WO2022148332A1 - 改造的免疫效应细胞及其用途 - Google Patents
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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
Description
Claims (66)
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 载体,其包含权利要求46-50中任一项所述的核酸分子。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
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