CN118255895B - Bispecific antibody against BAFF and hIL21 and application thereof - Google Patents

Abstract

The present invention belongs to the field of genetic engineering and antibody drugs, and specifically relates to anti-BAFF and hIL21 bispecific antibodies and their applications. The bispecific antibody comprises a first antigen-binding fragment that binds to BAFF and a second antigen-binding fragment that binds to hIL21, wherein the first antigen-binding fragment comprises a variable region CDR sequence of a belimumab monoclonal antibody and a variable region CDR sequence of a belimumab-scFv single-chain antibody, and the second antigen-binding fragment comprises a variable region CDR sequence of an h18B10 monoclonal antibody; its Fc domain is obtained by knob-into-hole mutation. The amino acid sequences of the heavy chain 1 and heavy chain 2 of the bispecific antibody are shown in SEQ ID NO.1 and SEQ ID NO.5, respectively, and the amino acid sequences of the light chain 1 and light chain 2 are shown in SEQ ID NO.3 and SEQ ID NO.7, respectively. It can inhibit BAFF and IL21 simultaneously, and exhibits a synergistic effect in inhibiting excessive immune responses. Therefore, it has certain prospects and value in the preparation of therapeutic drugs for preventing, neutralizing and/or treating various autoimmune diseases related to overexpression of BAFF and IL21.

Description

Anti-BAFF and hIL21 bispecific antibodies and their applications

Technical Field

The invention belongs to the field of genetic engineering and antibody drugs, and specifically relates to an anti-BAFF and hIL21 bispecific antibody and an application thereof.

Background Art

B cell activating factor (BAFF) is a cytokine that stimulates B cell survival, proliferation and differentiation, and is closely related to B cell activation and the production of autoantibodies. High expression of BAFF has been detected in patients with a variety of autoimmune diseases, such as systemic lupus erythematosus (SLE), lupus nephritis (LN), rheumatoid arthritis, etc. Among them, the expression of BAFF in SLE patients is also positively correlated with disease activity. The anti-BAFF monoclonal antibody belimumab is the first biological agent approved by the FDA for the treatment of SLE. It can significantly reduce the titer of circulating autoantibodies, improve the disease remission rate, and reduce the risk of serious complications. Clinical trials using belimumab to treat a variety of autoimmune diseases are also underway, such as LN, idiopathic myositis, vasculitis, etc. Some data suggest that anti-BAFF treatment has a certain effect. Although belimumab was approved for the clinical treatment of SLE 13 years ago, its clinical use over the years has shown that belimumab has limited effects, and even failed to achieve the primary endpoint in some clinical trials for the treatment of SLE. This also indirectly shows that inhibiting BAFF alone is far from meeting the needs of controlling autoimmune diseases.

IL21 can stimulate CD40 on the surface of B cells to bind to CD40L on the surface of helper T (Th) cells, promoting B cell proliferation and differentiation, antibody production and antibody class switching. At the same time, IL21 promotes T cell differentiation into Tfh and Th17 cells. The proportion and cell function status of these two T cell subtypes in SLE patients are much higher than those in healthy people. They are the "criminal cells" of various autoimmune diseases. IL21 is significantly upregulated in patients with various immune diseases such as SLE, LN, inflammatory bowel disease, rheumatoid arthritis, and psoriasis. The genetic polymorphism of IL-21 and its receptor is also associated with the onset of SLE and rheumatoid arthritis. For various lupus models, such as BXSB-Yaa and MRL-lpr mouse models, after using IL-21R-Fc to block the binding of IL-21 to its receptor, the level of dsDNA autoantibodies decreased, and proteinuria, IgG glomerular deposition, glomerular basal thickening and lupus skin lesions were all alleviated, indicating that blocking the IL21 signaling pathway can alleviate the inflammatory progression of lupus mice. Inhibition of the IL21 signaling pathway can significantly improve the disease phenotype of the collagen-induced rheumatoid arthritis mouse model, such as joint swelling and increased levels of anti-citrullinated antibodies. Avizakimab (BOS161721), an anti-IL21 monoclonal antibody developed by Boston Pharmaceuticals, has completed a Phase II clinical trial for SLE. Compared with the placebo group, the high-dose treatment group significantly reduced the level of circulating dsDNA, reduced the disease relapse rate, and delayed multiple organ damage. However, the treatment group still did not reach the primary treatment endpoint.

Therefore, autoimmune diseases are in urgent need of alternative treatments, especially for diseases with complex pathogenesis such as SLE and LN. Using two different biological agents for co-treatment (such as the current clinical combination of Rituximab and belimumab for SLE) is one of the alternative therapies. Although the simultaneous use of two biological agents may further improve the efficacy of treatment with a single biological agent, the use of two biological agents is not convenient for patients and increases the cost of medication. It is quite challenging or almost impossible to design a preparation containing two different antibodies at the same time to ensure that its physical and chemical properties do not change during storage and use.

Given that both BAFF and IL21 significantly affect the survival, proliferation and differentiation of B cells, and IL21 also promotes the activation and differentiation of autoimmune T cells, using a bispecific antibody to simultaneously block the activity of BAFF and IL21 is expected to simultaneously inhibit T and B cell activation and reduce autoantibody secretion, becoming a preferred alternative therapy.

Summary of the invention

The technical problem to be solved by the present invention is to provide an anti-BAFF and hIL21 bispecific antibody in view of the deficiencies in the prior art.

The present invention also aims to solve the technical problem of providing the application of the above-mentioned bispecific antibody.

In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

A bispecific antibody against BAFF and hIL21, comprising a first antigen-binding fragment that binds to BAFF and a second antigen-binding fragment that binds to hIL21;

Wherein, the first antigen-binding fragment comprises the variable region CDR sequence of the belimumab monoclonal antibody and the variable region CDR sequence of the belimumab-scFv single-chain antibody;

Wherein, the second antigen-binding fragment comprises the variable region CDR sequence of the h18B10 monoclonal antibody.

Specifically, the variable region CDR sequence of the belimumab monoclonal antibody is shown in SEQ ID NOs. 9 to 14, the variable region CDR sequence of the h18B10 monoclonal antibody is shown in SEQ ID NOs. 15 to 20, and the variable region CDR sequence of the belimumab-scFv single-chain antibody is shown in SEQ ID NOs.: 9 to 14.

Further, the amino acid sequence of the heavy chain CDR1 of the belimumab monoclonal antibody is shown in SEQ ID NO.9, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.10, the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO.11, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.12, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.13, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO.14;

The amino acid sequence of the heavy chain CDR1 of the h18B10 monoclonal antibody is shown in SEQ ID NO.15, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.16, the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO.17, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.18, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.19, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO.20;

The amino acid sequence of the heavy chain CDR1 of the belimumab-scFv single-chain antibody is shown in SEQ ID NO.9, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.10, the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO.11, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.12, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.13, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO.14.

Among them, the detailed information of the belimumab monoclonal antibody has been disclosed in patent WO 2015173782 A1; the detailed information of the h18B10 monoclonal antibody has been disclosed in patent CN116554323A.

The first antigen-binding fragment contains the Fab region, the scFv region and the first Fc region of the belimumab monoclonal antibody, and the second antigen-binding fragment contains the Fab region and the second Fc region of the h18B10 monoclonal antibody.

Specifically, the C-terminus of the Fab region of the belimumab monoclonal antibody in the first antigen-binding fragment is connected to the N-terminus of the first Fc region, the C-terminus of the scFv region of the belimumab monoclonal antibody in the first antigen-binding fragment is connected to the N-terminus of the h18B10 monoclonal antibody, and the C-terminus of the Fab region of the h18B10 monoclonal antibody is connected to the N-terminus of the second Fc region.

Specifically, the first Fc region and the second Fc region are connected via a knob-into-hole structure.

Furthermore, the first Fc region and the second Fc region are both derived from human IgG4; wherein the first Fc region has a T366W mutation, the second Fc region has T366S, L368A and Y407V mutations, and the first Fc region and the second Fc region have disulfide bond S354C and Y349C mutations between the chains.

Furthermore, the first Fc region has an amino acid sequence as shown in SEQ ID NO.21, and the second Fc region has an amino acid sequence as shown in SEQ ID NO.22.

Among them, the amino acid sequence of the heavy chain 1 of the bispecific antibody is shown as SEQ ID NO.1, the amino acid sequence of the heavy chain 2 is shown as SEQ ID NO.5, the amino acid sequence of the light chain 1 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain 2 is shown as SEQ ID NO.7.

Specifically, for the bispecific antibody, the corresponding nucleotide sequence of the heavy chain 1 is shown as SEQ ID NO.2, the nucleotide sequence of the heavy chain 2 is shown as SEQ ID NO.6, the nucleotide sequence of the light chain 1 is shown as SEQ ID NO.4, and the nucleotide sequence of the light chain 2 is shown as SEQ ID NO.8.

The nucleic acid molecules encoding the above-mentioned bispecific antibodies against BAFF and hIL21 are also within the scope of protection of the present invention.

The expression vector containing the above nucleic acid molecule is also within the scope of protection of the present invention.

Recombinant cells containing the above nucleic acid molecules or the above expression vectors are also within the scope of protection of the present invention.

The pharmaceutical composition comprising the above nucleic acid molecule or the above expression vector or the above recombinant cell is also within the scope of protection of the present invention.

The use of the above-mentioned anti-BAFF and hIL21 bispecific antibody, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned recombinant cell, or the above-mentioned pharmaceutical composition in the preparation of drugs for treating or preventing immune diseases is also within the scope of protection of the present invention.

Wherein, the immune disease includes any one of systemic lupus erythematosus, lupus nephritis and graft-versus-host disease.

The use of the above-mentioned anti-BAFF and hIL21 bispecific antibody, the above-mentioned nucleic acid molecule, the above-mentioned expression vector, the above-mentioned recombinant cell, or the above-mentioned pharmaceutical composition in the preparation of a kit is also within the scope of protection of the present invention.

The kit is used to detect any one or a combination of belimumab and h18B10.

Beneficial effects:

This patent provides a bispecific antibody that can target BAFF and IL21 - anti-BAFF×hIL21 bispecific antibody. Compared with monoclonal antibodies, this bispecific antibody can inhibit BAFF and IL21 at the same time, and shows a synergistic effect in inhibiting excessive immune responses. Its immunosuppressive effect is better than that of a single monoclonal antibody targeting BAFF or a single monoclonal antibody targeting IL21. Therefore, this bispecific antibody is helpful for the preparation of therapeutic drugs for the prevention, neutralization and/or treatment of various autoimmune diseases related to the overexpression of BAFF and IL21 (such as systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, etc.), and provides more detection options for clinical and scientific research, and has certain application prospects and value.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and/or other advantages of the present invention will become more clear.

Figure 1: Molecular configuration of anti-BAFF×hIL21 bispecific antibody, where Anti-Blys represents anti-BAFF and Anti-IL21 represents anti-hIL21.

Figure 2: Elisa measured the affinity of anti-BAFF×hIL21 bispecific antibodies to BAFF.

Figure 3: Elisa measured the affinity of anti-BAFF×hIL21 bispecific antibodies to hIL21.

Figure 4: Inhibitory effect of anti-BAFF×hIL21 bispecific antibody on hIL21 signal transduction, wherein BAFF×hIL21 bishole2 represents anti-BAFF×hIL21 bispecific antibody.

Figure 5: Inhibitory effect of anti-BAFF×hIL21 bispecific antibody on BAFF signal transduction, wherein BAFF×hIL21 bishole2 represents anti-BAFF×hIL21 bispecific antibody.

Figure 6: Flow cytometry gate diagram of antibody secreting cells. Belimumab*h18B10 bishole2 represents anti-BAFF×hIL21 bispecific antibody.

Figure 7: Inhibitory effects of anti-BAFF×hIL21 bishole2, h18B10 monoclonal antibody and belimumab monoclonal antibody on differentiation of antibody-secreting cells. BAFF×hIL21 bishole2 refers to anti-BAFF×hIL21 bishole2.

Figure 8: Anti-BAFF×hIL21 bispecific antibody, h18B10 monoclonal antibody and belimumab monoclonal antibody improve urine protein in GVHD model mice. 1 to 5 represent no antibody, Anti-KLH antibody, anti-BAFF×hIL21 bispecific antibody, h18B10 monoclonal antibody and belimumab monoclonal antibody, respectively.

Figure 9: Anti-BAFF×hIL21 bispecific antibody, h18B10 monoclonal antibody and belimumab monoclonal antibody improve the anti-dsDNA autoantibody in GVHD model mice. 1 to 5 represent no antibody, Anti-KLH antibody, anti-BAFF×hIL21 bispecific antibody, h18B10 monoclonal antibody and belimumab monoclonal antibody, respectively.

Figure 10: The inhibition results of anti-BAFF×hIL21 bispecific antibody, h18B10 monoclonal antibody and belimumab monoclonal antibody on mIL6 and hIFNγ in GVHD model mice, respectively. A is the inhibition result of mIL6 in GVHD model mice; B is the inhibition result of hIFNγ in GVHD model mice; 1 to 5 represent no antibody, Anti-KLH antibody, anti-BAFF×hIL21 bispecific antibody, h18B10 monoclonal antibody, belimumab monoclonal antibody, respectively.

DETAILED DESCRIPTION

The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

In the following embodiments, the detailed information of the belimumab monoclonal antibody has been disclosed in patent WO2015173782A1; the detailed information of the h18B10 monoclonal antibody has been disclosed in patent CN116554323A.

In the following examples, all buffers and culture media involved are as follows: PBS buffer (pH 7.4), PBST buffer (PBS buffer + 0.05% Twen-20), carbonate coating solution (pH 9.6 _{Na 2} CO ₃ 1.59 g + NaHCO ₃ 2.93 g + distilled water 1000 mL), RPMI-1640 complete medium (RPMI-1640 purchased from Shanghai Peiyuan + 10% fetal bovine serum), DMEM complete medium (DMEM purchased from Shanghai Peiyuan + 10% fetal bovine serum).

Example 1: Construction, expression and purification of anti-BAFF×hIL21 bispecific antibody molecules

In this example, belimumab monoclonal antibody is used as the BAFF domain of the bispecific antibody, and h18B10 monoclonal antibody is used as the hIL21 binding domain of the bispecific antibody to construct an anti-BAFF×hIL21 bispecific antibody. The molecular configuration of the anti-BAFF×hIL21 bispecific antibody is shown in FIG1 .

The construction of the bispecific antibody was completed by Conoya Biopharmaceutical Technology Co., Ltd. Specifically, the bispecific antibody: (a) the first antigen-binding portion specifically binds to human BAFF, and is composed of the Fab region of the belimumab monoclonal antibody and the belimumab-scFv single-chain antibody (formed by the heavy chain variable region and the light chain variable region of the belimumab monoclonal antibody connected by a connecting peptide GGGGSGGGGSGGGGS), which is contained in the amino acid sequence of the heavy chain 1 as shown in SEQ ID NO.1 and the amino acid sequence of the light chain 1 as shown in SEQ ID NO.3; (b) the second antigen-binding portion specifically binds to human IL21 through the Fab configuration, and is contained in the amino acid sequence of the heavy chain 2 as shown in SEQ ID NO.5 and the amino acid sequence of the light chain 2 as shown in SEQ ID NO.7. Among them, the Fab arm of the second antigen-binding fragment is connected to the belimumab-scFv single-chain antibody in the first antigen-binding fragment through the connecting peptide GGGGSGGGGS; (c) in the Fc domain, the knob chain (first Fc region, derived from human IgG4) T366W, the hole chain (second Fc region, derived from human IgG4) T366S, L368A and Y407V, and the interchain disulfide bonds S354C and Y349C are mutated by knob-into-hole, thereby effectively achieving the correct pairing of heterodimers between heavy chains. The specific heavy chain, light chain and corresponding CDR region sequences of the anti-BAFF×hIL21 bispecific antibody are shown in Tables 1 and 2.

Table 1 Heavy chain and light chain sequences of anti-BAFF×hIL21 bispecific antibodies

Anti-BAFF×hIL21 bispecific antibody Amino acid sequence Nucleotide sequence Heavy chain 1 SEQ ID NO.1 SEQ ID NO.2 Light chain 1 SEQ ID NO.3 SEQ ID NO.4 Heavy chain 2 SEQ ID NO.5 SEQ ID NO.6 Light chain 2 SEQ ID NO.7 SEQ ID NO.8

Table 2 CDR and Fc region sequences of anti-BAFF×hIL21 bispecific antibodies

The anti-BAFF×hIL21 bispecific antibody plasmids include four plasmids, all of which were constructed by Conoya Biopharmaceutical Technology Co., Ltd. The simple construction process is as follows:

(1) Heavy chain 1: The belimumab heavy chain variable region derivative was cloned into the PHCT4S vector containing the T366W mutant heavy chain constant region and regulatory elements independently developed by Conoya to obtain the PHCT4S-belimumabknob plasmid expressing heavy chain 1;

(2) Heavy chain 2: The scFv-linked h18B10 variable region derivative was cloned into the PHCT4S vector containing T366S, L368A and Y407V mutant heavy chain constant regions and regulatory elements independently developed by Conoya to obtain the PHCT4S-Bis2 hole plasmid expressing heavy chain 2;

(3) Light chain 1: The belimumab light chain variable region derivative was cloned into the pHCT2 vector containing the human IgGκ light chain constant region and regulatory elements independently developed by Conoya to obtain the pHCT2-belimumabV1 plasmid expressing light chain 1;

(4) Light chain 2: The h18B10 light chain variable region derivative was cloned into the pHCT2 vector containing the human IgGκ light chain constant region and regulatory elements independently developed by Conoya, to obtain the pHCT2-h18B10V1 plasmid expressing light chain 2.

Four plasmids of anti-BAFF×hIL21 bispecific antibody were transferred into ExpiCHO cells (purchased from ATCC) using a transfection reagent at a transfection ratio of heavy chain 1: light chain 1: heavy chain 2: light chain 2 = 1:2:1:2, and cultured for 7 days at 37°C, 8% CO ₂ , and 80% humidity to obtain fermentation broth. After the culture was completed, the fermentation broth was centrifuged at 4°C, 4500 rpm, for 30 min, and the supernatant was filtered with a 0.22μm syringe filter. Protein A was first used for gravity column purification, and the broth was prepared by equilibration (PBS buffer, pH 7.4 to equilibrate Protein A filler) - loading (adding filtered fermentation broth) - elution (elution solution was 50 mM acetic acid-sodium acetate, pH 5.5) - elution (elution solution was 0.1 M acetic acid-sodium acetate, pH 3.8). Subsequently, the protein eluted by Protein A was subjected to gradient elution using CEX with Capto S ImpAct column as carrier to remove antibody aggregates and fragments to improve the purity of protein monomer. The quality of BAFF×IL21 was detected by SEC and mass spectrometry (Table 3). As can be seen from Table 3, a batch of high-quality, mismatch-free anti-BAFF×hIL21 bispecific antibodies were successfully prepared through the above steps, and the monomer purity was as high as 99.7%.

Table 3 Expression yield and quality of anti-BAFF×hIL21 bispecific antibody

Example 2 Identification of the Binding Activity of Anti-BAFF×hIL21 Bispecific Antibody to BAFF

Construction, expression and purification of BAFF-His protein: The BAFF gene was synthesized according to the sequence provided by NCBI (the accession number of the BAFF gene in NCBI is NC_000013.11), and a 6×His tag was added to its C-terminus. It was constructed into a CMV eukaryotic expression vector (purchased from Guangzhou Funeng Gene Co., Ltd.) through the two restriction sites of EcoRI and HindIII, and then transiently transfected into HEK-293E cells (purchased from ATCC) using the biohub kit PEI to express and purify the BAFF-His protein.

In order to detect the affinity of the anti-BAFF×hIL21 bispecific antibody to the BAFF antigen, the BAFF-His protein was diluted to 1 μg/mL with a carbonate coating solution (Na ₂ CO ₃ 1.59 g + NaHCO ₃ 2.93 g + distilled water 1000 mL) at pH 9.6, and then 100 μl/well was added to the ELISA plate; incubated overnight at 4°C; the plate was washed three times with PBST buffer (pH 7.4) the next day; 3% BSA buffer was added to each well for blocking, and the blocking was carried out at room temperature for 1 h; the plate was washed three times with PBST buffer (pH 7.4); then the antibody to be tested was added in a gradient dilution with 1% BSA buffer, with an initial concentration of 200 nM, and 12 gradients of 5-fold dilution were performed stepwise. Incubate at room temperature for 1 hour; wash the plate five times with PBST buffer (pH 7.4), add the secondary antibody HRP-labeled goat anti-human Fc antibody (purchased from Thermo Fisher), and incubate at room temperature for another 1 hour; wash the plate five times with PBST buffer (pH 7.4) and pat dry, add 100 μl TMB solution (purchased from Sigma) to each well, and place it at 37°C in the dark for 10 minutes; add 50 μl 2M H ₂ SO ₄ stop solution to each well to terminate the substrate reaction, read the OD value at 450nm on an enzyme reader, and use GraphPad Prism for data analysis, plotting and calculating EC50 (Figure 2). Figure 2 shows that the anti-BAFF×hIL21 bispecific antibody can bind to the BAFF antigen, with an EC50 of 0.22nM.

Example 3 Identification of the Binding Activity of Anti-BAFF×hIL21 Bispecific Antibody to Human IL21

Construction, expression and purification of IL21-His protein: The IL21 gene was synthesized according to the sequence provided by NCBI (the NCBI accession number of the IL21 gene is NC_000004.12), and a 6×His tag was added to its C-terminus. It was constructed into a CMV eukaryotic expression vector (purchased from Guangzhou Funeng Gene Co., Ltd.) through the two restriction sites of EcoRI and HindIII, and then transiently transfected into HEK-293E cells (purchased from ATCC) using the biohub kit PEI to express and purify the IL21-His protein.

In order to detect the affinity of the anti-BAFF×hIL21 bispecific antibody to the IL21 antigen, the IL21-His protein was diluted to 1 μg/mL with a carbonate coating solution (Na ₂ CO ₃ 1.59 g + NaHCO ₃ 2.93 g + distilled water 1000 mL) at pH 9.6, and then 100 μl/well was added to the ELISA plate; incubated overnight at 4°C; the plate was washed three times with PBST buffer (pH 7.4) the next day; 3% BSA buffer was added to each well for blocking, and the blocking was carried out at room temperature for 1 h; the plate was washed three times with PBST buffer (pH 7.4); then the antibody to be detected was added in a gradient dilution with 1% BSA buffer, with an initial concentration of 200 nM, and 12 gradients of 5-fold dilution were performed stepwise. Incubate at room temperature for 1 hour; wash the plate five times with PBST buffer (pH 7.4), add the secondary antibody HRP-labeled goat anti-human Fc antibody (purchased from Thermo Fisher), and incubate at room temperature for another 1 hour; wash the plate five times with PBST buffer (pH 7.4) and pat dry, add 100 μl TMB solution (purchased from Sigma) to each well, and place it at 37°C in the dark for 10 minutes; add 50 μl 2M H ₂ SO ₄ stop solution to each well to terminate the substrate reaction, read the OD value at 450nm on an enzyme reader, and use GraphPad Prism for data analysis, plotting and calculating EC50 (Figure 3). Figure 3 shows that the anti-BAFF×hIL21 bispecific antibody can bind to human IL21, with an EC50 of 3.67nM.

Example 4 Identification of anti-BAFF×hIL21 bispecific antibody inhibiting activation of Ba/F3-hIL21R-STAT3-lucia reporter cells stimulated by human IL21

Construction of reporter cells: The Ba/F3-STAT3-lucia stable cell line independently developed and constructed by Conoya Biotechnology Co., Ltd. was incubated at 37°C in 5% _CO2 , and cultured in RPMI-1640 medium containing 10% fetal bovine serum, 2mM L-glutamine, 50μg/mL penicillin-streptomycin, 50mg/mL hygromycin and 10ng/mL mouse IL-3 for standby use. The lentiviral expression vector expressing human IL21R (Guangzhou Funeng Gene Co., Ltd., LPP-H0023-Lv187-A00) was packaged with lentivirus and transfected into the Ba/F3-STAT3-lucia mother cell line through the steps on the Lenti-PacHIV lentiviral packaging kit (purchased from Guangzhou Funeng Gene Co., Ltd.), and then 10μg/mL blasticidin was added for resistance screening to obtain the reporter cell line Ba/F3-hIL21R-STAT3-lucia that can stably express the human IL21 receptor.

The reporter cells were diluted to 6×10 ⁵ /mL with RPMI-1640 complete medium, added to a 96-well plate, and incubated overnight at 37°C in 5% CO ₂ for later use. The anti-BAFF×hIL21 bispecific antibody was then serially diluted 4-fold from 200 nM to 0.06 nM with RPMI-1640 complete medium, and the gradient diluted antibodies were incubated with IL-21 ligand (Novoprotein, CC45) at a final concentration of 5 ng/mL for 30 minutes, and 50 μL per well was added to the prepared reporter cells. After incubation for 6 hours at 37°C in 5% _CO2 , Bio-LiteTM reagent (Vazyme Biotech, DD1201-02) was added to the plate at 100 μl/well, and luciferase activity was measured using a SpectraMax multi-label plate reader (Molecular Devices) to test the inhibition of IL-21 signaling by the anti-BAFF×hIL21 bispecific antibody.

The results are shown in FIG4 , from which it can be seen that the anti-BAFF×hIL21 bispecific antibody can significantly inhibit the activity of hIL21.

Example 5 Identification of BAFF×hIL2 bispecific antibody inhibiting human BAFF-stimulated HEK293-BCMA-NFκB-lucia reporter cell activation

Construction of reporter cells: The HEK293-NFκB-lucia stable cell line independently developed and constructed by Conoya Biotechnology Co., Ltd. was incubated at 37°C in 5% _CO2 , and cultured in DMEM medium containing 10% fetal bovine serum, 50μg/mL penicillin-streptomycin, and 50mg/mL hygromycin for use. The lentiviral expression vector expressing human BCMA (Guangzhou Funeng Gene Co., Ltd., EX-A3720-Lv105) was packaged with lentivirus and transfected into the HEK293-NFκB-lucia mother cell line through the steps on the Lenti-PacHIV lentiviral packaging kit (purchased from Guangzhou Funeng Gene Co., Ltd.), and then 10μg/mL blasticidin was added for resistance screening to obtain the reporter cell line HEK293-BCMA-NFκB-lucia that can stably express human BCMA.

The reporter cells were diluted to 6×10 ⁵ /mL with DMEM complete medium (+10% fetal bovine serum), added to a 96-well plate, and incubated overnight at 37°C in 5% CO ₂ for later use. The anti-BAFF×hIL21 bispecific antibody was then serially diluted 4-fold from 200 nM to 0.06 nM with DMEM complete medium, and the gradient diluted antibodies were incubated with BAFF ligand (purchased from Conoya and expressed independently by Conoya) at a final concentration of 10 ng/mL for 30 minutes, and 50 μL was added to each well to the prepared reporter cells. After incubation for 6 hours at 37°C and 5% _CO2 , Bio-LiteTM reagent (Vazyme Biotech, DD1201-02) was added to the plate at 100 μl/well, and luciferase activity was measured using a SpectraMax multi-label plate reader (Molecular Devices) to test the inhibition of BAFF signaling by the anti-BAFF×hIL21 bispecific antibody.

The results are shown in FIG5 , from which it can be seen that the anti-BAFF×hIL2 bispecific antibody can significantly inhibit the activity of BAFF.

Example 6 Anti-BAFF×hIL21 bispecific antibody inhibits human antibody secreting cell differentiation and antibody secretion

Human peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole blood by density gradient centrifugation. Specifically, whole blood was diluted with PBS buffer at a ratio of 1:1, and then added to a centrifuge tube containing Ficoll-Paque PLUS density gradient centrifugation medium (purchased from Univi) and centrifuged to separate PBMCs. The upper layer containing PBMCs was transferred to a new centrifuge tube and washed twice with PBS buffer. The PBMCs were then resuspended in a complete medium of RPMI 1640 + 10% fetal bovine serum + 50 μm β-mercaptoethanol (purchased from Merck) + 10 ng/mL IL21 + 1 μg/mL anti-CD40 antibody (biolegend) + 50 ng/mL BAFF at a concentration of approximately 2 × 10 ⁶ cells/mL, and plated in a 96-well U-shaped plate (100 μL cells/well; approximately 20,000 cells/well). Subsequently, anti-BAFF×hIL21 bispecific antibody, h18B10 monoclonal antibody, and belimumab monoclonal antibody diluted in the same medium were added to the above-seeded cells, and no antibody was added as a control. The final concentration of the above antibodies was 20nM, and 100μl was added to each well. Incubate at 37°C, 5% _CO2 for 5 days, and collect cells by centrifugation on the 5th day. Wash the collected cells twice with flow buffer (PBS buffer containing 4% calf serum), 200μl per well. 50 μl of blocking solution (PBS buffer + 4% calf serum + 100 μg/mL hIgG) was added to the cells, blocked on ice for 10 minutes, centrifuged at 300g for 3 minutes and the supernatant was discarded, 1:200 anti-human CD19 FITC (biolegend), anti-human CD38 APC (BD) and anti-human CD27 Percp-cy5.5 (biolegend) antibody dilution mixture was added to each well, 50 μl, and blocked on ice for 30 minutes; centrifuged at 300g for 3 minutes and the supernatant was discarded, and the cells were washed twice with flow cytometry buffer, 200 μl per well; PI staining solution was added to the cells, 100 μl per well, ice-bathed and protected from light for 5 minutes, centrifuged at 300g for 3 minutes and the supernatant was discarded, the cells were washed twice with 200 μl flow cytometry buffer per well, and the cells were resuspended with 100 μl PBS buffer per well. Flow cytometry was used to measure the ratio of antibody-secreting cells (ASC gate defined as singlet live cells with CD27 ⁺ CD38 ⁺ CD20 ⁺ ) to B cells (gate defined as singlet live cells with CD20 ⁺ ).

The above experiment was repeated in blood samples from 8 different people. The flow gate diagram is shown in Figure 6, from which the detailed gate logic can be seen. The inhibition rate ((ASC ratio of the well without antibody addition - ASC ratio of the well with antibody addition)/ASC ratio of the well without antibody addition) was calculated, and the results were summarized and analyzed using GraphPad Prism. The graph is shown in Figure 7. From Figure 7, it can be seen that the anti-BAFF×hIL21 bispecific antibody inhibits the differentiation activity of antibody-secreting cells much more strongly than each monoclonal antibody.

Example 7 In vivo model for evaluating the blocking activity of anti-BAFF×hIL21 bispecific antibodies as therapeutic treatment: Antibody testing in a xenogeneic acute graft-versus-host disease model

To determine the effects of anti-BAFF×hIL21 bispecific antibodies in a relevant in vivo model, a xenogeneic acute graft-versus-host disease (GvHD) study was performed. Briefly, human peripheral blood mononuclear cells (human PBMCs) were injected into NSG mice (NOD-scid IL2rγnull, purchased from Biocytogen) to induce GvHD in mice. After transplantation, human immune cells recognized the mouse host as a xenogeneic and mounted a strong immune response against its tissues.

Specifically, on the day of injection, human PBMC ( Biotechnologies) was thawed in RPMI-1640 medium supplemented with 10% fetal bovine serum, and then the cells were washed in PBS buffer, and then resuspended in PBS buffer (pH7.4) at 10 million cells/100 μL. 10 million human PBMCs were injected into the tail vein of NSG mice (purchased from Biocytogen) using a syringe. 100 μL of PBS buffer was injected into the tail vein of the blank control group. After the tail vein injection of human PBMC, the four groups of human PBMC-transplanted NSG mice were subcutaneously injected with 10 mg/kg anti-BAFF×hIL21 bispecific antibody, h18B10 monoclonal antibody, belimumab monoclonal antibody or isotype control antibody (anti-KLH, expressed independently by Conoya) starting 2 weeks later, and then once every 4 days, a total of 4 injections were given and observed, with no antibody subcutaneous injection as the control. The experiment was terminated by killing the remaining mice on the 161st day after human PBMC transplantation. The experimental dosing and treatment schemes of each group of mice are shown in Table 4.

Table 4 Experimental drug administration and treatment scheme for each group of mice

Group Number of NSG mice Human PBMC injection Antibody 1 4 none none 2 8 10 million Anti-KLH Antibody 3 6 10 million Anti-BAFF×hIL21 bispecific antibody 4 6 10 million h18B10 monoclonal antibody 5 6 10 million Belimumab monoclonal antibody

Throughout the experiment, mice were monitored twice a week for weight loss and death (to assess the effect of therapeutic antibodies on survival). Before killing the mice, the levels of inflammatory factors and anti-dsDNA in the blood were assessed, and urine was collected and measured for micro-urinary protein to assess the extent of kidney involvement in the mice.

Specific experimental steps:

1. Detection of mouse urine microalbumin using the mouse urine microalbumin ELISA detection kit (Elabscience): Collect 20 μl of each mouse urine and perform the test according to the operating steps. Specifically, dilute each mouse urine 1000 times with the standard/sample diluent, add 100 μl to each well of the 96-well ELISA plate that has been coated in the kit, incubate at 37°C for 90 minutes, dry and add 100 μl of biotinylated antibody working solution to each well, incubate at 37°C for 1 hour. Wash 4 times, add 100 μl of HRP enzyme conjugate working solution to each well, incubate at 37°C for 30 minutes, wash 4 times, add 90 μl of color development solution, place at 37°C for 10 minutes, add 50 μl of stop solution, and immediately place in the microplate reader to read the OD ₄₅₀ value. The results are shown in Figure 8. The anti-BAFF×hIL21 bispecific antibody significantly improved the urine albumin in the model mice and alleviated the further deterioration of the renal function of the mice (P<0.05), and had a more significant trend than the monoclonal antibodies.

2. ELISA detection of anti-dsDNA levels in mouse plasma: 50-100 μl of blood from each mouse was collected, centrifuged at 4500 rpm, and the supernatant was frozen at -80°C for subsequent detection. UltraPureTM salmon sperm DNA solution (Invitrogen) was coated on a 96-well ELISA plate at 100 μg/mL and incubated overnight at 4°C. The next day, after washing three times with PBS buffer (pH 7.2), the plate was blocked with 10% BSA buffer (Sangong) at 37°C for 1 hour, and then washed three times with PBS buffer (pH 7.2). Then, plasma samples diluted 50 times with 1% BSA buffer were added, 100 μl per well, incubated at 37°C for 90 minutes, and then washed five times with PBS buffer (pH 7.2). HRP-labeled Fc-specific anti-mouse IgG1 (abclonal) was added at a ratio of 1:4000. Incubate at room temperature for 1 hour, wash 7 times with PBS buffer (pH 7.2), add 100 μl of colorimetric solution (TMB solution, purchased from Sigma, catalog number T2885), place at 37°C for 10 minutes, add 50 μl of 2M concentrated sulfuric acid solution to terminate the reaction, and immediately place in a microplate reader to read the OD ₄₅₀ value. As shown in Figure 9, the anti-BAFF×hIL21 bispecific antibody significantly improved the level of anti-dsDNA antibodies in the blood of the model mice (P<0.05), and had a more significant trend than each monoclonal antibody.

3. ELISA detection of mIL6 and IFNγ levels in mouse plasma: The above two inflammatory factors were detected using the mouse IL6 ELISA kit (BD No. 550950) and the human IFNγ kit (BD No. 555142), respectively. The test was carried out according to the kit instructions. The operating steps of the two inflammatory factor kits are the same.

The specific operation steps are as follows: 50-100 μl of each mouse blood was collected, centrifuged at 4500rpm, and the supernatant was frozen at -80°C for subsequent detection. The capture antibody was coated on a 96-well ELISA plate at 1:250 and incubated overnight at 4°C. The next day, after washing 3 times with PBS buffer, it was blocked with 10% fetal bovine serum at room temperature for 1 hour, and then washed 3 times with PBS buffer, and 100 μl of sample (cell supernatant) and standard were added respectively, incubated at room temperature for 2 hours, and then washed 5 times with PBS buffer, and 5AV-HRP was added to the diluent containing 0.4% detection antibody at a ratio of 1:250. Finally, incubated at room temperature for 1 hour, washed 7 times with PBS buffer, added 100 μl of color development solution (TMB solution, purchased from Sigma, item number T2885), placed at 37°C for 10 minutes, and then 50 μl of 2M concentrated sulfuric acid solution was added to terminate the reaction, and immediately placed in an ELISA reader to read the OD ₄₅₀ value. As shown in Figure 10A, the anti-BAFF×hIL21 bispecific antibody significantly improved the level of mIL6 in the blood of the model mice (P<0.05), which was more significant than the monoclonal antibodies. Figure 10B shows that the anti-BAFF×hIL21 bispecific antibody, h18B10 monoclonal antibody and belimumab monoclonal antibody all inhibited the secretion of IFNγ in the model, among which the anti-BAFF×hIL21 bispecific antibody and h18B10 monoclonal antibody had more obvious inhibitory effects.

In summary, this patent creatively invented a bispecific antibody that simultaneously targets BAFF and hIL21. This antibody inhibits the activity of BAFF and hIL21, while inhibiting T and B cell activation, reducing autoantibody secretion, improving mouse renal function, and reducing urinary protein, and is expected to become a therapeutic antibody for autoimmune diseases.

The present invention provides an anti-BAFF and hIL21 bispecific antibody and its application ideas and methods. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims (11)

1. A bispecific antibody against BAFF and hIL21, comprising a first antigen-binding fragment that binds BAFF and a second antigen-binding fragment that binds hIL 21;

the first antigen binding fragment comprises the variable region CDR sequence of belimumab monoclonal antibody and the variable region CDR sequence of belimumab-scFv single chain antibody,

The second antigen binding fragment comprises the variable region CDR sequences of an h18B10 monoclonal antibody,

The variable region CDR sequence of the belimumab monoclonal antibody is shown as SEQ ID NO. 9-14, the variable region CDR sequence of the h18B10 monoclonal antibody is shown as SEQ ID NO. 15-20, and the variable region CDR sequence of the belimumab-scFv single-chain antibody is shown as SEQ ID NO. 9-14;

Wherein the first antigen binding fragment consists of a Fab region of belimumab monoclonal antibody and a belimumab-scFv single chain antibody,

Specifically, the belimumab-scFv single-chain antibody is formed by connecting a heavy chain variable region and a light chain variable region of a belimumab monoclonal antibody through a connecting peptide GGGGSGGGGSGGGGS;

Wherein the second antigen binding fragment is the Fab region of the h18B10 monoclonal antibody,

Specifically, the Fab region of the h18B10 monoclonal antibody has its Fab arm linked to the belimumab-scFv single-chain antibody in the first antigen-binding fragment via the linker peptide GGGGSGGGGS;

wherein the bispecific antibody comprises an Fc domain region,

Specifically, the Fc domain region consists of a first Fc region and a second Fc region, which are linked by a knob-intoo-hole structure;

Wherein the C-terminal of the Fab region of the belimumab monoclonal antibody in the first antigen-binding fragment is connected with the N-terminal of the first Fc region, the C-terminal of the belimumab-scFv single-chain antibody in the first antigen-binding fragment is connected with the N-terminal of the h18B10 monoclonal antibody, and the C-terminal of the Fab region of the h18B10 monoclonal antibody is connected with the N-terminal of the second Fc region.

2. The bispecific antibody of claim 1, wherein the bispecific antibody consists of a first antigen-binding fragment that binds BAFF and a second antigen-binding fragment that binds hIL21,

The first antigen binding fragment comprises a variable region CDR sequence of belimumab monoclonal antibody and a variable region CDR sequence of belimumab-scFv single chain antibody,

The second antigen binding fragment comprises the variable region CDR sequences of an h18B10 monoclonal antibody,

Wherein the amino acid sequence of the heavy chain CDR1 of the belimumab monoclonal antibody is shown as SEQ ID NO.9, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.10, the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO.11, the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.12, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.13, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 14;

The amino acid sequence of the heavy chain CDR1 of the h18B10 monoclonal antibody is shown as SEQ ID NO.15, the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.16, the amino acid sequence of the heavy chain CDR3 is shown as SEQ ID NO.17, the amino acid sequence of the light chain CDR1 is shown as SEQ ID NO.18, the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.19, and the amino acid sequence of the light chain CDR3 is shown as SEQ ID NO. 20;

The amino acid sequence of a heavy chain CDR1 of the belimumab-scFv single-chain antibody is shown as SEQ ID NO.9, the amino acid sequence of a heavy chain CDR2 is shown as SEQ ID NO.10, the amino acid sequence of a heavy chain CDR3 is shown as SEQ ID NO.11, the amino acid sequence of a light chain CDR1 is shown as SEQ ID NO.12, the amino acid sequence of a light chain CDR2 is shown as SEQ ID NO.13, and the amino acid sequence of a light chain CDR3 is shown as SEQ ID NO. 14.

3. The bispecific antibody of claim 1, wherein the first Fc region and the second Fc region are each derived from human IgG4; wherein the first Fc region has a T366W mutation, the second Fc region has a T366S, L a and Y407V mutation, and the first and second Fc regions have inter-chain disulfide S354C and Y349C mutations.

4. The bispecific antibody of claim 1, wherein the amino acid sequence of the first Fc region is shown in SEQ ID No.21 and the amino acid sequence of the second Fc region is shown in SEQ ID No. 22.

5. The bispecific antibody of claim 1, wherein the bispecific antibody consists of heavy chain 1, heavy chain 2, light chain 1 and light chain 2;

The amino acid sequence of the heavy chain 1 is shown as SEQ ID NO.1, the amino acid sequence of the heavy chain 2 is shown as SEQ ID NO.5, the amino acid sequence of the light chain 1 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain 2 is shown as SEQ ID NO. 7.

6. A nucleic acid molecule encoding the bispecific antibody against BAFF and hIL21 of any one of claims 1-5.

7. An expression vector comprising the nucleic acid molecule of claim 6.

8. A recombinant cell comprising the nucleic acid molecule of claim 6 or the expression vector of claim 7.

9. A pharmaceutical composition comprising the nucleic acid molecule of claim 6 or the expression vector of claim 7 or the recombinant cell of claim 8.

10. Use of a bispecific antibody against BAFF and hIL21 according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, an expression vector according to claim 7, a recombinant cell according to claim 8 or a pharmaceutical composition according to claim 9 for the preparation of a medicament for the treatment or prophylaxis of an immune disorder,

Wherein the immune disease is graft versus host disease.

11. Use of a bispecific antibody against BAFF and hIL21 according to any one of claims 1 to 5, a nucleic acid molecule according to claim 6, an expression vector according to claim 7, a recombinant cell according to claim 8 or a pharmaceutical composition according to claim 9 for the preparation of a kit,

Wherein the kit is for use in identifying binding activity to BAFF or hIL 21.