Anti-CD40 / PD1 bispecific antibody and application thereof

Abstract

The invention discloses an anti-CD40 / PD1 bispecific antibody and application thereof, the bispecific antibody is obtained by connecting an immunoglobulin antibody lgG and two same single-chain variable fragments scFv, and the bispecific antibody has a brand new sequence, and can specifically bind CD40 and PD1, block a signal channel, activate T cells and APC cells, and inhibit and kill tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an anti-CD40 / PD1 bispecific antibody and applications of the bispecific antibody. Background Art

[0002] CD40 is a co-stimulatory protein with a molecular weight of 50kD located on the cell membrane surface. It belongs to the tumor necrosis factor receptor (TNFR) family. It is constitutively expressed in antigen presenting cells (including dendritic cells, B cells, monocytes and macrophages), and is also expressed in many other cells, such as endothelial cells, fibroblasts, thymic epithelial cells, etc. In addition, CD40 has been found to be expressed in a variety of tumor cells (such as chronic lymphocytic leukemia, multiple myeloma, renal cancer, lung cancer, etc.). CD40 forms a trimer on the cell surface, and the corresponding ligand CD40L (i.e., CD154) is mainly expressed on the surface of activated T cells. The interaction between CD40 and CD40L is a co-stimulatory signal for T cell activation. The binding of CD40L to CD40 on T cells can activate multiple pathways, including the NF-κB (nuclear factor κB) signaling pathway. Apexigen Pharmaceuticals' CD40 antibody APX005M has been granted orphan drug designation by the FDA and has the potential to treat metastatic pancreatic cancer (ductal carcinoma), with a disease control rate of 90%, but with significant side effects.

[0003] PD1 is a receptor protein expressed on the surface of T cells. When T cells are activated, the expression of PD1 on their surface is upregulated. When PD1 binds to its ligand PD-L1, PD1 begins to exert an inhibitory effect, causing T cells to be exhausted and no longer able to play an immune killing role. That is, after PD1 binds to PD-L1, it can provide inhibitory signals, induce T cell apoptosis, and inhibit T cell activation and proliferation.

[0004] Bispecific antibodies (BsAb) are artificially produced special antibodies that can bind to two different antigens or antigenic epitopes at the same time. The main mechanisms of action of bispecific antibodies include: mediating immune cell killing, blocking dual-target signals, and promoting the formation of functional complexes of proteins. This gives bispecific antibodies unique advantages over traditional monoclonal antibodies.

[0005] At present, the clinical trial application (IND) for the YH008 bispecific antibody submitted by Biocytogen's wholly-owned subsidiary, Youhe Pharmaceutical, has been approved by the China National Medical Products Administration (NMPA). YH008 was independently developed by Biocytogen and is the world's first PD1 / CD40 bispecific antibody. The present invention is dedicated to clarifying a CD40 / PD1 bispecific antibody with a different sequence from the YH008 bispecific antibody and different drugability in the future. Summary of the invention

[0006] In view of this, the present invention provides an anti-CD40 / PD1 bispecific antibody, which has a completely new sequence, has verified preliminary drugability, has low toxicity and side effects, and has verified efficacy.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention first provides an anti-CD40 / PD1 bispecific antibody, which includes an immunoglobulin antibody IgG and two identical single-chain variable fragments scFv, each scFv comprising a heavy chain variable region VH and a light chain variable region VL;

[0009] Wherein, the heavy chain variable region VH comprises complementary determining regions CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences thereof are shown in SEQ ID NO.1-3 respectively;

[0010] The light chain variable region VL comprises complementary determining regions CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences thereof are shown in SEQ ID NO.5-7 respectively;

[0011] The heavy chain variable region of the IgG comprises complementary determining regions CDR-H4, CDR-H5 and CDR-H6, ​​and the amino acid sequences thereof are shown in SEQ ID NOs.10-12, respectively;

[0012] The light chain variable region of the IgG comprises complementary determining regions CDR-L4, CDR-L5 and CDR-L6, and their amino acid sequences are shown in SEQ ID NOs.14-16 respectively.

[0013] In a further embodiment, the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.4; the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.8;

[0014] The amino acid sequence of the heavy chain variable region of the IgG is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the IgG is shown in SEQ ID NO.17.

[0015] In a further embodiment, the constant region of the IgG is from a human antibody;

[0016] Preferably, the heavy chain constant region sequence of the IgG is the heavy chain constant region of human IgG1, and the light chain constant region sequence of the IgG is the light chain constant region of human IgG1.

[0017] In a further embodiment, the heavy chain variable region VH and the light chain variable region VL of the single-chain variable fragment scFv are connected via a peptide linker L1, and the N-terminus of each single-chain variable fragment scFv is connected to the C-terminus of the IgG heavy chain via a peptide linker L2;

[0018] Preferably, the amino acid sequence of the peptide linker L1 and the peptide linker L2 is (GGGGS)n, wherein n is a positive integer.

[0019] In a further embodiment, the heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO.21; the light chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO.23.

[0020] The present invention further provides a nucleotide molecule encoding the bispecific antibody as described above;

[0021] Preferably, the nucleotide sequence encoding the heavy chain of the bispecific antibody is SEQ ID NO.22, and the nucleotide sequence encoding the light chain is SEQ ID NO.24.

[0022] The present invention further provides an expression vector, which contains the nucleotide molecule as described above.

[0023] The present invention further provides a host cell, which contains the expression vector as described above.

[0024] The present invention further provides a pharmaceutical composition comprising the bispecific antibody as described above and any one or more pharmaceutically acceptable excipients.

[0025] The present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating tumors.

[0026] Beneficial effects of the present invention:

[0027] This anti-CD40 / PD1 bispecific antibody has a brand-new bispecific sequence. This bispecific antibody can specifically bind to CD40 and PD1 antigens, block signal pathways, activate T cells and APC cells, and inhibit the killing of tumor cells.

[0028] In addition, the bispecific antibody is composed of the IgG molecule of the anti-CD40 antibody and the scFv domain of the anti-PD1 antibody. Both monoclonal antibody monomers have efficacy that exceeds that of products on the market. The combined bispecific antibody structure can effectively improve the stability of the entire fusion protein while retaining the affinity of the IgG molecule itself. The preparation of bispecific antibodies based on IgG molecules can effectively reduce the uncertainty in stability and immunogenicity caused by excessive changes in molecular structure, which has significant advantages. The combined bispecific antibodies have verified the initial drugability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results of the target protein purity identification by SDS-PAGE gel electrophoresis in Example 1, wherein M: Marker, NOn-reduced: 5 μg CD40 / PD1, Reduced: 5 μg CD40 / PD1;

[0030] Figure 2 This is the SDS-PAGE quality inspection analysis in Example 2, wherein M: Molecular weight marker, 1: Reduce sample, 2: NOn-reduce sample;

[0031] Figure 3 The SEC-HPLC test results in Example 2;

[0032] Figure 4 This is the result of dual antibody activity detection in Example 3. DETAILED DESCRIPTION

[0033] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. In addition, unless otherwise specified, methods that do not specifically record conditions or steps are conventional methods, and the reagents and materials used can be obtained from commercial sources.

[0035] The first aspect of the present invention provides an anti-CD40 / PD1 bispecific antibody, wherein the bispecific antibody can specifically bind to CD40 and PD1, and the bispecific antibody comprises an anti-CD40 immunoglobulin antibody IgG and two identical anti-PD1 single-chain variable fragments scFv. Specifically, the immunoglobulin antibody IgG and the two identical single-chain variable fragments scFv are connected, and the connection method can be a conventional bispecific antibody connection method in the art.

[0036] Wherein, each of the single-chain variable fragments scFv comprises a heavy chain variable region VH and a light chain variable region VL. As is well known in the art, the binding region of an antibody generally comprises a heavy chain variable region and a light chain variable region, and each variable region comprises three complementary determining regions CDR domains. The CDR domains of the heavy chain and light chain of an antibody are referred to herein as CDR-H and CDR-L, respectively.

[0037] The heavy chain variable region VH comprises a complementary determining region amino acid sequence such as CDR-H1 as shown in SEQ ID NO.1, CDR-H2 as shown in SEQ ID NO.2, and CDR-H3 as shown in SEQ ID NO.3;

[0038] The light chain variable region VL comprises a complementary determining region amino acid sequence such as CDR-L1 as shown in SEQ ID NO.5, CDR-L2 as shown in SEQ ID NO.6 and CDR-L3 as shown in SEQ ID NO.7.

[0039] In some specific embodiments of the present invention, the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.4; the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.8;

[0040] In some specific embodiments of the present invention, the molecular structure of the single-chain variable fragment scFv herein is VH-L1-VL, that is, the heavy chain variable region VH is connected to the light chain variable region VL through a peptide linker L1, wherein the peptide linker L1 can adopt a common connecting fragment in the art, such a connecting fragment can be (GGGGS)n, wherein n is a positive integer, which can be selected according to actual needs, preferably, n is 2, 3, 4, 5, etc.

[0041] In a typical embodiment of the present invention, n is 4, the peptide linker L1 is (GGGGS)4, its amino acid sequence is shown in SEQ ID NO.20, and the amino acid sequence of the single-chain variable fragment scFv is shown in SEQ ID NO.9.

[0042] Further, the heavy chain variable region of the IgG comprises a complementary determining region amino acid sequence such as CDR-H4 shown in SEQ ID NO.10, CDR-H5 shown in SEQ ID NO.11, and CDR-H6 shown in SEQ ID NO.12;

[0043] The light chain variable region of the IgG comprises a complementary determining region amino acid sequence such as CDR-L4 as shown in SEQ ID NO.14, CDR-L5 as shown in SEQ ID NO.15 and CDR-L6 as shown in SEQ ID NO.16.

[0044] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the IgG is shown as SEQ ID NO.13; the amino acid sequence of the light chain variable region of the IgG is shown as SEQ ID NO.17.

[0045] In some embodiments of the present invention, the constant region of the immunoglobulin antibody IgG is from a human antibody. Preferably, the heavy chain constant region sequence of the immunoglobulin antibody IgG is the heavy chain constant region of human IgG1 or human IgG4, and the light chain constant region sequence is the light chain constant region of human IgG1 or human IgG4; more preferably, the heavy chain constant region sequence of the immunoglobulin antibody IgG is the heavy chain constant region of human IgG1, and the light chain constant region sequence is the light chain constant region of human IgG1.

[0046] In a typical embodiment of the present invention, the heavy chain amino acid sequence of IgG is shown as SEQ ID NO.18; the light chain amino acid sequence of IgG is shown as SEQ ID NO.19.

[0047] In some specific embodiments of the present invention, in the bispecific antibody, the N-terminus of each single-chain variable fragment scFv is connected to the C-terminus of the IgG heavy chain through a peptide linker L2, wherein the peptide linker L2 can adopt a common connecting fragment in the art, such a connecting fragment can be (GGGGS)n, wherein n is a positive integer, which can be selected according to actual needs, preferably, n is 2, 3, 4, 5, etc.

[0048] In a typical embodiment of the present invention, n is 4, the peptide linker L2 is (GGGGS)4, and its amino acid sequence is shown in SEQ ID NO.20.

[0049] In a typical embodiment of the present invention, the heavy chain amino acid sequence of the bispecific antibody is shown as SEQ ID NO.21, and the light chain amino acid sequence is shown as SEQ ID NO.23.

[0050] The second aspect of the present invention provides a nucleotide molecule encoding the bispecific antibody as described in the first aspect of the present invention. It is understood that the preparation of the nucleotide molecule can be prepared by conventional preparation methods in the art, such as PCR method or artificial sequence synthesis, etc., which will not be described in detail here.

[0051] In a typical embodiment of the present invention, the nucleotide sequence encoding the heavy chain of the bispecific antibody is SEQ ID NO.22, and the nucleotide sequence encoding the light chain thereof is SEQ ID NO.24.

[0052] The third aspect of the present invention provides an expression vector, which contains the nucleotide molecule as described in the second aspect of the present invention. The expression vector can be a conventional vector in the art, which will not be described in detail here. Preferably, the expression vector is a plasmid.

[0053] The fourth aspect of the present invention provides a host cell, which contains the expression vector as described in the third aspect of the present invention. It is understood that the host cell can be selected conventionally in the art, as long as it can satisfy the requirement that the expression vector as described in the third aspect of the present invention can stably replicate itself and the nucleotide molecules carried can be effectively expressed. Specifically, it can be a eukaryotic expression cell or a prokaryotic expression cell. The specific host cell and its transformation method are conventional means in the art, which will not be described in detail.

[0054] The fifth aspect of the present invention provides a pharmaceutical composition, which contains the bispecific antibody as described in the first aspect of the present invention and any one or more pharmaceutically acceptable excipients. It is understood that the bispecific antibody in the pharmaceutical composition is based on a therapeutically effective amount. Specifically, the effective amount here refers to an amount sufficient to prevent or treat the symptoms or diseases of a medical condition. After being used for a specific patient or medical subject, the following changes may occur: the condition to be treated and the overall health of the patient / subject are improved. The effective amount can also be a dosage regimen below the maximum dose to avoid significant side effects or toxic effects. The pharmaceutically acceptable excipients include carriers, excipients, diluents, etc. The specific types can be adjusted according to the type of preparation of the pharmaceutical composition, so they are not described in detail here.

[0055] The sixth aspect of the present invention provides the use of the pharmaceutical composition as described in the fifth aspect of the present invention in the preparation of a drug, wherein the drug is used to prevent and / or treat tumors. The prevention and / or treatment of tumors described herein mainly has the effect of preventing and / or treating tumors, specifically including the delay of the development of tumor-related symptoms and / or the reduction of the severity of these symptoms, as well as the alleviation of existing tumor-associated symptoms and the prevention of the occurrence of other symptoms, and also includes reducing or preventing the metastasis of tumors, etc.

[0056] The tumors that can be prevented and / or treated by the drugs described herein specifically include but are not limited to pancreatic cancer, lung cancer, etc.

[0057] Example 1 Expression and purification of anti-CD40 / PD1 bispecific antibodies

[0058] 1. Reagents used:

[0059] (1) CHO-S cell expression supernatant;

[0060] (2) Sodium chloride (Da Mao, LOT: 20191201);

[0061] (3) 2M Tris pH 8.0 (Sinopharm, Lot: F802KA2653);

[0062] (4) 2 M NaCl (self-prepared);

[0063] (5) Buffer D: 10 mM Tris, 200 mM NaCl pH 8.0 (self-prepared);

[0064] (6) 0.1 M Glycine pH 2.7 (self-prepared);

[0065] (7) PBS (Source Biotechnology, LOTL: G210808)

[0066] (8) Precast gel (GenScript)

[0067] (9) Gel staining, decolorization and elution (GenScript, eStain L1)

[0068] 2. Experimental methods

[0069] (1) Harvesting the supernatant: General Biotech was commissioned to artificially synthesize the cDNA of the light chain and the cDNA of the heavy chain of the antibody, and the synthesized cDNA was cloned into the pTT5 plasmid respectively, and the plasmid construction was confirmed to be correct by sequencing. The sequenced plasmid was transfected, and fresh culture medium Freestyle 293 Gibco was added after transfection. Finally, 50 ml of culture medium was centrifuged at 1000 rpm for 10 minutes, and 0.5 ml of 2M Tris pH 8.0 mother solution and 0.3 g of NaCl powder were added to the supernatant, stirred evenly, and the supernatant was filtered through a 0.22 μm filter membrane.

[0070] (2) Sample loading: Equilibrate the Mabselect column with Buffer D for 5-10 column volumes and load the sample at a flow rate of 3 ml / min.

[0071] (3) Washing: After loading, use 80 ml of Buffer D to wash the column.

[0072] (4) Elution: Use 0.1 M Glycine pH 2.7 for direct elution. Collect the eluate in a 15 ml tube. Add 800 μl of 2 M Tris pH 8.0 stock solution and 900 μl of 2 M NaCl stock solution to the collection tube in advance. Mix the eluate quickly and evenly.

[0073] (5) Purity identification: SDS-PAGE gel electrophoresis (12.5% ​​SDS-Polyacrylamide gel) to identify the purity of the target protein. The results are shown in Figure 1 .

[0074] (6) Concentrate the sample to 1-2 ml, add PBS, and concentrate again. Repeat 3 times and measure the concentration. Dilute the CD40 / PD1 sample to 0.23 mg / ml, 500 μl, a total of 115 μg, freeze quickly in liquid nitrogen, and store at -80°C.

[0075] pass Figure 1 From the test results in , it can be seen that the purity of the anti-CD40 / PD1 bispecific antibody in this example exceeds 90%.

[0076] Example 2 Anti-CD40 / PD1 bispecific antibody protein electrophoresis analysis and HPLC quality inspection

[0077] 1. SDS-PAGE quality inspection analysis

[0078] SDS-PAGE electrophoresis was used for quality control analysis. Figure 2 .

[0079] 2. SEC-HPLC detection

[0080] 10 μl of the anti-CD40 / PD1 bispecific antibody obtained in Example 1 was loaded onto Agilent 1260. The chromatographic conditions were: TSKgel G3000SWXL, 5 μm, 7.8×300 mm column; mobile phase was PBS, pH 7.2; mobile phase flow rate was set to 1 ml / min; run for 20 min; detection method was UV280 nm. Results are shown in Figure 3 .

[0081] pass Figure 2 and Figure 3 It can be seen from the results in that the purity of the antibodies obtained in this article is more than 90%.

[0082] Example 3 Bis-antibody activity detection

[0083] In order to confirm the binding activity of the full-length antibody prepared in Example 1 of the present invention to PD1 and CD40, PD1 (Sino Biological, Cat: 10377-H08H) and CD40 protein (Sino Biological, Cat: 10774-H08H) were plated in an ELISA plate with a diluent CBS at an antigen concentration of 1.0 μg / ml, 50 μL / well. After overnight coating, the ELISA plate was washed three times with PBST at 300 μL / well. PBST blocking solution containing 1% BSA was added and the plate was kept at 37°C for 2 hours, and washed three times with PBST at 300 μL / well. Add 50 μL / well of the dilution-graded double antibody obtained in Example 1, incubate at 37°C for 1 hour, wash three times with PBST at 300 μl / well, add 50 μL / well of HRP-labeled anti-human IgG secondary antibody (goat anti-human IgG, Fcγ fragment specific, Jackson Laboratorise, lot number: 150445, item number: 109-035-098) to detect the double antibody binding content, and then incubate at 37°C for 30 minutes. Then wash the plate four times with PBST at 300 μL / well, add 50 μL / well of TMB (Neogen, Lot: 200707, Cat: 308177) color solution, incubate at room temperature in the dark for 5 minutes, and terminate the color reaction with 2M HCl (stock solution, Shanghai test, Lot: 20181023, Cat: 7647-01-0). The full wavelength microplate reader was used to read the data, and the wavelength was set to 450nm. The data were then sorted and plotted to calculate the EC50 value. The results are shown in Table 1 and Figure 4 As shown in .

[0084] Table 1

[0085]

[0086] Through Table 1 and Figure 4 It can be seen from the results that the bispecific antibodies obtained in this article can specifically bind to PD1 and CD40, activate the NF-KB signal downstream of CD40, and have a dose-effect relationship.

[0087] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0088] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An anti-CD40 / PD1 bispecific antibody, characterized in that: It includes an immunoglobulin antibody IgG and two identical single-chain variable fragments scFv, each scFv comprising a heavy chain variable region VH and a light chain variable region VL; Wherein, the heavy chain variable region VH comprises complementary determining regions CDR-H1, CDR-H2 and CDR-H3, whose amino acid sequences are shown in SEQ ID NOs.1-3, respectively; the light chain variable region VL comprises complementary determining regions CDR-L1, CDR-L2 and CDR-L3, whose amino acid sequences are shown in SEQ ID NOs.5-7, respectively; The heavy chain variable region of the IgG comprises complementary determining regions CDR-H4, CDR-H5 and CDR-H6, ​​and their amino acid sequences are shown in SEQ ID NOs.10-12, respectively; the light chain variable region of the IgG comprises complementary determining regions CDR-L4, CDR-L5 and CDR-L6, and their amino acid sequences are shown in SEQ ID NOs.14-16, respectively.

2. The anti-CD40 / PD1 bispecific antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.4; the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.8; The amino acid sequence of the heavy chain variable region of the IgG is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the IgG is shown in SEQ ID NO.

17.

3. The anti-CD40 / PD1 bispecific antibody according to claim 1, characterized in that: The constant region of the IgG is from a human antibody; Preferably, the heavy chain constant region sequence of the IgG is the heavy chain constant region of human IgG1, and the light chain constant region sequence of the IgG is the light chain constant region of human IgG1.

4. The anti-CD40 / PD1 bispecific antibody according to claim 1, characterized in that: The heavy chain variable region VH and the light chain variable region VL of the single-chain variable fragment scFv are connected via a peptide linker L1, and the N-terminus of each single-chain variable fragment scFv is connected to the C-terminus of the IgG heavy chain via a peptide linker L2; Preferably, the amino acid sequences of the peptide linker L1 and the peptide linker L2 are independently (GGGGS)n, wherein n is a positive integer.

5. The anti-CD40 / PD1 bispecific antibody according to claim 1, characterized in that: The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.21; the light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO.

23.

6. A nucleotide molecule, characterized in that It encodes the bispecific antibody according to any one of claims 1 to 5; Preferably, the nucleotide sequence encoding the heavy chain of the bispecific antibody is SEQ ID NO.22, and the nucleotide sequence encoding the light chain is SEQ ID NO.

24.

7. An expression vector, characterized in that: It contains the nucleotide molecule as claimed in claim 6.

8. A host cell, characterized in that It contains the expression vector as claimed in claim 7.

9. A pharmaceutical composition, characterized in that It contains the bispecific antibody according to any one of claims 1 to 5 and any one or more pharmaceutically acceptable excipients.

10. Use of the pharmaceutical composition according to claim 9 in preparing a drug, characterized in that: The medicine is used for preventing and / or treating tumors.

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