Anti-SEZ6 nano antibody or antigen binding fragment and application

Abstract

The invention belongs to the field of biological medicine, and relates to an anti-SEZ6 nano antibody or antigen binding fragment and application. The anti-SEZ6 nano antibody or the antigen binding fragment comprises three complementary determining regions, namely, CDR1, CDR2 and CDR3; the sequences of all the regions are respectively shown as SEQ ID NO: 1 to SEQ ID NO: 18. Compared with a conventional antibody, the anti-SEZ6 nano antibody is efficient in expression, simple in purification, small in molecular weight, high in affinity, relatively good in specificity, high in solubility and strong in tolerance, and can be widely applied to the fields of scientific research, diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an anti-SEZ6 nanobody or antigen-binding fragment and an application thereof. Background Art

[0002] Immunotherapy based on the interdependence and interaction between cancer cells and their microenvironment has become a research hotspot in the field of tumor treatment, including immune checkpoint inhibitors such as anti-CTLA-4, anti-PD-1 and anti-PD-L1, and chimeric antigen receptor (CAR) T cells. Many antibody drugs targeting different targets have been approved for clinical use and have achieved good results. Currently, drugs targeting more targets are under development. In the past 30 years, traditional monoclonal antibodies have occupied an important position in the research and development of therapeutic biomacromolecules. As antibody drugs are continuously applied to more therapeutic fields, the therapeutic mechanism of antibody drugs has become more complex, and the structure of antibody drugs has become more diverse. Therefore, new technologies need to be developed to address the defects of monoclonal antibodies.

[0003] The SEZ6 family includes SEZ6 (Seizure-related gene 6), SEZ6L (Seizure-related gene6-like) and SEZ6L2 (Seizure-related gene 6-like 2). All SEZ6 family members contain 2-3 CUB domains and 5 complement control protein (CCP) domains, indicating that they may be involved in complement regulation. SEZ6 is a type I transmembrane protein encoded by the SEZ6 gene located on chromosome 17 [17q11.2], which is involved in the normal dendritic branching of cerebral cortical neurons. Under normal physiological conditions, SEZ6 is mainly expressed in the brain, such as the cerebral cortex, cerebellum, basal ganglia, and hypothalamus. It is highly expressed in SCLC and other neuroendocrine tumors, but very little in most normal tissues.

[0004] SEZ6 (Seizure-related gene 6): SEZ6 is usually expressed in the central nervous system. SEZ6 protein plays an important role in the development of nerve cells and the formation of synapses. Synapses are the connection points between nerve cells, and they play a key role in information transmission. SEZ6 has a total of 994AA.

[0005] SEZ6L (Seizure-related gene 6-like): SEZ6L is highly similar to the SEZ6 gene, so it is called "SEZ6-like". The protein encoded by the SEZ6L gene is expressed in the nervous system and is involved in the process of synapse formation and nerve cell connection. SEZ6L and SEZ6 have some similarities in sequence, but they may differ in some details. SEZ6L has a total of 1024AA.

[0006] SEZ6L2 (Seizure-related gene 6-like 2): The protein encoded by the SEZ6L2 gene is highly expressed in the central nervous system, especially in the cortical area of ​​the brain. SEZ6L2 is associated with the migration of nerve cells, synapse formation, and the development and function of the nervous system. SEZ6L2 has a total of 910 AA.

[0007] Currently, there are targeted drugs for SEZ6 under development, including monoclonal antibodies and ADCs, but few antibodies have entered clinical trials. The preparation process of traditional monoclonal antibodies is complicated, the production cost is high, and the molecular weight is large, and the tissue penetration ability is poor. Nanobodies are the smallest fragments known to bind to antigens. They are only 1 / 10 the size of monoclonal antibodies and have structural stability and binding activity comparable to the original heavy chain antibodies. Compared with traditional antibodies, nanobodies have many unique advantages, such as good tissue penetration, rapid clearance, easy production and modification, high stability, low immunogenicity, etc., and are a new type of antibody molecule with great application prospects.

[0008] Nanobodies targeting SEZ6 have not been reported or clinically applied, and there is an urgent need in this field to develop new and effective specific nanobodies targeting SEZ6. Summary of the invention

[0009] definition

[0010] Unless otherwise indicated or defined, all terms used have the usual meaning in the art, which will be understood by those skilled in the art. In addition, unless otherwise specified, all methods, steps, techniques and operations not specifically described in detail can and have been performed in a manner known per se, which will be understood by those skilled in the art.

[0011] Unless otherwise indicated, the interchangeable terms "antibody" or "immunoglobulin" herein, whether referring to heavy chain antibodies or conventional four-chain antibodies, are used as general terms to include full-length antibodies, their individual chains, and all parts, domains or fragments thereof (including but not limited to antigen binding domains or fragments, such as VHH domains or VH / VL domains, respectively). In addition, the term "sequence" used herein (e.g., in terms of "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence", etc.) is generally understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the amino acid sequence, unless a more limited explanation is required herein.

[0012] The technical problem to be solved by the present invention is to provide a VHH chain of an anti-SEZ6 nanobody that can block the binding of SEZ6 to its ligand, and its further derivation and application.

[0013] In order to achieve the above-mentioned object, the first aspect of the present invention provides an anti-SEZ6 nanobody or antigen-binding fragment, which comprises three complementary determining regions CDR1, CDR2 and CDR3; wherein,

[0014] The amino acid sequence of CDR1 is a sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 6 and has the same function;

[0015] The amino acid sequence of CDR2 is a sequence as shown in any one of SEQ ID NO:7 to SEQ ID NO:12, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO:7 to SEQ ID NO:12 and functions the same;

[0016] The amino acid sequence of CDR3 is a sequence shown in one of SEQ ID NO: 13 to SEQ ID NO: 18, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of SEQ ID NO: 13 to SEQ ID NO: 18 and has the same function.

[0017] The meaning of "same function" in the present invention is: being able to bind to SEZ6 protein.

[0018] The position of the CDR in the antibody or nanobody sequence can be determined by those skilled in the art using prior art. Typically, the CDR can be identified by sequencing the DNA of the antibody or nanobody, and then using a dedicated database (such as the international ImMunoGeneTics database or IMGT) to analyze the sequence thus obtained.

[0019] In the sequences provided in the present invention, CDRs were mapped according to IMGT (https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results).

[0020] According to a preferred embodiment of the present invention, the anti-SEZ6 nanobody or antigen-binding fragment has any one of the following CDR sequence characteristics:

[0021] (1) the amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 13, or sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences and have the same functions;

[0022] (2) the amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 14, respectively, or sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences and have the same functions;

[0023] (3) the amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 15, respectively, or sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences and have the same functions;

[0024] (4) the amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO:4, SEQ ID NO:10, and SEQ ID NO:16, respectively, or sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences and function identically;

[0025] (5) the amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 17, respectively, or sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the above sequences and have the same functions;

[0026] (6) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 18, respectively, or sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above sequences and have the same functions.

[0027] According to the present invention, in addition to the above-mentioned complementary determining regions, the anti-SEZ6 nanobody or antigen-binding fragment further comprises four framework regions FR1, FR2, FR3 and FR4 arranged alternately with the three complementary determining regions, wherein the amino acid sequence of FR1 is a sequence shown in one of SEQ ID NO: 19 to SEQ ID NO: 24;

[0028] The amino acid sequence of FR2 is one of SEQ ID NOs: 25 to 30;

[0029] The amino acid sequence of FR3 is one of SEQ ID NOs: 31 to 36;

[0030] The amino acid sequence of FR4 is a sequence shown in any one of SEQ ID NO:37 to SEQ ID NO:39.

[0031] According to a preferred embodiment of the present invention, the anti-SEZ6 nanobody or antigen-binding fragment has any one of the following FR sequence characteristics:

[0032] (a) the amino acid sequences of FR1, FR2, FR3, and FR4 are SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 31, and SEQ ID NO: 37, respectively;

[0033] (b) the amino acid sequences of FR1, FR2, FR3, and FR4 are SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 37, respectively;

[0034] (c) the amino acid sequences of FR1, FR2, FR3, and FR4 are SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 37, respectively;

[0035] (d) the amino acid sequences of FR1, FR2, FR3, and FR4 are SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, and SEQ ID NO: 38, respectively;

[0036] (e) the amino acid sequences of FR1, FR2, FR3, and FR4 are SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, and SEQ ID NO: 37, respectively;

[0037] (f) The amino acid sequences of FR1, FR2, FR3 and FR4 are the sequences shown in SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36 and SEQ ID NO: 39, respectively.

[0038] The present invention includes all sequences that meet the above sequence characteristics, and preferably,

[0039] The Nanobody or antigen-binding fragment comprises one or more of the following sequences:

[0040] (i) an amino acid sequence as shown in any one of SEQ ID NO: 40 to SEQ ID NO: 45;

[0041] (ii) an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO:40 to SEQ ID NO:45 and functions the same;

[0042] (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence as shown in any one of SEQ ID NO: 40 to SEQ ID NO: 45, and which retains the function of the amino acid sequence as shown in any one of SEQ ID NO: 40 to SEQ ID NO: 45, i.e., is able to bind to the SEZ6 protein.

[0043] Ph-2-B3:QVKLEESGGGLVQAGGALNLSCVASGIIFSMYDMGWYRQGSGEARDIVAAIG KGGSTYYADAVKGRFTISRDNAQNTLYLQMNSLKPEDTSMYYCTTVEPYDY WGQGTQVTVSS(SEQ ID NO:40)

[0044] Ph-3-A7:AVQLVDSGGGLVQPGGSLRLSCTASDSRFIANIMGWYRQAPGKERELVVAISS GGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTDMYYCAFFEGGIPDG WGQGTQVTVSS(SEQ ID NO:41)

[0045] Ph-3-H7:AVQLVDSGGGLVQAGGSLRLSCAASGSIFSINTMGWYRQAPGKERELVAAISS HGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTTMYYCAASRDSDYDP GRGSWGQGTQVTVSS(SEQ ID NO:42)

[0046] Ph-2-C03:AVQLVESGGGLVQAGGSLRLSCAASGSIFSIEAMGWYRQAPGEVRELVAAISS GNSTYYADNVKGRFTISRDNANNTVYLQMTSLKPEDTAMYYCAAFESSRPW ALGKGALVTVSS(SEQ ID NO:43)

[0047] Ph-2-E05:QVKLEESGGGLVQAGGSLRLSCTASGSIFSFDALGWYRQAPGSERELVAAVGS GGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDVAMYYCAAFSPHSGIP RLWGQGTQVTVSS(SEQ ID NO:44)

[0048] Ph-2-E10:QVKLEESGGGLVQAGGSLRLSCAASGSIFSIDTMGWYRSAPGEERRLVAAIST GGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCAGFSSQSDPG VPNLLGQGTQVTVSS(SEQ ID NO:45)

[0049] The second aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned anti-SEZ6 nanobody or antigen-binding fragment.

[0050] The third aspect of the present invention provides a vector comprising the nucleic acid molecule of the second aspect.

[0051] The fourth aspect of the present invention provides a host cell, which contains the vector of the third aspect or the nucleic acid molecule of the second aspect.

[0052] The host cells include, but are not limited to, bacterial cells, fungal cells, animal cells, plant cells, or progeny of these cells.

[0053] The anti-SEZ6 nanobody of the present invention can be obtained by the following method:

[0054] (1) Human, monkey, and mouse SEZ6 proteins were used to immunize alpacas and prepare phage display libraries.

[0055] (2) Affinity screening of phage display libraries using human, monkey, and mouse SEZ6 proteins;

[0056] (3) ELISA and FACS identification of positive clones;

[0057] (4) Expression and purification of SEZ6 nanobody.

[0058] A fifth aspect of the present invention provides a method for engineering the generation of anti-SEZ6 nanobodies, comprising the following steps:

[0059] (a) culturing the host cell as described in the fourth aspect of the invention under conditions suitable for the production of nanobodies, thereby obtaining a culture containing the anti-SEZ6 nanobody; and

[0060] (b) isolating and / or recovering the anti-DLL3 Nanobody from the culture; and optionally

[0061] (c) purifying and / or modifying the anti-SEZ6 nanobody obtained in step (b).

[0062] The sixth aspect of the present invention provides an antibody-drug conjugate, comprising the anti-SEZ6 nanobody or antigen-binding fragment and an effector; preferably, the effector comprises at least one of a nuclide, a cytotoxic agent, a fluorescent group, an enzyme that catalyzes the color development of a substrate, a chemiluminescent reagent, and a nanoparticle marker.

[0063] The seventh aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned anti-SEZ6 nanobody or antigen-binding fragment, or the above-mentioned antibody-drug conjugate.

[0064] The eighth aspect of the present invention provides the following uses of the anti-SEZ6 nanobody or antigen-binding fragment:

[0065] (i) Use in the preparation of a reagent for detecting a human SERZ6-related disease; the reagent is preferably a kit for detecting a SERZ6-related disease;

[0066] (ii) Use in the preparation of drugs for treating SEZ6-related diseases.

[0067] The anti-SEZ6 nanobody or antigen-binding fragment of the present invention can also be used to treat SEZ6-related diseases.

[0068] The SEZ6-related diseases include but are not limited to cancer, and the cancer includes but is not limited to at least one of small cell lung cancer, neuroendocrine tumors, glioblastoma and high-grade central nervous system tumors. The anti-SEZ6 nanobody can be used as an immune checkpoint inhibitor, as a cancer treatment drug alone, or in combination with other anticancer drugs, so the active component of the drug includes the anti-SEZ6 nanobody and optionally other anticancer drugs.

[0069] Compared with conventional antibodies, the anti-SEZ6 nanoantibody of the present invention has high expression efficiency, simple purification, small molecular weight (about 15kDa), high affinity, good specificity, high solubility, strong tolerance, and can be widely used in scientific research, diagnosis and treatment. In addition, nanoantibodies are more likely to recognize antigens that traditional antibodies cannot capture, have better tissue penetration, can enter tumor tissues and cross the blood-brain barrier, and can be developed as immune checkpoint inhibitors to provide solutions for tumor imaging and treatment.

[0070] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0072] Figure 1 This is the gel electrophoresis diagram of the recombinant protein Reduced SDS-PAGE detection.

[0073] Figure 2a -b shows the amplification process of the yeast display library VHH fragments of 252# alpaca and 297-231# alpaca, respectively.

[0074] Figure 3a -b shows the QC results of the yeast display library of 252# alpaca and 297-231# alpaca, respectively.

[0075] Figure 4a-b shows the results of magnetic sorting of yeast display libraries of 252# alpaca and 297-231# alpaca, respectively.

[0076] Figure 5a -b shows the results of the second round of sorting of the yeast display library of 252# alpaca and 297-231# alpaca.

[0077] Figure 6a -d show the results of plate 1, plate 2, plate 3 and plate 4 of 252# alpaca monoclonal FACS detection respectively.

[0078] Figure 7a -c respectively show the results of plate 1, plate 2, and plate 3 of 297-231# alpaca monoclonal FACS detection.

[0079] Figure 8a -c shows the detection results of the second batch of candidate antibodies in the ELISA experiment to detect the binding of recombinant antibodies to target proteins.

[0080] Fig. 9 The results of FACS detection of the binding of the recombinant antibody to the target protein are shown.

[0081] Fig.10a -b shows the FACS EC of candidate antibodies against overexpressing cells at 37°C 50 Test results.

[0082] Fig.11a -b shows the FACS EC of candidate antibodies against overexpressing cells at 4°C 50 Test results.

[0083] Fig.12 The results of the candidate antibody internalization assay are shown.

[0084] FIG. 13 shows the results of affinity testing of candidate antibodies. DETAILED DESCRIPTION

[0085] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0086] Example

[0087] 1. Antigen Preparation

[0088] According to the amino acid sequence information of Cynomolgus SEZ6 and Mouse SEZ6 proteins in the uniprot database (Leu20-His927 (Cynomolgus SEZ6) [Accession|A0A2K5WPJ4] & (Leu20-His922) (MouseSEZ6) [Accession|Q7TSK2]), codon optimization was performed according to the codon preference of mammals. After synthesizing the antigen encoding nucleic acid sequence, the nucleic acid sequence was subcloned into the pCDNA3.4 vector and His Tag was added to the C-terminus to construct a eukaryotic expression vector; the two plasmids were transfected into 293F cells respectively, and the supernatant was collected and the target protein was purified using a nickel column. The amino acid sequence of the constructed Cynomolgus SEZ6 protein is shown in SEQ ID NO: 46, and the amino acid sequence of the constructed Mouse SEZ6 protein is shown in SEQ ID NO: 47. Human SEZ6-His protein was purchased from Kaixia Biotechnology (Shanghai) Co., Ltd.

[0089] The purity of the recombinant protein was detected by SDS-PAGE. Figure 1 As shown, the results show that the purity of Human SEZ6-His protein is >95%, which can be used for immunization and subsequent panning. The purity of CynoSEZ6-His and MouseSEZ6-His proteins is >90%, which can be used for cross-binding verification.

[0090] 2. ELISA binding activity detection of recombinant protein antigen and positive antibody

[0091] 1) Use sterile CBS to dilute SEZ6-His recombinant protein to a final concentration of 1 μg / mL, take a new 96-well plate, add 100 μL to each well, and coat overnight at 4°C; shake off the antigen coating solution, wash three times with PBST (containing 0.5% Tween); add 200 μL / well of 3% MPBS at 37°C for 2 hours; shake off the blocking buffer, and wash the plate three times with PBST;

[0092] 2) The positive control antibody hSEZ6VH+VL (expressed and prepared by Aikangde Biotechnology Co., Ltd., sequence information refers to WO 2019 / 232241A1) was diluted to 10 μg / mL with PBS, 5-fold dilution was made at 7 points, and 100 μL / well was added to the ELISA plate and incubated at room temperature for 1 hour. The control well was PBS;

[0093] 3) Shake off the liquid in the wells and wash three times with PBST;

[0094] 4) Add secondary antibody HRP-Protein A (1:10000 dilution) to the ELISA plate at 100 μL / well and incubate at room temperature for 1 hour;

[0095] 5) After shaking off the liquid in the wells, wash the plate three times with PBST;

[0096] 6) Add 100 μL / well TMB colorimetric solution;

[0097] 7) Incubate at room temperature in the dark for 15 minutes;

[0098] 8) Add 50 μL / well stop solution (2M HCl);

[0099] 9) Use a microplate reader to read the OD in the well 450 value.

[0100] The results are shown in Table 1 below:

[0101] Table 1

[0102]

[0103]

[0104] The results showed that the proteins of the three species all had good binding activity with the positive control antibody and could be used for subsequent experiments.

[0105] 3. Alpaca Immunity

[0106] The purchased Human SEZ6-His recombinant protein and the prepared Mouse SEZ6-His recombinant protein were used to immunize alpacas. The alpacas were numbered 297-231# and 252#, respectively. The immunization interval was 14 days. Starting from the second immunization, peripheral blood was collected seven days after each immunization to monitor the immune serum titer. The immunization progress table is shown in Table 2 below:

[0107] Table 2

[0108]

[0109] 4. Immune titer detection

[0110] The immune titer test steps include:

[0111] 1) After immunization, collect 5 mL of peripheral blood and place the centrifuge tube containing the blood sample in a 37°C incubator for 1 hour; then transfer the blood sample to 4°C overnight;

[0112] 2) Place the centrifuge tube containing the blood sample in a centrifuge and centrifuge at 5000 rpm for 20 min; separate the upper serum and transfer the serum to a new sterile centrifuge tube to collect the immune serum;

[0113] 3) Use sterile CBS (carbonate buffer) to dilute the target recombinant protein to a final concentration of 1 μg / mL; take a new 96-well ELISA plate, add 100 μL / well and coat overnight at 4°C;

[0114] 4) Remove the antigen coating solution and wash five times with PBST (containing 0.05% Tween 20);

[0115] 5) Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours;

[0116] 6) After removing the blocking buffer, wash the plate 5 times with PBST;

[0117] 7) Add 100 μL of serially diluted serum (100 μL / well) and incubate at room temperature for 1 hour. The control wells are PBS.

[0118] 8) Remove the liquid in the wells and wash 5 times with PBST;

[0119] 9) Add 100 μL HRP anti-Llama IgG (H+L) antibody (1:50,000 dilution) and incubate at room temperature for 1 hour;

[0120] 10) After removing the liquid in the wells, wash the plate 5 times with PBST;

[0121] 11) Add 100 μL / well TMB colorimetric solution;

[0122] 12) Incubate at room temperature in the dark for 10-15 minutes;

[0123] 13) Add 50 μL / well stop solution;

[0124] 14) Use a microplate reader to read the OD in the well 450 value.

[0125] The results of the immune titer test are shown in Tables 3 and 4 below. Two groups of samples were tested in parallel:

[0126] Table 3

[0127]

[0128] Table 4

[0129]

[0130] According to the ELISA test results, the immune serum can bind to the target recombinant protein, and with the gradient dilution of the immune serum, the OD value changes gradiently. The alpaca titer is significantly improved to meet the requirements for blood collection and library construction. 100mL of peripheral blood was collected for the construction of the antibody display library.

[0131] 5. Construction of Yeast Display Library

[0132] The amplification process of the yeast display library VHH fragments of 252# alpaca and 297-231# alpaca were as follows: Figure 2a and 2b As shown: peripheral blood was collected to separate PBMC cells, and a reverse transcription kit was used to prepare a cDNA library after RNA extraction; single domain antibody amplification primers were used for 1 round of PCR to obtain PCR bands of about 1000 bp and 750 bp, respectively, and the 750 bp fragment was recovered from the gel as a template for the second round of PCR; the second round of PCR obtained a band of about 400 bp, which was a VHH fragment. The VHH fragment was mixed with the linearized yeast display vector and electroporated into yeast cells to obtain a yeast library. The constructed 252# alpaca yeast library has a library capacity of 2.63×10 9 , the yeast library capacity of 297-231# alpaca is 1.0×10 9 The specific steps are as follows:

[0133] 5.1 PBMC isolation and VHH antibody fragment cloning

[0134] 1) After immunization, collect 100 mL of peripheral blood and separate PBMC using lymphocyte separation solution.

[0135] 2) Extract RNA using PrimeScript TM cDNA was prepared by reverse transcription using II 1st Strand cDNA Synthesis Kit.

[0136] 2-1) Prepare the following reaction mixture in 200 μL PCR:

[0137]

[0138] 2-2) Keep at 65℃ for 5 min, then cool rapidly on ice;

[0139] 2-3) Prepare the following reaction solution in the above PCR tube:

[0140]

[0141] 2-4) After mixing by pipetting, dispense into 80 μL / tube, place in a PCR instrument at 42°C for 1 hour, heat inactivate at 70°C for 15 minutes, and finally place the cDNA sample on ice or at -20°C for long-term storage.

[0142] 3) Amplification of VHH fragments

[0143] 3-1) Prepare the first round of PCR reaction system (50 μL / tube): the upstream primer binds to the signal peptide, the sequence is shown in SEQ ID NO: 48, and the downstream primer binds to the CH2 region, the sequence is shown in SEQ ID NO: 49.

[0144]

[0145] After configuring the PCR reaction system, set up the PCR instrument as follows:

[0146]

[0147] 3-2) Agarose electrophoresis of PCR products

[0148] 1% agarose gel electrophoresis was used to analyze the PCR products, and the fragments with a molecular weight of about 750 bp were separated. The PCR products were recovered using a gel recovery kit, and the concentration was determined using NanoDrop.

[0149] 3-3) Prepare a second-round PCR reaction system (50 μL / tube): the upstream primer binds to the antibody FR1 region, the sequence is shown in SEQ ID NO: 50, the downstream primer binds to the antibody Hinge and FR4 region, the sequence is shown in SEQ ID NO: 51, and the restriction site is SfiI.

[0150]

[0151] After configuring the PCR reaction system, set up the PCR instrument as follows:

[0152]

[0153] 3-4) Agarose electrophoresis analysis of the second-round PCR products

[0154] The PCR products were analyzed by electrophoresis using 1% agarose to separate the VHH fragments with a molecular weight of about 400 bp. The VHH PCR products were recovered using a gel recovery kit and the concentration was determined using NanoDrop.

[0155] 3-5) PCR product precipitation

[0156] The recovered second-round PCR products were divided into 200 μL in each 1.5 mL centrifuge tube, and 1 / 10 volume (20 μL) of 3M sodium acetate and 1 μg / μL glycogen were added. The mixture was mixed by pipetting, and 880 μL of anhydrous ethanol was added. The mixture was mixed by inversion, and the mixture was stored at -80°C.

[0157] 5.2 Linearization of yeast display vector pYDisplay, enzyme digestion system is as follows:

[0158]

[0159] 1) Use SfiI to digest the pYDisplay vector, aliquot 100 μL / tube, and digest at 50°C overnight;

[0160] 2) Separate the pYDisplay vector fragment using 1% agarose gel, cut out a 5000 bp vector fragment for gel recovery, and determine the concentration using NanoDrop;

[0161] 3) Dispense 200 μL of the recovered pYDisplay digestion product into each 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3M sodium acetate and 1 μg / μL glycogen, pipette to mix, add 880 μL of anhydrous ethanol, invert to mix, and freeze at -80°C.

[0162] 5.3 Electroporation to construct yeast display library

[0163] 1) Streak the yeast competent strain frozen at -80℃ onto a YPD solid medium plate and activate at 30℃ for 3-5 days;

[0164] 2) Inoculate a single colony of competent yeast into 50 mL of YPD medium and shake at 250 rpm and 30°C for 1-2 days;

[0165] 3) Preparation of yeast competent strain: linearized vector fragment and PCR product were mixed, added to the electroporation cup, and electroporated; after electroporation, the yeast competent transfection culture bottle was shaken at 220 rpm and 30°C for 1 hour;

[0166] 4) Take 20 μL of the resuspension, dilute it 5000 times with SDCAA, take 100 μL, apply it to SDCAA plate, culture for 2-3 days, calculate the reservoir capacity, and continue to culture the rest of the bacterial solution for 24 hours;

[0167] 5) Preservation of bacteria: Collect the remaining bacterial liquid into a 50 mL centrifuge tube, centrifuge at 3000 × g for 5 min, discard the supernatant, add 10 mL SDCAA to resuspend, mix with 50% glycerol: resuspension solution = 1:1, and freeze at -80°C.

[0168] Results: The QC of yeast display libraries of 252# alpaca and 297-231# alpaca were as follows: Figure 3a and 3b As shown: Single clones were randomly picked for sequencing analysis, the overlapping stop codon sequences were removed, there were no empty sequences and repeated sequences, and the sequences were highly different, with good library diversity.

[0169] 6. Magnetic Sorting of Yeast Display Library

[0170] 1) The yeast cultured in SDCAA was added to a 250 mL shake flask containing 50 mL SGCAA medium and cultured in a shaking incubator at 30°C and 240 rpm for 16 h.

[0171] 2) After centrifugation, discard the supernatant, resuspend with 1 mL 0.5% PBSA, add to a 1.5 mL centrifuge tube, centrifuge at 3000×g for 5 min, discard the supernatant, and wash again with 0.5% PBSA.

[0172] 3) The streptavidin magnetic beads incubated with the antigen were washed twice with 0.5% PBSA (rotating and incubating at 4°C for 5 minutes each time), placed on a magnetic stand for 5 minutes, and the supernatant was discarded.

[0173] 4) Add the yeast solution to the magnetic beads bound to the antigen, incubate with rotation at 4°C for 60 minutes, and place on a magnetic stand for 15 minutes.

[0174] 5) Discard the yeast solution and keep the magnetic beads, and wash them three times with 0.5% PBSA (rotating and incubating at 4°C for 5 minutes each time).

[0175] 6) Resuspend the magnetic beads with 1 mL of SDCAA medium, pipette 0.5-5 μL of the resuspension into 100 μL of SDCAA medium and spread on a plate. Divide the resuspension into two portions, add 500 μL of 50% glycerol (stored at -80°C) to one portion; add the other portion to a shaking tube, add 2 mL of SDCAA medium, and culture at 30°C, 240 rpm for 16 h.

[0176] 7) Transfer the bacterial solution in the shake tube to 50 mL SDCAA medium (250 mL shake flask) and culture overnight at 30°C and 240 rpm.

[0177] 8) Measure the OD of bacterial solution 600 Value, according to OD 600 Centrifuge a portion of the bacterial suspension, resuspend it in SGCAA, and transfer it to 50 mL SGCAA medium to a final OD 600 The value was 1, cultured overnight at 30°C and 240rpm, and the remaining bacterial solution was resuspended in SDCAA:50% glycerol = 1:1 and frozen at -80°C.

[0178] Figure 4a and 4b The results of magnetic sorting of yeast display libraries of 252# alpaca and 297-231# alpaca are shown respectively.

[0179] Figure 4aA: NC group; B: original library: primary antibody: V5-FITC; C: 1MACS: primary antibody: Biotin-Human-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC; D: 1MACS: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC. After using Bio-Human-SEZ6-His protein to sort the yeast library by magnetic separation, the positive proportion of Human SEZ6 was 4.88%, and the positive proportion of Mouse SEZ6 was 1.44%, and two rounds of flow sorting were arranged.

[0180] Figure 4b A: NC group; B: original library: primary antibody: V5-FITC; C: 1MACS: primary antibody: Biotin-Human-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC; D: 1MACS: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC. After using Bio-Human-SEZ6-His protein to sort the yeast library by magnetic separation, the positive proportion of Human SEZ6 was 1.18%, and the positive proportion of Mouse SEZ6 was 1.88%, and two rounds of flow sorting were arranged.

[0181] 7. Yeast display library second round sorting

[0182] 1) Take 1 mL of SGCAA-cultured yeast solution into a 1.5 mL centrifuge tube, centrifuge and remove the supernatant, wash twice with 1 mL PBS, resuspend with 1 mL PBS, take 100 μL of the bacterial solution into a new 1.5 mL centrifuge tube (as NC), centrifuge and remove the supernatant.

[0183] 2) Dilute the Biotin-antigen protein to a concentration of 10 μg / mL with 100 μL PBS, resuspend the experimental group bacteria, incubate with rotation at 4°C for 60 min, centrifuge at 3000×g for 3 min, discard the supernatant, and wash twice with 1 mL PBS.

[0184] 3) Dilute APC-Streptavidin at 1:1000 and resuspend the experimental group bacteria, incubate with rotation at 4°C for 60 min, centrifuge at 3000×g for 3 min, discard the supernatant, wash twice with 1 mL PBS, and resuspend with 1 mL PBS.

[0185] 4) Add the resuspended bacteria to the flow cytometry tube for flow cytometry.

[0186] 5) Prepare 1 mL of SDCAA medium in a 15 mL centrifuge tube as a collection tube.

[0187] 6) Put the flow cytometry machine on, adjust the gate with the NC group, and sort the APC-positive yeast.

[0188] Figure 5a and 5b The results of the second round of sorting of the yeast display library of 252# alpaca and 297-231# alpaca are shown respectively.

[0189] Figure 5a A: NC group; B: 1MACS: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC. P3 was sorted, and a total of 19006 yeast cells were obtained. Plates were arranged, and yeast monoclonal amplification and induction were selected for yeast monoclonal flow cytometry detection.

[0190] Figure 5b In the middle, A: NC group; B: 1MACS: primary antibody: Biotin-Human-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC; C: 1MACS: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC. P3 was sorted, 45046 cells were obtained by protein sorting of Human SEZ6, and 19602 cells were obtained by protein sorting of Mouse SEZ6. Plates were arranged for coating, and yeast monoclonal amplification and induction were selected for flow cytometry detection of yeast monoclonal.

[0191] 8. Flow cytometry detection of yeast monoclonal

[0192] After sorting, the yeast liquid was coated on SDCAA plates, and a single clone was picked for culture. After 48 hours of induction expression, it was incubated with Biotin-antigen, and PE-Streptavidin was used as the secondary antibody. After incubation, flow cytometry was performed. The yeast clones bound to the target antigen were resuspended with 0.2% SDS, incubated at 95°C for 10 minutes, lysed, centrifuged to obtain the supernatant, and 0.5 μL was taken as a template for PCR amplification and sent for testing (the remaining bacterial liquid was stored at -20°C).

[0193] Monoclonal cells were randomly selected from the enriched yeast display library, and after amplification and induction, they were tested with antigens to determine the binding of the single-domain antibody displayed on the surface of the monoclonal yeast cells to the target antigen. Figure 6a -d, Figure 7a -c.

[0194] According to the experimental results, positive clones with cross-binding between Human SEZ6 and Mouse SEZ6 proteins were selected for PCR testing, and the obtained differential sequences were used to arrange vector construction.

[0195] 9. Construction of antibody eukaryotic expression vector

[0196] The positive yeast clones were subjected to PCR to obtain the antibody sequence, and a His tag was added to the C-terminus. After SfiI digestion, the antibody expression vector was connected to the eukaryotic expression vector pcDNA3.4-human IgG1Fc to construct the antibody expression vector. After verification by Sanger sequencing, the plasmid was extracted.

[0197] 10. Expression and purification of candidate single domain antibodies

[0198] 1) Take out LVTransm transfection reagent and pcDNA3.4-human IgG1Fc antibody expression vector from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out PBS buffer and warm it to room temperature. Take 2mL PBS to one well of a 6-well plate, add 20μg of antibody eukaryotic expression vector, mix thoroughly by pipetting up and down, add 60μLLVTransm, and immediately mix thoroughly by pipetting up and down, and let stand at room temperature for 10 minutes.

[0199] 2) Add the above DNA / LVTransm complex to 20 mL of 293F cells, shake gently to mix thoroughly, and continue culturing the cells in an incubator at 37°C, 5% CO2, 130 rpm.

[0200] 3) After continuous culture for 5-7 days, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, the filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using a Protein A column.

[0201] 11. ELISA to detect the binding of recombinant antibodies to target proteins

[0202] 1) Use sterile CBS to dilute the recombinant protein to a final concentration of 1 μg / mL. Take a new 96-well ELISA plate and add 100 μL / well to coat overnight at 4°C.

[0203] 2) Remove the antigen coating solution and wash five times with PBST (containing 0.05% Tween 20).

[0204] 3) Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours;

[0205] 4) After removing the blocking buffer, wash the plate 5 times with PBST;

[0206] 5) Add the expressed recombinant antibody, 50 μL / well of transfection supernatant or purified antibody (starting concentration is 10 μg / mL, 3-fold gradient dilution 7 points, 100 μL / well), incubate at room temperature for 1 hour, and the control well is PBS;

[0207] 6) Remove the liquid in the wells and wash 5 times with PBST;

[0208] 7) Add 100 μL / well HRP-Protein A antibody (1:50000 dilution) and incubate at room temperature for 1 hour;

[0209] 8) After removing the liquid in the wells, wash the plate 5 times with PBST;

[0210] 9) Add 100 μL / well TMB colorimetric solution;

[0211] 10) Incubate at room temperature in the dark for 10-15 minutes;

[0212] 11) Add 50 μL / well stop solution;

[0213] 12) Use a microplate reader to read the OD in the well 450 value.

[0214] The test results of the first batch of candidate antibodies are shown in Tables 5 to 7.

[0215] Table 5

[0216]

[0217] Human-SEZ6-His (2 μg / mL) was coated to detect the binding of candidate antibodies to Human-SEZ6-His. According to the test results, only the 297-231-2-A10 candidate antibody was weakly bound to the target protein.

[0218] Table 6

[0219]

[0220] Mouse-SEZ6-His (2 μg / mL) was coated to detect the binding of candidate antibodies to Mouse-SEZ6-His. According to the test results, only the 297-231-2-A10 candidate antibody was weakly bound to the target protein.

[0221] Table 7

[0222]

[0223]

[0224] Cyno-SEZ6-His (2 μg / mL) was coated to detect the binding of candidate antibodies to Cyno-SEZ6-His. According to the test results, only the 297-231-2-A10 candidate antibody was weakly bound to the target protein.

[0225] The second batch of candidate antibody tests, the results are as follows Figure 8a-8c shown.

[0226] As shown in 8a, Human-SEZ6-His (2 μg / mL) was coated to detect the binding of candidate antibodies to Human-SEZ6-His. According to the test results, candidate antibodies Ph-2-B3, Ph-3-A7, Ph-3-H7, Ph-1-A03, Ph-2-C02, Ph-2-C03, Ph-2-E05, and Ph-2-E10 bound to the target protein.

[0227] As shown in 8b, Mouse-SEZ6-His (2 μg / mL) was coated to detect the binding of candidate antibodies to Mouse-SEZ6-His. According to the test results, only Ph-1-A03 and Ph-2-E05 bound to the target protein.

[0228] like Figure 8c As shown, Cyno-SEZ6-His (2 μg / mL) was coated to detect the binding of candidate antibodies to Cyno-SEZ6-His. According to the test results, only Ph-3-A7, Ph-1-A03, Ph-2-E05, and Ph-2-E10 candidate antibodies bound to the target protein.

[0229] 12. FACS detection of the binding of recombinant antibodies to target proteins

[0230] 1) Resuscitate 293T and 293T-SEZ6 cell lines from liquid nitrogen and adjust the cell state to the logarithmic growth phase;

[0231] 2) Divide the cells into several portions, with the number of cells in each portion being 3×10 5 cells;

[0232] 3) Incubate the candidate antibody (starting concentration of 30 μg / mL, 3-fold gradient dilution with 11 points, 100 μL / well) or 100 μL of transfection supernatant with the target cells, mix thoroughly, and incubate at room temperature for 1 hour;

[0233] 4) Centrifuge at 800 × g for 3 minutes at room temperature, remove the supernatant containing antibodies, and wash the cells three times with PBS;

[0234] 5) Add secondary antibody PE anti human IgG (1:500 dilution), mix thoroughly, and incubate at room temperature in the dark for 30 minutes;

[0235] 6) Centrifuge at 800 × g for 3 minutes at room temperature, remove the supernatant containing the secondary antibody, and wash the cells three times with PBS;

[0236] 7) Resuspend the cells in 500 μL PBS and perform flow cytometry analysis.

[0237] The results are as follows Fig. 9 As shown, Fig. 9 The candidate antibodies (252-1-B03, Ph-2-B3, Ph-3-A7, Ph-3-H7, Ph-1-A03, Ph-2-C02, Ph-2-C03, Ph-2-E05, and Ph-2-E10) with red color were specifically bound to the overexpressing cell line 293T-SEZ6, and the above antibodies were arranged for FACS EC 50 Detection.

[0238] FACS EC analysis of candidate antibodies against overexpressing cells at 37°C 50 Test results such as Fig.10a , Fig.10b As shown in Table 8: The five candidate antibodies Ph-2-B3, Ph-3-H7, Ph-2-C03, Ph-2-E05, and Ph-2-E10 bound strongly to the cells; among them, Ph-2-C03 and Ph-2-E05 bound to the positive antibodies at the same level, and the binding of Ph-2-E10 antibody was stronger than that of the positive antibodies.

[0239] Table 8

[0240]

[0241] Note: The test result of 252-1-B03 was non-binding and is not shown in the table.

[0242] FACS EC analysis of candidate antibodies against overexpressing cells at 4°C 50 Test results such as Fig.11a , Fig.11b , and as shown in Table 9:

[0243] Table 9

[0244]

[0245] According to the test results, the five candidate antibodies Ph-2-B3, Ph-3-H7, Ph-2-C03, Ph-2-E05, and Ph-2-E10 bind strongly to cells; among them, Ph-2-C03 and Ph-2-E05 bind to the positive antibodies at the same level, and Ph-2-E10 binds stronger than the positive antibodies.

[0246] 13. Internalization detection of candidate antibodies

[0247] 1) Prepare target cells 293T-human SEZ6, 5×10 cells per well 5 cells;

[0248] 2) Add 10 μg / mL, 100 μL / well single domain antibody and incubate at 4°C for 30 min;

[0249] 3) Centrifuge at 800 × g for 3 min at 4°C, remove the supernatant containing antibodies, and wash the cells three times with pre-cooled 2% FBS;

[0250] 4) The cells were evenly divided into 2 groups and incubated at 4°C and 37°C for 240 min, respectively. After the incubation, 2% FBS in an ice bath was immediately added to terminate the endocytosis experiment. The cells were centrifuged at 4°C, 800×g for 3 min, and the cells were washed 3 times with pre-cooled 2% FBS.

[0251] 5) Join MonoRab now TM Rabbit Anti-Camelid VHH Cocktail [iFluor 647] (candidate antibody) and PE-Goat anti-Human IgG Fc (invitrogen, Cat#: 12-4998-82) (positive antibody) secondary antibodies (1:1000 dilution), incubated at 4°C for 30 min;

[0252] 6) Centrifuge at 800 × g for 3 min at 4°C, remove the supernatant containing antibodies, and wash the cells three times with pre-cooled 2% FBS;

[0253] 7) Use 200 μL pre-cooled 2% FBS to resuspend the cells, prepare a corresponding number of 1.5 mL EP tubes, add 300 μL pre-cooled 2% FBS to each EP tube, transfer the corresponding samples to the EP tubes, place them on ice, and perform flow cytometry analysis.

[0254] 8) Calculation of cell surface bound antibody internalization level:

[0255] MFI% at time tx = MFI of sample incubated at 37°C / MFI of control sample incubated at 4°C

[0256] Internalization percentage at tx time point = 100% - MFI% at tx time point

[0257] The results are as follows Fig.12 As shown in Table 10:

[0258] Table 10

[0259]

[0260]

[0261] According to the experimental data, except for Ph-1-F11 and 252-1-B03, which basically did not bind to the cell lines, the other antibodies all had internalization effects, and Ph-3-A7, Ph-3-H7, Ph-1-A03, Ph-2-C02, Ph-2-C03, Ph-2-E05, and Ph-2-E10 antibodies had good internalization effects, among which the internalization effect of Ph-2-E10 antibody was equivalent to that of the positive antibody.

[0262] 14. Affinity testing of candidate antibodies

[0263] 1) The antibody affinity was determined using the ForteBio OCTET R2 instrument, and Biotin-Human TSLP (R127A, R130A)-C-His was immobilized using the SA sensor at a concentration of 5 μg / mL and a curing time of 60 s.

[0264] 2) The buffer was PBST (PBS+0.02% tween 20), and the candidate antibody was diluted to 5, 2.5, 1.25, 0.625, 0.3125, 0 nM.

[0265] 3) Affinity detection: equilibrium 60 s, binding 180 s, dissociation 180 s, detection temperature 25°C.

[0266] 4) Kinetic characterization analysis was performed using the ForteBio OCTET R2 system.

[0267] The test results are shown in Figure 13. It can be seen that Ph-2-C03, Ph-3-A7, Ph-3-H7, Ph-2-B3, Ph-2-E05, and Ph-2-E10 all have good affinity.

[0268] 15. Identification of candidate antibody epitopes

[0269] Coated with hSEZ6 (positive antibody) (1 μg / mL); primary antibody: SEZ6 candidate antibody (final concentration 10 μg / mL, 3-fold gradient dilution 7 points), 50 μL / well, and Biotin-SEZ6-His antigen (final concentration 1 μg / mL), 50 μL / well, incubated at 37°C for 30 minutes, then added to the coated plate together, incubated at 37°C for 30 minutes. Secondary antibody: HRP-Streptavidin (1:10000 dilution), to detect whether the candidate antibody and the positive antibody are the same epitope.

[0270] The results are shown in Tables 11 and 12.

[0271] Table 11

[0272]

[0273] Table 12

[0274]

[0275] From the above results, it can be seen that the candidate antibodies screened and the positive antibodies are of different epitopes.

[0276] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. An anti-SEZ6 nanobody or antigen-binding fragment, characterized in that: It contains three complementarity determining regions CDR1, CDR2 and CDR3; among them, The amino acid sequence of CDR1 is the sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 6 and has the same function; The amino acid sequence of CDR2 is one of SEQ ID NO:7 to SEQ ID NO:12, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of SEQ ID NO:7 to SEQ ID NO:12 and functions the same; The amino acid sequence of CDR3 is a sequence shown in one of SEQ ID NO: 13 to SEQ ID NO: 18, or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of SEQ ID NO: 13 to SEQ ID NO: 18 and has the same function.

2. The anti-SEZ6 nanobody or antigen-binding fragment according to claim 1, characterized in that It also comprises four framework regions FR1, FR2, FR3 and FR4 arranged alternately with the three complementary determining regions, wherein: The amino acid sequence of FR1 is one of SEQ ID NOs: 19 to 24; The amino acid sequence of FR2 is one of SEQ ID NOs: 25 to 30; The amino acid sequence of FR3 is one of SEQ ID NOs: 31 to 36; The amino acid sequence of FR4 is a sequence shown in any one of SEQ ID NO:37 to SEQ ID NO:

39.

3. The anti-SEZ6 nanobody or antigen-binding fragment according to claim 1, characterized in that The Nanobody or antigen-binding fragment comprises one or more of the following sequences: (i) an amino acid sequence as shown in any one of SEQ ID NO: 40 to SEQ ID NO: 45; (ii) an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO:40 to SEQ ID NO:45 and functions the same; (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence as shown in any one of SEQ ID NO: 40 to SEQ ID NO: 45, and which retains the function of the amino acid sequence as shown in any one of SEQ ID NO: 40 to SEQ ID NO:

45.

4. A nucleic acid molecule, characterized in that It encodes the anti-SEZ6 nanobody or antigen-binding fragment as described in any one of claims 1 to 3.

5. A carrier, characterized in that It comprises the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that It contains the vector according to claim 5 or the nucleic acid molecule according to claim 4; the host cell is preferably a bacterial cell, a fungal cell, an animal cell, a plant cell or a progeny cell of these cells.

7. A method for producing an anti-SEZ6 nanobody, characterized in that: The following steps are involved: (a) culturing the host cell according to claim 6 under conditions suitable for the production of nanobodies, thereby obtaining a culture containing the anti-SEZ6 nanobody; (b) isolating and / or recovering the anti-DLL3 Nanobody from the culture; and optionally (c) purifying and / or modifying the anti-SEZ6 nanobody obtained in step (b).

8. An antibody-drug conjugate, characterized in that: It comprises the anti-SEZ6 nanobody or antigen-binding fragment according to any one of claims 1 to 3 and an effector; preferably, the effector comprises at least one of a nuclide, a cytotoxic agent, a fluorescent group, an enzyme that catalyzes the color development of a substrate, a chemiluminescent reagent and a nanoparticle marker.

9. A pharmaceutical composition, characterized in that Contains the anti-SEZ6 nanobody or antigen-binding fragment according to any one of claims 1 to 3, or the antibody-drug conjugate according to claim 8.

10. The following use of the anti-SEZ6 nanobody or antigen-binding fragment according to any one of claims 1 to 3: (i) Use in the preparation of a reagent for detecting a human SERZ6-related disease; the reagent is preferably a kit for detecting a SERZ6-related disease; (ii) Use in the preparation of a drug for treating a SEZ6-related disease; the SEZ6-related disease is preferably cancer; the cancer is preferably at least one of small cell lung cancer, neuroendocrine tumors, glioblastoma and high-grade central nervous system tumors.

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