An anti-SEZ6 nanobody or antigen-binding fragment and its application

Abstract

The present invention belongs to the field of biomedicine and relates to an anti-SEZ6 nanobody or antigen-binding fragment and its application. The anti-SEZ6 nanobody or antigen-binding fragment comprises three complementarity-determining regions CDR1, CDR2 and CDR3; the sequences of each region are shown in SEQ ID NO: 1 to SEQ ID NO: 18 respectively. Compared with conventional antibodies, the anti-SEZ6 nanobody of the present invention has high expression efficiency, simple purification, small molecular weight, high affinity, good specificity, high solubility and strong tolerance, and can be widely applied in 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 its application. Background Art

[0002] Immunotherapy based on specific targets in 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, as well as chimeric antigen receptor (CAR) T cells, etc. Numerous antibody drugs targeting different targets have now been approved for clinical use and achieved good results, and 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 biologic macromolecule drugs. As antibody drugs are continuously applied to more therapeutic fields, the therapeutic mechanism of antibody drugs has become more complex, and at the same time, the structure of antibody drugs has become more diverse. Therefore, new technologies are needed to solve 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 members of the SEZ6 family 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], and it is involved in the normal dendritic branching of cortical neurons. Under normal physiological conditions, SEZ6 is mainly expressed in the brain, such as in the cerebral cortex, cerebellum, basal ganglia, and hypothalamus. It is highly expressed in SCLC and other neuroendocrine tumors, while it is rarely expressed in most normal tissues.

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

[0005] SEZ6L (Seizure-related gene 6-like): SEZ6L is highly similar to the SEZ6 gene and is thus called "SEZ6-like". The protein encoded by the SEZ6L gene is expressed in the nervous system and is involved in the processes of synapse formation and nerve cell connection. There are some sequence similarities between SEZ6L and SEZ6, but there may be differences in some details. SEZ6L has a total of 1024 amino acids (AA).

[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 regions 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 research, including monoclonal antibody drugs and antibody-drug conjugates (ADCs), etc., but few antibodies have entered clinical trials. The preparation process of traditional monoclonal antibodies is relatively complex, with high production costs and large molecular weights, resulting in poor tissue penetration ability. Nanobodies are the smallest fragments known to date that can bind antigens, only 1 / 10 the size of monoclonal antibodies, and have structural stability and binding activity comparable to those of the original heavy-chain antibodies. Compared with traditional antibodies, nanobodies have many unique advantages, such as good tissue penetration ability, rapid clearance, easy production and modification, high stability, and low immunogenicity, and are a very promising new type of antibody molecule.

[0008] There have been no reports or clinical applications of nanobodies targeting the SEZ6 target, and there is an urgent need in this field to develop new specific nanobodies effectively targeting the SEZ6 target. Summary of the Invention

[0009] Definitions

[0010] Unless otherwise indicated or defined, all terms used herein have their ordinary meanings in the art, which will be understood by those skilled in the art. In addition, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed can and have been carried out in a manner known per se, which will be understood by those skilled in the art.

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

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

[0014] 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 having 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 having the same function;

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

[0016] The amino acid sequence of CDR3 is the sequence shown in any one of SEQ ID NO: 13 to SEQ ID NO: 18, or a sequence having 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 having the same function.

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

[0018] The positions of the CDRs in the antibody or nanobody sequences can be determined by those skilled in the art using existing techniques. Generally, the CDRs can be identified by sequencing the DNA of the antibody or nanobody, and then analyzing the resulting sequences using a dedicated database (such as the international ImMunoGeneTics database or IMGT).

[0019] In the sequences provided by the present invention, the CDRs are 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 the CDR sequence characteristics of any one of the following:

[0021] (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 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and having the same function;

[0022] (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 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and having the same function;

[0023] (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 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the above sequences and having the same function;

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

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

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

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

[0028] The amino acid sequence of FR2 is one of the sequences shown in SEQ ID NO: 25 to SEQ ID NO: 30;

[0029] The amino acid sequence of FR3 is one of the sequences shown in SEQ ID NO: 31 to SEQ ID NO: 36;

[0030] The amino acid sequence of FR4 is one of the sequences shown in 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 the FR sequence characteristics of any one of the following:

[0032] (a) The amino acid sequences of FR1, FR2, FR3, and FR4 are the sequences shown in 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 the sequences shown in 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 the sequences shown in 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 the sequences shown in 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 the sequences shown in 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 satisfying the above sequence characteristics. Specifically and preferably,

[0039] the nanobody or antigen-binding fragment contains one or more of the following sequences:

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

[0041] (ii) An amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in any one of SEQ ID NO: 40 to SEQ ID NO: 45 and having the same function;

[0042] (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 which retains the function of the amino acid sequence shown in any one of SEQ ID NO: 40 to SEQ ID NO: 45, that is, it can bind to the SEZ6 protein.

[0043] Ph-2-B3: QVKLEESGGGLVQAGGALNLSCVASGIIFSMYDMGWYRQGSGEARDIVAAIGKGGSTYYADAVKGRFTISRDNAQNTLYLQMNSLKPEDTSMYYCTTVEPYDYWGQGTQVTVSS (SEQ ID NO: 40)

[0044] Ph-3-A7: AVQLVDSGGGLVQPGGSLRLSCTASDSRFIANIMGWYRQAPGKERELVVAISSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTDMYYCAFFEGGIPDGWGQGTQVTVSS (SEQ ID NO: 41)

[0045] Ph-3-H7: AVQLVDSGGGLVQAGGSLRLSCAASGSIFSINTMGWYRQAPGKERELVAAISSHGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTTMYYCAASRDSDYDPGRGSWGQGTQVTVSS (SEQ ID NO: 42)

[0046] Ph-2-C03: AVQLVESGGGLVQAGGSLRLSCAASGSIFSIEAMGWYRQAPGEVRELVAAISSGNSTYYADNVKGRFTISRDNANNTVYLQMTSLKPEDTAMYYCAAFESSRPWALGKGALVTVSS (SEQ ID NO: 43)

[0047] Ph-2-E05: QVKLEESGGGLVQAGGSLRLSCTASGSIFSFDALGWYRQAPGSERELVAAVGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDVAMYYCAAFSPHSGIPRLWGQGTQVTVSS (SEQ ID NO: 44)

[0048] Ph-2-E10: QVKLEESGGGLVQAGGSLRLSCAASGSIFSIDTMGWYRSAPGEERRLVAAISTGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCAGFSSQSDPGVPNLLGQGTQVTVSS (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 above.

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

[0052] The host cell includes but is not limited to bacterial cells, fungal cells, animal cells, plant cells or descendant cells of these cells.

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

[0054] (1) Immunize alpacas with human, monkey, and mouse SEZ6 proteins to prepare a phage display library.

[0055] (2) Affinity screen the phage display library with human, monkey, and mouse SEZ6 proteins;

[0056] (3) Identify positive clones by ELISA and FACS;

[0057] (4) Express and purify the SEZ6 nanobody.

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

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

[0060] (b) Isolate and / or recover the anti-SEZ6 nanobody from the culture; and optionally

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

[0062] The sixth aspect of the present invention provides an antibody-drug conjugate, comprising the above-mentioned anti-SEZ6 nanobody or antigen-binding fragment and an effector; preferably, the effector includes at least one of radionuclides, cytotoxic agents, fluorescent groups, enzymes that catalyze substrate chromogenesis, chemiluminescent reagents, and nanoparticle-based markers.

[0063] The seventh aspect of the present invention provides a pharmaceutical composition containing 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 human SEZ6-related diseases; the reagent is preferably a kit for detecting SEZ6-related diseases;

[0066] (ii) Application in the preparation of a drug 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, used alone as a cancer treatment drug, or in combination with other anti-cancer drugs. Therefore, the active ingredient of the drug includes the anti-SEZ6 nanobody and optionally other anti-cancer drugs.

[0069] Compared with conventional antibodies, the anti-SEZ6 nanobody of the present invention has high expression efficiency, simple purification, small molecular weight (about 15 kDa), high affinity, good specificity, high solubility, and strong tolerance, and can be widely used in the fields of scientific research, diagnosis, and treatment. In addition, nanobodies are more likely to recognize antigens that cannot be captured by traditional antibodies, 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 specific implementation manner section. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] By describing the exemplary embodiments of the present invention in more detail in conjunction with the accompanying drawings, the above and other objects, features, and advantages of the present invention will become more apparent.

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

[0073] Figure 2a -b respectively show the amplification processes of the VHH fragments of the yeast display libraries of alpaca 252# and alpaca 297-231#.

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

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

[0076] Figure 5a -b shows the results of two-round fluorescence-activated cell sorting (FACS) of the yeast display libraries of alpacas #252 and #297-231, respectively.

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

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

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

[0080] Figure 9 Shows the results of FACS detection of the binding of recombinant antibodies to the target protein.

[0081] Figure 10a -b shows the FACS EC of candidate antibodies with overexpressing cells at 37°C 50 detection results.

[0082] Figure 11a -b shows the FACS EC of candidate antibodies with overexpressing cells at 4°C 50 detection results.

[0083] Figure 12 Shows the detection results of candidate antibody endocytosis.

[0084] Figure 13 shows the detection results of candidate antibody affinity. Detailed implementation manners

[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 by 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) (Mouse SEZ6) [Accession|Q7TSK2]), codon optimization was carried out 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 by nickel column. The amino acid sequence of the constructed Cynomolgus SEZ6 protein is shown as SEQ ID NO: 46, and the amino acid sequence of the constructed Mouse SEZ6 protein is shown as SEQ ID NO: 47. Human SEZ6-His protein was purchased from Kaikai Biotech Co., Ltd. (Shanghai).

[0089] The purity of the recombinant protein was detected by SDS-PAGE, and the results are as Figure 1 shown, where M: Protein marker, Lane1: Human SEZ6-His, Lane 2: Cyno SEZ6-His, Lane 3: Mouse SEZ6-His, Lane 4: pcDNA3.4-hSEZ6_VL-hIgKc + pcDNA3.4-hSEZ6_VL-hIgG1 (positive antibody). The results showed that: the purity of Human SEZ6-His protein > 95%, which can be used for immunization and subsequent panning. The purity of Cyno SEZ6-His and Mouse SEZ6-His proteins > 90%, which can be used for the verification of cross-binding.

[0090] 2. Detection of ELISA Binding Activity between Recombinant Protein Antigen and Positive Antibody

[0091] 1) Dilute the SEZ6-His recombinant protein with sterile CBS to a final concentration of 1 μg / mL. Take a new 96-well plate and 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; after discarding the blocking buffer, wash the well plate 3 times with PBST;

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

[0093] 3) Discard the liquid in the wells and wash 3 times with PBST;

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

[0095] 5) After discarding the liquid in the wells, wash the plate 3 times with PBST;

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

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

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

[0099] 9) Read the OD 450 value in the wells using an ELISA reader.

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

[0101] Table 1

[0102]

[0103] The results show that the proteins of the three species all have good binding activity with the positive control antibody and can be used for subsequent experiments.

[0104] 3. Alpaca Immunization

[0105] The purchased Human SEZ6-His recombinant protein and the above-prepared Mouse SEZ6-His recombinant protein were used to immunize alpacas with numbers 297-231# and 252# respectively. The immunization interval was 14 days. Starting from the second immunization, peripheral blood was collected 7 days after each immunization to monitor the titer of immune serum. The immunization schedule is shown in Table 2 below:

[0106] Table 2

[0107]

[0108] 4. Detection of Immune Titer

[0109] The steps for detecting the immunization titer include:

[0110] 1) After immunization, collect 5 mL of peripheral blood, place the centrifuge tube containing the blood sample in an incubator at 37 °C for 1 hour; then transfer the blood sample to 4 °C overnight;

[0111] 2) Place the centrifuge tube containing the blood sample in a centrifuge and centrifuge at 5000 rpm for 20 min; separate the upper serum, transfer the serum to a new sterile centrifuge tube, and collect the immune serum;

[0112] 3) Dilute the target recombinant protein with sterile CBS (carbonate buffer solution) to a final concentration of 1 μg / mL; take a new 96-well ELISA plate and add 100 μL / well for coating overnight at 4 °C;

[0113] 4) Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20);

[0114] 5) Add 200 μL / well of 3% MPBS and block at 37 °C for 2 hours;

[0115] 6) After removing the blocking buffer, wash the plate 5 times with PBST;

[0116] 7) Add 100 μL of serially diluted serum (100 μL / well), incubate at room temperature for 1 hour, and the control well is PBS;

[0117] 8) Remove the liquid in the well and wash 5 times with PBST;

[0118] 9) Add 100 μL of HRP anti-Llama IgG (H+L) antibody (diluted 1:50000) and incubate at room temperature for 1 hour;

[0119] 10) After removing the liquid in the well, wash the plate 5 times with PBST;

[0120] 11) Add 100 μL / well of TMB chromogenic solution;

[0121] 12) Incubate at room temperature in the dark for 10 - 15 minutes;

[0122] 13) Add 50 μL / well of stop solution;

[0123] 14) Use an ELISA reader to read the OD 450 value.

[0124] The results of the immune titer detection are shown in Tables 3 and 4 below. The samples were tested in parallel in 2 groups:

[0125] Table 3

[0126]

[0127] Table 4

[0128]

[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, and the alpaca titer is significantly improved to meet the requirements for blood collection and library construction. 100 mL 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 isolate PBMC cells, RNA was extracted, and a reverse transcription kit was used to prepare a cDNA library. Single-domain antibody amplification primers were used for the first round of PCR, obtaining PCR bands of approximately 1000 bp and 750 bp, respectively. The 750 bp fragment was recovered from the gel 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 a VHH fragment. The VHH fragment was mixed with a linearized yeast display vector and electroporated into yeast cells to obtain a yeast library. The constructed 252# alpaca yeast library has a storage capacity of 2.63×10 9 The yeast library of 297-231# alpaca has a storage capacity of 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 PBMCs using lymphocyte separation medium.

[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°C for 5 minutes and then cool rapidly on ice;

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

[0140]

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

[0142] 3) Amplification of VHH fragment

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

[0144]

[0145] After preparing the PCR reaction system, set the PCR instrument according to the following program:

[0146]

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

[0148] Use 1% agarose for electrophoresis analysis of PCR products to separate fragments with a molecular weight of about 750 bp. Use a gel extraction kit to recover the PCR products and measure the concentration with NanoDrop.

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

[0150]

[0151]

[0152] After preparing the PCR reaction system, set the PCR instrument according to the following program:

[0153]

[0154] 3 - 4) Agarose gel electrophoresis analysis of the second - round PCR products

[0155] Use 1% agarose for electrophoresis analysis of PCR products to separate VHH fragments with a molecular weight of about 400 bp; use a gel extraction kit to recover the VHH PCR products and measure the concentration with NanoDrop.

[0156] 3 - 5) Precipitation of PCR products

[0157] Aliquot 200 μL of the recycled second-round PCR product into each 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3 M sodium acetate and 1 μg / μL glycogen, pipette and mix well, add 880 μL of absolute ethanol, invert to mix, and store at -80 °C.

[0158] 5.2 Linearize the yeast display vector pYDisplay. The digestion system is as follows:

[0159]

[0160] 1) Digest the pYDisplay vector with SfiI, aliquot 100 μL per tube, and digest overnight at 50 °C;

[0161] 2) Separate the pYDisplay vector fragments using 1% agarose gel, excise the 5000 bp vector fragment for gel extraction, and measure the concentration with NanoDrop;

[0162] 3) Aliquot 200 μL of the recycled pYDisplay digestion product into each 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3 M sodium acetate and 1 μg / μL glycogen, pipette and mix well, add 880 μL of absolute ethanol, invert to mix, and store at -80 °C.

[0163] 5.3 Construct the yeast display library by electroporation

[0164] 1) Streak the yeast competent cells stored at -80 °C onto a YPD solid medium plate and activate at 30 °C for 3 - 5 days;

[0165] 2) Inoculate a single colony of yeast competent cells into 50 mL of YPD medium and shake culture at 250 rpm and 30 °C for 1 - 2 days;

[0166] 3) Prepare yeast competent cells: Mix the linearized vector fragment and the PCR product, add them to the electroporation cuvette, and perform electroporation; Transfer the electroporated yeast competent cells to a culture flask, shake culture at 220 rpm and 30 °C for 1 h;

[0167] 4) Take 20 μL of the resuspension, dilute it 5000 - fold with SDCAA, pipette 100 μL, spread it on an SDCAA plate, culture for 2 - 3 days, calculate the library capacity, and continue to culture the remaining bacterial solution for 24 h;

[0168] 5) Preserve the bacteria: Collect the remaining bacterial solution into a 50 mL centrifuge tube, centrifuge at 3000 × g for 5 min, discard the supernatant, add 10 mL of SDCAA to resuspend, mix with 50% glycerol:resuspension = 1:1, and store at -80 °C.

[0169] Results: The yeast display library QC of 252 alpacas and 297 - 231 alpacas was as follows Figure 3a and 3b shown as follows: Single colonies were randomly picked for sequencing analysis. Sequences with overlapping peaks and stop codons were removed. There were no empty vector sequences or repeated sequences, and the sequence differences were large, indicating good library diversity.

[0170] 6. Magnetic Sorting of Yeast Display Library

[0171] 1) Yeast cultured in SDCAA was added to a 250 mL shake flask containing 50 mL of SGCAA medium and cultured on a shaker at 30 °C and 240 rpm for 16 h.

[0172] 2) After centrifugation, the supernatant was discarded, and the pellet was resuspended in 1 mL of 0.5% PBSA and transferred to a 1.5 mL centrifuge tube. It was centrifuged at 3000 × g for 5 min, and the supernatant was discarded. It was washed once more with 0.5% PBSA.

[0173] 3) The streptavidin magnetic beads incubated with the antigen were washed twice with 0.5% PBSA (rotary incubation at 4 °C for 5 minutes each time), placed on a magnetic stand for 5 min, and the supernatant was discarded.

[0174] 4) The yeast cell suspension was added to the magnetic beads conjugated with the antigen, and incubated with rotation at 4 °C for 60 minutes, then placed on a magnetic stand for 15 min.

[0175] 5) The yeast cell suspension was discarded, leaving the magnetic beads, which were washed three times with 0.5% PBSA (rotary incubation at 4 °C for 5 minutes each time).

[0176] 6) The magnetic beads were resuspended in 1 mL of SDCAA medium. 0.5 - 5 μL of the resuspended solution was pipetted into 100 μL of SDCAA medium for plating. The resuspended solution was divided into two equal parts. One part was added with 500 μL of 50% glycerol (stored at -80 °C); the other part was added to a shake flask, supplemented with 2 mL of SDCAA medium, and cultured at 30 °C and 240 rpm for 16 h.

[0177] 7) The bacterial suspension in the shake flask was transferred to 50 mL of SDCAA medium (250 mL shake flask) and cultured overnight at 30 °C and 240 rpm.

[0178] 8) Measure the OD 600 value of the bacterial suspension. According to the OD 600 value, a portion of the bacterial suspension was centrifuged, resuspended in SGCAA, and transferred to 50 mL of SGCAA medium to make the final OD 600 value 1, and cultured overnight at 30 °C and 240 rpm. The remaining bacterial suspension was resuspended with SDCAA:50% glycerol = 1:1 and stored at -80 °C.

[0179] Figure 4a and4b The magnetic sorting results of the yeast display libraries of alpacas #252 and #297 - 231 are shown respectively.

[0180] Figure 4a Among them, 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 by magnetic separation, the positive proportion of Human SEZ6 was 4.88%, and the positive proportion of Mouse SEZ6 was 1.44%. Two rounds of fluorescence - activated cell sorting were arranged.

[0181] Figure 4b Among them, 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 by magnetic separation, the positive proportion of Human SEZ6 was 1.18%, and the positive proportion of Mouse SEZ6 was 1.88%. Two rounds of fluorescence - activated cell sorting were arranged.

[0182] 7. Two-Round Flow Sorting of Yeast Display Library

[0183] 1) Take 1 mL of the yeast solution cultured in SGCAA into a 1.5 mL centrifuge tube, centrifuge to remove the supernatant, wash twice with 1 mL of PBS, resuspend with 1 mL of PBS, take 100 μL of the bacterial solution into a new 1.5 mL centrifuge tube (as NC), centrifuge, and remove the supernatant.

[0184] 2) Dilute the Biotin - antigen protein to a concentration of 10 μg / mL with 100 μL of PBS, resuspend the bacterial cells in the experimental group, 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 of PBS.

[0185] 3) Dilute APC - Streptavidin at a ratio of 1:1000 and resuspend the bacterial cells in the experimental group, incubate with rotation at 4°C for 60 min, centrifuge at 3000×g for 3 min, discard the supernatant, wash twice with 1 mL of PBS, and resuspend with 1 mL of PBS.

[0186] 4) Resuspend the bacterial cells and add them to a flow cytometry sorting tube for flow cytometry sorting.

[0187] 5) Prepare 1 mL of SDCAA medium in a 15 mL centrifuge tube as the collection tube.

[0188] 6) Perform flow cytometry sorting on the machine. Use the NC group to set the gates and sort for APC-positive yeast.

[0189] Figure 5a and 5b respectively show the results of the second-round flow cytometry sorting of the yeast display libraries of alpaca #252 and alpaca #297-231.

[0190] Figure 5a In, A: NC group; B: 1 MACS: Primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC. Sort P3, a total of 19,006 yeast cells were obtained, arrange for plating, pick yeast monoclonal colonies, amplify and induce them, and then perform yeast monoclonal flow cytometry detection.

[0191] Figure 5b In, 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. Sort P3, 45,046 cells were obtained using the protein of Human SEZ6 for sorting, and 19,602 cells were obtained using the protein of Mouse SEZ6 for sorting. Arrange for plating, pick yeast monoclonal colonies, amplify and induce them, and then perform yeast monoclonal flow cytometry detection.

[0192] 8. Flow Detection of Yeast Monoclonal

[0193] After sorting, spread the yeast cell suspension on SDCAA plates, pick monoclonal colonies for culture, after inducing expression for 48 h, incubate with Biotin-antigen, use PE-Streptavidin as the secondary antibody, and perform flow cytometry detection after incubation. Resuspend the yeast clones that bind to the target antigen with 0.2% SDS, incubate at 95 °C for 10 min to lyse, centrifuge to take the supernatant of the bacterial liquid, take 0.5 μL as a template for PCR amplification and send for testing (the remaining bacterial liquid is stored at -20 °C).

[0194] Randomly pick monoclonal cells from the enriched yeast display library, amplify and induce them, and then detect them with antigens respectively to determine the binding of monoclonal yeast cell surface-displayed single-domain antibodies to the target antigen. Results Figure 6a -d, Figure 7a-c as shown. Figure 6a , 6b In 6c and 6d, left figure: primary antibody: Biotin-Human-SEZ6-His, secondary antibody: APC-streptavidin; right figure: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin. Figure 7a In it, left figure: primary antibody: Biotin-Human-SEZ6-His, secondary antibody: APC-streptavidin; right figure: A1: NC, C5: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC, F4: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC. Figure 7b In it, left figure: primary antibody: Biotin-Human-SEZ6-His, secondary antibody: APC-streptavidin; right figure: A1: NC, A8 A10 A12 C10 D4 E3 F9: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC. Figure 7c In it, left figure: primary antibody: Biotin-Human-SEZ6-His, secondary antibody: APC-streptavidin; right figure: A1: NC, B10, D3: primary antibody: Biotin-Mouse-SEZ6-His, secondary antibody: APC-streptavidin, V5-FITC.

[0195] According to the experimental results, positive clones with cross-binding of Human SEZ6 and Mouse SEZ6 proteins were selected for PCR and sent for testing, and the obtained differential sequences were arranged for vector construction.

[0196] 9. Construction of Antibody Eukaryotic Expression Vector

[0197] The positive yeast clones were 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 to the eukaryotic expression vector pcDNA3.4-human IgG1Fc to construct an antibody expression vector. After verification by Sanger sequencing, plasmid extraction was performed.

[0198] 10. Expression and Purification of Candidate Single-Domain Antibody

[0199] 1) Take out the LVTransm transfection reagent and the pcDNA3.4-human IgG1Fc antibody expression vector from the refrigerator. After thawing at room temperature, pipette up and down to mix well completely. Take out the PBS buffer and warm it to room temperature. Add 2 mL of PBS to one well of a 6-well plate, add 20 μg of the antibody eukaryotic expression vector respectively, pipette up and down to mix well, then add 60 μL of LVTransm, immediately pipette up and down to mix well, and let it stand at room temperature for 10 minutes.

[0200] 2) Add the above DNA / LVTransm complex to 20 mL of 293F cells, and gently shake to mix well. Place the cells in an incubator at 37 °C, 5% CO2, and 130 rpm for continued culture.

[0201] 3) After continuous culture for 5 - 7 days, centrifuge to collect the culture medium supernatant, filter it through a 0.45 μm filter membrane, transfer the filtrate to a sterile centrifuge tube, and purify the antibody using a Protein A column.

[0202] 11. Detection of Binding between Recombinant Antibody and Target Protein by ELISA

[0203] 1) Use sterile CBS to dilute the recombinant protein to a final concentration of 1 μg / mL. Take a new 96-well enzyme-linked immunosorbent assay (ELISA) plate, add 100 μL / well and coat overnight at 4 °C.

[0204] 2) Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20).

[0205] 3) Add 200 μL / well of 3% MPBS and block at 37 °C for 2 hours;

[0206] 4) After removing the blocking buffer, wash the plate 5 times with PBST;

[0207] 5) Add the expressed recombinant antibody, transfection supernatant 50 μL / well or purified antibody (starting concentration 10 μg / mL, 7 points diluted in 3-fold gradient, 100 μL / well), incubate at room temperature for 1 hour, and the control well is PBS;

[0208] 6) Remove the liquid in the well and wash 5 times with PBST;

[0209] 7) Add 100 μL / well of HRP-Protein A antibody (diluted 1:50000) and incubate at room temperature for 1 hour;

[0210] 8) After removing the liquid in the well, wash the plate 5 times with PBST;

[0211] 9) Add 100 μL / well of TMB chromogenic solution;

[0212] 10) Incubate in the dark at room temperature for 10 - 15 minutes;

[0213] 11) Add 50 μL / well of stop solution;

[0214] 12) Use an ELISA reader to read the OD 450 value in the wells.

[0215] The detection results of the first batch of candidate antibodies are shown in Tables 5 - 7.

[0216] Table 5

[0217]

[0218] Coated with Human-SEZ6-His (2 μg / mL) to detect the binding of candidate antibodies to Human-SEZ6-His. According to the detection results, only the candidate antibody 297 - 231 - 2 - A10 weakly binds to the target protein.

[0219] Table 6

[0220]

[0221] Coated with Mouse-SEZ6-His (2 μg / mL) to detect the binding of candidate antibodies to Mouse-SEZ6-His. According to the detection results, only the candidate antibody 297 - 231 - 2 - A10 weakly binds to the target protein.

[0222] Table 7

[0223]

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

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

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

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

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

[0229] 12. Detection of Binding between Recombinant Antibody and Target Protein by FACS

[0230] 1) Resuscitate the 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 aliquots, with 3×10 5 cells per aliquot;

[0232] 3) Incubate the candidate antibodies (starting concentration 30 μg / mL, 11 points diluted in 3-fold gradient, 100 μL / well) or 100 μL of transfection supernatant with the target cells. After thorough mixing, incubate at room temperature for 1 hour;

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

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

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

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

[0237] The results are as Figure 9 shown, Figure 9 The candidate antibodies marked in red (252-1-B03, Ph-2-B3, Ph-3-A7, Ph-3-H7, Ph-1-A03, Ph-2-C02, Ph-2-C03, Ph-2-E05, Ph-2-E10) specifically bind to the overexpressing cell line 293T-SEZ6. Arrange for FACS EC 50 testing of these antibodies.

[0238] The FACS EC of the candidate antibodies with overexpressing cells at 37°C 50 The detection results are as shown in Figure 10a , Figure 10b , and Table 8: Among the 5 candidate antibodies, namely Ph-2-B3, Ph-3-H7, Ph-2-C03, Ph-2-E05, and Ph-2-E10, the binding to cells is relatively strong; among them, the binding of Ph-2-C03 and Ph-2-E05 is comparable to that of the positive antibody, and the binding of the Ph-2-E10 antibody is stronger than that of the positive antibody.

[0239] Table 8

[0240]

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

[0242] The FACS EC of the candidate antibodies with overexpressing cells at 4°C 50 The detection results are as shown in Figure 11a , Figure 11b , and Table 9:

[0243] Table 9

[0244]

[0245]

[0246] According to the detection results, among the 5 candidate antibodies, namely Ph-2-B3, Ph-3-H7, Ph-2-C03, Ph-2-E05, and Ph-2-E10, the binding to cells is relatively strong; among them, the binding of Ph-2-C03 and Ph-2-E05 is comparable to that of the positive antibody, and the binding of the Ph-2-E10 antibody is stronger than that of the positive antibody.

[0247] 13. Detection of Candidate Antibody Endocytosis

[0248] 1) Prepare target cells 293T-human SEZ6, with 5×10 5 cells per well;

[0249] 2) Add 10 μg / mL, 100 μL / well of single-domain antibody, and incubate at 4°C for 30 min;

[0250] 3) Centrifuge at 800×g for 3 min at 4°C, discard the supernatant containing the antibody, and wash the cells 3 times with pre-cooled 2% FBS.

[0251] 4) Divide the cells into two groups evenly and incubate them at 4°C and 37°C for 240 min respectively. After the incubation, immediately add ice-cold 2% FBS to terminate the endocytosis experiment, centrifuge at 4°C, 800×g for 3 min, and wash the cells three times with pre-cooled 2% FBS.

[0252] 5) Immediately add MonoRab 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 (diluted 1:1000), and incubate at 4°C for 30 min.

[0253] 6) Centrifuge at 4°C, 800×g for 3 min, discard the supernatant containing the antibody, and wash the cells three times with pre-cooled 2% FBS.

[0254] 7) Resuspend the cells with 200 μL of pre-cooled 2% FBS, prepare the corresponding number of 1.5 mL EP tubes, add 300 μL of pre-cooled 2% FBS to each EP tube, transfer the corresponding samples to the EP tubes respectively, place them on ice, and perform flow cytometry analysis.

[0255] 8) Calculation of the internalization level of cell surface-bound antibodies:

[0256] MFI% at tx time point = MFI of the sample incubated at 37°C / MFI of the control sample incubated at 4°C

[0257] Internalization percentage at tx time point = 100% - MFI% at tx time point

[0258] The results are as Figure 12 shown in

[0259] Table 10

[0260]

[0261] According to the experimental data, except that Ph-1-F11 and 252-1-B03 hardly bind to the cell line; the other antibodies all have endocytosis effects, and Ph-3-A7, Ph-3-H7, Ph-1-A03, Ph-2-C02, Ph-2-C03, Ph-2-E05, and Ph-2-E10 have good endocytosis effects, among which the endocytosis effect of Ph-2-E10 is comparable to that of the positive antibody.

[0262] 14. Detection of Candidate Antibody Affinity

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

[0264] 2) The buffer was PBST (PBS + 0.02% tween20). The candidate antibodies were diluted to 5, 2.5, 1.25, 0.625, 0.3125, 0 nM.

[0265] 3) Affinity detection: equilibration for 60 s, binding for 180 s, dissociation for 180 s, and the detection temperature was 25 °C.

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

[0267] The detection 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. Epitope Identification of Candidate Antibody

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

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

Claims

1. An anti-SEZ6 nanobody, 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 SEQ ID NO: 6; the amino acid sequence of CDR2 is the sequence shown in SEQ ID NO: 12; the amino acid sequence of CDR3 is the sequence shown in SEQ ID NO:

18.

2. The anti-SEZ6 nanobody according to claim 1, characterized in that, It also contains four framework regions FR1, FR2, FR3, and FR4 alternately arranged with the three complementarity-determining regions, among which, 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 one of the sequences shown in SEQ ID NO: 25 to SEQ ID NO: 30; the amino acid sequence of FR3 is one of the sequences shown in SEQ ID NO: 31 to SEQ ID NO: 36; the amino acid sequence of FR4 is one of the sequences shown in SEQ ID NO: 37 to SEQ ID NO:

39.

3. The anti-SEZ6 nanobody according to claim 1, wherein The nanobody contains one of the following sequences: (i) the amino acid sequence shown in SEQ ID NO: 45; (ii) an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 45 and having the same function.

4. The anti-SEZ6 nanobody according to claim 3, characterized in that, The nanobody contains one of the following sequences: an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 45 and having the same function.

5. The anti-SEZ6 nanobody according to claim 4, characterized in that, The nanobody contains one of the following sequences: an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 45 and having the same function.

6. The anti-SEZ6 nanobody according to claim 5, characterized in that, The nanobody contains one of the following sequences: an amino acid sequence having at least 91% identity with the amino acid sequence shown in SEQ ID NO: 45 and having the same function.

7. The anti-SEZ6 nanobody according to claim 6, characterized in that, The nanobody contains one of the following sequences: an amino acid sequence having at least 92% identity with the amino acid sequence shown in SEQ ID NO: 45 and having the same function.

8. The anti-SEZ6 nanobody according to claim 7, characterized in that, The nanobody contains one of the following sequences: an amino acid sequence having at least 93% identity with the amino acid sequence shown in SEQ ID NO: 45 and having the same function.

9. The anti-SEZ6 nanobody according to claim 8, wherein, The nanobody contains one of the following sequences: an amino acid sequence having at least 94% identity with the amino acid sequence shown in SEQ ID NO: 45 and having the same function.

10. The anti-SEZ6 nanobody according to claim 9, characterized in that, The nanobody contains one of the following sequences: an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO: 45 and having the same function.

11. The anti-SEZ6 nanobody according to claim 10, characterized in that, The nanobody contains one of the following sequences: an amino acid sequence having at least 96% identity with the amino acid sequence shown in SEQ ID NO: 45 and having the same function.

12. The anti-SEZ6 nanobody according to claim 11, wherein The nanobody contains one of the following sequences: an amino acid sequence having at least 97% identity with the amino acid sequence shown in SEQ ID NO: 45 and having the same function.

13. The anti-SEZ6 nanobody according to claim 12, characterized in that, The nanobody contains one of the following sequences: An amino acid sequence having at least 98% identity to the amino acid sequence shown in SEQ ID NO: 45 and having the same function.

14. The anti-SEZ6 nanobody according to claim 13, wherein, The nanobody comprises one of the following sequences: An amino acid sequence having at least 99% identity to the amino acid sequence shown in SEQ ID NO: 45 and having the same function.

15. A nucleic acid molecule, characterized in that, It encodes the anti-SEZ6 nanobody according to any one of claims 1-14.

16. A carrier, characterized in that, It comprises the nucleic acid molecule according to claim 15.

17. A host cell, characterized in that, It contains the vector according to claim 16 or the nucleic acid molecule according to claim 15, and the host cell is a non-plant cell.

18. The host cell according to claim 17, wherein, The host cell is a bacterial cell, a fungal cell or an animal cell.

19. A method for generating anti-SEZ6 nanobodies, characterized in that, Comprising the following steps: (a) Culturing the host cell according to claim 17 or 18 under conditions suitable for producing the nanobody, thereby obtaining a culture containing the anti-SEZ6 nanobody; (b) Isolating and / or recovering the anti-SEZ6 nanobody from the culture; and optionally (c) Purifying and / or modifying the anti-SEZ6 nanobody obtained in step (b).

20. An antibody-drug conjugate, characterized in that, Comprising the anti-SEZ6 nanobody according to any one of claims 1-14 and an effector; the effector comprises at least one of a radionuclide, a fluorophore, an enzyme that catalyzes substrate color development, a chemiluminescent reagent, and a nanoparticle-based label.

21. A pharmaceutical composition, characterized in that, Containing the anti-SEZ6 nanobody according to any one of claims 1-14, or the antibody-drug conjugate according to claim 20.

22. The following uses of the anti-SEZ6 nanobody according to any one of claims 1-14: (i) Use in the preparation of a reagent for detecting human SEZ6-related diseases; (ii) Application in the preparation of a drug for treating SEZ6-related diseases; The SEZ6-related disease is a neuroendocrine tumor.

23. The use according to claim 22, characterized in that, The SEZ6-related disease is small cell lung cancer.

24. The use according to claim 22, wherein The reagent is a kit for detecting SEZ6-related diseases.

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