Anti-siglec-6 nanobodies and methods of making and uses thereof

Abstract

The application provides an anti-Siglec-6 nanobody and a preparation method and application thereof, and belongs to the technical field of biological medicines. The specific nanobody of anti-Siglec-6 is screened from a double-peak camel VHH immune library, and can specifically combine with the Siglec-6 antigen. ‑14 ~10 ‑11 M order of magnitude, and the affinity is high. The nanobody can specifically combine with the Siglec-6 of human origin, can be used for preparing medicines for preventing or treating various diseases such as acute myeloid leukemia, allergy and inflammation, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an anti-Siglec-6 nanobody and a preparation method and use thereof. BACKGROUND

[0002] With the rapid development of immunotherapy, CAR-T cell therapy (chimeric antigen receptor T cell therapy) has become an important breakthrough in the field of tumor immunotherapy. Traditional CAR-T cell therapy mainly targets common tumor markers such as CD19, however, this therapy also faces some significant limitations. For example, tumor cells can escape immune surveillance through antigen heterogeneity, CAR-T cells can have off-target effects, and damage to normal cells of the target can lead to the occurrence of side effects. Therefore, developing new targeting strategies to overcome these limitations has become an important direction of current tumor immunotherapy research.

[0003] As a natural immune cell, γδT cell has unique anti-tumor function. Unlike traditional αβT cells, γδT cells can recognize a wide spectrum of tumor-associated antigens and do not depend on HLA molecules for antigen presentation, avoiding the possibility of tumor cells escaping immune attack by changing HLA molecules. Therefore, γδT cells have great application potential in tumor immunotherapy, especially in dealing with antigen heterogeneity or HLA low-expression tumors.

[0004] Siglec-6 is a glycosylated antigen that is mainly abnormally expressed in various malignant tumors, especially highly expressed in acute myeloid leukemia (AML) and other hematological tumors. As a tumor-specific marker, Siglec-6 provides a potential targeting opportunity for tumor immunotherapy. However, existing researches mostly focus on the targeting treatment of Siglec-6 using traditional αβT cells or monoclonal antibodies, and there is no systematic research on targeting Siglec-6 using CAR technology combined with γδT cells. In particular, there is no reported research on CAR-γδT cells targeting human Siglec-6.

[0005] Therefore, developing CAR-γδT cells targeting human Siglec-6 has important research value for treating acute myeloid leukemia and other hematological tumors that highly express Siglec-6. SUMMARY

[0006] In order to overcome the problems existing in the prior art, the purpose of the present application is to provide an anti-Siglec-6 nanobody and a preparation method and use thereof.

[0007] The application provides an anti-Siglec-6 nanobody or antigen binding fragment, a heavy chain variable region of the anti-Siglec-6 nanobody comprising CDR1, CDR2 and CDR3, wherein the amino acid sequences of the CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or the amino acid sequences of the CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or the amino acid sequences of the CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or the amino acid sequences of the CDR1, CDR2 and CDR3 are respectively shown as SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18.

[0008] Further, the anti-Siglec-6 nanobody is at least one of a monovalent nanobody, a multivalent nanobody, a multispecific antibody or a fusion nanobody.

[0009] Further, the anti-Siglec-6 nanobody is a monovalent nanobody, and the heavy chain variable region of the anti-Siglec-6 nanobody further comprises a framework region, and the structure of the heavy chain variable region of the anti-Siglec-6 nanobody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0010] Further, the amino acid sequence of the heavy chain variable region or the antigen binding fragment of the anti-Siglec-6 nanobody is shown as SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14 or SEQ ID NO:19.

[0011] The application further provides a gene fragment encoding the anti-Siglec-6 nanobody or antigen binding fragment, and the nucleotide sequence of the gene fragment is shown as SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:15 or SEQ ID NO:20.

[0012] The application further provides an antibody comprising the anti-Siglec-6 nanobody or antigen binding fragment.

[0013] Further, the antibody is any one of a full-length antibody, a heavy chain antibody, a chimeric antibody, a multispecific antibody, a murine antibody, a humanized antibody or an antigen binding fragment.

[0014] Further, the antigen binding fragment comprises any one selected from F(ab')2, Fab', Fab, Fv or scFv of an antibody.

[0015] The present application also provides a recombinant vector comprising a gene fragment encoding the anti-Siglec-6 Nanobody or antigen binding fragment, antibody as above.

[0016] Further, the recombinant vector is a plasmid or a virus; the virus is an adenovirus, an adeno-associated virus, a retrovirus, a lentivirus or an oncolytic virus.

[0017] The present application also provides a host cell comprising the recombinant vector as above.

[0018] The present application also provides a chimeric antigen receptor, wherein the antigen binding domain of the chimeric antigen receptor comprises at least one of the anti-Siglec-6 Nanobody or antigen binding fragment, antibody as above.

[0019] The present application also provides a CAR-γδT cell comprising the chimeric antigen receptor as above.

[0020] The present application also provides a recombinant protein comprising at least one of the anti-Siglec-6 Nanobody or antigen binding fragment, antibody as above, or a pharmaceutical composition thereof.

[0021] Further, the pharmaceutical composition further comprises an active agent; the active agent comprises at least one of an immune checkpoint related agent, an antibody conjugate drug, a bispecific antibody, a multispecific antibody, a radionuclide or a kinase inhibitor.

[0022] The present application also provides a use of the anti-Siglec-6 Nanobody or antigen binding fragment, gene fragment, antibody, recombinant vector, host cell, chimeric antigen receptor, CAR-γδT cell, recombinant protein or pharmaceutical composition thereof as above in the preparation of a medicament for preventing and / or treating a tumor or an autoimmune disease.

[0023] Further, the tumor comprises at least one of brain glioma, neuroblastoma, medulloblastoma, meningioma, lung cancer, esophageal cancer, pancreatic cancer, liver cancer, cholangiocarcinoma, renal cancer, bladder cancer, ureter cancer, prostate cancer, skin cancer, melanoma, ovarian cancer, endometrial cancer, cervical cancer, soft tissue sarcoma, acute and chronic leukemia, Hodgkin and non-Hodgkin lymphoma, gastric cancer or head and neck tumor; the autoimmune disease comprises at least one of allergy, lupus erythematosus, ankylosing spondylitis, multiple sclerosis, psoriasis, antiphospholipid antibody syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, autoimmune hepatitis, arthritis, rheumatoid arthritis, pemphigus, Guillain-Barre syndrome, Crohn's disease, vasculitis or autoimmune diabetes.

[0024] Further, the drug includes the following forms: immune cells, reagents, kits or compositions.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] The specific nanobody of anti-Siglec-6 screened from the double-peak camel VHH immune library can specifically bind to the Siglec-6 antigen, and through the determination of antibody affinity, it can be known that the nanobody obtained by the present application has high affinity between 10 -14 ~ 10 -11 M orders of magnitude, and the nanobody of the present application can specifically bind to human Siglec-6, and can be used for preparing drugs for preventing or treating acute myeloid leukemia, allergy, inflammation and various diseases, and has a wide application prospect.

[0027] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modification, replacement or change can be made without departing from the above technical idea of the present application.

[0028] The above content of the present application will be further described in detail through the specific embodiments below. However, it should not be understood that the above subject matter of the present application is limited to the following embodiments. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 ELISA was used to identify positive reaction clones after phage enrichment.

[0030] Figure 2 SDS-PAGE was used to identify the purified nanobody.

[0031] Figure 3 ELISA was used to identify the reactivity of recombinant anti-Siglec-6 nanobody with human Siglec-6 antigen.

[0032] Figure 4 The specific binding of recombinant anti-Siglec-6 nanobody to Siglec-6+-HeLa cells was identified.

[0033] Figure 5 The in vivo anti-tumor activity of CAR-gammadelta T targeting Siglec-6 was evaluated by using an immunodeficient mouse to construct an AML xenotransplantation model. DETAILED DESCRIPTION

[0034] The raw materials and equipment used in the present application are known products, which can be obtained by purchasing commercially available products.

[0035] Preparation of anti-Siglec-6 nanobodies

[0036] 1. Preparation of Siglec-6 recombinant protein

[0037] The purchased human pGEM-Siglec-6 plasmid was used as a template for PCR amplification, and the extracellular domain (ECD) fragment of Siglec-6 was obtained by agarose gel electrophoresis. The ECD fragment of Siglec-6 was then cloned into the C-terminal pcDNA3.1 expression vector carrying Fc or His tags. Then, 293FT cells were transiently transfected, and the Siglec-6 recombinant protein carrying Fc or His tags was purified by Protein A / G or nickel column affinity chromatography and molecular sieve chromatography. TM The Siglec-6 recombinant protein carrying Fc or His tags was purified by Protein A / G or nickel column affinity chromatography and molecular sieve chromatography.

[0038] 2. Construction, panning, and ELISA preliminary screening of phage nanobody library

[0039] (1) Immunization of Bactrian camels

[0040] 2 mg of purified Siglec-6 recombinant protein obtained in step 1 was added to 2 mL Freund's complete adjuvant, and then emulsified using an emulsifier. The Bactrian camel was immunized subcutaneously at multiple points on the neck, and then immunized every two weeks (2 mg of purified Siglec-6 recombinant protein) using Freund's incomplete adjuvant, for a total of 4 times. Peripheral blood was collected after the last immunization to test the titer, and if the target titer was not reached, the above immunization steps were repeated. When the camel reached a certain immune titer, it was given a final boost (2 mg of purified Siglec-6 recombinant protein), and 7 days later, 200 mL of peripheral blood was collected using a blood collection bag for lymphocyte separation.

[0041] (2) Construction of nanobody library

[0042] The isolated lymphocytes in step 2.(2) were subjected to RNA extraction according to the RNA extraction kit procedure of Promega. The RNA of the lymphocytes was immediately subjected to reverse transcription cDNA using the TaKaRa reverse transcription kit, and then the VHH gene was amplified using nested PCR. The amplified VHH gene was inserted into the pMECS phage display vector, and the TG1 competent cells were electroporated. The culture broth after electroporation was subjected to dilution (10-fold dilution) using LB / Amp-GLU medium, and then 10 -4 , 10 -5 , 10 -6,10 -7 Dilution of dilution 100 μL coated on LB / Amp-GLU plate, 37°C inverted culture, culture 8h after counting different dilution bacteria, to calculate the capacity of the antibody library, namely 6.56 x 10 9 At the same time, randomly selected 50 similar size of the morphology of the colony culture 4-6h, bacteria liquid PCR, to identify the positive rate of the library, namely the insertion rate of the library reached 97%.

[0043] (3) Screening of anti-Siglec-6 nanobodies

[0044] Nanobody phage library screening: ① antigen coating: Siglec-6 recombinant protein was diluted with PBS, 20 μg per well (the antigen coating amount of the following two rounds of screening was 10 μg / well, 5 μg / well, respectively) coated in 96-well enzyme-labeled plate, 4℃ overnight coating; ② washing: after overnight coating, discard the liquid in the hole, wash each hole with 200 μL PBST for 5 times; ③ blocking: add 200 μL 5% skimmed milk powder to each well, and place it at 37℃ for 1h; ④ washing: discard the liquid in the hole, wash each hole with 200 μL PBST for 3 times; ⑤ incubation of recombinant phage: dilute the recombinant phage with 5% skimmed milk powder to 5 x 10 11 pfu / mL, add 100 μL to each well, incubate at room temperature for 2h; ⑥ washing: discard the liquid in the hole, wash each hole with 200 μL PBST for 15 times. Add 100 μL of freshly prepared 0.1M triethylamine to each well, and place it at room temperature for 10min, then elute the solution into a 1.5mL centrifuge tube and quickly add an equal volume of 1M Tris-HCl (pH=7.4) for neutralization; ⑦ determination of recombinant phage titer: collect the neutralized phage solution and determine the phage titer; the remaining phage solution was used to infect 2mL of TG1 in logarithmic growth phase, and incubated at 37℃ for 30min; add 8mL of 2 x YT / Amp GLU medium, and incubate at 37℃ at 220rpm until logarithmic growth; ⑧ rescue: add 8mL of 2 x YT ampicillin-resistant medium and 4% glucose, and incubate at 37℃ at 220rpm; ⑨ phage concentration; ⑩ repeat the above steps ①-⑨ to perform the second and third rounds of screening.

[0045] (4) Detection of specific recombinant phage enrichment

[0046] Antigen coating: Dilute the two antigens with PBS, 400 ng per well, coat in 96-well enzyme-labeled plate at 4°C overnight. Washing: After overnight coating, discard the liquid in the well, wash each well with 200 μL PBST for three times. Blocking: Add 200 μL 5% skim milk powder per well, and place it at 37°C for 1 h. Washing: Discard the liquid in the well, wash each well with 200 μL PBST for three times. Incubation of recombinant phage: Dilute the phage concentrate (1:10), add 100 μL per well, and incubate at 37°C for 1 h. Washing: Discard the liquid in the well, wash each well with 200 μL PBST for three times. Secondary antibody: HRP-labeled mouse anti-M13 secondary antibody is diluted 1:2000, 100 μL per well, and incubate at 37°C for 1 h. Washing: Discard the liquid in the well, wash each well with 200 μL PBST for three times. Color development: Add 100 μL TMB color developing solution per well, and place it at room temperature in the dark for 10-15 min. Termination and reading: After color development, add 50 μL 2M H2SO4 per well to terminate the reaction; read the absorbance value at 450 nm. Analyze the data.

[0047] (5) Sequencing analysis of specific nanobodies

[0048] Step 2. (4) ELISA detection results are shown in Table 1, and clones more than 3 times the negative value are determined as positive, and bacterial liquid sequencing, alignment analysis, and finally 4 anti-Siglec-6 nanobody sequences are obtained, as shown in Table 1. Figure 1

[0049] Table 1 Amino acid sequence of anti-Siglec-6 nanobody

[0050]

[0051]

[0052]

[0053]

[0054] Example 2 Expression, purification and reactivity of anti-Siglec-6 nanobodies with antigens

[0055] ​Based on the pcDNA3.1 eukaryotic expression vector, the nucleotide sequences of the four recombinant anti-Siglec-6 Nanobodies (nucleotide sequences as shown in SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO: 20) were cloned into the pcDNA3.1-hFc-His vector. The constructed expression vector was expressed and purified using the HEK293T eukaryotic protein expression system. The SDS-PAGE results showed that four high-purity Siglec-6 Nanobody-hFc fusion proteins were obtained after affinity chromatography purification, and the results are shown in Figure 2

[0056] To identify the reactivity of Siglec-6 Nanobody-hFc fusion protein (hereinafter referred to as recombinant anti-Siglec-6 Nanobody) with antigen, 200 ng / well of Siglec-6 recombinant protein (i.e. Siglec-6 antigen) was coated on an enzyme-labeled plate, and after overnight incubation at 4°C, the plate was blocked and different amounts of recombinant anti-Siglec-6 Nanobody (dilution: 10 -5 2 μg / mL) were added. After washing with secondary antibody, color development, and termination of the reaction, the optical density (OD450) at 450 nm was measured using an enzyme-labeled instrument, and the binding capacity was determined using four-parameter nonlinear regression curve fitting. The results are shown in Figure 3

[0057] Experimental Example 1 Binding experiment of recombinant anti-Siglec-6 Nanobody with Siglec-6 + -Hela cells

[0058] First, Hela cells were infected with a lentivirus containing the full-length gene of Siglec-6, and high-purity Siglec-6 + -Hela cells stably expressing Siglec-6 gene were obtained by flow sorting, and then the recombinant anti-Siglec-6 Nanobody prepared in Example 2 was incubated with Siglec-6 + -Hela cells at 37°C for 40 min, washed with PBS for 3 times, and then incubated with APC@goat anti-human secondary antibody, washed with PBS for 3 times, and detected by flow cytometry. The results are shown in Figure 4 + -Hela cells. ​​​​

[0059] Experimental Example 2 Siglec-6 Nanobody Affinity Determination

[0060] The affinity of the recombinant anti-Siglec-6 nanobody of the present invention was verified by surface plasmon resonance, and its binding kinetic constant (KD) was determined. TM 8K instrument, the anti-mouse IgG antibody is fixed on the surface of the CM5 chip using the coupling buffer in the amino coupling kit, and the Siglec-6 recombinant protein is captured on the CM5 chip in a 2-fold serial dilution; the purified recombinant anti-Siglec-6 nanobody is then allowed to flow over the surface of the chip, and the machine reads Ka (1 / M*s), kd (1 / s), and KD (M), that is, the affinity of the recombinant anti-Siglec-6 nanobody of the present invention is measured. The affinity measurement results show that the four candidate humanized recombinant anti-Siglec-6 nanobodies of the present invention can all specifically bind to the Siglec-6 recombinant protein, with an affinity of 10 -14 ~10 -11 The affinity between the orders of magnitude of M are all high-affinity antibodies. The kinetic characteristics show that the four humanized recombinant anti-Siglec-6 nanoantibodies all have relatively slow dissociation rates. The specific data of the detection are shown in Table 2.

[0061] Table 2 Affinity of recombinant anti-Siglec-6 nanobody

[0062] Antibody Ka (1 / M*s) kd (1 / s) KD (M) Nb45 1.20 x 10 5 ]] 6 x 10 -9 ]]> 4.9 x 10 -14 ]]> Nb46 1.40 x 10 4 ]]> 1.09 x 10 -6 ]]> 7.7 x 10 -11 ]] Nb49 1.14 x 10 5 ]]> 3.32 x 10 -9 ]] 2.91 x 10 -14 ]]> Nb50 1.81 x 10 5 ]]> 9.11 x 10 -8 ]]> 5.03 x 10 -13 ]]>

[0063] Experimental Example 3 In vivo anti-tumor activity of allogeneic CAR-γδT cells

[0064] Peripheral blood 10 mL of healthy adult donors was collected in a sterile anticoagulant tube. First, dilute the blood 1:1 with an equal volume of PBS, mix well upside down. Add 3 mL of lymphocyte separation medium to each separation tube, centrifuge at room temperature 1500 rpm for 1 min, after taking out, add the diluted blood to the separation tube, centrifuge at room temperature 1500 rpm for 15 min (the ascending speed gear is adjusted to 2, and the descending speed gear is adjusted to 2). The separation tube after centrifugation is divided into three layers, the uppermost layer is plasma, the middle layer is a circle of milky white which is lymphocytes, and the lowermost layer is red blood cells; insert the gun tip into the milky white layer, slowly suck the lymphocytes into a new 15 ml centrifuge tube, add an appropriate amount of PBS, mix well and centrifuge at 1500 rpm for 5 min. Discard the supernatant, resuspend with an appropriate amount of PBS, mix well upside down, and centrifuge at 1500 rpm for 5 min. Repeat the washing for 1 time. After discarding the supernatant, resuspend the cells with an appropriate amount of RPMI1640 complete medium containing zoledronate at a concentration of 1.00 μg / ml-4.00 μg / ml (serum inactivation treatment), and use a cell counter to count the lymphocytes. After counting, adjust to the appropriate density, add to the cell culture system, stimulate the proliferation and activation of γδT cells.

[0065] Siglec-6-CAR was constructed with the nucleotide sequence of the four anti-Siglec-6 nanobodies of the application (i.e. Siglec-6(Nb)) respectively, and the structure was as follows: CD8α signal peptide-Siglec-6(Nb)-CD8α hinge-CD28αTm-4-1BB-CD3ζ-P2A-EGFP. HEK293T cells were used as lentivirus packaging cells, and a three-plasmid packaging system (psPAX2, pMD2.G, CAR-γδT vector) was used for lentivirus packaging. The supernatant virus liquid was collected after 48 h, concentrated by ultracentrifugation, and RetroNectin protein was coated in advance and added to the concentrated virus to infect γδT cells. After 48 h of infection, the transfection efficiency of Siglec-6 CAR-γδT cells was evaluated by flow cytometry to be more than 80.00%. The obtained Siglec-6 CAR-γδT cells were named 45#CAR-γδT cells, 46#CAR-γδT cells, 49#CAR-γδT cells and 50#CAR-γδT cells respectively according to the nucleotide sequence of the anti-Siglec-6 nanobodies used.

[0066] The in vivo anti-tumor activity of CAR-γδT targeting Siglec-6 was evaluated by a xenograft mouse model. The NCG mouse acute myeloid leukemia model was established using the THP-1 cell line for evaluation.

[0067] 5×10 6THP-1-mCherry.ffLuc cells were used to establish AML mouse tumor models to verify the in vivo effect of Siglec-6 CAR-γδT. On the 5th day after inoculation, NCG mice were randomly divided into 6 groups, 5 in each group, and 1×10 7 NTγδT cells (untreated γδT cells), 1×10 7 Siglec-6 CAR-γδT cells (45#CAR-γδT, 46#CAR-γδT, 49#CAR-γδT or 50#CAR-γδT, respectively) were injected into the tail vein, and a control group injected with PBS was set. The growth state and survival of the mice were observed every day, and the survival time was recorded. The results are shown in Figure 5 Fig. 6, and the Siglec-6 CAR-γδT treatment group observed a significant delay in tumor progression, and the survival time of tumor-bearing mice was significantly prolonged.

[0068] In summary, the specific nanobody against Siglec-6 screened from the Bactrian camel VHH immune library can specifically bind to the Siglec-6 antigen. Through antibody affinity determination, it can be known that the nanobody obtained by the application has a nanobody affinity between 10 -14 ~ 10 -11 M orders of magnitude, and has high affinity. The nanobody of the application can specifically bind to human Siglec-6, and can be used for preparing drugs for preventing or treating acute myeloid leukemia, allergy, inflammation and various diseases, and has a wide application prospect.

Claims

1. An anti-Siglec-6 nanobody or an antigen-binding fragment thereof, characterized in that: The heavy chain variable region of the anti-Siglec-6 nanobody comprises CDR1, CDR2 and CDR3; the amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, respectively; or, the amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, respectively; or, the amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, respectively; or, the amino acid sequences of the CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, respectively.

2. The anti-Siglec-6 nanobody or antigen-binding fragment thereof according to claim 1, characterized in that: The anti-Siglec-6 nanobody is at least one of a monovalent nanobody, a multivalent nanobody, a multispecific nanobody or a fusion nanobody.

3. The anti-Siglec-6 nanobody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The anti-Siglec-6 nanobody is a monovalent nanobody, and the heavy chain variable region of the anti-Siglec-6 nanobody further includes a framework region; The heavy chain variable region structure of the anti-Siglec-6 nanobody is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

4. The anti-Siglec-6 nanobody or antigen-binding fragment thereof according to claim 3, characterized in that: The amino acid sequence of the heavy chain variable region or antigen-binding fragment of the anti-Siglec-6 nanobody is shown in SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 14 or SEQ ID NO:

19.

5. A gene fragment encoding the anti-Siglec-6 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The nucleotide sequence of the gene fragment is shown in SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 15 or SEQ ID NO:

20.

6. A recombinant vector comprising a gene fragment encoding the anti-Siglec-6 nanobody or its antigen-binding fragment according to any one of claims 1 to 4.

7. The recombinant vector according to claim 6, characterized in that: The recombinant vector is a plasmid or a virus; the virus is an adenovirus, an adeno-associated virus, a retrovirus, a lentivirus or an oncolytic virus.

8. A host cell comprising the recombinant vector according to claim 6 or 7.

9. A chimeric antigen receptor, characterized in that: The antigen binding domain of the chimeric antigen receptor includes the anti-Siglec-6 nanobody or its antigen binding fragment according to claim 1. 10 . A CAR-γδT cell comprising the chimeric antigen receptor according to claim 9 .

11. A recombinant protein or pharmaceutical composition comprising the anti-Siglec-6 nanobody or antigen-binding fragment thereof according to any one of claims 1 to 4.

12. The recombinant protein or pharmaceutical composition thereof according to claim 11, characterized in that: The pharmaceutical composition further comprises an active agent; the active agent comprises at least one of an immune checkpoint-related preparation, an antibody-drug conjugate, a multispecific antibody, a radionuclide or a kinase inhibitor.

13. The recombinant protein or pharmaceutical composition thereof according to claim 11, characterized in that: The pharmaceutical composition further comprises an active agent; the active agent is a bispecific antibody.

14. Use of the anti-Siglec-6 nanobody or its antigen-binding fragment according to any one of claims 1-4, the gene fragment according to claim 5, the recombinant vector according to claim 6 or 7, the host cell according to claim 8, the chimeric antigen receptor according to claim 9, the CAR-γδT cell according to claim 10, the recombinant protein according to any one of claims 11-13, or its pharmaceutical composition in the preparation of a medicament for preventing and / or treating acute and chronic leukemia.

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