Anti-Siglec-6 nano antibody as well as preparation method and application thereof

Abstract

The invention provides an anti-Siglec-6 nano antibody as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The anti-Siglec-6 specific nano antibody screened from a bactrian camel VHH immune library can be specifically combined with a Siglec-6 antigen, and it can be known through antibody affinity determination that the affinity of the obtained nano antibody ranges from 10 <-14 > M to 10 <-11 > M order of magnitude, and the affinity is high. The nano antibody disclosed by the invention can be specifically combined with human Siglec-6, can be used for preparing medicines for preventing or treating various diseases such as acute myelogenous leukemia, allergy, inflammation and the like, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-Siglec-6 nano antibody and a preparation method and use thereof. Background Art

[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, but this therapy also faces some significant limitations. For example, tumor cells may escape immune surveillance through antigen heterogeneity, CAR-T cells may have off-target effects, and damage to normal cells at the target site can lead to side effects. Therefore, developing new targeting strategies to overcome these limitations has become an important direction of current tumor immunotherapy research.

[0003] As natural immune cells, γδT cells have unique anti-tumor functions. Unlike traditional αβT cells, γδT cells can not only recognize a wide spectrum of tumor-associated antigens, but also do not rely on HLA molecules for antigen presentation, thus avoiding the possibility of tumor cells escaping immune attacks by changing HLA molecules. Therefore, γδT cells have great application potential in tumor immunotherapy, especially in dealing with antigen heterogeneity or HLA low-expressing tumors.

[0004] Siglec-6 is a glycosylated antigen that is abnormally expressed in a variety of malignant tumors, especially in acute myeloid leukemia (AML) and other blood tumors. As a tumor-specific marker, Siglec-6 provides a potential targeting opportunity for tumor immunotherapy. However, most existing studies focus on the targeted treatment of Siglec-6 using traditional αβT cells or monoclonal antibodies, and there has been no systematic study on the use of CAR technology combined with γδT cells to target Siglec-6. In particular, research on CAR-γδT cells targeting human Siglec-6 has not been reported.

[0005] Therefore, the development of CAR-γδT cells targeting human Siglec-6 has important research value for the treatment of acute myeloid leukemia and other blood tumors that highly express Siglec-6. Summary of the invention

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

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

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

[0009] Furthermore, the anti-Siglec-6 nanobody is a monovalent nanobody, and the heavy chain variable region of the anti-Siglec-6 nanobody also includes a framework region; the heavy chain variable region structure of the anti-Siglec-6 nanobody is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0010] Furthermore, 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.

[0011] The present invention also provides a gene fragment encoding the above-mentioned anti-Siglec-6 nanobody or antigen-binding fragment, 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.

[0012] The present invention also provides an antibody, which comprises the above-mentioned anti-Siglec-6 nanobody or antigen-binding fragment.

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

[0014] Furthermore, the antigen-binding fragment comprises a F(ab')2 , Fab', Fab, Fv or scFv.

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

[0016] Furthermore, 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 invention also provides a host cell comprising the above recombinant vector.

[0018] The present invention also provides a chimeric antigen receptor, the antigen binding domain of which includes at least one of the above-mentioned anti-Siglec-6 nanoantibodies or antigen binding fragments, and antibodies.

[0019] The present invention also provides a CAR-γδT cell, which comprises the above-mentioned chimeric antigen receptor.

[0020] The present invention also provides a recombinant protein or a pharmaceutical composition thereof comprising at least one of the above-mentioned anti-Siglec-6 nanobodies, antigen-binding fragments, and antibodies.

[0021] Furthermore, 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 bispecific antibody, a multispecific antibody, a radionuclide or a kinase inhibitor.

[0022] The present invention also provides the use of the above-mentioned anti-Siglec-6 nanobody or antigen-binding fragment, gene fragment, antibody, recombinant vector, host cell, chimeric antigen receptor, CAR-γδT cell, recombinant protein or its pharmaceutical composition in the preparation of drugs for preventing and / or treating tumors or autoimmune diseases.

[0023] Furthermore, the tumor includes at least one of brain glioma, neuroblastoma, medulloblastoma, meningioma, lung cancer, esophageal cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, bladder cancer, ureteral cancer, prostate cancer, skin cancer, melanoma, ovarian cancer, endometrial cancer, cervical cancer, soft tissue sarcoma, acute and chronic leukemia, Hodgkin's and non-Hodgkin's lymphoma, gastric cancer or head and neck tumors; the autoimmune disease includes 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] Furthermore, the medicine includes the following forms: immune cells, reagents, kits or compositions.

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

[0026] The specific nanoantibody against Siglec-6 screened from the Bactrian camel VHH immune library of the present invention can specifically bind to Siglec-6 antigen. The affinity of the nanoantibody obtained by the present invention is 10 -14 ~10 -11 The nanoantibody of the present invention can specifically bind to human Siglec-6, and can be used to prepare drugs for preventing or treating various diseases such as acute myeloid leukemia, allergies, inflammation, etc., and has broad application prospects.

[0027] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0028] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0030] Figure 2 The purified nanobody was identified by SDS-PAGE.

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

[0032] Figure 4 Identification of the specific binding of recombinant anti-Siglec-6 nanobody to Siglec-6+-HeLa cells.

[0033] Figure 5 To establish an AML xenograft model using immunodeficient mice to evaluate the in vivo anti-tumor activity of CAR-γδT targeting Siglec-6. DETAILED DESCRIPTION

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

[0035] Example 1 Preparation of anti-Siglec-6 nanobody

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

[0037] The purchased human pGEM-Siglec-6 plasmid was used as a template for PCR amplification. The extracellular domain (ECD) fragment of Siglec-6 was obtained by agarose gel electrophoresis. The extracellular domain fragment of Siglec-6 was cloned into the pcDNA3.1 expression vector with an Fc or His tag at the C-terminus using the pcDNA3.1 vector as a backbone. Then, 293FT cells were transiently transfected and FreeStyle TM The cells were cultured in shake flasks in serum-free medium (Life Technologies) for 5-7 days, and the supernatant was collected and purified by ProteinA / G or nickel column affinity chromatography and molecular sieve chromatography column to purify the Siglec-6 recombinant protein carrying Fc or His tag.

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

[0039] (1) Bactrian camel immunization

[0040] Take 2 mg of the purified Siglec-6 recombinant protein obtained in step 1, add 2 mL of Freund's complete adjuvant, and fully emulsify it using an emulsifier; immunize the Bactrian camel by subcutaneous injection at multiple points in the neck, and then immunize once every two weeks (2 mg of purified Siglec-6 recombinant protein), and use Freund's incomplete adjuvant, for a total of 4 immunizations. After the last immunization, collect peripheral blood to test the titer. If the target titer is not reached, repeat the above immunization steps. When the camel reaches a certain immune titer, perform the last impact immunization (2 mg of purified Siglec-6 recombinant protein), and collect 200 mL of Bactrian camel peripheral blood in a blood collection bag 7 days later for lymphocyte separation.

[0041] (2) Construction of Nanobody Library

[0042] Take out the lymphocytes separated in step 2.(2) and extract RNA according to the steps of Promega RNA extraction kit. Immediately after the RNA of lymphocytes is extracted, reverse transcribe cDNA using TaKaRa reverse transcription kit, and then amplify VHH gene using nested PCR; insert the amplified VHH gene into pMECS phage display vector and electrotransform TG1 competent cells. Take the culture liquid after electrotransformation and dilute it in multiples (10 times dilution) using LB / Amp-GLU medium, and then take 10 -4 , 10 -5 , 10 -6 , 10 -7100 μL of the dilution solution was spread on LB / Amp-GLU plates and incubated at 37°C. After 8 h of incubation, the number of colonies at different dilutions was counted to calculate the capacity of the antibody library, which was 6.56×10 9 At the same time, 50 colonies with similar morphology and size were randomly selected and cultured for 4-6 hours, and PCR was performed on the bacterial solution to identify the positive rate of the library, that is, the insertion rate of the library reached 97%.

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

[0044] Panning of nanoantibody phage library: ① Antigen coating: After diluting Siglec-6 recombinant protein with PBS, 20 μg per well (the antigen coating amount for the subsequent two rounds of panning was 10 μg / well and 5 μg / well, respectively) was coated in a 96-well ELISA plate and coated overnight at 4°C; ② Washing: After overnight coating, the liquid in the well was discarded and each well was washed 5 times with 200 μL PBST; ③ Blocking: 200 μL 5% skim milk powder was added to each well and placed at 37°C for 1 hour; ④ Washing: The liquid in the well was discarded and each well was washed 3 times with 200 μL PBST; ⑤ Incubation of recombinant phage: The recombinant phage was diluted to 5×10 with 5% skim milk powder. 11 pfu / mL, add 100 μL to each well and incubate at room temperature for 2 h; ⑥ Washing: discard the liquid in the wells and wash each well with 200 μL PBST for 15 times. Add 100 μL of freshly prepared 0.1 M triethylamine to each well, let it stand at room temperature for 10 min, aspirate the eluate into a 1.5 mL centrifuge tube and quickly add an equal volume of 1 M Tris-HCl (pH = 7.4) for neutralization; ⑦ Determination of recombinant phage titer: collect the neutralized phage solution and determine the phage titer; infect 2 mL of TG1 in the logarithmic growth phase with the remaining phage solution, let it stand at 37°C for 30 min; add 8 mL of 2×YT / AmpGLU culture medium, and culture at 37°C 220 rpm until the logarithmic growth phase; ⑧ Rescue: add 8 mL of 2×YT ampicillin resistance culture medium, add 4% glucose, and culture at 37°C 220 rpm; ⑨ Phage concentration; ⑩ Repeat the above steps ①-⑨ for the second and third rounds of panning.

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

[0046] Antigen coating: After diluting the two antigens with PBS, 400 ng per well was coated in a 96-well ELISA plate and coated overnight at 4°C. Washing: After overnight coating, the liquid in the wells was discarded and each well was washed three times with 200 μL PBST. Blocking: Add 200 μL 5% skim milk powder to each well and place it at 37°C for 1 hour. Washing: Discard the liquid in the wells and wash each well three times with 200 μL PBST. Incubation of recombinant phage: Dilute the phage concentrate (1:10), add 100 μL to each well, and incubate at 37°C for 1 hour. Washing: Discard the liquid in the wells and wash each well three times with 200 μL PBST. Secondary antibody: HRP-labeled mouse anti-M13 secondary antibody was diluted 1:2000, 100 μL / well, and incubated at 37°C for 1 hour. Washing: Discard the liquid in the wells and wash each well three times with 200 μL PBST. Color development: Add 100 μL of TMB color development solution to each well and place in the dark at room temperature for 10-15 minutes. Stop and read: After color development, add 2M H 2 SO 4 50 μL, stop the reaction; read the absorbance at 450 nm. Analyze the data.

[0047] (5) Sequencing analysis of specific nanobodies

[0048] Step 2. (4) ELISA test results are as follows Figure 1 As shown, clones with a value greater than 3 times the negative value were determined to be positive, and the bacterial solution was sent for sequencing and comparison analysis, and finally 4 anti-Siglec-6 nanobody sequences were obtained, and the sequences are shown in Table 1.

[0049] Table 1 Amino acid sequences of anti-Siglec-6 nanobodies

[0050]

[0051]

[0052]

[0053]

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

[0055] A Nanobody-hFc fusion protein expression platform was constructed based on the pcDNA3.1 eukaryotic expression vector, and the nucleotide sequences of 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 Siglec-6 nanobody-hFc fusion proteins with relatively high purity were obtained after affinity chromatography purification. The results are as follows Figure 2 As shown, the band sizes are consistent with expectations.

[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 the ELISA plate in advance, the plate was blocked after overnight at 4°C, and different amounts of recombinant anti-Siglec-6 nanobody (dilution: 10 -5 ~10 2 μg / mL), add secondary antibody for washing, color development, terminate the reaction, and measure the optical density (OD450) at 450nm using a microplate reader. Use four-parameter nonlinear regression curve fitting to determine the binding capacity. The results are shown in Figure 3 As shown, the four recombinant anti-Siglec-6 nanobodies of the present invention can bind to the Siglec-6 recombinant protein with high specificity.

[0057] Experimental Example 1 Recombinant anti-Siglec-6 nanobody and Siglec-6 + -Hela cell binding assay

[0058] First, Hela cells were infected with a lentivirus containing the full-length Siglec-6 gene, and high-purity Siglec-6 cells stably expressing the Siglec-6 gene were obtained by flow cytometry. + -Hela cells, and then the recombinant anti-Siglec-6 nanobody prepared in Example 2 was combined with Siglec-6 + -Hela cells were incubated at 37°C for 40 min, washed 3 times with PBS, and then incubated with APC@goatanti-human secondary antibody. After washing 3 times with PBS, the cells were detected by flow cytometry. The results are shown in Figure 4 As shown, the recombinant anti-Siglec-6 nanoantibodies Nb45, Nb46, Nb49 and Nb50 of the present invention can bind to Siglec-6 + -HeLa cells bind well.

[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 was fixed on the surface of CM5 chip using the coupling buffer in the amino coupling kit, and then the Siglec-6 recombinant protein was captured on the CM5 chip in a 2-fold serial dilution; then the purified recombinant anti-Siglec-6 nanobody was allowed to flow over the surface of the chip, and the machine read 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 was measured. The affinity measurement results showed 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 kinetic characteristics show that the four humanized recombinant anti-Siglec-6 nanoantibodies all have a relatively slow dissociation rate. 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 <![CDATA[1.20×10 5 ]]> <![CDATA[6×10 -9 ]]> <![CDATA[4.9×10 -14 ]]> Nb46 <![CDATA[1.40×10 4 ]]> <![CDATA[1.09×10 -6 ]]> <![CDATA[7.7×10 -11 ]]> Nb49 <![CDATA[1.14×10 5 ]]> <![CDATA[3.32×10 -9 ]]> <![CDATA[2.91×10 -14 ]]> Nb50 <![CDATA[1.81×10 5 ]]> <![CDATA[9.11×10 -8 ]]> <![CDATA[5.03×10 -13 ]]>

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

[0064] 10mL of peripheral blood from healthy adult donors was collected in a sterile anticoagulant tube. First, the blood was diluted 1:1 with an equal volume of PBS and mixed by inverting. 3mL of lymphocyte separation solution was added to each separation tube, centrifuged at room temperature at 1500rpm for 1min, and the diluted blood was added to the separation tube after removal, and centrifuged at room temperature at 1500rpm for 15min (the speed increase gear was adjusted to 2, and the speed decrease gear was adjusted to 2). The centrifuged separation tube was divided into three layers, the top layer was plasma, the milky white circle in the middle layer was lymphocytes, and the bottom layer was red blood cells; insert the gun tip into the milky white layer, slowly draw lymphocytes into a new 15ml centrifuge tube, add an appropriate amount of PBS to the tube, mix well, and centrifuge at 1500rpm for 5min. Discard the supernatant, add an appropriate amount of PBS to resuspend, mix well by inverting, and centrifuge at 1500rpm for 5min. Repeat the wash once. After discarding the supernatant, the cells were resuspended in an appropriate amount of RPMI1640 complete medium (serum inactivated) containing 1.00 μg / ml to 4.00 μg / ml of zoledronic acid, and the lymphocytes were counted using a cell counter. After counting, the cells were adjusted to an appropriate density and added to the cell culture system to stimulate the proliferation and activation of γδT cells.

[0065] Siglec-6-CAR was constructed with the nucleotide sequences of the four anti-Siglec-6 nanoantibodies of the present invention (i.e., Siglec-6 (Nb)), and the structure was as follows: CD8αsignalpeptide-Siglec-6 (Nb)-CD8αhinge-CD28αTm-4-1BB-CD3ζ-P2A-EGFP. HEK293T cells were used as cells for lentiviral packaging, and lentiviral packaging was performed using a three-plasmid packaging system (psPAX2, pMD2.G, CAR-γδT vector). After 48 hours, the supernatant virus solution was collected, concentrated by ultracentrifugation, coated with RetroNectin protein in advance, and concentrated virus was added to infect γδT cells. After 48 hours of infection, the transfection efficiency of Siglec-6 CAR-γδT cells was evaluated by flow cytometry to be above 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 according to the nucleotide sequence of the anti-Siglec-6 nanoantibody used.

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

[0067] 5 × 10 NCG mice were injected via tail vein 6The AML mouse tumor model was established with THP-1-mCherry.ffLuc cells 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, with 5 mice in each group, and injected with 1×10 7 NTγδT cells (untreated γδT cells), 1×10 7 The mice were injected with Siglec-6CAR-γδT cells (45#CAR-γδT, 46#CAR-γδT, 49#CAR-γδT or 50#CAR-γδT), and a control group injected with PBS was set up. The growth status and survival of the mice were observed every day, and the survival period was recorded. Figure 5 As shown, a significant delay in tumor progression was observed in the Siglec-6 CAR-γδT treatment group, and the survival time of tumor-bearing mice was significantly prolonged.

[0068] In summary, the specific nanoantibodies against Siglec-6 screened from the Bactrian camel VHH immune library of the present invention can specifically bind to Siglec-6 antigen. The affinity of the nanoantibodies obtained by the present invention is 10 -14 ~10 -11 The nanoantibody of the present invention can specifically bind to human Siglec-6, and can be used to prepare drugs for preventing or treating various diseases such as acute myeloid leukemia, allergies, inflammation, etc., and has broad application prospects.

Claims

1. An anti-Siglec-6 nanobody or antigen-binding fragment, 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, and 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, and 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, and 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, and SEQ ID NO:18, respectively.

2. The anti-Siglec-6 nanobody or antigen-binding fragment 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 antibody or a fusion nanobody.

3. The anti-Siglec-6 nanobody or antigen-binding fragment 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 also includes a framework region; The heavy chain variable region structure of the anti-Siglec-6 nanobody is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; preferably, the amino acid sequence of the heavy chain variable region or antigen-binding fragment of the anti-Siglec-6 nanobody is as shown in SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:14 or SEQ ID NO:

19.

4. A gene fragment encoding the anti-Siglec-6 nanobody or antigen-binding fragment according to any one of claims 1 to 3, 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.

5. An antibody, characterized in that: It comprises the anti-Siglec-6 nanobody or antigen-binding fragment described in any one of claims 1-3.

6. The antibody according to claim 5, characterized in that: 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; preferably, the antigen-binding fragment comprises any one selected from the group consisting of F(ab')2, Fab', Fab, Fv or scFv of an antibody.

7. A recombinant vector comprising a gene fragment encoding the anti-Siglec-6 nanobody or antigen-binding fragment described in any one of claims 1-3, or the antibody described in any one of claims 5-6; preferably, 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 7.

9. A chimeric antigen receptor, characterized in that: The antigen binding domain of the chimeric antigen receptor includes at least one of the anti-Siglec-6 nanobody or antigen binding fragment as described in any one of claims 1-3 and the antibody as described in any one of claims 5-6.

10. A CAR-γδT cell, characterized in that: It comprises the chimeric antigen receptor as claimed in claim 9.

11. A recombinant protein or a pharmaceutical composition thereof comprising at least one of the anti-Siglec-6 nanobody or antigen-binding fragment described in any one of claims 1 to 3 and the antibody described in any one of claims 5 to 6; preferably, the pharmaceutical composition further comprises an active agent; the active agent includes at least one of an immune checkpoint-related preparation, an antibody-drug conjugate, a bispecific antibody, a multispecific antibody, a radionuclide or a kinase inhibitor.

12. Use of the anti-Siglec-6 nanobody or antigen-binding fragment according to any one of claims 1 to 3, the gene fragment according to claim 4, the antibody according to any one of claims 5 to 6, the recombinant vector according to claim 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 claim 11 or a pharmaceutical composition thereof in the preparation of a medicament for preventing and / or treating tumors or autoimmune diseases; preferably, the tumor includes glioma, neuroblastoma, medulloblastoma, meningioma, lung cancer, esophageal cancer, pancreatic cancer, liver cancer, bile duct cancer, kidney cancer, bladder cancer The invention relates to a method for treating a leukemia or a degenerative disease of the present invention, wherein the leukemia or the degenerative disease is at least one of: cancer, ureteral cancer, prostate cancer, skin cancer, melanoma, ovarian cancer, endometrial cancer, cervical cancer, soft tissue sarcoma, acute and chronic leukemia, Hodgkin's and non-Hodgkin's lymphoma, gastric cancer or head and neck tumors; 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; more preferably, the drug comprises the following forms: immune cells, reagents, kits or compositions.

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