Antigen binding fragment specifically targeting CLL1, chimeric antigen receptor and application of antigen binding fragment and chimeric antigen receptor in treatment of leukemia

Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses an antigen binding fragment of a specific targeting CLL1, a chimeric antigen receptor and application of the antigen binding fragment and the chimeric antigen receptor in treatment of leukemia. On one hand, an extra heavy chain variable region is provided with Lwj001 antigen binding fragments of VHCDR1: DTYMY, VHCDR2: RIDPANGNTKYDTKFQG and VHCDR3: FLRD, and on the other hand, a heavy chain variable region is provided with 6e8002 antigen binding fragments of VHCDR1: GYYMH, VHCDR2: RINPYNGAIIYNQNFKD and VHCDR3: DYGSGDYAMDY. The invention also provides application of the antigen binding fragment in treatment of leukemia. In the invention, a novel CAR-T cell targeting CLL1 is successfully developed and optimized, so that the targeting property and killing property of the CAR-T cell are enhanced, and a new reliable means is provided for CAR-T cell treatment of leukemia.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to an antigen binding fragment specifically targeting CLL1, a chimeric antigen receptor and application thereof in treating leukemia. BACKGROUND

[0002] Leukemia is a group of hematopoietic malignancies that seriously endanger human health, which can be mainly divided into myeloid and lymphoid categories. Among them, acute myeloid leukemia (AML) is one of the most common acute leukemias in adults, characterized by high heterogeneity, low long-term survival rate and high relapse tendency. Although traditional chemotherapy regimens are effective for some patients, the five-year survival rate is still not satisfactory, which highlights the urgent need to develop new and efficient treatment strategies. Chimeric antigen receptor T cell (CAR-T) therapy has achieved revolutionary success in B-cell malignancies, significantly improving the remission rate and survival of patients, providing a new idea for the treatment of AML. However, to successfully apply CAR-T therapy in the field of acute myeloid leukemia (AML), researchers and clinicians still face many complex and challenging challenges. Currently, the main obstacle of AML CAR-T therapy is the lack of truly ideal tumor-specific targets, which makes it difficult to ensure the precision and safety of treatment. In traditional research, some common AML targets such as CD33 and CD123 are highly expressed on the surface of AML cancer cells, but these targets are also widely present on normal hematopoietic stem cells (HSCs). This feature poses a serious potential risk: when CAR-T cells are designed to target these antigens, they can effectively kill tumor cells, but also cause significant damage to normal hematopoietic function. This damage is usually severe and long-term, and can even lead to life-threatening complications such as bone marrow failure. In fact, such adverse reactions have been observed in some early clinical trials, and in some cases even caused fatal events, which sounded the alarm for the safety of treatment. Therefore, exploring a new target that is highly specifically expressed in AML cells but almost not expressed or expressed at very low levels in healthy hematopoietic cells has become a core task for developing safe and effective AML chimeric antigen receptor T cell (CAR-T) therapy. Only by identifying such a target can we ensure that CAR-T cells can minimize damage to normal tissues when attacking cancer cells, thereby significantly reducing treatment-related side effects and improving the overall feasibility and clinical application value of the therapy. This requires not only more in-depth research on the molecular biological characteristics of AML, but also the combination of advanced gene editing technology and high-throughput screening methods to identify truly potential targets from a large number of candidate targets. This process is undoubtedly challenging, but it is a key link to promote the clinical success of AML CAR-T therapy.

[0003] C-type lectin-like molecule-1 (CLL1, also known as CLEC12A) is considered as a highly potential CAR-T therapeutic target as a myeloid lineage antigen. Studies have shown that CLL1 is highly expressed in more than 90% of AML patients' malignant cells, including chemotherapy-resistant leukemia stem cells (LSCs), while there is no expression on healthy hematopoietic stem cells (HSCs). This unique expression pattern makes CLL1 an ideal therapeutic target, which is expected to effectively eliminate AML cells while significantly reducing damage to normal hematopoietic function, thereby avoiding the long-term bone marrow suppression risk caused by traditional targets, and providing a safer and more effective treatment plan for a wider AML patient population.

[0004] Currently, although some progress has been made in the field of CAR (chimeric antigen receptor) research targeting CLL1, it is undeniable that there is still much room for improvement and an urgent need in many key aspects. For example, in the design of CAR structure, how to optimize the selection of scFv (single-chain variable region) sequence, how to determine the optimal length and structural characteristics of the hinge region, the type selection of the transmembrane region and the combination strategy of the costimulatory signal domain have not been completely solved. The design of each component will have an important impact on the functional performance of CAR-T cells, and the reasonable arrangement of these factors is directly related to the treatment effect. At the same time, in the evaluation of killing efficiency in vitro experiments and animal models, the existing research results have not reached the ideal level, and both the specific recognition ability of tumor cells and the killing efficiency need to be further improved. Based on this status quo, the present application is dedicated to developing a new anti-CLL1 CAR structure, which has better targeting and can significantly improve the therapeutic efficacy compared to the prior art, thereby providing a more reliable and effective means for the treatment of related diseases. SUMMARY

[0005] The present application provides antigen-binding fragments, chimeric antigen receptors that specifically target CLL1 and their use in the treatment of leukemia, mainly to solve the problem that the current CAR targeting CLL1 and treating AML still needs to be improved.

[0006] To solve the above problems, the technical scheme adopted by the present application is as follows:

[0007] The first aspect of the present application provides an antigen-binding fragment capable of specifically targeting CLL1. In the content described in this part, there are various forms of antigen-binding fragments, which can be antigen-binding fragments containing only a heavy chain variable region, or antigen-binding fragments containing both a heavy chain variable region and a light chain variable region.

[0008] Regarding antigen-binding fragments containing only the heavy chain variable region, the heavy chain variable region is considered to contain heavy chain complementarity-determining regions (CDMs). As long as it contains the following CDMs, it should be considered within the scope of this invention. Further, the heavy chain variable region contains three CDMs. When the antigen-binding fragment is Lwj001, these three CDMs are: VHCDR1: DTYMY, VHCDR2: RIDPANGNTKYDTKFQG, VHCDR3: FLRD; when the antigen-binding fragment is 6e8002, the three CDMs are: VHCDR1: GYYMH, VHCDR2: RINPYNGAIIYNQNFKD, VHCDR3: DYGSGDYAMDY. Specifically, if the three CDMs of the heavy chain variable region of the antigen-binding fragment match the contents listed in this paragraph, it should be considered within the scope of this invention.

[0009] For antigen-binding fragments that simultaneously encompass both heavy chain variable regions and light chain variable regions, the reference standard for both the heavy chain variable region and the light chain variable region is whether they respectively contain heavy chain complementarity-determining regions and light chain complementarity-determining regions. As long as the following heavy chain complementarity-determining regions and light chain complementarity-determining regions are included, it should be considered within the scope of this invention. This antigen-binding fragment specifically targeting CLL1 typically includes a light chain variable region in addition to the heavy chain variable region (primarily based on the heavy chain complementarity-determining region). The antigen-binding fragment possesses a light chain variable region containing three light chain complementarity-determining regions. When the antigen-binding fragment is Lwj001, the three light chain complementarity-determining regions (CDRs) are: VLCDR1: KSSQSLLDSDGKTYLN, VLCDR2: QVSKQDS, VLCDR3: WQGTHFPYT; when the antigen-binding fragment is 6e8002, the three light chain CDRs are: VLCDR1: SASQGISNYLN, VLCDR2: YTSSLHS, VLCDR3: QQYSKFPLT. Similarly, if the three CDRs of the variable regions of the light chains of an antigen-binding fragment are consistent with those listed in this paragraph, they should be considered within the scope of this invention.

[0010] In each group, the CDR and FR regions alternate, folding together to form a stable three-dimensional structure. The FR region provides robust support for the CDR ring through its β-sheet structure, ensuring that the CDR ring can be exposed on the molecular surface with the correct spatial orientation and configuration, thereby effectively recognizing and binding antigens. The following sections will further introduce the heavy chain variable region and light chain variable region in conjunction with specific FR regions. Regarding the antigen-binding fragment specifically targeting CLL1, the heavy chain variable region sequence of the Lwj001 antigen-binding fragment is: EIQLQQSGAELVKPGASVKLSCTASGFNIK DTYMYWVKQRPEQGLEWIG RIDPANGNTKYDTKFQ G KATITADTSSNTVYLQLSSLTSEDTAVYYCAL FLRD The light chain variable region sequence of the WGQGTTLTVSS;Lwj001 antigen-binding fragment is: DVVMTQTPLTLSVIIGQPASISC KSSQSLLDSDGKTYLN WLFQRPGQSPKRLIY QVSKQDS GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPYT The heavy chain variable region sequence of the FGGGTKLEIK;6e8002 antigen-binding fragment is: EVQLQQSGPELVKPGASVKISCKASGYSFT GYYMH WVKQSHIKSLEWIG RINPYNGAIIYNQNFKD KASLTVEKSSSTAYMEFYSLTSEDSAVYYCAR DYGSGDYAMDY The light chain variable region sequence of the WGQGTSVTVSS;6e8002 antigen-binding fragment is: DIQMTQTTSSLSASLGDRVTISC SASQGISNYLN WYQQKPDGAVKLLIY YTSSLHS GVPSRFSGSGSGTDYSLTISNLEPEDIATYYC QQYSKFPLT FGAGTKLELK.

[0011] The heavy chain variable regions and light chain variable regions of antigen-binding fragments Lwj001 and 6e8002 are linked by linker peptides. Preferably, the linker peptide is a flexible peptide linker, a rigid linker peptide, or a naturally derived peptide. More preferably, the flexible peptide linker sequence is at least one of GGGGSGGGGSGGGGS, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, the rigid linker peptide is EAAAKEAAAKEAAAK, and the naturally derived peptide is EPKSCDKTHTCPPCP. In some cases, the sequences of the heavy chain variable regions and light chain variable regions of antigen-binding fragments Lwj001 and 6e8002 may be as shown in SEQ ID NO. 5 to SEQ ID NO. 8.

[0012] A second aspect of the present invention provides a chimeric antigen receptor that specifically targets CLL1, comprising an antigen-binding fragment, a hinge region, a transmembrane domain, and an intracellular signal transduction domain; wherein the antigen-binding fragment is any of the antigen-binding fragments specifically targeting CLL1 mentioned above.

[0013] Regarding the interpretation of antigen-binding fragments, the antigen-binding fragments referred to here should still primarily be based on whether or not they contain VHCDR or VLCDR. Any antigen-binding fragment that specifically targets CLL1 and contains any of the aforementioned forms should be within the scope of this invention.

[0014] Regarding the hinge region, transmembrane domain, and intracellular signal transduction domain: The hinge region sequence is: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY; the transmembrane domain sequence is: IWAPLAGTCGVLLLSLVITLYC or FWVLVVVGGVLACYSLLVTVAFIIFWV; the intracellular signal transduction domain sequence is: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR or RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0015] Based on the above, when antigen-binding fragments are included, in some cases: the amino acid sequence of the chimeric antigen receptor Lwj001 that specifically targets CLL1 is shown as SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequence of the chimeric antigen receptor 6e8002 that specifically targets CLL1 is shown as SEQ ID NO.3 or SEQ ID NO.4.

[0016] A third aspect of the present invention provides a nucleic acid and an expression vector comprising the nucleic acid. This nucleic acid encodes any of the chimeric antigen receptors specifically targeting CLL1 described above. It should be understood that all nucleic acids capable of encoding any of the chimeric antigen receptors specifically targeting CLL1 described in the second aspect above should be within the scope of the present invention. The complete sequence of the nucleic acid may vary depending on the hinge region, transmembrane domain, and intracellular signal transduction domain, but should still be within the scope of the present invention. Specifically, when the amino acid sequence of the chimeric antigen receptor Lwj001 specifically targeting CLL1 is as shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the amino acid sequence of the chimeric antigen receptor 6e8002 specifically targeting CLL1 is as shown in SEQ ID NO. 3 or SEQ ID NO. 4, the full-length nucleotide sequence of the Lwj001 CAR is correspondingly shown in SEQ ID NO. 9 and SEQ ID NO. 10, and the full-length nucleotide sequence of the 6e8002 CAR is correspondingly shown in SEQ ID NO. 11 and SEQ ID NO. 12. The expression vector contains a nucleic acid that can encode any of the chimeric antigen receptors that specifically target CLL1 in the second aspect mentioned above. Specifically, it can be a lentivirus, a lipid nanoparticle expression vector, etc. The specific form is not particularly limited, but it is based on containing the above-mentioned nucleic acid.

[0017] A fourth aspect of this invention provides CLL1 CAR-T cells. These CLL1 CAR-T cells contain any of the nucleic acids described in the third aspect above, or their cell membrane surface expresses any of the chimeric antigen receptors specifically targeting CLL1 described in the third aspect above. CLL1 CAR-T cells are primarily obtained by transfecting T cells with any of the aforementioned CARs specifically targeting CLL1. The CAR gene is introduced into T cells, thereby performing "genetic modification" and "functional upgrade." The modified CAR-T cells, due to receiving different types of CAR transfection, contain any of the nucleic acids described in the third aspect (but are not limited to this, mainly based on the complementarity-determining region described in the first aspect above); or, after transfection with different types of CARs, their cell membrane surface expresses any of the aforementioned chimeric antigen receptors specifically targeting CLL1 described in the third aspect above. In this process, the modified CAR-T cells can accurately recognize and efficiently kill tumor cells.

[0018] The fifth aspect of this invention provides the application of any of the aforementioned chimeric antigen receptors specifically targeting CLL1 or any of the aforementioned CLL1 CAR-T cells in the treatment of leukemia or the preparation of drugs for treating leukemia. It mainly includes the following: the application of chimeric antigen receptors specifically targeting CLL1 in the treatment of leukemia or the preparation of drugs for treating leukemia; and the application of CLL1 CAR-T cells in the treatment of leukemia or the preparation of drugs for treating leukemia. The application in the treatment of leukemia indicates that it can be directly used to treat the disease, while the application in the preparation of drugs for treating leukemia indicates that it is primarily used to prepare drugs for treating leukemia, and the dosage form of the drugs for treating leukemia is not limited, generally being CAR-T therapy drugs. In specific applications, chimeric antigen receptors specifically targeting CLL1 often exert their side effects in the form of a pre-existing "drug," such as in the fourth aspect, where T cells are transfected with chimeric antigen receptors, resulting in "genetic modification" and "functional upgrades" of the T cells, enabling them to target and kill tumors. CLL1 CAR-T cells can be directly used to treat leukemia, and their specific mode of action can be prepared into a drug formulation that meets clinical standards. However, this section does not require that CAR-T cells be prepared in vitro. Where conditions permit, directly injecting a vector carrying the CAR gene into the patient to obtain CAR-T cells in vivo should not be excluded from the scope of this invention. Specifically, any chimeric antigen receptor that specifically targets CLL1 should be within the scope of this invention. The leukemia mentioned in this paragraph is often acute myeloid leukemia, but other treatable leukemias with similar effects are not excluded.

[0019] This disclosure reveals the successful development and continuous optimization of a novel CAR-T cell specifically targeting CLL1. This CAR-T cell exhibits significantly enhanced ability to recognize specific targets and kill cancer cells. This brings hope to leukemia patients (especially AML patients), provides a more reliable new approach to CAR-T cell therapy, adds a powerful tool to the fight against the disease, and is expected to improve treatment efficacy and enhance patient survival and quality of life. Attached Figure Description

[0020] Figure 1 A schematic diagram of the structure of a chimeric antigen receptor targeting CLL1;

[0021] Figure 2 ELISA results after three rounds of cloning and ELISA screening in hybridoma selection and cloning culture;

[0022] Figure 3 The images show lentiviral expression vectors containing the Lwj001 gene and lentiviral expression vectors containing the 6e8002 gene.

[0023] Figure 4 This shows the expression of CAR+ on T cells;

[0024] Figure 5 Flow cytometry diagram of hybridoma cell supernatant binding to target cells;

[0025] Figure 6 For monoclonal antibody subtype determination;

[0026] Figure 7 The killing rate of CLL1-targeting CAR-T cells against Thrp-1 cells. Detailed Implementation

[0027] The invention will be further described below with reference to specific research examples.

[0028] I. Experimental Methods

[0029] (1) Mouse immunization steps and determination of immune effect

[0030] a. Take BALB / c mice that are 6 to 8 weeks old and immunize them 6 to 10 weeks before the planned fusion. Collect serum or plasma samples before immunization as a baseline control for antibody screening. Collect blood samples from the mice by orbital blood collection or by tail vein. Store the serum or plasma with 0.1% sodium azide at -20°C for later use.

[0031] b. For initial immunization, take 100ug of commercial CLL1 protein, add Freund's complete adjuvant and mix thoroughly to prepare a stable emulsion, and inject subcutaneously at multiple points;

[0032] c. A second immunization is administered 2-3 weeks later, with the same dosage as above, but with Freund's incomplete adjuvant administered subcutaneously.

[0033] d. Two to three weeks later, administer the third immunization at the same dose as above, without adjuvant, via intraperitoneal injection;

[0034] e. After 5 to 7 days, orbital blood was collected from mice to measure the titer, and plasma antibody titers were detected by ELISA;

[0035] f. After 2 to 3 weeks, administer a booster immunization at a dose of 100 to 200 μg, without adjuvant, via intraperitoneal or intravenous injection.

[0036] g. The spleen was harvested and fused 3 days later.

[0037] (2) Hybridoma fusion

[0038] a. The total number of cells is 1×10 8 Spleen cells and myeloma SP2 / 0 cells (2×10⁻⁶) 7 ~5×10 7 Mix, add the remaining 1640 culture medium to 30 ml, and mix thoroughly;

[0039] a. Centrifuge at 1000 rpm / min for 5–10 minutes, then discard the supernatant;

[0040] b. Gently tap the bottom of the test tube to loosen and evenly distribute the cell precipitate, and preheat it in a 40°C water bath;

[0041] c. Insert a 1ml pipette into the bottom of the tube and add 1ml of 50% polyethylene glycol (PEG) preheated to 40°C to the cell pellet over about 45 seconds. Add the PEG dropwise into the centrifuge tube while gently stirring with the pipette tip to ensure that the cells and PEG are fully mixed. Let it stand for 90 seconds.

[0042] d. Add 10 ml of incomplete 1640 medium to terminate the fusion reaction. Add the first 5 ml at a rate of 20 s / ml, starting slowly and gradually increasing the speed, while stirring continuously. Add the remaining 5 ml along the cell wall over 2-4 minutes. The resulting cell suspension does not require further mixing.

[0043] e. Centrifuge at 1000 rpm / min for 5 min, then discard the supernatant;

[0044] f. Gently blow up the bottom fusion cells with 2ml of fusion cell culture medium (containing 10% hybridoma addition factor and 10% FBS), and bring the volume to 100ml with culture medium. Distribute the mixture evenly into 96-well plates and incubate in an incubator.

[0045] g. After 3 days, add 2 drops of solution to each well. Colonies can be observed after 5-6 days. After 10 days, change the solution and perform ELISA detection.

[0046] (3) Hybridoma selection and clonal culture

[0047] a. Preliminary screening: 12-15 days after fusion, when the cells have grown to fill 1 / 4 to 1 / 2 of the wells, the culture supernatant is detected by free ELISA to screen for positive clones; the microplate is coated with goat anti-mouse polyclonal antibody (0.5 μg / well) and incubated overnight at 4°C, then blocked with 5% milk powder at 37°C for 2 h, washed 5 times with washing buffer for 3 min each time, the liquid is patted dry, 50 μL of cell culture supernatant and 50 μL of 0.2% PBST are added, mouse immune serum is used as the positive control, SP2 / 0 culture supernatant is used as the negative control, and washing buffer is used as the blank control, and incubated at 37°C for 1 h; the microplate is incubated with biotin-labeled goat anti-mouse IgG antibody at 37°C for 1 h, then washed, 100 μL of Beyotime chromogenic solution is added per well, reacted at room temperature in the dark for 15 min, and 100 μL of stop solution is added per well to stop the reaction, and the absorbance value at 450 nm is measured by microplate reader;

[0048] b. Re-screening: Two days later, a re-screening is performed. Wells with ODs > 1.5 from the previous step are selected for re-screening; the remaining wells are discarded. Figure 2 );

[0049] c. Results showed that by immunizing three female Ba1b / c mice with CLL1 protein as the antigen and performing one fusion, two hybridoma cell lines that could secrete high-affinity CLL1 antibodies were obtained. After three cloning and ELISA screenings, hybridoma cell lines that secreted CLL1 monoclonal antibodies were obtained. These hybridoma cells were cryopreserved several times and cultured in vitro for more than 3 months, and they were able to stably secrete CLL1 monoclonal antibodies. Finally, they were frozen at -80°C.

[0050] (4) Detection of hybridoma antibody binding to natural CLL1 epitope of tumor cells

[0051] a. Take the supernatant of hybridoma cells and incubate it with CLL1-expressing leukemia cells (such as AML cells) at 4°C for 40 min;

[0052] b. Wash once with PBS, then add 0.5 μL of goat anti-mouse flow cytometry antibody;

[0053] c. Wash once with PBS and incubate at room temperature in the dark for 15 minutes;

[0054] d. Flow cytometry testing.

[0055] (5) Sequencing of hybridoma antibody

[0056] a. RNA extraction after hybridoma cell lysis;

[0057] b. Reverse transcription: Using the extracted RNA as a template, cDNA was obtained in two steps using the Yiqiao Technology Reverse Transcription Kit (by first opening the secondary structure of the RNA and then allowing the primers to bind to the RNA and polymerize the cDNA to obtain cDNA).

[0058] c. Using cDNA obtained through reverse transcription as a template, the amplified fragment is inserted into an expression vector or commercial vector by PCR amplification. The ligation product is then transformed into E. coli, and the antibody sequence is obtained through cloning identification and sequencing.

[0059] (6) Construction of CAR plasmid

[0060] a. such as Figure 1 As shown, a full-length CAR gene fragment encoding anti-CLL1 scFv, CD8α hinge and transmembrane region, 4-1BB co-stimulatory domain and CD3ζ signaling domain was obtained by gene synthesis, and AgeI and NheI restriction sites were introduced at both ends of the fragment.

[0061] Full-length amino acid sequence of Lwj001 CAR - 1 (SEQ ID NO.1): MALPVTALLLPLALLLHAARPDVVMTQTPLTLSVIIGQPASISCKSSQSLLDSDGKTYLNWLFQRPGQSPKRLIYQVSKQDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIKGGGGSGGGGSGGGGSEIQLQQSGAELVKPGASVKLSCTASGFNIKDTYMYWVKQRPEQGLEWIGRIDPANGNTKYDTKFQGKATITADTSSNTVYLQLSSLTSEDTAVYYCALFLRDWGQGTTLTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

[0062] Full-length amino acid sequence of Lwj001 CAR - 2 (SEQ ID NO.2): MALPVTALLLPLALLLHAARPEIQLQQSGAELVKPGASVKLSCTASGFNIKDTYMYWVKQRPEQGLEWIGRIDPANGNTKYDTKFQGKATITADTSSNTVYLQLSSLTSEDTAVYYCALFLRDWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQTPLTLSVIIGQPASISCKSSQSLLDSDGKTYLNWLFQRPGQSPKRLIYQVSKQDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

[0063] 6e8002 Full-length Amino Acid Sequence of CAR-1 (SEQ ID NO.3): MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGAVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKFPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKISCKASGYSFTGYYMHWVKQSHIKSLEWIGRINPYNGAIIYNQNFKDKASLTVEKSSSTAYMEFYSLTSEDSAVYYCARDYGSGDYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

[0064] 6e8002 CAR full-length amino acid sequence-2 (SEQ ID NO.4):MALPVTALLLPLALLLHAARPEVQLQQSGPELVKPGASVKISCKASGYSFTGYYMHWVKQSHIKSLEWIGRINPYNGAIIYNQNFKDKASLTVEKSSSTAYMEFYSLTSEDSAVYYC ARDYGSGDYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGAVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIA TYYCQQYSKFPLTFGAGTKLELKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR FPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

[0065] The full-length nucleotide sequence of Lwj001 CAR is shown in SEQ ID NO.9 and SEQ ID NO.10, and the full-length nucleotide sequence of 6e8002 CAR is shown in SEQ ID NO.11 and SEQ ID NO.12.

[0066] b. The pLVX lentiviral vector backbone was double-digested with the same restriction enzyme and purified by gel electrophoresis;

[0067] c. The CAR fragment and the linearized vector were ligated using T4 DNA ligase, and the ligation product was transformed into Stbl3 chemocompetent cells and plated on ampicillin-resistant plates to screen for positive clones.

[0068] d. After selecting single-clone colonies for expansion culture, positive clones are initially screened by plasmid mini-extraction, enzyme digestion identification, and colony PCR;

[0069] e. After verifying the complete CAR expression cassette using Sanger sequencing to ensure sequence accuracy, high-purity plasmids were prepared using an endotoxin-free plasmid large-scale extraction kit for subsequent virus packaging.

[0070] (7) Construction of lentiviral expression vector (pLVX-puro-CLL1 CAR LWJ, pLVX-puro- CLL1 CAR 6E8) Figure 3(8) Isolation of human peripheral T cells and preparation of CAR-T cells

[0071] During the construction process, combined with Figure 4 The lentiviral expression vector map shown is constructed using the following method:

[0072] a. Carrier design and preparation;

[0073] b. Obtaining the target gene and performing enzyme digestion and ligation;

[0074] c. Transformation and clone screening;

[0075] d. Enzyme digestion verification and sequencing;

[0076] e. Virus packaging.

[0077] (9) Determination of CAR-T cell killing function

[0078] a. Centrifuge to separate plasma at 1500 rpm for 5 min at a decreasing speed of 5. You can place the plasma in a blood collection tube for centrifugation, remove the plasma, and then dilute with PBS equal to the original blood volume.

[0079] b. Ficol centrifugation procedure, Ficol volume = volume of diluted PB, 400g for 30min, temperature 12℃, with a 6°C increase and 3°C decrease.

[0080] c. Absorb the white membrane layer, dilute with at least 5 times the volume of PBS, 1500 rpm for 3 min, and remove the supernatant;

[0081] d. The cell pellet was clearly red. Add 1 ml of red blood cell lysis buffer to resuspend the pellet, incubate at room temperature for 5 min, and then centrifuge to remove the supernatant.

[0082] e. Add an appropriate volume of PBS to resuspend the count, and repeat every 10... 7 Add 100 μL of MACS buffer to each cell;

[0083] f. Vortex CD3 microbeads for 30 seconds, every 10 7 Add 10 μL of magnetic beads to each cell, mix the sample thoroughly, and incubate at 4°C for 15 minutes;

[0084] g. per 10 7 Add 2 mL of MACS buffer to each cell and centrifuge at 1500 rpm for 5 minutes at 4°C.

[0085] h. Connect the LS column to a compatible MACS separator to prepare for cell separation. Place a 15 mL sterile centrifuge tube under the column and add 3 mL of MACS buffer to the column;

[0086] i. After centrifugation, aspirate and discard the supernatant. Resuspend the cells in 500 μL of cold MACS buffer;

[0087] j. Load the cell suspension onto the prepared LS column;

[0088] k. Thoroughly wash the column with 3 mL of MACS buffer. Repeat this step twice.

[0089] 1. Discard the liquid that has flowed through and place a new 15 mL sterile centrifuge tube under the column;

[0090] m. Apply 5 mL of MACS buffer and immediately place it on the plunger. Maintain moderate, continuous pushing to recover the labeled pan-T cells, then perform cell counting;

[0091] n. cells at a rate of 1×10 6 The cells were plated in a pre-prepared plate and activated for 48 hours. Lentiviral transfection was then performed. The expression of CAR+ on T cells was measured on day 5 before and after transfection. Figure 5 ).

[0092] Figure 5

[0093] a. Mix CFSE-labeled target cells with effector CAR-T cells at different effector-to-target ratios (E:T ratio) (commonly 2:1, 1:1, 1:2, 1:4);

[0094] b. Volume: 200 μL / well (96-well plate), culture conditions: 37℃, 5% CO2. Various controls were established: target cells cultured alone (background apoptosis); target cells + non-transduced T cells (non-specific killing control), incubated for 18–24 hours.

[0095] c. Add an appropriate amount of PI, incubate at room temperature in the dark for 15 minutes, and then perform flow cytometry analysis.

[0096] II. Experimental Results and Result Analysis

[0097] Result 1: Two CLL1-resistant hybridomas were successfully obtained.

[0098] Balb / C mice were immunized with the extracellular CLL1 protein as an antigen. Plasma was collected before and after immunization. CLL1 protein was plated, and the titer of anti-CLL1 antibodies in the plasma was detected by ELISA. After the third immunization, the mouse plasma was diluted 100k times, and the presence of anti-CLL1 antibodies was still detectable. Three days after the mice were boosted with tail vein and intraperitoneal immunization, the mouse spleen was fused with SP2 / 0 myeloma cells. After selection in HAT medium and semi-solid medium, the cells were cultured as single clones. The cell culture supernatant was repeatedly tested for CLL1 by ELISA, and two specific anti-CLL1 hybridoma cell lines were finally obtained, which were named Lwj001 and 6e8002, respectively.

[0099] Results showed that anti-CLL1 antibody recognized and bound to both Thrp-1 cells and Hl-60 cells.

[0100] To demonstrate the binding ability of the two hybridoma cell lines obtained above to CLL1 protein, Thrp-1 cells and HL-60 cells were incubated sequentially with hybridoma cell supernatant and secondary antibody PE goat anti-mouse antibody. Flow cytometry results showed that the antibodies produced by the two hybridoma cell lines could bind to CLL1+ Thrp-1 cells and HL-60 cells, and were comparable to those of the commercially available anti-CLL1 antibody (Biolegend 353603(50C1)). Figure 6 The above results preliminarily confirm that both Lwj001 and 6e8002 can recognize and bind to natural CLL1 epitopes. Figure 7 The staining effects of commercially available antibodies, Lwj001, and 6e8002 on the HL-60 / THP-1 cell line are shown. Antibody 6e8002: 92.7% binding rate to HL-60 cells and 99.4% binding rate to THP-1 cells; Antibody Lwj001: 92.7% binding rate to HL-60 cells and 99.4% binding rate to THP-1 cells.

[0101] Results of anti-CLL1 antibody subtype identification

[0102] Subtype identification was performed using the Proteintech subtype identification kit, and the results are as follows: ​ As shown, the Lwj001 monoclonal antibody heavy chain belongs to the IgG2 type, and the light chain is of the Kappa type; the 6e8002 monoclonal antibody heavy chain belongs to the IgG1 type, and the light chain is of the Kappa type. It has strong effector function characteristics, can strongly activate the immune system, and efficiently kill CLL1+ leukemia cells.

[0103] Result 4: Hybridoma cell sequencing results

[0104] Anti-CLL1 hybridoma cells were lysed, and the cell genome was obtained by reverse transcription PCR. Then, antibody variable region PCR was performed, and the antibody amino acid sequence was finally obtained by sequencing analysis and comparison with database.

[0105] The heavy and light chain amino acid sequences of the hybridoma antibody are as follows:

[0106] Full-length amino acid sequence of Lwj001 light chain (SEQ ID NO.5): MRSRDQFLFLLVLWIRETNGDVVMTQTPLTLSVIIGQPASISCKSSQSLLDSDGKTYLNWLFQRPGQSPKRLIYQVSKQDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC*

[0107] Full-length amino acid sequence of Lwj001 heavy chain (SEQ ID NO.6): MKCSWVIFFLMAVVTGVNSEIQLQQSGAELVKPGASVKLSCTASGFNIKDTYMYWVKQRPEQGLEWIGRIDPANGNTKYDTKFQGKATITADTSSNTVYLQLSSLTSEDTAVYYCALFLRDWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK-

[0108] 6e8002 light chain full-length amino acid sequence (SEQ ID NO.7):MMSSAQLLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGAVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKF PLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC-

[0109] 6e8002 heavy chain full-length amino acid sequence (SEQ ID NO.8):MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKASGYSFTGYYMHWVKQSHIKSLEWIGRINPYNGAIIYNQNFKDKASLTVEKSSSTAYMEFYSLTSEDSA VYYCARDYGSGDYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASS TKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPA PIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK-

[0110] As a result, 5CLL1 CAR-T cells successfully recognized and killed Thrp-1 cells in vitro.

[0111] Using flow cytometry, NTD-T cells (normal T cells not transduced with CAR), Lwj001 CAR-T cells, or 6e8002 CAR-T cells were co-incubated with Thrp-1 cells at effector-target ratios of 2:1, 1:1, 1:2, and 1:4 for 18 hours. Results are as follows: ​As shown, both Lwj001 CAR-T cells and 6e8002 CAR-T cells exhibited strong killing power.

[0112] III. Analysis of Research Findings

[0113] This research represents a significant breakthrough in the field of CAR-T cell therapy for leukemia. A novel CLL1-targeting CAR-T cell was successfully developed and optimized, and its in vitro killing ability was evaluated. This study successfully developed CLL1 CAR-T cells with strong antigen-specific killing activity against leukemia cell lines and primary leukemia cells from patients. This research not only confirms the feasibility of CLL1 as an effective target for CAR-T therapy in leukemia (especially AML), but also improves its therapeutic efficacy through optimized CAR design. It provides solid scientific support for the clinical translation of CAR-T therapy for leukemia, especially its advantages in addressing myelosuppression, which holds promise for reshaping the treatment landscape of leukemia.

[0114] IV. Application Prospects and Effects

[0115] This study on CLL1-targeted CAR-T cell therapy in leukemia (especially AML) demonstrates great potential for clinical application and is expected to bring safer and more effective new treatment options to leukemia patients.

[0116] (1) Improved treatment safety: The most prominent advantage of CLL1 is that it is highly expressed in malignant cells in more than 90% of AML patients, but not in healthy hematopoietic stem cells (HSCs). This means that CAR-T cells targeting CLL1 can more accurately identify and kill tumor cells while minimizing the impact on normal cells.

[0117] (2) Accelerating clinical translation: The results of this study and other related preclinical and early clinical trials provide a solid scientific basis for clinical trials of anti-CLL1CAR-T cells in a larger population.

[0118] In conclusion, CLL1-targeted CAR-T cell therapy represents a significant advancement in leukemia treatment. Its high specificity, high efficacy, and low off-target toxicity make it a highly promising treatment modality. With ongoing research and the accumulation of clinical experience, CLL1 CAR-T therapy is expected to become an important component of standard treatment regimens for leukemia patients in the near future.

[0119] Those skilled in the art will appreciate that various modifications to the above embodiments can be made without departing from the overall spirit and concept of the present invention. For any aspects not detailed herein, reference can be made to the prior art. All such modifications fall within the protection scope of the present invention. The protection scheme of the present invention is defined by the appended claims.

Claims

1. An antigen-binding fragment that specifically targets CLL1, characterized in that, The antigen-binding fragment has a heavy chain variable region, which includes three heavy chain complementarity-determining regions: When the antigen-binding fragment is the Lwj001 antigen-binding fragment, the three heavy chain complementarity-determining regions are: VHCDR1: DTYMY, VHCDR2: RIDPANGNTKYDTKFQG, VHCDR3: FLRD; When the antigen-binding fragment is the 6e8002 antigen-binding fragment, the three heavy chain complementarity-determining regions are: VHCDR1: GYYMH, VHCDR2: RINPYNGAIIYNQNFKD, and VHCDR3: DYGSGDYAMDY.

2. The antigen-binding fragment specifically targeting CLL1 according to claim 1, characterized in that, The antigen-binding fragment has a light chain variable region, which includes three light chain complementarity-determining regions: When the antigen-binding fragment is the Lwj001 antigen-binding fragment, the three light chain complementarity-determining regions are: VLCDR1: KSSQSLLDSDGKTYLN, VLCDR2: QVSKQDS, VLCDR3: WQGTHFPYT; When the antigen-binding fragment is the 6e8002 antigen-binding fragment, the three light chain complementarity-determining regions are: VLCDR1: SASQGISNYLN, VLCDR2: YTSSLHS, VLCDR3: QQYSKFPLT.

3. The antigen-binding fragment specifically targeting CLL1 according to claim 2, characterized in that, The heavy chain variable region sequence of the Lwj001 antigen-binding fragment is: EIQLQQSGAELVKPGASVKLSCTA SGFNIKDTYMYWVKQRPEQGLEWIGRIDPANGNTKYDTKFQGKATITADTSS NTVYLQLSSLTSEDTAVYYCALFLRDWGQGTTLTVSS. The light chain variable region sequence of the Lwj001 antigen-binding fragment is: DVVMTQTPLTLSVIIGQPASIS CKSSQSLLDSDGKTYLNWLFQRPGQSPKRLIYQVSKQDSGVPDRFTGSGSGT DFTLKISRVEAEDLGVYYCWQGTHFPYTFGGGTKLEIK; The heavy chain variable region sequence of the 6e8002 antigen-binding fragment is: EVQLQQSGPELVKPGASVKIS CKASGYSFTGYYMHWVKQSHIKSLEWIGRINPYNGAIIYNQNFKDKASLTVE KSSSTAYMEFYSLTSEDSAVYYCARDYGSGDYAMDYWGQGTSVTVSS; The light chain variable region sequence of the 6e8002 antigen-binding fragment is: DIQMTQTTSSLSASLGDRVTIS CSASQGISNYLNWYQQKPDGAVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTI SNLEPEDIATYYCQQYSKFPLTFGAGTKLELK; In antigen-binding fragments Lwj001 and 6e8002, both the heavy chain variable region and the light chain variable region are linked by linker peptides; preferably, the linker peptide is a flexible peptide linker, a rigid linker peptide, or a naturally derived peptide; more preferably, the flexible peptide linker sequence is at least one of GGGGSGGGGSGGGGS, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, the rigid linker peptide is EAAAKEAAAKEAAAK, and the naturally derived peptide is EPKSCDKTHTCPPCP.

4. A chimeric antigen receptor that specifically targets CLL1, characterized in that, It includes an antigen-binding fragment, a hinge region, a transmembrane domain, and an intracellular signal transduction domain; wherein the antigen-binding fragment is the antigen-binding fragment specifically targeting CLL1 as described in any one of claims 1 to 3.

5. The chimeric antigen receptor specifically targeting CLL1 according to claim 4, characterized in that, The hinge region sequence is: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIY; The transmembrane domains are: IWAPLAGTCGVLLLSLVITLYC or FWVLVVVGGVLACYS LLVTVAFIIFWV; The intracellular signaling domain is: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR or RSKRSRLLHSDYMNMTPR RPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

6. The chimeric antigen receptor specifically targeting CLL1 according to claim 4, characterized in that, The amino acid sequence of the chimeric antigen receptor Lwj001 that specifically targets CLL1 is shown in SEQ ID NO.1 or SEQ ID NO.2, and the amino acid sequence of the chimeric antigen receptor 6e8002 that specifically targets CLL1 is shown in SEQ ID NO.3 or SEQ ID NO.

4.

7. Nucleic acid, characterized in that, The nucleic acid encodes the chimeric antigen receptor that specifically targets CLL1 as described in any one of claims 4 to 6.

8. An expression vector containing the nucleic acid of claim 7.

9. CLL1 CAR-T cells, characterized in that, It contains the nucleic acid of claim 7, or the cell membrane surface expresses the chimeric antigen receptor that specifically targets CLL1 as described in any one of claims 4 to 6.

10. The use of the chimeric antigen receptor specifically targeting CLL1 as described in any one of claims 4 to 6 or the CLL1 CAR-T cell of claim 9 in the treatment of leukemia or in the preparation of a drug for treating leukemia; preferably, the leukemia is acute myeloid leukemia.

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