Siglec-9 targeting nano antibody and application thereof
By developing nanobodies targeting Siglec-9, the problems of tumor penetration and functional limitation of traditional monoclonal antibodies have been solved, achieving efficient tumor penetration and T cell activation, synergistically reversing the immunosuppressive microenvironment, and enhancing therapeutic efficacy.
Patent Information
- Application Number
- CN202510978452.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-16
- Publication Date
- 2025-11-28
AI Technical Summary
Existing traditional monoclonal antibodies targeting Siglec-9 suffer from problems such as large molecular weight leading to poor penetration into tumor tissues, immunogenicity risks, and limited functionality, making it difficult to achieve integrated diagnosis and treatment or synergistic effects with other therapies.
Develop nanobodies targeting Siglec-9, obtain the CDR region through genetic engineering, and construct multi-epitope or multi-specific nanobodies by combining them with flexible linker peptides. Utilize the small molecular weight and strong tissue penetration of nanobodies to activate T cell anti-tumor activity when used in combination with PD-1 inhibitors.
This study achieved high affinity recognition of the Siglec-9 protein by nanobodies, improved tumor penetration efficiency, enabled precise delivery of therapeutic molecules, enhanced antibody-dependent effector function, synergistically activated T cell anti-tumor activity, and reversed the immunosuppressive microenvironment.
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Figure CN121021693A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological medicine, and particularly relates to a nano antibody targeting Siglec-9 and application thereof. BACKGROUND
[0002] Siglec-9 (sialic acid-binding immunoglobulin-like lectin 9) is an important member of the immunoglobulin superfamily, which activates the immune inhibitory signaling pathway by recognizing the over-sialylated glycan structure on the surface of tumor cells, and promotes tumor immune escape. As a core regulatory molecule of the Siglec receptor family, Siglec-9 is widely expressed on the surface of immune suppressive cells such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in the tumor microenvironment (TME), and transmits inhibitory signals through the intracellular ITIM motif, significantly weakening the anti-tumor activity of T cells and NK cells, and becoming a key target that urgently needs to be broken through in the field of tumor immunotherapy.
[0003] At present, the intervention strategy for Siglec-9 mainly depends on traditional monoclonal antibodies, but it has the following limitations: 1. The molecular weight is too large (about 150 kDa), which leads to poor tumor tissue permeability and makes it difficult to effectively reach the target; 2. Immunogenicity risk, mouse or chimeric antibodies are easy to cause human anti-drug antibody (HAMA) reaction; 3. Single function, it is difficult to realize the integration of diagnosis and treatment or synergistic effect with other therapies. SUMMARY
[0004] Based on the technical problems existing in the background art, the present application provides a nano antibody targeting Siglec-9 and application thereof. The camelid nano antibody targeting the extracellular region of human Siglec-9 has nanomolar binding efficiency, is suitable for developing immunotherapy drugs, and can be used for the treatment or detection of diseases related to abnormal expression of Siglec-9 such as tumors. The nano antibody is obtained by genetic engineering technology, and has the characteristics of small molecular weight and strong tissue penetration.
[0005] The present application provides a CDR region of a nano antibody targeting Siglec-9, which includes any one of the following:
[0006] a. In the CDR region, the amino acid sequences of CDR1, CDR2 and CDR3 are sequentially shown as SEQ ID NO: 1-3;
[0007] b. In the CDR region, the amino acid sequences of CDR1, CDR2 and CDR3 are sequentially shown as SEQ ID NO: 4-6;
[0008] c. In the CDR region, the amino acid sequences of CDR1, CDR2 and CDR3 are sequentially shown as SEQ ID NO: 7-9;
[0009] d. an amino acid sequence having greater than or equal to 80% homology to at least one of the amino acid sequences of SEQ ID NOs: 1-9 and still having the function of targeting the Siglec-9 protein;
[0010] e. an amino acid sequence having one or more amino acids added, deleted, modified and / or replaced to at least one of the amino acid sequences of SEQ ID NOs: 1-9 and still having the function of targeting the Siglec-9 protein.
[0011] In 1993, Hamers-Casterman et al. first reported that they isolated homodimers of heavy chains from the serum of Camelidae, which were called heavy chain antibodies (HCAbs). In order to distinguish from the heavy chain variable region (VH) of traditional antibodies, the single variable domain at the N-terminus of HCAbs with functional antigen binding properties was called heavy chain antibody variable region fragment (VHH), with a molecular weight of about 15 kDa, also known as nanobodies (Nbs). In addition to Camelidae, similar to HCAbs, immunoglobulin new antigen receptors (IgNARs) were also found in shark serum.
[0012] Traditional antibodies are composed of two identical heavy chains (H chains) and two identical light chains (L chains), the L chain includes two domains, while the H chain is folded into four domains, the sequences of the N-terminal domains of the H chain and the L chain are different for antibodies, i.e. the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody. Compared with traditional antibodies, HCAbs isolated from Camelidae serum lack light chains (VL and CL) and a constant region (CH1) of heavy chains, and contain a variable region (VHH) and two constant regions (CH2 and CH3). VHH has the same domain structure as VH of traditional antibodies, i.e. four conserved framework regions (FRs) and three complementarity determining regions (CDRs). The "CDR region" can recognize specific antigens (such as Siglec-9 protein) and has good affinity, and has a targeting effect on specific antigens.
[0013] The above-mentioned Siglec-9 protein is the extracellular domain of the Siglec-9 protein, and the amino acid sequence is shown as SEQ ID NO: 26.
[0014] The application also provides a nanobody, which comprises the CDR region of the above-mentioned Siglec-9-targeting nanobody.
[0015] In addition to containing the CDR region (i.e. the complementarity determining region), the nanobody of the application also contains the FR region (i.e. the conserved framework region), which exists in four FR regions (i.e. FR1, FR2, FR3, FR4).
[0016] Preferably, the nanobody is a monovalent nanobody, a multivalent nanobody, a multi-epitope nanobody or a multi-specific nanobody; more preferably a monovalent nanobody or a multi-epitope nanobody.
[0017] A monovalent nanobody is a monomer form composed of a single nanobody domain, which only binds to a single epitope of an antigen.
[0018] A multivalent nanobody is a series of nanobodies recognizing the same epitope, such as dimers, trimers, etc., which repeatedly bind to the same epitope of an antigen.
[0019] A multi-epitope nanobody is a series of nanobodies recognizing different epitopes of the same antigen, which synchronously bind to two or more non-overlapping epitopes of the same antigen.
[0020] A multi-specific nanobody is a fusion of nanobodies recognizing different antigens, which simultaneously binds to two or more independent antigens or cells.
[0021] Preferably, the amino acid sequence of the nanobody includes any one of the following:
[0022] a. as shown in one of SEQ ID NOs: 10-14;
[0023] b. an amino acid sequence having a homology of 80% or more with at least one of the amino acid sequences of SEQ ID NOs: 10-14, and still having a function of targeting a Siglec-9 protein;
[0024] c. an amino acid sequence having a function of targeting a Siglec-9 protein after one or more amino acids of at least one of the amino acid sequences of SEQ ID NOs: 10-14 are added, deleted, modified and / or replaced.
[0025] The nanobodies having the above-mentioned amino acid sequences as shown in SEQ ID NOs: 10-14 are sequentially named as nanobody 1H8, nanobody 5B4, nanobody 8B6, bi-epitope nanobody 5-1, bi-epitope nanobody 5-8.
[0026] The above-mentioned bi-epitope nanobody 5-1 is obtained by connecting the C-terminus of nanobody 5B4 with the N-terminus of nanobody 1H8 through a flexible linker peptide.
[0027] The above-mentioned bi-epitope nanobody 5-8 is obtained by connecting the C-terminus of nanobody 5B4 with the N-terminus of nanobody 8B6 through a flexible linker peptide.
[0028] The amino acid sequence of the flexible linker peptide is as shown in SEQ ID NO: 16.
[0029] The application also provides an antibody targeting Siglec-9, comprising: the CDR region of the above-mentioned nanobody targeting Siglec-9 or the above-mentioned nanobody.
[0030] Preferably, the antibody targeting Siglec-9 further comprises: a TEV enzyme cleavage site and a Fc domain.
[0031] Preferably, the Fc domain is a human IgG1 Fc domain.
[0032] Preferably, the Fc domain is connected to the C-terminal of the amino acid sequence of the CDR region or the nanobody through the TEV enzyme cleavage site.
[0033] Preferably, the amino acid sequence of the Fc domain is as shown in SEQ ID NO: 15; or an amino acid sequence with homology greater than or equal to 80% with the amino acid sequence shown in SEQ ID NO: 15.
[0034] Preferably, the amino acid sequence of the TEV enzyme cleavage site is as shown in SEQ ID NO: 17; or a DNA sequence with homology greater than or equal to 80% with the DNA sequence shown in SEQ ID NO: 17.
[0035] Preferably, the antibody targeting Siglec-9 is a heavy chain antibody, a multi-epitope antibody or a multi-specific antibody; more preferably, it is a heavy chain antibody or a multi-epitope heavy chain antibody.
[0036] The heavy chain antibody of the application refers to a homodimer with only heavy chains, and each heavy chain of the heavy chain antibody of the application comprises one variable region (VHH) and two constant regions (CH2 and CH3); by fusion with the constant region of human IgG1 Fc, the heavy chain antibody can have CH2 and CH3 of human IgG1 Fc.
[0037] The multi-epitope antibody is a single antibody molecule that binds two or more non-overlapping epitopes on the same antigen molecule.
[0038] The multi-specific antibody is an antibody that can bind two different antigens (such as antigen A and antigen B), or the same epitope of the same antigen.
[0039] Preferably, the amino acid sequence of the antibody targeting Siglec-9 comprises any one of the following:
[0040] a. as shown in one of SEQ ID NO: 18-22;
[0041] b. an amino acid sequence with homology greater than or equal to 80% with the amino acid sequence shown in SEQ ID NO: 18-22, and still having the function of targeting Siglec-9 protein;
[0042] c. one or more amino acids of the amino acid sequences shown in SEQ ID NOs: 18-22 are added, deleted, modified and / or replaced, and the amino acid sequence still has the function of targeting Siglec-9 protein.
[0043] The above-mentioned amino acid sequences of the antibodies targeting Siglec-9 shown in SEQ ID NOs: 18-22 are sequentially named as antibody 1H8-Fc targeting Siglec-9, antibody 5B4-Fc targeting Siglec-9, antibody 8B6-Fc targeting Siglec-9, bi-epitope antibody 5-1-Fc targeting Siglec-9, and bi-epitope antibody 5-8-Fc targeting Siglec-9.
[0044] The present application also provides a polynucleotide encoding the CDR region of the above-mentioned nanobody targeting Siglec-9, or encoding the above-mentioned nanobody, or encoding the above-mentioned antibody targeting Siglec-9.
[0045] The above-mentioned polynucleotide refers to a chain compound polymerized by nucleotides; and the above-mentioned polynucleotide includes DNA or RNA.
[0046] The DNA sequences encoding the above-mentioned nanobody 1H8, nanobody 5B4, and nanobody 8B6 are shown in SEQ ID NOs: 23-25.
[0047] The above-mentioned homology is greater than or equal to 80%, and the homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
[0048] The present application also provides an expression vector containing the above-mentioned polynucleotide.
[0049] The present application also provides a host cell containing the above-mentioned expression vector or having the above-mentioned polynucleotide integrated into the genome of the host cell.
[0050] Preferably, the host cell is a cell capable of expressing an exogenous protein.
[0051] Preferably, the host cell is a bacterium, a yeast, an insect cell, or a mammalian cell.
[0052] The present application also provides an antibody conjugate, which comprises:
[0053] a. the above-mentioned nanobody or the above-mentioned antibody targeting Siglec-9; and
[0054] b. a conjugated moiety selected from any one or a combination of the following: a detectable label, an enzyme, a drug, a therapeutic radioisotope, a toxin, a cytokine, and a solid carrier.
[0055] Antibodies can be labeled and detected, for example, by being labeled with detectable markers or enzymes that produce detectable markers. These detectable markers include: radioactive isotopes for detection, luminescent substances, colored substances, colloidal gold markers, polyethylene glycol, streptavidin, or biotin, etc.; the luminescent substances can be fluorescent agents, etc.
[0056] The radioactive isotopes used for the above-mentioned detection include: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, etc.
[0057] The aforementioned radioactive isotopes used for treatment include: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, Ir-192, Re-186, Sm-153, Ru-106, Yb-169, Yb-177, etc.
[0058] The aforementioned drugs can be cytotoxic drugs, immunomodulators, and proteasome inhibitors. The cytotoxic drugs can be doxorubicin, paclitaxel, oxaliplatin, gemcitabine, etoposide, etc.; the immunomodulators can be thalidomide, lenalidomide, pomalidomide, interferon-α, interleukin-2, etc.; and the proteasome inhibitors can be bortezomib, carfilzomib, ixazomib, etc.
[0059] The aforementioned solid support can be polystyrene plates, beads, etc.
[0060] This invention also proposes applications of the CDR region of the above-mentioned Siglec-9-targeting nanobody, the above-mentioned nanobody, the above-mentioned Siglec-9-targeting antibody, and the above-mentioned antibody conjugate, the applications of which include:
[0061] 1) Application in the preparation of reagents and / or kits for detecting Siglec-9, and reagents and / or kits for diagnosing Siglec-9-related diseases;
[0062] 2) Application in the preparation of drugs targeting Siglec-9;
[0063] 3) Use in the preparation of medicines for the prevention and / or treatment of Siglec-9 related diseases.
[0064] Preferably, the diseases associated with Siglec-9 are tumors, sepsis, chronic inflammatory diseases, or autoimmune diseases.
[0065] Preferably, the tumor includes: colon cancer, breast cancer, ovarian cancer, cervical cancer, lung cancer, liver cancer, bladder cancer, prostate cancer, brain glioma or melanoma.
[0066] The application also provides a pharmaceutical composition comprising one of the CDR regions of the Siglec-9-targeting nanobody, the nanobody, the Siglec-9-targeting antibody or the antibody conjugate.
[0067] The pharmaceutical composition can be in the form of a gastrointestinal administration form (such as a powder, a tablet, a granule, a capsule, a solution, an emulsion, a suspension, etc.), an injection administration form (such as an injection, including subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, intracavitary injection or intralesional injection, etc.), a respiratory tract administration form (such as a spray, an aerosol, a powder mist, etc.), a skin administration form (such as a topical solution, a lotion, a liniment, an ointment, a plaster, a paste, a patch, etc.), a mucous membrane administration form (such as eye drops, nose drops, an eye ointment, a gargle, a sublingual tablet, a sticking tablet and a film patch, etc.), and a cavity administration form (such as a suppository, an aerosol, an effervescent tablet, drops and drop pills, etc.).
[0068] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
[0069] The pharmaceutically acceptable excipient includes at least one of a diluent, an excipient, a stabilizer, an antioxidant, a coloring agent, a sustained-release agent, etc., such as physiological saline, starch, lipids, wax, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, inorganic salts, hydroxypropyl methylcellulose, hydroxyethyl cellulose, etc.
[0070] The application develops a Siglec-9-targeting nanobody, which has high affinity and specific recognition performance for the Siglec-9 protein, has a molecular weight of only about 15 kDa, is reduced by 90% compared with a traditional antibody, and significantly improves the tumor penetration efficiency; the CDR region thereof is screened by directional evolution, has a binding affinity for the Siglec-9 protein of sub-nanomolar level, and can be used to construct a bispecific antibody or an antibody-drug conjugate through a flexible connecting peptide to realize precise delivery of a therapeutic molecule in a tumor microenvironment; the nanobody can be used to prepare a pharmaceutical composition containing the nanobody, or a recombinant antibody form in which the nanobody is coupled with an Fc domain, which can enhance antibody-dependent effect functions; the nanobody has the advantages of small molecular weight, strong penetration, strong antigen recognition ability, easy engineering modification and low production cost, etc.
[0071] In addition, the Siglec-9-targeting nanobody can be applied to immune combination therapy, and can be used in combination with a PD-1 inhibitor to synergistically activate T cell anti-tumor activity and reverse an immunosuppressive microenvironment.
[0072] The application obtains nanobodies with a brand-new CDR topological structure through phage display library screening, guided rational design, sequence specificity, and a space epitope binding mode, and a structure-function synergistic optimization strategy form a complete intellectual property protection system. BRIEF DESCRIPTION OF DRAWINGS
[0073] Figure 1 Molecular sieve chromatography results and reduced SDS-PAGE analysis results of Siglec-9 protein, wherein A is the molecular sieve chromatography result, and B is the reduced SDS-PAGE analysis result.
[0074] Figure 2 ELISA test results of monoclonal phage on Siglec-9 protein, wherein PBS is a control, and Siglec9-ECD is an extracellular domain of Siglec-9 protein.
[0075] Figure 3 Detection results of five nanobody-Fc fusion proteins and five nanobodies, wherein C-G are, in sequence, Protein A affinity chromatography elution curves of 8B6-Fc, 5B4-Fc, 1H8-Fc, bi-epitope 5-1-Fc, and bi-epitope 5-8-Fc fusion proteins, with an elution pH of 3.0; H is a reduced SDS-PAGE analysis result; and I is a schematic diagram of a molecular structure of a bi-epitope nanobody-Fc fusion protein.
[0076] Figure 4 A is a competitive ELISA detection result of 5B4-Fc fusion protein, nanobody 8B6, and nanobody 1H8 binding epitopes.
[0077] Figure 4 B-D are ELISA detection results of 5B4-Fc, 8B6-Fc, and 1H8-Fc fusion proteins on binding activities of three epitope domains in an extracellular domain of Siglec-9.
[0078] Figure 5 A is an ELISA detection result of 5B4-Fc, 8B6-Fc, 1H8-Fc, bi-epitope 5-1-Fc, and bi-epitope 5-8-Fc fusion proteins on binding activities of recombinant human Siglec-9 ECD protein (His tag).
[0079] Figure 5 B is a flow cytometry detection result of five nanobody-Fc fusion proteins on binding activities of HEK293T cells overexpressing human Siglec-9, wherein Blank is a blank control, and Control is IgG1-Fc.
[0080] Figure 5C. The kinetic parameters of five fusion proteins to immobilized Siglec-9 ECD were determined by surface plasmon resonance technology.
[0081] Figure 5 D. The inhibition rates of five nanobodies to the binding of Siglec-9 to ligands on the surface of three tumor cells (SKOV3, SUM159, HCT116) were detected by competitive ELISA.
[0082] Figure 6 Figure 4 is the results of the anti-tumor effects of 5B4-Fc, 8B6-Fc and 5-8-Fc fusion proteins in a mouse subcutaneous tumor model, wherein A is a schematic diagram of a human ovarian cancer mouse subcutaneous tumor model construction and treatment strategy, B is a tumor volume growth curve during treatment, C is a tumor peel real object diagram after treatment, and D is a mouse body weight change curve during treatment. DETAILED DESCRIPTION
[0083] Hereinafter, the technical solutions of the present application will be described in detail through specific embodiments, but it should be clear that these embodiments are used for illustration, but not to be interpreted as limiting the scope of the present application.
[0084] Example 1 Construction and screening of Siglec-9 nanobody phage library
[0085] 1. Preparation of antigen and animal immunization
[0086] The amino acid sequence shown in SEQ ID NO: 26, which is the extracellular domain amino acid sequence of Siglec-9 protein, was constructed into a vector plasmid with a His tag, then transfected into HEK293F cells (ATCC CBP60437) for expression and purification to obtain Siglec-9-His protein; the results are shown in Figure 1
[0087] Figure 1 Figure 2 is the results of molecular sieve chromatography and reduced SDS-PAGE analysis of Siglec-9 protein, wherein A is the results of molecular sieve chromatography, and B is the results of reduced SDS-PAGE analysis.
[0088] As can be seen from Figure 1 It can be seen that: Siglec-9 protein shows part of single elution peak after molecular sieve chromatography, suggesting that Siglec-9 protein exists in dimeric conformation, but mainly exists in monomeric conformation; reduced SDS-PAGE analysis confirms that the molecular weight of recombinant Siglec-9 protein is about 60 kDa (the theoretical value is 36 kDa), and the glycosylation of recombinant Siglec-9 protein is serious.
[0089] Healthy adult camels were selected for the first immunization at day 0, and immunized every 14 days; the first to third immunization (day 0, 14, 28), the Siglec-9-His protein and Freund's complete adjuvant (Sigma, F5508) were mixed in equal volume, and the camels were subcutaneously injected, the amount of Siglec-9-His protein used for each immunization was 500 μg / time; two weeks later, the peripheral blood lymphocytes were separated by Ficoll gradient centrifugation (Solebao), and the total RNA was obtained by Omega Biotek RNA extraction kit, and the cDNA was synthesized by PrimeScript TM II cDNA Synthesis Kit (Takara) after DNAse treatment.
[0090] 2. Construction of phage library
[0091] The VHH-specific primer was used to amplify the nanobody gene fragment with the cDNA as a template, and the Gibson assembly method was used to clone the fragment into the phagemid pR2 (NcoI / NotI site) to obtain the assembly product; the assembly product was electroporated into TG1 competent cells (10% glycerol washing method) and then coated on a 2×YT agar plate (formula: 16 g / L tryptone, 10 g / L yeast powder, 5 g / L NaCl) containing 2% glucose and 100 μg / ml ampicillin, and cultured at 37°C overnight, then the colonies were scraped and inoculated into 2×YT liquid medium (containing 2% glucose and 100 μg / ml ampicillin), and cultured to the logarithmic phase, then KM13 helper phage (1×10 12 pfu) was added and incubated at 37°C for 45 min, then centrifuged and resuspended in 2×YT medium containing 0.1% glucose, 50 μg / ml kanamycin and 100 μg / ml ampicillin, and cultured at 25°C for 20 h, then the phage was enriched by PEG / NaCl precipitation, resuspended in PBS and stored at -80°C to obtain the phage library.
[0092] 3. Screening of phage library
[0093] The Siglec-9-His protein prepared in step 1 was diluted with PBS to a concentration of 0.1 mg / mL and coated into a 96-well plate (Nunc MaxiSorp), and then blocked with 5% skim milk / PBS at 4°C for 2 h; then the phage library (1×10 11pfu / well), washed 30 times with PBST (0.1% Tween-20) after incubation at room temperature for 1 h, eluted by 0.5 mg / mL trypsin (Macklin P913714) for 1 h, and collected by infecting TG1 strain, then plated and amplified to complete the first round of screening; for the second round of screening, the Siglec-9 protein concentration was reduced to 0.02 mg / mL, the washing strength was increased to 30 times of PBST (0.2% Tween-20), and the rest of the steps were the same as the first round of screening, to obtain monoclonal phages.
[0094] 4. Preparation and identification of monoclonal phages
[0095] The monoclonal phages obtained by screening were randomly picked and inoculated into 96-well plates containing 2xYT medium (containing 2% glucose and 100 μg / mL ampicillin), and cultured at 37°C with shaking until the logarithmic phase. Then, KM13 helper phages (4x10 8 After infection at 25°C overnight, the supernatant was mixed with MPBS at a ratio of 1:3, and the mixture was recorded as phage supernatant dilution;
[0096] The phage supernatant dilution was subjected to ELISA detection, and the specific steps were as follows: the Siglec-9-His protein (1 μg / mL) prepared in step 1 was used to coat a 96-well ELISA plate, and after blocking, the phage supernatant dilution was added for incubation. After washing, HRP-anti M13 antibody (1:8000, Yiqiao Shenzhou) was added for incubation, followed by washing, TMB color development, and then determination of OD 450nm values, and the clones with OD 450nm values greater than 1 were sequenced, and three nanobodies with specific recognition and binding performance to Siglec-9 protein were obtained, which were named nanobody 5B4, nanobody 8B6, and nanobody 1H8, respectively. All of them have CDR regions targeting Siglec-9 protein.
[0097] The CDR1 amino acid sequence in the CDR region of nanobody 1H8 is shown as SEQ ID NO: 1, the CDR2 amino acid sequence is shown as SEQ ID NO: 2, and the CDR3 amino acid sequence is shown as SEQ ID NO: 3; the amino acid sequence of nanobody 1H8 is shown as SEQ ID NO: 10, and the DNA sequence is shown as SEQ ID NO: 23.
[0098] The CDR1 amino acid sequence in the CDR region of nanobody 5B4 is shown as SEQ ID NO: 4, the CDR2 amino acid sequence is shown as SEQ ID NO: 5, and the CDR3 amino acid sequence is shown as SEQ ID NO: 6; the amino acid sequence of nanobody 5B4 is shown as SEQ ID NO: 11, and the DNA sequence is shown as SEQ ID NO: 24.
[0099] The CDR1 amino acid sequence in the CDR region of the nanobody 8B6 is shown as SEQ ID NO: 7, the CDR2 amino acid sequence is shown as SEQ ID NO: 8, and the CDR3 amino acid sequence is shown as SEQ ID NO: 9; the amino acid sequence of the nanobody 8B6 is shown as SEQ ID NO: 12, and the DNA sequence is shown as SEQ ID NO: 25.
[0100] The results of the ELISA detection are shown in Table 1. Figure 2 Table 1: ELISA test results of the monoclonal phage against Siglec-9 protein, wherein PBS is the control, and Siglec9-ECD is the extracellular domain of Siglec-9 protein. Figure 2 Table 1: ELISA test results of the monoclonal phage against Siglec-9 protein, wherein PBS is the control, and Siglec9-ECD is the extracellular domain of Siglec-9 protein.
[0101] Figure 2 The results of the verification of the targeting binding ability of the anti-Siglec-9 monoclonal phage obtained by screening based on the phage display technology are shown.
[0102] Example 2: Expression and purification of nanobody-Fc fusion protein
[0103] 1. Recombinant plasmid construction: a human IgG1 Fc fragment (the amino acid sequence of which is shown as SEQ ID NO: 15) is fused to the C-terminus of the amino acid sequence of the nanobody 5B4, 8B6, and 1H8 through a TEV enzyme cleavage site (the amino acid sequence of which is shown as SEQ ID NO: 17), respectively; then, each is cloned into a mammalian expression vector pTT5 (NRC Biotechnology Research Institute) to obtain each recombinant plasmid.
[0104] 2. Protein expression and preliminary purification: each recombinant plasmid is transiently transfected into HEK293F cells (ATCC CBP60437) through PEI, respectively, and cultured at 37°C, 5% CO2 for 4 days, respectively; the cell supernatant is collected and purified through a Protein A affinity chromatography column to obtain 5B4-Fc fusion protein, 8B6-Fc fusion protein, and 1H8-Fc fusion protein, each with a purity of >95%.
[0105] 3. Expression and purification of bi-epitope nanobody-Fc fusion protein:
[0106] It was found by the following Example 3 that the nanobodies 5B4, 8B6, 1H8 bind to different epitopes of the Siglec-9 extracellular domain, respectively, so the C-terminus of the nanobody 5B4 was connected to the N-terminus of the nanobody 1H8 with a flexible linker peptide (the amino acid sequence of which is SEQ ID NO: 16), denoted as the biparatopic nanobody 5-1 (the amino acid sequence of the biparatopic nanobody 5-1 is SEQ ID NO: 13); the C-terminus of the nanobody 5B4 was connected to the N-terminus of the nanobody 8B6 with a flexible linker peptide (the amino acid sequence of which is SEQ ID NO: 16), denoted as the biparatopic nanobody 5-8 (the amino acid sequence of the biparatopic nanobody 5-8 is SEQ ID NO: 14);
[0107] The human IgG1 Fc fragment (the amino acid sequence of which is SEQ ID NO: 15) was fused to the C-terminus of the amino acid sequence of the biparatopic nanobodies 5-1, 5-8, respectively, through a TEV enzyme cleavage site (the amino acid sequence of which is SEQ ID NO: 17) (the specific connection mode is shown in the following figure) Figure 3 I); then cloned into the mammalian expression vector pTT5 (NRC Biotechnology Research Institute), respectively, to obtain each recombinant plasmid; then the biparatopic 5-1-Fc fusion protein and the biparatopic 5-8-Fc fusion protein were obtained according to Step 2, both with a purity of >95%.
[0108] 4. TEV enzyme cleavage and secondary purification: the 5B4-Fc fusion protein, the 8B6-Fc fusion protein, the 1H8-Fc fusion protein, the biparatopic 5-1-Fc fusion protein, and the biparatopic 5-8-Fc fusion protein were incubated with 6xHis-tagged TEV protease at 4°C for 24h, respectively, to obtain the cleavage products; each cleavage product was sequentially passed through a Protein A column (to capture the uncut Fc fragment) and a nickel column (to bind 6xHis-TEV enzyme and impurities), and the flow-through was collected, concentrated by ultrafiltration, to obtain high-purity nanobodies 5B4, 8B6, 1H8, biparatopic nanobody 5-1, and biparatopic nanobody 5-8, respectively, all with a purity of >98%.
[0109] In the amino acid sequences of the above-mentioned nanobody-Fc fusion proteins, a short-chain flexible linker peptide can also be contained between the nanobody and the TEV enzyme cleavage site, and the amino acid sequence of the short-chain flexible linker peptide can be SRGS; a short-chain flexible linker peptide can also be contained between the TEV enzyme cleavage site and the Fc domain, and the amino acid sequence of the short-chain flexible linker peptide can be GS.
[0110] The obtained five 5B4-Fc, 8B6-Fc, 1H8-Fc, bispecific 5-1-Fc and bispecific 5-8-Fc fusion proteins were detected after enzymatic digestion, and the obtained nanobodies 5B4, 8B6, 1H8, bispecific nanobody 5-1 and bispecific nanobody 5-8 were detected in turn, and the results are shown in Figure 3
[0111] Figure 3 The detection results of the five nanobody-Fc fusion proteins and the five nanobodies, wherein C-G are the elution curves of Protein A affinity chromatography of 8B6-Fc, 5B4-Fc, 1H8-Fc, bispecific 5-1-Fc and bispecific 5-8-Fc fusion proteins, and the elution pH is 3.0; H is the result of reduced SDS-PAGE analysis; I is a schematic diagram of the molecular structure of the bispecific nanobody-Fc fusion protein.
[0112] Figure 3 It can be seen that the molecular weight of 8B6-Fc, 5B4-Fc and 1H8-Fc fusion proteins is about 42 kDa, and the molecular weight of bispecific 5-1-Fc and bispecific 5-8-Fc fusion proteins is about 58 kDa; the molecular weight of nanobodies 5B4, 8B6 and 1H8 is about 14 kDa, and the molecular weight of bispecific nanobodies 5-1 and 5-8 is about 28 kDa; the electrophoretic band uniformity is > 98%, and there is no dimer or degradation fragment.
[0113] The above molecular weights are the molecular weights containing glycosylation.
[0114] The amino acid sequence of the 1H8-Fc fusion protein (i.e., the antibody 1H8-Fc targeting Siglec-9) is shown in SEQ ID NO: 18.
[0115] The amino acid sequence of the 5B4-Fc fusion protein (i.e., the antibody 5B4-Fc targeting Siglec-9) is shown in SEQ ID NO: 19.
[0116] The amino acid sequence of the 8B6-Fc fusion protein (i.e., the antibody 8B6-Fc targeting Siglec-9) is shown in SEQ ID NO: 20.
[0117] The amino acid sequence of the bispecific 5-1-Fc fusion protein (i.e., the bispecific antibody 5-1-Fc targeting Siglec-9) is shown in SEQ ID NO: 21.
[0118] The amino acid sequence of the bispecific 5-8-Fc fusion protein (i.e., the bispecific antibody 5-8-Fc targeting Siglec-9) is shown in SEQ ID NO: 22.
[0119] ELISA detection of the binding properties and competition of the nanobody-Fc fusion proteins with different epitope domains in the Siglec-9 extracellular domain
[0120] 1. Specific binding detection of different epitope domains
[0121] Three different epitope domain sequences in the Siglec-9 extracellular domain were selected to prepare three epitope domains, which were sequentially designated as Ig1, Ig2, and Ig3 (the amino acid sequences of Ig1, Ig2, and Ig3 are sequentially shown as SEQ ID NOs: 27-29); each was diluted with PBS to 2 μg / mL, and added to an enzyme-labeled plate in an amount of 100 μL / well, and coated overnight at 4°C. After PBST washing for 3 times, 5% skimmed milk-PBS blocking solution was added to each well, and blocked at room temperature for 1 h. Then, the 5B4-Fc, 8B6-Fc, and 1H8-Fc fusion proteins were gradiently diluted at 1:4, and added to the wells corresponding to the coated epitope domains in an amount of 100 μL / well, and incubated at room temperature for 1 h. After PBST washing, HRP-labeled anti-human IgG1 Fc secondary antibody (Beijing Yiqioshen State) was added, and incubated at room temperature for 1 h. After TMB color development and termination, the OD 450 values were detected. The results are shown in Figure 4 B-D,
[0122] Figure 4 B-D are the ELISA detection results of the binding activities of the 5B4-Fc, 8B6-Fc, and 1H8-Fc fusion proteins with the three epitope domains in the Siglec-9 extracellular domain.
[0123] As can be seen from Figure 4 B-D, the 5B4-Fc and 8B6-Fc fusion proteins can simultaneously bind to the Ig1 and Ig2 epitope domains, and the 1H8-Fc fusion protein can specifically bind to the Ig2 epitope domain.
[0124] 2. Antibody competitive binding analysis
[0125] The Siglec-9-His protein (2 μg / mL) prepared in Example 1 was used to coat and block an enzyme-labeled plate. The concentration of the 5B4-Fc fusion protein was fixed at 0.4 nM, and was mixed with gradiently diluted nanobodies 8B6 and 1H8 (1:4 dilution), and then added to the wells in an amount of 100 μL / well for incubation for 1 h. After PBST washing, HRP-labeled anti-human IgG1 Fc secondary antibody (Beijing Yiqioshen State) was added, and incubated at room temperature for 1 h. After TMB color development and termination, the OD 450 values were detected. The results are shown in Figure 4 A.
[0126] Figure 4A competitive ELISA assay result of 5B4-Fc fusion protein, nanobody 8B6, nanobody 1H8 binding to the epitope.
[0127] From Figure 4 As can be seen from A, the OD 450 values of 5B4-Fc fusion protein, nanobody 8B6, nanobody 1H8 did not rise significantly, indicating that the three bind to different epitopes of Siglec-9 extracellular domain.
[0128] Example 4 ELISA assay of five nanobody-Fc fusion proteins binding to Siglec-9
[0129] 1. Protein coating and blocking: the Siglec-9-His protein prepared in Example 1 was diluted with PBS buffer to 2 μg / mL, and added to a 96-well enzyme-labeled plate (BIOFIL) at an amount of 100 μL / well, coated overnight at 4°C; then the coating solution was discarded, washed twice with PBS buffer containing 0.1% Tween-20, and 5% skim milk-PBS blocking solution was added to each well, and incubated at room temperature for 1 h;
[0130] 2. Antibody incubation and detection: 5B4-Fc, 8B6-Fc, 1H8-Fc, bi-epitope 5-1-Fc and bi-epitope 5-8-Fc fusion proteins were respectively diluted with MPBS at a gradient of 1:4, 100 μL of each gradient was added to the corresponding well, and incubated at room temperature for 1 h, washed 3 times with PBST, then HRP-labeled anti-human IgG1 Fc secondary antibody (Beijing Yiqiao God) was added to each well, and incubated at room temperature for 1 h, washed, then TMB color developing solution was added and reacted for 5 min, 1 M sulfuric acid was added to stop the reaction, and the absorbance (OD 450 ) at 450 nm was measured.
[0131] 3. Data analysis: the half effective binding concentration (EC 50 ) was calculated by fitting the OD 450 values with the antibody concentration, and the results are shown in Figure 5 A.
[0132] Figure 5 A ELISA assay result of 5B4-Fc, 8B6-Fc, 1H8-Fc, bi-epitope 5-1-Fc and bi-epitope 5-8-Fc fusion proteins binding to recombinant human Siglec-9 ECD protein (His tag).
[0133] From Figure 5 As can be seen from A, the EC 500.6444 ± 0.1689 nM, 0.9361 ± 0.1268 nM, 1.0756 ± 0.2653 nM, 0.8243 ± 0.6466 nM, 0.7242 ± 0.090 nM; indicating that the above five kinds of fusion proteins have a binding affinity to Siglec-9 reaching the nanomolar level.
[0134] Example 5 Flow cytometry detection of the binding ability of five kinds of nanobody-Fc fusion proteins to Siglec-9
[0135] The interaction of five kinds of nanobody-Fc fusion proteins with cell surface natural conformation Siglec-9 was evaluated by flow cytometry, including the following steps:
[0136] The full-length human Siglec-9 expression plasmid was constructed, and the lipo8000 transfection reagent (Beyotime) was used to transfect into HEK293T cells (ATCC CBP60437). After 48 h of culture in a 6 cm culture dish, the transfected cells were collected, washed with pre-cooled PBS, and then divided into 7 groups for surface labeling experiment. Each group was added with 100 nM concentration of 5B4-Fc fusion protein, 8B6-Fc fusion protein, 1H8-Fc fusion protein, dual epitope 5-1-Fc fusion protein, dual epitope 5-8-Fc fusion protein, IgG1-Fc and blank control, respectively, and incubated at 4°C for 1 h, followed by two times of cold PBS washing. Then, 0.5 μL of FITC-labeled goat anti-human IgG (H+L) secondary antibody (Beyotime) was added to each group, and incubated in the dark for 1 h, and then washed and resuspended in 200 μL of PBS for flow cytometry detection. The results are shown in Figure 5 B.
[0137] Figure 5 B is the flow cytometry detection result of the binding activity of five kinds of nanobody-Fc fusion proteins to HEK293T cells overexpressing human Siglec-9, wherein Blank is a blank control, and Control is IgG1-Fc.
[0138] From Figure 5 It can be seen from B that 5B4-Fc, 8B6-Fc, 1H8-Fc, dual epitope 5-1-Fc and dual epitope 5-8-Fc fusion proteins all show specific binding to cell surface Siglec-9 protein, confirming that the five kinds of fusion proteins can recognize the active antigen (i.e. Siglec-9 protein) epitope in the natural state.
[0139] Example 6
[0140] Surface plasmon resonance detection of the binding ability of five kinds of nanobody-Fc fusion proteins to Siglec-9
[0141] 1. SPR (Surface Plasmon Resonance) detection system construction:
[0142] Using Biacore 8K system, human Siglec-9 extracellular domain protein was immobilized on the surface of CM5 sensor chip, and PBST buffer (containing 0.05% surfactant Tween-20) was used as the mobile phase.
[0143] The fusion proteins (i.e. 5B4-Fc, 8B6-Fc, 1H8-Fc, bi-epitope 5-1-Fc and bi-epitope 5-8-Fc fusion proteins) were diluted in a two-fold gradient (initial concentrations were 50, 200, 1000, 100, 100 nM, respectively), and their binding and dissociation kinetics were detected.
[0144] 2. Data acquisition and analysis:
[0145] The binding and dissociation times were set to 120 seconds and 300 seconds, respectively, the flow rate was 30 μL / min, and the 1:1 Langmuir model was used to calculate the equilibrium dissociation constant (KD). The results are shown in Figure 5 C.
[0146] Figure 5 C is the kinetic parameter of five fusion proteins and immobilized Siglec-9 ECD determined by surface plasmon resonance technology.
[0147] As can be seen from Figure 5 C, the KD of 5B4-Fc, 8B6-Fc, 1H8-Fc, bi-epitope 5-1-Fc and bi-epitope 5-8-Fc fusion proteins is 0.655 nM, 6.11 nM, 5.49 nM, 0.12 nM, 3.16 nM, respectively; the KD of bi-epitope 5-1-Fc fusion protein is 5.5 times higher than that of 5B4-Fc; indicating that the five fusion proteins have high affinity properties to Siglec-9 ECD (i.e. Siglec-9 extracellular domain protein).
[0148] Example 7
[0149] Detection of nanobody-Fc fusion protein blocking Siglec-9 and tumor cell surface ligand binding based on ELISA method
[0150] 1. Tumor cell culture and fixation treatment:
[0151] SKOV3, SUM159, HCT116 cells (density > 90%) were evenly spread on two 96-well cell culture plates (BIOFIL), and the cell density in each well was 5 x 10 4Cells were cultured overnight at 37°C and 5% CO2. The culture medium in the cell culture plate was then discarded. 220 μL of fixative (2 mL of 25% glutaraldehyde aqueous solution was mixed with 48 mL of pre-cooled PBS buffer to obtain 1% glutaraldehyde fixative) was added to each well. The cells were incubated at room temperature for 15 min. The fixative was then discarded, and the cells were washed twice with PBS buffer (PBST) containing 0.1% Tween-20 for 5 min each time.
[0152] 2. Blocking and antibody incubation:
[0153] The coding sequence of the extracellular domain of Siglec-9 was cloned into the mammalian expression vector pTT5, and HEK293F cells (ATCC CBP60437) were transiently transfected with PEI. After 4 days of culture, the supernatant was collected and purified by Protein A affinity chromatography to obtain Siglec-9-Fc protein.
[0154] Add 5% skim milk-PBS blocking solution (MPBS) to each well and block at room temperature for 2 h; then dilute Siglec-9-Fc protein to 3 nM and serially dilute it with nanobodies (i.e., 5B4, 8B6, 1H8, biepitaxy 5-1 and biepitaxy 5-8 nanobodies) at a ratio of 1:3. Take 100 μL of each gradient and add it to the wells coated with tumor cells and incubate at room temperature for 1 h.
[0155] 3. Detection and Analysis:
[0156] After washing each well three times with PBST, add HRP-labeled anti-human IgG1 Fc secondary antibody (Beijing Yiqiao Shenzhou) to each well and incubate at room temperature for 1 hour. After washing, add TMB chromogenic solution and react for 5 minutes. Stop the reaction with 1M sulfuric acid and measure the absorbance (OD) at 450 nm. 450 ); through OD 450 The half-maximal inhibitory concentration (IC50) was calculated by fitting the value to the antibody concentration. 50 The result is as follows Figure 5 As shown in D.
[0157] Figure 5 D represents the inhibitory rate of five nanobodies on the binding of Siglec-9 to ligands on the surface of three tumor cell lines (SKOV3, SUM159, and HCT116) by competitive ELISA.
[0158] Depend on Figure 5 D shows that the IC50 of 5B4, 8B6, 1H8, biepitaxy 5-1, and biepitaxy 5-8 nanobodies against SKOV3 tumor cells is significant. 50 The IC50 values for SUM159 tumor cells were 1.719, 498.6, 3.130, 1.608, and 1.641 nM, respectively. 500.8371, 715.6, 31.44, 1.426 and 1.346 nM, respectively, for HCT116 tumor cells 50 1.105, 531.8, 17.25, 1.407 and 1.254 nM, respectively; the IC 50 values of the bi-specific 5-8 nanobody were similar to those of the mono-specific nanobody, but the inhibition rate of the bi-specific 5-8 nanobody was significantly better than that of the mono-specific nanobody.
[0159] Example 8 Evaluation of therapeutic effect of nanobody-Fc fusion proteins on mouse tumors
[0160] Since the bi-specific 5-8-Fc fusion protein was found to have good blocking function in vitro in Example 7, the therapeutic effect of 5B4-Fc, 8B6-Fc and 5-8-Fc fusion proteins on mouse tumors was explored. This experiment was carried out in a SPF animal laboratory, aiming to evaluate the therapeutic effect of each fusion protein on tumors in vivo in NCG mice (Jiangsu Jicui Yekang Biotechnology Co., Ltd., strain number T001475). The specific steps include the following:
[0161] First, a mouse subcutaneous tumor model of human ovarian cancer was constructed, and after the tumor grew to a specific volume, the experimental animals were divided into three drug administration groups (n = 5) and a control group (n = 5); each drug administration group was injected intraperitoneally with a dose of 10 mg / kg of nanobody fusion protein (total volume of 100 μL), and the control group was injected with the same volume of PBS (a total of 5 intraperitoneal injections in each group, with injection times of 8, 11, 14, 18 and 22 days); every two days during the treatment period, the mouse tumor volume was measured using a vernier caliper, and when the tumor grew to the preset endpoint volume, the mice were euthanized and killed, and the tumor mass was obtained for photographing and recording. The results are shown in Figure 6 .
[0162] Figure 6 The anti-tumor effects of 5B4-Fc, 8B6-Fc and 5-8-Fc fusion proteins in a mouse subcutaneous tumor model, where A is a schematic diagram of the construction of a human ovarian cancer mouse subcutaneous tumor model and the treatment strategy, B is a tumor volume growth curve during the treatment period, C is a tumor peeling real object diagram after the treatment, and D is a mouse body weight change curve during the treatment period.
[0163] From the above results, it can be seen that the 5B4-Fc, 8B6-Fc and 5-8-Fc fusion proteins have good anti-tumor effects in vivo in NCG mice, and the 5-8-Fc fusion protein has the best anti-tumor effect. Figure 6It can be seen that the tumor growth of 5B4-Fc, 8B6-Fc and 5-8-Fc fusion protein groups all showed different degrees of slowing trend, among which the 5B4-Fc group showed significant difference with the control group since the 9th day of treatment (*P<0.05, two way-ANOVA); the 8B6-Fc group and the 5-8-Fc group showed significant difference in volume difference with the control group since the 21st day of treatment (*P<0.05, two way-ANOVA); statistical analysis was performed by two way-ANOVA, the sample size of this experiment was n=5 / group, and the experimental period was 25 days.
[0164] The weight fluctuation range of mice in each group during treatment was ±10% of the baseline value, and there was no abnormal behavior or drug-related toxicity reaction (such as diarrhea and activity inhibition), indicating that the 5B4-Fc, 8B6-Fc and 5-8-Fc fusion proteins had good in vivo tolerance. The current findings have clearly demonstrated the feasibility and potential value of the technical solutions described in the present application. * indicates that the significance difference P is less than 0.05, ** indicates that the significance difference P is less than 0.01, and *** indicates that the significance difference P is less than 0.0001.
[0165] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can make equivalent replacement or change according to the technical solutions and inventive concept of the present application within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.
Claims
1. A CDR region of a nanobody targeting Siglec-9, characterized in that, The CDR area includes any of the following: a. In the CDR region, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 1-3, respectively; b. In the CDR region, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 4-6, respectively; c. In the CDR region, the amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7-9, respectively; d. An amino acid sequence that has greater than or equal to 80% homology with at least one of the amino acid sequences in SEQ ID NO: 1-9 and still has the function of targeting the Siglec-9 protein; e. An amino acid sequence that still has the function of targeting the Siglec-9 protein after adding, deleting, modifying and / or replacing one or more amino acids in at least one of the amino acid sequences in SEQ ID NO: 1-9.
2. A nanobody, characterized in that, Includes the CDR region of the nanobody targeting Siglec-9 as described in claim 1; Preferably, the nanobody is a monovalent nanobody, a multivalent nanobody, a multi-epitope nanobody, or a multi-specific nanobody; Preferably, the amino acid sequence of the nanobody includes any of the following: a. As shown in one of SEQ ID NO: 10-14; b. An amino acid sequence that has greater than or equal to 80% homology with at least one of the amino acid sequences in SEQ ID NO: 10-14 and still has the function of targeting Siglec-9 protein; c. An amino acid sequence that still has the function of targeting Siglec-9 protein after adding, deleting, modifying and / or replacing one or more amino acids in at least one of the amino acid sequences in SEQ ID NO: 10-14.
3. An antibody targeting Siglec-9, characterized in that, include: The CDR region of the nanobody targeting Siglec-9 as described in claim 1 or the nanobody as described in claim 2; Preferably, the antibody targeting Siglec-9 further includes: a TEV restriction site and an Fc domain; preferably, the Fc domain is a human IgG1 Fc domain; preferably, the Fc domain is linked to the CDR region or the C-terminus of the amino acid sequence of the nanobody via the TEV restriction site. Preferably, the amino acid sequence of the Fc domain is as shown in SEQ ID NO: 15; or an amino acid sequence with greater than or equal to 80% homology to the amino acid sequence shown in SEQ ID NO:
15. Preferably, the amino acid sequence of the TEV restriction site is as shown in SEQ ID NO: 17; or a DNA sequence with greater than or equal to 80% homology to the DNA sequence shown in SEQ ID NO:
17. Preferably, the antibody targeting Siglec-9 is a heavy chain antibody, a multi-epitope antibody, or a multi-specific antibody.
4. The antibody targeting Siglec-9 according to claim 3, characterized in that, The amino acid sequence of an antibody targeting Siglec-9 includes any of the following: a. As shown in one of SEQ ID NO: 18-22; b. An amino acid sequence that has greater than or equal to 80% homology with the amino acid sequence shown in SEQ ID NO: 18-22 and still has the function of targeting Siglec-9 protein; c. An amino acid sequence that still has the function of targeting Siglec-9 protein after adding, deleting, modifying and / or replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO: 18-22.
5. A polynucleotide, characterized in that, The polynucleotide encodes the CDR region of the Siglec-9-targeting nanobody of claim 1, or the nanobody of claim 2, or the antibody targeting Siglec-9 of claim 3 or 4.
6. An expression carrier, characterized in that, The expression vector contains the polynucleotide of claim 5.
7. A host cell, characterized in that, The host cell contains the expression vector of claim 6 or the host cell genome integrates the polynucleotide of claim 5; preferably, the host cell is a cell capable of expressing exogenous proteins; preferably, the host cell is a bacterium, yeast, insect cell or mammalian cell.
8. An antibody conjugate, characterized in that, Antibody conjugates include: a. The nanobody of claim 2 or the antibody targeting Siglec-9 of claim 3 or 4; and b. A coupling portion selected from any one or a combination of the following: detectable markers, enzymes, drugs, therapeutic radioisotopes, toxins, cytokines, and solid-phase carriers.
9. The application of the CDR region of the Siglec-9-targeting nanobody as described in claim 1, the nanobody as described in claim 2, the antibody as described in claim 3 or 4, or the antibody conjugate as described in claim 8, characterized in that, The applications include: 1) Application in the preparation of reagents and / or kits for detecting Siglec-9, and reagents and / or kits for diagnosing Siglec-9-related diseases; 2) Application in the preparation of drugs targeting Siglec-9; 3) Use in the preparation of drugs for the prevention and / or treatment of Siglec-9 related diseases; Preferably, the Siglec-9-related diseases are tumors, sepsis, chronic inflammatory diseases, or autoimmune diseases; preferably, tumors include: colon cancer, breast cancer, ovarian cancer, cervical cancer, lung cancer, liver cancer, bladder cancer, prostate cancer, glioma, or melanoma.
10. A pharmaceutical composition, characterized in that, include: The pharmaceutical composition comprises, preferably, one of the following: the CDR region of the Siglec-9-targeting nanobody of claim 1, the nanobody of claim 2, the antibody targeting Siglec-9 of claim 3 or 4, or the antibody conjugate of claim 8; preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.