Anti-pdl1 antibodies and their use in the preparation of antitumor formulations

Abstract

The present application relates to anti-PDL1 antibodies and their use in the preparation of anti-tumor preparations, a series of anti-PDL1 antibodies are screened, which have outstanding binding capacity with PDL1; the anti-PDL1 antibodies are combined with human immunoglobulin to form single-chain antibodies, which can efficiently block the binding effect of PD1 / PDL1 protein; further constructed CAR-T cells capable of secreting anti-PDL1 antibodies, which can relieve the inhibition of the PD1-PDL1 signal pathway in the tumor microenvironment on the CAR-T cells themselves, improve the activation degree of T cells, and greatly improve the killing effect of CAR-T cells on tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an anti-PDL1 antibody and its application in the preparation of anti-tumor agents. Background Technology

[0002] Immunotherapy for malignant tumors has developed rapidly in recent years, achieving remarkable clinical efficacy. In recent years, immune checkpoint monoclonal antibody therapy (such as PD-1 and PDL-1 monoclonal antibodies) has reactivated the killing effect on tumor-specific cytotoxic T cells by relieving the inhibition of various tumor microenvironment inhibitors. The overall efficacy rate for various malignant tumors can reach approximately 30%, and many patients, once they respond, can establish immune memory against tumor killing, maintaining a long-term killing effect. Chimeric antigen receptor (CAR) therapy, or CAR-T cell therapy, involves obtaining the patient's T cells, genetically modifying them in vitro to rapidly obtain tumor antigen-specific T cells, and then adoptively infusing them for treatment. Currently, several CAR-T therapy products for B-cell leukemia and myeloma have been approved globally, with complete remission rates exceeding 90% for relapsed / refractory B-cell leukemia. CAR-T cells are genetically modified with CAR genes and endowed with the ability to recognize tumor antigens (generally cell membrane proteins) and initiate killing or proliferation. Because CAR-T cells recognize antigens expressed on the cell membrane, rather than antigens that are presented to the cell surface after binding to MHC molecules, they can bypass the MHC restriction of T cells.

[0003] Currently, several CAR-T cell therapies targeting solid tumors are undergoing clinical trials, including GPC3, Mesothelin, GD2, HER2, EGFR, EGFRvIII, IL-13Rα2, PSMA, and CEA. While some clinical trials have yielded relatively promising results, overall, the efficacy of CAR-T therapy for solid tumors still lags significantly behind that for hematologic malignancies. This is primarily because solid tumor tissues possess an immunosuppressive microenvironment compared to hematologic malignancies. This microenvironment includes Treg cells, cancer-associated fibroblasts (CAFs), and tumor-associated macrophages (TAMs). These cells and their secreted cytokines can suppress T cell function directly or indirectly.

[0004] To disrupt the immunosuppressive microenvironment of solid tumors and enhance the therapeutic effect of CAR-T therapy, clinical approaches include: disrupting the tumor microenvironment through radiotherapy and chemotherapy to create an inflammatory environment and activate innate immunity; combining this with immune checkpoint antibody therapy (such as PD1 / PDL1); specifically blocking related signaling pathways with negative immune regulators (such as IDO small molecule inhibitors); and overexpressing positive immune regulators (such as IL2, IL7, and IL-12). These strategies can all act on the immunosuppressive microenvironment of solid tumors, improving the survival and killing effect of infiltrating CAR-T cells. However, current clinical data on CAR-T therapy in the treatment of solid tumors show that these methods have not achieved ideal clinical results. The treatment of solid tumors with CAR-T therapy still has a long way to go. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an anti-PDL1 antibody and its application in the preparation of antitumor agents.

[0006] The technical solution adopted in this invention is: an anti-PDL1 antibody, named antibody D19, antibody F62 or antibody G47;

[0007] The variable region of antibody D19 light chain includes LCDR1 with the sequence shown in SEQ ID No. 1, LCDR2 with the sequence shown in SEQ ID No. 2, and LCDR3 with the sequence shown in SEQ ID No. 3; the variable region of heavy chain includes HCDR1 with the sequence shown in SEQ ID No. 4, HCDR2 with the sequence shown in SEQ ID No. 5, and HCDR3 with the sequence shown in SEQ ID No. 6.

[0008] The variable region of the light chain of antibody F62 includes LCDR1 as shown in SEQ ID No. 11, LCDR2 as shown in SEQ ID No. 12, and LCDR3 as shown in SEQ ID No. 13; the variable region of the heavy chain includes HCDR1 as shown in SEQ ID No. 14, HCDR2 as shown in SEQ ID No. 15, and HCDR3 as shown in SEQ ID No. 16.

[0009] The variable region of the light chain of antibody G47 includes LCDR1 as shown in SEQ ID No. 21, LCDR2 as shown in SEQ ID No. 22, and LCDR3 as shown in SEQ ID No. 23; the variable region of the heavy chain includes HCDR1 as shown in SEQ ID No. 24, HCDR2 as shown in SEQ ID No. 25, and HCDR3 as shown in SEQ ID No. 26.

[0010] Preferably, the amino acid sequence of the light chain variable region of antibody D19 is shown in SEQ ID No. 7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 9;

[0011] The amino acid sequence of the light chain variable region of antibody F62 is shown in SEQ ID No. 17, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 19.

[0012] The amino acid sequence of the light chain variable region of antibody G47 is shown in SEQ ID No. 27, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 29.

[0013] Preferably, the nucleotide sequence encoding the variable region of the light chain of antibody D19 is shown in SEQ ID No. 8, and the nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID No. 10;

[0014] The nucleotide sequence encoding the light chain variable region of antibody F62 is shown in SEQ ID No. 18, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 20.

[0015] The nucleotide sequence encoding the light chain variable region of antibody G47 is shown in SEQ ID No. 28, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No. 30.

[0016] A recombinant antibody containing an anti-PDL1 antibody, which is used as an scFv molecule.

[0017] Preferably, the light chain variable region and the heavy chain variable region are connected by a linking sequence, as shown in SEQ ID No. 31.

[0018] Preferably, the Fc portion is human immunoglobulin.

[0019] Preferably, the human immunoglobulin is IgG1.

[0020] A CAR-T cell that secretes PDL1 antibody, wherein the CAR-T cell contains an expression vector capable of expressing a recombinant antibody containing anti-PDL1 antibody.

[0021] Application of recombinant antibodies containing anti-PDL1 antibodies or CAR-T cells that secrete PDL1 antibodies in the preparation of antitumor agents.

[0022] Preferred for use in anti-brain tumor preparations.

[0023] Preferably, the anti-tumor effect is achieved by blocking the PD1-PDL1 signaling pathway and activating T cell function.

[0024] The advantages and positive effects of this invention are: it provides a series of highly efficient anti-PDL1 antibodies that bind to PDL1, which can block the binding of PD1-PDL1; when constructing CAR-T cells that express the anti-PDL1 antibody and using them to kill tumor cells, the competitive binding of the anti-PDL1 antibody to PDL1 blocks the PD1-PDL1 signaling pathway in the tumor microenvironment, reduces the inhibitory effect on T cells, activates T cell function, and significantly improves the therapeutic effect of tumor treatment; especially for solid tumors with complex microenvironments, it can significantly improve the therapeutic effect of CAR-T. Attached Figure Description

[0025] Figure 1 A schematic diagram of the structure of the expressed anti-PDL1 scFv-Fc antibody molecule;

[0026] Figure 2 SDS-PAGE results of three anti-PDL1 scFv-Fc antibodies;

[0027] Figure 3 The expression of PDL1 in the U87 cell line was analyzed by flow cytometry after staining with anti-PDL1 antibody and isotype.

[0028] Figure 4 The blocking effect of 100 ng / ml anti-PDL1 antibody molecules on PDL1 in U87 cells;

[0029] Figure 5 The effect of 10 ng / ml anti-PDL1 antibody molecules on PDL1 blocking in U87 cells;

[0030] Figure 6 The effect of 1 ng / ml anti-PDL1 antibody molecule on PDL1 blocking in U87 cells;

[0031] Figure 7 The CAR molecular structure of an autocrine anti-PDL1 scFv antibody targeting IL13Rα2;

[0032] Figure 8 The expression of PDL1 in the U251 cell line was analyzed by flow cytometry after staining with anti-PDL1 antibody and isotype.

[0033] Figure 9 Effector cells: In vitro killing experiment of IL13Rα2 CAR-T cells and U251-GFP tumor cells co-incubated under a target cell ratio of 3:1;

[0034] Figure 10In vitro killing experiment of IL13Rα2 CAR-T cells and U251-GFP tumor cells co-incubated under the condition of effector cell:target cell ratio of 1:1. Detailed Implementation

[0035] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0036] This invention relates to anti-PDL1 antibodies and their application in the preparation of antitumor agents. Anti-PDL1 antibodies belong to the category of immune checkpoint inhibitors. They work by blocking the binding of PD-L1 protein to T cells, thereby relieving the suppression of the immune system by tumors and activating the body's own immunity to kill tumor cells. Specifically, anti-PDL1 antibodies can specifically bind to PD-L1 on the surface of tumor cells or immune cells, blocking the binding of PD-L1 to the PD-1 receptor on T cells, and restoring the antitumor activity of T cells.

[0037] This invention provides three anti-PDL1 antibodies, named Antibody D19, Antibody F62, and Antibody G47, all of which are single-chain antibodies. The light chain variable region of Antibody D19 includes LCDR1, LCDR2, and LCDR3, with the LCDR1 sequence shown in SEQ ID No. 1, the LCDR2 sequence shown in SEQ ID No. 2, and the LCDR3 sequence shown in SEQ ID No. 3, and also includes a frame region sequence outside each complementarity-determining region; the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, with the HCDR1 sequence shown in SEQ ID No. 4, the HCDR2 sequence shown in SEQ ID No. 5, and the HCDR3 sequence shown in SEQ ID No. 6, and also includes a frame region sequence outside each complementarity-determining region. The amino acid sequence of the light chain variable region of antibody D19 is shown in SEQ ID No. 7; it can be obtained by translation and expression of the nucleotide sequence shown in SEQ ID No. 8, or by translation and expression of a similar nucleotide sequence with codon optimization; the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 9; it can be obtained by translation and expression of the nucleotide sequence shown in SEQ ID No. 10, or by translation and expression of a similar nucleotide sequence with codon optimization.

[0038] SEQ ID No. 1: TGTSSDVGGYNYVS

[0039] SEQ ID No. 2: EVSKRPS

[0040] SEQ ID No. 3: SSYAGSNNLGV

[0041] SEQ ID No.4: SYAIS

[0042] SEQ ID No.5:GIIPIFGTANYAQKFQG

[0043] SEQ ID No.6:LAAAGTLDY

[0044] SEQ ID No.7:

[0045] QSALTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGSNNLGVFGGGTKVTVL

[0046] SEQ ID No.8:

[0047] CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGAGGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCAGCTCATATGCAGGCAGCAACAATTTGGGAGTATTCGGCGGAGGGACCAAGGTGACCGTCCTC

[0048] SEQ ID No.9:

[0049] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARLAAAGTLDYWGQGTLVTVSS

[0050] SEQ ID No.10:

[0051] CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAAC TACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGACTAGCAGCAGCTGGTACTCTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA

[0052] The light chain variable region of antibody F62 includes LCDR1, LCDR2, and LCDR3. The sequence of LCDR1 is shown in SEQ ID No. 11, the sequence of LCDR2 is shown in SEQ ID No. 12, and the sequence of LCDR3 is shown in SEQ ID No. 13. It also includes frame region sequences outside the complementarity-determining regions. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. The sequence of HCDR1 is shown in SEQ ID No. 14, the sequence of HCDR2 is shown in SEQ ID No. 15, and the sequence of HCDR3 is shown in SEQ ID No. 16. It also includes frame region sequences outside the complementarity-determining regions. The amino acid sequence of the light chain variable region of antibody F62 is shown in SEQ ID No. 17; it can be obtained by translation and expression of the nucleotide sequence shown in SEQ ID No. 18, or by translation and expression of a similar codon-optimized nucleotide sequence. The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 19; it can be obtained by translation and expression of the nucleotide sequence shown in SEQ ID No. 20, or by translation and expression of a similar codon-optimized nucleotide sequence.

[0053] SEQ ID No. 11: SGSTSNIGSNYVY

[0054] SEQ ID No. 12: RNNQRPS

[0055] SEQ ID No. 13: AAWDDSLSGLV

[0056] SEQ ID No. 14: SYAIS

[0057] SEQ ID No.15:GIIPIFGTANYAQKFQG

[0058] SEQ ID No.16:SQGYGSGTYFDY

[0059] SEQ ID No.17:

[0060] QPVLTQPPSASGTPGQRVTISCSGSTSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGLVFGGGTKLTVL

[0061] SEQ ID No.18:

[0062] CAGCCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCACCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTCTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA

[0063] SEQ ID No.19:

[0064] QVQLVQSGAEVKKPGSSVKVSCKASGCTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCASSQGYGSGTYFDYWGQGTLVTVSS

[0065] SEQ ID No.20:

[0066] CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGATGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACG CACAGAAGTTCCAGGGCAGAGTCACGATTACCGCAGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGTTCTCAGGGCTATGGTTCGGGGACCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCA

[0067] The light chain variable region of antibody G47 includes LCDR1, LCDR2, and LCDR3. The sequence of LCDR1 is shown in SEQ ID No. 21, the sequence of LCDR2 is shown in SEQ ID No. 22, and the sequence of LCDR3 is shown in SEQ ID No. 23. It also includes frame region sequences outside the complementarity-determining regions. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3. The sequence of HCDR1 is shown in SEQ ID No. 24, the sequence of HCDR2 is shown in SEQ ID No. 25, and the sequence of HCDR3 is shown in SEQ ID No. 26. It also includes frame region sequences outside the complementarity-determining regions. The amino acid sequence of the light chain variable region of antibody G47 is shown in SEQ ID No. 27; it can be obtained by translation and expression of the nucleotide sequence shown in SEQ ID No. 28, or by translation and expression of a similar codon-optimized nucleotide sequence. The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 29; it can be obtained by translation and expression of the nucleotide sequence shown in SEQ ID No. 30, or by translation and expression of a similar codon-optimized nucleotide sequence.

[0068] SEQ ID No. 21: SGSSSNIGADYDVR

[0069] SEQ ID No. 22: GNSNRPS

[0070] SEQ ID No. 23: QSYDSILSGSL

[0071] SEQ ID No. 24: SYAIS

[0072] SEQ ID No.25:MIIPIFGIANYAQKFQG

[0073] SEQ ID No.26:APYRGYSYGYFDY

[0074] SEQ ID No.27:

[0075] QSVLTQPPSVSGAPGQRVTISCSGSSSNIGADYDVRWYQHLPGTPPILLIYGNSNRPSGVPDRFSGSKSGTSTSLAITGLHAEDEDDYYCQSYDSILSGSLFGGGTKVTVL

[0076] SEQ ID No.28:

[0077] CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGACAGAGGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATCGGGGCAGATTATGATGTACGCTGGTACCAGCACCTTCCAGGAACACCCCCCATACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAACCTCCCTGGCCATCACTGGGCTCCATGCTGAGGATGAGGATGATTATTACTGCCAGTCTTATGACAGCATCCTGAGTGGTTCGCTCTTCGGCGGAGGGACCAAGGTCACCGTCCTA

[0078] SEQ ID No.29:

[0079] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGMIIPIFGIANYAQKFQGRVTITADKSTSTAYMELSILRSEDTAVYYCASAPYRGYSYGYFDYWGQGTLVTVSS

[0080] SEQ ID No.30:

[0081] CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCTGGACAAGGGCTTGAGTGGATGGGAATGATCATCCCTATCTTTGGTATAGCAAACTACGCA CAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACGGCCTACATGGAGCTGAGCATCCTGAGATCTGAGGACACTGCCGTGTATTACTGTGCGAGTGCCCCCTACCGTGGATACAGCTATGGGTACTTTGACTACTGGGGCCAGGGAACTCTGGTCACCGTCTCCTCA

[0082] When preparing antibodies D19, F62, or G47, expression vectors capable of expressing amino acid sequences as shown in SEQ ID No. 7 and SEQ ID No. 9, SEQ ID No. 17 and SEQ ID No. 19, or SEQ ID No. 27 and SEQ ID No. 29 can be pre-constructed, wherein the corresponding light chain variable region and heavy chain variable region are connected by a linker sequence; in some embodiments of the present invention, the linker sequence is shown in SEQ ID No. 31.

[0083] SEQ ID No. 31 GGGGSGGGGSGGGGS

[0084] The constructed expression vector was transfected into host cells, and after induction culture, antibodies D19, F62, or G47 were extracted and purified from the engineered strain product. Detection and verification showed that antibodies D19, F62, and G47 could all bind to PD-L1 with high specificity.

[0085] This invention also provides anti-PDL1 scFv-Fc recombinant proteins, which are formed by combining antibody D19, antibody F62, or antibody G47 as scFv molecules with Fc molecules to form antibody D19-Fc, antibody F62-Fc, or antibody G47-Fc. To prepare proteins capable of acting on immune cells, in some embodiments of this invention, human immunoglobulin is used as the Fc molecule and linked to one of the three anti-PDL1 antibody scFv molecules, for example, lgG1 is used as the Fc molecule and linked to antibody D19, antibody F62, or antibody G47 to form antibody D19-Fc, antibody F62-Fc, and antibody G47-Fc. The amino acid sequence of lgG1 is shown in SEQ ID No. 32 and is encoded by the nucleic acid sequence shown in SEQ ID No. 33.

[0086] SEQ ID No. 32

[0087] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0088] SEQ ID No. 33

[0089] GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCC ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGTCCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

[0090] The anti-PDL1 scFv-Fc recombinant protein can bind to the PDL1 protein, thereby effectively blocking the PD1 / PDL1 protein binding effect and preventing the inhibition of T cell activation. Experimental verification showed that all three anti-PDL1 scFv-Fc proteins could achieve outstanding blocking effects on PD1 / PDL1 protein binding, and even at a protein concentration of 1 ng / ml, they had a strong blocking effect on PD1 / PDL1 binding.

[0091] This invention also provides a CAR-T cell capable of self-secreting anti-PDL1 antibodies. Nucleic acid fragments expressing antibodies D19-Fc, F62-Fc, and G47-Fc are constructed into CAR expression lentiviral vectors via PCR amplification and homologous recombination seamless ligation technology, thereby constructing CAR-T cells capable of self-secreting anti-PDL1 antibodies. These cells can express anti-PDL1 antibodies and bind to PD1 molecules on T cells, thus mediating the inhibition of T cell function and enhancing the killing effect of CAR-T. Especially for CAR-T cells targeting solid tumors, expressing anti-PDL1 antibodies that disrupt the inhibitory effect of the tumor microenvironment, and by relieving the inhibitory effect of the PD1-PDL1 signaling pathway on CAR-T cells themselves in the tumor microenvironment, can effectively overcome the challenges of CAR-T therapy for solid tumors and significantly improve efficacy. In addition, the anti-PDL1 antibody secreted by CAR-T cells can effectively activate tumor antigen-reactive T cells that already exist around tumor cells, which can further enhance the synergistic killing effect on tumor cells. At the same time, it can potentially solve the problem that CAR-T cells cannot recognize and kill these tumor cells due to the loss of tumor antigens.

[0092] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0093] Example 1: Screening for anti-PDL1 scFv sequences

[0094] Anti-PDL1 scFv sequences were screened from a fully human phage display library.

[0095] To construct a primitive phage library, an appropriate amount of the original phage library culture was inoculated into a 500 ml Erlenmeyer flask containing 2×YT medium, with an initial OD value of approximately 0.1. 100 μg / mL ampicillin and 2% glucose were added. The culture was incubated at 37°C and 250 rpm for approximately 3 hours until the OD600 value reached approximately 0.5. Approximately 3×10⁻⁶ cells were then collected. 12M13KO7 helper phage was added to the above bacterial culture and incubated at 37°C for 30 minutes. The infected culture was then transferred to a 50ml centrifuge tube and centrifuged at 2400g, 12°C for 30 minutes, discarding the supernatant. The pellet was resuspended in 600ml of 2×YT medium, and ampicillin (100 μg / mL), kanamycin (60 μg / mL), and IPTG (100 μM) were added to a final concentration. The culture was incubated overnight at 30°C, 200 rpm. The culture was collected and centrifuged at 2400g, 12°C for 30 minutes. 36ml of the supernatant was transferred to a new 50ml centrifuge tube, and 9ml of PEG / NaCl was added. The mixture was thoroughly mixed and incubated on ice for 1-2 hours. After 1-2 hours, the mixture was centrifuged at 4000 rpm, 4°C for 30 minutes. Discard the supernatant gently, resuspend the phage pellet in 3 ml of 1×PBS / 5% FBS solution, and store at 4°C for later use.

[0096] The purified phage antibody library was added to a pre-prepared 96-well plate coated with PDL1-His protein. The plate was incubated at 4°C for 60 minutes. The plates were washed 10-30 times with 5% FBS / PBS to remove as much supernatant as possible. The phages were eluted with 500 μl of glycine (0.1 M, pH 2.2) and incubated at room temperature for 10 minutes. After centrifugation, the supernatant was transferred to a new centrifuge tube, and 50 μl of Tris-HCl (pH 9.5) was added to neutralize the pH. The eluted phages were then transferred to a new 15 ml centrifuge tube for TG1 infection. The panning process was repeated twice. The resulting monoclonal phages were analyzed by phage ELISA to identify three strains: D19, F62, and G47.

[0097] The three selected strains were subjected to PCR amplification. The forward primer sequence is shown in SEQ ID No. 34, and the reaction primer sequence is shown in SEQ ID No. 35. Finally, the scFv sequence fragment was obtained.

[0098] SEQ ID No.34: GACTGGAAAGCGGGCAGTGAGC

[0099] SEQ ID No.35: CCTTATTAGCGTTTGCCATCT

[0100] Example 2: Expression and purification of anti-PDL1-scFv-Fc protein

[0101] The anti-PDL1 scFv sequence was linked to IgG1 to form an anti-PDL1-scFv-Fc antibody molecule, with the structure shown below. Figure 1As shown in the figure. The three PDL1 scFv sequences obtained in Example 1 were inserted into the HindIII and BamHI restriction sites to construct transient expression plasmid vectors fused with Fc. The transient expression plasmid vector sequences are shown in SEQ ID No. 36, forming expression vectors for D19-Fc, F62-Fc and G47-Fc recombinant proteins, respectively.

[0102] SEQ ID No. 36:

[0103]

[0104] The expression vector was transformed into 293T suspension cells, which served as the expression host. After cell resuscitation, the cells were continuously passaged for expansion, maintaining a viable cell density of 0.5 × 10⁻⁶ cells / year. 6 ~3.0×10 6 Cells / mL, cell viability ≥95%, logarithmic growth phase cells were used for transfection. Before transfection, the density of suspension 293T cells was adjusted to 1.0 × 10⁶ cells / mL. 6 ~2.0×10 6 Cells / mL. Prepare a DNA mixture according to a total plasmid concentration of 1 μg / mL. Prepare a transfection reagent dilution solution according to a DNA:transfection reagent mass ratio of 1:2. After incubation at room temperature, mix the DNA dilution solution with the DNA dilution solution and let stand at room temperature for 10–20 min to form a transfection complex. Add the complex dropwise to the cell culture, mix gently, and continue to culture on a shaker. 18–24 h after transfection, add special feed and expression enhancer according to the cell growth status; continue culture for 5 days, and terminate the culture when the cell viability drops to about 70%. Collect the culture supernatant, centrifuge at 8000×g at 4℃ for 15–20 min to remove cell debris, clarify the supernatant through a 0.22 μm filter membrane, and purify by Protein A column affinity chromatography. The purified protein was concentrated by ultrafiltration and buffer replacement using a 30 kDa ultrafiltration system, centrifuged at 3000–4000 × g at 4 °C. The solution was then replaced 3–5 times with PBS (pH 7.4) buffer to remove salts, small molecule impurities, and elution reagents, yielding a high-purity antibody solution. Following the above steps, proteins D19-Fc, F62-Fc, and G47-Fc were prepared and purified, respectively.

[0105] The purified antibody was detected by SDS-PAGE and Coomassie Brilliant Blue staining, respectively, to assess the denaturing and non-denaturing states of proteins D19-Fc, F62-Fc, and G47-Fc. The results are as follows: Figure 2 As shown, the molecular weights of the three antibody proteins all meet expectations.

[0106] Example 3: PD-L1 antibody blocking PD1 / PDL1 binding assay

[0107] Using the human glioblastoma (GBM) cell line U87 as a model, the blocking effect of three anti-PDL1 scFv-Fc antibodies on PD1 / PDL1 protein binding was detected.

[0108] Human glioma cell line U87 was cultured in RPMI-1640 or DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody, and passaged approximately every 2-3 days. After staining with commercial anti-PDL1 antibody and isotype, flow cytometry was used to analyze PDL1 expression in U87 cells. The results are as follows: Figure 3 As shown, U87 cells highly express PDL1 protein, making them suitable for detecting the blocking effect of three anti-PDL1 scFv-Fc antibodies on PD1 / PDL1 protein binding.

[0109] Take 0.5×10 6 U87 cells were added to 96-well plates, and then D19-Fc, F62-Fc, or G47-Fc proteins were added to the wells at final concentrations of 1 ng / ml, 10 ng / ml, or 100 ng / ml, respectively. The plates were incubated at 4°C for 30 minutes. After 30 minutes, the cells were centrifuged, the supernatant was discarded, and the cells were washed once with PBS. Then, PE-labeled PD1-Fc protein was added, and the plates were incubated at 4°C for 30 minutes. After 30 minutes, the cells were centrifuged, the supernatant was discarded, and the cells were washed once with PBS. Flow cytometry analysis was performed, and the results are shown below. Figures 4-6 As shown, the three anti-PDL1 scFv-Fc antibodies, D19-Fc, F62-Fc, and G47-Fc, can effectively block the binding of PD1 and PDL1, and the blocking effect is better with the increase of antibody concentration.

[0110] Example 4: Preparation of CAR-T cells that secrete anti-PDL1 antibodies

[0111] The three anti-PDL1 scFv antibody fragments obtained in Example 1 were constructed into CAR expression lentiviral vectors using PCR amplification and homologous recombination seamless ligation technology, respectively, to obtain CAR molecules targeting IL13Rα2 that can self-secrete anti-PDL1 scFv antibodies. The structures are as follows: Figure 7 The fragment shown was ligated into the vector with the sequence shown in SEQ ID No. 36. Lentiviral suspensions were prepared by transient transfection of 293T cells with PEI, and the lentiviruses were further concentrated by ultracentrifugation at 25000g for 3 hours to prepare lentiviruses expressing D19, F62 or G47 antibodies, respectively; the lentiviruses were stored at -80℃ for later use.

[0112] On day 1, T cells were activated using anti-CD3 / CD28 magnetic beads, and the cell density was adjusted to 1×10⁻⁶. 6 / ml. On day 2, add the corresponding volume of lentivirus to T cells at MOI=2 for infection. On day 5, remove the anti-CD3 / CD28 magnetic beads, perform cell counting, passage, and fluid replacement. On day 9, collect 3×10⁶ cells. 5T cells were stained with anti-EGFR antibody and analyzed by flow cytometry to detect the CAR positivity rate. CAR-T cells were continuously cultured for approximately 11 days, then harvested and cryopreserved for later use.

[0113] Example 5: In vitro cytotoxicity assay of CAR-T cells secreting anti-PDL1 antibody

[0114] Using the human glioma cell line U251 as a model, the cytotoxicity of CAR-T cells prepared in Example 4 was tested.

[0115] U251 cells were cultured in RPMI-1640 or DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody, and passaged approximately every 2-3 days. After staining with commercial anti-PDL1 antibody and isotype, flow cytometry was used to analyze PDL1 expression in U251 cells. The results are as follows: Figure 8 As shown, U251 cells highly express PDL1 protein; U251 cells naturally express IL13Rα2 protein at high levels, and IL13Rα2 CAR-T cells can target U251 cells. Therefore, it can be used for subsequent in vitro killing experiments to verify whether autocrine PDL1 antibodies can relieve the inhibition of T cell function mediated by tumor cells expressing PDL1 molecules and binding to T cell PD1 molecules.

[0116] Twelve hours before co-culturing CAR-T cells with tumor target cells in the cytotoxicity assay, U251 cells were seeded into flat-bottomed 96-well plates at 5000 cells / 100 μL per well. After 8–12 hours, once the cells had fully adhered, the CAR-T cells were diluted to an appropriate cell density and then co-incubated with U251-GFP tumor cells at different effector-to-target ratios (e.g., E:T = 3:1 and 1:1). The 96-well plates were placed in an InCucyte S3 scanner, and scanning parameters were set. Four days after scanning, the total cumulative green fluorescence intensity (GCU x µm² / well) was analyzed to calculate the tumor cell killing efficiency. Results are shown in Figure 9. Figure 10 As shown, the killing effect of IL13Rα2 CAR-T cells with three autocrine PDL1 antibody molecules was superior to that of conventional IL13Rα2 CAR-T cells; among them, the PDL1 antibody F62 had the strongest effect in promoting CAR-T killing.

[0117] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. An anti-PDL1 antibody, characterized in that: It was named antibody D19, antibody F62, or antibody G47. The variable region of antibody D19 light chain includes LCDR1 with the sequence shown in SEQ ID No. 1, LCDR2 with the sequence shown in SEQ ID No. 2, and LCDR3 with the sequence shown in SEQ ID No. 3; the variable region of heavy chain includes HCDR1 with the sequence shown in SEQ ID No. 4, HCDR2 with the sequence shown in SEQ ID No. 5, and HCDR3 with the sequence shown in SEQ ID No.

6. The variable region of the light chain of antibody F62 includes LCDR1 as shown in SEQ ID No. 11, LCDR2 as shown in SEQ ID No. 12, and LCDR3 as shown in SEQ ID No. 13; the variable region of the heavy chain includes HCDR1 as shown in SEQ ID No. 14, HCDR2 as shown in SEQ ID No. 15, and HCDR3 as shown in SEQ ID No.

16. The variable region of the light chain of antibody G47 includes LCDR1 as shown in SEQ ID No. 21, LCDR2 as shown in SEQ ID No. 22, and LCDR3 as shown in SEQ ID No. 23; the variable region of the heavy chain includes HCDR1 as shown in SEQ ID No. 24, HCDR2 as shown in SEQ ID No. 25, and HCDR3 as shown in SEQ ID No.

26.

2. The anti-PDL1 antibody according to claim 1, characterized in that: The amino acid sequence of the light chain variable region of antibody D19 is shown in SEQ ID No. 7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

9. The amino acid sequence of the light chain variable region of antibody F62 is shown in SEQ ID No. 17, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

19. The amino acid sequence of the light chain variable region of antibody G47 is shown in SEQ ID No. 27, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

29.

3. The anti-PDL1 antibody according to claim 2, characterized in that: The nucleotide sequence encoding the light chain variable region of antibody D19 is shown in SEQ ID No. 8, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No.

10. The nucleotide sequence encoding the light chain variable region of antibody F62 is shown in SEQ ID No. 18, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No.

20. The nucleotide sequence encoding the light chain variable region of antibody G47 is shown in SEQ ID No. 28, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No.

30.

4. A recombinant antibody containing an anti-PDL1 antibody, characterized in that: Anti-PDL1 antibody as an scFv molecule.

5. The recombinant antibody containing anti-PDL1 antibody according to claim 4, characterized in that: The light chain variable region and the heavy chain variable region are connected by a linking sequence, as shown in SEQ ID No.

31.

6. The recombinant antibody containing anti-PDL1 antibody according to claim 5, characterized in that: The Fc portion is human immunoglobulin.

7. The recombinant antibody containing anti-PDL1 antibody according to claim 6, characterized in that: Human immunoglobulin is IgG1.

8. A CAR-T cell that secretes its own PDL1 antibody, characterized in that: The CAR-T cells contain an expression vector capable of expressing the recombinant antibody containing the anti-PDL1 antibody as described in any of claims 4-7.

9. The use of the recombinant antibody containing anti-PDL1 antibody as described in any one of claims 4-7 or the CAR-T cells containing autocrine PDL1 antibody as described in claim 8 in the preparation of an anti-brain tumor agent.

10. The application according to claim 9, characterized in that: By blocking the PD1-PDL1 signaling pathway, T cell function is activated, thereby achieving anti-tumor effects.

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