An anti-sez6 nanobody or antigen binding fragment and uses thereof

Abstract

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

Description

[0001] This application is a divisional application of the invention patent application filed on October 18, 2024, with application number 202411461154.0 and invention title "An anti-SEZ6 nanobody or antigen-binding fragment and its application". Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to an anti-SEZ6 nanobody or antigen-binding fragment and its application. Background Technology

[0003] Immunotherapy targeting specific targets within the interdependence and interaction between cancer cells and their microenvironment has become a research hotspot in cancer treatment. This includes immune checkpoint inhibitors such as anti-CTLA-4, anti-PD-1, and anti-PD-L1, as well as chimeric antigen receptor (CAR) T cells. Numerous antibody drugs targeting different targets have been approved for clinical use with promising results, and drugs targeting even more targets are currently under development. Over the past 30 years, traditional monoclonal antibodies have played a crucial role in the development of therapeutic biopharmaceuticals. As antibody drugs are increasingly applied to more therapeutic areas, their therapeutic mechanisms have become more complex, and their structures have become more diverse. Therefore, new technologies are needed to address the limitations of monoclonal antibodies.

[0004] The SEZ6 family includes SEZ6 (Seizure-related gene 6), SEZ6L (Seizure-related gene 6-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, suggesting their potential involvement in complement regulation. SEZ6 is a type I transmembrane protein encoded by the SEZ6 gene located on chromosome 17 [17q11.2], and it participates in the normal dendritic branching of neurons in the cerebral cortex. Under normal physiological conditions, SEZ6 is primarily expressed in the brain, including the cerebral cortex, cerebellum, basal ganglia, and hypothalamus. It is highly expressed in SCLC and other neuroendocrine tumors, but rarely in most normal tissues.

[0005] SEZ6 (Seizure-related gene 6): SEZ6 is normally expressed in the central nervous system. SEZ6 protein plays an important role in nerve cell development and synapse formation. Synapses are the connection points between nerve cells, and they play a crucial role in information transmission. SEZ6 contains 994 amino acids (AAs).

[0006] SEZ6L (Seizure-related gene 6-like): SEZ6L is highly similar to the SEZ6 gene, hence the name "SEZ6-like". The protein encoded by the SEZ6L gene is expressed in the nervous system and participates in synapse formation and nerve cell connections. SEZ6L and SEZ6 share some sequence similarities, but they may differ in certain details. SEZ6L contains 1024 amino acids (AAs).

[0007] SEZ6L2 (Seizure-related gene 6-like 2): The protein encoded by the SEZ6L2 gene is highly expressed in the central nervous system, particularly in the cortical regions of the brain. SEZ6L2 is associated with nerve cell migration, synapse formation, and the development and function of the nervous system. SEZ6L2 contains a total of 910 amino acids (AAs).

[0008] Currently, there are targeted drugs for SEZ6 under development, including monoclonal antibodies and antibody-drug conjugates (ADCs), but very few antibodies have entered clinical trials. Traditional monoclonal antibodies have complex preparation processes, high production costs, and large molecular weights, resulting in poor tissue penetration. Nanobodies are the smallest known fragments capable of binding antigens, only 1 / 10 the size of monoclonal antibodies, and possess structural stability and binding activity comparable to proto-heavy-chain antibodies. Compared to traditional antibodies, nanobodies have several unique advantages, such as excellent tissue penetration, rapid clearance, ease of production and modification, high stability, and low immunogenicity, making them a promising new type of antibody molecule.

[0009] There are no reports or clinical applications of nanobodies targeting SEZ6, and there is an urgent need in this field to develop new and effective specific nanobodies targeting SEZ6. Summary of the Invention

[0010] definition

[0011] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art as would be understood by those skilled in the art. Furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as would be understood by those skilled in the art.

[0012] Unless otherwise stated, the interchangeable terms “antibody” or “immunoglobulin” used herein, whether referring to heavy-chain antibodies or conventional four-chain antibodies, are used generally to include full-length antibodies, their individual chains, and all their portions, domains, or fragments (including, but not limited to, antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively). Furthermore, the term “sequence” as used herein (e.g., in the terms “antibody sequence,” “single variable domain sequence,” “VHH sequence,” or “protein sequence,” etc.) should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding said amino acid sequence, unless a more specific interpretation is required herein.

[0013] 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.

[0014] To achieve the above objectives, a first aspect of the present invention provides an anti-SEZ6 nanobody or antigen-binding fragment comprising three complementarity-determining regions CDR1, CDR2, and CDR3; wherein...

[0015] The amino acid sequence of CDR1 is the sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6, or is a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 1 to SEQ ID NO: 6 and has the same function;

[0016] The amino acid sequence of CDR2 is the sequence shown in one of SEQ ID NO: 7 to SEQ ID NO: 12, or a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with one of SEQ ID NO: 7 to SEQ ID NO: 12 and has the same function;

[0017] The amino acid sequence of CDR3 is the sequence shown in any one of SEQ ID NO: 13 to SEQ ID NO: 18, or is a sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO: 13 to SEQ ID NO: 18 and has the same function.

[0018] In this invention, "having the same function" means being able to bind to the SEZ6 protein.

[0019] The location of the CDR in the antibody or nanobody sequence can be determined by those skilled in the art using existing techniques. Typically, the CDR can be identified by sequencing the DNA of the antibody or nanobody, and the resulting sequence can then be analyzed using a dedicated database (such as the international ImMunoGeneTics database or IMGT).

[0020] In the sequence provided by this invention, CDR is drawn according to IMGT (https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results).

[0021] According to a preferred embodiment of the present invention, the anti-SEZ6 nanobody or antigen-binding fragment has any of the following CDR sequence characteristics:

[0022] (1) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 13, respectively, or sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and have the same function;

[0023] (2) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 14, respectively, or sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and have the same function;

[0024] (3) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 3, SEQ ID NO: 9, and SEQ ID NO: 15, respectively, or sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and have the same function;

[0025] (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 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and have the same function;

[0026] (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 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and have the same function;

[0027] (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 have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the above sequences and have the same function.

[0028] According to the present invention, in addition to the above-mentioned complementary determinant regions, the anti-SEZ6 nanobody or antigen-binding fragment further comprises four frame regions FR1, FR2, FR3 and FR4 alternately arranged with the three complementary determinant regions, wherein the amino acid sequence of FR1 is the sequence shown in one of SEQ ID NO: 19 to SEQ ID NO: 24;

[0029] The amino acid sequence of FR2 is the sequence shown in one of SEQ ID NO: 25 to SEQ ID NO: 30;

[0030] The amino acid sequence of FR3 is shown in one of SEQ ID NO: 31 to SEQ ID NO: 36;

[0031] The amino acid sequence of FR4 is shown in one of SEQ ID NO: 37 to SEQ ID NO: 39.

[0032] According to a preferred embodiment of the present invention, the anti-SEZ6 nanobody or antigen-binding fragment has any of the following FR sequence characteristics:

[0033] (a) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 31, and SEQ ID NO: 37, respectively;

[0034] (b) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 37, respectively;

[0035] (c) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 37, respectively;

[0036] (d) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, and SEQ ID NO: 38, respectively;

[0037] (e) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, and SEQ ID NO: 37, respectively;

[0038] (f) The amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36 and SEQ ID NO: 39, respectively.

[0039] This invention includes all sequences that satisfy the above sequence characteristics, and more preferably,

[0040] The nanobody or antigen-binding fragment comprises one or more of the following sequences:

[0041] (i) An amino acid sequence as shown in any one of SEQ ID NO: 40 to SEQ ID NO: 45;

[0042] (ii) An amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 40 to SEQ ID NO: 45 and having the same function;

[0043] (iii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in any one of SEQ ID NO: 40 to SEQ ID NO: 45, and retains the function of the amino acid sequence shown in any one of SEQ ID NO: 40 to SEQ ID NO: 45, i.e., it is able to bind to the SEZ6 protein.

[0044] Ph-2-B3:QVKLEESGGGLVQAGGALNLSCVASGIIFSMYDMGWYRQGSGEARDIVAAIGKGGSTYYADAVKGRFTISRDNAQNTLYLQMNSLKPEDTSMYYCTTVEPYDYWGQGTQVTVSS(SEQ ID NO:40)

[0045] Ph-3-A7:AVQLVDSGGGLVQPGGSLRLSCTASDSRFIANIMGWYRQAPGKERELVVAISSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTDMYYCAFFEGGIPDGWGQGTQVTVSS(SEQ ID NO:41)

[0046] Ph-3-H7:AVQLVDSGGGLVQAGGSLRLSCAASGSIFSINTMGWYRQAPGKERELVAAISSHGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTTMYYCAASRDSDYDPGRGSWGQGTQVTVSS(SEQ ID NO:42)

[0047] Ph-2-C03:AVQLVESGGGLVQAGGSLRLSCAASGSIFSIEAMGWYRQAPGEVRELVAAISSGNSTYYADNVKGRFTISRDNANNTVYLQMTSLKPEDTAMYYCAAFESSRPWALGKGALVTVSS(SEQ ID NO:43)

[0048] Ph-2-E05:QVKLEESGGGLVQAGGSLRLSCTASGSIFSFDALGWYRQAPGSERELVAAVGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDVAMYYCAAFSPHSGIPRLWGQGTQVTVSS(SEQ ID NO:44)

[0049] Ph-2-E10:QVKLEESGGGLVQAGGSLRLSCAASGSIFSIDTMGWYRSAPGEERRLVAAISTGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCAGFSSQSDPGVPNLLGQGTQVTVSS(SEQ ID NO:45)

[0050] A second aspect of the present invention provides a nucleic acid molecule encoding the aforementioned anti-SEZ6 nanobody or antigen-binding fragment.

[0051] A third aspect of the present invention provides a carrier comprising the nucleic acid molecule described in the second aspect.

[0052] A fourth aspect of the present invention provides a host cell containing the vector described in the third aspect or the nucleic acid molecule described in the second aspect.

[0053] The host cells include, but are not limited to, bacterial cells, fungal cells, animal cells, plant cells, or their progeny cells.

[0054] The anti-SEZ6 nanobody of the present invention can be obtained by the following methods:

[0055] (1) Phage display library was prepared by immunizing alpacas with human, monkey and mouse SEZ6 protein.

[0056] (2) Phage display libraries were screened using human, monkey, and mouse SEZ6 protein affinity screening;

[0057] (3) ELISA and FACS were used to identify positive clones;

[0058] (4) Expression and purification of SEZ6 nanobodies.

[0059] A fifth aspect of the present invention provides a method for engineered production of anti-SEZ6 nanobodies, comprising the following steps:

[0060] (a) Culturing host cells as described in the fourth aspect of the invention under conditions suitable for generating nanobodies, thereby obtaining a culture containing the anti-SEZ6 nanobodies; and

[0061] (b) Isolating and / or recovering the anti-SEZ6 nanobody from the culture; and optionally...

[0062] (c) Purification and / or modification of the anti-SEZ6 nanobody obtained in step (b).

[0063] A sixth aspect of the present invention provides an antibody-drug conjugate comprising the aforementioned anti-SEZ6 nanobody or antigen-binding fragment and an effector; preferably, the effector comprises at least one of a radionuclide, a cytotoxic agent, a fluorescent group, an enzyme that catalyzes substrate color development, a chemiluminescent reagent, and a nanoparticle-based label.

[0064] A seventh aspect of the present invention provides a pharmaceutical composition comprising the above-described anti-SEZ6 nanobody or antigen-binding fragment, or the above-described antibody-drug conjugate.

[0065] The eighth aspect of the present invention provides the following uses of the anti-SEZ6 nanobody or antigen-binding fragment:

[0066] (i) Use in the preparation of a reagent for detecting SEZ6-related diseases in humans; said reagent is preferably a kit for detecting SEZ6-related diseases;

[0067] (ii) Application in the preparation of medicines for the treatment of SEZ6-related diseases.

[0068] The anti-SEZ6 nanobody or antigen-binding fragment of the present invention can also be used to treat SEZ6-related diseases.

[0069] The SEZ6-related diseases include, but are not limited to, cancers, including, but 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, either alone or in combination with other anticancer drugs, and therefore the active component of the drug includes the anti-SEZ6 nanobody and optionally other anticancer drugs.

[0070] Compared to conventional antibodies, the anti-SEZ6 nanobody of this invention exhibits high expression efficiency, simple purification, small molecular weight (approximately 15 kDa), high affinity, good specificity, high solubility, and strong tolerability, making it widely applicable in scientific research, diagnosis, and therapeutic fields. Furthermore, nanobodies more readily recognize antigens that traditional antibodies cannot capture, exhibit better tissue penetration, can enter tumor tissue and cross the blood-brain barrier, and can be developed as immune checkpoint inhibitors, providing solutions for tumor imaging and treatment.

[0071] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0072] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0073] Figure 1 Gel electrophoresis image of reducible protein detected by SDS-PAGE.

[0074] Figure 2a -b shows the amplification process of the yeast display library VHH fragment for alpacas #252 and #297-231, respectively.

[0075] Figure 3a -b shows the QC results of the yeast display library for alpaca #252 and alpacas #297-231, respectively.

[0076] Figure 4a-b shows the magnetic sorting results of the yeast display library for alpaca #252 and alpacas #297-231, respectively.

[0077] Figure 5a -b shows the results of the two-round sorting of yeast display libraries for alpacas #252 and #297-231, respectively.

[0078] Figure 6a -d shows the results of FACS testing on plates 1, 2, 3, and 4 of the 252# alpaca monoclonal antibody.

[0079] Figure 7a -c shows the results of plate 1, plate 2, and plate 3 of the FACS test for 297-231# alpaca monoclonal antibodies.

[0080] Figure 8a -c shows the detection results of the second batch of candidate antibodies in the ELISA assay for detecting the binding of recombinant antibodies to the target protein.

[0081] Figure 9 The results of FACS detection of recombinant antibody binding to target protein are shown.

[0082] Figure 10a -b shows the interaction of the candidate antibody with FACS EC overexpressing cells at 37°C. 50 Test results.

[0083] Figure 11a -b shows the interaction of the candidate antibody with FACS EC overexpressing cells at 4°C. 50 Test results.

[0084] Figure 12 The results of the candidate antibody endocytosis assay are shown.

[0085] Figure 13 shows the results of the candidate antibody affinity test. Detailed Implementation

[0086] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0087] Example

[0088] 1. Antigen preparation

[0089] Based on the amino acid sequence information of Cynomolgus SEZ6 and Mouse SEZ6 proteins from the uniprot database (Leu20-His927(Cynomolgus SEZ6)[Accession|A0A2K5WPJ4]&(Leu20-His922)(MouseSEZ6)[Accession|Q7TSK2]), codon optimization was performed according to mammalian codon preferences. After synthesizing the antigen-encoding nucleic acid sequence, the nucleic acid sequence was subcloned into the pCDNA3.4 vector, and a His tag was added to the C-terminus to construct a eukaryotic expression vector. The two plasmids were transfected into 293F cells, 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 Kaika Biotechnology (Shanghai) Co., Ltd.

[0090] The purity of the recombinant protein was determined by SDS-PAGE, and the results are as follows: Figure 1 As shown, the results indicate that the Human SEZ6-His protein has a purity >95% and can be used for immunization and subsequent panning. The Cyno SEZ6-His and Mouse SEZ6-His proteins have a purity >90% and can be used for cross-binding verification.

[0091] 2. ELISA detection of the binding activity between recombinant protein antigen and positive antibody

[0092] 1) Dilute SEZ6-His recombinant protein to a final concentration of 1 μg / mL using sterile CBS. Take a new 96-well plate, add 100 μL to each well, and coat overnight at 4°C. Discard the antigen coating solution and wash 3 times with PBST (containing 0.5% Tween). Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours. Discard the blocking buffer and wash the plate 3 times with PBST.

[0093] 2) Positive control antibody hSEZ6VH+VL (expressed and prepared by Aikon Biotechnology Co., Ltd., sequence information refers to WO 2019 / 232241A1) was diluted to 10 μg / mL with PBS, 5-fold diluted for 7 spots, and 100 μL / well was added to the microplate and incubated at room temperature for 1 hour. The control wells were prepared with PBS.

[0094] 3) Discard the liquid from the well and wash three times with PBST;

[0095] 4) Add secondary antibody HRP-Protein A (1:10000 dilution) to the microplate at a rate of 100 μL / well and incubate at room temperature for 1 hour;

[0096] 5) After removing the liquid from the wells, wash the plate three times with PBST;

[0097] 6) Add 100 μL / well of TMB colorimetric solution;

[0098] 7) Incubate at room temperature in the dark for 15 minutes;

[0099] 8) Add 50 μL / well stop solution (2M HCl);

[0100] 9) Use an ELISA reader to read the OD in the wells. 450 value.

[0101] The results are shown in Table 1 below:

[0102] Table 1

[0103]

[0104]

[0105] The results showed that the proteins from all three genera had good binding activity with the positive control antibody and could be used for subsequent experiments.

[0106] 3. Alpaca Immunization

[0107] Alpaca were immunized using the purchased Human SEZ6-His recombinant protein and the Mouse SEZ6-His recombinant protein prepared above. The alpaca numbers were 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 is shown in Table 2 below:

[0108] Table 2

[0109]

[0110] 4. Immunotiter testing

[0111] The steps for immunogenicity testing include:

[0112] 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.

[0113] 2) Place the centrifuge tube containing the blood sample in a centrifuge and centrifuge at 5000 rpm for 20 min; separate the upper serum layer and transfer the serum to a new sterile centrifuge tube to collect the immune serum;

[0114] 3) Dilute the target recombinant protein to a final concentration of 1 μg / mL using sterile CBS (carbonate buffer); take a new 96-well microplate, add 100 μL / well, and coat overnight at 4°C;

[0115] 4) Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20);

[0116] 5) Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours;

[0117] 6) After removing the blocking buffer, wash the plate 5 times with PBST;

[0118] 7) Add 100 μL of serially diluted serum (100 μL / well), incubate at room temperature for 1 hour, and use PBS for the control wells;

[0119] 8) Remove the liquid from the well and wash 5 times with PBST;

[0120] 9) Add 100 μL of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 hour;

[0121] 10) After removing the liquid from the wells, wash the plate 5 times with PBST;

[0122] 11) Add 100 μL / well of TMB colorimetric solution;

[0123] 12) Incubate at room temperature in the dark for 10-15 minutes;

[0124] 13) Add 50 μL / well stop solution;

[0125] 14) Use a microplate reader to read the OD in the wells. 450 value.

[0126] The results of the immunogenicity test are shown in Tables 3 and 4 below. Two groups of samples were tested in parallel.

[0127] Table 3

[0128]

[0129] Table 4

[0130]

[0131]

[0132] Based on the ELISA test results, the immune serum can bind to the target recombinant protein, and the OD value changes in a gradient with the gradient dilution of the immune serum. The alpaca titer is significantly improved, meeting the requirements for blood collection and library construction. 100 mL of peripheral blood was collected from each to construct the antibody display library.

[0133] 5. Construction of the yeast display library

[0134] The amplification processes of the VHH fragment in the yeast display library of alpacas #252 and #297-231 are as follows: Figure 2a and 2b As shown: Peripheral blood was collected to isolate PBMCs, and RNA was extracted. A cDNA library was prepared using a reverse transcription kit. One round of PCR was performed using single-domain antibody amplification primers, yielding PCR bands of approximately 1000 bp and 750 bp, respectively. The 750 bp fragment was recovered by gel electroporation and used as a template for the second round of PCR. The second round of PCR yielded a band of approximately 400 bp, which was the VHH fragment. The VHH fragment was mixed with a linearized yeast display vector and electroporated into yeast cells to obtain the yeast library. The constructed yeast library of alpaca #252 had a volume of 2.63 × 10⁻⁶. 9 The yeast storage capacity of alpacas #297-231 is 1.0 × 10⁻⁶. 9 The specific steps are as follows:

[0135] 5.1 PBMC isolation and VHH antibody fragment cloning

[0136] 1) After immunization, 100 mL of peripheral blood was collected and PBMCs were separated using lymphocyte separation solution.

[0137] 2) Extract RNA using PrimeScript. TM II. The 1st Strand cDNA Synthesis Kit was used for reverse transcription to prepare cDNA.

[0138] 2-1) Prepare the following reaction mixture in 200 μL of PCR medium:

[0139]

[0140]

[0141] 2-2) After holding at 65℃ for 5 minutes, rapidly cool on ice;

[0142] 2-3) Prepare the following reaction solutions in the PCR tubes described above.

[0143]

[0144] 2-4) After mixing by pipetting, dispense 80 μL / tube and place in a PCR instrument at 42℃ for 1 hour, then heat-inactivate at 70℃ for 15 minutes. Finally, store the cDNA sample on ice or at -20℃ for long-term storage.

[0145] 3) Amplification of the VHH fragment

[0146] 3-1) Prepare the first round of PCR reaction system (50 μL / tube): the upstream primer binds to the signal peptide, the sequence of which is shown in SEQ ID NO: 48, and the downstream primer binds to the CH2 region, the sequence of which is shown in SEQ ID NO: 49.

[0147]

[0148] After configuring the PCR reaction system, set up the PCR instrument according to the following procedure:

[0149]

[0150] 3-2) Agarose gel electrophoresis of PCR products

[0151] PCR products were analyzed by electrophoresis using 1% agarose gel to separate fragments with a molecular weight of approximately 750 bp. PCR products were recovered using a gel extraction kit, and their concentrations were determined using NanoDrop.

[0152] 3-3) Prepare a two-round PCR reaction system (50 μL / tube): The upstream primer binds to the FR1 region of the antibody, and the sequence is shown in SEQ ID NO: 50. The downstream primer binds to the Hinge and FR4 regions of the antibody, and the sequence is shown in SEQ ID NO: 51. The restriction enzyme site is SfiI.

[0153]

[0154] After configuring the PCR reaction system, set up the PCR instrument according to the following procedure:

[0155]

[0156] 3-4) Agarose gel electrophoresis analysis of the second round of PCR products

[0157] The PCR products were analyzed by electrophoresis using 1% agarose gel to separate the VHH fragment with a molecular weight of approximately 400 bp. The VHH PCR products were recovered using a gel extraction kit and their concentration was determined using NanoDrop.

[0158] 3-5) PCR product precipitation

[0159] Aliquot 200 μL of the recovered second-round PCR product into each 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3M sodium acetate and 1 μg / μL of glycogen, mix by pipetting and aspiration, add 880 μL of anhydrous ethanol, mix by inversion, and store at -80℃.

[0160] 5.2 Linearization of the yeast display vector pYDisplay, the enzyme digestion system is as follows:

[0161]

[0162] 1) Digest the pYDisplay vector with SfiI enzyme, aliquot 100 μL / tube, and incubate overnight at 50°C;

[0163] 2) The pYDisplay vector fragment was separated using a 1% agarose gel, a 5000bp fragment was excised and recovered from the gel, and the concentration was determined using NanoDrop.

[0164] 3) Aliquot 200 μL of the recovered pYDisplay enzyme digestion product into each 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3M sodium acetate and 1 μg / μL of glycogen, mix by pipetting and aspiration, add 880 μL of anhydrous ethanol, mix by inversion, and freeze at -80℃.

[0165] 5.3 Construction of a yeast display library by electroconversion

[0166] 1) Streak the competent yeast strains frozen at -80℃ onto YPD solid medium plates and activate them at 30℃ for 3-5 days;

[0167] 2) Inoculate single colony yeast competent cells into 50 mL of YPD medium and incubate at 250 rpm and 30°C for 1-2 days;

[0168] 3) Preparation of competent yeast strains: After mixing the linearized vector fragment and PCR product, add it to an electroporation cuvette and electroporate; after electroporation, the competent yeast cells are transfected into culture flasks and incubated at 220 rpm and 30℃ for 1 h.

[0169] 4) Take 20 μL of the resuspension, dilute it 5000 times with SDCAA, take 100 μL, spread it on an SDCAA plate, incubate for 2-3 days, calculate the volume of the culture, and continue to incubate the remaining bacterial culture for 24 hours.

[0170] 5) Preservation of bacteria: Collect the remaining bacterial culture into a 50mL centrifuge tube, centrifuge at 3000×g for 5min, discard the supernatant, add 10mL of SDCAA to resuspend, mix with 50% glycerol: resuspension = 1:1, and freeze at -80℃.

[0171] Results: The QC of the yeast display library for alpaca #252 and alpacas #297-231 were as follows: Figure 3a and 3b As shown: Randomly selected single clones were sequenced and analyzed. The peak-stacking stop codon sequences were removed. There were no empty or repetitive sequences, and the sequences showed great differences, indicating good library diversity.

[0172] 6. Magnetic sorting of yeast display library

[0173] 1) Add the yeast cultured in SDCAA to a 250mL shake flask containing 50mL SGCAA medium and culture at 30℃ and 240rpm for 16h.

[0174] 2) After centrifugation, discard the supernatant, resuspend in 1 mL of 0.5% PBSA, add to a 1.5 mL centrifuge tube, centrifuge at 3000×g for 5 min, and discard the supernatant. Wash again with 0.5% PBSA.

[0175] 3) Wash the streptavidin magnetic beads that have been incubated with the antigen twice with 0.5% PBSA (incubate at 4°C for 5 minutes each time), place them on a magnetic rack, incubate for 5 minutes, and then discard the supernatant.

[0176] 4) Add the yeast culture to the magnetic beads that have been bound to the antigen, incubate at 4°C for 60 minutes by rotating, and then place on a magnetic rack for 15 minutes.

[0177] 5) Discard the yeast culture and keep the magnetic beads. Wash three times with 0.5% PBSA (incubate at 4°C for 5 minutes each time).

[0178] 6) Resuspend the magnetic beads in 1 mL of SDCAA medium, and transfer 0.5-5 μL of the resuspension into 100 μL of SDCAA medium for plating. Divide the resuspension into two portions. Add 500 μL of 50% glycerol to one portion (store at -80℃); add the other portion to a shaker tube, add 2 mL of SDCAA medium, and incubate at 30℃ and 240 rpm for 16 h.

[0179] 7) Transfer the bacterial culture from the shake tube to 50 mL of SDCAA medium (250 mL shake flask) and incubate overnight at 30°C and 240 rpm.

[0180] 8) Measure the OD of the bacterial solution 600 Value, based on OD 600 Centrifuge a portion of the bacterial culture, resuspend in SGCAA, and transfer to 50 mL of SGCAA medium to allow the final OD to reach the target value. 600 The bacterial culture was incubated overnight at 30°C and 240 rpm with a value of 1. The remaining bacterial culture was resuspended in SDCAA:50% glycerol = 1:1 and stored at -80°C.

[0181] Figure 4a and4b The results of magnetic sorting of yeast display libraries for alpacas #252 and #297-231 are shown respectively.

[0182] Figure 4a In the diagram, A: NC group; B: original library: primary antibody: V5-FITC; C: 1 MACS: primary antibody: Biotin-Human-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC; D: 1 MACS: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC. After sorting the yeast library using the Bio-Human-SEZ6-His protein via magnetic separation, the positive rate for Human SEZ6 was 4.88%, and the positive rate for Mouse SEZ6 was 1.44%. A two-round sorting process was then implemented.

[0183] Figure 4b In the diagram, A: NC group; B: Original library: primary antibody: V5-FITC; C: 1 MACS: primary antibody: Biotin-Human-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC; D: 1 MACS: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC. After sorting the yeast library using the Bio-Human-SEZ6-His protein via magnetic separation, the positive rate for Human SEZ6 was 1.18%, and the positive rate for Mouse SEZ6 was 1.88%. A two-round sorting process was then implemented.

[0184] 7. Two-round sorting of yeast display library

[0185] 1) Take 1 mL of SGCAA-cultured yeast culture into a 1.5 mL centrifuge tube, centrifuge to remove the supernatant, wash twice with 1 mL PBS, resuspend with 1 mL PBS, take 100 μL of the culture into a new 1.5 mL centrifuge tube (as NC), centrifuge, and remove the supernatant.

[0186] 2) Dilute the Biotin-antigen protein to a concentration of 10 μg / mL with 100 μL PBS, resuspend the bacterial cells in the experimental group, incubate at 4℃ for 60 min by rotation, centrifuge at 3000×g for 3 min, discard the supernatant, and wash twice with 1 mL PBS.

[0187] 3) Dilute APC-Streptavidin at 1:1000 and resuspend the bacterial cells in the experimental group. Incubate at 4°C with rotation 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.

[0188] 4) Add the resuspended bacterial cells to the flow cytometry tube for flow cytometry separation.

[0189] 5) Prepare 1 mL of SDCAA culture medium into a 15 mL centrifuge tube as a collection tube.

[0190] 6) Use the machine for flow sorting and NC group gates to sort APC-positive yeast.

[0191] Figure 5a and 5b The results of the two-round sorting of yeast display libraries for alpacas #252 and #297-231 are shown respectively.

[0192] Figure 5a In the study, A: NC group; B: 1MACS: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC. P3 was sorted, yielding a total of 19006 yeast cells. These were plated, and yeast monoclonal antibodies were selected for amplification and induction, followed by flow cytometry analysis.

[0193] Figure 5b In the study, A: NC group; B: 1 MACS: primary antibody: Biotin-Human-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC; C: 1 MACS: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC. Cells were sorted at P3 level. Human SEZ6 protein sorting yielded 45,046 cells, and Mouse SEZ6 protein sorting yielded 19,602 cells. Cells were plated, and yeast single clones were selected for amplification and induction, followed by yeast single-clone flow cytometry analysis.

[0194] 8. Yeast monoclonal flow cytometry detection

[0195] After sorting, the yeast culture was plated on SDCAA plates, and single clones were picked and cultured. After induced expression for 48 h, the culture was incubated with biotin-antigen. PE-Streptavidin was used as the secondary antibody. Flow cytometry was performed after incubation. The yeast clones that bound to the target antigen were resuspended in 0.2% SDS, incubated at 95 °C for 10 min for lysis, and the supernatant was collected by centrifugation. 0.5 μL of the supernatant was used as a template for PCR amplification and assay (the remaining culture was stored at -20 °C).

[0196] Monoclonal cells were randomly selected from the enriched yeast display library, amplified and induced, and then detected using antigens to determine the binding of single-domain antibodies on the surface of the monoclonal yeast cells to the target antigens. Results Figure 6a -d、 Figure 7a-c is shown.

[0197] Based on the experimental results, positive clones that cross-linked Human SEZ6 and Mouse SEZ6 proteins were selected for PCR testing, and the differential sequences obtained were used to construct vectors.

[0198] 9. Construction of antibody eukaryotic expression vector

[0199] The positive yeast clone was subjected to PCR to obtain the antibody sequence, a His tag was added to the C-terminus, and after digestion with SfiI, it was ligated into the eukaryotic expression vector pcDNA3.4-human IgG1Fc to construct the antibody expression vector. After Sanger sequencing confirmed that the sequence was correct, plasmid extraction was performed.

[0200] 10. Expression and purification of candidate single-domain antibodies

[0201] 1) Remove the LVTransm transfection reagent and pcDNA3.4-human IgG1Fc antibody expression vector from the freezer. After thawing at room temperature, mix thoroughly by pipetting. Remove the PBS buffer and warm it to room temperature. Transfer 2 mL of PBS to one well of a 6-well plate, add 20 μg of the eukaryotic antibody expression vector, mix thoroughly by pipetting, then add 60 μL of LVTransm, immediately mix by pipetting, and let stand at room temperature for 10 minutes.

[0202] 2) Add the above DNA / LVTransm complex to 20 mL of 293F cells and gently shake to mix thoroughly. Incubate the cells at 37°C, 5% CO2, 130 rpm.

[0203] 3) After continuous culture for 5-7 days, centrifuge to collect the supernatant of the culture medium, filter it through a 0.45μm filter membrane, transfer the filtrate to a sterile centrifuge tube, and purify the antibody using a Protein A column.

[0204] 11. ELISA detection of the binding of recombinant antibody to target protein

[0205] 1) Dilute the recombinant protein to a final concentration of 1 μg / mL using sterile CBS. Take a new 96-well microplate, add 100 μL / well, and coat overnight at 4°C.

[0206] 2) Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20).

[0207] 3) Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours;

[0208] 4) After removing the blocking buffer, wash the plate 5 times with PBST;

[0209] 5) Add the expressed recombinant antibody, transfect 50 μL of supernatant / well or purified antibody (starting concentration of 10 μg / mL, serially diluted 3 times for 7 spots, 100 μL / well), incubate at room temperature for 1 hour, and use PBS for control wells;

[0210] 6) Remove the liquid from the well and wash 5 times with PBST;

[0211] 7) Add 100 μL / well of HRP-Protein A antibody (1:50000 dilution) and incubate at room temperature for 1 hour;

[0212] 8) After removing the liquid from the wells, wash the plate 5 times with PBST;

[0213] 9) Add 100 μL / well of TMB colorimetric solution;

[0214] 10) Incubate at room temperature in the dark for 10-15 minutes;

[0215] 11) Add 50 μL / well of stop solution;

[0216] 12) Use a microplate reader to read the OD in the wells. 450 value.

[0217] The results of the first batch of candidate antibody tests are shown in Tables 5 to 7.

[0218] Table 5

[0219]

[0220] The antibody was coated with Human-SEZ6-His (2 μg / mL), and the binding of candidate antibodies to Human-SEZ6-His was detected. The results showed that only candidate antibody 297-231-2-A10 showed weak binding to the target protein.

[0221] Table 6

[0222]

[0223] The candidate antibodies were coated with Mouse-SEZ6-His (2 μg / mL), and the binding of the candidate antibodies to Mouse-SEZ6-His was detected. The results showed that only candidate antibody 297-231-2-A10 showed weak binding to the target protein.

[0224] Table 7

[0225]

[0226]

[0227] The candidate antibodies were coated with Cyno-SEZ6-His (2 μg / mL), and the binding of the candidate antibodies to Cyno-SEZ6-His was detected. The results showed that only candidate antibodies 297-231-2-A10 exhibited weak binding to the target protein.

[0228] The results of the second batch of candidate antibody testing are as follows: Figures 8a-8c As shown.

[0229] As shown in Figure 8a, Human-SEZ6-His (2 μg / mL) was coated, and the binding of candidate antibodies to Human-SEZ6-His was detected. The results showed that candidate antibodies Ph-2-B3, Ph-3-A7, Ph-3-H7, Ph-1-A03, Ph-2-CO2, Ph-2-CO3, Ph-2-E05, and Ph-2-E10 bound to the target protein.

[0230] As shown in Figure 8b, Mouse-SEZ6-His (2 μg / mL) was coated, and the binding of the candidate antibody to Mouse-SEZ6-His was detected. The results showed that only Ph-1-A03 and Ph-2-E05 bound to the target protein.

[0231] like Figure 8c As shown, Cyno-SEZ6-His (2 μg / mL) was coated, and the binding of candidate antibodies to Cyno-SEZ6-His was detected. The results showed that only Ph-3-A7, Ph-1-A03, Ph-2-E05, and Ph-2-E10 candidate antibodies bound to the target protein.

[0232] 12. FACS detection of the binding of recombinant antibodies to target proteins

[0233] 1) Resuscitate 293T and 293T-SEZ6 cell lines from liquid nitrogen and adjust the cell state to the logarithmic growth phase;

[0234] 2) Divide the cells into several portions, with each portion containing 3 × 10 cells. 5 One cell;

[0235] 3) Incubate the candidate antibody (starting concentration of 30 μg / mL, serially diluted 3 times for 11 spots, 100 μL / well) or 100 μL of transfection supernatant with the target cells, mix thoroughly, and incubate at room temperature for 1 hour.

[0236] 4) Centrifuge at 800×g at room temperature for 3 minutes, remove the supernatant containing antibodies, and wash the cells 3 times with PBS;

[0237] 5) Add secondary antibody PE anti-human IgG (1:500 dilution), mix thoroughly, and incubate at room temperature in the dark for 30 minutes;

[0238] 6) Centrifuge at 800×g at room temperature for 3 minutes, remove the supernatant containing the secondary antibody, and wash the cells 3 times with PBS;

[0239] 7) Resuspend the cells in 500 μL PBS and perform flow cytometry analysis.

[0240] The results are as follows Figure 9 As shown, Figure 9 Candidate antibodies labeled with red (252-1-B03, Ph-2-B3, Ph-3-A7, Ph-3-H7, Ph-1-A03, Ph-2-CO2, Ph-2-CO3, Ph-2-E05, Ph-2-E10) specifically bound to the overexpressing cell line 293T-SEZ6. These antibodies were then used in FACS ECMO. 50 Testing.

[0241] Candidate antibodies were reacted with FACS EC overexpressing cells at 37°C. 50 Test results as follows Figure 10a , Figure 10b As shown in Table 8, the five candidate antibodies Ph-2-B3, Ph-3-H7, Ph-2-CO3, Ph-2-E05, and Ph-2-E10 showed strong binding to cells; among them, Ph-2-CO3 and Ph-2-E05 bound to positive antibodies similarly, while Ph-2-E10 bound to positive antibodies more strongly.

[0242] Table 8

[0243]

[0244] Note: The test result for 252-1-B03 was non-binding and is not shown in the table.

[0245] Candidate antibodies were reacted with FACS EC of overexpressing cells at 4°C. 50 Test results as follows Figure 11a , Figure 11b As shown in Table 9:

[0246] Table 9

[0247]

[0248] According to the test results, the five candidate antibodies Ph-2-B3, Ph-3-H7, Ph-2-CO3, Ph-2-E05, and Ph-2-E10 have strong binding to cells; among them, Ph-2-CO3 and Ph-2-E05 have similar binding to positive antibodies, while Ph-2-E10 has a stronger binding than positive antibodies.

[0249] 13. Detection of candidate antibody endocytosis

[0250] 1) Prepare target cells: 293T-human SEZ6, 5 × 10⁶ cells per well. 5 One cell;

[0251] 2) Add 10 μg / mL, 100 μL / well single-domain antibody and incubate at 4℃ for 30 min;

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

[0253] 4) Divide the cells into two groups and incubate them at 4℃ and 37℃ for 240 min respectively. After incubation, immediately add 2% FBS in an ice bath to stop the endocytosis experiment. Centrifuge at 800×g for 3 min at 4℃. Wash the cells three times with pre-cooled 2% FBS.

[0254] 5) Join MonoRab immediately 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℃ for 30 min;

[0255] 6) Centrifuge at 800×g for 3 min at 4℃, remove the supernatant containing antibodies, and wash the cells 3 times with pre-cooled 2% FBS;

[0256] 7) Resuspend the cells in 200 μL of pre-chilled 2% FBS, prepare the corresponding number of 1.5 mL EP tubes, add 300 μL of pre-chilled 2% FBS to each EP tube, transfer the corresponding samples to the EP tubes, place them on ice, and perform flow cytometry analysis.

[0257] 8) Calculation of the level of antibody internalization on the cell surface:

[0258] tx time point MFI% = MFI of sample incubated at 37℃ / MFI of control sample incubated at 4℃

[0259] Internalization percentage at time point tx = 100% - MFI% at time point tx

[0260] The results are as follows Figure 12 As shown in Table 10:

[0261] Table 10

[0262]

[0263]

[0264] According to the experimental data, except for Ph-1-F11 and 252-1-B03 which basically do not bind to the cell line, the remaining antibodies all have endocytosis effects. Among them, the endocytosis effects of Ph-3-A7, Ph-3-H7, Ph-1-A03, Ph-2-C02, Ph-2-C03, Ph-2-E05, and Ph-2-E10 antibodies are good, and the endocytosis effect of Ph-2-E10 antibody is comparable to that of positive antibodies.

[0265] 14. Affinity detection of candidate antibodies

[0266] 1) Antibody affinity was determined using a ForteBio OCTET R2 instrument. Biotin-Human TSLP(R127A,R130A)-C-His was immobilized using an SA sensor at a concentration of 5 μg / mL for 60 s.

[0267] 2) The buffer solution was PBST (PBS + 0.02% Tween 20), and the candidate antibody was diluted to 5, 2.5, 1.25, 0.625, 0.3125, and 0 nM.

[0268] 3) Affinity test: Equilibrium 60s, binding 180s, dissociation 180s, test temperature 25℃.

[0269] 4) Dynamic characterization analysis was performed using the ForteBio OCTET R2 system.

[0270] The test results are shown in Figure 13. It can be seen that Ph-2-CO3, Ph-3-A7, Ph-3-H7, Ph-2-B3, Ph-2-E05, and Ph-2-E10 all have good affinity.

[0271] 15. Identification of candidate antibody epitopes

[0272] The plate was coated with hSEZ6 (positive antibody) (1 μg / mL); primary antibody: SEZ6 candidate antibody (final concentration 10 μg / mL, serially diluted 3-fold for 7 spots), 50 μL / well, and Biotin-SEZ6-His antigen (final concentration 1 μg / mL), 50 μL / well. After co-incubation at 37°C for 30 min, both were added to the coated plate and incubated at 37°C for 30 min. Secondary antibody: HRP-Streptavidin (1:10000 dilution), used to detect whether the candidate antibody and positive antibody share the same epitope.

[0273] The results are shown in Tables 11 and 12.

[0274] Table 11

[0275]

[0276]

[0277] Table 12

[0278]

[0279] The results above show that the candidate antibodies and positive antibodies obtained through screening have different epitopes.

[0280] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations 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 its antigen-binding fragment, characterized in that, The nanobody or its antigen-binding fragment contains three complementarity-determining regions CDR1, CDR2 and CDR3; the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 5, SEQ ID NO: 11 and SEQ ID NO: 17, respectively.

2. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 1, characterized in that, It also includes four frame regions FR1, FR2, FR3, and FR4, which are alternately set with the three complementary determinant regions. The amino acid sequence of FR1 is one of the sequences shown in SEQ ID NO: 19 to SEQ ID NO: 24; The amino acid sequence of FR2 is the sequence shown in one of SEQ ID NO: 25 to SEQ ID NO: 30; The amino acid sequence of FR3 is shown in one of SEQ ID NO: 31 to SEQ ID NO: 36; The amino acid sequence of FR4 is shown in one of SEQ ID NO: 37 to SEQ ID NO:

39.

3. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 1, characterized in that, The nanobody or its antigen-binding fragment is selected from the following sequences: The amino acid sequence has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 44 and has the same function.

4. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 3, characterized in that, The nanobody or its antigen-binding fragment is selected from the following sequences: The amino acid sequence has at least 85% identity with the amino acid sequence shown in SEQ ID NO: 44 and has the same function.

5. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 4, characterized in that, The nanobody or its antigen-binding fragment is selected from the following sequences: The amino acid sequence has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 44 and has the same function.

6. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 5, characterized in that, The nanobody or its antigen-binding fragment is selected from the following sequences: The amino acid sequence has at least 91% identity with the amino acid sequence shown in SEQ ID NO: 44 and is functionally identical.

7. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 6, characterized in that, The nanobody or its antigen-binding fragment is selected from the following sequences: The amino acid sequence has at least 92% identity with the amino acid sequence shown in SEQ ID NO: 44 and has the same function.

8. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 7, characterized in that, The nanobody or its antigen-binding fragment is selected from the following sequences: The amino acid sequence has at least 93% identity with the amino acid sequence shown in SEQ ID NO: 44 and has the same function.

9. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 8, characterized in that, The nanobody or its antigen-binding fragment is selected from the following sequences: The amino acid sequence has at least 94% identity with the amino acid sequence shown in SEQ ID NO: 44 and is functionally identical.

10. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 9, characterized in that, The nanobody or its antigen-binding fragment is selected from the following sequences: The amino acid sequence has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 44 and has the same function.

11. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 10, characterized in that, The nanobody or its antigen-binding fragment is selected from the following sequences: The amino acid sequence has at least 96% identity with the amino acid sequence shown in SEQ ID NO: 44 and has the same function.

12. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 11, characterized in that, The nanobody or its antigen-binding fragment is selected from the following sequences: The amino acid sequence has at least 97% identity with the amino acid sequence shown in SEQ ID NO: 44 and is functionally identical.

13. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 12, characterized in that, The nanobody or its antigen-binding fragment is selected from the following sequences: The amino acid sequence has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 44 and is functionally identical.

14. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 13, characterized in that, The nanobody or its antigen-binding fragment is selected from the following sequences: The amino acid sequence has at least 99% identity with the amino acid sequence shown in SEQ ID NO: 44 and has the same function.

15. The anti-SEZ6 nanobody or its antigen-binding fragment according to claim 1, characterized in that, The nanobody or its antigen-binding fragment has the amino acid sequence shown in SEQ ID NO:

44.

16. A nucleic acid molecule, characterized in that, Its encoding is the anti-SEZ6 nanobody or its antigen-binding fragment as described in any one of claims 1-15.

17. A carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 16.

18. A host cell, characterized in that, It contains the vector of claim 17 or the nucleic acid molecule of claim 16.

19. The host cell according to claim 18, characterized in that, The host cell can be a bacterial cell, a fungal cell, or an animal cell.

20. A method for generating anti-SEZ6 nanobodies, characterized in that, Includes the following steps: (a) Under conditions suitable for the production of nanobodies, host cells as described in claim 18 or 19 are cultured to obtain a culture containing the anti-SEZ6 nanobodies; (b) Isolating and / or recovering the anti-SEZ6 nanobody from the culture; and optionally... (c) Purification and / or modification of the anti-SEZ6 nanobody obtained in step (b).

21. An antibody conjugate, characterized in that, It includes the anti-SEZ6 nanobody or its antigen-binding fragment and effector as described in any one of claims 1-15; the effector is selected from at least one of radionuclides, fluorescent groups, enzymes that catalyze substrate color development, chemiluminescent reagents and nanoparticle markers.

22. Use of the anti-SEZ6 nanobody or its antigen-binding fragment according to any one of claims 1-15 in the preparation of a reagent for detecting human SEZ6 expression levels.

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