CLL1 targeting nano antibody and application thereof

Abstract

The invention provides a nano antibody targeting CLL1 and application of the nano antibody. The nano antibody comprises a heavy chain variable region VHH, the heavy chain variable region VHH comprises CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is shown as SEQ ID NO.1, the amino acid sequence of the CDR2 is shown as SEQ ID NO.2, and the amino acid sequence of the CDR3 is shown as SEQ ID NO.3. The invention further provides a preparation method of the nano antibody. The CAR-T cell provided by the invention can identify and eliminate recurrent / refractory AML (acute myeloid leukemia) with weak expression or partial expression of CLL1 antigen, supports proliferation and activation of the CAR-T cell, reduces exhaustion of the CAR-T cell, and has higher durability and better treatment effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to a nanobody targeting CLL1 and application thereof. BACKGROUND

[0002] The conventional treatment methods for acute myeloid leukemia (AML) include chemotherapy, targeted therapy and hematopoietic stem cell transplantation, but most patients face the risk of relapse. At present, CAR-T cell therapy in AML is not mature, and the overall efficacy is poor. The early target points of CAR-T cell therapy for AML include CD33 and CD123, but they have the shortcomings of high toxicity or poor efficacy, which limits their clinical application. Other potential target points include CLL1, FLT3, NKG2D, LewisY, CD44v6 and CD38, etc.

[0003] CLL1 (C-type lectin-like molecule-1) is a C-type lectin-like receptor, which is highly expressed on leukemia stem cells (about 45%) and leukemia progenitor cells (77.5-92%), so CLL1 is an ideal target for treating AML. Tashiro et al. first constructed CLL1 CAR-T cells, which selectively kill leukemia progenitor cells and leukemia cells. CLL1 CAR-T constructed by Wang et al. showed good AML killing in vitro and in mice. In 2022, Zhang et al. first reported that a 10-year-old child with secondary AML achieved complete remission after treatment with CLL1 CAR-T cells.

[0004] However, we found that the expression level of CLL1 antigen on AML tumor cells is uneven, the killing power of CLL1 CAR-T cells on AML tumor cells with low expression of CLL1 is insufficient, the AML cells of patients who relapsed after CLL1 CAR-T treatment weakly express target antigens, which may escape the recognition of CLL1 CAR-T, and the poor efficacy of patients treated with CLL1 CAR-T cells may be related to the state of CAR-T cells themselves, and the CAR-T cells of patients who failed treatment have a high exhaustion and terminal differentiation phenotype, and regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) can all affect the efficacy of CAR-T cells. SUMMARY

[0005] Therefore, the present application aims to overcome the defects in the prior art and provides a nanobody targeting CLL1 and application thereof.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows: This invention provides a nanobody targeting CLL1, wherein the nanobody includes a heavy chain variable region VHH, the heavy chain variable region VHH includes CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2 and the amino acid sequence of CDR3 is shown in SEQ ID NO.3.

[0007] Furthermore, the amino acid sequence of the nanobody is shown in SEQ ID NO.4.

[0008] The present invention also provides a nucleic acid molecule that encodes the nanobody.

[0009] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.5.

[0010] The present invention also provides a chimeric antigen receptor, wherein the antigen recognition domain of the chimeric antigen receptor includes the nanobody, and the chimeric antigen receptor further includes a signal peptide, a hinge region, a transmembrane region and a signal transduction domain.

[0011] Furthermore, the chimeric antigen receptor also includes a tag protein, which is RQR8.

[0012] Furthermore, the chimeric antigen receptor also includes a self-cleaving peptide, wherein the self-cleaving peptide is T2A.

[0013] Furthermore, the signal transduction domains include intracellular domains of 4-1BB and CD3zeta.

[0014] The present invention also provides a CAR-T cell comprising the chimeric antigen receptor described above.

[0015] The present invention also provides the use of the aforementioned CLL1-targeting nanobody, the aforementioned nucleic acid molecule, the aforementioned chimeric antigen receptor, and the aforementioned CAR-T cells in the preparation of a drug for treating acute myeloid leukemia.

[0016] Compared with the prior art, the present invention has the following advantages: The CAR-T cells described in this invention can recognize and eliminate relapsed / refractory AML with weak or partial expression of CLL1 antigen, support CAR-T cell proliferation and activation, reduce CAR-T cell depletion, and have higher durability and better therapeutic effect. Attached Figure Description

[0017] Figure 1 The binding ability of the monoclonal antibody to the FAP antigen described in the embodiments of the present invention; Figure 2 This is a schematic diagram of the structure of the chimeric antigen receptor described in an embodiment of the present invention; Figure 3 This is the expression vector map described in the embodiments of the present invention; Figure 4 CLL1 expression in AML patients as described in this embodiment of the invention; Figure 5 The CLL1 described in this embodiment of the invention is expressed in AML cell lines MOLM13, THP1, HL60 and CML cell line K562; Figure 6 Flow cytometry plots showing the cytotoxic effects of CLL1 CAR-T and CLL1 VHH CAR-T cells on in vitro AML cell lines as described in embodiments of the present invention; Figure 7 The effects of CLL1 CAR-T and CLL1 VHH CAR-T cells on MOLM13, HL60, IFN-γ and TNF-α cytokines as described in the embodiments of the present invention; Figure 8 The killing power of CLL1 CAR-T and CLL1 VHH CAR-T cells against AML cells as described in the embodiments of the present invention; Figure 9 The effects of CLL1 CAR-T and CLL1 VHH CAR-T cells on PD-1, CTLA4, TIM-3 and LAG-3 exhaustion markers described in the embodiments of the present invention; Figure 10 The effects of CLL1 CAR-T and CLL1 VHH CAR-T cells on Tcm, Tn, Teff and Tem cells as described in the embodiments of the present invention; Figure 11 This is a diagram showing the in vivo experimental results of CLL1 CAR-T and CLL1 VHH CAR-T cell therapy in mice as described in this embodiment of the invention; Figure 12 The differential distribution of transcriptome sequencing analysis of CLL1 CAR-T and CLL1 VHH CAR-T cells described in the embodiments of the present invention. Detailed Implementation

[0018] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0019] The present invention will be described in detail below with reference to the embodiments.

[0020] Example 1 Experimental Method 1. Construction of the variable region VHH of the heavy chain of a nanobody targeting CLL1: (1) Animal immunization An alpaca was immunized with recombinant human CLL1 protein. For immunization, the antigen was formulated into an emulsion using either CFA (primary immunization) or IFA (booster immunization). The antigen was administered intramuscularly via a two-point injection in the neck. The animal received two weekly injections of the emulsion containing 100 μg of CLL1 protein, followed by four weekly injections containing 50 μg of CLL1 protein. At different time points during immunization, 10 ml blood samples were collected from the animals, and serum was prepared. Conventional IgG (IgG1) and heavy chain antibodies (HCAb, IgG2, and IgG3) were fractionated from the pre-immunization and post-immunization serum. Using the fractionated IgG1, IgG2, and IgG3, the induction of antigen-specific humoral immune responses was validated using immobilized human and cynomolgus monkey CLL1 in experiments based on enzyme-linked immunosorbent assay (ELISA).

[0021] (2) Construction of phage display library Total RNA was extracted from lymphocytes of immunized alpacas using reagents. cDNA was synthesized based on the RNA template using the PRIMESCRIPT™ 1st Strand cDNA Synthesis Kit with oligo(dT)20 primers. VHH was amplified from the alpaca cDNA, purified, and ligated into an internally prepared phagemid vector. The ligation product was used to transform competent cells. The resulting library was supplemented with 20% glycerol and stored at 80°C.

[0022] Building the Alpaca sdAb Library The size of the library is estimated to be greater than 10. 8 Sequencing was performed on more than 48 randomly selected clones. Insertion rate, the percentage of clones containing sdAb inserts, was 100%. Frame fit rate, the percentage of clones containing inserted sdAb DNA that could be correctly translated into the sdAb amino acid sequence, was 100%.

[0023] (4) Binder separation and high-throughput screening Following a standard protocol, an immune sdAb phage library was rescued, and at least one round of panning was performed using solid-phase panning until CLL1-specific antibodies were significantly enriched. The total number of exported clones, the percentage of CLL1-positive clones (by ELISA), and the sequence diversity of CLL1-specific binders were analyzed in each round. Based on these results, the optimal panning output was selected for high-throughput screening. After one round of panning of the immune alpaca library, CLL1-specific binders were significantly enriched. Several clones were selected. The selected exported phages were used to infect exponentially growing *E. coli* cells. The double-stranded DNA of the exported phages was extracted, the sdAb insert was cut from the phage vector, and inserted into an antibody fragment expression vector for high-throughput screening.

[0024] (5) Single-clone validation: The enriched positive clones were cultured as single clones and their binding ability to FAP antigen was verified again by FACS (e.g., Figure 1 (As shown). Clones with strong binding signals were selected for gene sequencing to obtain the amino acid sequence of VHH.

[0025] (6) Affinity and specificity: The binding strength (MFI value) and specificity of VHH clones to FAP were evaluated by methods such as flow cytometry.

[0026] 2. Design of CLL1 VHH CAR-T by Figure 2 An exemplary anti-CLL1 CAR construct was designed in this format. The sequences of these CARs, following this pattern, from N-terminus to C-terminus, are: leader sequence, RQR8 tag, T2A peptide, target-binding region (i.e., anti-CLL sdAb), CD8α hinge, CD8a transmembrane (TM) region, cytoplasmic portion of the 4-1BB (CD137) molecule, and cytoplasmic portion of the CD3ζ molecule. The DNA encoding each CAR construct was codon-optimized and synthesized. The CAR sequences were ligated into lentiviral vector plasmids for expression. CAR molecules typically consist of extracellular, transmembrane, and intracellular regions. Figure 3 As shown, the vector used is a pCDH-based lentiviral transfer vector (commonly used for gene overexpression or knockdown), which integrates LTRs, packaging signals, regulatory elements, etc., to achieve stable transduction and expression of exogenous genes. The composition and corresponding sequences are shown in Table 1.

[0027] Table 1 Sequence

[0028] Experimental results suggest that the design of its VHH CAR-T may be intended to address specific problems: Combating Exhaustion: Data showed that CLL1 VHH CAR-T cells, compared to conventional CLL1 CAR-T cells, expressed lower levels of exhaustion markers (such as PD-1 and LAG-3) and had a higher proportion of central memory T cells (Tcm). This suggests that the VHH or final CAR structure selected may be more conducive to maintaining T cells in a "young" and sustained fighting state.

[0029] Enhanced killing of cells with low antigen density: Due to their small size and unique structure, nanobodies can sometimes reach epitopes that are difficult for conventional scFv to access, which may help identify and eliminate AML cells with low CLL1 expression and prevent relapse.

[0030] 3. Plasmid synthesis: Plasmids were synthesized using the pLVX vector. CLL1 CAR-T and CLL1 VHH CAR-T were synthesized by Sangon Biotech. The vector and the synthesized target sequence were digested with enzymes. The target sequence was then inserted into the vector using homologous recombination. Finally, double enzyme digestion was performed for verification. The insertion was observed by agarose gel electrophoresis. 4. Virus packaging: The constructed CLL1 CAR-T and CLL1 VHH CAR-T plasmids, along with the viral packaging plasmids (VSVG and SPAX2), were incubated with PEI transfection reagent at a ratio of 5:3:2 (total 10 μg) and then transfected into 293T cells (e.g., in T-75 culture flasks, until confluence reached 70-85%). The DMEM complete medium was replaced 6 h after transfection. 5. Virus harvesting and concentration: Cell culture supernatants were collected at 48 and 72 hours, respectively. After centrifugation at 4000 rpm for 15 min at 4°C, the supernatant was filtered through a 0.45 μm filter membrane and ultracentrifuged at 22000 rpm for 2 hours. The supernatant was then discarded, and the precipitate was resuspended in fresh DMEM medium to obtain a concentrated virus solution for later use (e.g., stored at -80°C). Viral RNA was extracted, and the viral titer was detected by Real-time PCR. 6. T cell isolation and CAR-T cell preparation CAR-T cells were cultured from peripheral blood T cells collected from healthy donors and patients with relapsed / refractory acute myeloid leukemia enrolled in clinical trials.

[0031] Peripheral blood mononuclear cells (PBMCs) were extracted using Ficoll, and CD3+ were sorted from the mononuclear cells using magnetic beads. + T cells. Obtained CD3+ T cells at 5 × 10 5 The concentration was [value missing] mL, placed in T cell culture medium containing IL-2, and treated with CD3 / CD28-stimulated magnetic beads for 48 h. The suspended cells were then counted and seeded into six-well plates at a density of 3 × 10⁶ cells per well. 6 CAR-T cells were collected and infected with the prepared virus at an MOI of 5. After 8-12 days, the cells were harvested. QBEND-10 monoclonal CD34 antibody (Abcam) and VHH antibody were added to the lentivirus-infected T cells, respectively. After mixing, the cells were incubated at 4°C for 30 min. After washing with PBS, the cells were analyzed by flow cytometry.

[0032] In 90 patients, CLL1 was positively expressed in 86% of AML patients, with a median expression rate of 63% (range 0%–100%); CD123 and CLL1 were co-expressed in 80% of AML patients (e.g., CD123 and CLL1 were co-expressed). Figure 4 (As shown). Flow cytometry analysis showed that CLL1 was expressed in AML cell lines MOLM13, THP1, HL60, and CML cell line K562 (as shown). Figure 5 (As shown).

[0033] 7. Detection of CAR-T cell killing effect on primary AML cells Isolation of Bone Marrow Mononuclear Cells (BMMCs): Bone marrow specimens from patients with a tumor burden greater than 50% were selected, and BMMCs were isolated using the same method as Ficoll isolation of PBMCs. The isolated BMMCs were counted and CFSE stained: BMMCs were resuspended in CFSE working solution (10-100 × 10⁻⁶). 6 Cells / mL), incubate in the dark for 20 minutes to stain, then stop staining. Centrifuge at 500g for 5 minutes, add 1ml of 1640 medium containing 10% FBS and incubate for another 10 minutes. Centrifuge again, and resuspend in the medium before use. Co-incubate the constructed CLL1 CAR-T and CLL1 VHH CAR-T or untransfected T cells with CFSE-stained BMMCs at a ratio of 5:1. After 24 hours, take a certain volume of cells and detect the number of tumor cells by flow cytometry. Calculate the killing effect of CAR-T cells on tumor cells using the following formula: (Number of tumor cells in blank control wells - Number of tumor cells in experimental wells) / Number of tumor cells in blank control wells × 100%.

[0034] 8. Cytokine assessment: The levels of cytokines IL-6, TNF-α, and IFNγ in mouse serum were measured using the CBA kit and analyzed using a NovoCyte flow cytometer (Agilent).

[0035] 9. Flow cytometry: The corresponding antibodies are used to assess the cellular characteristics of CAR-T cells. The indicators assessed include T cell subsets and immune checkpoint marker expression.

[0036] 10. Mouse Experiment: The effects of CLL1 CAR-T and CLL1 VHH CAR-T were verified using in vivo mouse experiments. Six 6-8 week old male NOG mice purchased from Spifort (Beijing) Biotechnology Co., Ltd. were randomly divided into three groups of three mice each. Each mouse was injected with 5 × 10⁻⁵ CAR-T via the tail vein. 6 Primary tumor cells weakly expressing CLL1 antigen. On day 3, 5 × 10⁶ cells were injected. 6 Treatment was administered with CLL1CAR-T and CLL1 VHH CAR-T cells. The following parameters were measured at 7, 14, 21, and 28 days post-tumor cell infusion: (1) Tumor burden detection in mice: 3 mg of D-fluorescein (Sigma, USA) was injected into the peritoneum of mice. After 10 min, the tumor burden of mice was assessed using IVIS Spectrum in vivo imaging system (Caliper Life Sciences, USA) to evaluate the therapeutic effect of CAR-T cells.

[0037] (2) Mouse survival: Plot mouse survival curves and observe mouse survival to evaluate the efficacy of CAR-T therapy.

[0038] (3) Cytokine detection: Serum was collected from the tail vein to detect the secretion level of mouse cytokines.

[0039] (4) CAR-T amplification level in mice: Blood was collected from the tail vein and the amplification level of CAR-T in mice was detected by flow cytometry.

[0040] 11. Transcriptomics sequencing (1) Select CLL1 CAR-T and CLL1 VHH CAR-T infusions that are effective and ineffective in treating R / R AML respectively, wash the cells 2-3 times with pre-cooled PBS, centrifuge at 1000g for 1 min at 4℃, discard the supernatant, place the cell pellet in a 2ml centrifuge tube, freeze in liquid nitrogen for 15 min, and store at -80℃. (2) RNA was extracted, and mRNA with polyA tails was aggregated using Oligo (dT) magnetic beads to construct a library. After library construction, it was initially quantified using a Qubit 2.0 Fluorometer, and the library was diluted to 1.5 ng / ul according to the initial quantification result. Subsequently, the insert size of the library was detected (using the Agilent 2100 bioanalyzer), and the library concentration was requantified using qRT-PCR. Different libraries were pooled and sequenced using Illumina, followed by bioinformatics analysis. (3) First, the gene expression level of each sample was quantitatively analyzed, and the obtained gene data were statistically analyzed to screen out genes with significant differences in expression. Then, the ClusterProfiler software was used to perform GO functional enrichment analysis and KEGG pathway enrichment analysis on the screened differentially expressed genes. GO includes biological process (BP), cellular component (CC), and molecular function (MF). The KEGG database integrates genomic, chemical, and systemic functional information. P<0.05 is the threshold for significant enrichment.

[0041] Example 2 Affinity of Nanobodies The affinity-related kinetic parameters of the CLL1-targeting nanobody (Nb044-02-hFc) binding to the antigen CLL1-His are shown in Table 2.

[0042] Table 2 Affinity

[0043] The antigen (target) used to assess its affinity for CLL1 binding is the CLL1-His antigen, a histidine-tagged fusion protein of CLL1 (CLL1-His), which mimics the binding epitope of natural CLL1. k a The higher the value of the (1 / Ms) binding rate constant (the rate at which the antibody binds to the antigen), the faster the antibody binds to the antigen, reflecting the kinetic efficiency of the binding process. k d The smaller the (1 / s) dissociation rate constant (the rate at which the antibody dissociates from the antigen), the slower the dissociation rate and the higher the stability of the binding. K D (M) dissociation constant ( K D = k d / k a The smaller the affinity value, the stronger the affinity between the antibody and the antigen (the stronger the binding and the higher the specificity).

[0044] As shown in Table 2: (1) Affinity strength: This nanobody (Nb044-02-hFc) has a strong affinity for CLL1. K D The value was 5.27, which is in the nanomolar (or even sub-nanomolar) range, indicating that it has a high affinity for binding to CLL1.

[0045] (2) Binding / dissociation kinetics: Binding rate constant k a = 1.31 (fast binding speed), dissociation rate constant k d = 6.87 (slow dissociation speed), further verifying the high efficiency and stability of the binding.

[0046] (3) Related to technical advantages: In conjunction with the previous description that "the VHH sequence is smaller, so the transfection rate is higher", the VHH nanobody used in this invention not only has high affinity (which is conducive to the target recognition of CLL1), but also shows higher efficiency in gene transfection (such as vector transfection when constructing CAR-T) due to its short sequence and compact structure. This provides a key molecular basis for the preparation and application of CLL1 VHH CAR-T cells (such as cytotoxicity assessment).

[0047] Example 3: Affinity and toxicity of CLL1 VHH CAR-T To accurately evaluate the cytotoxic effects of CLL1 CAR-T and CLL1 VHH CAR-T cells on in vitro AML cell lines, we used CLL1... + AML cell lines were co-cultured with CAR-T or uninfected T cells at an effector-to-target ratio (E:T) of 1:3. After 8 hours of co-culture, flow cytometry analysis showed that CLL1 VHH CAR-T cells exhibited stronger cytotoxicity than CLL1 CAR-T cells (e.g., Figure 6 (As shown).

[0048] Furthermore, compared to CLL1 CAR-T cells, CLL1 VHH CAR-T cells significantly upregulated the levels of IFN-γ and TNF-α cytokines in MOLM13 and HL60 at an E:T ratio of 1:3 (e.g., Figure 7(As shown). These results indicate that CLL1 VHH CAR-T cells exhibit higher recognition and cytotoxicity against AML compared to CLL1 CAR-T cells, resulting in better therapeutic efficacy. Furthermore, CLL1 VHH CAR-T cells also demonstrate more durable anti-tumor activity, maintaining the ability to eliminate tumor cells for an extended period.

[0049] To further evaluate the cytotoxicity of CLL1 VHH CAR-T and CLL1 CAR-T cells, we performed in vitro cytotoxicity assays using bone marrow mononuclear cells (BMMCs) isolated from relapsed / refractory AML patients. Cell-killing efficiency was assessed by incubating CLL1 VHHCAR-T and CLL1 CAR-T cells with primary AML cells at a 3:1 E:T ratio for 6 and 24 hours. Figure 8 As shown in AC, CLL1 VHH CAR-T cells were significantly more effective against primary AML cells than CLL1 CAR-T cells. Furthermore, co-culturing CLL1 VHH CAR-T cells with primary AML cells resulted in a significant increase in IL-6 and IFN-γ levels in the supernatant. Figure 8 DF).

[0050] We observed the production of exhausted phenotype cells during CAR-T cell culture after two rounds of CAR-T cell co-culture with MOLM13. We performed multiple rounds of repeated experiments to assess the expression levels of immune checkpoint markers PD-1, CTLA4, TIM-3, and LAG-3 on the surface of CAR-T cells. We found that the expression levels of exhaustion-related markers PD-1, LAG-3, and TIM-3 in CLL1 VHH CAR-T cells were all higher than those in CLL1 CAR-T cells (e.g., ...). Figure 9 (as shown in the figure), but only the expression level of CTLA4 showed a significant difference between the two (P<0.005).

[0051] The results of cell differentiation subset statistics showed that, compared with CLL1 CAR-T, the differentiation subset of CLL1 VHH CAR-T cells had a higher proportion of Tcm (central memory T cells) and Tn (naïve T cells) (e.g., Figure 10 (As shown); the proportions of Teff (effective T cells) and Tem (effective memory T cells) were low (P<0.005).

[0052] To evaluate and compare the effects of CLL1 VHH CAR-T and CLL1 CAR-T on AML progenitor cells in vivo, we established a xenograft model in NSG mice. On day 0, mice were intravenously injected with 2×10⁻⁶ CAR-T cells. 6MOLM-13-LGP cells were intravenously injected at a dose of 2.5 × 10⁻⁶ cells on day 3 post-tumor inoculation. 6 CLL1 VHH CAR-T cells and CLL1 CAR-T cells. Leukemia proliferation was assessed weekly using bioluminescence imaging (BLI). Figure 11 Figure A illustrates the changes in tumor burden over time in different groups of mice. Compared with the control group receiving untransfected T-cell therapy, mice treated with CLL1 CAR-T and CLL1 VHH CAR-T showed significantly reduced tumor invasion. Simultaneously, mouse survival analysis indicated that CLL1 VHH CAR-T and CLL1 CAR-T significantly prolonged mouse lifespan. Figure 11 B). Furthermore, comparison of the percentage of CAR-T cells among CD3-positive T cells in peripheral blood indicated a peak in expansion around day 14 post-injection, with CLL1 VHH CAR-T cells showing higher expansion levels than CLL1 CAR-T cells (B). Figure 11 D). Furthermore, cytokine levels showed that CLL1 VHH CAR-T cells released significantly higher levels of TNF-α, IFN-γ, and IL-6 on day 14 compared to CLL1 CAR-T cells. Figure 11 C).

[0053] The volcano plot provides a visual overview of the differential distribution of transcriptome sequencing analysis between the two groups. CLL1 CAR-T cells and CLL1 VHH CAR-T cells differentially expressed a total of 4373 genes (log2FC > 2, padj < 0.05). Figure 12 Compared to CLL1 CAR-T cells, CLL1 VHH CAR-T cells downregulated genes including exhaustion-related genes such as LTA, BATF3, GZMB, LAG3, and JUNB; and upregulated genes including genes related to naïve and memory differentiation, such as IL7R, LEF1, CD44, and KLF3. Figure 12 B).

[0054] In this invention, we successfully constructed a CLL1 VHH CAR-T cell line, achieving high efficiency and stability in binding to CLL1 antigens with low or partial expression. Results showed that, in in vitro experiments, CLL1 VHH CAR-T cells exhibited higher recognition and cytotoxicity against AML cell lines and primary leukemia cells compared to CLL1 CAR-T cells. Furthermore, in an AML xenograft model, CLL1 VHH CAR-T cells demonstrated greater durability and better long-term survival. This structure enables CAR-T cells to precisely recognize specific tumor antigens and activate downstream signaling pathways to release anti-tumor effector molecules. Simultaneously, transcriptome sequencing analysis revealed that, compared to CLL1 CAR-T cells, CLL1 VHH CAR-T cells showed downregulation of exhaustion-related genes and upregulation of genes related to naïve and memory differentiation, indicating stronger durability of CLL1 VHH CAR-T cells.

[0055] Future research should focus on optimizing and enhancing the specificity of CAR-T cells to minimize potential off-target effects. This strategy holds promise for improving disease control and enhancing safety, thereby effectively addressing a significant challenge currently facing the treatment of AML.

[0056] In summary, this invention screens and identifies high-quality nanobodies targeting specific targets (FAP / CLL1), integrates VHH into the CAR backbone in the optimal form (single / double), selects appropriate hinges, transmembrane and signaling domains to optimize function, and comprehensively evaluates the efficacy and safety of CAR-T cells through in vitro killing experiments (such as RTCA, flow cytometry), cytokine release detection and in vivo animal models.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanobody targeting CLL1, characterized in that: The nanobody includes a heavy chain variable region VHH, which includes CDR1, CDR2 and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2 and the amino acid sequence of CDR3 is shown in SEQ ID NO.

3.

2. The nanobody targeting CLL1 according to claim 1, characterized in that: The amino acid sequence of the nanobody is shown in SEQ ID NO.

4.

3. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the nanobody as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

5.

5. A chimeric antigen receptor, characterized in that: The chimeric antigen receptor's antigen recognition domain includes the nanobody described in claim 1 or 2, and the chimeric antigen receptor further includes a signal peptide, a hinge region, a transmembrane region, and a signal transduction domain.

6. The chimeric antigen receptor according to claim 5, characterized in that: The chimeric antigen receptor also includes a tag protein, which is RQR8.

7. The chimeric antigen receptor according to claim 5, characterized in that: The chimeric antigen receptor also includes a self-cleaving peptide, wherein the self-cleaving peptide is T2A.

8. The chimeric antigen receptor according to claim 5, characterized in that: The signal transduction domains include the intracellular domains of 4-1BB and CD3zeta.

9. A CAR-T cell, characterized in that: Includes the chimeric antigen receptor according to any one of claims 5-8.

10. Use of the CLL1-targeting nanobody of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, the chimeric antigen receptor of any one of claims 5-8, and the CAR-T cell of claim 9 in the preparation of a medicament for treating acute myeloid leukemia.

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