Nanobodies against ny-eso-1 and uses thereof

Abstract

This invention discloses an anti-NY-ESO-1 nanobody and its applications, relating to the field of immunology. The anti-NY-ESO-1 nanobody provided by this invention exhibits specific recognition and binding ability to the NY-ESO-1 antigen. The nanobody has a minimum affinity constant of 68.1 nM, demonstrating highly specific binding activity, and is expected to be used for the treatment of NY-ESO-1-expressing tumors or for the diagnostic detection of NY-ESO-1 protein.

Description

Technical Field

[0001] This invention relates to the field of immunology, and in particular to an anti-NY-ESO-1 nanobody and its application. Background Technology

[0002] NY-ESO-1 is a member of the tumor-testis antigen family. It was first discovered by Chen et al. using SEREX technology to screen cDNA libraries from the serum of esophageal squamous cell carcinoma patients, and is a tumor-shared antigen that can induce humoral immunity. NY-ESO-1 has high expression levels in neuroblastoma (82%), synovial sarcoma (80%), melanoma (46%), esophageal cancer (32%), and lung cancer (13%), and its expression level increases with disease progression. In rectal cancer and lymphoma, its expression is very low or even absent. However, its expression in normal tissues is limited to germ cells and placental cells. In principle, it belongs to tumor-associated antigens. However, germ cells and placental cells do not express HLA class I molecules and cannot present antigenic peptides to the cell surface, so they cannot be recognized by specific T cells. Therefore, the complex formed by the peptides produced by the degradation of NY-ESO-1 and HLA molecules can be considered a tumor-specific antigen. Studies have found that NY-ESO-1 can simultaneously induce humoral and cellular immunity. Serum IgG positivity is positively correlated with the production of CD8+ T cells against this antigen, making it the most immunogenic tumor-specific antigen discovered to date (Raza A, Merhi M, Inchakalody VP, et al. Unleashing the immune response to NY-ESO-1 cancer testis antigen as a potential target for cancer immunotherapy. J Transl Med, 2020, 18(1): 140.). Because the off-target toxicity of NY-ESO-1-targeted therapy is very limited, it is expected to become a candidate target with extraordinary potential in tumor immunotherapy. Currently, research on this target mainly focuses on tumor vaccines, TCR-like antibodies, and adoptive T-cell therapy (TCR-T). There are no nanobody drugs targeting this target under development.

[0003] A unique type of antibody found in the serum of camels, lacking a light chain, is called a heavy chain antibody (HCAb). Heavy chain antibodies consist only of the heavy chain variable region and the heavy chain constant regions CH2 and CH3. The heavy chain variable region is called VHH. With a relative molecular weight of 15 kDa and molecular dimensions of 4 nm * 2.5 nm * 3 nm, this type of antibody was named nanobodies (Nbs) by Ablynx. Nanobodies naturally evolve to have only three complementarity-determining regions (CDRs), significantly fewer than conventional antibodies. However, the average length of the CDR3 loop of nanobodies is longer than that of the VH in conventional antibodies, which can expand the contact area with the target antigen to a certain extent (Peter B, Julia H, Friedrich K N. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Front Immunol, 2017, 8: 1603.). At the same time, the CDR3 loop of the VHH can naturally ring to form a finger-like protrusion that can be inserted into the cavity of the antigen epitope. This allows nanobodies to bind to uncommon or unique epitopes that cannot be reached by conventional monoclonal antibodies (Stijlemans B, Caljon G, Natesan S, et al. High Affinity Nanobodies against the Trypanosome brucei VSG Are Potent Trypanolytic Agents that Block Endocytosis. Plos Pathogens, 2011, 7(6).), such as enzyme active sites. Due to their small molecular size, nanobodies possess highly efficient tissue distribution capabilities, making them more effective in treating dense solid tumors and showing great promise for clinical applications. Furthermore, nanobodies offer advantages such as water solubility, high stability, and low production costs, enabling their widespread use in disease diagnosis and treatment. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention aims to provide an anti-NY-ESO-1 nanobody and its application, wherein the nanobody has a strong affinity and specificity for NY-ESO-1.

[0005] In a first aspect, the present invention provides a nanobody against NY-ESO-1, wherein the VHH chain of the nanobody includes three complementarity-determining regions CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is any one of SEQ ID NO.1, SEQ ID NO.6, SEQ ID NO.11, and SEQ ID NO.16; the amino acid sequence of CDR2 is any one of SEQ ID NO.2, SEQ ID NO.7, SEQ ID NO.12, and SEQ ID NO.17; and the amino acid sequence of CDR3 is any one of SEQ ID NO.3, SEQ ID NO.8, SEQ ID NO.13, and SEQ ID NO.18.

[0006] Preferably, the VHH chain of the nanobody includes three complementary determinant regions CDR1, CDR2 and CDR3 of any one of the following: (1) the amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3; (2) the amino acid sequence of CDR1 is shown in SEQ ID NO.6, the amino acid sequence of CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 is shown in SEQ ID NO.8; (3) the amino acid sequence of CDR1 is shown in SEQ ID NO.11, the amino acid sequence of CDR2 is shown in SEQ ID NO.12, and the amino acid sequence of CDR3 is shown in SEQ ID NO.13; (4) the amino acid sequence of CDR1 is shown in SEQ ID NO.16, the amino acid sequence of CDR2 is shown in SEQ ID NO.17, and the amino acid sequence of CDR3 is shown in SEQ ID NO.18. The amino acid sequence of the VHH chain is any one of SEQ ID NO.4, SEQ ID NO.9, SEQ ID NO.14, and SEQ ID NO.19.

[0007] In another preferred embodiment, CDR1 has an amino acid sequence that is at least 80% identical to SEQ ID NO.1, SEQ ID NO.6, SEQ ID NO.11, and SEQ ID NO.16; and / or CDR2 has an amino acid sequence that is at least 80% identical to SEQ ID NO.2, SEQ ID NO.7, SEQ ID NO.12, and SEQ ID NO.17; and / or CDR3 has an amino acid sequence that is at least 80% identical to SEQ ID NO.3, SEQ ID NO.8, SEQ ID NO.13, and SEQ ID NO.18.

[0008] In a second aspect, the present invention provides a multivalent nanobody complex comprising at least two nanobody molecules, wherein the nanobody is the aforementioned nanobody. In a third aspect, the present invention provides a nucleic acid molecule encoding the nanobody. The nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID NO. 5, SEQ ID NO. 10, SEQ ID NO. 15, and SEQ ID NO. 20. In a fourth aspect, the present invention provides an expression vector containing the aforementioned nucleic acid molecule. The expression vector is pET21a. In a fifth aspect, the present invention provides a host cell containing the aforementioned expression vector, or having the aforementioned nucleic acid molecule integrated into its genome. The host cell is *Escherichia coli* BL21. In a sixth aspect, the present invention provides an immunoconjugate comprising: (a) the aforementioned nanobody; and (b) a conjugation moiety selected from at least one of the following: a detectable marker, a drug, a toxin, a cytokine, a therapeutic agent, a PK-modified moiety, or an enzyme.

[0009] Preferably, the therapeutic agent is a CAR; more preferably, the therapeutic agent is an anti-CD3 antibody.

[0010] In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g., bivalent) anti-NY-ESO-1 nanobody as described in the first aspect of the invention, and a multivalent nanobody complex as described in the second aspect of the invention. The term multivalent means that the amino acid sequence of the immunoconjugate contains a plurality of repeating anti-NY-ESO-1 nanobodies as described in the first aspect of the invention and multivalent nanobody complexes as described in the second aspect of the invention.

[0011] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the nanobody, the nucleic acid molecule, or the immunoconjugate, and a pharmaceutically acceptable carrier. In an eighth aspect, the present invention provides applications of the nanobody, the nucleic acid molecule, the host cell, or the immunoconjugate, wherein the application is any of the following: (1) in the preparation of a product for detecting NY-ESO-1; (2) in the preparation of a product that binds to NY-ESO-1; (3) in the preparation of a product for diagnosing or treating tumors expressing NY-ESO-1. The applications are non-diagnostic and non-therapeutic. The products are pharmaceuticals, reagents, detection plates, or kits, etc. The NY-ESO-1 specific nanobody of the present invention can be used to treat any NY-ESO-1-related disease presenting the NY-ESO-1 antigen short peptide SLLMWITQC-HLAA0201 complex. Including but not limited to tumors, preferably, the tumors include but are not limited to: neuroblastoma, sarcoma, melanoma, prostate cancer, bladder cancer, breast cancer, multiple myeloma, hepatocellular carcinoma, oral squamous cell carcinoma, esophageal cancer, gastric cancer, lung cancer, head and neck squamous cell carcinoma, colon cancer, or ovarian cancer, etc.

[0012] The beneficial effects of this invention are:

[0013] The anti-NY-ESO-1 nanobody provided by this invention has a specific recognition and binding ability to the NY-ESO-1 antigen. The nanobody has a minimum affinity constant of 68.1 nM, which shows that the nanobody provided by this invention has highly specific binding activity and is expected to be used for the treatment of NY-ESO-1 expressing tumors or for the diagnostic detection of NY-ESO-1 protein. Attached Figure Description

[0014] Figure 1 SDS-PAGE gel image of the purified inclusion bodies; lane 2 is HLA-A2 and lane 3 is β2m.

[0015] Figure 2 This is an SDS-PAGE gel image of the SLLMWITQC-HLA A0201 complex obtained by molecular sieve purification; lane 1 is the polymer, lane 2 is β2m, and lanes 3-8 are the SLLMWITQC-HLA A0201 complex.

[0016] Figure 3 The image shows the ELISA identification results of the SLLMWITQC-HLA A0201 complex.

[0017] Figure 4 The expression rate and positive rate of nanoantibodies after magnetic sorting.

[0018] Figure 5The change in the proportion of double-positive cells in four-round cell sorting.

[0019] Figure 6 , Figure 7 , Figure 8 , Figure 9 The binding ability of VHH-1, VHH-4, VHH-5, and VHH-9 monoclonal antibodies to positive and negative antigens was compared.

[0020] Figure 10 BIAcore kinetics of the binding of VHH-1, VHH-4, VHH-5, VHH-9 to the SLLMWITQC-HLA A0201 complex.

[0021] Figure 11 SDS-PAGE gel images of dimers and tetramers.

[0022] Figure 12 This compares the binding ability of the dimer to positive and negative antigens.

[0023] Figure 13 This compares the binding ability of the tetramer to positive and negative antigens. Detailed Implementation

[0024] the term

[0025] Nanobodies, also known as single-domain antibodies, are naturally occurring antibodies lacking the light chain, found in alpaca peripheral blood. These antibodies contain only a single heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. The VHH structure, cloned and expressed independently, possesses structural stability and antigen-binding activity comparable to the original heavy chain antibody. It is the smallest known unit capable of binding to large target antigens, hence the name nanobody (Nb).

[0026] MHC molecules are proteins of the immunoglobulin superfamily, and can be either class I or class II MHC molecules. Therefore, they are specific for antigen presentation; different individuals have different MHCs, capable of presenting different short peptides of a protein antigen to the surface of their respective APC cells. In humans, MHCs are commonly referred to as HLA genes or the HLA complex.

[0027] An anti-CD3 antibody is an antibody that specifically binds to a single CD3 chain (e.g., a CD3(γ) chain, a CD3(δ) chain, or a CD3(ε) chain) or a complex formed by two or more single CD3 chains (e.g., a complex of one or more CD3(ε) chains, a complex of CD3(γ) and CD3(ε) chains, or a complex of CD3(δ) and CD3(ε) chains). In some embodiments, the anti-CD3 antibody specifically binds to CD3(γ), CD3(δ), or CD3(ε), or any combination thereof; more preferably, it specifically binds to CD3(ε). Human-derived CD3 is referred to as hCD3; therefore, "antibody against human CD3" and "anti-hCD3 antibody" refer to antibodies that specifically bind to human-derived CD3.

[0028] The "Fc" of an antibody refers to the portion of the antibody composed of the second (CH2) and third (CH3) constant regions of the first heavy chain bonded to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc portion of the antibody is responsible for various effector functions, such as ADCC and CDC, but does not participate in antigen binding. A tumor is a disease characterized by pathological proliferation of cells or tissues, and its subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, neither inducing nor inhibiting the proliferation of normal cells. The NY-ESO-1 specific nanobody of the present invention can be used to treat any NY-ESO-1 related disease that presents the NY-ESO-1 antigen short peptide complex SLLMWITQC-HLAA0201. This includes, but is not limited to, tumors, preferably including neuroblastoma, sarcoma, melanoma, prostate cancer, bladder cancer, breast cancer, multiple myeloma, hepatocellular carcinoma, oral squamous cell carcinoma, esophageal cancer, as well as gastric cancer, lung cancer, head and neck squamous cell carcinoma, colon cancer, ovarian cancer, etc.

[0029] Example 1: Preparation of the SLLMWITQC-HLA A0201 complex

[0030] 1.1 Purification of Inclusion Bodies

[0031] Collect bacterial culture induced to express HLA-A2 and β2m, centrifuge at 4000 rpm for 30 min, discard the supernatant, resuspend the precipitate in 40 mL re-suspension buffer (25% Sucrose, 2 M Tris pH 8.0, 1 mM EDTA, 1 mM PMSF, 1 mM DTT), add to 30 mL centrifuge tube, and store at -80 °C.

[0032] After removing the bacterial culture from the -80℃ freezer, allow it to thaw at room temperature. At 4℃, add 400 μL of 1 MM MgCl2, 400 μL of 10 mg / mL DNase, and 400 μL of 100 mM DTT sequentially. Stir for 10 min, then disrupt the cell walls using a cell disruptor. Centrifuge at 20000×g for 20 min at 4℃. Resuspend the precipitate in 40 mL of detergent buffer (0.1% NP-40, 0.2 M NaCl, 20 mM Tris pH 7.5, 2 mM EDTA) and wash. Then wash twice each with 40 mL of wash buffer-I (0.5% Triton X-100, 50 mM Tris pH 8.0, 100 mM NaCl) and wash buffer-II (100 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA). The precipitate was resuspended in 1 mL of distilled water and then 4 mL of denaturing buffer (10 M Urea, 62.5 mM MES pH 6.5, 0.125 mM EDTA, 0.125 mM DTT) was added. The centrifuge tube was inverted for 30 min using a rotary mixer to completely denature and dissolve the protein. After centrifugation using the same method, the protein supernatant was taken to determine the concentration and corrected using the extinction coefficient. The actual concentration (mg / mL) = reading (mg / mL) / ε280. (A2: ε280 = 2.28, β2m: ε280 = 1.56). The SDS-PAGE results are shown below. Figure 1 As shown, the HLA-A2 and β2m protein bands are in line with the expected molecular weight and have good purity.

[0033] 1.2 pMHC renaturation

[0034] 1.54g of glutathione (reduced) and 0.31g of glutathione (oxidized), 5mL of 100mM PMSF, were dissolved in 1L of folding buffer (400mM L-arginine, 100mM Tris, pH 8.0, 2mM EDTA). NY-ESO-1 157-165 The (SLLMWITQC) peptide was dissolved in DMF to a concentration of 20 mg / mL, and 0.5 mL was added to the reaction solution. β2m was prepared into a 5 mL solution using folding buffer, and this solution was added to the reaction system in three portions. Next, HLA-A2 was prepared into a 5 mL solution using injection buffer (3M guanidine HCl, 10mM sodium acetate, 10mM EDTA), and this solution was added to the reaction system in three portions. The renaturation process was carried out at 4°C for 2 days.

[0035] 1.3 Biotinylation

[0036] Centrifuge the refolded solution at 20,000 × g for 10 min at 4 °C, and filter the supernatant through a 0.22 μm microporous membrane. Concentrate the liquid using a Millipore 30 kD ultrafiltration membrane at 4 °C and 4500 rpm, replacing the supernatant with 1 × PBS to ensure the PBS content is above 80%, until the final volume is approximately 5 mL. Add 5 mM MgCl2, 5 mM ATP, 100 μM Biotin, and 10 μg / mL BirA to the reaction system in that order, and incubate at 30 °C for 1 h. Centrifuge at 20,000 × g for 5 min at 4 °C, aspirate the supernatant, and continue ultrafiltration using a 30 kD ultrafiltration membrane to a final volume of 600 μL.

[0037] 1.4 Molecular sieve purification of refolded products

[0038] Using the AKTApure 25L1 protein purification system, 600 μL of biotinylated pMHC molecules were loaded onto a Superdex 75increase 10 / 300GL column (GE Healthcare Life Sciences) and then eluted with PBS at a flow rate of 0.75 mL / min. Biotinylated pMHC molecules appeared after approximately 10 mL. The collected fraction was identified by SDS-PAGE, and the results are as follows. Figure 2 As shown, the pMHC bands obtained by refolding are in line with the expected molecular weight and have good purity.

[0039] 1.5pMHC ELISA identification

[0040] Dilute streptavidin to 1 μg / mL with coating buffer, add 200 μL to each well, and incubate overnight at 4°C. After coating, discard the liquid in the plate, rinse 4 times with water, add 200 μL of blocking buffer to each well, and incubate at 37°C for 1.5 h. Discard the liquid in the plate, add 100 μL of sample or denatured sample to each well, and incubate at 37°C for 1.5 h. Rinse 4 times with water, add 100 μL of W6 / 32 antibody to each well, and incubate at 37°C for 1.5 h. Rinse 6 times with water, add 100 μL of HRP-labeled goat anti-mouse IgG antibody to each well, and incubate at 37°C for 1 h. Rinse 9 times with water, add 100 μL of chromogenic solution to each well in the dark, incubate at 37°C for 30 min, and stop the reaction by adding 100 μL of 2M H2SO4 to each well. Read the plate at 450 nm using a microplate reader. ELISA results are shown below. Figure 3 The undenatured sample showed significant binding to W6 / 32 compared to the blank and denatured samples, indicating that the pMHC conformation obtained after refolding was correct.

[0041] Example 2: Screening of Nanobodies

[0042] Construction of yeast display library for nanobody:

[0043] A DNA library of nanobody was constructed using a two-step overlap extension PCR method. A set of ten primers, P1_for, P2_rev...P10_rev, was dissolved at 100 μM and mixed in equimolar ratios to prepare three mixed libraries, each containing 10 μM of primers. The three mixed libraries, "short mix," "medium mix," and "long mix," differed in the P9 primer, using P9a_for, P9b_for, or P9c_for to introduce CDR3 regions of varying lengths of 7, 11, or 15 random residues, respectively. Subsequently, 1 μL of each mixed library, serially diluted 5-fold at a concentration of 10 μM, was used to prepare 50 μL of overlap extension PCR reaction using a high-fidelity polymerase. The full-length nanobody DNA product from each library was mixed with the short / medium / long CDR3 regions in a 1:2:1 molar ratio, representing the length distribution frequency observed in the camel VHH domain. These synthetic nanobodies were simply validated in E. coli by amplifying the resulting mixture using primers pET26b_NbLib_GA_for and pET26b_NbLib_GA_rev and cloning it into pET26b.

[0044] Nanobody DNA libraries were sequentially amplified using primers pYDSFor1-pYDSRev1, pYDSFor2-pYDSRev2, and pYDSFor3-pYDSRev2 for yeast transformation. When 500 mL of BJ5465 yeast reached OD600 = 1.5, the yeast was electroporated with 245 μg of nanobody insert DNA and 50 μg of pYDS649 plasmid, followed by digestion with NheI-HF and BamHI-HF. The transformed yeast dilutions were then plated as single colonies on tryptophan-free selective medium to estimate library diversity. The primers used are shown in Table 1.

[0045] Table 1 Primer sequence list

[0046]

[0047] 2.1 Nanobody Yeast Display Library Induced Expression

[0048] The yeast display library of nanobodies was removed from -80℃ and thawed in a 30℃ water bath. It was centrifuged at 2000×g for 5 min at 4℃, the supernatant was discarded, and the cell pellet was resuspended in a small amount of growth medium. The resuspended yeast was added to 1 L of growth medium and incubated overnight at 220 rpm in a shaker at 30℃ until the yeast library concentration reached OD600 = 2-3. It was then centrifuged at 2000×g for 5 min at 4℃, the supernatant was discarded, the cell pellet was resuspended in induction medium, the OD600 was diluted to 1, and it was incubated at 220 rpm at 25℃ for 18-24 h. Finally, the initial expression level of the nanobodies (ER1) was assessed using flow cytometry.

[0049] 2.2 First round of magnetic sorting

[0050] Wash the magnetic beads twice with 1 mL of selection buffer, discard the supernatant, and resuspend the magnetic beads (Invitrogen, 11205D) in 400 μL of selection buffer. Centrifuge the bacterial culture (2000 OD 600) at 2000 × g for 5 min at 4 °C, discard the supernatant, resuspend in 20 mL of selection buffer, centrifuge again, discard the supernatant, and resuspend in 10 mL of selection buffer. Add 200 μL of magnetic beads to the resuspended yeast, mix well by pipetting, and incubate at 4 °C with a rotary mixer for 30 min. Add the mixture to 1.5 mL centrifuge tubes (1 mL per tube), place on a magnetic rack, and wait 2-3 min. Aspirate the unbound yeast cells into 30 mL centrifuge tubes. Wash the magnetic beads with selection buffer and combine with the washed cells in the same centrifuge tube. Centrifuge at 2000 × g for 5 min at 4 °C, discard the supernatant, and resuspend the yeast in 5 mL of selection buffer.

[0051] Add NY-ESO-1 antigen to resuspend yeast at a final concentration of 200 nM and incubate at 4°C for 1 hour using a rotary mixer. Centrifuge at 2000 × g for 5 minutes at 4°C, aspirate the supernatant, resuspend the yeast in 10 mL of selection buffer, centrifuge again, and resuspend the yeast in 5 mL of selection buffer. Add 200 μL of magnetic beads to the resuspended yeast, mix well, and incubate at 4°C for 30 minutes using a rotary mixer. Transfer the mixture to centrifuge tubes, place them on a magnetic rack, and wait 2-3 minutes. Aspirate any unbound yeast and wash the magnetic beads with 1 mL / tube of selection buffer, repeating twice. Resuspend the magnetic beads in growth medium and transfer to a 50 mL Erlenmeyer flask, adding growth medium until the total volume reaches 15 mL. Incubate overnight at 30°C and 220 rpm until OD600 = 2-3. Centrifuge the yeast at 2000 × g for 5 minutes at 4°C until OD600 reaches 50%. Discard the culture medium, resuspend the yeast in 20 mL of induction medium, centrifuge again, remove the supernatant, resuspend the yeast in induction medium, dilute OD600 to 1, and incubate overnight at 25°C and 220 rpm. Flow cytometry was used to assess the expression level (ER2) and positive rate of nanobody in the first round of magnetically sorted yeast cells. Results are shown below. Figure 4 The initial HA expression rate of the yeast library was 17.97%, but after one round of magnetic sorting, cells expressing nanobodies did not accumulate, and the proportion of cells binding to the NY-ESO-1 antigen was 0.29%. Although the results were not as expected, magnetic sorting can rapidly reduce the library size by 2-3 orders of magnitude.

[0052] 2.3 Second round of magnetic sorting

[0053] Change the yeast culture volume to 100 OD600, the magnetic bead volume to 80 μL, and the antigen concentration to 100 nM. For other operations, refer to 2.2.

[0054] 2.4 First-round cell sorting

[0055] The expression levels and positivity rates of nanobodies in the second round of magnetic yeast were determined by flow cytometry using APC-labeled anti-HA antibody (Invitrogen, 26183-A647) and FITC-labeled Streptavidin protein (Abcam, ab136201). APC and FITC-positive cells were isolated and transferred to flow cytometry tubes containing 1.5 mL of growth medium. The sorted yeast cells were spread onto solid growth medium and incubated at 30°C for 1-2 days. After a large number of colonies had grown, the yeast cells on the solid medium were washed off with growth medium, centrifuged at 2000×g for 5 min at 4°C, the supernatant was removed, the yeast was resuspended in 5 mL of induction medium, centrifuged again, and the OD600 was diluted to 1 with induction medium. The mixture was then incubated overnight at 25°C with shaking at 220 rpm.

[0056] 2.5 Second Round of Flow Cytometry Cell Sorting and Detection

[0057] The antigen was changed to TP53-R273H, and the antigen concentration, grouping, and operation were the same as in 2.4.

[0058] 2.6 Third Round of Flow Cytometry Cell Sorting and Detection

[0059] The antigen was changed to NY-ESO-1, and the antigen concentration was 50 nM. The grouping and operation were the same as in 2.4.

[0060] 2.7 Fourth Round of Flow Cytometry Cell Sorting and Detection

[0061] The antigen, concentration, grouping, and operating procedures are the same as in 2.6.

[0062] The results of the four-round cell sorting are shown below Figure 5 After four rounds of screening, the expression rate of nanobody increased from 12.7% to 74.2%, and the proportion of double-positive cells increased to 32.5%, showing a significant enrichment effect.

[0063] Example 3: Identification and Analysis of Specific Yeast Clones

[0064] 3.1 Yeast single-clone sequence analysis

[0065] Take 5 μL of the yeast from the fourth round of sorting in Example 2, which has been cultured overnight, and add it to 1 mL of growth medium. Mix thoroughly, and then spread 50 μL onto a solid growth medium. Incubate for 1-2 days. Randomly select 10 single clones and add them to 1 mL of growth medium. Incubate in a shaker at 30°C and 180 rpm for 24 h. Send the 10 yeast single clones to Qingke Biotechnology for sequencing. Sequencing yielded four different nanobodies, as shown in Table 2.

[0066] Table 2

[0067]

[0068]

[0069] 3.2 Detection of yeast monoclonal antigen binding specificity

[0070] Four monoclonal strains were added to 1 mL of growth medium and incubated on a shaker at 30°C and 180 rpm for 24 h. After centrifugation at 2000 × g for 5 min at 4°C, the supernatant was discarded, and the yeast was resuspended in 2 mL of induction medium and induced at 25°C and 180 rpm for 24 h. Flow cytometry was used for detection, and staining with APC-labeled anti-HA antibody and FITC-labeled streptavidin protein was performed. The results are shown below. Figure 6-9 The four monoclonal antibodies VHH-1, VHH-4, VHH-5, and VHH-9 showed strong binding affinity to the positive antigen NY-ESO-1, but almost no binding affinity to other negative antigens, indicating that the four selected monoclonal antibodies have strong binding specificity to the NY-ESO-1 antigen.

[0071] Example 4: SPR determination of nanobody affinity

[0072] The carboxyl groups on the chip surface were activated with 0.4M EDC and 0.1M NHS, followed by coupling with streptavidin protein and blocking with 1M ethanolamine. The SLLMWITQC-HLA A0201 complex protein flowed through the activated CM5 chip, immobilizing the ligands on the chip surface. A gradient concentration of nanobodies was sequentially introduced, and signal changes were observed. The BIAcore kinetics of the binding of VHH-1, VHH-4, VHH-5, and VHH-9 to the SLLMWITQC-HLA A0201 complex are shown below. Figure 10 Affinity data are shown in Table 3.

[0073] Table 3. Affinity data for VHH-1, VHH-4, VHH-5, and VHH-9

[0074] name Affinity value VHH-1 96.6 nM VHH-4 102nM VHH-5 68.1 nM VHH-9 1170nM

[0075] Example 5: Specificity Validation of VHH-5 Nanobody

[0076] a) Expression of dimer and tetramer proteins

[0077] Constructing the dimer expression plasmid: VHH-GGGGS-Fc, and the tetramer expression plasmid:

[0078] VHH-(GGGGS)3-CH1-Fc and VHH-(GGGGS)3-CL were transfected into HEK293F cells for 3-4 days. After centrifugation at 4000×g for 20 min, the cell supernatant was collected, filtered through a 0.22μm filter membrane, and purified using an AKTA protein purifier and a HiTrap proteinA affinity column. The purification steps are as follows: a. Turn on the AKTA protein purifier and the connected control computer. After the instrument is connected to the computer, set the pressure parameters (high pressure 0.25MPa); b. Place pump heads A and B in pure water filtered through a 0.45μm filter membrane, set the flow rate (3mL / min) and the rinsing ratio of pumps A and B (50% B). After the pure water reaches conductivity equilibrium (approximately 60mL), connect the HiTrap protein A affinity column (5mL) to the AKTA purifier and continue rinsing with pure water for at least three column volumes; c. Change the rinsing ratio of pumps A and B to 0% B, and replace pump A with 50mM Tris-HCl (pH 7.4) loading buffer. After conductivity equilibrium, replace pump A with the supernatant of the culture medium to be purified. After loading, replace pump A with loading buffer until conductivity equilibrium is reached; then change the ratio of pumps A and B to 100% B, and replace pump B with 1mol / L sodium acetate (pH 7.4). 3.0) The target protein was obtained by elution in elution buffer. The AKTA purification system was rinsed with pure water to conductivity equilibrium and the entire system was stored in 20% ethanol (v / v). Tetramer and dimer proteins were ultrafiltered using a 50 kDa ultrafiltration membrane. After replacing the solvent with PBS buffer, the protein concentration was determined using a Nanodrop ND-1000, and the proteins were aliquoted and stored at -80°C for later use. The purity of the proteins was analyzed by SDS-PAGE, and the results are as follows: Figure 11 As shown, the dimer and tetramer protein bands are in line with the expected molecular weight and have good purity.

[0079] b) VHH-5 nanobody specific affinity assay

[0080] Collect T2 cells in the logarithmic growth phase, centrifuge at 1000 rpm for 5 min, discard the supernatant, wash once with PBS, and resuspend in serum-free IMDM medium. Repeat at 5 × 10⁻⁶ cells / day. 5Seed cells per well in a 12-well plate, 1 mL / well. Add peptide to a final concentration of 25 μg / mL. Mix well and incubate at 37°C, 5% CO2, and saturated humidity for 2 h. After incubation, centrifuge at 500g for 5 min to collect cells, wash once with PBS, add 200 μL of dimer or tetramer protein (100 nM), and incubate at 4°C for 30 min. Wash twice with PBS, add 200 μL of 1:500 diluted goat anti-human IgG antibody (Beyotime, A0556), and incubate at 4°C for 30 min. Wash twice with PBS, resuspend cells in 300 μL of PBS, and use ACEANovoCyte. TM Flow cytometry was used to detect FITC levels, and the results are as follows: Figure 12 and Figure 13 As shown, the dimer and tetramer proteins bind strongly to the positive antigen NY-ESO-1, but hardly bind to other negative antigens, indicating that the VHH-5 nanobody has strong binding specificity to NY-ESO-1.

Claims

1. A nanobody against NY-ESO-1, characterized in that, The VHH chain of the nanobody includes three complementarity-determining regions CDR1, CDR2, and CDR3 of any of the following: (1) The amino acid sequence of CDR1 is shown in SEQ ID NO.11, the amino acid sequence of CDR2 is shown in SEQ ID NO.12, and the amino acid sequence of CDR3 is shown in SEQ ID NO.13; (2) The amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3; (3) The amino acid sequence of CDR1 is shown in SEQ ID NO.6, the amino acid sequence of CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 is shown in SEQ ID NO.8; (4) The amino acid sequence of CDR1 is shown in SEQ ID NO.16, the amino acid sequence of CDR2 is shown in SEQ ID NO.17, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

18.

2. The nanobody according to claim 1, characterized in that, The amino acid sequence of the VHH chain is any one of SEQ ID NO. 14, SEQ ID NO. 4, SEQ ID NO. 9, and SEQ ID NO.

19.

3. A nucleic acid molecule encoding the nanobody of claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in any one of SEQ ID NO. 15, SEQ ID NO. 5, SEQ ID NO. 10, and SEQ ID NO.

20.

5. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 3.

6. A host cell, characterized in that, The host cell contains the expression vector of claim 5, or the nucleic acid molecule of claim 4 is integrated into its genome.

7. A pharmaceutical composition, characterized in that, The composition contains the nanobody of claim 1 or 2 or the nucleic acid molecule of claim 3, and a pharmaceutically acceptable carrier.

8. The application of the nanobody according to claim 1 or 2, the nucleic acid molecule according to claim 3, or the host cell according to claim 6, characterized in that, The application is in the preparation of reagents for the detection of NY-ESO-1.

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