Anti-ny-ESO-1 nanobody and use thereof

Abstract

An anti-NY-ESO-1 nanobody and a use thereof, relating to the field of immunology. The anti-NY-ESO-1 nanobody has specific recognition and binding capabilities for the NY-ESO-1 antigen. The nanobody has a minimum affinity constant of 68.1 nM, which shows that the nanobody has highly specific binding activity. It is expected that the nanobody will be used in the treatment of tumors expressing NY-ESO-1, or in diagnostic detection of an NY-ESO-1 protein.

Description

A nanobody against NY-ESO-1 and its application Technical Field

[0001] The present invention relates to the field of immunology, and in particular to an anti-NY-ESO-1 nanobody and applications thereof. Background Art

[0002] NY-ESO-1, a member of the tumor-testis antigen family, was first discovered by Chen et al. using the SEREX technique to immunoscreen a cDNA library from serum samples collected from patients with esophageal squamous cell carcinoma. NY-ESO-1 is highly expressed in neuroblastoma (82%), synovial sarcoma (80%), melanoma (46%), esophageal cancer (32%), and lung cancer (13%), with expression increasing with disease progression. It is expressed at very low or even absent levels in colorectal cancer and lymphoma. However, expression in normal tissues is restricted to germ cells and placental cells, making it a tumor-associated antigen. However, germ cells and placental cells do not express HLA class I molecules, preventing them from presenting antigenic peptides on the cell surface and thus eluding specific T cells. Therefore, the complexes formed by the peptides produced by NY-ESO-1 degradation and HLA molecules can be considered tumor-specific antigens. Studies have found that NY-ESO-1 can induce both humoral and cellular immunity. Positive IgG in serum is positively correlated with the production of CD8+ T cells against the antigen. It is 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, the research on this target mainly includes tumor vaccines, TCR-like antibodies, and adoptive T cell therapy TCR-T. There are no nano-antibody drugs targeting this target under development.

[0003] A unique type of antibody found in the serum of camelids lacking a light chain is called a heavy chain antibody (HCAb). Heavy chain antibodies consist solely 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 15kD and a molecular size of 4nm*2.5nm*3nm. Therefore, this type of antibody was named nanobodies (Nbs) by Ablynx. Nanobodies naturally evolve to have only three complementary determining regions (CDRs), which are significantly fewer than traditional antibodies. However, the CDR3 loop of nanobodies is on average longer than the VH of traditional 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 VHH can naturally form a finger-like protrusion that can be inserted into the cavity of the antigen epitope, which allows nanobodies to bind to uncommon or unique epitopes that conventional monoclonal antibodies cannot reach (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 efficient tissue distribution, making them more easily accessible to dense solid tumors for therapeutic effects, and thus hold great promise for clinical application. Furthermore, nanobodies possess advantages such as water solubility, strong stability, and low production costs, making them widely used in the diagnosis and treatment of diseases. Summary of the Invention

[0004] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide an anti-NY-ESO-1 nanobody and its application, wherein the nanobody has strong affinity and specificity for NY-ESO-1.

[0005] In the first aspect, the present invention provides an anti-NY-ESO-1 nanobody, the VHH chain of the nanobody comprising three complementary determining regions CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 being 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 the CDR2 being 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 the CDR3 being 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 comprises three complementary determining 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 example, the CDR1 has an amino acid sequence with at least 80% sequence identity with SEQ ID NO.1, SEQ ID NO.6, SEQ ID NO.11, and SEQ ID NO.16; and / or the CDR2 has an amino acid sequence with at least 80% sequence identity with SEQ ID NO.2, SEQ ID NO.7, SEQ ID NO.12, and SEQ ID NO.17; and / or the CDR3 has an amino acid sequence with at least 80% sequence identity with SEQ ID NO.3, SEQ ID NO.8, SEQ ID NO.13, and SEQ ID NO.18.

[0008] In the second aspect, the present invention provides a multivalent nanobody complex comprising at least two nanobody molecules, wherein the nanobody is the nanobody described above. In the 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 the fourth aspect, the present invention provides an expression vector comprising the nucleic acid molecule. The expression vector is pET21a. In the fifth aspect, the present invention provides a host cell comprising the expression vector or having the nucleic acid molecule integrated into its genome. The host cell is Escherichia coli BL21. In the sixth aspect, the present invention provides an immunoconjugate comprising: (a) the nanobody; and (b) at least one coupling portion selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a therapeutic agent, a PK modifying portion, or an enzyme.

[0009] Preferably, the therapeutic agent is 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 present invention, or a multivalent Nanobody complex as described in the second aspect of the present invention. The multivalency means that the amino acid sequence of the immunoconjugate contains multiple repeats of the anti-NY-ESO-1 Nanobody as described in the first aspect of the present invention, or a multivalent Nanobody complex as described in the second aspect of the present 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 uses of the Nanobody, the nucleic acid molecule, the host cell, or the immunoconjugate, wherein the uses are 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 that express NY-ESO-1. The uses are non-diagnostic and non-therapeutic. The products are pharmaceutical agents, reagents, test plates, or kits. The NY-ESO-1-specific Nanobodies of the present invention can be used to treat any NY-ESO-1-related disease that presents 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] Beneficial effects of the present invention:

[0013] The anti-NY-ESO-1 nanobody provided by the present invention has specific recognition and binding ability to the NY-ESO-1 antigen. The minimum affinity constant of the nanobody is 68.1 nM, indicating that the nanobody provided by the present invention has highly specific binding activity and is expected to be used for the treatment of tumors expressing NY-ESO-1 or for the diagnostic detection of NY-ESO-1 protein. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0015] FIG2 is an SDS-PAGE gel image of the SLLMWITQC-HLA A0201 complex obtained by molecular sieve purification; wherein lane 1 is the polymer, lane 2 is β2m, and lanes 3-8 are the SLLMWITQC-HLAA0201 complex.

[0016] FIG3 is a diagram showing the results of ELISA identification of the SLLMWITQC-HLAA0201 complex.

[0017] Figure 4 shows the expression rate and positive rate of nanoantibodies after magnetic separation.

[0018] Figure 5 shows the changes in the proportion of double-positive cells after four rounds of flow cytometry sorting.

[0019] Figures 6, 7, 8, and 9 are comparisons of the binding abilities of VHH-1, VHH-4, VHH-5, and VHH-9 monoclonals to positive and negative antigens, respectively.

[0020] Figure 10 is a BIAcore kinetic profile of the binding of VHH-1, VHH-4, VHH-5, and VHH-9 to the SLLMWITQC-HLA A0201 complex.

[0021] FIG11 is an SDS-PAGE gel image of the dimer and tetramer.

[0022] FIG12 is a comparison of the binding ability of the dimer to positive antigens and negative antigens.

[0023] FIG13 is a comparison of the binding ability of tetramers to positive antigens and negative antigens. DETAILED DESCRIPTION

[0024] the term

[0025] Nanobodies, also known as single-domain antibodies, are naturally light-chain-deficient antibodies found in the peripheral blood of alpacas. They contain only a single heavy-chain variable region (VHH) and two conventional CH2 and CH3 domains. The individually cloned and expressed VHH structure possesses comparable structural stability and antigen-binding activity to the original heavy-chain antibody, making it the smallest known unit capable of binding to a target large antigen, hence the name Nanobody (Nb).

[0026] MHC molecules are proteins of the immunoglobulin superfamily and can be either class I or class II. Therefore, they are specific for antigen presentation. Different individuals have different MHCs, capable of presenting different short peptides from a protein antigen on their APC cell surfaces. The human MHC is often referred to as the HLA gene or HLA complex.

[0027] An anti-CD3 antibody refers to an antibody that specifically binds to a single CD3 chain (e.g., the CD3(γ) chain, the CD3(δ) chain, or the 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 a CD3(γ) chain and a CD3(ε) chain, or a complex of a CD3(δ) chain and a CD3(ε) chain). In certain embodiments, the anti-CD3 antibody specifically binds to CD3(γ), CD3(δ), or CD3(ε), or any combination thereof, more preferably, to CD3(ε). Human CD3 is denoted as hCD3; therefore, "anti-human CD3 antibodies" and "anti-hCD3 antibodies" refer to antibodies that specifically bind to human CD3.

[0028] The "Fc" of an antibody refers to the portion of the antibody consisting of the second (CH2) and third (CH3) constant regions of the first heavy chain bound to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc portion of an antibody is responsible for a variety of 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 their subsequent migration or invasion of other tissues or organs. Tumor growth is typically uncontrolled and progressive, and does not induce or inhibit the proliferation of normal cells. The NY-ESO-1-specific Nanobodies of the present invention can be used to treat any NY-ESO-1-related disease that presents the NY-ESO-1 antigen short peptide SLLMWITQC-HLAA0201 complex. These include, but are 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, and the like.

[0029] Example 1 Preparation of SLLMWITQC-HLA A0201 Complex

[0030] 1.1 Inclusion body purification

[0031] The bacterial suspension induced to express HLA-A2 and β2m was collected and centrifuged at 4000 rpm for 30 min. The supernatant was discarded and the precipitate was resuspended in 40 mL re-suspension buffer (25% Sucrose, 2 M Tris pH 8.0, 1 mM EDTA, 1 mM PMSF, 1 mM DTT) and added to a 30 mL centrifuge tube and frozen at -80°C.

[0032] After removing the bacterial suspension from the -80°C freezer, allow it to thaw at room temperature. Add 400 μL of 1M MgCl2, 400 μL of 10mg / mL DNAse, and 400 μL of 100mM DTT at 4°C, stir for 10 minutes, and then disrupt the cells using a cell disruptor. Centrifuge at 20,000×g for 20 minutes at 4°C. Resuspend the pellet 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 twice. Wash twice 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 pellet 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 tube was inverted on a rotary mixer for 30 minutes to completely denature and dissolve the protein. Following centrifugation using the same method, the supernatant was collected for concentration measurement and corrected using the extinction coefficient: actual concentration (mg / mL) = reading (mg / mL) / ε280 (A2: ε280 = 2.28, β2m: ε280 = 1.56). The SDS-PAGE results are shown in Figure 1. The HLA-A2 and β2m protein bands met the expected molecular weights and were of good purity.

[0033] 1.2 pMHC renaturation

[0034] 1.54g glutathione (reduced form) and 0.31g glutathione (oxidized form) and 5mL 100mM PMSF were dissolved in 1L folding buffer (400mM L-arginine, 100mM Tris pH 8.0, 2mM EDTA). 157-165 Dissolve the (SLLMWITQC) peptide in DMF to 20 mg / mL, and add 0.5 mL to the reaction mixture. Prepare a 5 mL solution of B2m in folding buffer and add this solution to the reaction system in three equal portions. Next, prepare a 5 mL solution of HLA-A2 in injection buffer (3M guanidine HCl, 10 mM sodium acetate, 10 mM EDTA) and add this solution to the reaction system in three equal portions. The renaturation process was carried out at 4°C for 2 days.

[0035] 1.3 Biotinylation

[0036] The refolding solution was centrifuged at 20,000 × g for 10 min at 4°C, and the supernatant was filtered through a 0.22 μm microporous filter. The solution was concentrated using a Millipore 30 kD ultrafiltration membrane at 4500 rpm at 4°C and replaced with 1× PBS to a final volume of approximately 5 mL. 5 mM MgCl2, 5 mM ATP, 100 μM Biotin, and 10 μg / mL BirA were added to the reaction system in this order and incubated in a 30°C water bath for 1 h. The solution was centrifuged at 20,000 × g for 5 min at 4°C, the supernatant was aspirated, and ultrafiltration was continued using a 30 kD ultrafiltration membrane to a final volume of 600 μL.

[0037] 1.4 Molecular sieve purification of renatured products

[0038] Using an AKTA pure 25 L1 protein purification system, 600 μL of biotinylated pMHC molecules were loaded onto a Superdex 75 increase 10 / 300GL column (GE Healthcare Life Sciences). Elution was performed with PBS at a flow rate of 0.75 mL / min. Biotinylated pMHC molecules appeared at approximately 10 mL. The collected fractions were characterized by SDS-PAGE. The results, shown in Figure 2, show that the renatured pMHC bands met the expected molecular weight and were of good purity.

[0039] 1.5 pMHC ELISA identification

[0040] Dilute streptavidin to 1 μg / mL in coating buffer, add 200 μL to each well, and incubate overnight at 4°C. After coating, shake off the liquid from the plate, rinse four times with water, add 200 μL of blocking buffer to each well, and incubate at 37°C for 1.5 hours. Shake off the liquid from the plate, add 100 μL of sample or denatured sample to each well, and incubate at 37°C for 1.5 hours. Rinse four times with water, add 100 μL of W6 / 32 antibody to each well, and incubate at 37°C for 1.5 hours. Rinse six times with water, add 100 μL of HRP-conjugated goat anti-mouse IgG antibody to each well, and incubate at 37°C for 1 hour. Rinse nine times with water, add 100 μL of color development solution to each well, incubate at 37°C for 30 minutes in the dark, 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 in Figure 3. The non-denatured sample had a significant binding to W6 / 32 compared with the blank and denatured samples, indicating that the pMHC obtained by renaturation had a correct conformation.

[0041] Example 2 Screening of Nanobodies

[0042] Nanobody yeast display library construction:

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

[0044] Nanobody DNA library was continuously amplified with pYDSFor1-pYDSRev1, pYDSFor2-pYDSRev2 and pYDSFor3-pYDSRev2 primers for yeast transformation. When 500 mL of BJ5465 yeast was grown to OD600 = 1.5, 245 μg of nanobody insert DNA and 50 μg of pYDS649 plasmid were electroporated into the yeast and digested with NheI-HF and BamHI-HF. Dilutions of the transformed yeast were then plated as single colonies on selective medium without tryptophan for estimation of library diversity. The primers used are shown in Table 1.

[0045] Table 1 Primer sequence list

[0046] 2.1 Nanobody Yeast Display Library Induced Expression

[0047] The nanobody yeast display library was taken out from -80°C and placed in a 30°C constant temperature water bath to melt. Centrifuge at 4°C, 2000×g for 5 minutes, discard the supernatant, and resuspend the bacterial pellet in a small amount of growth medium. Add the resuspended yeast to 1 L of growth medium and culture it in a shaker at 220rpm at 30°C overnight until the yeast library concentration reaches OD600 = 2-3. Then centrifuge at 4°C, 2000×g for 5 minutes, discard the supernatant, resuspend the bacterial cell in induction medium, dilute OD600 to 1, and culture at 25°C at 220rpm for 18-24 hours. Finally, flow cytometry was used to evaluate the initial expression level of nanobody ER1.

[0048] 2.2 First round of magnetic separation

[0049] Wash the magnetic beads twice with 1 mL of selection buffer, aspirate the supernatant, and resuspend the beads in 400 μL of selection buffer (Invitrogen, 11205D). Centrifuge the bacterial suspension at 2000 OD600 at 2000 × g for 5 minutes at 4°C. Discard the supernatant, resuspend in 20 mL of selection buffer, and centrifuge again. Discard the supernatant and resuspend in 10 mL of selection buffer. Add 200 μL of magnetic beads to the resuspended yeast by pipetting and mixing thoroughly. Rotate the mixture on a rotary mixer at 4°C for 30 minutes. Add 1 mL of the mixture to each 1.5 mL centrifuge tube, place on a magnetic rack, and wait 2-3 minutes. Aspirate unbound yeast cells into a 30 mL centrifuge tube. Wash the magnetic beads with selection buffer and combine them in the same centrifuge tube. Centrifuge at 2000 × g for 5 minutes at 4°C. Discard the supernatant and resuspend the yeast in 5 mL of selection buffer.

[0050] Add NY-ESO-1 antigen to a final concentration of 200 nM and resuspend the yeast at 4°C on a rotary mixer for 1 hour. Centrifuge at 2000 × g at 4°C for 5 minutes, 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 thoroughly, and rotate on a rotary mixer at 4°C for 30 minutes. Transfer the mixture to a centrifuge tube, place on a magnetic stand, and wait 2-3 minutes. Aspirate the yeast that has not bound to the beads and wash the beads twice with 1 mL of selection buffer per tube. Resuspend the beads in growth medium and transfer to a 50 mL Erlenmeyer flask. Add growth medium to a total volume of 15 mL. Incubate overnight at 30°C at 220 rpm until the OD600 reaches 2-3. Centrifuge 50 OD600 yeast at 2000 × g at 4°C for 5 minutes. The culture medium was discarded, the yeast was resuspended in 20 mL of induction medium and centrifuged again, the upper culture medium was aspirated, the yeast was resuspended in induction medium, the OD600 was diluted to 1, and cultured overnight at 25°C and 220 rpm. The nanobody expression level (ER2) and positive rate of the first round of magnetically sorted yeast cells were evaluated by flow cytometry, and the results are shown in Figure 4. The initial HA expression rate of the yeast library was 17.97%, but after one round of magnetic sorting, the cells expressing nanobodies were not enriched, 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 quickly reduce the library capacity by 2-3 orders of magnitude.

[0051] 2.3 Second round of magnetic separation

[0052] Change the amount of yeast solution to 100 OD600, the amount of magnetic beads to 80 μL, and the antigen concentration to 100 nM. For other operations, refer to 2.2.

[0053] 2.4 First round of flow cytometry sorting

[0054] The cells were stained with an APC-labeled anti-HA antibody (Invitrogen, 26183-A647) and FITC-labeled Streptavidin protein (Abcam, ab136201). The expression level and positive rate of the nanobodies in the second round of magnetic yeast were determined on a flow cytometer. APC- and FITC-positive cells were isolated and transferred to a flow cytometer tube containing 1.5 mL of growth medium. The sorted target yeast cells were spread on solid growth medium. Incubate at 30°C for 1-2 days. After a large number of colonies have grown, the yeast cells on the solid medium were rinsed with growth medium and centrifuged at 2000 × g for 5 minutes at 4°C. The supernatant of the growth medium was aspirated and the yeast was resuspended in 5 mL of induction medium and centrifuged again. The OD600 was diluted to 1 with induction medium and shaken at 220 rpm at 25°C overnight.

[0055] 2.5 Second round of flow cytometry sorting and detection

[0056] The antigen was changed to TP53-R273H, and the antigen concentration, grouping, and operation were the same as in 2.4.

[0057] 2.6 The third round of flow cytometry sorting and detection

[0058] The antigen was changed to NY-ESO-1, the antigen concentration was 50 nM, and the grouping and operation were the same as 2.4.

[0059] 2.7 Fourth round of flow cytometry sorting and detection

[0060] Antigen, concentration, grouping and operation steps are the same as 2.6.

[0061] The results of four rounds of flow cytometry sorting are shown in Figure 5. After four rounds of screening, the expression rate of nanobodies increased from 12.7% to 74.2%, and the proportion of double-positive cells increased to 32.5%, indicating a significant enrichment effect.

[0062] Example 3 Identification and Analysis of Specific Yeast Clones

[0063] 3.1 Yeast monoclonal sequence analysis

[0064] 5 μL of the overnight culture of yeast from the fourth flow sort in Example 2 was added to 1 mL of growth medium, mixed thoroughly, and 50 μL was spread onto solid growth medium. Cultured for 1-2 days. Ten single clones were randomly selected and added to 1 mL of growth medium. The cells were grown on a shaker at 30°C and 180 rpm for 24 hours. The 10 yeast clones were sent to Qingke Bio for sequencing. Sequencing yielded four different nanobodies, as shown in Table 2.

[0065] Table 2

[0066] 3.2 Yeast monoclonal antigen binding specificity detection

[0067] The four monoclonal clones were added to 1 mL of growth medium and grown on a shaker at 30°C, 180 rpm for 24 hours. The mixture was centrifuged at 4°C, 2000 × g for 5 minutes, the supernatant discarded, and the yeast resuspended in 2 mL of induction medium. The cells were induced at 25°C, 180 rpm for 24 hours. Flow cytometry was used to analyze the cells using APC-labeled anti-HA antibodies and FITC-labeled streptavidin staining. The results are shown in Figures 6-9. The VHH-1, VHH-4, VHH-5, and VHH-9 monoclonal clones all showed strong binding to the positive antigen NY-ESO-1, but showed little binding to other negative antigens, indicating that the four selected monoclonal clones have strong binding specificity for the NY-ESO-1 antigen.

[0068] Example 4 SPR determination of nanobody affinity

[0069] The chip surface carboxyl groups were activated with 0.4M EDC and 0.1M NHS, followed by streptavidin coupling and blocking with 1M ethanolamine. The SLLMWITQC-HLAA0201 complex protein was flowed over the activated CM5 chip, immobilizing the ligand on the chip surface. A gradient of nanoantibodies was applied sequentially, and signal changes were observed. The BIAcore kinetic profiles of the binding of VHH-1, VHH-4, VHH-5, and VHH-9 to the SLLMWITQC-HLAA0201 complex are shown in Figure 10. Affinity data are shown in Table 3.

[0070] Table 3. Affinity data of VHH-1, VHH-4, VHH-5, and VHH-9

[0071] Example 5 Validation of VHH-5 Nanobody Specificity

[0072] a) Dimeric and tetrameric protein expression

[0073] A dimer expression plasmid, VHH-GGGGS-Fc, and a tetramer expression plasmid, VHH-(GGGGS)3-CH1-Fc and VHH-(GGGGS)3-CL, were constructed. The expression plasmids were transfected into HEK293F cells for 3-4 days, respectively. The cells were centrifuged at 4000×g for 20 min, and the cell supernatant was collected and filtered through a 0.22 μm filter membrane. The cells were then purified using an AKTA protein purifier and a HiTrap protein A 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 the A and B pump heads in pure water filtered through a 0.45μm filter membrane, set the flow rate (3mL / min) and the flushing ratio of the AB pump (50%B). After the pure water is flushed to conductivity equilibrium (about 60mL), connect the HiTrap proteinA affinity column (5mL) to the AKTA purifier and continue to flush with pure water for at least three column volumes; c. Change the flushing ratio of the AB pump to 0%B, and replace the A pump with 50mM Tris-HCl (PH7.4) loading buffer. After conductivity equilibrium, replace the A pump with the culture supernatant to be purified. After loading is completed, replace the A pump with the loading buffer until conductivity equilibrium is achieved; then change the AB pump ratio to 100%B, and replace the B pump with 1mol / L sodium acetate (PH 3.0) Elution buffer was added to obtain the target protein. The AKTA purifier was flushed with pure water to conductivity equilibrium and the entire system was preserved with 20% ethanol (v / v). The tetramer and dimer proteins were ultrafiltered using a 50 kDa pore size ultrafiltration membrane. After replacing the solvent with PBS buffer, the protein concentration was determined using a Nanodrop ND-1000. Aliquots were stored frozen at -80°C until further use. The protein was analyzed for purity by SDS-PAGE. The results are shown in Figure 11. The dimer and tetramer protein bands met the expected molecular weight and were of good purity.

[0074] b) VHH-5 nanobody specific affinity detection

[0075] 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. 5Cells were plated in 12-well plates, 1 mL / well. Peptide was added to a final concentration of 25 μg / mL. After mixing evenly, the cells were placed in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity for 2 hours. After incubation, the cells were collected by centrifugation at 500g for 5 minutes, washed once with PBS, and 200uL of dimer or tetramer protein (100nM) was added and incubated at 4°C for 30 minutes. The cells were washed twice with PBS, and 200μL of 1:500 diluted goat anti-human IgG antibody (Biyuntian, A0556) was added and incubated at 4°C for 30 minutes. The cells were washed twice with PBS, and 300μL of PBS was added to resuspend the cells and the cells were incubated with ACEA NovoCyte TM FITC intensity was detected by flow cytometry, and the results are shown in Figures 12 and 13. The dimer and tetramer proteins had a high binding intensity to the positive antigen NY-ESO-1 and almost no binding 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, wherein the VHH chain of the nanobody comprises three complementary determining regions CDR1, CDR2 and CDR3, characterized in that: The amino acid sequence of the CDR1 is any one of SEQ ID NO.11, SEQ ID NO.1, SEQ ID NO.6, and SEQ ID NO.16; The amino acid sequence of the CDR2 is any one of SEQ ID NO.12, SEQ ID NO.2, SEQ ID NO.7, and SEQ ID NO.17; The amino acid sequence of the CDR3 is any one of SEQ ID NO.13, SEQ ID NO.3, SEQ ID NO.8, and SEQ ID NO.

18.

2. The Nanobody according to claim 1, characterized in that The VHH chain of the Nanobody comprises three complementary determining regions CDR1, CDR2 and CDR3 of any one 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.

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3. 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.

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4. A nucleic acid molecule encoding the Nanobody according to any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that It has a nucleotide sequence as shown in any one of SEQ ID NO.15, SEQ ID NO.5, SEQ ID NO.10, and SEQ ID NO.

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6. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 4.

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

8. An immunoconjugate, characterized in that: The immunoconjugate contains: (a) a Nanobody according to any one of claims 1 to 3; and (b) at least one coupling portion selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a therapeutic agent, a PK modifying portion or an enzyme.

9. A pharmaceutical composition, characterized in that The composition contains the Nanobody described in any one of claims 1-3, the nucleic acid molecule described in claim 4 or the immunoconjugate described in claim 8, and a pharmaceutically acceptable carrier.

10. Use of the Nanobody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the host cell according to claim 7 or the immunoconjugate according to claim 8, characterized in that: The application is any of the following: (1) Application in the preparation of products for detecting NY-ESO-1; (2) Use in the preparation of products combined with NY-ESO-1; (3) Use in the preparation of products for diagnosing or treating tumors expressing NY-ESO-1.

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