染293细胞，37℃、5％CO 2培养48小时后收取上清，0.45μM过滤后，使用贝克曼超速离心机和SW28转头，以25000RPM的速度离心2小时以浓缩病毒，即为含有pCDH-MSCVEF-20BBZ的病毒，含有pCDH-MSCVEF-Ab10BBZ的病毒，含有pCDH-MSCVEF-GC33BBZ的病毒，含有pCDH-MSCVEF-GC179BBZ的病毒和含有pCDH-MSCVEF-M3C11BBZ的病毒(简称20BBZ病毒，Ab10BBZ病毒，GC33BBZ病毒，GC179BBZ病毒和M3C11BBZ病毒)，用于后续CAR-T细胞生产。将所得病毒感染293细胞，使用anti-mouse Fab抗体(Jackson ImmunoResearch#115-605-006)用流式检测的方法测病毒滴度。图2中显示的是在添加1μL、3μL、9μL的所述病毒的时候的流式检测结果，以不添加病毒作为空白对照。结果显示，随着所加病毒剂量的增加，所述CAR：20BBZ，Ab10BBZ，GC33BBZ，GC179BBZ和M3C11BBZ的CAR表达量也随之增加。
制备抗CD20的CAR-T细胞(20BBZ CAR-T细胞)，抗CLDIN18.2CAR-T细胞(Ab10BBZ CAR-T细胞)和抗GPC-3CAR-T细胞(GC33BBZ CAR-T细胞，GC179BBZ CAR-T细胞和 M3C11BBZ CAR-T细胞)。将人PBMC经过Stemcell T细胞分离试剂盒(购自stem cell Catlog#19671)纯化后，接种到anti-hCD3(购自Bioxcell#BE0001-2)和anti-hCD28(购自Bioxcell#BE0248)包被的96孔培养板，2天后，按照MOI(感染复数，即病毒量与细胞数的比值)＝10-20感染本实施例制备的20BBZ病毒，Ab10BBZ病毒，GC33BBZ病毒，GC179BBZ病毒和M3C11BBZ病毒，1天后换液继续细胞培养，培养基为含10％FBS的RPMI完全培养基，IL2(50IU/ml)，IL21(4ng/ml)，按照每6天使用人工抗原呈递细胞(X射线100Gray辐照后的Raji-CLDN18.2细胞)或anti-hCD3(0.1μg/ml)或anti-hCD28(0.25μg/ml)刺激，经过2轮刺激后，所得细胞即为20BBZ CAR-T细胞，Ab10BBZ CAR-T细胞，GC33BBZ CAR-T细胞，GC179BBZ CAR-T细胞和M3C11BBZ CAR-T细胞，使用Alexa
Figure PCTCN2022070012-appb-000001
647 AffiniPure F(ab') 2Fragment Goat Anti-Mouse IgG,Fab fragment specific抗体染色和流式分析，其结果如图3所示，结果显示，所得细胞均为CAR阳性。
实施例2抗CD20 Bcl-2 CAR-T细胞、抗CLDN18.2 Bcl-2 CAR-T细胞和抗GPC-3 Bcl-2 CAR-T细胞的制备
按照实施例1的方法制备表达Bcl-2的靶向CD20的CAR(20BBZ-Bcl-2，结构如图1所示)，CLDN18.2的CAR(Ab10BBZ-Bcl-2，结构如图1所示)，GPC-3的CAR(GC33BBZ-Bcl-2、GC179BBZ-Bcl-2和M3C11BBZ-Bcl-2，结构如图1所示)，和表达Bcl-2的抗CD20的CAR-T病毒(20BBZ-Bcl-2病毒)，CLDN18.2的CAR-T病毒(Ab10BBZ-Bcl-2病毒)，GPC-3的CAR-T病毒(GC33BBZ-Bcl-2病毒、GC179BBZ-Bcl-2病毒和M3C11BBZ-Bcl-2病毒)。
将20BBZ，Ab10BBZ，GC33BBZ，GC179BBZ和M3C11BBZ去除终止密码子，连接片段2A、Bcl-2，通过overlap PCR，分子克隆，及病毒生产获得pCDH-MSCVEF-20BBZ-Bcl-2病毒、pCDH-MSCVEF-Ab10BBZ-Bcl-2病毒、pCDH-MSCVEF-GC33BBZ-Bcl-2病毒、pCDH-MSCVEF-GC179BBZ-Bcl-2病毒和pCDH-MSCVEF-M3C11BBZ-Bcl-2病毒(简称20BBZ-Bcl-2病毒、Ab10BBZ-Bcl-2病毒、GC33BBZ-Bcl-2病毒、GC179BBZ-Bcl-2病毒和M3C11BBZ-Bcl-2病毒)。用实施例1中流式检测的方法测病毒滴度。图2显示的是在添加1μL、3μL、9μL的所述病毒的时候的流式检测结果，以不添加病毒作为空白对照。结果显示，随着所加病毒剂量的增加，所述CAR：20BBZ-Bcl-2、Ab10BBZ-Bcl-2、GC33BBZ-Bcl-2、GC179BBZ-Bcl-2和M3C11BBZ-Bcl-2的CAR表达量也随之增加。
制备表达Bcl-2的抗CD20的CAR-T细胞(20BBZ-Bcl-2 CAR-T细胞)，抗CLDIN18.2CAR-T细胞(Ab10BBZ-Bcl-2 CAR-T细胞)和抗GPC-3CAR-T细胞(GC33BBZ-Bcl-2 CAR-T细 胞，GC179BBZ-Bcl-2 CAR-T细胞和M3C11BBZ-Bcl-2 CAR-T细胞)。将人PBMC来源的T细胞经过纯化，活化，使用20BBZ-Bcl-2病毒、Ab10BBZ-Bcl-2病毒、GC33BBZ-Bcl-2病毒、GC179BBZ-Bcl-2病毒、M3C11BBZ-Bcl-2病毒感染和扩增后，分别获得20BBZ-Bcl-2 CAR-T细胞，Ab10BBZ-Bcl-2 CAR-T细胞，GC33BBZ-Bcl-2 CAR-T细胞，GC179BBZ-Bcl-2 CAR-T细胞和M3C11BBZ-Bcl-2 CAR-T细胞，通过流式染色，用Alexa
Figure PCTCN2022070012-appb-000002
647 AffiniPure F(ab') 2Fragment Goat Anti-Mouse IgG,Fab fragment specific抗体染色，其结果如图3所示。结果显示，所得细胞均为CAR阳性。
实施例3抗CD20 Bcl-2 CAR-T细胞的扩增能力
将实施例1制备所得的CD20BBZ CAR-T细胞，实施例2制备所得的CD20BBZ-Bcl-2 CAR-T细胞连续培养，每隔6天用人工抗原呈递细胞刺激一次，细胞计数，其结果如图4所示。由图4可知，CD20BBZ-Bcl-2 CAR-T细胞相比CD20BBZ CAR-T细胞有更强的扩增能力。
实施例4抗CLDN18.2 Bcl-2 CAR-T细胞的扩增能力
将实施例1制备所得的Ab10BBZ CAR-T细胞和实施例2制备所得的Ab10BBZ-Bcl-2 CAR-T细胞，连续培养，每隔6天用人工抗原呈递细胞刺激一次，细胞计数，其结果如图5所示。由图5可知，Ab10BBBZ-Bcl-2 CAR-T细胞相比Ab10BBZ CAR-T细胞有着更强的体外扩增能力。
实施例5抗GPC-3Bcl-2 CAR-T细胞的扩增能力
将实施例1制备所得的GC33BBZ CAR-T细胞和实施例2制备所得的GC33BBZ-Bcl-2 CAR-T细胞，连续培养，每隔6天用人工抗原呈递细胞刺激一次，细胞计数，其结果如图6所示。由图6可知，GC33BBZ-Bcl-2 CAR-T细胞相比GC33BBZ CAR-T细胞有着更强的体外扩增能力。
实施例6抗CD20 Bcl-2 CAR-T细胞的体外肿瘤杀伤能力
将实施例1制备所得的20BBZ CAR-T细胞，实施例2制备所得的20BBZ-Bcl-2 CAR-T细胞，接种到96孔板，按照CAR-T：肿瘤细胞比例1:1，0.5:1和0.25:1加入CD20阳性肿瘤细胞(Raji)，24小时后流式细胞仪检测Raji的存活。对体外肿瘤杀伤的效果检测如图7所示，20BBZ-Bcl-2 CAR-T细胞比20BBZ CAR-T细胞有着相似的体外肿瘤杀伤能力。
实施例7抗CD20 Bcl-2 CAR-T细胞的体内肿瘤杀伤能力
将3x10 6Raji细胞静脉注射接种到B-NDG小鼠，6天后给予10 7CD20BBZ CAR-T细胞或CD20BBZ-Bcl-2 CAR-T细胞治疗，给予PBS作为空白对照，测量小鼠的骨髓中T细胞和Raji细胞的比例，其结果分别如图8A所示，在骨髓中CD20BBZ-Bcl-2 CAR-T细胞与CD20BBZ CAR-T细胞的比例具有显著性统计学差异(P<0.01)。与对照组CD20BBZ CAR-T细胞治疗相比，CD20BBZ-Bcl-2 CAR-T细胞治疗使骨髓中Raji细胞比例显著减少，且具有显著性统计学差异(P<0.001)。
将3x10 6Raji细胞静脉注射接种到B-NDG小鼠，6天后给予10 7CD20BBZ CAR-T细胞或CD20BBZ-Bcl-2 CAR-T细胞治疗，给予PBS作为空白对照，绘制小鼠的生存曲线，其结果如图8B所示，与对照组CD20BBZ CAR-T细胞治疗相比，CD20BBZ-Bcl-2 CAR-T细胞治疗后的小鼠生存时间更长，最长可达48天，证明CD20BBZ-Bcl-2 CAR-T细胞在体外有着显著的抗肿瘤能力。
实施例8抗CLDN18.2 Bcl-2 CAR-T细胞的体内肿瘤杀伤能力
将3x10 6CFPAC-1肿瘤细胞皮下接种到B-NDG小鼠，6天后给予10 7Ab10BBZ CAR-T细胞或Ab10BBZ-Bcl-2 CAR-T细胞治疗，测量小鼠的肿瘤负荷，其结果如图9所示，Ab10BBZ-Bcl-2 CAR-T细胞相对Ab10BBZ CAR-T细胞能够更好的控制肿瘤负荷。结果显示，Ab10BBZ-Bcl-2 CAR-T细胞治疗小鼠的肿瘤在第16天后开始缩小，而对照组Ab10BBZ CAR-T细胞治疗的肿瘤却不断快速生长，证明Ab10BBZ-Bcl-2 CAR-T细胞在体内有着明显的抗肿瘤能力。
实施例9抗GPC-3Bcl-2 CAR-T细胞的体内肿瘤杀伤能力
将3x10 6Huh-7肿瘤细胞皮下接种到B-NDG小鼠，7天后给予10 7GC33BBZ CAR-T细胞或GC33BBZ-Bcl-2 CAR-T细胞治疗，测量小鼠的肿瘤负荷，其结果如图10所示，GC33BBZ-Bcl-2 CAR-T细胞相对GC33BBZ CAR-T细胞能够更好的控制肿瘤负荷，抑制肿瘤生长，证明GC33BBZ-Bcl-2 CAR-T细胞在体内有着明显的抗肿瘤能力。

Claims (66)

  1. 免疫效应细胞，其包含和/或表达嵌合抗原受体(CAR)，以及Bcl-2蛋白或其功能活性片段。
  2. 根据权利要求1所述的免疫效应细胞，其中所述CAR包含抗原结合结构域，且所述抗原结合结构域包含特异性结合CD20的抗体或其抗原结合片段。
  3. 根据权利要求2所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含HCDR3，且所述HCDR3包含SEQ ID NO:1所示的氨基酸序列。
  4. 根据权利要求2-3中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含HCDR2，且所述HCDR2包含SEQ ID NO:2所示的氨基酸序列。
  5. 根据权利要求2-4中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含HCDR1，且所述HCDR1包含SEQ ID NO:3所示的氨基酸序列。
  6. 根据权利要求2-5中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含LCDR3，且所述LCDR3包含SEQ ID NO:4所示的氨基酸序列。
  7. 根据权利要求2-6中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含LCDR2，且所述LCDR2包含SEQ ID NO:5所示的氨基酸序列。
  8. 根据权利要求2-7中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含LCDR1，且所述LCDR1包含SEQ ID NO:6所示的氨基酸序列。
  9. 根据权利要求2-8中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含VH，所述VH包含SEQ ID NO:7所示的氨基酸序列。
  10. 根据权利要求2-9中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含VL，所述VL包含SEQ ID NO:8所示的氨基酸序列。
  11. 根据权利要求2-10中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含SEQ ID NO:10所示的氨基酸序列。
  12. 根据权利要求1所述的免疫效应细胞，其中所述CAR包含抗原结合结构域，且所述抗原结合结构域包含特异性结合CLDN18.2的抗体或其抗原结合片段。
  13. 根据权利要求12所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含HCDR3，且所述HCDR3包含SEQ ID NO:11所示的氨基酸序列。
  14. 根据权利要求12-13中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含HCDR2，且所述HCDR2包含SEQ ID NO:12所示的氨基酸序列。
  15. 根据权利要求12-14中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含HCDR1，且所述HCDR1包含SEQ ID NO:13所示的氨基酸序列。
  16. 根据权利要求12-15中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包 含LCDR3，且所述LCDR3包含SEQ ID NO:14所示的氨基酸序列。
  17. 根据权利要求12-16中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含LCDR2，且所述LCDR2包含SEQ ID NO:15所示的氨基酸序列。
  18. 根据权利要求12-17中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含LCDR1，且所述LCDR1包含SEQ ID NO:16所示的氨基酸序列。
  19. 根据权利要求12-18中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含VH，所述VH包含SEQ ID NO:17所示的氨基酸序列。
  20. 根据权利要求12-19中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含VL，所述VL包含SEQ ID NO:18所示的氨基酸序列。
  21. 根据权利要求12-20中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含SEQ ID NO:19所示的氨基酸序列。
  22. 根据权利要求1所述的免疫效应细胞，其中所述CAR包含抗原结合结构域，且所述抗原结合结构域包含特异性结合GPC-3的抗体或其抗原结合片段。
  23. 根据权利要求22所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含HCDR3，且所述HCDR3包含SEQ ID NO:20、SEQ ID NO:29和SEQ ID NO:38中任一项所示的氨基酸序列。
  24. 根据权利要求22-23中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含HCDR2，且所述HCDR2包含SEQ ID NO:21、SEQ ID NO:30和SEQ ID NO:39中任一项所示的氨基酸序列。
  25. 根据权利要求22-24中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含HCDR1，且所述HCDR1包含SEQ ID NO:22、SEQ ID NO:31和SEQ ID NO:40中任一项所示的氨基酸序列。
  26. 根据权利要求22-25中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含LCDR3，且所述LCDR3包含SEQ ID NO:23、SEQ ID NO:32和SEQ ID NO:41中任一项所示的氨基酸序列。
  27. 根据权利要求22-26中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含LCDR2，且所述LCDR2包含SEQ ID NO:24、SEQ ID NO:33和SEQ ID NO:42中任一项所示的氨基酸序列。
  28. 根据权利要求22-27中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含LCDR1，且所述LCDR1包含SEQ ID NO:25、SEQ ID NO:34和SEQ ID NO:43中任一项所示的氨基酸序列。
  29. 根据权利要求22-28中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含VH，所述VH包含SEQ ID NO:26、SEQ ID NO:35和SEQ ID NO:44中任一项所示的氨基酸序列。
  30. 根据权利要求22-29中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含VL，所述VL包含SEQ ID NO:27、SEQ ID NO:36和SEQ ID NO:45中任一项所示的氨基酸序列。
  31. 根据权利要求22-30中任一项所述的免疫效应细胞，其中所述抗体或其抗原结合片段包含SEQ ID NO:28、SEQ ID NO:37和SEQ ID NO:46中任一项所示的氨基酸序列。
  32. 根据权利要求2-31中任一项所述的免疫效应细胞，其中所述抗体包含单链抗体。
  33. 根据权利要求1-32中任一项所述的免疫效应细胞，其中所述CAR包含跨膜结构域，所述跨膜结构域包含源自选自下述蛋白的跨膜结构域：CD28、CD3e、CD45、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
  34. 根据权利要求33所述的免疫效应细胞，其中所述跨膜结构域包含如SEQ ID NO:47所示的氨基酸序列。
  35. 根据权利要求1-34中任一项所述的免疫效应细胞，其中所述CAR包含共刺激域，且所述共刺激域包含一种或多种选自下述蛋白的共刺激域：CD28、4-1BB、CD40L、TIM1、CD226、DR3、SLAM、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD27、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT和DAP12中的共刺激信号传导区。
  36. 根据权利要求35所述的免疫效应细胞，其中所述共刺激域包含如SEQ ID NO:48所示的氨基酸序列。
  37. 根据权利要求1-36中任一项所述的免疫效应细胞，其中所述CAR包含胞内信号传导域，且所述胞内信号传导域包含源自CD3ζ的胞内信号传导域。
  38. 根据权利要求37所述的免疫效应细胞，其中所述胞内信号传导域包含如SEQ ID NO:49所示的氨基酸序列。
  39. 根据权利要求1-38中任一项所述的免疫效应细胞，其中所述CAR包含铰链区，所述铰链区位于所述抗原结合结构域和所述跨膜结构域之间。
  40. 根据权利要求39所述的免疫效应细胞，其中所述铰链区包含以下至少一项：CD8、CD28、4-1BB、CD4、CD27、CD7和PD-1的铰链区。
  41. 根据权利要求38-39中任一项所述的免疫效应细胞，其中所述铰链区包含如SEQ ID  NO:50所示的氨基酸序列。
  42. 根据权利要求1-41中任一项所述的免疫效应细胞，其中所述CAR包含如SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59和SEQ ID NO:61中任一项所示的氨基酸序列。
  43. 根据权利要求1-42中任一项所述的免疫效应细胞，其中所述Bcl-2蛋白或其功能活性片段为外源Bcl-2蛋白或其功能活性片段。
  44. 根据权利要求1-43中任一项所述的免疫效应细胞，其中所述免疫效应细胞包括T细胞。
  45. 根据权利要求1-44中任一项所述的免疫效应细胞，其中所述Bcl-2蛋白或其功能活性片段包含SEQ ID NO:52所示的氨基酸序列。
  46. 核酸分子，所述核酸分子编码权利要求1-45中任一项所述的CAR和权利要求1-44中任一项所述的Bcl-2蛋白或其功能活性片段。
  47. 根据权利要求46所述的核酸分子，其包含编码自剪切肽的序列，所述编码自剪切肽的序列位于编码所述CAR的序列和编码所述Bcl-2蛋白的序列之间。
  48. 根据权利要求47所述的核酸分子，其中所述自剪切肽包含2A肽。
  49. 根据权利要求48所述的核酸分子，其中所述2A肽为选自下述的一种或多种：P2A，T2A，E2A和F2A。
  50. 根据权利要求48-49中任一项所述的核酸分子，其中所述2A肽包含如SEQ ID NO:51所示的氨基酸序列。
  51. 载体，其包含权利要求46-50中任一项所述的核酸分子。
  52. 根据权利要求51所述的载体，其选自质粒、逆转录病毒载体和慢病毒载体中的一种或多种。
  53. 免疫效应细胞，其包含权利要求46-50中任一项所述的核酸分子或权利要求51-52中任一项所述的载体。
  54. 制备权利要求1-45或权利要求53中任一项所述的免疫效应细胞的方法，所述方法包括在使得所述嵌合抗原受体表达的条件下，培养权利要求53所述的免疫效应细胞。
  55. 组合物，其包含权利要求1-45或权利要求53中任一项所述的免疫效应细胞。
  56. 权利要求1-45或权利要求53中任一项所述的免疫效应细胞、权利要求46-50中任一项所述的核酸分子、权利要求51-52中任一项所述的载体或权利要求55所述的组合物用于制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
  57. 根据权利要求56所述的用途，其中所述疾病和/或病症与CD20表达相关。
  58. 根据权利要求56所述的用途，其中所述疾病和/或病症与CLDN18.2表达相关。
  59. 根据权利要求56所述的用途，其中所述疾病和/或病症与GPC-3表达相关。
  60. 根据权利要求56-59中任一项所述的用途，其中所述疾病和/或病症包括肿瘤。
  61. 根据权利要求60所述的用途，其中所述肿瘤包括实体瘤和/或血液瘤。
  62. 根据权利要求60-61中任一项所述的用途，其中所述肿瘤包括CD20阳性肿瘤。
  63. 根据权利要求60-61中任一项所述的用途，其中所述肿瘤包括CLDN18.2阳性肿瘤。
  64. 根据权利要求60-61中任一项所述的用途，其中所述肿瘤包括GPC-3阳性肿瘤。
  65. 根据权利要求60-64中任一项所述的用途，其中所述肿瘤包括淋巴瘤。
  66. 根据权利要求60-64中任一项所述的用途，其中所述肿瘤包括胰腺癌。
PCT/CN2022/070012 2021-01-07 2022-01-04 改造的免疫效应细胞及其用途 Ceased WO2022148332A1 (zh)

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