CN118459585A - BAFF-R binding molecules and their applications - Google Patents

Abstract

The present disclosure relates to a BAFF-R binding molecule and application thereof, in particular, the present disclosure obtains monoclonal cell lines B7G10, B12E9 by mouse immunity, cell fusion and antibody screening technology, and obtains murine antibodies, chimeric antibodies, humanized antibodies and antibody molecules without fucose modification with high affinity by antibody purification and identification, antibody grafting technology, humanized technology and glycoengineering technology.

Description

BAFF-R binding molecules and their applications

Technical Field

The present disclosure belongs to the field of biomedicine technology, and specifically, relates to a BAFF-R binding molecule and an application thereof.

Background Art

BAFF belongs to the tumor necrosis factor (TNF) ligand family and plays an important role in B cell dynamic balance, immune tolerance and carcinogenesis. BAFF can bind to three different TNF receptors, namely TACI, BCMA and BAFF-R, and BAFF-R is the main receptor of BAFF. Gene knockout experiments in mice have also confirmed that only knockout of BAFF-R can completely mimic the phenotype of BAFF deficiency (Yan, M., J. R. Brady, B. Chan, W. P. Lee, B. Hsu, S. Harless, M. Cancro, I. S. Grewal, and V. M. Dixit. 2001. Identification of a novel receptor for B lymphocyte stimulator that is mutated in a mouse strain with severe B cell deficiency. Curr. Biol. 11: 1547, Schiemann, B., J. L. Gommerman, K. Vora, T. G. Cachero, S. Shulga-Morskaya, M. Dobles, E. Frew, and M. L. Scott. 2001. An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Science 293:211, Schneider, P., H. Takatsuka, A. Wilson, F. Mackay, A. Tardivel, S. Lens, T. G. Cachero, D. Finke, F. Beermann, and J. Tschopp. 2001. Maturation of marginal zone and follicular B cells requires B cell activating factor of the tumor necrosis factor family and is independent of B cell maturation antigen. J. Exp. Med. 194:1691). The BAFF/BAFF-R signaling pathway is essential for the survival and growth of normal and cancerous B cells. BAFF is expressed on the cell surface and can be released from the cell surface and become soluble.

B cells develop from hematopoietic stem cells in the bone marrow. The main steps of this process have been studied more clearly. The heavy chain (H-chain) D and J gene rearrangement that occurs in the total recombination activation gene RAG1/2 in the germline cells promotes the formation of pro-B cells (pro-B). In the early stage of pre-B cells, V gene rearrangement further occurs. The functional heavy chain pairs with the transitional V-preB/l-like polypeptide to form pre-BCR. Pre-BCR turns off the rearrangement of allelic heavy chain genes and starts the rearrangement of light chain genes. The k and l light chains replace V-preB/l5 and form a complex IgM with the heavy chain. The formation of IgM marks the entry of cell development into the immature B cell (iB) stage. iB cells migrate into the spleen and are called transitional B cells (transitional B). From then on, tB cells receive survival signals through BAFF-R and complete the first stage of B cell development: developing into marginal zone B (MZ B) cells or follicular B cells, and further forming plasma cells or memory B cells on this basis (Pieper K, J Allergy Clin Immunol 2013). BAFF-R begins to express as early as when B cells develop in the bone marrow, and the expression level is further increased in the transitional B cell stage. In the study of Baff-r or Baff mouse gene knockout, the total amount of B cells was significantly reduced, and B cell development was significantly blocked in the transitional T2 stage (Thompson JS, Science 2001; 293: 2108-11; Sasaki Y, J Immunology 2004; 173: 2245-52). In addition, Hu S et al. confirmed that BAFF can also promote the survival of isolated and cultured T cells, among which CD3+CD4+, CD4+CD25+, CD4+CD154+ and CD4+CD69+ subpopulations increased, while CD4+CD62L+CD subpopulations decreased. Further studies confirmed that BAFF/BAFF-R induced T cell activation by activating the PI3K-Akt signaling pathway (Hu, S et al., BAFF Promotes T Cell Activation Through the BAFF-BAFF-R-PI3K-Akt Signaling Pathway).

B cell non-Hodgkin lymphoma (B-NHL), various types of chronic lymphocytic leukemia (CLL), etc. usually abnormally overexpress BAFF. This abnormal overexpression (autocrine or paracrine) promotes tumor cell survival and protects against spontaneous or drug-induced apoptosis by activating the NF-κB signal transduction pathway. Designing drugs to antagonize the BAFF/BAFF-R pathway may have special clinical therapeutic value (Yang, S. et al 2014Role of BAFF/BAFF-R Axis in B-cell non-Hodgkin Lymphoma). Emily M.McWilliams et al. reported that a fully human antibody VAY-736 targets BAFF-R, blocks BAFF/BAFF-R binding, and can effectively inhibit the survival of chronic lymphocytic tumor cells mediated by this signaling pathway. Furthermore, the drug has the potential to be used in combination with BTK inhibitor drugs (Anti-BAFF-Rantibody VAY-736 demonstrates promising preclinical activity in CLL and enhances effectiveness of ibrutinib.McWILLIAMS et al 12FEBRUARY 2019, VOLUME3, NUMBER 3, Blood Advances). Qin H et al. developed a CAR T cell targeting BAFF-R, which can effectively target B cell-mediated carcinogenesis that relapses due to resistance to CAR T targeting CD19. Studies have shown that after CD19 targeted therapy, primary acute lymphoblastic leukemia ALL will evolve into a tumor that loses the CD19 antigen and is no longer recognized by CD19-CAR T cells, but these tumor cells carry BAFF-R. Therefore, BAFF-R-CAR can effectively overcome the drug resistance caused by antigen loss (Qin H. et al., 2019. CAR T Cells Targeting BAFF-R Can Overcome CD19 Antigen Loss in B Cell Malignancies). We infer that the development of antibodies with high affinity that can block BAFF/BAFF-R binding has great application potential in the treatment of B cell-mediated malignancies.

Summary of the invention

The present invention provides a BAFF-R binding molecule, which is generally an antibody or an antigen-binding fragment thereof that immunospecifically binds to BAFF-R and comprises a heavy chain variable region and a light chain variable region.

In a preferred embodiment of the present invention, the present invention provides a BAFF-R binding molecule wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 38, 39 and 40, respectively.

In a preferred embodiment of the present invention, the present invention provides a BAFF-R binding molecule, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 41, 42 and 43 respectively.

In a preferred embodiment of the present invention, the present invention provides a BAFF-R binding molecule, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 44, 45 and 46, respectively.

In a preferred embodiment of the present invention, the present invention provides a BAFF-R binding molecule, wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 47, 48 and 49, respectively.

In a preferred embodiment of the present invention, the present invention provides a BAFF-R binding molecule, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 38, 39, and 40, respectively; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 41, 42, and 43, respectively.

In a preferred embodiment of the present invention, the present invention provides a BAFF-R binding molecule, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 44, 45, and 46, respectively; and/or, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NOs: 47, 48, and 49, respectively.

In a preferred embodiment of the present invention, the BAFF-R binding molecule provided by the present invention is a murine antibody or a fragment thereof.

In a preferred embodiment of the present invention, the BAFF-R binding molecule provided by the present invention is a chimeric antibody or a fragment thereof.

In a preferred embodiment of the present invention, the BAFF-R chimeric antibody or fragment thereof provided by the present invention, wherein the sequence of the heavy chain variable region of the chimeric antibody is: SEQ ID NO: 9 or 11.

In a preferred embodiment of the present invention, the BAFF-R chimeric antibody or fragment thereof provided by the present invention, wherein the sequence of the light chain variable region of the chimeric antibody is: SEQ ID NO: 10 or 12.

In a preferred embodiment of the present invention, the BAFF-R chimeric antibody or fragment thereof provided according to the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprises a heavy chain constant region of human IgG1 or IgG4, and more preferably comprises a heavy chain constant region of human IgG1 or a variant thereof.

In a preferred embodiment of the present invention, the BAFF-R chimeric antibody or fragment thereof provided by the present invention further comprises a light chain constant region of human κ, λ chain or a variant thereof, preferably a light chain constant region of human κ chain or a variant thereof.

In a preferred embodiment of the present invention, the BAFF-R binding molecule provided by the present invention is a humanized antibody or a fragment thereof.

In a preferred embodiment of the present invention, according to the BAFF-R humanized antibody or fragment thereof provided by the present invention, the heavy chain FR region sequence on the heavy chain variable region of the humanized antibody is derived from the human germline heavy chain IGHV3-21.

In a preferred embodiment of the present invention, according to the BAFF-R humanized antibody or fragment thereof provided by the present invention, the humanized antibody heavy chain variable region sequence is selected from the sequence shown in SEQ ID NO: 23, 24, 25, 26, 31, 32, 33 or a variant thereof;

In a preferred embodiment of the present invention, the BAFF-R humanized antibody or fragment thereof provided according to the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprises a human IgG1 or IgG4 heavy chain constant region, more preferably comprises a human IgG1 constant region.

In a preferred embodiment of the present invention, the BAFF-R humanized antibody or fragment thereof provided by the present invention, wherein the FR region sequence on the light chain variable region of the humanized antibody is derived from human germline light chain IGKV2-30 or/and IGKV2-29.

In a preferred embodiment of the present invention, the BAFF-R humanized antibody or fragment thereof provided by the present invention, wherein the humanized antibody light chain variable region sequence is selected from the sequence shown in SEQ ID NO: 27, 28, 29, 30, 34, 35, 36, 37 or a variant thereof.

In a preferred embodiment of the present invention, the BAFF-R humanized antibody or fragment thereof provided according to the present invention further comprises a light chain constant region of human κ, λ chain or a variant thereof, preferably a light chain constant region of human κ chain or a variant thereof.

In a preferred embodiment of the present invention, the BAFF-R binding molecule provided by the present invention is an antibody or an antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fab', Fv, sFv, F(ab')2.

In a preferred embodiment of the present invention, the BAFF-R binding molecule provided by the present invention is an antibody or an antigen-binding fragment thereof that is glycoengineered.

The present invention further provides a nucleic acid sequence or combination encoding a BAFF-R binding molecule as described above.

The present invention further provides an expression vector containing the nucleotide sequence or combination as described above.

The present invention further provides a host cell transformed with the expression vector as described above. The host cell includes a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell, preferably a mammalian cell, more preferably a HEK293F cell, an ExpiCHO S cell or a CHO-K1 cell, wherein the CHO-K1 cell is preferably a FUT8 biallelic knockout cell.

The present invention also provides a medicine or a pharmaceutical composition, which contains the anti-BAFF-R binding molecule as described above and one or more pharmaceutically acceptable carriers, diluents or excipients.

The present invention provides a method for preparing a drug for treating cancer and/or autoimmune diseases comprising an anti-BAFF-R binding molecule as described above, or a pharmaceutical composition as described above, or a nucleotide sequence or combination as described above;

Preferably, the cancer disease includes non-Hodgkin's lymphoma (B-NHL), various types of chronic lymphocytic leukemia (CLL), primary acute lymphocytic leukemia (ALL), and multiple myeloma; the autoimmune disease includes: systemic lupus erythematosus, idiopathic pulmonary fibrosis, rheumatoid arthritis (RA), primary Sjögren's syndrome (PSS), autoimmune hepatitis, multiple sclerosis, myasthenia gravis, IgA nephropathy, neuromyelitis optica, granulomatosis with polyangiitis, microscopic polyangiitis, immune thrombocytopenic purpura, and idiopathic thrombocytopenic purpura.

Beneficial effects:

The BAFF-R binding molecule provided by the present invention has a binding epitope different from that of MOR6654-hG1 (VAY-736), and has significantly higher affinity and in vitro pharmacodynamic activity than those of MOR6654-hG1 (VAY-736).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Comparison of binding activity of mouse B7G10, B12E9 antibodies and MOR6654-mG2a antibody in ELISA-binding assay in Example 3

Figure 2: Comparison of blocking activity of mouse B7G10, B12E9 antibodies and MOR6654-mG2a/hG1 antibody in ELISA-binding assay in Example 3

Figure 3: Comparison of binding activity of murine B7G10, B12E9 antibodies and MOR6654-mG2a antibody in FACS-binding assay in Example 3

Figure 4: Comparison of blocking activity of murine B7G10, B12E9 antibodies and MOR6654-mG2a antibody in FACS-binding assay in Example 3

Figure 5: Comparison of cross-reactivity between mouse B7G10 and B12E9 antibodies and Mus-BAFFR in ELISA-binding assay in Example 3

FIG6A : Example 3 Antibody Competition Binding ELISA Assay (Biotin-labeled B12E9 added during primary antibody incubation)

FIG6B : Example 3 Antibody competition binding ELISA test (biotin-labeled B7G10 was added during primary antibody incubation)

Figure 7: Example 5 ELISA test - Comparison of antibody binding activity of B7G10-CHI, B12E9-CHI and MOR6654-hG1

Figure 8: Comparison of antibody blocking activity of B7G10-CHI, B12E9-CHI and MOR6654-hG1 detected by ELISA in Example 5

Figure 9: Comparison of antibody binding activity of B7G10-CHI, B12E9-CHI and MOR6654-hG1 detected by FACS in Example 5

Figure 10A: Example 7B-7G10 humanized antibody binding activity evaluation

Figure 10B: Example 7B-12E9 humanized antibody binding activity evaluation

Figure 11A: Example 7B-7G10 humanized antibody blocking activity evaluation

Figure 11B: Example 7B-12E9 humanized antibody blocking activity evaluation

Figure 12: Example 8 Screening of B7G10CHI based on PBMC ADCC activity method

Figure 13: Example 8: Screening of B7G10CHI by reporter gene assay for ADCC activity

Figure 14: Example 8: Screening of Hu7G10 molecules by measuring ADCC activity using reporter gene method

Figure 15: Example 8: Reporter gene method to measure ADCC activity of Hu7G10-22AF

Figure 16: Example 8: Reporter gene method for measuring ADCC activity of Hu7G10-22AF and Hu7G10-22 mixed in different ratios

DETAILED DESCRIPTION

1. Terminology

In the present invention, the CDR is a complementarity-determining region; the ScFv is a single-chain fragment variable; the HEK293F cell is a human embryonic kidney 293F cell; the CHO cell is a Chinese hamster ovary cell; the ExpiCHO S cell (Thermo Fisher) is a CHO cell grown in suspension; and the CHO-K1 is a CHO cell grown in adherence.

The term "antigen-binding fragment" in the present invention refers to an antibody fragment formed by an antibody fragment containing one or more CDRs or any other antibody fragment that binds to an antigen but does not have a complete native antibody structure. In certain embodiments, the antibody described in the present application is an antigen-binding fragment.

The "Fab" fragment of an antibody refers to a monovalent antigen-binding fragment of an antibody, which consists of a light chain (including a variable region and a constant region) and a variable region and a first constant region of a heavy chain bound by a disulfide bond. Fab can be obtained by digesting with papain at the residues near the N-terminus of the disulfide bond between the heavy chains in the hinge region of the antibody.

"F(ab)2" refers to a dimer of Fab, which comprises two light chains and a portion of two heavy chains.

The "Fv" fragment of an antibody refers to the smallest antibody fragment that contains a complete antigen binding site. The Fv fragment consists of the variable region of one light chain bound to the variable region of one heavy chain.

"scFv" refers to an engineered antibody composed of a light chain variable region connected directly to a heavy chain variable region or connected through a polypeptide linker sequence.

2. Example

Example 1 Preparation of BAFF-R, BAFF and Control Antibodies (Can be Simplified)

1.1 Construction, expression and purification of eukaryotic expression vector

According to the public BAFF-R (Uniprot: Q96RJ3) sequence, its extracellular domain (ECD) was intercepted, SEQ ID NO: 1, reversely translated into a coding DNA sequence, SEQ ID NO: 2-4, and gene synthesis was performed using the overlapping PCR method, and constructed into the pHr expression vector. By design, the expressed BAFF-R-ECD protein was equipped with different tags, namely BAFF-R-ECD-Hisx6, BAFF-R-ECD-mFc and BAFF-R-ECD-hFc. The C-terminus of BAFF-R-ECD was connected to Hisx6, mFc or hFc tags for easy purification and detection. Among them, Hisx6 represents a continuous 6 histidine peptide segment, mFc is the Fc segment of mouse IgG2a, and hFc is the Fc segment of human IgG1. In the same way, the BAFF extracellular domain/soluble fragment was intercepted, cloned and constructed, and the sequence is shown in SEQ ID NO: 5 and 6.

1.1.1 BAFF/-R-ECD-mFc/-hFc protein expression and purification

As described by the manufacturer, pHr-BAFF-R-ECD-mFc or pHr-BAFF-R-ECD-hFc plasmid DNA encoding the BAFF-R extracellular domain protein was transiently transfected into ExpiCHO S cells (Thermo Fisher) using the ExpiFectamine CHO transfection kit (Gibco). The cells were cultured in ExpiCHO-S expression medium for 10 days and the cell supernatant was collected for protein purification. The CHO cell supernatant was centrifuged at 6,000 × g for 10 min and filtered through a 0.45 μm filter membrane for later use. Hitrap MabSelectSuReProtein A prepacked column (GE, #11-0034-93-GEC) was equilibrated with 10 volumes of buffer 1# (PBS, pH 7.4). The sample was loaded using an AKTA instrument at a flow rate of 1 ml/min. Buffer 1# was used for washing, about 30 ml, until the A280 value stabilized. Keep the rate constant, use buffer 3# (100mM glycine, 150mM NaCl, pH3.0) to elute the protein bound to the column, and collect the peak with A280 value above 30. Add 15μL/ml neutralization buffer (1M Tris-HCl, pH9.0) to the collection tube to keep the pH value of the obtained antibody solution neutral (pH=7.0-8.0). Use 10KD ultrafiltration tube to replace and concentrate the eluate, replace the buffer with PBS, and detect the protein concentration by Nanodrop and the protein purity by SDS-PAGE.

Nanodrop was used to detect the light absorption of the eluted BAFF-R-ECD-mFc/Fc protein solution at a wavelength of 280 nm, and the obtained value was divided by the theoretical absorption coefficient to estimate its concentration. SDS-PAGE non-reducing electrophoresis and Coomassie brilliant blue staining were used to detect protein purity, which was greater than 95%.

1.1.2 BAFF-R-ECD-HIS protein expression and purification

The pHr-BAFF-R-ECD–Hisx6 plasmid DNA encoding the BAFF-R-ECD protein was transiently transfected into K293 F cells (Zhuhai Kairui Biotechnology Co., Ltd.) using a K293 transfection kit (Zhuhai Kairui Biotechnology Co., Ltd.) as described by the manufacturer, and the cells were cultured in K293 expression medium for 7 days, and the cell supernatant was collected for protein purification.

The supernatant of 293 cells cultured for 7 days was collected, centrifuged at 6,000×g for 10 minutes, and filtered with a 0.45μm filter membrane for later use. The HIS-excel pre-packed column (GE, 17-3712-05) was washed with 10 times the column volume of deionized water, and the column was balanced with 10 times the column volume of PBS buffer. The AKTA instrument was used to load the sample at a flow rate of 1mL/min. 50mL PBS-10 imidazole was used to wash the impurities, and the target protein was eluted with PBS-500 imidazole buffer. The eluate was buffer exchanged and concentrated using a 10KD ultrafiltration tube, and the buffer was replaced with PBS. The crude protein was chromatographed again by DEAE to obtain a pure product, which was electrophoresed by SDS-PAGE and stained with Coomassie Brilliant Blue, and its purity was determined to be 80%.

1.2 Construction, expression and purification of eukaryotic expression vectors for control antibodies MOR6654-mG2a and MOR6654-hG1

In some embodiments, comparative antibodies against BAFF-R (MOR6654-mG2a, MOR6654-hG1) were used, and the patented anti-BAFFR antibody preparation MOR6654 (patent application publication number: CN 104363920 A) was cited, and its VH and VL sequences were cloned into pHr-hG1/pHr-hkappa and pHr-mG2a/pHr-mkappa expression vectors. One-step purification was performed by Protein A affinity chromatography to obtain a sample with a purity greater than 95%.

Example 2 Animal immunization and antibody screening

2.1 Immunization of mice

Select 6-8 week old SJL female mice (Jackson Lab), generally about 20 grams, healthy and disease-free. Freund's complete adjuvant was used for the first immunization, and the ratio of BAFF-R-ECD-hFc immune antigen to immune adjuvant was 1:1. Before immunization, the antigen and adjuvant only needed to be gently mixed. The abdominal subcutaneous multi-point injection method was 50μg/mouse. After an interval of two weeks, booster immunization was performed, using Freund's incomplete adjuvant, 30μg/mouse. A total of 3 booster immunizations were performed. Ten days after the last booster immunization, blood was collected from the medial canthal sinus of the orbit for ELISA to detect antibody titers. Then a sprint immunization was performed. At this time, no adjuvant was added and only the antigen was used for intraperitoneal immunization, 50μg/mouse. Cell fusion was performed 3 days after the sprint immunization.

2.2 ELISA method to detect antibody titer

ELISA plate wells were coated with BAFF-R-ECD-Hisx6 at a concentration of 2μg/mL, 50μl/well. After blocking, 50μl of gradient diluted mouse serum to be tested was added to each well, and negative control wells (non-immunized mouse serum) were set up at the same time, and incubated at 37℃ for 1h; after rinsing, anti-mouse-IgG-HRP enzyme-labeled secondary antibody was added, 50μl per well, and incubated at 37℃ for 1h; after washing, 50μl of substrate solution TMB was added, color development was performed at 37℃ for 2-15min, 50μl 2mol/LH ₂ SO ₄ was used to terminate the reaction, and the OD ₄₅₀ value was read by a microplate reader. According to the experimental results, the antibody titer reached 10 to the 4th power or more, and cell fusion could be performed.

Table 1 Mouse titer test results

Dilution multiple 200 400 800 1600 3200 6400 12800 25600 51200 102400 blank Negative B-h-1 1.644 1.572 1.576 1.624 1.52 1.428 1.424 1.231 0.952 0.707 0.06 0.059 B-h-3 1.691 1.573 1.593 1.639 1.593 1.396 1.326 1.263 0.912 0.715 0.077 0.069 B-h-4 1.61 1.555 1.545 1.508 1.498 1.329 1.229 1.077 0.73 0.518 0.062 0.067 B-h-5 1.646 1.653 1.55 1.538 1.54 1.429 1.285 1.136 0.82 0.566 0.056 0.064

2.3 Cell fusion and initial screening of positive hybridoma cells and cloning cell fusion

SP2/0 myeloma cells in logarithmic growth phase were gently blown off from the flask wall tumor with an elbow dropper, washed twice with 1640 culture medium, and then resuspended in 10ml 1640 culture medium, mixed, and counted with a blood cell counting plate. The mice were killed by pulling the neck, dissected, the spleen was taken and ground, spleen cells were collected, and counted. Splenocytes and myeloma cells SP2/0-Ag14 were mixed in a ratio of 5:1-2:1, PEG1450 (Sigma) was used for cell fusion, 400ml HAT culture medium (1640 culture medium, 20% serum, double antibody 5ml, HAT storage solution 5ml, fixed to 500ml) was added, and the cells were divided into 20 96-well plates, cultured in a 37°C, 5% CO2 incubator for 7 days, and then the cell supernatant was taken for antibody screening detection.

2.4 Enzyme-linked immunosorbent assay (ELISA) screening of positive hybridoma wells

Coat with BAFF-ECD-hFc antigen at a concentration of 2 μg/mL, add 50 μl of a 1:1 mixture of the culture supernatant of the hybridoma cells to be tested and BAFF-R-ECD-mFc (2 μg/ml) blocking detection mixed reaction solution to each well, and set up negative control wells (PBS+1% BSA) and positive control wells (MOR6654-mG2a, concentration of 20 μg/mL) for incubation at 37°C for 1 hour; after washing, add anti-mouse-IgG-HRP enzyme-labeled secondary antibody, 50 μl/well, and incubate at 37°C for 1 hour; after washing, add 50 μl of color development solution TMB, develop at 37°C for 2-15 minutes, terminate the reaction with 50 μl/well 2mol/L H ₂ SO ₄ , and read the OD ₄₅₀ value with an enzyme reader.

Coat with BAFF-R-ECD-hFc antigen at a concentration of 2 μg/mL, add 50 μl of the culture supernatant of the hybridoma cells to be tested to each well, and set up negative control wells (PBST+1% BSA) and positive control wells (MOR6654-mG2a, concentration of 20 μg/mL) at the same time, and incubate at 37°C for 1 hour; after washing, add anti-mouse-IgG-HRP enzyme-labeled secondary antibody, 50 μl per well, and incubate at 37°C for 1 hour; after washing, add 50 μl of color development solution TMB, develop at 37°C for 2-15 minutes, terminate the reaction with 50 μl/well 2mol/L H ₂ SO ₄ , and read the OD ₄₅₀ value with a microplate reader.

Two antibody strains with higher hybridoma supernatant than positive antibody strains and higher blocking efficiency were screened: B7G10 and B12E9.

The B7G10 and B12E9 hybridoma cells were subcloned by limiting dilution method, and monoclonal cell lines B7G10 and B12E9 were obtained by enzyme-linked immunosorbent assay (ELISA) screening.

2.5 Production, purification and identification of mouse monoclonal antibodies

Take the expanded cultured and counted B7G10-C2, B7G10-D2, B12E9-B2, and B12E9-B5, with a cell number of 5×10 ⁶ , and transfer them into T75 bottles. Put 20 ml of culture medium (CD Hybridoma + 1% P/S + 8mM L-Glu) in each bottle, with a density of 2.5×10 ⁵ cells/mL. Culture at 37°C and 5% CO ₂ for 5 days, and then collect the cell supernatant for antibody purification. Use MabSelect SuRe (GE, #11-0034-93-GEC) Protein A pre-packed column to affinity purify mouse monoclonal antibodies B7G10 and B12E9. The purity is determined to be greater than 95% by SDS-PAGE and Coomassie Brilliant Blue staining. Use Nanodrop instrument to perform light absorption and concentration calculation, and freeze in a -80°C refrigerator after labeling.

Example 3 Determination of biochemical activity of mouse B7G10 and B12E9 monoclonal antibodies

The activities of mouse B7G10, B12E9 antibodies (B7G10-C2, B7G10-D2, B12E9-B2, B12E9-B5) and MOR6654-mG2a antibody were compared by ELISA-binding, blocking and FACS-binding, blocking experiments.

3.1 ELISA-binding activity analysis

BAFFR-hFc coating solution was diluted to 2μg/mL, and added to the wells of the ELISA plate at 50μL/well for incubation. Then 200μL of blocking solution (5% skim milk) was added to each well and placed at 4°C overnight. The next day, the wells were washed 5 times with PBST (PBS, 0.05% Tween 20) washing solution 200μl/well. Next, the antigen-antibody reaction was performed, that is, 50μL of B7G10-C2, B7G10-D2, B12E9-B2, B12E9-B5 and MOR6654-mG2a were added. The maximum concentration of each test product was 20μg/mL, and a gradient dilution was performed at a ratio of 5 times. At the same time, a blank control was set up; incubated at 37°C for 60min. After the reaction, the plate was rinsed, and the second antibody Goat-anti-mouse-IgG-Fc-Secondary-Antibody (1:10000 dilution, Jackson Immun) was added and incubated; after the secondary antibody reaction was completed, the plate was rinsed again, and then the color developing solution was added, and the light absorption OD ₄₅₀ value was read after incubation for 15 minutes. EC ₅₀ of B7G10-C2 = 12.74, EC ₅₀ of B7G10-D2 = 13.97, EC ₅₀ of B12E9-B2 = 5.358, EC ₅₀ of B12E9-B5 = 8.811, and EC ₅₀ of MOR6654-mG2a = 3.837 (all in ng/mL).

The results are shown in Figure 1. At the ELISA binding level, the binding activity of B7G10 and B12E9 was higher than that of the positive reference MOR6654-mG2a.

3.2 ELISA-blocking activity analysis

Dilute BAFF-ECD-hFc coating solution to 2μg/mL for coating. Add 50μL/well of coating to the wells of the ELISA plate and incubate at 37℃ for 2 hours. Discard the liquid in the wells and wash 5 times with a plate washer at 200μl/well of washing solution. Add 200μL of blocking solution to each well and incubate overnight at 4℃. Discard the liquid in the wells and wash 5 times with a plate washer at 200μl/well of washing solution. 25 μL BAFF-R-ECD-mFc (4 μg/mL) and 25 μL B7G10-C2, B7G10-D2, B12E9-B2, B12E9-B5 or MOR6654-mG2a, MOR6654-hG1 were added to each well, with the concentration of 20 μg/mL in the first well, and then diluted 5 times in sequence, with 7 different concentrations, and a blank control was set at the same time; pre-incubated at 37°C for 30 minutes, transferred to a plate, and incubated at 37°C for 120 minutes; the liquid in the well was discarded, and the plate was washed 5 times with a plate washer and 200 μl/well of washing solution. 50 μL of the second antibody Goat-anti-Mouse-IgG-Fc-Secondary-Antibody (1:10000 dilution) was added to each well, incubated at 37°C for 60 minutes; the liquid in the well was discarded, and the plate was washed 5 times with a plate washer and 200 μl/well of washing solution. Add 50 μL/well of colorimetric solution and incubate at 37°C for 15 min. Add 50 μL/well of 2 mol/L H ₂ SO ₄ to terminate the reaction and read the OD ₄₅₀ value on a microplate reader. The results are shown in Figure 2. At the ELISA-blocking level, the blocking activity of B7G10 and B12E9 was higher than that of the positive references MOR6654-mG2a and MOR6654-hG1.

3.3 FACS-binding activity comparison

Raji cell counting, 3E5 cells were added to each well, washed twice with PBS; B7G10-C2, B7G10-D2, B12E9-B2, B12E9-B5 or MOR6654-mG2a were added to the wells at a concentration of 20 μg/mL in the first well, diluted 5 times in sequence, 7 gradient antibodies, and blank controls were set at the same time; incubated at 4°C for 30 minutes; centrifuged at 1000 rpm for 2 minutes, the liquid in the well was discarded, PBS was washed, and 200 μl/well was washed twice. 50 μL of PE-anti-Mouse-IgG-Fc-Secondary-Antibody (1:200 dilution) was added to each well, incubated at 4°C for 30 minutes; centrifuged at 1000 rpm for 2 minutes, the liquid in the well was discarded, PBS was washed, and 200 μl wells were washed twice, centrifuged at 1000 rpm for 2 minutes; 200 μL/well PBS was added, the cells were resuspended, and the flow cytometer was read. The results are shown in Figure 3. At the FACS-binding level, the binding activity of B7G10 and B12E9 was higher than that of the positive reference MOR6654-mG2a.

3.4 Comparison of FACS blocking activity

Raji cell counting, 3E5 cells were added to each well, washed twice with PBS; 25 μL BAFF-ECD-hFc (concentration of 20 μg/mL) + 25 μL B7G10-C2, B12E9-B5 or MOR6654-mG2a at a concentration of 20 μg/mL, 5-fold dilution, 7 gradient antibodies were added to the well, and a blank control was set at the same time; incubated at 4°C for 30 minutes; centrifuged at 1000 rpm for 2 minutes, the liquid in the well was discarded, and the PBS wash solution was washed twice with 200 μl/time. 50 μL PE-labeled anti-Human-IgG-Fc (1:200 dilution) secondary antibody was added to each well, incubated at 4°C for 30 minutes; centrifuged at 1000 rpm for 2 minutes, the liquid in the well was discarded, and the PBS wash solution was washed twice with 200 μl/time, and centrifuged at 1000 rpm for 2 minutes; 200 μL/well PBS solution was added, the cells were resuspended, and the flow cytometer was read. The results are shown in Figure 4: At the FACS-blocking level, the blocking activity of B7G10-C2 and B12E9-B5 was equivalent to the positive reference MOR6654-mG2a, and their blocking activity gradually increased with increasing doses, improving the controllability of the dose used.

3.5 Cross-reactivity

Dilute Mus-BAFFR coating solution to 1μg/mL, add 50μL/well of coating to the ELISA plate wells, and incubate at 37℃ for 2 hours. Discard the liquid in the wells, wash 5 times with 200μl/well of washing solution using a plate washer. Add 200μL of blocking solution to each well and incubate overnight at 4℃. Discard the liquid in the wells, wash 5 times with 200μl/well of washing solution using a plate washer. Add 50μL of B7G10-C2, B7G10-D2, B12E9-B2, B12E9-B5 at a concentration of 20μg/mL, 5-fold dilution, 7 gradient antibodies to each well, incubate at 37℃ for 60min; discard the liquid in the wells, wash 5 times with 200μl of washing solution using a plate washer. Add 50 μL of Goat-anti-mouse-IgG-Fc-Secondary-Antibody (1:10000 dilution) to each well and incubate at 37°C for 60 min; discard the liquid in the well and wash 5 times with a plate washer, 200 μL/time of washing solution. Add 50 μL/well of colorimetric solution and incubate at 37°C for 15 min. Add 50 μL/well of 2 mol/L H ₂ SO ₄ to terminate the reaction and read the OD ₄₅₀ value on an ELISA reader. The results are shown in Figure 5: B-12E9-B2 and B-12E9-B5 have cross-reactions with Mus; B-7G10-C2 and B-7G10-D2 have no cross-reactions with Mus.

3.6 Competition binding between antibodies

To explore whether the binding epitopes of B7G10, B12E9 and the reference antibody are consistent, we performed a competition ELISA experiment between antibodies. Antigen coating, antibody binding and secondary antibody detection were consistent with normal, except that 100ng of biotin-labeled B7G10 or B12E9 was added during the primary antibody incubation. The results are shown in Figures 6A and 6B: MOR6654-mG2a does not compete with B7G10 or B12E9 antibodies for binding to BAFF-R, while B7G10 and B12E9 can compete with each other for binding to BAFF-R. This indicates that B7G10 and B12E9 bind to the same or similar epitopes on the BAFF-R-ECD molecule, but are different from the binding epitopes of the reference antibody.

Example 4 Sequencing of variable regions of B7G10 and B12E9 antibodies and construction of B7G10-CHI and B12E9-CHI chimeric antibodies

5×106 B7G10 and B12E9 hybridoma cells were collected, washed twice with PBS, and RNA was extracted according to RNAprep Pure cultured cell/bacterial total RNA extraction kit (Tiangen, DP430). Nanodrop was used for quantification. Reverse transcription was performed according to the instructions of High Capacity RNA-to-cDNA Kit (Thermo fisher, 4387406). Primers were designed according to the method of Dubel, S., and the variable regions of H and K chains of B7G10 and B12E9-F11 cell lines were amplified by PCR, and the PCR products were directly sequenced (Dubel, S. Isolation of IgG antibody Fv-DNA from various mouse and rat hybridoma cell lines using the polymerase chain reaction with a simple set of primers. Journal of Immunological Methods 175 (1): 89-95, 1994). The DNA sequence obtained by Sanger sequencing was compared and analyzed in the IMGT-V quest database (Brochet, X. et al., Nucl. Acids Res. 36, W503-508 (2008). PMID: 18503082.), confirming that the two monoclonal strains B7G10-C2 and D2 obtained by subcloning of B7G10 were single sequences, and their DNA sequences were SEQ ID 7 and SEQ ID 8. After comparison and translation, their amino acid sequences should be SEQ ID 9 and SEQ ID 10; similarly, the two monoclonal strains of B12E9 also had the same sequence, and their heavy chain and light chain variable region DNA sequences were SEQ ID 11 and SEQ ID 12, and the amino acid sequences after comparison and translation were SEQ ID 13 and SEQ ID 14. The amino acid sequences of the light and heavy chain V regions of B7G10 and B12E9 were grafted to the constant regions of human κ and G1, and the sequences were shown in SEQ ID 15, SEQ ID 16, SEQ ID 17 and SEQ ID 18. The amino acid sequences were reverse translated into DNA and then gene synthesized, and the sequences were shown in SEQ ID 19, SEQ ID 20, SEQ ID 21 and SEQ ID 22. The chimeric antibodies B7G10-CHI and B12E9-CHI were obtained by constructing them onto a pHr vector using genetic engineering technology, and then transiently transfected into an ExpiCHO S cell (Thermo Fisher) expression system for protein expression. The expressed chimeric antibodies were affinity chromatographed using a Hitrap MabSelect SuRe chromatography column to obtain purified antibodies with a purity greater than 95%.

Example 5 Evaluation of the activity of B7G10-CHI and B12E9-CHI chimeric antibodies

The affinity of antibodies B7G10-CHI and B12E9-CHI to the antigen BAFFR was tested by ELISA-binding, ELISA-blocking and FACS-binding assays to compare the activities of B7G10-CHI, B12E9-CHI and MOR6654-hG1 antibodies.

5.1 ELISA-Binding Activity Comparison

Dilute BAFFR-mFc coating solution to 2μg/mL, add 50μL/well of coating to the ELISA plate wells, and incubate at 37℃ for 2 hours. Discard the liquid in the wells and wash 5 times with 200μl/well of washing solution using a plate washer. Add 200μL of blocking solution to each well at 4℃ overnight. Discard the liquid in the wells and wash 5 times with 200μl/well of washing solution using a plate washer. Add 50μL of B7G10, B12E9-CHI or MOR6654-hG1 at a concentration of 20μg/mL, 5-fold dilution, 7 gradient antibodies to each well, and set up a blank control at the same time; incubate at 37℃ for 60min; discard the liquid in the wells and wash 5 times with 200μl/well of washing solution using a plate washer. Add 50 μL of Goat-anti-human-IgG-Fc-Secondary-Antibody (1:10000 dilution) to each well and incubate at 37°C for 60 min; discard the liquid in the well and wash 5 times with a plate washer, 200 μL/well of washing solution. Add 50 μL/well of colorimetric solution and incubate at 37°C for 15 min. Add 50 μL/well of 2 mol/L H ₂ SO ₄ to terminate the reaction and read the OD ₄₅₀ value on an ELISA reader. The results are shown in Figure 7: At the ELISA-binding level, the binding activity of B7G10-CHI and B12E9-CHI was higher than that of the positive reference MOR6654-hG1.

5.2 ELISA-blocking activity comparison

BAFF-hFc coating solution was diluted to 2μg/mL, and 50μL/well of coating was added to the ELISA plate wells, and incubated at 37℃ for 2 hours. Discard the liquid in the wells, and wash 5 times with 200μl/well of washing solution using a plate washer. Add 200μL of blocking solution to each well at 4℃ overnight. Discard the liquid in the wells, and wash 5 times with 200μl/well of washing solution using a plate washer. Add 25μLBAFFR-mFc (4μg/mL) + 25μL of 7G10-CHI, 12E9-CHI or MOR6654-hG1 to each well, with a concentration of 20μg/mL in the first well, and dilute 5 times in sequence, 7 gradient antibodies, and set up a blank control at the same time; incubate at 37℃ for 120min; discard the liquid in the wells, and wash 5 times with 200μl/time of washing solution using a plate washer. Add 50 μL of Goat-anti-Mouse-IgG-Fc-Secondary-Antibody (1:10000 dilution) to each well and incubate at 37°C for 60 min; discard the liquid in the well and wash 5 times with a plate washer, 200 μL of washing solution per time. Add 50 μL/well of colorimetric solution, incubate at 37°C for 15 min, add 50 μL/well of 2 mol/L H ₂ SO ₄ to terminate the reaction, and read the OD ₄₅₀ value on an ELISA reader. The results are shown in Figure 8: At the ELISA-binding level, the blocking activity of B7G10-CHI and B12E9-CHI was higher than that of the positive reference MOR6654-hG1.

5.3 Comparison of binding activity by FACS

Raji cell counting, 3E5 cells were added to each well, washed twice with PBS; B7G10-CHI, B12E9-CHI or MOR6654-hG1 was added to the well at a concentration of 20μg/mL, 5-fold dilution, 7 gradient antibodies, and a blank control was set at the same time; incubated at 4℃ for 30min; centrifuged at 1000rpm for 2min, the liquid in the well was discarded, PBS was washed twice with 200μl/well. 50μL of PE-anti-Mouse-IgG-Fc-Secondary-Antibody (1:200 dilution) was added to each well, incubated at 4℃ for 30min; centrifuged at 1000rpm for 2min, the liquid in the well was discarded, PBS was washed twice with 200μl/well, centrifuged at 1000rpm for 2min; 200μL/well PBS was added, the cells were resuspended, and the flow cytometer was read. The results are shown in FIG9 : at the FACS-binding level, the binding activities of B7G10-CHI and B12E9-CHI were higher than that of the positive reference MOR6654-hG1.

Example 6 Construction and purification of B7G10 and B12E9 humanized antibody expression vectors

5×10 ⁶ B7G10 and B12E9 hybridoma cells were collected and washed twice with PBS. RNA was extracted according to the RNAprep Pure cultured cell/bacterial total RNA extraction kit (Tiangen, DP430). Nanodrop was used for quantification. Reverse transcription was performed according to the instructions of HighCapacity RNA-to-cDNA Kit (Thermo fisher, 4387406), and the variable regions of the H and K chains of B7G10 and B12E9 cell lines were amplified by PCR and sequenced. The proteins were ligated to the eukaryotic expression vectors PHR-XK and PHR-XG by restriction endonucleases, and the expiCHO S cell expression system (Thermo Fisher) was used for protein expression, and the Hitrap MabSelectSuRe chromatography column was used for protein purification.

The protein concentration of Hu-7G10-02 detected by Nanodrop was 0.8811 mg/ml, and the protein purity was detected by 12% SDS-PAGE, which was greater than 95%; the protein concentration of Hu-7G10-22 detected by Nanodrop was 1.2911 mg/ml, and the protein purity was detected by 12% SDS-PAGE, which was greater than 95%; the protein concentration of Hu-7G10-32 detected by Nanodrop was 1.1859 mg/ml, and the protein purity was detected by 12% SDS-PAGE, which was greater than 95%; the protein concentration of Hu-7G10-42 detected by Nanodrop was 1.7346 mg/ml, and the protein purity was detected by 12% SDS-PAGE, which was greater than 95%; the protein concentration of Hu-7G10-42 detected by Nanodrop was 0.6432 mg/ml, and the protein purity was detected by 12% SDS-PAGE, which was greater than 95%.

The protein concentration of Hu-12E9-04 detected by Nanodrop was 1.1189 mg/ml, and the protein purity was detected by 12% SDS-PAGE, which was greater than 95%; the protein concentration of Hu-12E9-20 detected by Nanodrop was 1.5504 mg/ml, and the protein purity was detected by 12% SDS-PAGE, which was greater than 95%; the protein concentration of Hu-12E9-22 detected by Nanodrop was 1.4660 mg/ml, and the protein purity was detected by 12% SDS-PAGE, which was greater than 95%; the protein concentration of Hu-12E9-23 detected by Nanodrop was 1.5578 mg/ml, and the protein purity was detected by 12% SDS-PAGE, which was greater than 95%; The protein purity was detected by SDS-PAGE, and the purity was greater than 95%; the protein concentration of Hu-12E9-24 detected by Nanodrop was 1.6026 mg/ml, and the protein purity was detected by 12% SDS-PAGE, and the purity was greater than 95%; the protein concentration of Hu-12E9-30 detected by Nanodrop was 1.4539 mg/ml, and the protein purity was detected by 12% SDS-PAGE, and the purity was greater than 95%; the protein concentration of Hu-12E9-32 detected by Nanodrop was 1.1169 mg/ml, and the protein purity was detected by 12% SDS-PAGE, and the purity was greater than 95%; the protein concentration of Hu-12E9-33 detected by Nanodrop was 0.9420 mg/ml, and the protein purity was detected by 12% SDS-PAGE, and the purity was greater than 95%; the protein concentration of Hu-12E9-34 detected by Nanodrop was 1.3293 mg/ml, and the protein purity was detected by 12% SDS-PAGE, and the purity was greater than 95%.

Example 7 Humanization transformation and screening of B7G10 and B12E9

Homologous sequence modeling-antibody complementary determining region (CDRs) transplantation-framework region (FR) key amino acid back mutation technology was used to humanize the murine antibodies B7G10 and B12E9. First, through sequence alignment, the most similar antibody germline sequences to the light and heavy chains of B7G10 and B12E9 were found in the known database, as shown in Table 2.

Table 2

The light and heavy chain genes of the mouse antibody are transplanted to the human antibody germline sequence with the highest sequence homology, and then the known antibodies with the most similar structures are searched in the PDB database, and structural modeling analysis is performed to find the key amino acids in the FR region. These key amino acids are involved in the interaction between the light and heavy chains, affecting the structural stability, and then affecting antigen binding. Therefore, they need to be consistent with the corresponding positions of the FR of the parent (mouse) antibody, and the amino acid sites are carefully selected to mutate back to the parent amino acid residues. The more back mutation sites there are, the lower the degree of humanization will be. Therefore, it is necessary to test the activity of antibodies with different degrees of back mutation sequences, and select the antibodies with the best activity and the least back mutation sites as the preferred ones. The back mutation sites were determined by CDR transplantation and structural modeling, and the alternative antibody light and heavy chain sequences are shown in Table 3 below.

Table 3: Alternative antibody light and heavy chain sequences

The alternative light and heavy chain variable region sequences were reverse translated into DNA sequences, and the heavy chain and human IgG1 constant region, and the light chain and human κ constant region coding sequences were concatenated together, and then gene synthesis was performed, and the complete antibody light and heavy chain coding genes were constructed into the pHr vector. Then, ExpiCHO cells were transiently transfected for antibody expression, wherein, during transfection, different light and heavy chain alternative sequences were paired and introduced into the expression cells together, as shown in Tables 4A and B below.

Table 4A: B7G10 humanized antibody combinations and numbers

VH0 VH1 VH2 VH3 VH4 VL0 Hu-7G10-00 Hu-7G10-10 Hu-7G10-20 Hu-7G10-30 Hu-7G10-40 VL1 Hu-7G10-01 7G10-CHI Hu-7G10-21 Hu-7G10-31 Hu-7G10-41 VL2 Hu-7G10-02 Hu-7G10-12 Hu-7G10-22 Hu-7G10-32 Hu-7G10-42 VL3 Hu-7G10-03 Hu-7G10-13 Hu-7G10-23 Hu-7G10-33 Hu-7G10-43 VL4 Hu-7G10-04 Hu-7G10-14 Hu-7G10-24 Hu-7G10-34 Hu-7G10-44

Table 4B: B12E9 humanized antibody combinations and numbers

VH0 VH1 VH2 VH3 VL0 Hu-12E9-00 Hu-12E9-10 Hu-12E9-20 Hu-12E9-30 VL1 Hu-12E9-01 12E9-CHI Hu-12E9-21 Hu-12E9-31 VL2 Hu-12E9-02 Hu-12E9-12 Hu-12E9-22 Hu-12E9-32 VL3 Hu-12E9-03 Hu-12E9-13 Hu-12E9-23 Hu-12E9-33 VL4 Hu-12E9-04 Hu-12E9-14 Hu-12E9-24 Hu-12E9-34

7.1 ELISA-Binding Activity Assessment

7.1.1 7G10 humanized antibody binding activity evaluation (ELISA)

Dilute BAFFR-mFc coating solution to 2μg/mL, add 50μL/well of coating to the ELISA plate wells, and incubate at 37℃ for 2 hours. Discard the liquid in the wells and wash 5 times with 200μl/well of washing solution using a plate washer. Add 200μL of blocking solution to each well at 4℃ overnight. Discard the liquid in the wells and wash 5 times with 200μl/well of washing solution using a plate washer. Add 50μL of Hu-7G10-02, Hu-7G10-22, Hu-7G10-32, Hu-7G10-42, Hu-7G10-44, or 7G10-CHI to each well with a concentration of 20μg/mL in the first well, 5-fold dilution, 7 gradient antibodies, and set up a blank control at the same time; incubate at 37℃ for 60min; discard the liquid in the wells and wash 5 times with 200μl/well of washing solution using a plate washer. Add 50 μL of Goat-anti-human-IgG-Fc-Secondary-Antibody (1:10000 dilution) to each well and incubate at 37°C for 60 min; discard the liquid in the well and wash 5 times with a plate washer, 200 μL/well of washing solution. Add 50 μL/well of color development solution and incubate at 37°C for 15 min. Add 50 μL/well of 2 mol/L H ₂ SO ₄ to terminate the reaction and read the OD ₄₅₀ value on an ELISA reader. The results are shown in Figure 10A.

7.1.2 12E9 humanized antibody binding activity assessment (ELISA)

Dilute BAFFR-mFc coating solution to 2μg/mL, add 50μL/well of coating to the wells of the ELISA plate, and incubate at 37℃ for 2 hours. Discard the liquid in the wells and wash 5 times with a plate washer at 200μl/well of washing solution. Add 200μL of blocking solution to each well and incubate overnight at 4℃. Discard the liquid in the wells and wash 5 times with a plate washer at 200μl/well of washing solution. Add 50 μL of Hu-12E9-04, Hu-12E9-20, Hu-12E9-22, Hu-12E9-23, Hu-12E9-24, Hu-12E9-30, Hu-12E9-32, Hu-12E9-33, Hu-12E9-34 or 12E9-CHI to each well, with a concentration of 20 μg/mL in the first well, 5-fold dilution, 7 gradient antibodies, and set up a blank control at the same time; incubate at 37°C for 60 minutes; discard the liquid in the well, wash 5 times with a plate washer, 200 μl/well of washing solution. Add 50 μL of Goat-anti-human-IgG-Fc-Secondary-Antibody (1:10000 dilution) to each well, incubate at 37°C for 60 minutes; discard the liquid in the well, wash 5 times with a plate washer, 200 μl/well of washing solution. Add 50 μL/well of colorimetric solution and incubate at 37°C for 15 min. Add 50 μL/well of 2 mol/L H ₂ SO ₄ to terminate the reaction and read the OD ₄₅₀ value on a microplate reader. The results are shown in FIG10B .

7.2 ELISA-Blocking Activity Assessment

7.2.1 Evaluation of blocking activity of 7G10 humanized antibody (ELISA)

Dilute BAFF-hFc coating solution to 2μg/mL, add 50μL/well of coating to the wells of the ELISA plate, and incubate at 37℃ for 2 hours. Discard the liquid in the wells, wash 5 times with a plate washer, 200μl/well of washing solution. Add 200μL of blocking solution to each well and incubate overnight at 4℃. Discard the liquid in the wells, wash 5 times with a plate washer, 200μl/well of washing solution. Add 25μL BAFFR-mFc (4μg/mL) + 25μL Hu-7G10-02, Hu-7G10-22, Hu-7G10-32, Hu-7G10-42, Hu-7G10-44, or 7G10-CHI to each well, with a concentration of 20μg/mL in the first well, and dilute 5 times in sequence, 7 gradient antibodies, and set up a blank control at the same time; incubate at 37℃ for 120min; discard the liquid in the well, use a plate washer, wash 5 times with 200μl/time of washing solution. Add 50μL of Goat-anti-Mouse-IgG-Fc-Secondary-Antibody (1:10000 dilution) to each well, incubate at 37℃ for 60min; discard the liquid in the well, use a plate washer, wash 5 times with 200μl/well of washing solution. 50 μL/well of colorimetric solution was added, incubated at 37°C for 15 min, 50 μL/well of 2 mol/L H ₂ SO ₄ was added to terminate the reaction, and the OD ₄₅₀ value was read on an ELISA reader. The results are shown in FIG11A .

7.2.2 Evaluation of blocking activity of 12E9 humanized antibody (ELISA)

Dilute BAFF-hFc coating solution to 2μg/mL, add 50μL/well of coating to the wells of the ELISA plate, and incubate at 37℃ for 2 hours. Discard the liquid in the wells, wash 5 times with a plate washer, 200μl/well of washing solution. Add 200μL of blocking solution to each well and incubate overnight at 4℃. Discard the liquid in the wells, wash 5 times with a plate washer, 200μl/well of washing solution. To each well, add 25 μL of BAFFR-mFc (4 μg/mL) + 25 μL of Hu-12E9-04, Hu-12E9-20, Hu-12E9-22, Hu-12E9-23, Hu-12E9-24, Hu-12E9-30, Hu-12E9-32, Hu-12E9-33, Hu-12E9-34 or 12E9-CHI at a concentration of 20 μg/mL in the first well, followed by 5-fold dilutions, 7 gradients of antibodies, and set up a blank control; incubate at 37°C for 120 min; discard the liquid in the wells and wash 5 times with a plate washer at 200 μl/well of washing solution. Add 50 μL of Goat-anti-Mouse-IgG-Fc-Secondary-Antibody (1:10000 dilution) to each well and incubate at 37°C for 60 min; discard the liquid in the well and wash 5 times with a plate washer, 200 μL/well of washing solution. Add 50 μL/well of color development solution, incubate at 37°C for 15 min, add 50 μL/well of 2 mol/L H ₂ SO ₄ to terminate the reaction, and read the OD ₄₅₀ value on an ELISA reader. The results are shown in Figure 11B.

Example 8 In vitro efficacy evaluation (ADCC) of B7G10 and B12E9 antibodies

8.1 Screening of B7G10CHI by PBMC-based ADCC Activity Method

One of the mechanisms of action of this type of antibody drugs is that antibodies recognize targets through the Fab region and mediate specific targeted killing (ADCC) through the interaction of the Fc segment with effector cell receptors. We took freshly prepared or frozen PBMCs and incubated them with target cells, and added different concentrations of antibodies to test the ADCC activity of the preferred antibody molecules. In the experiment, the frozen PBMCs were revived one day in advance and cultured overnight. The next day, Raji cells in the logarithmic growth phase were collected, the cells were washed once with culture medium, the cell density was adjusted to 1×10 ⁶ /ml, 1.5μl DELFIABATDA was added to 2ml of cell suspension, and incubated at 37℃ for 20min. The cells were washed 3 times with culture medium, resuspended, the cell density was adjusted to 5×10 ⁴ /ml, and 100μl was added to each well of a 96-well cell culture plate. The test antibodies B7G10CHI and B12E9CHI, as well as the control antibody MOR6654-hG1 were diluted to 200 g/ml, 40 μg/ml and 8 μg/ml with culture medium, and 50 μl of each was added to the corresponding wells. 50 μl of 1×10 ⁷ /ml lymphocytes (E:T=100:1) were added to each well, and the spontaneous release and maximum release wells were supplemented with culture medium to 200 μl. At the same time, 10 μl of lysis solution was added to the target cell maximum release wells, and incubated at 37°C for 2 hours. The well plate was placed in a well plate centrifuge and centrifuged at 500 rpm for 5 minutes. 20 μl of supernatant was taken from each well and transferred to another 96 opaque flat-bottom white plate, and 200 μl of europium solution was added. After shaking and incubating on a shaker at room temperature for 15 minutes, the fluorescence intensity (TRF) was measured. The results are shown in Figure 12.

8.2 Screening of B7G10CHI by reporter gene assay for ADCC activity

ADCC killing occurs from the activation of effector cells. Artificially modified Jurkat cells can be used as a model for effector cell activation. The Fc receptor CD16A subunit and the DNA action element NFAT that starts gene transcription after receptor activation are transferred into this Jurkat cell. This element controls the transcription of luciferase. In this test, the modified Jurkat cells are incubated with target cells, and different concentrations of the test object (antibody) are added. The fluorescence signals at different concentrations are collected, and the fitting curve can distinguish the activity of antibody-activated effector cells. The specific operation is as follows: Collect Raji cells in the logarithmic growth phase, centrifuge to remove the supernatant, resuspend and count, adjust the cell density to 6×10 ⁶ /ml, and spread 25μl per well in a 96-well opaque cell culture plate. Dilute the test antibody and control antibody with culture medium to 60μg/ml as the starting concentration, dilute 8 concentration points by 5 times, take 25μl of each concentration point and add it to the 96-well plate, and set up duplicate wells. Incubate in a 37℃, 5% CO2 incubator for 45min. Jurkat cells were collected, and 25 μl of cells at a density of 1×106/ml were added to the well plate according to the effector-target ratio of 1:6. After incubation at 37°C, 5% CO2 incubator for 6 hours, the cells were equilibrated at room temperature for 15 minutes. 75 μl of fluorescence detection reagent was added to each well, and the cells were incubated at room temperature in the dark for 5 minutes. The fluorescence signal was detected, and the results are shown in Figure 13.

8.3 Screening of Hu7G10 molecules by measuring ADCC activity using reporter gene assay

B7G10 is the parent antibody, and its humanized molecules are multiple, among which at least 5 molecules have similar ELISA binding and blocking activities, and need to be further distinguished by ADCC reporter gene method. The specific operation is as follows: collect Raji cells in the logarithmic growth phase, centrifuge to remove the supernatant, resuspend and count, adjust the cell density to 6×10 ⁶ /ml, and spread 25μl per well in a 96-well opaque cell culture plate. Dilute the test antibody and control antibody with culture medium to 60μg/ml as the starting concentration, 5-fold gradient dilution to 8 concentration points, take 25μl of each concentration point and add it to the 96-well plate, set up duplicate wells. Incubate in a 37℃5% CO2 incubator for 45min. Collect ADCC Report Cell, take 25μl of 1×10 ⁶ /ml density cells according to the effect-target ratio of 1:6 and add them to the well plate, incubate in a 37℃, 5% CO2 incubator for 6h, and equilibrate at room temperature for 15min. 75 μl of fluorescence detection reagent was added to each well, incubated at room temperature in the dark for 5 min, and the fluorescence signal was detected. The results are shown in FIG14 .

8.4 Reporter gene assay for ADCC activity of Hu7G10-22AF

Hu7G10 was expressed in CHO-K1 cells with FUT8 biallelic knockout to obtain the non-fucose-modified molecule Hu7G10AF. Non-fucose-modified antibodies generally increase their affinity for Fc receptors, thereby activating effector cells with higher efficiency. This example uses the reporter gene method to test the ADCC activity of Hu7G10-22AF. The operation is as follows: Raji cells in the logarithmic growth phase are collected and centrifuged to remove the supernatant, resuspended and counted, the cell density is adjusted to 6×10 ⁶ /ml, and 25μl is plated in each well of a 96-well opaque cell culture plate. The test antibody and the control antibody are diluted with culture medium to 15μg/ml as the starting concentration, 5-fold gradient dilution of 8 concentration points, 25μl of each concentration point is added to a 96-well plate, and duplicate wells are set. Incubate in a 37°C 5% CO2 incubator for 45min. Collect ADCC Report Cells, and take 25μl of cells with a density of 1×106/ml according to the effect-target ratio of 1:6 and add them to the well plate. After incubation in a 37°C 5% CO2 incubator for 6 hours, the cells were equilibrated at room temperature for 15 minutes. 75 μl of fluorescence detection reagent was added to each well, and the cells were incubated at room temperature in the dark for 5 minutes. The fluorescence signal was detected. The results are shown in FIG15 .

Hu7G10-22AF and Hu7G10-22 were mixed and diluted in different proportions, and tested according to the above method. The results are shown in Figure 16.

The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.

Claims (10)

1. An antibody or antigen-binding fragment thereof that specifically binds to BAFF-R, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 44, 45, and 46, respectively, The light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 47, 48, and 49, respectively. 2. An antibody or antigen-binding fragment thereof that specifically binds to BAFF-R, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region sequence is selected from the amino acid sequence shown in any one of SEQ ID NOs: 13, 31, 32, and 33, The light chain variable region sequence is selected from the amino acid sequence shown in any one of SEQ ID NOs: 14, 34, 35, 36, and 37. 3. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment is a murine antibody or a fragment thereof, a chimeric antibody or a fragment thereof, or a humanized antibody or a fragment thereof. 4. The antibody or antigen-binding fragment according to claim 2, characterized in that the antigen-binding fragment is one or any combination of Fab, Fab', Fv, sFv, F(ab')2. 5. A nucleic acid sequence encoding the antibody or antigen-binding fragment according to any one of claims 1 to 4. 6. An expression vector containing the nucleotide sequence according to claim 5. 7. A host cell transformed by the expression vector according to claim 6, wherein the host cell is selected from prokaryotic cells, yeast cells, insect cells or mammalian cells. 8. The host cell of claim 7, wherein the host cell is selected from HEK293F cells, ExpiCHO S cells or CHO-K1 cells, wherein the CHO-K1 cells are FUT8 biallelic knockout cells. 9. A medicament or pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 4 and one or more pharmaceutically acceptable carriers, diluents or excipients. 10. A use of the antibody or antigen-binding fragment according to any one of claims 1 to 4, or the pharmaceutical composition according to claim 9, or the nucleotide sequence according to claim 5 in the preparation of a medicament for treating cancer and/or autoimmune diseases, wherein the cancer disease is selected from non-Hodgkin's lymphoma (B-NHL), various types of chronic lymphocytic leukemia (CLL), primary acute lymphocytic leukemia (ALL), and multiple myeloma; and the autoimmune disease is selected from one of systemic lupus erythematosus, idiopathic pulmonary fibrosis, rheumatoid arthritis (RA), primary Sjögren's syndrome (PSS), autoimmune hepatitis, multiple sclerosis, myasthenia gravis, IgA nephropathy, neuromyelitis optica, granulomatosis with polyangiitis, microscopic polyangiitis, and immune thrombocytopenic purpura.