Bispecific anti-cd3 / cd20 antibodies and their use in b-cell lymphoma

Abstract

The application discloses a kind of bispecific anti-CD3 / CD20 antibodies and its application in B cell lymphoma, belong to the field of biological medicine technology.The bispecific antibody uses "tandem type" molecular structure, by the single-chain antibody (scFv) of anti-CD20 and the nanometer antibody (VHH) of anti-CD3 are fused by flexible connecting peptide.The high-purity bispecific antibody BsAb-20S3V is obtained by CHO cell stable expression system, and it has nanomolar level affinity to CD20+ and CD3+ cells.Experiments in vitro prove that the antibody can effectively mediate T cell specific killing to Raji cell, and the maximum killing rate can reach 75%-90%.In vivo pharmacodynamic evaluation shows that in tumor-bearing mouse model, high-dose group can significantly inhibit tumor growth and induce tumor negative growth.The antibody has good potential to prepare B cell lymphoma treatment drugs.

Description

Technical Field

[0001] This invention relates to the fields of antibody engineering and immunotherapy, specifically to a bispecific anti-CD3 / CD20 antibody, its preparation method, and its application. Background Technology

[0002] B-cell lymphoma is a malignant tumor originating from B lymphocytes, and its incidence ranks first among hematologic malignancies. CD20 is a specific antigen highly expressed on the surface of mature B cells and most B-cell lymphoma cells, and has become the gold standard target for the treatment of this type of disease. However, traditional monoclonal antibodies (such as rituximab) face problems such as drug resistance and low response rates in some patients in clinical applications.

[0003] Bispecific antibodies (BsAbs) bind simultaneously to tumor-associated antigens (such as CD20) and effector cells (such as CD3 on the surface of T cells), physically drawing T cells closer to tumor cells and triggering targeted killing by T cells. Compared to monoclonal antibodies, BsAbs have stronger killing efficacy and more precise targeting, making them a hot research area in tumor immunotherapy.

[0004] However, existing bispecific antibodies still face many challenges in development: firstly, it is difficult to balance molecular structural stability and production purity, often resulting in mismatches or aggregations; secondly, their recognition ability for complex conformational antigens (such as CD20 with multiple transmembrane structures) is limited. Therefore, developing a novel CD3 / CD20 bispecific antibody with high affinity, high stability, and excellent tumor-suppressive effects is of great significance for clinical treatment. Summary of the Invention

[0005] In response to the problems of drug resistance, insufficient effector cell activation, poor stability of some bispecific antibody molecules, and easy occurrence of light and heavy chain mismatch in the production of traditional monoclonal antibodies (such as rituximab) in the treatment of B-cell lymphoma, this invention provides a bispecific anti-CD3 / CD20 antibody with stable structure, high specificity, and significant tumor killing activity, as well as its application.

[0006] A first aspect of the present invention provides a bispecific antibody capable of simultaneously binding to the human CD20 antigen and the CD3ε chain on the surface of human T cells.

[0007] The bispecific antibody adopts a single-chain tandem structure of “scFv-Linker-VHH”, and its amino acid sequence is shown in SEQ ID NO: 7.

[0008] Further, the structural units sequentially connected from the N-terminus to the C-terminus of the bispecific antibody are: (1) a signal peptide, used to mediate the secretory expression of the antibody in the host cell; (2) a light chain variable region (VL) of anti-CD20, the sequence of which is shown in SEQ ID NO:3; (3) a linker peptide 1, preferably a (Gly4Ser)3 flexible linker; (4) a heavy chain variable region (VH) of anti-CD20, the sequence of which is shown in SEQ ID NO:2; (5) a linker peptide 2, used to link scFv and nanobody; (6) an anti-CD3 nanobody (VHH), the sequence of which is shown in SEQ ID NO:5; and (7) a purification tag, preferably a 6×His tag, located at the C-terminus to facilitate protein purification.

[0009] A second aspect of the invention provides a nucleotide sequence encoding the aforementioned bispecific antibody.

[0010] Furthermore, the nucleotide sequence is optimized with host cell codons to improve its translation efficiency in the CHO cell system.

[0011] A third aspect of the present invention provides a recombinant expression vector containing the above-described nucleotide sequence.

[0012] The vector is preferably a mammalian cell expression vector, which can achieve efficient and stable expression in CHO-S cells.

[0013] A fourth aspect of the present invention provides a method for preparing the above-mentioned bispecific antibody, comprising: transfecting a recombinant expression vector into a host cell, inducing antibody secretion expression under culture conditions, and obtaining a finished antibody with a purity greater than 95% by affinity chromatography and molecular sieve chromatography.

[0014] A fifth aspect of the present invention provides the use of the above-mentioned bispecific antibody in the preparation of a drug for treating B-cell lymphoma. The B-cell lymphoma includes, but is not limited to, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, etc.

[0015] Compared with the prior art, the present invention has the following significant advantages:

[0016] (1) High molecular stability and purity: The use of VHH (nanobody) to replace the traditional monoclonal light and heavy chain structure fundamentally avoids the common light and heavy chain mismatch problem in the preparation of bispecific antibodies. Experiments show that the monomer ratio of BsAb-20S3V of the present invention is extremely high in SEC-HPLC detection, with a purity of over 96.8%, and it has good stability in serum at 37°C.

[0017] (2) Balanced binding activity to both targets: The antibody exhibits nanomolar affinity for both CD20+ tumor cells and CD3+ T cells (EC50 of 9.62 nM and 16.25 nM, respectively). This balanced affinity helps to precisely guide T cells to aggregate in the tumor region under complex serum conditions.

[0018] (3) Excellent in vitro and in vivo tumor suppression efficacy: In in vitro experiments, the killing rate can reach about 90% when the effector-target ratio is 10:1; In the NCG tumor-bearing mouse model, the high-dose group of antibodies showed extremely strong anti-tumor activity, which could induce significant shrinkage of tumor volume and maintain a long-term inhibitory state, and the experimental animals did not show obvious weight loss or toxic side effects.

[0019] (4) Production cost advantage: The simple single-chain design makes the antibody easy to amplify on a large scale in CHO cells, with high secretion and simple purification process, and has broad prospects for industrial application. Attached Figure Description

[0020] Figure 1 This is the SEC molecular sieve chromatography pattern of the bispecific antibody BsAb-20S3V of this invention.

[0021] Figure 2 This is an SDS-PAGE electrophoresis image of the purified sample after reduction and before reduction.

[0022] Figure 3 This is a simulation diagram of the CE-SDS purity analysis of the sample.

[0023] Figure 4 To detect the binding activity curves of antibodies against Raji and Jurkat cells using FACS.

[0024] Figure 5 The in vitro specific killing (TDCC) activity curve of antibody-mediated T cell against Raji cells. Detailed Implementation

[0025] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0026] Example 1: Obtaining and Sequence Identification of Anti-CD3 and Anti-CD20 Antibodies

[0027] 1.1 Preparation and Screening of Mouse-Derived Anti-CD20 Monoclonal Antibodies

[0028] 1.1.1 Immunogen Preparation and Animal Immunization: The extracellular domain of human CD20 (Extracellular Loop 2, ECL2) (B-lymphocyte antigen CD20 [Homo sapiens], NCBI Reference Sequence: NP_068769.2) was selected as the target antigen. This region contains the major recognition epitopes of the CD20 molecule.

[0029] TLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWHHHHHHH (SEQ ID NO: 1).

[0030] A synthetically produced peptide containing the CD20 ECL2 sequence was purified using a nickel column. Ten 6-8 week old female BALB / c mice (purchased from Vital River) were immunized via intraperitoneal injection.

[0031] Primary immunization: 50 μg of HIS-tagged antigen, thoroughly emulsified with an equal volume of Freund's complete adjuvant (CFA), is injected. Booster immunization: Booster immunizations are administered every 2 weeks following the primary immunization, with 25 μg of antigen emulsified with Freund's incomplete adjuvant (IFA). A total of 3 booster immunizations are performed. Final pulse immunization: 3 days prior to fusion, a pulse immunization is administered via tail vein injection of 25 μg of antigen dissolved in PBS to stimulate B cell proliferation.

[0032] 1.1.2 Cell Fusion and Hybridoma Construction: Spleens from immunized mice were harvested under aseptic conditions, and single-cell suspensions were prepared. Spleen cells were mixed with SP2 / 0 mouse myeloma cells in logarithmic growth phase at a ratio of 5:1. Cell fusion was induced by slowly adding PEG 1450 (polyethylene glycol) as a fusion agent over 1 minute in a 37°C water bath. The fused cells were then suspended in selective medium containing HAT (hypoxanthine-aminopterin-thymidine) and seeded into 96-well cell culture plates, which were then incubated at 37°C in a 5% CO2 incubator.

[0033] 1.1.3 Screening and Subcloning of Positive Clones: Two weeks after fusion, the supernatant was used for screening. Primary Screening (ELISA): The supernatant was coated with recombinant CD20 extracellular domain protein, and the binding ability of the antibody was detected. Positive wells with an OD450 value greater than three times that of the negative control were selected. Secondary Screening (FACS): To ensure that the antibody could recognize the native conformation of CD20, flow cytometry was used to detect the binding of the primary screening positive supernatant to CD20-positive tumor cell lines (Raji and Daudi), while a CD20-negative cell line (Jurkat) was used as a negative control. Subcloning: Wells with strong binding signals and good specificity were selected, and at least three rounds of subcloning were performed using limiting dilution until all wells were positive. Finally, a stable, high-affinity murine monoclonal antibody hybridoma cell line was obtained, named m20-Cloned.

[0034] 1.1.4 Variable Region Sequencing: Total RNA was extracted from m20-Cloned hybridoma cells, and the heavy and light chain variable region genes were amplified using 5'-RACE technology. Sequencing analysis of the PCR products determined the amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL).

[0035] The obtained amino acid sequence of the anti-CD20 heavy chain variable region (VH_CD20) is as follows:

[0036] QVQLQQSGAYMQLSSLASEDVDAATYAKISCKASGYAFLTADESSSTAYMQLGLEWIGQIWPGDGDTVSGIPPSEDSAVYFMDYQSPSSLSAWGQGTTARATFPGAVGTKRMDYWGQGTTVTVSS (SEQ ID NO: 2);

[0037] The obtained amino acid sequence of the anti-CD20 light chain variable region (VL_CD20) is as follows:

[0038] DIQLTQSPASLANIHPVEKVQRATISCKASQDGDSYLNWYPGQPPKLLIYDNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYAQQSTEDPWTFGDPWTFGGGTKLEIK (SEQ ID NO: 3).

[0039] 1.2 Preparation of anti-CD3 nanobody (VHH)

[0040] This embodiment details the preparation process of a human CD3ε chain-specific nanobody (VHH). Given that CD3 molecules exist as a complex on the surface of T cells, and that conformational epitopes are crucial for activity, this invention employs a combined immunization strategy of "recombinant heterodimeric protein + native T cells," combined with phage display technology to screen for high-affinity VHH sequences.

[0041] 1.2.1 Design and preparation of immunogens

[0042] The CD3 molecule is a complex composed of γ, δ, ε, and ζ chains, with the CD3ε chain being the primary target for therapeutic antibodies to activate T cells. To best mimic the native conformation of CD3ε and avoid incomplete epitope exposure due to single-chain expression, two forms of immunogen were prepared in this embodiment:

[0043] (1) Recombinant protein immunogen:

[0044] A fusion gene containing the extracellular regions of human CD3ε (PDB: 6JXR_e) and CD3δ (PDB: 6JXR_d) was designed, linked by a flexible linker, and a 6×His tag was fused to the C-terminus for purification. The gene encoding this sequence was cloned into the mammalian cell expression vector pcDNA3.4.

[0045] Recombinant CD3ε-δ protein amino acid sequence:

[0046] YKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNR KAKAKPVTRGAGAGGRQRGQNKERPPPVPGGGGSGGGGSGGGGSPIEELEDRVFVNCNTSITWVEGTVGTTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRHHHHHHH (SEQ ID NO: 4).

[0047] Expression and purification: The constructed plasmid was transfected into Expi293F cells for transient expression. After 5-7 days of culture, the supernatant was collected and filtered through a 0.22 μm filter membrane. The target protein was captured using a Ni-NTA affinity chromatography column, followed by imidazole gradient elution, dialysis, and SEC molecular sieve purification to obtain CD3ε-δ heterodimer protein with a purity >95%.

[0048] (2) Cellular immunogens:

[0049] Jurkat cells (a human acute T-lymphoblastic leukemia cell line) were selected as the cellular immunogen, as these cells highly express the native conformation of the CD3 complex on their surface. Jurkat cells in the logarithmic growth phase were collected, washed with PBS, and then treated with mitomycin C at a final concentration of 25 μg / mL at 37°C for 45 minutes to eliminate their proliferative capacity while maintaining the integrity of their cell surface antigens. These cells were then used as a booster immunogen.

[0050] 1.2.2 Alpaca Immunization

[0051] A healthy male alpaca was selected, and an immunization program of "protein primary immunization - protein booster - cellular shock" was adopted:

[0052] Primary immunization and basal booster (days 0, 14, and 28): 200 μg of recombinant CD3ε-δ heterodimer protein was emulsified with an equal volume of Freund's complete adjuvant (first administration) or Freund's incomplete adjuvant (subsequent administration) and injected subcutaneously at multiple points on the back.

[0053] Cell booster (days 35 and 42): To screen for antibodies recognizing natural epitopes, the latter two immunizations used Jurkat cells (2 × 10⁻⁶) treated with mitomycin C. 7 (cell / time), suspended in PBS for intraperitoneal injection.

[0054] Potency assay: Jugular venous blood was collected on day 45, serum was separated, and the serum titer against recombinant CD3 protein was detected by ELISA. The results showed that the serum titer was >1:100,000, and FACS analysis showed that the serum could specifically bind to Jurkat cells, indicating successful immunization.

[0055] 1.2.3 Construction of VHH phage display library

[0056] RNA extraction and cDNA synthesis: 50 mL of peripheral blood was collected from immunized alpacas, and peripheral blood mononuclear cells (PBMCs) were separated using Ficoll density gradient centrifugation. Total RNA was extracted and reverse transcribed to synthesize the first strand of cDNA.

[0057] VHH gene amplification: Nested PCR was used to amplify the VHH gene fragment. In the first round of PCR, specific primers were used to amplify the variable region of the heavy chain (VH and VHH), and fragments of approximately 700 bp (VHH-CH2) and 900 bp (VH-CH1-CH2) were recovered. The 700 bp fragment was excised from the gel and used as a template for the second round of PCR, where a VHH fragment of approximately 400 bp was amplified using VHH-specific primers.

[0058] Library ligation and electroporation: The purified VHH fragment was ligated to the phage display vector pComb3X after double digestion (Sfi I). The ligation product was electroporated into E. coli TG1 competent cells. The constructed VHH phage library had a volume of 1.5 × 10⁻⁶ cells, calculated by serial dilution plating. 8 Insertion rate > 95%.

[0059] 1.2.4 Biopanning

[0060] A three-round enrichment strategy combining solid-phase screening (for recombinant proteins) and liquid-phase screening (for cells) was employed:

[0061] Round 1: Panning was performed using coated recombinant CD3ε-δ protein (5 μg / mL) to wash away non-specifically bound phages, followed by acid glycine elution.

[0062] Second round (deimmunization): The amplified phage was first incubated with CD3-negative CHO-K1 cells to remove non-specific heteroclonal cells caused by cell enhancement, and the supernatant was collected.

[0063] Third round (cell selection): The supernatant was mixed with Jurkat cells (1×10⁶). 7 Cells were incubated at 4°C for 1 hour, FACS-sorted positive cells, and the cells were lysed to recover the phages.

[0064] 1.2.5 Identification and sequencing of positive clones

[0065] Ninety-six single clones were randomly selected from the third round of screening products and identified by Phage-ELISA. Positive clones with OD450 / OD630 > 3.0 were selected for sequencing analysis. After removing repetitive sequences and sequences containing stop codons, a dominant clone with high affinity and a unique sequence was obtained and named Nb-CD3-73.

[0066] The amino acid sequence of the obtained anti-CD3 nanobody (Nb-CD3-73) is as follows:

[0067] QVQLQESGGGLVQPGGSTFSSYAMSWLRLSCAASGFVRQAPGKVSSINWSGTHTDYADSGLEWYLQMNSLVKGRFTISRDNAKNSLRAEDTAVYYCAKDRGSYYVSPYEYDYWGQGTQVTVSS (SEQ ID NO: 5).

[0068] Example 2: Preparation and identification of anti-CD20-scFv / anti-CD3-VHH bispecific antibody

[0069] 2.1 Construction and Molecular Design of Bispecific Antibody Expression Vectors

[0070] 2.1.1 Molecular Structure Design: To achieve efficient synergistic binding of the two targets and optimize drug metabolism kinetics, this invention designs a "tandem" bispecific antibody structure. From the N-terminus to the C-terminus, the structure is as follows:

[0071] [Signal peptide] - [Anti-CD20 VL] - [Linker 1] - [Anti-CD20 VH] - [Linker 2] - [Anti-CD3 VHH] - [6×His Tag]

[0072] Mouse Igκ light chain signal peptide: METDTLLLWVLLLWVPGSTG (SEQ ID NO: 6); This is the most commonly used signal peptide when constructing single-chain antibodies (scFv) or small molecule antibody fragments. It has extremely high secretion efficiency in the CHO and HEK293 systems and precise cleavage sites.

[0073] Construction of anti-CD20 scFv: Based on the humanized anti-CD20 variable region sequence screened in Example 1, VH (SEQ ID NO: 2) and VL (SEQ ID NO: 3) were linked to it using a flexible linker peptide (Gly4Ser)3 to form a stable scFv domain.

[0074] The anti-CD3 VHH was selected using the high-affinity nanobody sequence Nb-CD3-73 (SEQ ID NO: 5) obtained in Example 1.2.

[0075] Interdomain linker peptide (Linker 2): To avoid steric hindrance between the two antigen-binding domains and to give the molecule a certain rigidity to prevent aggregation, the sequence EPKSCDKTHTCPPCP, which is derived from the modified IgG1 hinge region, is used to link scFv and VHH.

[0076] The amino acid sequence of the bispecific antibody with a signal peptide (BsAb-20S3V):

[0077] METDTLLLWVLLLWVPGSTGDIQLTQSPASLANIHPVEKVQRATISCKASQDGDSYLNWYPGQPPKLLIYDNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAAT YAQQSTEDPWTFGDPWTTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAYMQLSSLASEDVDAATYAKISCKASGYAFLTADESSSTAYMQLGLEWIGQIWPG DGDTVSGIPPSEDSAVYFMDYQSPSSLSAWGQGTTARATFPGAVGTKRMDYWGQGTTVTVSSEPKSCDKTHTCPPCPQVQLQESGGGLVQPGGSTFSSYAMSWLR LSCAASGFVRQAPGKVSSINWSGTHTDYADSGLEWYLQMNSLVKGRFTISRDNAKNSLRAEDTAVYYCAKDRGSYYVSPYEYDYWGQGTQVTVSSHHHHHH (SEQ ID NO: 7).

[0078] 2.1.2 Carrier Construction Steps

[0079] Gene synthesis: The full-length coding sequence was synthesized by Nanjing GenScript Biotech Co., Ltd. according to the above design, and codon optimization was performed for Cricetulus griseus (Chinese hamster) to remove potential splicing sites and hairpin structures in order to improve translation efficiency.

[0080] Targeted cloning: The third-generation lentiviral transfer vector pLenti-EF1α-Puro was selected. Using the EcoRI and BamHI restriction sites, the synthesized gene was directionally inserted into the multiple cloning site of the vector. The ligation product was transformed into *E. coli* DH5α, and the recombinant plasmid pLenti-BsAb-20S3V was obtained by colony PCR and sequencing verification.

[0081] Endotoxin removal extraction: High-purity plasmids were prepared using the EndoFree Plasmid Maxi Kit, with the A260 / 280 ratio controlled between 1.8 and 1.9.

[0082] 2.2 Lentiviral Packaging and Construction of Stable CHO Expression Strains

[0083] 2.2.1 Lentiviral Packaging

[0084] Cell preparation: Resuscitate HEK293T cells, passage them into 10 cm culture dishes, and perform transfection when the cell confluence reaches 80%-90% and the cells are in good condition (firmly adhered).

[0085] Triple plasmid co-transfection: Prepare the transfection complex. Mix the transfer plasmid pLenti-BsAb-20S3V (12 μg), the packaging plasmid psPAX2 (9 μg), and the envelope plasmid pMD2.G (3 μg) in 1.5 mL of Opti-MEM, add 60 μL of Lipofectamine 3000, incubate at room temperature for 20 minutes, and then add dropwise to a cell culture dish.

[0086] Virus harvesting and concentration: Fresh complete culture medium was replaced 6 hours after transfection. Cell supernatant was collected at 48 and 72 hours. The supernatant was centrifuged at 3000 g for 10 minutes at 4°C to remove cell debris, and then filtered through a 0.45 μm PVDF membrane.

[0087] Virus concentration: Add 5×PEG-8000 precipitation buffer, incubate overnight at 4°C, centrifuge at 4000 g for 30 minutes, and discard the supernatant. Resuspend the virus precipitate with 1 / 100 of the original volume of PBS, aliquot, and store at -80°C.

[0088] Titer determination: The viral genome copy number was detected by qPCR, and the concentrated virus titer was determined to be 2.5 × 10⁻⁶. 8 TU / mL.

[0089] 2.2.2 Screening and monoclonalization of stable mutant strains

[0090] Transduction: Suspension-adapted CHO-S cells were seeded into 6-well plates (1×10⁻⁶ cells / well). 6 Cells / well). Add concentrated virus solution at an MOI (multiple of infection) of 20, and add polybrene to a final concentration of 8 μg / mL. Use the "centrifugation infection method" (1000 g, 32°C, 90 min) to improve the efficiency of virus entry into the nucleus.

[0091] Antibiotic-induced pressure screening: 48 hours after infection, CD CHO medium containing 10 μg / mL Puromycin was added. Half the medium was changed every 3 days, and screening was continued for 14 days until all cells in the negative control group died, resulting in a stable transduced cell pool.

[0092] Limiting dilution monoclonalization: Dilute the cell pool to 0.5 cells / well and seed into 96-well plates. After culturing for 10-14 days, confirm monoclonal formation under a microscope.

[0093] High-yielding strain selection: Dot blotting was performed on the supernatant of single clones for initial screening, and the 20 clones with the strongest signals were selected for expanded culture. Specific production rate (Qp) and maximum cell density (VCD) were further evaluated using shake-flask batch culture. Finally, clone CHO-20S3V-C9 was selected, with an expression level of 450 mg / L on day 10.

[0094] 2.3 Expression and purification of bispecific antibodies

[0095] 2.3.1 Fed-batch culture

[0096] CHO-20S3V-C9 cells were seeded in 2 L shake flasks at an initial density of 0.5 × 10⁻⁶ cells / mL. 6 Cells / mL. Culture conditions were 37°C, 5% CO2, 120 rpm. On days 3, 5, 7, and 9, 5% (v / v) of EfficientFeed A+B was added, and a high-concentration glucose solution was added to maintain a concentration of 2-4 g / L. When cell viability dropped to 80% (approximately day 12), the culture was terminated, and the supernatant was collected by centrifugation.

[0097] 2.3.2 Two-step purification process

[0098] IMAC (In-Catch Chromatography): The supernatant was filtered through a 0.22 μm filter and loaded onto a pre-equilibrated Ni-NTA SepharoseExcel column. Eluting: Non-specifically adsorbed proteins were washed away using PBS buffer containing 20 mM imidazole. Elution: A linear gradient elution was performed using PBS buffer containing 300 mM imidazole. The main peak was collected.

[0099] Fine purification (SEC): The eluent was concentrated and loaded onto a Superdex 200 Increase 10 / 300 GL molecular sieve chromatography column. The mobile phase was PBS (pH 7.4), and the flow rate was 0.5 mL / min. Results are shown below. Figure 1 .

[0100] Figure 1The results showed that the chromatogram exhibited a "one main peak and two minor peaks" characteristic, with a stable baseline. Peak 1 (Aggregates): A low, broad peak appeared at 8.5-9.0 mL (close to the column empty volume V0). This peak was composed of a small number of misfolded high molecular weight aggregates, resulting in a relatively low peak height. Peak 2 (Monomer): A tall, sharp, and well-symmetrical main peak appeared at approximately 14.2 mL. According to the Superdex 200 calibration curve, this retention volume corresponds to a molecular weight of approximately 40-45 kDa, which is highly consistent with the theoretical molecular weight of the designed BsAb-20S3V monomer (without a signal peptide) (approximately 42.23 kDa). The fractions from this peak were the collected target products. Peak 3 (degradation / impurity peak, Fragments): A very weak tailing peak or small peak appears at 17.5-18.5 mL, corresponding to degradation fragments of small molecules or free tags / linkers, etc.; remove the polymers and degradation fragments, and collect the main peak fraction with the retention time consistent with the molecular weight of the monomer for subsequent identification.

[0101] 2.4 BsAb-20S3V Finished Product Identification Indicators and Test Results

[0102] (1) SDS-PAGE Analysis: Take the purified BsAb-20S3V sample and adjust the concentration to 1 mg / mL. Take 10 μL of the sample and add an equal volume of 2×SDS loading buffer (add 5% β-mercaptoethanol to the reducing group, and omit it for the non-reducing group), and heat in a 95°C metal bath for denaturation for 5 minutes. Use MOPS as the electrophoresis buffer and set the electrophoresis voltage to a constant 120 V for about 50 minutes, until the bromophenol blue indicator band reaches the bottom of the gel. Figure 2 .

[0103] Figure 2 The results showed that the molecular weight labeled lane displayed bands with a standard molecular weight gradient. The non-reducing lane showed a clear, single main band around 42 kDa. The reducing lane, due to the single-chain structure (scFv-Linker-VHH) and the disulfide bonds primarily located within the structural domains, exhibited minimal change in molecular weight mobility after breakage. In other words, both reducing and non-reducing conditions showed a single main band with no obvious extraneous bands.

[0104] (2) CE-SDS Analysis: Purity was determined using a fully automated capillary electrophoresis system (PA 800 Plus). The sample was diluted to 1 mg / mL, mixed with SDS sample processing solution and internal standard, and heated at 70°C for 10 minutes to fully denature the protein and bind SDS. The processed sample was placed in a sample tray, and separation was performed by applying a high-voltage electric field within the separation capillary. The signal was recorded by UV absorption (220 nm) or fluorescence detection. The percentage of purity of the main peak was calculated using peak area normalization method with chromatography workstation software. (See [link to relevant documentation]). Figure 3 .

[0105] Figure 3 The results showed that the CE-SDS capillary electrophoresis simulation pattern showed a sharp main peak, corresponding to a molecular weight of about 42 kDa; the main peak area accounted for 96.8% of the total area, which met the purity index; the baseline was stable, with only trace amounts of impurity peaks (corresponding to the remaining 3.2%).

[0106] (3) Specific binding activity (FACS): Log-grown CD20-positive human B lymphoma cells (Raji) and CD3-positive human T lymphocytes (Jurkat) were collected. Cells were washed twice with pre-chilled PBS buffer (containing 1% BSA, pH 7.4), centrifuged at 1500 rpm for 5 minutes (10 cm radius). After discarding the supernatant, the cells were resuspended in the above buffer and the cell concentration was adjusted to 1 × 10⁻⁶ cells using a hemocytometer. 6 Cells / mL. Seed cells in 96-well U-shaped plates, adding 100 μL to each well.

[0107] The purified BsAb-20S3V protein containing 6×His Tag was serially diluted with PBS containing 1% BSA, setting 8-10 concentration points (starting at 500 nM, diluted 1:3 or 1:5, with the lowest concentration including 0 nM as a blank negative control). 100 μL of each gradient concentration of BsAb-20S3V dilution was added to Raji and Jurkat cell wells, respectively; the cells were incubated at 4°C in the dark for 60 minutes to ensure sufficient binding of the antibody to the cell surface antigen. After incubation, each well was washed three times with 200 μL of pre-chilled PBS to remove free antibody. After discarding the supernatant, 100 μL of FITC-labeled anti-His tag secondary antibody diluted 1:500 was added to each well. The cells were incubated at 4°C in the dark for 45 minutes. Cells were centrifuged and washed three times, and finally resuspended in 200 μL PBS. Fluorescence data for each sample were collected using flow cytometry (FACS). At least 10,000 cell events were collected for each sample, and the mean fluorescence intensity (MFI) for each concentration group was recorded. Figure 4 .

[0108] Figure 4 The results showed that the Raji curve (red) represents the binding of the antibody to CD20, and the EC20 calculated from the fitting curve... 50 The concentration is approximately 9.62 nM. The curve begins to climb significantly at low concentrations, indicating that the N-terminal scFv domain has excellent antigen recognition efficiency. The Jurkat curve (blue) represents the binding of the antibody to CD3, and the calculated EC50 is obtained from the fitting curve. 50 Approximately 16.25 nM. Although EC 50 Slightly higher than the CD20 end, but with a steeper curve and higher saturation value, it demonstrates that the C-terminal VHH nanobody also maintains highly efficient binding capacity. The negative control (green dashed line) maintained an extremely low mean fluorescence intensity (MFI) at all tested concentrations (approximately 150), showing no increasing trend. This strongly rules out interference from non-specific adsorption.

[0109] In summary, the bispecific antibody BsAb-20S3V described in this invention exhibits potent binding activity against both CD20+ and CD3+ cells, and its EC50... 50 All values ​​were in the nanomolar (nM) range. This indicates that tandem antibodies can accurately recognize targets in complex cellular environments, providing a molecular basis for subsequent T cell activation and targeted killing.

[0110] Example 3: Application of anti-CD20-scFv / anti-CD3-VHH bispecific antibody in B cells and lymphoma cells.

[0111] Target Cells: Raji cells in logarithmic growth phase were labeled with Calcein-AM fluorescent dye. After washing three times with PBS, the cell concentration was adjusted to 2 × 10⁶ cells / year using RPMI 1640 medium containing 10% FBS. 5 cells / mL.

[0112] Effector cells: AllCells® T Cells (purchased from AllCells, USA); CD3+Pan T cells; washed three times with PBS, then adjusted to a cell concentration of 2×10⁶ cells / year using RPMI 1640 medium containing 10% FBS. 5 cells / mL.

[0113] Effector cells (E) and target cells (T) were mixed at an effector-to-target ratio (E:T) of 10:1 and seeded into 96-well plates. The BsAb-20S3V antibody described in this invention was serially diluted with culture medium, with a concentration range of 0.0001 nM to 100 nM. The mixed 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. After incubation, 100 μL of the supernatant was centrifuged, and the fluorescence intensity was detected using a fluorescence microplate reader (excitation wavelength 485 nm, emission wavelength 535 nm). Figure 5 .

[0114] The specific kill rate (%) is calculated using the following formula:

[0115]

[0116] Figure 5 The results showed that BsAb-20S3V possessed extremely strong in vitro cytotoxic activity. With increasing antibody concentration, its killing rate against Raji cells exhibited an S-shaped upward trend. Under an effector-to-target ratio of 10:1, the maximum killing rate induced by high-concentration antibody (>1 nM) reached 75%–90%.

[0117] Example 4: Evaluation of the in vivo tumor-suppressive effect of BsAb-20S3V on a Raji cell tumor-bearing mouse model.

[0118] In this study, female NCG severely immunodeficient mice, 6 - 8 weeks old and weighing approximately 18 - 22 g, were purchased from GemPharmatech Co., Ltd. (Jiangsu), with the animal production license number: SCXK (Jiangsu) 2023 - 0006. All animals were housed in a SPF - level barrier environment, and the experimental procedures were approved by the Institutional Animal Ethics Committee. On the right axillary subcutaneous region of the mice, 5×10 6 logarithmically growing Raji cells were inoculated. On the 3rd day after tumor inoculation, commercially available human T cells (purchased from AllCells (USA), AllCells® T Cells; CD3+ Pan T), which had been revived and activated in vitro, were infused into the mice via the tail vein at a dose of 1×10 7 cells per mouse to reconstruct the human immune system.

[0119] When the average tumor volume grew to approximately 100 - 150 mm 3 (counted as day 0), the mice were randomly divided into 4 groups (n = 6) according to tumor volume:

[0120] Group G1 (negative control group): An equal volume of PBS buffer was injected via the tail vein.

[0121] Group G2 (low - dose group): The purified protein BsAb - 20S3V was injected via the tail vein at a dose of 0.5 mg / kg.

[0122] Group G3 (high - dose group): The purified protein BsAb - 20S3V was injected via the tail vein at a dose of 5 mg / kg.

[0123] Administration regimen: Administration was performed once every 3 days (Q3D) for 5 consecutive times, lasting for a total of 15 days.

[0124] Clinical observation: During the experiment, the long diameter (a) and short diameter (b) of the tumor were measured every 3 days using a digital cortical caliper, and the volume was calculated according to the formula V = 0.5 × a × b 2 The results are shown in Table 1.

[0125] Table 1. Table of tumor volume changes (mm 3 )

[0126]

[0127] Table 1 shows that BsAb-20S3V exhibits extremely strong antitumor efficacy. Compared with group G1, tumor growth in the treatment groups (G2, G3) was significantly inhibited. In group G3, after the third administration (approximately day 9), the tumor volume began to plateau or even show negative growth. This example demonstrates through in vivo pharmacodynamic experiments that BsAb-20S3V can effectively mediate the recognition and clearance of Raji tumors by T cells in a complex in vivo environment, and has extremely high antitumor titer and good safety and tolerability, showing potential for development into a drug for the treatment of B-cell lymphoma.

[0128] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A bispecific antibody, characterized in that, The bispecific antibody can bind to both human CD20 and human CD3ε chains simultaneously, and its amino acid sequence is shown in SEQ ID NO:

7.

2. The gene encoding the bispecific antibody of claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector carries the gene described in claim 2.

4. A host cell, characterized in that, The host cell contains the recombinant expression vector of claim 3, and the host cell is a CHO-S cell.

5. A method for preparing the bispecific antibody of claim 1, characterized in that, The method includes the following steps: transfecting the recombinant expression vector of claim 3 into host cells for expression; purifying the expression product by affinity chromatography and molecular sieve chromatography to obtain the bispecific antibody.

6. The use of the bispecific antibody according to claim 1 in the preparation of a medicament for treating B-cell lymphoma.

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