Bispecific antibodies targeting PD-L1 and IL-1 and their use

Abstract

The present invention provides a bispecific antibody comprising a first portion targeting PD-L1 and a second portion targeting IL-1, wherein the first portion comprises an anti-PD-L1 antibody or its antigen-binding fragment, and the second portion comprises the extracellular binding domain of the IL-1 receptor (IL-1R) or a variant thereof, or an anti-IL-1β antibody or its antigen-binding fragment. The invention also provides the use of the bispecific antibody in the treatment of tumors.

Description

[Technical Field]

[0001] This invention relates to a bispecific antibody, particularly a bispecific antibody comprising a first portion targeting PD-L1 and a second portion targeting IL-1. This specification further relates to the use of the bispecific antibody in the treatment of tumors. [Background technology]

[0002] The mechanism of action involves simultaneously targeting PD-L1 on the surface of tumor cells and IL-1R1 or IL-1β in the tumor microenvironment, thereby achieving superior therapeutic effects compared to monotherapy or combination therapy.

[0003] According to existing research, PD-L1 is highly expressed on the surface of various tumor cells, and when PD-L1 on the surface of tumor cells binds to PD-1 on the surface of T cells, it suppresses T cell-mediated tumor cell killing and CD8 + It has been shown that it reduces T cell proliferation while simultaneously reducing Treg apoptosis. Antibodies targeting PD-L1 inhibit the binding of PD-L1 to PD-1, restoring the tumor-killing effect of T cells and showing significant therapeutic effects in the treatment of patients with various tumors, including malignant melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, and gastric cancer.

[0004] L-1β is a member of the IL-1 cytokine family. After IL-1α or IL-1β binds to IL-1R1, it binds to IL-1R accessory protein (IL-1RAcP) to form a complex. This complex activates IL-1R-related kinase (IRAK), which in turn activates nuclear genes via NF-κB and other transcription factors. Studies have shown that IL-1β is fully upregulated in the tumor microenvironment (TME) of various cancers, including breast, gastric, pancreatic, and lung cancer. This is because it can alter the TME's composition by recruiting immunosuppressive cells such as MDSCs, tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), and Th17 cells, which themselves are sources of IL-1β. Recent studies have also shown that tumors themselves are sources of IL-1β, establishing autostimulatory cycles that maintain IL-1β synthesis and release, and that tumor-derived IL-1β promotes the expression of angiogenic factors such as VEGF. In short, TME-derived IL-1β and tumor-derived IL-1β not only promote tumor growth and migration, but are also involved in the development of tumor resistance by inducing anti-apoptotic signals. Antibodies targeting IL-1β suppress tumor growth and migration and CD8 in tumor tissue. + It has been shown to enhance T cell infiltration and reduce immunosuppression. Recent preclinical and clinical data indicate that combinations of immune checkpoint inhibitors and agents that negatively modulate IL-1 signaling are a promising therapeutic strategy for solid tumors. Antibody fusion proteins or bispecific antibodies that simultaneously target PD-L1 and IL-1R1, or PD-L1 and IL-1β, offer advantages over monotherapy, including higher specificity and targeting, greater safety, easier administration, and lower development costs. [Overview of the Initiative]

[0005] In one respect, this specification provides a bispecific antibody comprising a first portion targeting PD-L1 and a second portion targeting IL-1, wherein the first portion comprises an anti-PD-L1 antibody or its antigen-binding fragment, and the anti-PD-L1 antibody comprises HCDR1, HCDR2 and HCDR3 derived from the heavy chain variable region (VH) shown in SEQ ID NO: 17 and LCDR1, LCDR2 and LCDR3 derived from the light chain variable region (VL) shown in SEQ ID NO: 18.

[0006] In some embodiments, the anti-PD-L1 antibody includes the sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 90% sequence identity thereto, and the sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 90% sequence identity thereto.

[0007] In some embodiments, the heavy chain constant region of the anti-PD-L1 antibody is derived from IgG1, and the light chain constant region of the anti-PD-L1 antibody is derived from the κ light chain.

[0008] In some embodiments, the second portion comprises the extracellular binding domain of the IL-1 receptor (IL-1R) or a variant thereof.

[0009] In some embodiments, the second portion includes the sequence shown in Sequence ID No. 14 or an amino acid sequence having at least 90% sequence identity thereto.

[0010] In some embodiments, the second portion consists of two parts, each linked by the same or different linking sequences to the N-terminus of the VH portion of the anti-PD-L1 antibody, the N-terminus of the VL portion of the anti-PD-L1 antibody, the C-terminus of the heavy chain constant region of the anti-PD-L1 antibody, or the C-terminus of the light chain constant region of the anti-PD-L1 antibody.

[0011] In some embodiments, the second portion is one and is linked by a linking sequence to the N-terminus of the VH portion of the anti-PD-L1 antibody, the N-terminus of the VL portion of the anti-PD-L1 antibody, the C-terminus of the heavy chain constant region of the anti-PD-L1 antibody, or the C-terminus of the light chain constant region of the anti-PD-L1 antibody.

[0012] In some embodiments, the first portion is further ligated by a linking sequence to the C-terminus of the heavy chain constant region of the anti-PD-L1 antibody, to which an antigen-binding fragment in Fab form of the anti-PD-L1 antibody is attached.

[0013] In some embodiments, the second portion is an anti-IL-1β antibody or its antigen-binding fragment.

[0014] In some embodiments, the anti-IL-1β antibody comprises VH HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 15, and VL LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 16.

[0015] In some embodiments, the anti-IL-1β antibody includes the sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence identity thereto, and the sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 90% sequence identity thereto.

[0016] In some embodiments, the heavy chain constant region of the anti-IL-1β antibody is derived from IgG1, and the light chain constant region of the anti-PD-L1 antibody is derived from the κ light chain.

[0017] In some embodiments, the antigen-binding fragment of the anti-IL-1β antibody is in the form of a Fab or scFv.

[0018] In some embodiments, the heavy chain constant region of the anti-PD-L1 antibody and / or the heavy chain constant region of the anti-IL-1β antibody can be mutated and linked together in a manner that forms a knob-hole between the heavy chain constant regions.

[0019] In some embodiments, the first part and the second part are linked by the same or different linking sequences, and preferably, the linking sequence is selected from (G4S)4G, (G4S)3G, (G4S)4, and (G4S)3.

[0020] In some embodiments, the heavy chain of the anti-PD-L1 antibody comprises the sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 90% sequence identity thereto, and the light chain of the anti-PD-L1 antibody comprises the sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 90% sequence identity thereto.

[0021] In some embodiments, the heavy chain of the anti-IL-1β antibody comprises the sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 90% sequence identity thereto, and the light chain of the anti-IL-1β antibody comprises the sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 90% sequence identity thereto.

[0022] In some embodiments, the antigen-binding fragment of the anti-PD-L1 antibody in scFv form comprises the sequence shown in SEQ ID NO: 66 or an amino acid sequence having at least 90% sequence identity thereto.

[0023] In some embodiments, the antigen-binding fragment of the anti-IL-1β antibody in scFv form comprises the sequence shown in SEQ ID NO: 64 or 65 or an amino acid sequence having at least 90% sequence identity thereto.

[0024] In some embodiments, the bispecific antibody has a schematic antibody structure as shown in any one of FIGS. 1A-1I.

[0025] In some embodiments, the bispecific antibody has a schematic antibody structure as shown in any one of FIGS. 2A-2J.

[0026] In some embodiments, the extracellular binding domain of the IL-1 receptor (IL-1R) or a variant thereof has an Fc fragment ligated to its C-terminus.

[0027] In some embodiments, the heavy chain constant region and / or Fc fragment comprises the N297A mutation.

[0028] In some embodiments, the heavy chain constant region and / or Fc fragment is derived from human IgG1.

[0029] In some embodiments, the heavy chain constant region includes the sequence shown in SEQ ID NO: 68, and the Fc fragment includes the sequence shown in SEQ ID NO: 67.

[0030] In some embodiments, the second portion includes the sequence shown in sequence number 10 or 66.

[0031] In some embodiments, the bispecific antibody comprises a polypeptide chain listed in any row of Table 1.

[0032] In some embodiments, the bispecific antibody is 1) The polypeptide chain of the sequence shown in Sequence ID No. 25 and the polypeptide chain of the sequence shown in Sequence ID No. 26, 2) The polypeptide chain of the sequence shown in Sequence ID No. 25 and the polypeptide chain of the sequence shown in Sequence ID No. 30, 3) The polypeptide chain of the sequence shown in Sequence ID No. 32 and the polypeptide chain of the sequence shown in Sequence ID No. 28, 4) Polypeptide chain of the sequence shown in Sequence ID No. 40, Polypeptide chain of the sequence shown in Sequence ID No. 39, and Polypeptide chain of the sequence shown in Sequence ID No. 28, 5) The polypeptide chain of the sequence shown in Sequence ID No. 43 and the polypeptide chain of the sequence shown in Sequence ID No. 44, 6) The polypeptide chain of the sequence shown in Sequence ID No. 45 and the polypeptide chain of the sequence shown in Sequence ID No. 46, 7) Polypeptide chain of the sequence shown in SEQ ID NO: 33, Polypeptide chain of the sequence shown in SEQ ID NO: 28, Polypeptide chain of the sequence shown in SEQ ID NO: 50, and Polypeptide chain of the sequence shown in SEQ ID NO: 51 8) The polypeptide chain of the sequence shown in Sequence ID No. 62 and the polypeptide chain of the sequence shown in Sequence ID No. 63, 9) The polypeptide chain of the sequence shown in Sequence ID No. 69 and the polypeptide chain of the sequence shown in Sequence ID No. 30, 10) A polypeptide chain of the sequence shown in Sequence ID No. 70 and a polypeptide chain of the sequence shown in Sequence ID No. 28, 11) A polypeptide chain of the sequence shown in Sequence ID No. 71 and a polypeptide chain of the sequence shown in Sequence ID No. 46, or 12) Polypeptide chain of the sequence shown in Sequence ID No. 72 and polypeptide chain of the sequence shown in Sequence ID No. 63 Includes.

[0033] In another respect, the present invention provides a nucleic acid molecule encoding the bispecific antibody or its polypeptide chain.

[0034] In another respect, the present invention provides an expression vector containing the above-mentioned nucleic acid molecule.

[0035] In another respect, the present invention is 1) The above bispecific antibody, nucleic acid molecule or expression vector, and 2) To provide a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0036] In another respect, the present invention provides the use of the above-mentioned bispecific antibody, nucleic acid molecule, or expression vector in the manufacture of a pharmaceutical for treating tumors.

[0037] In some embodiments, the tumor is selected from malignant melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, gastric cancer, breast cancer, pancreatic cancer, and lung cancer.

[0038] In another respect, the present invention provides a method for treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the above-mentioned bispecific antibody, nucleic acid molecule, expression vector, or pharmaceutical composition.

[0039] In some embodiments, the tumor is selected from malignant melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, gastric cancer, breast cancer, pancreatic cancer, and lung cancer. [Brief explanation of the drawing]

[0040] [Figure 1] This is a schematic diagram of the structure of the bispecific antibody of the present invention, which includes Anakinra. [Figure 2] This is a schematic diagram of the structure of the bispecific antibody of the present invention, which includes an anti-IL-1β antibody or its antigen-binding fragment. [Figure 3] The results of the binding affinity of the bispecific antibody of the present invention to huPD-L1-His are shown. [Figure 4] The results of the binding affinity of the bispecific antibodies of the present invention to huIL-1R1-His or IL-1β-His are shown. [Figure 5] The results of blocking IL-1β / IL-1R1 activity by the bispecific antibody of the present invention are shown. [Figure 6] The results of the binding affinity of the bispecific antibody of the present invention to huPD-L1-CHO-K cells are shown. [Figure 7] The results of blocking PD-1 / PD-L1 binding activity by the bispecific antibody of the present invention are shown. [Figure 8] The results of blocking the PD-1 / PD-L1 signaling pathway by the bispecific antibody of the present invention are shown. [Figure 9] The results of the binding ability of the bispecific antibody of the present invention to both huPD-L1 and IL-1R1, or to both IL-1β, are shown. [Figure 10] The SDS-PAGE results for the bispecific antibody and reference substance IPI of the present invention are shown. [Figure 11] The SEC-HPLC results for the bispecific antibody of the present invention are shown. [Modes for carrying out the invention]

[0041] Unless otherwise stated, all technical and scientific terms used herein have meanings that are generally understood by those skilled in the art.

[0042] IL-1β and IL-1R1 are the most important inflammatory factors and their receptors in the tumor microenvironment, and blocking their signaling pathway has been shown to suppress tumor growth and metastasis, as well as contribute to the deactivation of immune checkpoint inhibition in the tumor microenvironment. Combination therapy with immune checkpoint inhibitors and agents that block the IL-1 signaling pathway is a novel and promising therapeutic strategy for solid tumors. Bispecific antibody fusion proteins or bispecific antibodies offer advantages over monotherapy, including higher targeting, greater safety, easier administration, lower development costs, reduced tumor escape, and the potential to overcome resistance.

[0043] The present invention, as generally described herein, will be more readily understood by referring to the following examples, which are provided as examples and are not intended to limit the invention. In the following examples, experimental methods not described in specific conditions were carried out according to common methods and conditions or according to the product description.

[0044] Example 1: Manufacturing of raw materials 1.1 Antigen Production The IL-1 used in this invention is IL-1β, and the main receptor used is IL-1R1. Human IgG1 Fc fragments (SEQ ID NO: 4) or 6×His tags (SEQ ID NO: 5, etc.) were fused to the C-terminus of the human IL-1β extracellular domain (UNIPROT: P01584, SEQ ID NO: 1), human IL-1R1 extracellular domain (UNIPROT: P14778, SEQ ID NO: 2), and human PD-L1 extracellular domain (UNIPROT: Q9NZQ7, SEQ ID NO: 3), respectively, to produce huIL-1β-Fc, huIL-1β-His, huIL-1R1-Fc, huIL-1R1-His, huPD-L1-Fc, and huPD-L1-His antigen proteins. After converting the above amino acid sequence into a gene sequence, target fragment genes were synthesized at General Biol Co., Ltd. Each target fragment was amplified by PCR and constructed into a pcDNA3.4 (Invitrogen) expression vector using homologous recombination. Each constructed recombinant protein expression vector was transformed into E. coli SS320, cultured overnight at 37°C, and the plasmid was extracted using the Endo-Free Plasmid Mini Kit (OMEGA, D6950-01) to obtain endotoxin-free plasmids for eukaryotic expression. The obtained expression vectors were expressed in the Expi293 transient expression system (ThermoFisher, A14635), and the transient expression method was described in the Expi293® Expression System USER GUIDE.

[0045] Five to seven days after transfection, the cell expression supernatant was purified using different tags. For the His-tagged protein supernatant, affinity purification was performed using Ni Smart Beads 6FF (Changzhou smart-Lifesciences Biotechnology Co., Ltd., SA036050), and the target protein was eluted with a concentration gradient of imidazole. Each eluted protein was then purged with buffer in an ultrafiltration tube (Millipore, UFC901096), followed by identification and activity evaluation by SDS-PAGE. After passing these tests, the proteins were frozen and stored at -80°C. For the Fc-tagged protein supernatant, filtration was performed using a 0.22 μm filter, followed by purification by a Protein A / G affinity chromatography column. After purification, the target protein was eluted with 100 mM glycine salt (pH 3.0), concentrated, and purged. After passing SDS-PAGE and activity testing, the proteins were frozen and stored at -80°C.

[0046] 1.2 Preparation of positive control molecules In this embodiment, the IL-1 activity-blocking positive control antibody produced was canakinumab, and the anti-PD-L1 positive control antibody was atezolizumab (Genentech). Both were synthesized based on the sequences disclosed in international application WO2001053353A2 and U.S. patent US8217149B (the light and heavy chains of canakinumab are sequence numbers 6 and 7, respectively, and the light and heavy chains of atezolizumab are sequence numbers 8 and 9, respectively). Plasmids containing the canakinumab light chain gene, the canakinumab heavy chain gene, the atezolizumab light chain gene, and the atezolizumab heavy chain gene were constructed by molecular cloning. The antibodies were expressed using the ExpiCHO transient expression system, and the resulting supernatant was filtered through a 0.22 μm filter. After purification by the Protein A / G affinity method, the antibodies were eluted with 100 mM glycine salt (pH 3.0) to obtain the positive control antibodies canakinumab and atezolizumab.

[0047] In this example, the positive control proteins Anakinra-Fc (SEQ ID NO: 10) and Anakinra-Fc (N297A) (SEQ ID NO: 66) were produced using the same method.

[0048] 1.3 Construction of cell lines 1.3.1 Construction of the huIL-1R1-HEK293 cell line A recombinant vector plasmid expressing full-length human IL-1R1 (UniProtKB-P14778, SEQ ID NO: 11) was constructed, and the plasmid was introduced into HEK293 cells (ATCC® CRL-1573™) by electroporation. Using positive control anakinra-Fc or IL-1β-Fc cells, cell lines with high human IL-1R1 expression were screened and identified by flow cytometry (Beckman, CytoFLEX AOO-1-1102), and these were named Hu-IL-1R1-FL-HEK293 cells.

[0049] 1.3.2 Construction of the IL-1R1-NF-κB-HEK293 cell line A recombinant vector plasmid expressing the full-length human IL-1R1 gene and an NF-κB luciferase reporter plasmid (containing a luciferase reporting gene driven and expressed by an NF-κB response element) were constructed, and these plasmids were co-introduced into HEK293 cells (ATCC® CRL-1573®) using electroporation. After screening monoclonal cell lines with a final concentration of 2 μg / mL of puromycin, cell lines with high human IL-1R1 expression were screened and identified by flow cytometry (Beckman, CytoFLEX AOO-1-1102) using positive control anakinra-Fc or IL-1β-Fc, and IL-1R1-NF-κB-HEK293 cells were obtained by screening by luciferase detection.

[0050] 1.3.3 Construction of the huPD-L1-CHO-K cell line A full-length human PD-L1 DNA fragment (UniProtKB-Q9NZQ7, SEQ ID NO: 12) was synthesized using gene synthesis technology and cloned into an expression vector. It was introduced into E. coli by chemical conversion, and after selecting the E. coli monoclonals, sequencing was performed to obtain the correct plasmid clone. The plasmid was then extracted and sequenced again for confirmation. CHO-K (Thermo, A1461801) cells were cultured in serum-free CD-CHO medium (Gibco, 10743029). The day before electroporation, cells were cultured in 5 × 10⁶ units. 6 The cells were subcultured at a rate of cells / mL, and the following day, the constructed plasmid was introduced into CHO-K cells using Invitrogen's electroporation kit (catalog number: MPK10096) and electroporator (catalog number: MP922947). After electroporation, the cells were transferred to CD-CHO medium and cultured for 48 hours in a 37°C cell culture incubator. After electroporation, CHO-K cells were seeded at a rate of 2000 cells / well in a 96-well plate, and 30 μM MSX (Millipore, GSS-1015-F) and GS supplement (Sigma, 58672C-100 ml) were added. The cells were cultured in a 37°C carbon dioxide incubator, and after 10 days, medium containing 30 μM MSX and 1×GS supplement was added. Single cell clones grown in 96-well plates were collected, transferred to 24-well culture plates, and continuously cultured for growth. Subsequently, FACS assays successfully obtained cell lines stably transfected with huPD-L1-CHO-K.

[0051] 1.3.4 Construction of the CD3L-PD-L1-CHO cell line Based on a CHO cell line (CD3L-CHO cell) that stably expresses CD3L (membrane-immobilized anti-CD3-scFv, capable of directly stimulating CD3 signaling) already manufactured by the applicant, a CD3L-PD-L1-CHO cell line was constructed. Specifically, a plasmid expressing the full length of human PD-L1 was introduced into CD3L-CHO cells using electroporation, monoclonal cell lines were screened using antibiotics, and finally, a cell line that stably expresses human PD-L1 was identified by FACS and named the CD3L-PD-L1-CHO cell line.

[0052] 1.3.5 Construction of the Jurkat-PD-1-NFAT cell line Based on a Jurkat cell line (Jurkat-NFAT cells) stably transfeed with an NFAT luciferase reporter gene plasmid (containing a luciferase reporter gene driven and expressed by an NFAT response element), which was already manufactured by the applicant, the Jurkat-PD-1-NFAT cell line was constructed. Specifically, a plasmid expressing full-length human PD-1 (UNIPROT:Q15116, SEQ ID NO: 13) was introduced into Jurkat-NFAT cells using electroporation, and monoclonal cell lines were screened using a final concentration of 2 μg / mL of puromycin. Subsequently, cell lines stably expressing human PD-1 were identified by FACS and named the Jurkat-PD-1-NFAT cell line.

[0053] Example 2: Configuration design and construction of anti-PD-L1 and IL-1 bispecific antibodies This example describes the construction of structures and expression vectors for bispecific antibodies that block exemplary anti-PD-L1 and IL-1 activity. Twenty-four construct structures were designed: the amino acid sequence of the antagonist protein that antagonizes IL-1R1 was derived from the antagonist protein anakinra (SEQ ID NO: 14), the amino acid sequence of the anti-IL-1 antibody was derived from the anti-IL-1β antibody canakinumab (VH and VL are SEQ ID NO: 15 and 16, respectively), the amino acid sequence of the anti-PD-L1 antibody was derived from atezolizumab (VH and VL are SEQ ID NO: 17 and 18, respectively), and the linker amino acid sequences in some constructs were (G4S)3G (SEQ ID NO: 19), (G4S)4G (SEQ ID NO: 20), (G4S)3 (SEQ ID NO: 21), (G4S)4 (SEQ ID NO: 22), TVAAPSVFIFPP (SEQ ID NO: 23), or ASTKGPSVFPLAP (SEQ ID NO: 24). The antibody arrangement is shown in Figures 1A-1I and 2A-2J, and the corresponding amino acid sequences are shown in Table 1. The specific antibody arrangement is as follows: Constructor BsAb1: Containing two identical first polypeptide chains, from the N-terminus to the C-terminus, it contains the VH domain of the anti-PD-L1 antibody atezolizumab and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Fc), and also contains two identical second polypeptide chains, from the N-terminus to the C-terminus, it contains the IL-1R1 antagonist protein anakinra, linker (G4S) 4G, the VL domain of atezolizumab, and the antibody κ light chain CL domain. BsAb1 has the morphology shown in Figure 1A.

[0054] Constructor BsAb2 contains two identical first polypeptide chains, from the N-terminus to the C-terminus, containing anakinra, the linker (G4S) 4G, the atezolizumab VH domain, and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Fc). It also contains two identical second polypeptide chains, from the N-terminus to the C-terminus, containing the atezolizumab VL domain and the antibody κ light chain CL domain. The difference between constructor BsAb3 and constructor BsAb2 lies in the linker in the first polypeptide chain; the linker in BsAb3 was (G4S) 3G. BsAb2 and BsAb3 have the morphology shown in Figure 1B.

[0055] Constructor BsAb4: Containing two identical first polypeptide chains, from the N-terminus to the C-terminus, it contains the atezolizumab VH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Fc), and two identical second polypeptide chains, from the N-terminus to the C-terminus, it contains the atezolizumab VL domain, antibody κ light chain CL domain, linker (G4S) 4G, and anakinra. BsAb4 has the morphology shown in Figure 1C.

[0056] Constructor BsAb5 contains two identical first polypeptide chains, from the N-terminus to the C-terminus, the atezolizumab VH domain, the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Fc), the linker (G4S) 4G, and anakinra, and two identical second polypeptide chains, from the N-terminus to the C-terminus, the atezolizumab VL domain and the antibody κ light chain CL domain. The difference between constructor BsAb6 and constructor BsAb5 lies in the linker in the first polypeptide chain; the linker of BsAb6 was (G4S) 3G. BsAb5 and BsAb6 have the morphology shown in Figure 1D.

[0057] Constructor BsAb7: Contains three polypeptide chains. The first polypeptide chain contains the atezolizumab VH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Knob mutation Fc) from the N-terminus to the C-terminus. The second polypeptide chain contains anakinra, linker (G4S) 4G, the atezolizumab VH domain, and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain hole mutation Fc) from the N-terminus to the C-terminus. Two identical third polypeptide chains contain the atezolizumab VL domain and the antibody κ light chain CL domain from the N-terminus to the C-terminus. BsAb7 has the morphology shown in Figure 1E.

[0058] Constructor BsAb8 contains four polypeptide chains. The first polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Knob mutation Fc). The second polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VL domain and the antibody κ light chain CL domain. The third polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VH domain, the antibody κ light chain CL domain, and the IgG1 heavy chain hole mutation Fc. The fourth polypeptide chain, from the N-terminus to the C-terminus, contains anakinra, linker (G4S) 4G, the atezolizumab VL domain, the antibody IgG1 heavy chain CH1, and the IgG1 heavy chain hinge region (EPKSC). BsAb8 has the morphology shown in Figure 1F.

[0059] Constructor BsAb9 contains four polypeptide chains. The first polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Knob mutation Fc). The second polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VL domain and the antibody κ light chain CL domain. The third polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VL domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain hole mutation Fc). The fourth polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VH domain, the antibody κ light chain CL domain, the linker (G4S) 4G, and anakinra. BsAb9 has the morphology shown in Figure 1G.

[0060] Constructor BsAb10 contains three polypeptide chains. The first polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Knob mutation Fc). The second polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VH domain, the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain hole mutation Fc), the linker (G4S) 4G, and anakinra. Two identical third polypeptide chains, from the N-terminus to the C-terminus, contain the atezolizumab VL domain and the antibody κ light chain CL domain. BsAb10 has the morphology shown in Figure 1H.

[0061] Constructor BsAb11: Contains three polypeptide chains. The first polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VH domain, the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, the IgG1 heavy chain hinge region, and the IgG1 heavy chain Knob mutation Fc), linker (G4S) 3, the atezolizumab VH domain, the IgG1 heavy chain CH1, and the IgG1 heavy chain hinge region (EPKSC). The second polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VH domain, the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, the IgG1 heavy chain hinge region, and the IgG1 heavy chain hole mutation Fc), linker (G4S) 4G, and anakinra. Three identical third polypeptide chains, from the N-terminus to the C-terminus, contain the atezolizumab VL domain and the antibody κ light chain CL domain. BsAb11 has the morphology shown in Figure 1I.

[0062] Constructor BsAb12: Contains two identical first polypeptide chains, from N-terminus to C-terminus, containing the canakinumab VH domain, linker (G4S)3, the atezolizumab VH domain, and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Fc). Also contains two identical second polypeptide chains, from N-terminus to C-terminus, containing the canakinumab VL domain, linker (G4S)3, the atezolizumab VL domain, and the antibody κ light chain CL domain. The difference between constructor BsAb13 and constructor BsAb12 lies in the linkers in the first and second polypeptide chains. In constructor BsAb13, the linker in the first polypeptide chain was ASTKGPSVFPLAP, and the linker in the second polypeptide chain was TVAAPSVFIFPP. BsAb12 and BsAb13 have the configurations shown in Figure 2A.

[0063] Constructor BsAb14: Contains two identical first polypeptide chains, from the N-terminus to the C-terminus, containing the atezolizumab VH domain, linker (G4S)3, the canakinumab VH domain and IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region and IgG1 heavy chain Fc), and contains two identical second polypeptide chains, from the N-terminus to the C-terminus, containing the atezolizumab VL domain, linker (G4S)3, the canakinumab VL domain and antibody κ light chain CL domain. BsAb14 has the morphology shown in Figure 2A.

[0064] Constructor BsAb15: Contains two identical first polypeptide chains, from the N-terminus to the C-terminus, containing the atezolizumab VH domain, the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Fc), the linker (G4S)3, and canakinumab scFv (VH-(G4S)3-VL). Also contains two identical second polypeptide chains, from the N-terminus to the C-terminus, containing the antibody atezolizumab VL domain and the antibody κ light chain CL domain. BsAb15 has the morphology shown in Figure 2B.

[0065] Constructor BsAb16: Containing four polypeptide chains, the first polypeptide chain contains the atezolizumab VH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Knob mutation Fc) from the N-terminus to the C-terminus, the second polypeptide chain contains the atezolizumab VL domain and the antibody κ light chain CL domain from the N-terminus to the C-terminus, the third polypeptide chain contains the canakinumab VL domain, the antibody κ light chain CL domain, and the IgG1 heavy chain hole mutation Fc from the N-terminus to the C-terminus, and the fourth polypeptide chain contains the canakinumab VH domain, IgG1 heavy chain CH1, and the IgG1 heavy chain hinge region (EPKSC) from the N-terminus to the C-terminus. BsAb16 has the morphology shown in Figure 2C.

[0066] Constructor BsAb17: Containing four polypeptide chains, the first polypeptide chain contains the atezolizumab VH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Knob mutation Fc) from the N-terminus to the C-terminus, the second polypeptide chain contains the atezolizumab VL domain and the antibody κ light chain CL domain from the N-terminus to the C-terminus, the third polypeptide chain contains the canakinumab VH domain, the antibody κ light chain CL domain, and the IgG1 heavy chain hole mutation Fc from the N-terminus to the C-terminus, and the fourth polypeptide chain contains the canakinumab VL domain, IgG1 heavy chain CH1, and the IgG1 heavy chain hinge region (EPKSC) from the N-terminus to the C-terminus. BsAb17 has the morphology shown in Figure 2D.

[0067] Constructor BsAb18: Containing four polypeptide chains, the first polypeptide chain contains the atezolizumab VH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Knob mutation Fc) from the N-terminus to the C-terminus, the second polypeptide chain contains the atezolizumab VL domain and the antibody κ light chain CL domain from the N-terminus to the C-terminus, the third polypeptide chain contains the canakinumab VL domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain hole mutation Fc) from the N-terminus to the C-terminus, and the fourth polypeptide chain contains the canakinumab VH domain and the antibody κ light chain CL domain from the N-terminus to the C-terminus. BsAb18 has the morphology shown in Figure 2E.

[0068] Constructor BsAb19: Containing three polypeptide chains, the first polypeptide chain contains the atezolizumab VH domain, the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Knob mutation Fc) from the N-terminus to the C-terminus, the linker (G4S)3, and canakinumab scFv (VH-(G4S)3-VL); the second polypeptide chain contains the atezolizumab VH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain hole mutation Fc) from the N-terminus to the C-terminus; and two identical third polypeptide chains contain the atezolizumab VL domain and the antibody κ light chain CL domain from the N-terminus to the C-terminus. BsAb19 has the morphology shown in Figure 2F.

[0069] Constructor BsAb20: Contains three polypeptide chains. The first polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Knob mutation Fc). The second polypeptide chain, from the N-terminus to the C-terminus, contains canakinumab scFv(VH-(G4S)3-VL), linker (G4S)3, IgG1 heavy chain hinge region, and IgG1 heavy chain hole mutation Fc. The third polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VL domain and the antibody κ light chain CL domain. BsAb20 has the morphology shown in Figure 2G.

[0070] Constructor BsAb21: Contains three polypeptide chains. The first polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VH domain, the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, the IgG1 heavy chain hinge region, and the IgG1 heavy chain Knob mutation Fc), the atezolizumab VH domain, the IgG1 heavy chain CH1, and the IgG1 heavy chain hinge region (EPKSC). The second polypeptide chain, from the N-terminus to the C-terminus, contains the atezolizumab VH domain, the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, the IgG1 heavy chain hinge region, and the IgG1 heavy chain hole mutation Fc), the linker (G4S)3, and canakinumab scFv (VH-(G4S)3-VL). Three identical third polypeptide chains, from the N-terminus to the C-terminus, contain the atezolizumab VL domain and the antibody κ light chain CL domain. BsAb21 has the morphology shown in Figure 2H.

[0071] Constructor BsAb22: Contains four polypeptide chains. The first polypeptide chain contains the atezolizumab VH domain and the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain F405L mutation Fc) from the N-terminus to the C-terminus; the second polypeptide chain contains the atezolizumab VL domain and the antibody κ light chain CL domain from the N-terminus to the C-terminus; the third polypeptide chain contains the canakinumab VH domain, the antibody κ light chain CL domain, and the IgG1 heavy chain K409R mutation Fc from the N-terminus to the C-terminus; and the fourth polypeptide chain contains the canakinumab VL domain, IgG1 heavy chain CH1, and the IgG1 heavy chain hinge region (EPKSC) from the N-terminus to the C-terminus. BsAb22 has the morphology shown in Figure 2I.

[0072] Constructor BsAb23: Contains two identical first polypeptide chains, from the N-terminus to the C-terminus, containing the atezolizumab VH domain, the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Fc), the linker (G4S)4, and canakinumab scFv (VH-(G4S)4-VL). Also contains two identical second polypeptide chains, from the N-terminus to the C-terminus, containing the atezolizumab VL domain and the antibody κ light chain CL domain. BsAb23 has the morphology shown in Figure 2J.

[0073] Constructor BsAb25: Contains two identical first polypeptide chains, from the N-terminus to the C-terminus, containing the canakinumab VH domain, the IgG1 heavy chain constant region domain (including IgG1 heavy chain CH1, IgG1 heavy chain hinge region, and IgG1 heavy chain Fc), the linker (G4S)4, and atezolizumab scFv (VH(G44C mutation)-(G4S)4-VL(Q100C mutation)), and contains two identical second polypeptide chains, from the N-terminus to the C-terminus, containing the canakinumab VL domain and the antibody κ light chain CL domain. BsAb25 has the morphology shown in Figure 2J.

[0074] Constructor BsAb26: Containing two identical first polypeptide chains, from the N-terminus to the C-terminus, it contains the atezolizumab VH domain and the human IgG1 heavy chain constant region domain (including human IgG1 heavy chain CH1, human IgG1 heavy chain hinge region, and human IgG1 heavy chain Fc(N297A)), and also contains two identical second polypeptide chains, from the N-terminus to the C-terminus, it contains the atezolizumab VL domain, antibody κ light chain CL domain, linker (G4S) 4G, and anakinra. BsAb26 has the morphology shown in Figure 1C.

[0075] Constructor BsAb27: Containing two identical first polypeptide chains, from the N-terminus to the C-terminus, it contains the atezolizumab VH domain, the human IgG1 heavy chain constant region domain (including human IgG1 heavy chain CH1, human IgG1 heavy chain hinge region, and human IgG1 heavy chain Fc(N297A)), and the linkers are (G4S)3G and anakinra, and it contains two identical second polypeptide chains, from the N-terminus to the C-terminus, it contains the atezolizumab VL domain and the antibody κ light chain CL domain. BsAb27 has the morphology shown in Figure 1D.

[0076] Constructor BsAb28: Contains two identical first polypeptide chains, from the N-terminus to the C-terminus, containing the atezolizumab VH domain, linker (G4S)3, the canakinumab VH domain, and the human IgG1 heavy chain constant region domain (including human IgG1 heavy chain CH1, human IgG1 heavy chain hinge region, and human IgG1 heavy chain Fc(N297A)). Contains two identical second polypeptide chains, from the N-terminus to the C-terminus, containing the atezolizumab VL domain, linker (G4S)3, the canakinumab VL domain, and the antibody κ light chain CL domain. BsAb28 has the morphology shown in Figure 2A.

[0077] Constructor BsAb29: Contains two identical first polypeptide chains, from the N-terminus to the C-terminus, containing the canakinumab VH domain, the human IgG1 heavy chain constant region domain (including human IgG1 heavy chain CH1, human IgG1 heavy chain hinge region, and human IgG1 heavy chain Fc(N297A)), the linker (G4S)4, and atezolizumab scFv (VH(G44C mutation)-(G4S)4-VL(Q100C mutation)), and contains two identical second polypeptide chains, from the N-terminus to the C-terminus, containing the canakinumab VL domain and the antibody κ light chain CL domain. BsAb29 has the morphology shown in Figure 2J.

[0078] Based on the structure of the construct, each fragment was amplified by PCR, and after ligating the fragments by overlap extension PCR, each fragment was incorporated into a modified eukaryotic expression vector plasmid pcDNA3.3-TOPO(Invitrogen) using homologous recombination to construct the complete construct polypeptide chain full-length gene. The vectors containing the constructed construct and the full-length polypeptide chain gene were transformed into E. coli SS320 and cultured overnight at 37°C. Plasmid extraction was performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain endotoxin-free construct polypeptide chain plasmids for eukaryotic expression.

[0079] [Table 1]

[0080] Example 3: Expression, purification, and analysis of physicochemical properties of anti-PD-L1 and anti-IL-1 bispecific antibodies. 3.1 Expression and purification of anti-PD-L1 and anti-IL-1 bispecific antibodies The antibody in Example 2 was expressed using the ExpiCHO transient expression system (Thermo Fisher, A29133), and the specific method is as follows: On the day of transfection, the CHO cell density was approximately 7 × 10⁶. 6 ~1 × 10 7 After confirming a cell / mL concentration and cell viability >98%, cells were then used in fresh ExpiCHO expression medium preheated to 37°C to a final concentration of 6 × 10⁶ cells. 6The concentration was adjusted to cells / mL. The target plasmid (1 μg of plasmid added to 1 mL of the medium) was diluted using OptiPRO™ SFM pre-cooled to 4°C, and at the same time, ExpiFectamine™ CHO was diluted with OptiPRO™ SFM. The two mixtures were then mixed in equivolume and gently pipettered to prepare the ExpiFectamine™ CHO / plasmid DNA mixture. This mixture was cultured at room temperature for 1-5 minutes, then slowly added to the prepared cell suspension while gently shaking. Finally, the mixture was transferred to a cell culture shaker and cultured at 37°C under 8% CO2 conditions. 18-22 hours after transfection, ExpiCHOTM Enhancer and ExpiCHOTM Feed were added to the medium, and the shake flask was kept in a shaker at 32°C under 5% CO2 conditions to continue culturing. On day 5 after transfection, the same volume of ExpiCHOTM Feed was added, and the cell suspension was gently shaken while slowly adding the feed. 7–15 days after transfection, the cell culture supernatant expressing the target protein was centrifuged at 15000g for 10 minutes at high speed. The resulting supernatant was affinity-purified with MabSelect SuRe LX (GE, 17547403), the target protein was then eluted with 100mM sodium acetate (pH 3.0), neutralized with 1M Tris-HCl, and finally the obtained protein was replaced with PBS buffer using an ultrafiltration tube (Millipore, UFC901096).

[0081] 3.2 Concentration measurement of anti-PD-L1 and anti-IL-1 bispecific antibodies The bispecific antibody purified in Example 3.1 was measured for concentration using an ultra-trace spectrophotometer (Hangzhou Allsheng Instruments Co., Ltd., Nano-300). The measured A280 value was divided by the theoretical extinction coefficient of the antibody to obtain the antibody concentration value used in subsequent studies. After passing quality control, the antibody was aliquoted and stored at -80°C.

[0082] 3.3 SDS-PAGE measurement of anti-PD-L1 and anti-IL-1 bispecific antibodies Preparation of non-reducing solution: 1 μg each of candidate antibody and reference IPI were taken, 5× SDS loading buffer and 40 mM iodoacetamide were added, and the mixture was heated in a 75°C dry bath for 10 minutes. After cooling to room temperature, the mixture was centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected.

[0083] Preparation of the reducing solution: Candidate antibody and reference IPI 2 μg were added to 5 × SDS loading buffer and 5 mM DTT, heated in a dry bath at 100°C for 10 minutes, then cooled to room temperature, centrifuged at 12000 rpm for 5 minutes, and the supernatant was collected.

[0084] The supernatant was added to a Bis-tris 4-15% gradient gel (purchased from GenScript), and gel electrophoresis was performed at a constant voltage of 110V. Protein bands were visualized by Coomassie brilliant blue staining. After destaining the protein gel, it was scanned with an EPSON V550 color scanner, and the purity of the reduced and unreduced bands was calculated using peak area normalization in ImageJ. The results are shown in Table 2 and Figures 10A-10I. The bands of the candidate bispecific antibodies and the reference IPI unreduced gel all matched the expected sizes, and the purity of BsAb12, BsAb13, BsAb14, BsAb16, BsAb22, BsAb25, BsAb28, and BsAb29 exceeded 95%.

[0085] 3.4 SEC-HPLC measurement of anti-PD-L1 and anti-IL-1 bispecific antibodies Preparation of materials: 1. Mobile phase: 150 mmol / L phosphate buffer, pH: 7.4; 2. Sample preparation: Each antibody and quality control IPI were diluted to 0.5 mg / mL in the mobile phase solution.

[0086] Using either an Agilent HPLC 1100 or a SHIMADZU LC2030C PLUS liquid gas chromatography system, the gas chromatography column was an XBridge BEH (SEC 3.5 μm, 7.8 mm ID × 30 cm), the Waters flow rate was 0.8 mL / min, the injection volume was 20 μL, and the VWD detector wavelengths were set to 280 nm and 214 nm. Blank solution, IPI quality control solution, and antibody sample solution were injected sequentially. The percentages of high molecular weight polymers, antibody monomers, and low molecular weight substances in the samples were calculated using area normalization. From the results, the individual purity of BsAb6, BsAb11, BsAb14, BsAb25, BsAb26, BsAb28, and BsAb29 all exceeded 90% (Table 2 and Figures 11A-11AD).

[0087] [Table 2]

[0088] JPEG2026521932000004.jpg78159

[0089] Example 4 Evaluation of antigen-binding and blocking activity of anti-PD-L1 and anti-IL-1 bispecific antibodies at the protein level 5.1 Binding activity of candidate bispecific antibodies at the protein level In this example, the antigen-binding ability of candidate bispecific antibody molecules to PD-L1 or IL-1 was evaluated, and the specific experimental method is as follows.

[0090] Human recombinant proteins huPD-L1-His, huIL-1R1-His, or huIL-1β-His were coated onto 96-well ELISA plates and left to stand overnight at 4°C. The following day, the well plates were washed three times with PBST, blocked with 5% skim milk for 2 hours, washed three times with PBST, and then incubated for 1 hour with different concentrations of test antibodies. Subsequently, the plates were washed three times with PBST, and the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 hour. After incubation was complete, the plates were washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added to induce color development. Based on the color development results, the reaction was stopped by adding 2M HCl, and the OD450 value was read using a microplate reader (Molecular Devices, SpecterMax 190). PRISM TM (GraphPad Software, San Diego, CA) was used to analyze the data and EC 50 The value (nM) was calculated.

[0091] The binding affinity of the candidate bispecific antibodies to huPD-L1-His is shown in Figures 3A-3H. Each candidate molecule bound to the antigen PD-L1 with high affinity. Here, BsAb4, BsAb6, BsAb26, BsAb27, and BsAb28 bound most strongly, comparable to the positive control antibody atezolizumab, while the others bound less strongly than the positive control antibody. Furthermore, as shown in Figures 4A-4H, the candidate bispecific antibody molecules showed the predetermined binding affinity to either huIL-1R1-His or IL-1β-His. Here, BsAb12, BsAb13, and BsAb26 bound most strongly, comparable to the positive control canakinumab, while the others bound less strongly than the positive control anakinra-Fc, canakinumab, or anakinra-Fc(N297A).

[0092] 5.2 IL-1β / IL-1R1 binding blocking activity of candidate bispecific antibody molecules The blocking activity of the obtained bispecific antibody against IL-1β / IL-1R1 was evaluated. By ELISA, it was detected whether the bispecific antibody blocked the binding of huIL-1β and huIL-1R1. The specific method was as follows: The 96-well ELISA plate was coated with human recombinant protein huIL-1R1-Fc and left standing overnight at 4°C. The next day, after washing the well plate three times with PBST, it was blocked with 5% skim milk for 2 hours at room temperature. After washing the plate three times with PBST, different concentrations of the test antibody were added and cultured at room temperature for 1 hour. After washing three times with PBST, huIL-1β-His was added to the 96-well ELISA plate and cultured at room temperature for 1 hour. Then, after washing three times with PBST, the secondary antibody Anti-6×His-HRP (proteintech, HRP-66005) was added and cultured at room temperature for 50 minutes. After the culture was completed, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added to develop color. Based on the color development result, 2M HCl was added to stop the reaction, and the OD450 value was read using a microplate reader (Molecular Devices, SpecterMax 190). PRISM TM (GraphPad Software, San Diego, CA) was used to analyze the data and calculate the IC 50 value (nM).

[0093] The blocking activities of the candidate bispecific antibodies against IL-1β / IL-1R1 were as shown in Figures 5A to 5F. All candidate molecules had blocking activity against IL-1β / IL-1R1 at the protein level, but were weaker than the positive controls anakinra-Fc or canakinumab.

[0094] Example 6 Evaluation of the binding and blocking activities of anti-PD-L1 and anti-IL-1 bispecific antibodies at the huPD-L1-CHO-K cell level 6.1 Binding activity of candidate bispecific antibodies at the huPD-L1-CHO-K cell level The binding of bispecific antibodies to huPD-L1-CHO-K cells was detected using the FACS method (expressed as average fluorescence intensity MFI). The specific test procedure is as follows: Well-cultured huPD-L1-CHO-K cells were collected and centrifuged at 300g, with the supernatant removed. The cells were resuspended in FACS buffer (PBS buffer with 2% FBS added), counted, and the cell suspension density was adjusted to 1 × 10⁶ cells per well. 5 Cells were added, and the supernatant was removed by further centrifugation. The corresponding wells were then filled with gradient-diluted test antibodies and the control antibody atezolizumab. After culturing at 4°C for 60 minutes, the cells were washed three times with FACS buffer, 0.5 μg of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596) was added, and the cells were cultured at 4°C for 30 minutes. Finally, the cultured cells were washed three times, resuspended in 200 μL of FACS buffer, and detected using a flow cytometer (Beckman, CytoFLEX AOO-1-1102).

[0095] The results are shown in Figures 6A-6H. All bispecific antibodies bound to huPD-L1-CHO-K overexpressing cells with high affinity. The binding activity of BsAb4 and BsAb6 was significantly stronger than that of the other candidate antibodies and the positive control antibody atezolizumab, while the binding activity of BsAb5, BsAb10, BsAb11, BsAb14, BsAb19, and BsAb23 was equivalent to that of the positive control antibody atezolizumab.

[0096] 6.2 Evaluation of the blocking activity of candidate bispecific antibodies at the PD-1 / PD-L1 cell level The activity of bispecific antibodies in blocking PD-1 / PD-L1 binding at the cellular level was evaluated using FACS (expressed as mean fluorescence intensity MFI). The specific test method is as follows: Well-cultured huPD-L1-CHO-K cells were collected and centrifuged at 300g, with the supernatant removed. The cells were resuspended in FACS buffer (PBS buffer with 2% FBS added), counted, and the cell suspension density was adjusted to 1 × 10⁶ cells per well. 5Cells were added, centrifuged, and the supernatant was removed. The corresponding wells were then filled with gradient-diluted test antibodies and the control antibody atezolizumab, and cultured at 4°C for 60 minutes. After washing the cells twice with FACS buffer, biotin-labeled PD-1-biotin protein was added, and the cells were cultured at 4°C for 60 minutes. Subsequently, the cells were washed twice with FACS buffer, PE-labeled streptavidin (Invitrogen, 12-4317-087) was added, and the cells were cultured at 4°C for 30 minutes. After washing the cultured cell mixture three times, the cells were resuspended in 200 μL of FACS buffer and measured using a flow cytometer (Beckman, CytoFLEX AOO-1-1102). Data were analyzed using PRISM™ (GraphPad Software, San Diego, CA), and IC50 was obtained. 50 The value was calculated.

[0097] The results are shown in Figures 7A-7I, where most bispecific antibodies exhibited PD-1 / PD-L1 binding blocking activity comparable to the control antibody atezolizumab. Here, the blocking activity of BsAb4, BsAb6, BsAb11, and BsAb25 was significantly stronger than that of the other candidate antibodies and the positive control antibody atezolizumab.

[0098] Example 7 Verification of the blocking activity of anti-PD-L1 and IL-1 bispecific antibodies against the PD-1 / PD-L1 signaling pathway. The blocking activity of bispecific antibodies against the PD-1 / PD-L1 signaling pathway was evaluated using luciferase reporter detection.

[0099] Specific experimental method: First, CD3L-huPD-L1-CHO-K and huPD-1-NF-AT-Jurkat cells in good proliferation state were cultured and collected. Next, the test bispecific antibody and the positive control atezolizumab were gradient diluted, and the diluted test antibody was added to a 96-well cell culture plate. The above two types of cells were washed in RPMI 1640 medium, resuspended, and then divided into 1 × 10⁶ cells each in a 5:1 ratio (huPD-1-NF-AT-Jurkat and CD3L-huPD-L1-CHO-K cells). 5Cells / well and 2 × 10 4 Cells were added to a 96-well cell culture plate containing the test antibody (cells / well) and cultured in a 37°C cell incubator for 6 hours. After culturing, 50 μL of luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well, and after shaking for 60 seconds, the fluorescence intensity was detected.

[0100] The results are shown in Figures 8A-8H. Most bispecific antibodies showed strong luciferase signaling activation activity. Here, BsAb4, BsAb6, BsAb11, and BsAb26 showed superior activation effects compared to the positive control antibody atezolizumab, suggesting that they possess excellent PD-1 / PD-L1 signaling pathway blocking activity and immunoactivating activity.

[0101] Example 8: Steric hindrance analysis of preferred bispecific antibody binding to biantigen targets at the protein level. In this example, we evaluate the ability of a preferred bispecific antibody molecule to simultaneously bind to PD-L1 and IL-1 antigens. The specific experimental method is as follows: Human recombinant proteins huPD-L1-Fc, huIL-1R1-Fc, or huIL-1β-Fc were coated onto 96-well ELISA plates and left to stand overnight at 4°C. The following day, the well plates were washed three times with PBST, blocked with 5% skim milk at room temperature for 2 hours, washed three times with PBST, and then incubated for 1 hour with different concentrations of test antibodies. After washing three times with PBST, another antigen, huIL-1R1-His or huIL-1β-His, or huPD-L1-His, was added and incubated for 1 hour at room temperature. Subsequently, the plates were washed three times with PBST, the secondary antibody Anti-6×His-HRP (proteintech, RP-66005) was added, and incubated for 50 minutes at room temperature. After incubation was complete, the plates were washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added for color development. Based on the color development results, the reaction was stopped by adding 2M HCl, and the OD450 value was read using a microplate reader (Molecular Devices, SpecterMax 190). PRISM TM (GraphPad Software, San Diego, CA) was used to analyze the data and EC 50 The value (nM) was calculated.

[0102] The binding ability of preferred bispecific antibodies to simultaneously bind huPD-L1 and IL-1R1 or IL-1β is shown in Figures 9A-9H (Figure 9A shows the result of coating huPD-L1-Fc and adding IL-1R1-His, Figure 9B shows the result of coating huIL-1R1-Fc and adding huPD-L1-His, and Figure 9C shows the result of coating huPD-L1-Fc and adding huIL-1β-His). As shown in Figure 9D (which shows the results of coating huIL-1β-Fc and adding huPD-L1-His), BsAb1, BsAb4, BsAb6, BsAb11, BsAb13, BsAb5, BsAb26, BsAb27, BsAb28, and BsAb29 bound simultaneously with PD-L1 and IL-1R1 or IL-1β antigen with high affinity.

[0103] The amino acid sequences referred to herein are as follows:

[0104]

Table 3

[0105] JPEG2026521932000006.jpg253165

[0106] JPEG2026521932000007.jpg254163

[0107] JPEG2026521932000008.jpg254164

[0108] JPEG2026521932000009.jpg255162

[0109] JPEG2026521932000010.jpg254164

[0110] JPEG2OTB521932000011.jpg254163

[0111] JPEG2026521932000012.jpg255162<00004US

[0112] JPEG2026521932000013.jpg254162

[0113] [[ID=4S]]JPEG2026521932000014.jpg255163

[0114] JPEG2026521932000015.jpg255163

[0115] JPEG2026521932000016.jpg255162

[0116] JPEG2026521932000017.jpg254164

[0117] JPEG2026521932000018.jpg254166

[0118] JPEG2026521932000019.jpg254164

[0119] JPEG2026521932000020.jpg255164

[0120] JPEG2026521932000021.jpg255163

[0121] JPEG2026521932000022.jpg255164

[0122] JPEG2026521932000023.jpg105164

Claims

1. A bispecific antibody comprising a first portion targeting PD-L1 and a second portion targeting IL-1, wherein the first portion comprises an anti-PD-L1 antibody or its antigen-binding fragment, and the anti-PD-L1 antibody comprises HCDR1, HCDR2, and HCDR3 derived from the heavy chain variable region (VH) shown in SEQ ID NO: 17, and LCDR1, LCDR2, and LCDR3 derived from the light chain variable region (VL) shown in SEQ ID NO:

18.

2. The bispecific antibody according to claim 1, wherein the anti-PD-L1 antibody comprises the sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least 90% sequence identity thereto, and the sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least 90% sequence identity thereto.

3. The bispecific antibody according to claim 1 or 2, wherein the heavy chain constant region of the anti-PD-L1 antibody is derived from IgG1, and the light chain constant region of the anti-PD-L1 antibody is derived from the κ light chain.

4. The bispecific antibody according to any one of claims 1 to 3, wherein the second portion comprises the extracellular binding domain of the IL-1 receptor (IL-1R) or a variant thereof.

5. The bispecific antibody according to any one of claims 1 to 4, wherein the second portion comprises the sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity thereto.

6. The bispecific antibody according to any one of claims 1 to 5, wherein the second portion comprises two portions, each linked by the same or different linking sequences to the N-terminus of the VH portion of the anti-PD-L1 antibody, the N-terminus of the VL portion of the anti-PD-L1 antibody, the C-terminus of the heavy chain constant region of the anti-PD-L1 antibody, or the C-terminus of the light chain constant region of the anti-PD-L1 antibody.

7. The bispecific antibody according to any one of claims 1 to 6, wherein the second portion is one and is linked by a linking sequence to the N-terminus of the VH portion of the anti-PD-L1 antibody, the N-terminus of the VL portion of the anti-PD-L1 antibody, the C-terminus of the heavy chain constant region of the anti-PD-L1 antibody, or the C-terminus of the light chain constant region of the anti-PD-L1 antibody.

8. The bispecific antibody according to any one of claims 1 to 7, wherein the first portion is further linked to the C-terminus of the heavy chain constant region of the anti-PD-L1 antibody by a linking sequence to an antigen-binding fragment in Fab form of the anti-PD-L1 antibody.

9. The bispecific antibody according to any one of claims 1 to 8, wherein the second portion is an anti-IL-1β antibody or an antigen-binding fragment thereof.

10. The anti-IL-1β antibody comprises VH HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 15, and VL LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 16, the bispecific antibody according to any one of claims 1 to 9.

11. The anti-IL-1β antibody comprises the sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence identity thereto, and the sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 90% sequence identity thereto, according to any one of claims 1 to 10, a bispecific antibody.

12. The bispecific antibody according to any one of claims 1 to 11, wherein the heavy chain constant region of the anti-IL-1β antibody is derived from IgG1, and the light chain constant region of the anti-PD-L1 antibody is derived from the κ light chain.

13. The bispecific antibody according to any one of claims 1 to 12, wherein the antigen-binding fragment of the anti-IL-1β antibody is in the form of Fab or scFv.

14. The bispecific antibody according to any one of claims 1 to 13, wherein the heavy chain constant region of the anti-PD-L1 antibody and / or the heavy chain constant region of the anti-IL-1β antibody can be mutated and conjugated in a manner that forms a knob hole between the heavy chain constant regions.

15. The first part and the second part are connected by the same or different connecting arrangement, preferably the connecting arrangement is (G ４ S) ４ G, (G ４ S) ３ G, (G ４ S) ４ and (G ４ S) ３ A bispecific antibody according to any one of claims 1 to 14, selected from the above.

16. The bispecific antibody according to any one of claims 1 to 15, wherein the heavy chain of the anti-PD-L1 antibody comprises the sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 90% sequence identity thereto, and the light chain of the anti-PD-L1 antibody comprises the sequence shown in SEQ ID NO: 8 or an amino acid sequence having at least 90% sequence identity thereto.

17. The bispecific antibody according to any one of claims 1 to 16, wherein the heavy chain of the anti-IL-1β antibody comprises the sequence shown in SEQ ID NO: 7 or an amino acid sequence having at least 90% sequence identity thereto, and the light chain of the anti-IL-1β antibody comprises the sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 90% sequence identity thereto.

18. The bispecific antibody according to any one of claims 1 to 17, wherein the antigen-binding fragment in scFv form of the anti-PD-L1 antibody comprises the sequence shown in SEQ ID NO: 65 or an amino acid sequence having at least 90% sequence identity thereto.

19. The bispecific antibody according to any one of claims 1 to 18, wherein the antigen-binding fragment in scFv form of the anti-IL-1β antibody comprises the sequence shown in SEQ ID NO: 64 or an amino acid sequence having at least 90% sequence identity thereto.

20. A bispecific antibody according to any one of claims 1 to 19, having a schematic antibody structure in any of Figures 1A to 1I.

21. A bispecific antibody according to any one of claims 1 to 19, having a schematic antibody structure in any of Figures 2A to 2J.

22. The bispecific antibody according to any one of claims 1 to 21, wherein the extracellular binding domain of the IL-1 receptor (IL-1R) or a variant thereof has an Fc fragment ligated to its C-terminus.

23. The bispecific antibody according to any one of claims 1 to 22, wherein the heavy chain constant region and / or Fc fragment comprises the N297A mutation.

24. The bispecific antibody according to any one of claims 1 to 23, wherein the heavy chain constant region and / or Fc fragment is derived from human IgG1.

25. The bispecific antibody according to any one of claims 1 to 24, wherein the heavy chain constant region comprises the sequence shown in SEQ ID NO: 68, and the Fc fragment comprises the sequence shown in SEQ ID NO:

67.

26. The second portion comprises the sequence shown in SEQ ID NO: 10 or 66, the bispecific antibody according to any one of claims 1 to 25.

27. The bispecific antibody according to any one of claims 1 to 26, wherein the bispecific antibody comprises a polypeptide chain listed in any row of Table 1.

28. The aforementioned bispecific antibody is 1) A polypeptide chain of the sequence shown in Sequence ID No. 25 and a polypeptide chain of the sequence shown in Sequence ID No. 26, 2) A polypeptide chain of the sequence shown in Sequence ID No. 25 and a polypeptide chain of the sequence shown in Sequence ID No. 30, 3) A polypeptide chain of the sequence shown in Sequence ID No. 32 and a polypeptide chain of the sequence shown in Sequence ID No. 28, 4) A polypeptide chain of the sequence shown in Sequence ID No. 40, a polypeptide chain of the sequence shown in Sequence ID No. 39, and a polypeptide chain of the sequence shown in Sequence ID No. 28, 5) A polypeptide chain of the sequence shown in Sequence ID No. 43 and a polypeptide chain of the sequence shown in Sequence ID No. 44, 6) The polypeptide chain of the sequence shown in Sequence ID No. 45 and the polypeptide chain of the sequence shown in Sequence ID No. 46, 7) A polypeptide chain of the sequence shown in SEQ ID NO: 33, a polypeptide chain of the sequence shown in SEQ ID NO: 28, a polypeptide chain of the sequence shown in SEQ ID NO: 50, and a polypeptide chain of the sequence shown in SEQ ID NO: 51, or 8) The polypeptide chain of the sequence shown in Sequence ID No. 62 and the polypeptide chain of the sequence shown in Sequence ID No. 63, 9) A polypeptide chain of the sequence shown in Sequence ID No. 69 and a polypeptide chain of the sequence shown in Sequence ID No. 30, 10) A polypeptide chain of the sequence shown in Sequence ID No. 70 and a polypeptide chain of the sequence shown in Sequence ID No. 28, 11) A polypeptide chain of the sequence shown in Sequence ID No. 71 and a polypeptide chain of the sequence shown in Sequence ID No. 46, or 12) Polypeptide chain of the sequence shown in Sequence ID No. 72 and polypeptide chain of the sequence shown in Sequence ID No. 63 A bispecific antibody according to any one of claims 1 to 27, comprising:

29. A nucleic acid molecule encoding a bispecific antibody or a polypeptide chain thereof according to any one of claims 1 to 28.

30. An expression vector comprising the nucleic acid molecule described in claim 29.

31. 1) A bispecific antibody according to any one of claims 1 to 28, a nucleic acid molecule according to claim 29, or an expression vector according to claim 30, and 2) Pharmaceutically acceptable carriers A pharmaceutical composition containing the following:

32. Use of a bispecific antibody according to any one of claims 1 to 28, a nucleic acid molecule according to claim 29, or an expression vector according to claim 30 in the manufacture of a pharmaceutical product for treating tumors.

33. The use according to claim 32, wherein the tumor is selected from malignant melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, gastric cancer, breast cancer, pancreatic cancer, and lung cancer.

34. A method for treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a bispecific antibody according to any one of claims 1 to 28, a nucleic acid molecule according to claim 29, an expression vector according to claim 30, or a pharmaceutical composition according to claim 31.

35. The method according to claim 34, wherein the tumor is selected from malignant melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, gastric cancer, breast cancer, pancreatic cancer, and lung cancer.

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