PD-L1-specific antibodies and anti-pd-l1-car-t cells

Abstract

The present invention relates to a monoclonal anti-human PD-L1 antibody or single chain variable fragment (scFv) comprising a V H H having the amino acid set forth in SEQ ID NO: 3 and a V L L having the amino acid set forth in SEQ ID NO: 5. The present invention also relates to a chimeric antigen receptor fusion protein comprising, from N-terminus to C-terminus: (i) a single chain variable fragment (scFv) of the present invention, (ii) a transmembrane domain, (iii) at least one costimulatory domain, and (iv) an activating domain. The inventors have shown that the PD-L1 CAR-T cells of the present invention are more effective than Avelumab PD-L1 CAR-T cells in killing several cancer cell lines. The PD-L1 CAR-T can be used alone or in combination with other drugs in immunotherapy.

Description

[0001] References to sequence lists, tables, or computer programs

[0002] The sequence list was submitted along with the specification via EFS-Web as an ASCII text file named Sequence Listing.txt, created on June 15, 2021, and is 31.1 kilobytes in size. The sequence list submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to PD-L1 (programmed death-ligand-1)-specific antibodies and anti-PD-L1-CAR-T cells, which are useful in the field of adoptive immunogene therapy for tumors. Background Technology

[0004] Immunotherapy is emerging as a promising cancer treatment approach. T cells, or T lymphocytes, are the armed forces of our immune system, constantly seeking out foreign antigens and distinguishing abnormal (cancer or infected cells) from normal cells. Genetic modification of T cells using CAR (chimeric antigen receptor) constructs has become the most common method for designing tumor-specific T cells. Infusing CAR-T cells that target tumor-associated antigens (TAAs) into a patient (called adoptive cell transfer or ACT) is an effective immunotherapy approach [1,2]. Compared to chemotherapy or antibodies, the advantage of CAR-T technology is that the reprogrammed engineered T cells can proliferate and persist in the patient's body ("a living drug") [1,3,4].

[0005] CARs typically consist of a single-chain variable fragment (scFv) derived from a monoclonal antibody at the N-terminus, a hinge, a transmembrane domain, and several intracellular co-stimulatory domains: (i) CD28, (ii) CD137 (4-1BB), CD27, or other co-stimulatory domains, tandemly linked with a CD3ζ domain. Figure 1 [1,2]. The evolution of CARs has gone from the first generation (without a costimulatory domain) to the second generation (with one costimulatory domain) and then to the third generation CARs (with several costimulatory domains). The generation of CARs with multiple costimulatory domains (the so-called third-generation CARs) has enhanced the cytotoxic activity of CAR-T cells, significantly improved the persistence of CAR-T cells, and thus enhanced their anti-tumor activity.

[0006] Figure 1The structure of CAR is shown. The left side shows the structure of the first generation (without costimulatory domain), the middle shows the structure of the second generation (with one costimulatory domain CD28 or 4-1BB), and the right side shows the structure of the third generation (with two or more costimulatory domains). This figure is from Golubovskaya, Wu, Cancers, 2016[5].

[0007] PD-L1, also known as differentiation cluster 274, CD274, or B7 homolog 1, B7-H1, is a 40 kDa transmembrane protein that plays a crucial role in suppressing the immune system during disease or other events. The binding of the PD-L1 domain to the PD-1 protein blocks the proliferation and activity of CD8+ T cells responsible for immune defense. The PD-L1 / PD-1 interaction plays a dominant role in suppressive T cell responses in vivo, particularly in the tumor microenvironment.

[0008] Several types of cancers overexpress PD-L1. Anti-PD-L1 monoclonal antibody (mAbs) and anti-PD-1 mAb immunotherapy have been tested in clinical trials [3]. PD-L1 is not normally expressed on the cell surface of normal tissues, but its expression is elevated in many tumor tissues. In addition, PD-L1 expression is significantly upregulated by immune cells, primarily through their production of IFN-γ.

[0009] In 2017, the U.S. Food and Drug Administration (FDA) granted accelerated approval to avelumab for the treatment of adults and children aged 12 years and older with metastatic Merkel cell tumors. Avelumab is a human IgG1λ monoclonal antibody that blocks PD-L1.

[0010] A highly specific and active anti-PD-L1 antibody is needed. Attached Figure Description

[0011] Figure 1 The structure of .CAR.

[0012] Figure 2 The structure of the .PD-L1 CAR construct, with CD28 (top) or 4-1BB (bottom) as the co-stimulatory domain. FLAG tags are optional.

[0013] Figure 3 PD-L1 antibody was detected by ELISA and Western blotting to detect PD-L1 antigen.

[0014] Figure 4 FACS detection of PD-L1 antibodies against different cancer cell lines. HepG2 and SKOV-3 express high levels of PD-L1.

[0015] Figures 5A to 5EPromab PD-L1-CAR-T cells (PMC159, CD28; PMC804, 4-1BB) exhibit high cytotoxicity against PD-L1-positive cancer cells. The effector cell to target cell ratio was 10:1. Figure 5A Target cells: PMC159, A1847; Figure 5B : PMC159, BxPC3 target cells; Figure 5C : PMC159, HeLa-CD19 target cells; Figure 5D : PMC159, SKOV target cells; Figure 5E : PMC804, A431 target cells.

[0016] Figures 6A to 6B Cytotoxic activity of avirumab PD-L1 scFv-CAR-T cells against target cancer cells (6A: BxPC3 cells; 6B: SKOV-3 cells). The ratio of effector cells to target cells was 10:1.

[0017] Figures 7A to 7B The combination of CD24-CAR-T cells and Promab-based PD-L1-CAR-T cells targeted cancer cells. This combination resulted in 100% killing in BxPC3 cells and >90% killing in SKOV-3 cells. Detailed Implementation

[0018] definition

[0019] As used herein, a "chimeric antigen receptor (CAR)" is a modified receptor protein that endows T cells with the new ability to target a specific protein. This receptor is chimeric because it combines antigen-binding and T-cell activation functions into a single receptor. A CAR is a fusion protein that includes an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is sometimes also referred to as a "chimeric receptor," a "T-body," or a "chimeric immune receptor (CIR)."

[0020] "Extracellular domains that can bind to antigens" refers to any oligopeptide or polypeptide that can bind to a particular antigen. "Intracellular domains" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit biological processes within the cell.

[0021] As used in this article, a “domain” refers to a region in a polypeptide that folds into a specific structure independently of other regions.

[0022] As used herein, a FLAG tag, or FLAG octapeptide, or FLAG epitope, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology and has the sequence motif DYKDDDDK (SEQ ID NO:1). It can be fused to the C-terminus or N-terminus of a protein or inserted into the protein.

[0023] As used herein, "single-chain variable fragment (scFv)" refers to a single-chain polypeptide derived from an antibody that retains its ability to bind to an antigen. An example of scFv includes an antibody polypeptide formed using recombinant DNA technology, wherein the Fv regions of the immunoglobulin heavy chain (H chain) and light chain (L chain) segments are linked by spacer sequences. Various methods for engineering scFv are known to those skilled in the art.

[0024] As used in this article, "tumor antigen" refers to an antigenic biomolecule whose expression leads to cancer.

[0025] The inventors have generated a mouse anti-human monoclonal antibody specifically targeting PD-L1 (Promab anti-PD-L1). The monoclonal anti-human PD-L1 antibody is generated from a purified recombinant fragment targeting human PD-L1.

[0026] The inventors have generated PD-L1-CAR-T cells to target cancer cells overexpressing the PD-L1 tumor antigen. The PD-L1-CAR-T cells of this invention exhibit high cytotoxic activity and in vivo antitumor activity against several cancer cell lines.

[0027] This invention relates to anti-human PD-L1 antibodies or antigen-binding fragments thereof, comprising a VH having the amino acid shown in SEQ ID NO:3 and a VL having the amino acid shown in SEQ ID NO:5. The antigen-binding fragment comprises a Fab monomer or Fab dimer (Fab')2 or scFv. In one embodiment, the monoclonal anti-human PD-L1 antibody is a single-chain variable fragment (scFv).

[0028] The present invention also relates to a chimeric antigen receptor fusion protein comprising, from the N-terminus to the C-terminus: (i) a single-chain variable fragment (scFv) targeting PD-L1, (ii) a transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) an activation domain.

[0029] Figure 2 Two structures of the PD-L1 CAR construct are shown. The second-generation CAR shown has CD28 (top) or 4-1BB (bottom) as a co-stimulatory domain. Abbreviations: Flag; TM.

[0030] In the PD-L1 CAR construct, ScFv can be either VH-connector-VL or VL-connector-VH.

[0031] In one embodiment, the co-stimulatory domain is selected from the group consisting of CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40, and DAP10. The preferred co-stimulatory domain is CD28.

[0032] The preferred activation domain is CD3zeta (CD3Z or CD3ζ).

[0033] The transmembrane domain can be derived from a natural polypeptide or can be artificially designed. Transmembrane domains derived from natural polypeptides can be obtained from any membrane-binding or transmembrane protein. For example, transmembrane domains of T cell receptor α or β chains, CD3ζ chains, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR can be used. Artificially designed transmembrane domains are polypeptides that primarily contain hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan, and valine is located at each end of the synthetic transmembrane domain. Optionally, short oligopeptide linkers or polypeptide linkers, for example, linkers having a length of 2 to 10 amino acids, can be placed between the transmembrane domain and the intracellular domain. In one embodiment, a linker sequence having a glycine-serine continuous sequence can be used.

[0034] This invention provides a nucleic acid encoding a PD-L1 CAR. The nucleic acid encoding the CAR can be prepared using conventional methods from the amino acid sequence of a specific CAR. For the amino acid sequence of each domain, the base sequence encoding the amino acid sequence can be obtained from an NCBI RefSeq ID or a GenBank accession number, and the nucleic acid of this invention can be prepared using standard molecular biology and / or chemical methods. For example, based on this base sequence, the nucleic acid can be synthesized, and the nucleic acid of this invention can be prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0035] The nucleic acid encoding the CAR of this invention can be inserted into a vector, and this vector can be introduced into cells. For example, viral vectors can be used, such as retroviral vectors (including oncogenic retroviral vectors, lentiviral vectors, and pseudotyped vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors or Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors. Viral vectors lacking replication capacity and thus unable to self-replicate in infected cells are preferred.

[0036] For example, when using retroviral vectors, suitable packaging cells can be selected based on the LTR sequence and the packaging signal sequence of the vector for preparing retroviral particles. Examples of packaging cells include PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP+E-86, GP+envAm-12, and Psi-Crip. Highly transfection-efficient 293 cells or 293T cells can also be used to prepare retroviral particles. Various retroviral vectors produced based on retroviruses and packaging cells that can be used to package retroviral vectors are widely available commercially from many companies.

[0037] CAR-T cells bind to specific antigens via the CAR, thereby transmitting signals into the cell and activating it. The activation of CAR-expressing cells varies depending on the host cell type and the intracellular domains of the CAR, and can be confirmed based on indicators such as cytokine release, improved cell proliferation rate, and changes in cell surface molecules. For example, the release of cytotoxic cytokines (tumor necrosis factor, lymphotoxin, etc.) from activated cells can lead to the destruction of target cells expressing the antigen. Furthermore, the release of cytokines or changes in cell surface molecules can stimulate other immune cells, such as B cells, dendritic cells, NK cells, and macrophages.

[0038] Cells expressing CARs can be used as therapeutic agents for diseases. The therapeutic agent contains CAR-expressing cells as the active ingredient, and it may further contain suitable excipients.

[0039] The inventors have generated PD-L1-CAR-T (PD-L1-CAR-T) cells that target different cancer cells (ovarian cancer, pancreatic cancer, and other cancers) overexpressing PD-L1. The inventors have provided data demonstrating the effective expression of PD-L1 in different types of cancer (ovarian cancer, pancreatic cancer, and others). Compared to untransduced T cells and mock-CAR-T cells, PD-L1-CAR-T cells exhibit higher cytotoxic activity against PL-1 positive cancer cells. In one embodiment, a FLAG tag is added to the C-terminus of the ScFv, which allows for the detection of CARs in the cells. PD-L1 antibodies have proven highly effective as therapeutic agents in many clinical applications.

[0040] The PD-L1 monoclonal antibody or PD-L1-ScFv of the present invention has the advantage over known anti-PD-L1 antibodies (such as avirumab) in that it exhibits high specificity against PD-L1-positive cancer cells (ovarian, pancreatic, and others). Furthermore, Promab PD-L1-CAR-T cells show higher activity than avirumab-PD-L1-CAR-T cells. The inventors have demonstrated that Promab PD-L1 CAR-T cells are more effective than avirumab PD-L1 CAR-T cells in killing several cancer cell lines.

[0041] The present invention uses a monoclonal mouse anti-human PD-L1 antibody to detect PD-L1 in PD-L1 positive cancer cells.

[0042] The PD-L1 antibody of the present invention can be used in immunotherapy applications: toxin / drug conjugated antibodies, monoclonal therapeutic antibodies, humanization of PD-L1 antibodies, and CAR-T cell immunotherapy.

[0043] PD-L1-CAR-T cells using the PD-L1 antibody of this invention can be effectively used to target the PD-L1 antigen in PD-L1 positive cell lines such as ovarian cancer, pancreatic cancer, and cervical cancer.

[0044] PD-L1-CAR-T can be used in combination with various chemotherapy therapies: checkpoint inhibitors; targeted therapies; small molecule inhibitors; and antibodies.

[0045] PD-L1 antibodies can be modified by site-directed mutagenesis for affinity regulation; they can be used for humanization and fully human antibody production.

[0046] PD-L1-CAR-T cells can be used clinically to target PD-L1 positive cells.

[0047] Co-activation domains: CD28, 4-1BB, and other modifications can be used to increase its power. Tag-coupled PD-L1scFv can be used for CAR generation.

[0048] Third-generation CAR-T or other co-stimulatory signaling domains can be used with the same PD-L1-scFv within the CAR.

[0049] Combining PD-L1 with other CARs that target other tumor antigens or the tumor microenvironment (VEGFR-1-3) or with bispecific scFv-CARs can be used to enhance the activity of monotherapy PD-L1-CAR.

[0050] It can generate bispecific antibodies with PD-L1 and CD3 or other antigens, which are used for treatment.

[0051] PD-L1 scFv, PD-L1 antibodies, or PD-L1 CAR-T cells can be used in conjunction with another CAR to enhance its activity. Dual targeting of PD-L1 and another tumor antigen can enhance treatment. Furthermore, co-transfection of PD-L1-CAR-T cells with other CAR-T cells can be used to inhibit checkpoint signaling and increase CAR-T cell activity. Combinations of CD24-CAR-T and PD-L1CAR-T showed similar killing activity in cytotoxicity assays of two different cell lines. This can be applied to the co-inhibition of both pathways in vivo when the PD-L1 pathway is activated in the tumor microenvironment. Additionally, bispecific CAR-T cells with two scFvs that bind via adaptors can be used to enhance the efficacy of single scFv CAR-T cells.

[0052] PD-L1 monoclonal antibodies can be used as a single agent or in combination with other therapies. This combination therapy will improve the efficacy of CAR-T therapy.

[0053] PD-L1-CAR-T cells can be used to target cancer stem cells that are most resistant to chemotherapy and form aggressive tumors.

[0054] PD-L1-CAR can be used to generate other cell types, such as CAR-natural killer (NK) cells, iPS (induced pluripotent)-NK or iPS-T cells, macrophages, γ-δT cells, and other hematopoietic cells, which can target PD-L1-positive cancers. This invention provides T cells, or NK cells, or macrophages, or hematopoietic cells modified to express PD-L1-CAR.

[0055] The following examples further illustrate the present invention. These examples are intended to illustrate the invention only and should not be construed as limiting the invention.

[0056] Example

[0057] Example 1. PD-L1 antibody and activity

[0058] We used a hybridoma (clone 7D2A10) to generate a mouse monoclonal anti-human PD-L1 antibody. The hybridoma was generated against a purified recombinant fragment (24 to 153 amino acid sequences) of human PD-L1 expressed in E. coli. The hybridoma technique is standard and described in [4]. The antibody detects the extracellular domain of PD-L1 and is of the IgG2b type.

[0059] Figure 3 This demonstrates the detection of the PD-L1 antigen using ELISA and Western blotting with this anti-human PD-L1 antibody. Figure 3Table A shows the ELISA results for the control antigen (100 ng), PD-L1 antigen (10 ng), PD-L1 antigen (50 ng), and PD-L1 antigen (100 ng) from bottom to top. The ELISA results indicate no binding to the control antigen (100 ng), and binding to the PD-L1 antigen (10 ng), PD-L1 antigen (50 ng), and PD-L1 antigen (100 ng) increases in a dose-dependent manner. Figure 3 In B, Western blot analysis showed that the antibody binds to the extracellular domain of PD-L1 (AA: 24 to 153, expected molecular weight 40.1 kDa). Figure 3 In C, Western blot analysis showed that the antibody did not bind to PD-L1 in (1) HEK293 cell lysates, but did bind to (2) HEK293 cell lysates transfected with the PD-L1 extracellular domain fused with human Fc (PD-L1-hFc).

[0060] This PD-L1 antibody was used to detect high levels of PD-L1 in tumor tissues and several cancer cell lines by flow cytometry.

[0061] This antibody detected moderate PD-L1 expression in liver cancer and some binding in normal liver, lung, uterus, and hypohysis. Most normal tissues (colon, duodenum, rectum, testis, esophagus, brain, muscle, pancreas, kidney, stomach, prostate, tonsils, and spleen) showed negative PD-L1 expression using this antibody.

[0062] The absence of staining in most normal tissues is advantageous for using this antibody in CAR-T form because it has lower non-targeting and non-tumor activity.

[0063] Example 2. PD-L1 VH, VL and scFv sequences

[0064] We sequenced the anti-PD-L1 antibody (which tested positive for PD-L1 antigen binding by ELISA) from the hybridoma clone (#7D2A10). The structure of the anti-PD-L1 scFv is: VH-linker-VL.

[0065] PD-L1 VH nucleotide sequence (SEQ ID NO:2)

[0066] CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAACCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGGTGGATAAACACCCACACTGGAGAGCCAACATATGCT GATGACTTCAAGGGACGGTTTGCCTTCTCTTCGGAAACCTCTGCCAGCTCTGCCTATTTGCAGATCAACAACCTCAAAAATGATGACATGGCTACATATTTCTGTGCAAAAGGTACCCACAGAGAAGAAATTCCGGCCTGGTTCGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0067] PD-L1 VH Protein Framework (SEQ ID NO:3) .

[0068] QIQLVQSGPELKNPGETVKIS CKASGYTFTNYGM NW VKQAPGKGLKWMGWINTHTGE PTYADDFKGRFAFSSE TS ASSAYLQINNLKNDDMATYFC AKGTHREEIPAWFAYW GQ GTLVTVSA

[0069] PD-L1 VL Protein Layer(SEQ ID NO:4)

[0070] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAGAGCTCCTGATCTACAAAGTTT CCAACCTATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG

[0071] PD-L1 VL amino acid sequence (SEQ ID NO:5)

[0072] DVLMTQTPLSLPVSLGDQASI SCRSSQSIVHSNG NT YLEWYLQKPGQSPELLIYKVS NLFSGVPDRFSGSGSG TD FTLKISRVEAEDLGVYYCFQG SHVPPTFGAGTKLELKR

[0073] Linker nucleotide sequence (SEQ ID NO:6)

[0074] GGTGGCGGTGGTTCT GGTGGCGGTGGTTCT GGTGGCGGTGGTTCT

[0075] Linker amino acid sequence (SEQ ID NO:7)

[0076]

[0077] PD-L1 scFv nucleotide sequence (SEQ ID NO:8)

[0078] The bold, larger font highlighted the V. H The nucleotide sequence; the underlined V is highlighted. L The nucleotide sequence is shown in the middle (italicized); the nucleotide sequence encoding the linker is shown in the middle (italicized).

[0079]

[0080]

[0081] PD-L1 scFv amino acid sequence (SEQ ID NO:9)

[0082]

[0083] Example 3. PD-L1 Lentiviral CAR Construct

[0084] The inventors generated a PD-L1 CAR construct within a lentiviral vector, cloning it into the Xba I and EcoRI sites of the lentiviral vector. The pCD510-FMC63-28z lentiviral CAR construct includes a PD-L1 ScFv-Flag tag-CD8 hinge, CD28 transmembrane / activation-CD3ζ insert (PMC159) between the Xba I and EcoRI cloning sites under the CMV promoter. A Flag tag was inserted for easier detection of CAR-positive T cells. The inventors also generated a PMC804 CAR with the same PD-L1-CAR scfv, whose scfv contains a 41BB co-stimulatory domain instead of CD28, lacks a Flag tag, and is regulated for higher CAR expression via the MNDU3 promoter.

[0085] Lentiviral cells were generated in 293T cells, and their titers were established by RT-PCR. Then, T cells were transduced with an equal dose of lentivirus.

[0086] Example 4A. PD-L1 CAR (PMC159) with CD28 as a co-stimulatory domain

[0087] The PD-L1-CAR construct scheme is as follows Figure 2 As shown. The lentiviral vector Lenti CMV-MCS-EF1a-puro was used for cloning all scFv CAR sequences.

[0088] The following nucleotide and amino acid sequences illustrate the PD-L1 ScFv Flag-CD8 hinge-TM28-CD28-CD3ζ of the present invention. This structure includes the human CD8 signaling peptide (CD8 leader) and PD-L1 scFv (V H -Connector 3x(G4S)-V L ), FLAG, CD8 hinge, CD28 transmembrane, activation domain, CD3ζ ( Figure 2 ).

[0089] <CD8 signal peptide>

[0090] Nucleotide sequence (SEQ ID NO: 10)

[0091] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

[0092] Amino acid sequence (SEQ ID NO: 11)

[0093] MALPVTALLLPLALLLHAARP

[0094] <Nhe restriction site I>

[0095] GCTAGC

[0096] Amino acid sequence

[0097] <as>

[0098] <PD-L1 scFV>

[0099] See Example 2, SEQ ID NO:8 and SEQ ID NO:9.

[0100] <flag>

[0101] Nucleotide sequence (SEQ ID NO: 12)

[0102] GACTACAAAGACGATGACGACAAG

[0103] Amino acid sequence (SEQ ID NO: 1)

[0104] DYKDDDDK

[0105] <XhoI restriction site>

[0106] Nucleotide sequence

[0107] CTCGAG

[0108] Amino acid sequence

[0109] LE

[0110] <CD8 hinge>

[0111] Nucleotide sequence (SEQ ID NO: 13)

[0112] AAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGAT

[0113] Amino acid sequence (SEQ ID NO: 14)

[0114] KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD

[0115] <Spacer>

[0116] Nucleotide sequence

[0117] aagccc

[0118] Amino acid sequence

[0119] KP

[0120] <CD28 TM / co-stimulation>

[0121] Nucleotide sequence (SEQ ID NO: 15)

[0122] TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC

[0123] Amino acid sequence (SEQ ID NO: 16)

[0124] FWVLVVVGGVLACYSLLVTVAFIIFWV / RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0125] <CD3ζ>

[0126] Nucleotide sequence (SEQ ID NO: 17)

[0127] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC TAA

[0128] Amino acid sequence (SEQ ID NO: 18)

[0129] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0130] Nucleotide sequence of PD-L1-CAR (PMC 159, FLAG underlined), SEQ ID NO: 19

[0131]

[0132] GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAGAGCTCCTGATCTACAAAGTTTCCAACCTATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG GACTACAAAGACGATGACGACAAG

[0133] ctcgagAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGATaagcccttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA

[0134] Amino acid sequence of PD-L1-CAR protein, PMC 159, SEQ ID NO: 20

[0135] M A L P V T A L L L P L A L L L H A A R P A S Q I Q L V Q S G P E L KN P G E T V K I S C K A S G Y T F T N Y G M N W V K Q A P G K G L K W M G W IN T H T G E P T Y A D D F K G R F A F S S E T S A S S A Y L Q I N N L K N D DM A T Y F C A K G T H R E E I P A W F A Y W G Q G T L V T V S A G G G G S G GG G S G G G G S D V L M T Q T P L S L P V S L G D Q A S I S C R S S Q S I V HS N G N T Y L E W Y L Q K P G Q S P E L L I Y K V S N L F S G V P D R F S G SG S G T D F T L K I S R V E A E D L G V Y Y C F Q G S H V P P T F G A G T K LE L K R D Y K D D D D K L E K P T T T P A P R P P T P A P T I A S Q P L S L RP E A S R P A A G G A V H T R G L D F A S D K P F W V L V V V G G V L A C Y SL L V T V A F I I F W V R S K R S R L L H S D Y M N M T P R R P G P T R K H YQ P Y A P P R D F A A Y R S R V K F S R S A D A P A Y Q Q G Q N Q L Y N E L NL G R R E E Y D V L D K R R G R D P E M G G K P Q R R K N P Q E G L Y N E L QK D K M A E A Y S E I G M K G E R R R G K G H D G L Y Q G L S T A T K D T Y DA L H MQALPP

[0136] Example 4B. PD-L1 CAR (PMC 804) with 4-1BB as a co-stimulatory domain

[0137] The nucleotide and amino acid sequences of this CAR are the same as those of Example 4A, except that this CAR does not have a FLAG tag and 4-1BB is used instead of CD28 as the co-stimulatory domain.

[0138] <41BB domain / costimulation>

[0139] Nucleotide sequence, SEQ ID NO:21

[0140] AAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0141] amino acid sequence, SEQ ID NO:22

[0142] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0143] Nucleotide sequence of PD-L1-CAR (PMC 804, 4-1BB is underlined), SEQ ID NO: 23 ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGgctagcCAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAACCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGGTGGATAAACACCCACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTCGGAAACCTCTGCCAGCTCTGCCTATTTGCAGATCAACAACCTCAAAAATGATGACATGGCTACATATTTCTGTGCAAAAGGTACCCACAGAGAAGAAATTCCGGCCTGGTTCGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGGTGGCGGTGGTTCTGGTGGCGGTGGTTCTGGTGGCGGTGGTTCTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAGAGCTCCTGATCTACAAAGTTTCCAACCTATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGctcgagAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCAGTGATaagcccttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg AAACGGGGCAGAAAGAAACTC CTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCC AGAAGAAGAAGAAGGAGGATGTGAACTG AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA

[0144] Amino acid sequence of PD-L1-CAR (PMC 804, 4-1BB is underlined), SEQ ID NO: 24

[0145] MALPVTALLLPLALLLHAARPASQIQLVQSGPELKNPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTHTGEPTYADDFKGRFAFSSETSASSAYLQINNLKNDDMATYFCAKGTHREEIPAWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPELLIYKVSNLFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPPTFGAGTKLELKRLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWV KRGRKKLLYIFKQPFMRPVQTTQEED GCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0146] Example 5 Production of CAR lentivirus

[0147] DNA encoding PD-L1 scFv was synthesized and subcloned into the third-generation lentiviral vector Lenti CMV-MCS-EF1a-puro by Syno Biological (Beijing, China). The lentiviral construct was bidirectionally sequenced to confirm the construct sequence and used for lentiviral production. 10 million growth-arrested HEK293FT cells (Thermo Fisher) were seeded into T75 flasks and cultured overnight, then transfected using a CalPhos transfection kit (Takara, Mountain View, CA) with pPACKH1 lentiviral vector packaging mixture (System Biosciences, Palo Alto, CA) and 10 μg of each lentiviral vector. The medium was replaced with fresh medium the next day, and the lentivir-containing medium was collected after 48 hours. Cell debris was removed from the medium by centrifugation at 2100g for 30 minutes. Viral particles were collected by centrifugation at 112,000 g for 100 minutes, suspended in DMEM or AIM V medium, aliquoted, and frozen at -80°C. The viral titers were determined by quantitative RT-PCR using a Lenti-X qRT-PCR kit (Takara) and a 7900HT thermal cycler (Thermo Fisher) according to the manufacturer's protocol. Lentiviral titers >1 × 10⁻⁶. 8 pfu / ml.

[0148] Example 6. Isolation of peripheral blood mononuclear cells (PBMCs) from whole blood.

[0149] Whole blood was collected in 10 mL fractions from individual or mixed donors (depending on the required blood volume) (Stanford Hospital Blood Center, Stanford, CA) and separated using Ficol-Paque PLUS. The cell layer containing peripheral blood mononuclear cells (PBMCs) seen at the diluted plasma / Ficoll interface was removed, avoiding any Ficoll. To ensure complete removal of Ficoll, platelets, and plasma proteins, the PBMCs were washed twice with PBS to a total volume of 40 mL and centrifuged at 200 × g for 10 minutes at room temperature. The cells were then counted using a hemocytometer. If the washed PBMCs were to be used immediately, they were washed once with CAR-T medium (AIM V-AlbuMAX (BSA) (Life Technologies) with 5% AB serum and 1.25 μg / mL amphotericin B (Gemini Bioproducts, Woodland, CA), 100 U / mL penicillin, and 100 μg / mL streptomycin). If you want to freeze PBMCs, suspend the washed cells in a transfer insulated vial at -80°C for 24 hours, and then store them in liquid nitrogen.

[0150] Example 7. T-cell activation from PBMCs

[0151] In the presence of human interleukin-2 300 U / mL (huIL-2, Invitrogen), at 5 × 10 5 Cells isolated from PBMCs were cultured in CAR-T medium (AIM V-AlbuMAX (BSA, Life Technologies) with 5% AB serum and 1.25 μg / mL amphotericin B (Gemini Bioproducts, Woodland, CA), 100 U / mL penicillin and 100 μg / mL streptomycin) at a concentration of 1 / mL. T cells were activated with CD3 / CD28 beads and incubated at 37°C for 24 hours in the presence of CO2, followed by CAR virus transduction.

[0152] Example 8. T-cell transduction and expansion

[0153] After PBMC activation, cells were incubated at 37°C and 5% CO2 for 24 hours. 1×10⁻⁶ cells were added to each well. 6 Cells, 5×10 6 Lentiviral virus and 2 μL / mL Transplus medium (Alstem, Richmond, CA) (final dilution 1:500). Cells were cultured for another 24 hours before repeated virus addition. Cells were then grown for 12 to 14 days in the continuous presence of 300 U / mL IL-2. Cell concentration was analyzed every 2 to 3 days, with medium added to dilute the cell suspension to 1 × 10⁻⁶. 6 Cells / mL. CAR expression can be verified using FLAG antibody or by detecting Flag-tagged scFv with anti-mouse F(ab)2 or by detecting untagged scFv expression.

[0154] Example 9. Cytotoxicity assay

[0155] Perform cytotoxicity assays using the ACEA machine according to the manufacturer's protocols listed below.

[0156] Adherent target cancer cells were distributed at a density of 1×10⁶ cells per well. 4 Cells were seeded into 96-well E plates (Acea Biosciences, San Diego, CA) and monitored overnight using the iCELLigence system (Acea Biosciences) for impedance-based real-time cell analysis (RTCA). The next day, the culture medium was removed and the cells were treated with a solution containing 10% FBS ± 1 × 10⁶ cells / well. 5 Effector cells (CAR-T cells or untransduced T cells) were replaced with AIM V-AlbuMAX medium, in triplicate. Cells in the E-plates were additionally monitored for 2 to 3 days using an RTCA system, and impedance plots were generated over time. Cell lysis was calculated as (impedance of target cells without effector cells - impedance of target cells with effector CAR-T cells) × 100 / impedance of target cells without effector cells.

[0157] Example 10. Expression of PD-L1 in different cancerous and normal tissues

[0158] Staining with PD-L1 monoclonal antibody confirmed high staining intensity in several cancer cell lines: ovarian cancer SKOV-3, hepatocellular carcinoma, and HepG2; and moderate staining intensity in the breast MCF-7 cell line. Figure 4 Normal HEK-293 cells, cancer cells: HT29, MDA-231, HCT116 and others were negative.

[0159] Example 11. Promab PD-L1-CAR exhibited high cytotoxic activity against PD-L1 positive cancer cells.

[0160] Real-time cytotoxicity assays demonstrated the high cytotoxic activity of Promab PD-L1-CD28-CD3 CAR cells (PMC159) against highly PD-L1 positive cancer cells: ovarian cancer A1847 cells, pancreatic cancer BxpC3 cells, cervical cancer HeLa-CD19 cells, and ovarian cancer SKOV-3 cells. Figures 5A to 5D ).

[0161] Promab PD-L1-CD28-CD3 CAR-T (PMC159) exhibits 100% killing activity against ovarian cancer A1847 cells. Figure 5A It exhibits almost 100% killing activity against the pancreatic cancer BxPC3 cell line. Figure 5B It exhibits >75% killing activity against cervical cancer HeLa-CD19 cells. Figure 5C ).

[0162] We also tested the effects of PD-L1-41BB-CD3 CAR-T cells (PMC804) on A431 epidermal cancer cells and found that they killed cancer cells ( Figure 5E Therefore, CAR-T cells with CD28 or 41BB co-stimulatory domains are active against cancer cells.

[0163] Example 12. Comparison of CAR-T cells with Promab PD-L1 scFv and CAR-T cells with Avirumab PD-L1-scFv

[0164] We compared the Promab PD-L1 scFv with a PD-L1 scFv from the published antibody avelumab, which is FDA-approved for the treatment of Merkel cell carcinoma. The published avelumab PD-L1 scFv sequence without a C-terminal FLAG tag was inserted into the CAR. The general structure of the avelumab PD-L1 CAR-T is as follows... Figure 2 As shown in the image. The sequence of avirumab PD-L1 scFv is shown below.

[0165] PD-L1 (Avelumab) VH, nucleotide sequence (SEQ ID NO:25)

[0166] gaagtgcagctgctggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggctttacctttagc agctatattatgatgtgggtgcgccaggcgccgggcaaaggcctggaatgggtgagcagcatttatccgagcggcggcattaccttttat gcggataccgtgaaaggccgctttaccattagccgcgataacagcaaaaacaccctgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgcgcattaaactgggcaccgtgaccaccgtggattattggggccagggcaccctggtgaccgtgagcagc

[0167] PD-L1 (Avelumab) VH, amino acid sequence (SEQ ID NO:26)

[0168] EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYIMMWVRQA PGKGLEWVSS IYPSGGITFYADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARIK LGTVTTVDYW GQGTLVTVSS

[0169] Linker nucleotide sequence (SEQ ID NO:27)

[0170] ggcggcggcggcagcggcggcggcggcagcggcggcggcggcagc

[0171] Linker amino acid sequence (SEQ ID NO:7)

[0172] GGGGS GGGGS GGGGS

[0173] PD-L1 (Avelumab) VL, nucleotide sequence (SEQ ID NO:28)

[0174] cagagcgcgctgacccagccggcgagcgtgagcggcagcccgggcgagcattaccattagctgcaccggcaccagcagcgatgtgggcggctataactatgtgagctggtatcagcagcatccgggcaaagcgccgaaactgatgatttatgatgtgagcaac cgcccgagcggcgtgagcaaccgctttagcggcagcaaaagcggcaacaccgcgagcctgaccattagcggcctgcaggcggagatgaagcggattattgcagcagctataccagcagcacccgcgtgtttggcaccggcaccaaagtgaccgtgctg

[0175] PD-L1(protein-labeled strain)VL, transcription factor (SEQ ID NO:29)

[0176] QSALTQPASVSGSPGQSITIS CTGTSSDVGGYNY VS WYQQHPGKAPKL With IYDVSNRPSGVSNRFSGSKSG NT AS LTISGLQAEDEADYYCSSYTS SSTRVFGTGTKVTVL

[0177] PD-L1(protein-free)ScFv,chain gene (SEQ ID NO:30)

[0178] gaagtgcagctgctggaaagcggcggcggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggctttacctttagcagctatattatgatgtgggtgcgccaggcgccgggcaaaggcctggaatgggtgagcagcatttatccgagcggcggcattaccttttatgcggataccgtgaaaggccgctttaccattagccgcgataacagcaaaaacaccctgtatctgcagatgaacagcctgcgcgcggaagataccgcggtgtattattgcgcgcgcattaaactgggcaccgtgaccaccgtggattattggggccagggcaccctggtgaccgtgagcagcggcggcggcggcagcggcggcggcggcagcggcggcggcggcagccagagcgcgctgacccagccggcgagcgtgagcggcagcccgggccagagcattaccattagctgcaccggcaccagcagcgatgtgggcggctataactatgtgagctggtatcagcagcatccgggcaaagcgccgaaactgatgatttatgatgtgagcaaccgcccgagcggcgtgagcaaccgctttagcggcagcaaaagcggcaacaccgcgagcctgaccattagcggcctgcaggcggaagatgaagcggattattattgcagcagctataccagcagcagcacccgcgtgtttggcaccggcaccaaagtgaccgtgctg

[0179] PD-L1 (Avelumab) ScFv, Amino Acid Sequence (SEQ ID NO: 31)

[0180] EVQLLESGGGLVQPGGSLRLS CAASGFTFSSYIM MW VRQAPGKGLEWVSSIYPSGGI TFYADTVKGRFTISRD NS KNTLYLQMNSLRAEDTAVYYC ARIKLGTVTTVDYWGQ GT LVTVSSGGGGSGGGGSGGGGS QSALTQPASVSGSPGQ SI TISCTGTSSDVGGYNYVSWYQ QHPGKAPKLMIYDVSN RP SGVSNRFSGSSKSGNTASLTIS GLQAEDEADYYCSSYT SS STRVFGTGTKVTVL

[0181] Avirumab PD-L1 CAR-T cells were generated according to Example 5.

[0182] Avirumab PD-L1 CAR-T cells were used for cytotoxicity assays (Figure 6). The results showed that Promab PD-L1 CAR-T cells killed the same cancer cell lines more effectively than avirumab PD-L1 CAR-T cells. Avirumab PD-L1 CAR-T cells exhibited approximately 25% killing activity against BxPC3 cells, while PMC159 PD-L1 CAR-T cells showed almost 100% killing activity against the same cell lines (see Example 11). Figure 5B Avirumab PD-L1 CAR-T cells exhibited approximately <35% cytotoxicity against SKOV-3 cells, while PMC159 PD-L1 CAR-T cells showed >67% cytotoxicity against the same cell type (see Example 11). Figure 5D ).

[0183] Example 13. Combination of CD24 and Promab PD-L1-CAR-T cells targeting cancer cells

[0184] Figures 7A to 7B This study demonstrates the combined targeting of CD24-CAR-T cells and PMC159 PD-L1-CAR-T cells to cancer cells. 100% killing was observed in BxPC3 cells, and >80% killing was observed in SKOV-3 cells.

[0185] The results indicate that PD-L1 CAR-T cells can be used in conjunction with other CAR-T cells. When the PD-L1 pathway is activated in the tumor microenvironment, the combination of PD-L1 CAR-T cells and CD24 CAR-T cells can be used to jointly inhibit both pathways in vivo.

[0186] References

[0187] 1.Maus, MV, Haas, AR, Beatty, GL, Albelda, SM, Levine, BL, Liu, X., Zhao, Y., Kalos, M., and June, CH (2013). T cells expressing chimeric antigenreceptors can cause anaphylaxis in humans. Cancer Immunol Res 1, 26-31.

[0188] 2.Maus, MV, Grupp, SA, Porter, DL, and June, CH (2014). Antibody-modified T cells: CARs take the front seat for hematologic malignancies. Blood123, 2625-2635.

[0189] 3.Sgambato,A.,Casaluce,F.,Sacco,PC,Palazzolo,G.,Maione,P.,Rossi,A.,Ciardiello,F.,and Gridelli,C.(2016).Anti PD-1and PD-L1 Immunotherapy in theTreatment ofAdvanced Non-Small Cell Lung Cancer(NSCLC):A Review on ToxicityProfile and its Management.Curr Drug Saf 11,62-68.

[0190] 4. Boeye, A. (1986). Clonal isolation of hybridomas by manual single-cell isolation. Methods Enzymol 121, 332-340.

[0191] 5.Goluboskaya V,Wu L.Different Subsets of T Cells,Memory,EffectorFunctions,and CAR-T Immunotherapy.Cancers(Basel).2016Mar 15;8(3).pii:E36.doi:10.3390 / cancers8030036.Review. sequence list <110> Prometheus Biotech Hunan Yuantai Biotechnology Co., Ltd. <120> PD-L1-specific antibodies and anti-PD-L1 CAR-T cells <130> 119995-8010.WO01 <150> 63 / 044,115 <151> 2020-06-25 <160> 31 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 1 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 2 <211> 366 <212> DNA <213> mice <400> 2 cagatccagt tggtgcagtc tggacctgag ctgaagaacc ctggagagac agtcaagatc 60 tcctgcaagg cttctgggta taccttcaca aactatggaa tgaactgggt gaagcaggct 120 ccaggaaagg gtttaaagtg gatggggtgg ataaacaccc acactggaga gccaacatat 180 gctgatgact tcaagggacg gtttgccttc tcttcggaaa cctctgccag ctctgcctat 240 ttgcagatca acaacctcaa aaatgatgac atggctacat atttctgtgc aaaaggtacc 300 cacagagaag aaattccggc ctggttcgct tactggggcc aagggactct ggtcactgtc 360 tctgca 366 <210> 3 <211> 122 <212> PRT <213> Mouse <400> 3 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Asn Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Ser Glu Thr Ser Ala Ser Ser Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Asp Asp Met Ala Thr Tyr Phe Cys 85 90 95 Ala Lys Gly Thr His Arg Glu Glu Ile Pro Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 4 <211> 339 <212> DNA <213> Mus musculus <400> 4 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120 tacctgcaga aaccaggcca gtctccagag ctcctgatct acaaagtttc caacctattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 300 cccacgttcg gtgctgggac caagctggag ctgaaacgg 339 <210> 5 <211> 113 <212> PRT <213> Mus musculus <400> 5 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Glu Leu Leu Ile Tyr Lys Val Ser Asn Leu Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 Angry <210> 6 <211> 45 <212> DNA <213> artificial sequence <220> <223> synthetic <400> 6 ggtggcggtg gttctggtgg cggtggttct ggtggcggtg gttct 45 <210> 7 <211> 15 <212> PRT <213> artificial sequence <220> <223> synthetic <400> 7 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 8 <211> 750 <212> DNA <213> Mus musculus <400> 8 cagatccagt tggtgcagtc tggacctgag ctgaagaacc ctggagagac agtcaagatc 60 tcctgcaagg cttctgggta taccttcaca aactatggaa tgaactgggt gaagcaggct 120 ccaggaaagg gtttaaagtg gatggggtgg ataaacaccc acactggaga gccaacatat 180 gctgatgact tcaagggacg gtttgccttc tcttcggaaa cctctgccag ctctgcctat 240 ttgcagatca acaacctcaa aaatgatgac atggctacat atttctgtgc aaaaggtacc 300 cacagagaag aaattccggc ctggttcgct tactggggcc aagggactct ggtcactgtc 360 tctgcaggtg gcggtggttc tggtggcggt ggttctggtg gcggtggttc tgatgttttg 420 atgacccaaa ctccactctc cctgcctgtc agtcttggag atcaagcctc catctcttgc 480 agatctagtc agagcattgt acatagtaat ggaaacacct atttagaatg gtacctgcag 540 aaaccaggcc agtctccaga gctcctgatc tacaaagttt ccaacctatt ttctggggtc 600 ccagacaggt tcagtggcag tggatcaggg acagatttca cactcaagat cagcagagtg 660 gaggctgagg atctgggagt ttattactgc tttcaaggtt cacatgttcc tcccacgttc 720 ggtgctggga ccaagctgga gctgaaacgg 750 <210> 9 <211> 250 <212> PRT <213> Mus musculus <400> 9 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Asn Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Ser Glu Thr Ser Ala Ser Ser Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Asp Asp Met Ala Thr Tyr Phe Cys 85 90 95 Ala Lys Gly Thr His Arg Glu Glu Ile Pro Ala Trp Phe Ala Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Leu Met Thr Gln Thr 130 135 140 Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys 145 150 155 160 Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 165 170 175 Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Glu Leu Leu Ile Tyr Lys 180 185 190 Val Ser Asn Leu Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp 210 215 220 Leu Gly Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Pro Thr Phe 225 230 235 240 Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 245 250 <210> 10 <211> 63 <212> DNA <213> Homo sapiens <400> 10 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 11 <211> 21 <212> PRT <213> Homo sapiens <400> 11 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 12 gactacaaag acgatgacga caag 24 <210> 13 <211> 141 <212> DNA <213> Homo sapiens <400> 13 aagcccacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 60 cccctgtccc tgcgcccaga ggcgagccgg ccagcggcgg ggggcgcagt gcacacgagg 120 gggctggact tcgccagtga t 141 <210> 14 <211> 47 <212> PRT <213> Homo sapiens <400> 14 Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr 1 5 10 15 Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala 20 25 30 Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Ser Asp 35 40 45 <210> 15 <211> 204 <212> DNA <213> Homo sapiens <400> 15 ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60 gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 120 aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180 cgcgacttcg cagcctatcg ctcc 204 <210> 16 <211> 68 <212> PRT <213> Homo sapiens <400> 16 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 20 25 30 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 35 40 45 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 50 55 60 Ala Tyr Arg Ser 65 <210> 17 <211> 342 <212> DNA <213> Homo sapiens <400> 17 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac 180 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240 cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300 acctacgacg cccttcacat gcaggccctg ccccctcgct aa 342 <210> 18 <211> 113 <212> PRT <213> Homo sapiens <400> 18 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 50 55 60 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 65 70 75 80 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 85 90 95 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 100 105 110 Arg <210> 19 <211> 1542 <212> DNA <213> Mus musculus <400> 19 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggctagcc agatccagtt ggtgcagtct ggacctgagc tgaagaaccc tggagagaca 120 gtcaagatct cctgcaaggc ttctgggtat accttcacaa actatggaat gaactgggtg 180 aagcaggctc caggaaaggg tttaaagtgg atggggtgga taaacaccca cactggagag 240 ccaacatatg ctgatgactt caagggacgg tttgccttct cttcggaaac ctctgccagc 300 tctgcctatt tgcagatcaa caacctcaaa aatgatgaca tggctacata tttctgtgca 360 aaaggtaccc acagagaaga aattccggcc tggttcgctt actggggcca agggactctg 420 gtcactgtct ctgcaggtgg cggtggttct ggtggcggtg gttctggtgg cggtggttct 480 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 540 atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 600 tacctgcaga aaccaggcca gtctccagag ctcctgatct acaaagtttc caacctattt 660 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 720 agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 780 cccacgttcg gtgctgggac caagctggag ctgaaacggg actacaaaga cgatgacgac 840 aagctcgaga agcccaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 900 gcgtcgcagc ccctgtccct gcgcccagag gcgagccggc cagcggcggg gggcgcagtg 960 cacacgagg ggctggactt cgccagtgat aagccctttt gggtgctggt ggtggttggt 1020 ggagtcctgg cttgctatag cttgctagta acagtggcct ttatatttt ctgggtgagg 1080 agtaagaga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 1140 cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1200 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 1260 1320 cgggaccctg agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac 1380 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaggcgag 1440 cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1500 acctacgacg cccttcacat gcaggccctg ccccctcgct aa 1542 <210> 20 <211> 512 <212> PRT <213> Mus musculus <400> 20 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ala Ser Gln Ile Gln Leu Val Gln Ser Gly Pro 20 25 30 Glu Leu Lys Asn Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser 35 40 45 Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro 50 55 60 Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr His Thr Gly Glu 65 70 75 80 Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Ser Glu 85 90 95 Thr Ser Ala Ser Ser Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Asp 100 105 110 Asp Met Ala Thr Tyr Phe Cys Ala Lys Gly Thr His Arg Glu Glu Ile 115 120 125 Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 130 135 140 Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 165 170 175 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 180 185 190 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 195 200 205 Pro Glu Leu Leu Ile Tyr Lys Val Ser Asn Leu Phe Ser Gly Val Pro 210 215 220 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 225 230 235 240 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 245 250 255 Ser His Val Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 260 265 270 Arg Asp Tyr Lys Asp Asp Asp Asp Lys Leu Glu Lys Pro Thr Thr Thr 275 280 285 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 290 295 300 Leu Ser Leu Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val 305 310 315 320 His Thr Arg Gly Leu Asp Phe Ala Ser Asp Lys Pro Phe Trp Val Leu 325 330 335 Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val 340 345 350 Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His 355 360 365 Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys 370 375 380 His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 385 390 395 400 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 405 410 415 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 420 425 430 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 435 440 445 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 450 455 460 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 465 470 475 480 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 485 490 495 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 500 505 510 <210> 21 <211> 126 <212> DNA <213> Homo sapiens <400> 21 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 22 <211> 42 <212> PRT <213> Homo sapiens <400> 22 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 23 <211> 1521 <212> DNA <213> Mus musculus <400> 23 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggctagcc agatccagtt ggtgcagtct ggacctgagc tgaagaaccc tggagagaca 120 gtcaagatct cctgcaaggc ttctgggtat accttcacaa actatggaat gaactgggtg 180 aagcaggctc caggaaaggg tttaaagtgg atggggtgga taaacaccca cactggagag 240 ccaacatatg ctgatgactt caagggacgg tttgccttct cttcggaaac ctctgccagc 300 tctgcctatt tgcagatcaa caacctcaaa aatgatgaca tggctacata tttctgtgca 360 aaaggtaccc acagagaaga aattccggcc tggttcgctt actggggcca agggactctg 420 gtcactgtct ctgcaggtgg cggtggttct ggtggcggtg gttctggtgg cggtggttct 480 gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 540 atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 600 tacctgcaga aaccaggcca gtctccagag ctcctgatct acaaagtttc caacctattt 660 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 720 agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttcct 780 cccacgttcg gtgctgggac caagctgggag ctgaaacggc tcgagaagcc caccacgacg 840 ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 900 ccagaggcga gccggccagc ggcggggggc gcagtgcaca cgaggggct ggacttcgcc 960 agtgataagc ccttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 1020 ctagtaacag tggcctttat tattttctgg gtgaaacggg gcaagaagaa actcctgtat 1080 atattcaaac aaccatttat gagaccagta caaactactc aagagaaga tggctgtagc 1140 1200 1260 1320 1380 atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1440 gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1500 caggccctgc cccctcgcta a 1521 <210> 24 <211> 506 <212> PRT <213> Mus musculus <400> 24 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Ala Ser Gln Ile Gln Leu Val Gln Ser Gly Pro 20 25 30 Glu Leu Lys Asn Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser 35 40 45 Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro 50 55 60 Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr His Thr Gly Glu 65 70 75 80 Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Ser Glu 85 90 95 Thr Ser Ala Ser Ser Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Asp 100 105 110 Asp Met Ala Thr Tyr Phe Cys Ala Lys Gly Thr His Arg Glu Glu Ile 115 120 125 Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 130 135 140 Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 165 170 175 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 180 185 190 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 195 200 205 Pro Glu Leu Leu Ile Tyr Lys Val Ser Asn Leu Phe Ser Gly Val Pro 210 215 220 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 225 230 235 240 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 245 250 255 Ser His Val Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 260 265 270 Arg Leu Glu Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 275 280 285 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Ser 290 295 300 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 305 310 315 320 Ser Asp Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 325 330 335 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys 340 345 350 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 355 360 365 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 370 375 380 Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser 385 390 395 400 Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 405 410 415 Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 420 425 430 Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro 435 440 445 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 450 455 460 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 465 470 475 480 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 485 490 495 Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 505 <210> 25 <211> 360 <212> DNA <213> Homo sapiens <400> 25 gaagtgcagc tgctggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggctt tacctttagc agctatatta tgatgtgggt gcgccaggcg 120 ccgggcaaag gcctggaatg ggtgagcagc atttatccga gcggcggcat taccttttat 180 gcggataccg tgaaaggccg ctttaccatt agccgcgata acagcaaaaa caccctgtat 240 ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gcgcattaaa 300 ctgggcaccg tgaccaccgt ggattattgg ggccagggca ccctggtgac cgtgagcagc 360 <210> 26 <211> 120 <212> PRT <213> Homo sapiens <400> 26 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 27 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Synthesized <400> 27 ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagc 45 <210> 28 <211> 330 <212> DNA <213> Homo sapiens <400> 28 cagagcgcgc tgacccagcc ggcgagcgtg agcggcagcc cgggccagag cattaccatt 60 agctgcaccg gcaccagcag cgatgtgggc ggctataact atgtgagctg gtatcagcag 120 catccgggca aagcgccgaa actgatgatt tatgatgtga gcaaccgccc gagcggcgtg 180 agcaaccgct ttagcggcag caaaagcggc aacaccgcga gcctgaccat tagcggcctg 240 caggcggaag atgaagcgga ttattattgc agcagctata ccagcagcag cacccgcgtg 300 tttggcaccg gcaccaaagt gaccgtgctg 330 <210> 29 <211> 112 <212> PRT <213> Homo sapiens <400> 29 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Glu Thr Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn 50 55 60 Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser 65 70 75 80 Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr 85 90 95 Ser Ser Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 30 <211> 735 <212> DNA <213> Homo sapiens <400> 30 gaagtgcagc tgctggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60 agctgcgcgg cgagcggctt tacctttagc agctatatta tgatgtgggt gcgccaggcg 120 ccgggcaaag gcctggaatg ggtgagcagc atttatccga gcggcggcat taccttttat 180 gcggataccg tgaaaggccg ctttaccatt agccgcgata acagcaaaaa caccctgtat 240 ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gcgcattaaa 300 ctgggcaccg tgaccaccgt ggattattgg ggccagggca ccctggtgac cgtgagcagc 360 ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagccagag cgcgctgacc 420 cagccggcga gcgtgagcgg cagcccgggc cagagcatta ccattagctg caccggcacc 480 agcagcgatg tgggcggcta taactatgtg agctggtatc agcagcatcc gggcaaagcg 540 ccgaaactga tgatttatga tgtgagcaac cgcccgagcg gcgtgagcaa ccgctttagc 600 ggcagcaaaa gcggcaacac cgcgagcctg accattagcg gcctgcaggc ggaagatgaa 660 gcggattatt attgcagcag ctataccagc agcagcaccc gcgtgtttgg caccggcacc 720 aaagtgaccg tgctg 735 <210> 31 <212> 245 <212> PRT <213> Homo sapiens <400> 31 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gln Ser Ala Leu Thr Gln Pro Ala Ser 130 135 140 Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr 145 150 155 160 Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His 165 170 175 Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Asp Val Ser Asn Arg Pro 180 185 190 Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala 195 200 205 Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr 210 215 220 Cys Ser Ser Tyr Thr Ser Ser Ser Thr Arg Val Phe Gly Thr Gly Thr 225 230 235 240 Light Val Thr Val Leu 245< / flag> < / as>

Claims

1. A monoclonal anti-human PD-L1 antibody or its antigen-binding fragment, comprising: V composed of the amino acids shown in SEQ ID NO: 3 H and V composed of the amino acids shown in SEQ ID NO: 5 L The antibody binds to the human PD-L1 protein.

2. A single-stranded variable fragment (scFv), comprising: V composed of the amino acids shown in SEQ ID NO: 3 H and V composed of the amino acids shown in SEQ ID NO: 5 L The scFv therein binds to the human PD-L1 protein.

3. The scFv according to claim 2, further comprising a location located at V H With V L The joint between them.

4. The scFv according to claim 2, having the amino acid sequence shown in SEQ ID NO:

9.

5. A chimeric antigen receptor fusion protein (CAR), said chimeric antigen receptor fusion protein comprising, from the N-terminus to the C-terminus: (i) the scFv as described in claim 2, (ii) Transmembrane domains, (iii) at least one co-stimulatory domain, and (iv) Activate the structural domain.

6. The CAR according to claim 5, wherein, The scFv further includes the location located at V H With V L The joint between them.

7. The CAR according to claim 5, wherein, The co-stimulatory domain is CD28 or 4-1BB.

8. The CAR according to claim 5, wherein, The activation domain is CD3ζ.

9. The CAR according to claim 5, wherein it is encoded by the nucleotide sequence shown in SEQ ID NO: 19 or SEQ ID NO:

23.

10. A nucleic acid encoding the CAR of claim 5.

11. T cells or natural killer cells modified to express the CAR of claim 5.

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