Antibody drug conjugate and application thereof

Abstract

The invention discloses an antibody drug conjugate and application thereof. The proportion of the dimerization antibody formed by the modification mode is obviously improved, and the dimerization antibody can be applied to dimerization of antibody fragments and dimerization of antibody coupling drugs in the future.

Description

[0001] Divisional application

[0002] This application is a divisional application of the patent application with application number 202310848813.5, application date July 11, 2023, and invention name “A dimer of antibody Fab fragment, its preparation method and application”. Technical Field

[0003] The present invention relates to the field of biotechnology, and in particular to an antibody-drug conjugate and application thereof. Background Art

[0004] Antibodies are produced by plasma cells generated by proliferation and differentiation of B cells after antigen stimulation, and have the function of specific binding to antigens. The structure of antibodies consists of the Fab region that determines the antigen binding ability and the Fc region that induces antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but the Fc fragment is prone to non-specific binding, which has an adverse effect on clinical diagnosis and treatment. Compared with complete antibodies, small molecule antibody drugs without Fc fragments have the following advantages: strong specificity; small molecular weight, easier to enter the tumor tissue; can be expressed in Escherichia coli, low cost, short cycle; weak immunogenicity, etc. Common monovalent small molecule antibodies include antigen-binding fragment Fab, single-chain variable region fragment scfv, variable region fragment Fv, single-domain antibodies, etc., but due to the disadvantages of low molecular weight, easy to be cleared, short half-life, and low affinity of monovalent small molecule antibodies, it is necessary to design multivalent small molecule antibodies in practical applications, such as double-chain antibodies (Diabody) and three-chain antibodies (Triabody). Multivalent antibodies can bind to multiple antigen epitopes, and have higher affinity and slower dissociation rate than monovalent antibodies. In addition, the multivalent binding of some antibodies can also cause receptor cross-linking, stimulate intracellular signal transduction, activate cells or induce apoptosis and other biological effects. The formation of multivalent antibodies can be connected by cross-linking, such as Fab segments can be connected into dimers, trimers, etc. through genetic engineering linkers, or formed through self-polymerization domains, ultimately increasing the size of antibody molecules and improving the half-life of antibody drugs in the body. With the in-depth research of small molecule antibody drugs, they are becoming more and more widely used in clinical applications. For example, linked toxin drugs can be used for the treatment of tumors. In addition, small molecule antibody drugs have also shown good application prospects in anti-infection treatment, organ transplantation, allergic diseases, etc. Therefore, converting monovalent small molecule antibodies into multivalent antibodies has become a trend in antibody drug research and development.

[0005] The prior art strategy for modifying antibody Fab fragments is to introduce a (CPP)3 sequence, but the ratio of dimerized antibodies obtained by this method is low and cannot meet the requirements of large-scale production. Therefore, it is urgent to find a new dimer of antibody Fab fragments to solve the above problems. Summary of the invention

[0006] Technical issues solved:

[0007] One aspect of the present invention is to provide a dimer of an antibody Fab fragment, a preparation method and application thereof, in view of the shortcomings of the prior art of dimerization methods of antibody Fab fragments, such as low dimer yield and weak affinity for target cells.

[0008] Specifically, the technical solution in the present disclosure is to H1 The C-terminal amino acid sequence (CP) n , its cysteine ​​is oxidized into a disulfide bond, thereby promoting the dimerization of the Fab fragment and ultimately forming a homologous bivalent antibody molecule.

[0009] The technical solution provided by the present invention is:

[0010] A dimer of an antibody Fab fragment, wherein the antibody Fab fragment comprises V L 、V H , C L and C H1 part, the dimer comprises two monomeric Fab fragments, the C H1 The C-terminus of the part is directly connected to the amino acid sequence (CP) n , or the C of the monomeric Fab fragment H1 The C-terminus of the moiety is indirectly connected to the amino acid sequence (CP) via at least one linker n ,

[0011] Wherein, n=1, 2, 3, 4 or 5.

[0012] Preferably, in certain embodiments of the present disclosure, the above n=1, 2 or 3. More preferably, in one embodiment of the present disclosure, the above n=3. In the present disclosure, the amino acid sequence (CP) n is an amino acid sequence comprising n CP repeating units, i.e., for example, CPCP, CPCCP, CPCCPCP, CPCCPCPCP, and at least one amino acid residue irrelevant to the dimerization effect may be inserted between each CP repeating unit. In certain embodiments of the present disclosure, the C H1 The C-terminus of the part is directly connected to the amino acid sequence (CP) n In other embodiments of the present disclosure, the C H1 The C-terminus of the part is indirectly connected to the amino acid sequence (CP) through a linker (Linker1) nThe purpose and means of the connection can be known in the prior art, for example, adding a flexible peptide linker rich in glycine (G) and serine (S) to increase the flexibility of the dimer, etc.

[0013] In certain embodiments of the present disclosure, the dimer may be a homodimer or a heterodimer. The homodimer or heterodimer may be spontaneously or artificially assembled into a dimer after the formation of the monomeric Fab fragment. The above assembly may be achieved using a suitable method in the prior art, such as, for example, expressing vectors containing gene sequences of different monomeric Fab fragments in the same expression system, and the expression product obtained contains heterodimers formed by the assembly of different monomeric Fab fragments.

[0014] In the present disclosure, the dimers formed by the monomeric Fab fragments can be linked by any suitable covalent bonds or non-covalent bonds. Preferably, in certain embodiments of the present disclosure, the monomeric Fab fragments are linked by disulfide bonds.

[0015] In certain embodiments of the present invention, exemplary technical solutions include, for example:

[0016] A dimer of an anti-CD19 antibody Fab fragment, wherein the antibody Fab fragment comprises V L 、V H , C L and C H1 part, the dimer comprises two monomeric Fab fragments, the C H1 The C-terminus of the part is directly connected to the amino acid sequence (CP) n , or the C of the monomeric Fab fragment H1 The C-terminus of the moiety is indirectly connected to the amino acid sequence (CP) via at least one linker n ,

[0017] Wherein, n=1, 2, 3, 4 or 5; preferably, n=3.

[0018] Another example:

[0019] A dimer of an anti-CD20 antibody Fab fragment, wherein the antibody Fab fragment comprises V L 、V H , C L and C H1 part, the dimer comprises two monomeric Fab fragments, the C H1 The C-terminus of the part is directly connected to the amino acid sequence (CP) n , or the C of the monomeric Fab fragment H1The C-terminus of the moiety is indirectly connected to the amino acid sequence (CP) via at least one linker n ,

[0020] Wherein, n=1, 2, 3, 4 or 5; preferably, n=3.

[0021] Another example:

[0022] A dimer of an anti-BCMA antibody Fab fragment, wherein the antibody Fab fragment comprises V L 、V H , C L and C H1 part, the dimer comprises two monomeric Fab fragments, the C H1 The C-terminus of the part is directly connected to the amino acid sequence (CP) n , or the C of the monomeric Fab fragment H1 The C-terminus of the moiety is indirectly connected to the amino acid sequence (CP) via at least one linker n ,

[0023] Wherein, n=1, 2, 3, 4 or 5; preferably, n=3.

[0024] In the present disclosure, the dimerization of antibody Fab fragments can be achieved through the above technical solution. In order to better exert certain effects of Fab segment dimers, in certain embodiments of the present disclosure, the amino acid sequence (CP) n The last proline residue of the amino acid sequence (CP) can also be directly linked to the effector molecule. n The terminal proline residue of can also be indirectly connected to the effector molecule through at least one linker (Linker 2). The purpose and means of connection through the linker (Linker 2) can be known in the prior art, for example, adding a flexible peptide linker rich in glycine (G) and serine (S) to increase the flexibility of the dimer, etc.

[0025] In certain embodiments of the present disclosure, the effector molecule can be an anticancer agent, an anti-inflammatory agent, an anti-autoimmune disease agent, an anti-infective agent, a toxin, a radiotherapeutic agent, a nucleotide or a cytokine. Preferably, in some embodiments of the present disclosure, the effector molecule is a member of the TNF family or the cofactor protein LDP in lidamycin. In the present disclosure, the above-mentioned effector molecule can be a protein, a nucleic acid or a small molecule compound, and the means of connecting it to the dimer can be a suitable method in the prior art. Exemplary examples of the effector molecule include TRAIL, the tumor necrosis factor-related apoptosis-inducing ligand TRAIL, also known as CD253 or TNF10, which is a member of the TNF superfamily. TRAIL is a type II glycoprotein composed of 281 amino acids, of which the 241 amino acids at the C-terminus are extracellular, and the extracellular part is degraded to form a functional soluble TRAIL. The 230th position of TRAIL is cysteine, which is cleaved by Zn 2+ It forms an active homotrimer and binds to DR4 and DR5 receptors. DR4 and DR5 are agonist receptors with cytoplasmic death domains. When TRAIL binds to them, it can induce the trimerization of the intracellular death domain to form a death signal complex, activate the apoptosis signal transduction pathway and induce tumor cell apoptosis. The strong anti-tumor property of TRAIL can be used as a toxic protein in antibody-drug conjugates and has great development prospects in clinical applications. In some embodiments of the present disclosure, by directly or indirectly combining TRAIL with the amino acid sequence (CP) of the dimer described in the present disclosure n The terminal proline residue of TRAIL was connected to achieve dimerization. Activity identification showed that the dimer has excellent target cell killing ability in vitro.

[0026] In the present disclosure, the antibody Fab fragment comprises V L and V H The V part of the antibody Fab fragment is used to specifically bind to the target protein. L and V H Parts may also be replaced with other protein fragments that specifically bind to the target protein. In certain embodiments of the present disclosure, the protein fragment is a ligand, receptor, scFv, Fv or dAb that specifically binds to the target protein, or a multivalent of the ligand, receptor, scFv, Fv or dAb.

[0027] Another aspect of the present invention is to provide a method for constructing an antibody Fab fragment dimerization vector, wherein the Fab fragment comprises a heavy chain constant region C located at the C-terminus of the fragment. H1 The dimer comprises two monomeric Fab fragments, a nucleotide sequence encoding the monomeric Fab fragment is set in the vector, and a coding (CP) is directly or indirectly set at the 3' end of the nucleotide sequence nThe nucleotide sequence of

[0028] Wherein, n=1, 2, 3, 4 or 5;

[0029] Wherein, the indirect is to set the coding (CP) via a nucleotide sequence encoding at least one connector n nucleotide sequence.

[0030] Another aspect of the present invention is to provide a method for dimerizing antibody Fab fragments, comprising:

[0031] Step a) obtaining a vector comprising a nucleotide sequence encoding the antibody Fab fragment using the above construction method;

[0032] Step b) expressing the vector obtained in step a) in an expression system and purifying it.

[0033] Another aspect of the present invention is to provide the use of the dimer in preparing a drug for treating tumors, inflammation, autoimmune diseases or infections.

[0034] Another aspect of the present invention is to provide use of the dimer in preparing antibody-drug conjugates.

[0035] Beneficial effects of the present invention:

[0036] The novel antibody dimerization modification method provided by the present invention is to modify the C H1 The amino acid sequence (CP)3 is introduced into the C-terminus of the Fab fragment, wherein the cysteine ​​is oxidized into a disulfide bond to promote dimerization of the Fab fragment, and finally form a homologous bivalent antibody molecule. Compared with the existing dimerization technology, i.e., the introduction of (CPP)3, the proportion of dimerized antibodies formed by the modification method of the present invention is significantly increased, and it can be applied to the dimerization of antibody fragments and the dimerization of antibody-drug conjugates in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1A to Figure 1E Schematic diagram of plasmid construction; VL and VH are the variable regions of antibody light and heavy chains, respectively; CL is the CL part of the human light chain constant region; and CH1 is the CH1 part of the human heavy chain constant region; Figure 1A PAYZ-Anti-Fab-(CP) n or PAYZ-Anti-Fab-(CPP) n n=1 or 2 or 3; Figure 1B for the monomer control plasmid PAYZ-Anti-Fab; Figure 1C is PAYZ-Anti-CD19Fab-(CP)3-G4S-LDP; Figure 1Dis PAYZ-Anti-CD19Fab-(CPP)3-G4S-LDP; Figure 1E is PAYZ-Anti-CD19Fab-G4S-LDP;

[0038] Figure 2A to Figure 2F This is a diagram showing the identification results of the modified dimerized protein; Figure 2A and Figure 2B To compare the dimer ratios of six remodeled proteins using the Coomassie Brilliant Blue method, Figure 2A For non-reducing conditions, Figure 2B The lanes are from left to right: Marker; CON; Anti-CD19Fab-LDP (control); No. 1 Anti-CD19Fab-(CPP)3; No. 2 Anti-CD19Fab-(CPP)2; No. 3 Anti-CD19Fab-CPP; No. 4 Anti-CD19Fab-(CP)3; No. 5 Anti-CD19Fab-(CP)2; No. 6 Anti-CD19Fab-CP; Figure 2C and Figure 2D To compare the dimer ratios of the three modified proteins using the Coomassie Brilliant Blue method, Figure 2C For non-reducing conditions, Figure 2D The conditions are reducing; lanes from left to right: Marker; Anti-CD19Fab-(CPP)3; Anti-CD19Fab-(CP)3; Anti-CD19Fab; Figure 2E This is the result of size exclusion chromatography identification of Anti-CD19Fab-(CP)3; Figure 2F This is a diagram of Anti-CD19Fab-(CP)3 liquid chromatography tandem mass spectrometry (LC-MS) identification results;

[0039] Figure 3A to Figure 3D The figure is the flow cytometry result of Anti-CD19Fab-(CPP)3, Anti-CD19Fab-(CP)3, Anti-CD19Fab and Raji direct binding; Figure 3A This is the flow cytometric graph when the antibody concentration is 0.135 μg / ml; Figure 3B The flow cytometry graph is for an antibody concentration of 0.675 μg / ml; Figure 3C This is the flow cytometric graph when the antibody concentration is 1.35 μg / m; Figure 3D Comparison of median values ​​of flow cytometry binding fluorescence intensity at different antibody concentrations; in the flow cytometry: a: Anti-CD19Fab-(CP)3; b: Anti-CD19Fab-(CPP)3; c: Anti-CD19Fab; d: secondary antibody control;

[0040] Figure 4A to Figure 4DThe flow cytometry competition results of Anti-CD19Fab-(CPP)3, Anti-CD19Fab-(CP)3, Anti-CD19Fab and CD19 parent mouse whole antibody are shown in Figure 1. Figure 4A This is the competitive flow cytometric graph of low concentration (9 μg / ml); Figure 4B This is the competition flow cytometric graph of the medium concentration (27 μg / ml); Figure 4C This is the competitive flow cytometric graph of high concentration (81 μg / ml); Figure 4D Comparison of competition percentages at different antibody concentrations; flow cytometry: a: Anti-CD19Fab-(CP)3; b: Anti-CD19Fab-(CPP)3; c: Anti-CD19Fab; d: positive control; e: secondary antibody control;

[0041] Figure 5A and Figure 5B The electrophoresis diagrams are Anti-CD20Fab-(CPP)3, Anti-CD20Fab-(CP)3, and Anti-CD20Fab; wherein, sample 1: Anti-CD20Fab-(CPP)3; 2: Anti-CD20Fab-(CP)3; 3: Anti-CD20Fab; Figure 5A For non-reducing conditions, Figure 5B is the reducing condition;

[0042] Figure 6A to Figure 6D The figure is the flow cytometry result of Anti-CD20Fab-(CPP)3, Anti-CD20Fab-(CP)3, and Anti-CD20Fab directly binding to Raji; Fig. 6A This is the flow cytometry graph of the antibody concentration of 0.135 μg / ml; Figure 6B The flow cytometry graph is for an antibody concentration of 0.675 μg / ml; Figure 6C This is the flow cytometry graph of the antibody concentration of 1.35 μg / ml; Fig.6D Comparison of median values ​​of flow cytometry binding fluorescence intensity at different antibody concentrations; in the flow cytometry: a: Anti-CD20Fab-(CP)3; b: Anti-CD20Fab-(CPP)3; c: Anti-CD20Fab; d: secondary antibody control;

[0043] Figure 7A to Figure 7D The flow cytometry competition results of Anti-CD20Fab-(CPP)3, Anti-CD20Fab-(CP)3, Anti-CD20Fab and CD20 parent mouse antibody are shown in Figure 1. Fig. 7A This is the competitive flow cytometric graph of low concentration (9 μg / ml); Figure 7B This is the competition flow cytometric graph of the medium concentration (27 μg / ml); Figure 7CThis is the competitive flow cytometric graph of high concentration (81 μg / ml); Fig.7D Comparison of competition percentages at different antibody concentrations; flow cytometry: a: Anti-CD20Fab-(CP)3; b: Anti-CD20Fab-(CPP)3; c: Anti-CD20Fab; d: positive control; e: secondary antibody control;

[0044] Fig. 8A and Figure 8B The electrophoresis diagrams are Anti-BCMAFab-(CPP)3, Anti-BCMAFab-(CP)3, and Anti-BCMA Fab; wherein, the loading sample 1: Anti-BCMAFab-(CPP)3; 2: Anti-BCMAFab-(CP)3; 3: Anti-BCMAFab; Fig. 8A For non-reducing conditions, Figure 8B For the reducing condition.

[0045] FIG. 9A to FIG. 9D The figure is the flow cytometry result of direct binding of Anti-BCMAFab-(CPP)3, Anti-BCMAFab-(CP)3, Anti-BCMA Fab and H929; Fig. 9A This is the flow cytometry graph of the antibody concentration of 0.135 μg / ml; Fig. 9B The flow cytometry graph is for an antibody concentration of 0.675 μg / ml; Fig. 9C This is the flow cytometry graph of the antibody concentration of 1.35 μg / ml; Fig.9D Comparison of median values ​​of flow cytometry binding fluorescence intensity at different antibody concentrations; in the flow cytometry: a: Anti-BCMAFab-(CP)3; b: Anti-BCMAFab-(CPP)3; c: Anti-BCMAFab; d: secondary antibody control;

[0046] FIG. 10A to FIG. 10D The flow cytometry competition results of Anti-BCMAFab-(CPP)3, Anti-BCMAFab-(CP)3, Anti-BCMAFab and BCMA parent mouse antibody are shown in Figure 1. Fig. 10A This is the competitive flow cytometric graph of low concentration (9 μg / ml); Fig. 10B This is the competition flow cytometric graph of the medium concentration (27 μg / ml); Fig. 10C This is the competitive flow cytometric graph of high concentration (81 μg / ml); Fig. 10D Comparison of competition percentages at different antibody concentrations; flow cytometry: a: Anti-BCMAFab-(CP)3; b: Anti-BCMAFab-(CPP)3; c: Anti-BCMA Fab; d: positive control; e: secondary antibody control;

[0047] Fig.11A and Fig. 11B The electrophoresis diagrams are of Anti-CD19Fab-(CPP)3-G4S-LDP, Anti-CD19Fab-(CP)3-G4S-LDP, and Anti-CD19Fab-G4S-LDP; wherein, sample 1: Anti-CD19Fab-(CPP)3-G4S-LDP; 2: Anti-CD19Fab-(CP)3-G4S-LDP; 3: Anti-CD19Fab-G4S-LDP; Fig.11A For non-reducing conditions, Fig. 11B is the reducing condition;

[0048] FIG. 12A to FIG. 12D Figure 2 is a graph showing the direct binding of Anti-CD19Fab-(CPP)3-G4S-LDP, Anti-CD19Fab-(CP)3-G4S-LDP, and Anti-CD19Fab-G4S-LDP to Raji; Fig. 12A This is the flow cytometry graph with an antibody concentration of 0.165 μg / ml; Fig. 12B This is the flow cytometry graph of the antibody concentration of 0.825 μg / ml; Fig. 12C The flow cytometry graph is for an antibody concentration of 1.65 μg / ml; Fig.12D Comparison of median values ​​of flow cytometry binding fluorescence intensity at different antibody concentrations; in the flow cytometry: a: Anti-CD19Fab-(CP)3-G4S-LDP; b: Anti-CD19Fab-(CPP)3-G4S-LDP; c: Anti-CD19Fab-G4S-LDP; d: secondary antibody control;

[0049] FIG. 13A to FIG. 13D The flow cytometry competition results of Anti-CD19Fab-(CPP)3-G4S-LDP, Anti-CD19Fab-(CP)3-G4S-LDP, Anti-CD19Fab-G4S-LDP and CD19 parental mouse antibody are shown in Figure 1. Fig.13A This is the competitive flow cytometric graph of low concentration (11 μg / ml); Fig. 13B The competition flow cytometric graph is for the medium concentration (33 μg / ml); Fig. 13C This is the competitive flow cytometric graph of high concentration (99 μg / ml); Fig.13D Comparison of competition percentages at different antibody concentrations; Flow cytometry: a: Anti-CD19Fab-(CP)3-G4S-LDP; b: Anti-CD19Fab-(CPP)3-G4S-LDP; c: Anti-CD19Fab-G4S-LDP; d: positive control; e: secondary antibody control;

[0050] Fig.14A and Fig. 14B Schematic diagram of plasmid construction; VL and VH are the variable regions of antibody light and heavy chains, respectively. CL is the CL part of the human light chain constant region, and CH1 is the CH1 part of the human heavy chain constant region; among them, Fig.14A It is a eukaryotic expression vector Anti-CD19Fab-(CP)3-TRAIL. Fig. 14B It is the eukaryotic expression vector Anti-CD19Fab-TRAIL;

[0051] Fig.15 The figure is the result of WB identification of Anti-CD19Fab-(CP)3-TRAIL and Anti-CD19Fab-TRAIL; wherein the lanes from left to right are: non-reduced Anti-CD19Fab-TRAIL, non-reduced Anti-CD19Fab-(CP)3-TRAIL; marker; reduced Anti-CD19Fab-TRAIL, reduced Anti-CD19Fab-(CP)3-TRAIL;

[0052] Fig.16 This is a graph showing the results of flow cytometry detection of the binding ability of Anti-CD19Fab-(CP)3-TRAIL to target cells.

[0053] Fig.17 This is the result of in vitro CCK8 assay to detect the killing ability of Anti-CD19Fab-(CP)3-TRAIL on CD19 positive cells Raji;

[0054] Fig.18 This is the result of in vitro CCK8 assay to detect the killing ability of Anti-CD19Fab-(CP)3-TRAIL on CD19-negative Jurkat cells. DETAILED DESCRIPTION

[0055] The present invention discloses a dimer of an antibody Fab fragment, a preparation method and application thereof, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the same. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention, and relevant personnel can obviously modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0056] In the present invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "include" or its transformations such as "include" or "include" etc. will be understood to include the elements or components stated, without excluding other elements or other components. The term "one (a)" ("a", "an" and "the") includes plural indicators. The term "multiple (multiple)" refers to two (kinds) or more (kinds). The terms "such as", "for example" etc. are intended to refer to exemplary embodiments, and are not intended to limit the scope of the present disclosure.

[0057] definition:

[0058] The term "Fab", "Fab fragment" or "Fab portion" refers to a polypeptide comprising the VH, CH1, VL and CL immunoglobulin domains. Typically, the Fab fragment comprises the entire light chain of an antibody. The Fab fragment can be used to define "one arm" of an immunoglobulin molecule. It comprises the epitope binding portion of the Ig. The Fab region of a native immunoglobulin can be obtained as a proteolytic fragment by papain digestion.

[0059] The term "dimer" refers broadly to a peptide comprising two monomer subunits. In the present disclosure, the dimer includes a homodimer or a heterodimer. The monomer subunits of a dimer can be connected at its C-terminus or N-terminus, or can be connected via internal amino acid residues. Each monomer subunit of a dimer can be connected by the same site, or each monomer subunit can be connected by different sites (e.g., C-terminus, N-terminus or internal sites).

[0060] The term "disulfide bond" refers to a covalent bond formed between two sulfur atoms, generally between the sulfur atoms of the side chains of two cysteine ​​residues in a polypeptide chain. It is important for maintaining the native conformation and stability of many protein molecules.

[0061] The term "effector molecule" refers to any natural or synthetically produced molecule, including small molecular weight molecules or chemical entities that can be chemically synthesized, and larger molecules or biological entities that require host cell fermentation to be produced and can confer new functions on target or antigen-binding specific immune ligands, and nucleic acid molecules (such as mRNA molecules).

[0062] The term "ligand" refers to a molecule that specifically binds to a receptor and causes a change in the receptor, which can cause the activity of the receptor to change or react in a cell expressing the receptor. A ligand can be a molecule or a complex thereof that acts as an agonist or antagonist of a receptor. A ligand can include natural and synthetic ligands, as well as small molecules, cytokines, and peptide mimetics of antibodies. The complex of a ligand and a receptor is referred to as a "ligand-receptor complex". A ligand can include a domain of a polyprotein or fusion protein (e.g., or a domain of an antibody / ligand fusion protein).

[0063] The term "receptor" refers to a class of proteins in the body that can bind to a specific signal molecule (ligand) outside the cell to cause a cell response. In the present disclosure, the receptor mainly plays the role of binding to the ligand.

[0064] The term "scFv" refers to a single-chain antibody and includes binding molecules consisting of one light chain variable domain (VL) or a portion thereof and one heavy chain variable domain (VH) or a portion thereof, wherein each variable domain (or a portion thereof) is derived from the same or different antibodies.

[0065] The term "Fv" refers to the smallest antibody fragment that is a part of the Fab (fragment antigen binding) region of an antibody, consisting of a heavy chain variable region (VH) and a light chain variable region (VL).

[0066] The term "dAb" refers to a single immunoglobulin variable domain (VH or VL) polypeptide that specifically binds an antigen. A "dAb" binds antigen independently of other V domains; however, a "dAb" may exist as a homo- or heteromultimer with other VH or VL domains, wherein the other domains are not required for antigen binding by the dAb, i.e., wherein the dAb binds antigen independently of other VH or VL domains.

[0067] The term "multivalent" refers to a recombinant molecule comprising two or more biologically active fragments. The protein fragments forming the multivalent molecule can be connected by a peptide linker, which connects the components without affecting their independent functions.

[0068] The term "vector" includes any genetic element, such as a plasmid, a phage, a transposon, a cosmid, a chromosome, an artificial chromosome, a virus, a virion, etc., which is capable of replication when associated with appropriate control elements and which can transfer gene sequences between cells. Thus, the term includes cloning vehicles and expression vehicles, as well as viral vectors.

[0069] The term "antibody drug conjugate" may also be referred to as "immunoconjugate" or ADCs (antibody drug conjugates), which is a conjugate consisting of an antibody or antibody derivative such as Fab conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents. The heterologous molecules may be proteins, nucleotides or small molecules.

[0070] Dimer of antibody Fab fragment:

[0071] One aspect of the present disclosure is to provide a dimer of an antibody Fab fragment. Its advantage is that the dimerization of the antibody Fab fragment can be achieved more effectively. Based on the strategy of immunotherapy, the polymerization of antibodies can increase the size of antibody molecules and improve the half-life of antibody drugs in the body. At the same time, compared with the complete IgG, the Fab fragment lacks the Fc segment and can selectively bind to the antigen but will not undergo a precipitation reaction; due to its low autoimmunogenicity, it will not be recognized by the immune cells of the living body, so it can significantly reduce the probability of hypersensitivity reactions and improve the safety of the product. Fab antibody fragments have the characteristics of small molecular weight, strong tissue distribution specificity, low immune prototype, and genetic engineering operation, making Fab one of the important members in the field of medical research. Fab drugs have a wide range of applications in prevention, diagnosis, treatment and other fields.

[0072] In the prior art, the preparation method of antibody Fab fragments may include enzymatic hydrolysis, preparation using an expression system, and preparation of a Fab antibody library. The enzymatic hydrolysis method is usually based on a monoclonal antibody, and human immunoglobulin G is degraded by papain or pepsin to obtain products such as F(ab')2, Fab, and Fc fragments. Its disadvantages are obvious, for example, the activity of Fab cannot be guaranteed. In certain embodiments of the present disclosure, an expression system is used for preparation. Since the Fc region is not present, recombinant Fab does not require post-translational modification and glycosylation, and can be expressed in both prokaryotic and mammalian systems. Fab fragments are produced using an expression system, usually using an Escherichia coli expression system and a mammalian expression system. At the same time, the expressed monomeric Fab fragments can smoothly form disulfide bonds, thereby preparing dimers of antibody Fab fragments.

[0073] The antibody Fab fragment dimerization strategy provided in the present disclosure can be applied to any suitable antibody for different disease targets. The above-mentioned diseases can be, for example, tumors, autoimmune diseases, inflammatory diseases, infections, etc. Examples of the above-mentioned targets, such as, CD19, CD20, BCMA, CD56, CD17, CD123, CD52, CD22, HER2, EGFR, VEGFR1, VEGFR2, FLT3, CD38, FGFR3, CD7, PD-1, PD-L1, CTLA4, PTK7, Notch3, FGF2, TNF-α, IL-1, IL-2, IL-6, IL-7, IL-17A, IL-12, IL-23, IL-36, TSLP, TSLPR, GRP78, integrin, adhesion protein-4, SDC1, AGS-16, MUC1. In certain embodiments of the present disclosure, the dimer can be monospecific or bispecific.

[0074] In order to achieve certain functions, in some embodiments of the present disclosure, the above-mentioned monomeric Fab fragments may also carry certain tags / markers, such as fluorescent markers, radioactive markers, enzyme markers, and exemplary examples include His tags, CBP tags, CYD tags, Streptococcus II tags, FLAG tags, HPC tags, and GST and MBP protein fusion tags.

[0075] The V of the dimer of the antibody Fab fragment provided in the present disclosure is L and / or V H part, one of its functions is to bind to the corresponding target protein through a covalent bond. Therefore, in certain embodiments of the present disclosure, the above V L and / or V H Parts can be replaced with molecules (protein fragments) having the same or similar functions, for example, ligands, receptors, scFv, Fv or dAbs that specifically bind to a target protein, or multivalents of said ligands, receptors, scFv, Fv or dAbs.

[0076] Dimer conjugate of antibody Fab fragment:

[0077] One aspect of the present disclosure is to provide a conjugate of a dimer of an antibody Fab fragment. Based on the dimerization strategy of the antibody Fab fragment in the present disclosure, the affinity of the formed dimerized antibody for the target cell / target protein is significantly enhanced. Therefore, the effector molecule can be delivered to the target cell / target protein through the dimer of the antibody Fab fragment in the present disclosure, so as to achieve more effective recognition of the target cell / target protein by the effector molecule.

[0078] In certain embodiments of the present disclosure, the effector molecule in the above conjugate can be directly linked to the amino acid sequence (CP).n The last proline residue of the amino acid sequence (CP) may also be connected to the amino acid sequence (CP) via at least one linker. n The preparation method thereof may be known in the prior art.

[0079] The effector molecule can be any suitable effector molecule, for example, an anti-cancer agent, an anti-inflammatory agent, an anti-autoimmune disease agent, an anti-infective agent, a toxin, a radiotherapeutic agent, a nucleotide, or a cytokine.

[0080] In certain embodiments of the present disclosure, the above effector molecules can be used to treat solid tumors, such as lung cancer, melanoma, liver cancer, gastric cancer, prostate cancer or breast cancer. "Treatment of solid tumors" or "treatment of solid tumors" as used in the present disclosure includes inhibiting the growth of tumors or tumor cells, reducing the size of tumors, killing tumor cells, or preventing the spread (metastasis) of tumor cells. In other embodiments of the present disclosure, the above effector molecules can be used to treat blood tumors, such as myeloma, lymphoma, leukemia.

[0081] In certain embodiments of the present disclosure, the above effector molecules can be used to treat autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, systemic progressive sclerosis and its variants, polymyositis and dermatomyositis, pernicious anemia, and autoimmune thrombocytopenia.

[0082] In certain embodiments of the present disclosure, the above-mentioned effector molecules can be used to treat inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, allergic airway diseases (such as asthma), chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases (such as Crohn's disease, ulcerative colitis).

[0083] In certain embodiments of the present disclosure, the above-mentioned effector molecules can be used to treat infectious diseases, including but not limited to any disease caused by infectious organisms. Infectious organisms can include viruses, (e.g., single-stranded RNA viruses, single-stranded DNA viruses, human immunodeficiency virus (HIV), hepatitis A, B and C viruses, herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), human papilloma virus (HPV)), parasites (e.g., protozoan pathogens and metazoan pathogens such as Plasmodium species, Leishmania species, Schistosoma species, Trypanosoma species), bacteria (e.g., mycobacteria, particularly Mycobacterium tuberculosis, Salmonella, Streptococcus, Escherichia coli, Staphylococcus), fungi (e.g., Candida species, Aspergillus species), Pneumocystis carinii, and prions. Hemolysin, Vero cytotoxin, CNF1, CNF2 or PMT.

[0084] In certain embodiments of the present disclosure, the effector molecules can be toxins, including protein toxins, bacterial toxins or plant toxins. For example, apoptosis proteins, Shiga toxins, Shiga-like toxins, Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE, full-length or truncated diphtheria toxin (DT), cholera toxin, α-sarcin, dianthin or saporin.

[0085] In certain embodiments of the present disclosure, the effector molecule can be a radiotherapeutic agent. Radiotherapeutic agents refer to a class of radioactive drugs that do not rely on the pharmacological effects of the drug itself, but produce therapeutic effects through the biological effects of ionizing radiation. They have the advantages of targeted therapeutic effects and small systemic effects. Commonly used radionuclide labels that emit beta rays, alpha rays and Auger electrons have potential therapeutic advantages.

[0086] In some embodiments of the present disclosure, the above-mentioned effector molecules can be nucleotides. For example, vectors, genes, transgenics that induce cell suicide, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense oligonucleotides (ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), DNA aptamers, RNA aptamers, mRNA, mini-circle DNA, peptide nucleic acids (PNA), phosphoramidate morpholino oligomers (PMO), locked nucleic acids (LNA), bridged nucleic acids (BNA), 2'-deoxy-2'-fluoroarabinose nucleic acids (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O, 4'-aminoethylene bridged nucleic acids, 3'-fluorohexitol nucleic acids (FHNA), plasmids, glycol nucleic acids (GNA) and threose nucleic acids (TNA), or their derivatives.

[0087] In certain embodiments of the present disclosure, the above-mentioned effector molecules can be cytokines, for example, TNF family members, such as tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), TNF-α-related apoptosis-inducing ligand (TRAIL), TNF-α-related activation-induced cytokine (TRANCE), TNF-α-related weak inducer of apoptosis (TWEAK), CD40 ligand (CD40L), LT-α, LT-β, ​​OX40L, CD40L, FasL, CD27L, CD30L, 4-1BBL, APRIL, LIGHT, TL1, TNFSF16, TNFSF17 or AITR-L.

[0088] Preferably, in certain embodiments of the present disclosure, an antibody dimer conjugate drug is provided, that is, the toxic protein Trail is connected to the tail end of Anti-CD19-Fab through a (CP) 3 structure. In vitro killing experiments have shown that Anti-CD19Fab-(CP) 3-TRAIL can target and kill CD19-positive lymphoma cells Raji that are insensitive to Trail, with an IC50 value of 0.049nM, and the killing effect is significantly better than Anti-CD19Fab-TRAIL (IC50: 0.25nM) and Trail protein (high-dose killing rate is only 25%). For Jurkat cells that are sensitive to Trail (IC50: 3.09nM) but CD19-negative, Anti-CD19Fab-(CP) 3-TRAIL can effectively reduce the toxic effect of Trail protein, and the high-dose killing rate is only 27%. The dimeric antibody-drug conjugate Anti-CD19Fab-(CP)3-TRAIL provided by the present invention formed by (CP)3 connection can effectively target and kill CD19-positive tumor cells and has great clinical application value.

[0089] Example:

[0090] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0091] Example 1: Construction of Anti-CD19Fab-(CP) n / (CPP) n Expression plasmid

[0092] The schematic diagram of the prokaryotic expression vector expression frame used in the present disclosure is shown in Figure 1A , Figure 1B , Figure 1C , Figure 1D , Figure 1E Based on the original variable region sequence of the Anti-CD19 monoclonal antibody of the applicant, the light and heavy chain variable regions were connected to the N-termini of the human antibody constant regions CL and CH1 to form Anti-CD19Fab ( Figure 1B As shown), a combination sequence of cysteine ​​and proline was introduced into the C-terminus of the Anti-CD19Fab heavy chain CH1: that is, the gene sequences of (CPP)3, (CPP)2, CPP, (CP)3, (CP)2, and CP were introduced to form the plasmid Anti-CD19Fab-(CP) n or Anti-CD19Fab-(CPP) n ( Figure 1AThe combined fragments were connected to the prokaryotic expression vector PAYZ by gene synthesis to form expression plasmids PAYZ-Anti-CD19Fab-(CPP)3, PAYZ-Anti-CD19Fab-(CPP)2, PAYZ-Anti-CD19Fab-CPP, PAYZ-Anti-CD19Fab-(CP)3, PAYZ-Anti-CD19Fab-(CP)2, PAYZ-Anti-CD19Fab-CP monomer control antibody plasmid was PAYZ-Anti-CD19Fab without (CP)3 or (CPP)3.

[0093] SEQ ID No.1: Anti-CD19Fab(VL+CL)

[0094] DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSAT

[0095] YRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKV

[0096] EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0097] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN

[0098] RGEC

[0099] SEQ ID No.2: Anti-CD19Fab(VH+CH1)

[0100] QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQI

[0101] YPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSV

[0102] VDFYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE

[0103] PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[0104] PSNTKVDKKVEPKSCDKTHT

[0105] Example 2: Anti-CD19Fab-(CP) n / (CPP) n Expression and purification

[0106] Take the expression plasmid PAYZ-Anti-CD19Fab-(CP) constructed in Example 1 n 、PAYZ-Anti-CD19Fab-(CPP) n 25ng of PAYZ-Anti-CD19Fab, 25ng each, were transformed into 16C9 competent cells by heat shock method, plated, cultured overnight at 37℃, and single clones were picked in 10ml 2×YT medium containing ampicillin, and cultured for 12-16h at 37℃ constant temperature shaker, 200rpm. Take 2ml of bacterial solution at a ratio of 1:100 and inoculate it into 200ml 2×YT medium containing ampicillin. After 8h of shaking culture at 37℃ and 200rpm, centrifuge at 4℃ and 8000rpm for 30min, discard the supernatant, and collect the bacteria. Resuspend the bacteria in 400ml AP5 medium containing ampicillin with 2 times the volume of 2xYT to induce expression, culture at 30℃ and 200rpm for 24h, centrifuge at 4℃ and 8000rpm for 30min, discard the supernatant, and collect the bacteria. The bacteria were frozen and thawed repeatedly for more than 3 times, 20 ml of bacterial periplasmic protein extract was added, mixed thoroughly, lysed at 100 rpm on ice for 1 hour, centrifuged at 8000 rpm at 4°C for 30 minutes, and the supernatant, which was the crude extract of the expression product, was collected. The crude extract was dialyzed with PBS at 4°C for 24-36 hours, and PBS was changed every 3 hours. After the dialysis, centrifuged at 4°C, 4000 rpm for 30 minutes, and the supernatant was filtered with a 0.45 μm filter membrane to remove the precipitate. The obtained sample was purified according to the GE purification manual using the AKTA protein purification system and a 1 ml ProteinG purification pre-packed column (cytiva, catalog number 17040401). The obtained antibody pure product was replaced with PBS solution in an ultrafiltration tube and concentrated to 1 ml. Pierce TMAfter quantification by BCA protein analysis kit (Thermo, catalog number 23225), the yields were 3.15 mg / L for Anti-CD19Fab-(CPP)3, 4.8 mg / L for Anti-CD19Fab-(CPP)2, 4.4 mg / L for Anti-CD19Fab-CPP, 3.95 mg / L for Anti-CD19Fab-(CP)3, 6.1 mg / L for Anti-CD19Fab-(CP)2, 6.7 mg / L for Anti-CD19Fab-CP, and 5.5 mg / L for Anti-CD19Fab.

[0107] Example 3: Identification of Anti-CD19Fab-(CP)n and Anti-CD19Fab-(CPP)n dimerization

[0108] In this example, Coomassie brilliant blue staining, size exclusion chromatography, and liquid chromatography-tandem mass spectrometry were used to identify the components and component ratios of the modified antibody protein.

[0109] Coomassie Brilliant Blue staining method to identify protein components:

[0110] 10 μg of each modified protein purified in Example 2 was mixed with the loading buffer and placed in a 95-100°C metal bath for 10 min. A final concentration of 2% mercaptoethanol was added to the reducing sample. After 12% SDS-PAGE, Coomassie Brilliant Blue R250 staining was performed for 5 min and decolorization was performed for more than 16 h. Clear bands were displayed. Figure 2A and Figure 2B .Depend on Figure 2A It can be seen that among the six modified proteins, No. 4 Anti-CD19Fab-(CP)3 has the highest proportion of high molecular weight polymers (~130KD position), followed by No. 1 CD19Fab-(CPP)3 and No. 3 CD19Fab-CPP. Based on the fact that No. 3 CD19Fab-CPP has a large number of low molecular weight (between 25KD and 15KD) unpaired light and heavy single chain bands in addition to the high molecular weight polymer band and the monomer band of about 40KD, No. 3 was excluded, and No. 4 Anti-CD19Fab-(CP)3 and No. 1 CD19Fab-(CPP)3 were selected for subsequent comparative studies.

[0111] 10 μg of purified protein Anti-CD19Fab-(CP)3, Anti-CD19Fab-(CPP)3, Anti-CD19Fab were mixed with loading buffer and placed in a 95-100°C metal bath for 10 min. A final concentration of 2% mercaptoethanol was added to the reducing sample. After 12% SDS-PAGE, Coomassie Brilliant Blue R250 staining was performed for 5 min, and decolorization was performed with decolorizing solution for more than 16 h, showing clear bands. The results are shown in Figure 2C and Figure 2DThe results show that the proportion of high molecular weight polymer components formed by the (CP)3 modification method of the present invention is significantly higher than that of the existing (CPP)3 modification method. The non-reduced electrophoresis diagram ( Figure 2C ) and analyzed, and roughly calculated that the high molecular weight component accounted for about 57.26% in the Anti-CD19Fab-(CP)3 sample and about 22.32% in the Anti-CD19Fab-(CPP)3 sample.

[0112] The relative molecular mass of the main components in the Anti-CD19Fab-(CP)3 protein sample was detected by size exclusion chromatography:

[0113] The specific method is: take 10μg of pure Anti-CD19Fab-(CP)3, centrifuge at 12000rpm, 4℃ for 10min, take the supernatant to a 250μl liner tube, set the liquid chromatography detection conditions as column temperature of 25℃ (chromatographic column BioCore SEC-300 5μm, 7.8×300mm), flow rate of 0.7ml / min, mobile phase of 150mmol phosphate (8.99g anhydrous NaH2PO4 10.65g anhydrous Na2HPO4 to 1L water), detection wavelength of UV280nm, calculate the corresponding molecular weight according to the peak time of each component. Results are shown in Figure 2E and Table 1. Among them, Figure 2E The corresponding ①② in the equation is Figure 2C ①② in. It is known that the number of amino acids in Anti-CD19Fab-(CP)3 monomer is 450, and the theoretical molecular weight is 48543.30Da. Comprehensive analysis shows that Figure 2C The high molecular weight component ① is in the form of a dimer, and ② is in the form of a monomer.

[0114] Table 1 Size exclusion chromatography identification results

[0115]

[0116] Liquid chromatography tandem mass spectrometry is used to accurately separate the components in the protein sample and perform mass spectrometry to obtain the precise molecular weight of each component and the content ratio in the total sample:

[0117] The specific method is as follows: take pure Anti-CD19Fab-(CP)3, centrifuge at 4°C and 12000rcf for 10 min, collect the supernatant into a new centrifuge tube for later use.

[0118] The ultra-high performance liquid chromatography conditions were set as follows: Column: ACQUITY UPLC Protein BEH C4 Column ( 1.7μm, 2.1mm×50mm), mobile phase A: 0.1% formic acid FA (Sigma-Aldrich), H2O, mobile phase B: 0.1% formic acid FA (Sigma-Aldrich), acetonitrile ACN (Fisher Chemical), flow rate: 0.3ml / min, liquid chromatography gradient: 15min, specific settings are shown in Table 2. Set the mass spectrometry conditions as follows: scan range selection: m / z 400–5000, Output Mass: M, S / N Threshold: 3, Rel.Abundance Threshold (%): 0, Charge Range: 2-100, Min.NumDetected Charge: 2, Isotope Table: Protein. LC-MS data are shown in Figure 2F and Table 3. The results show that most of Anti-CD19Fab-(CP)3 is in the form of dimer, with the exact molecular weight consistent with the theoretical molecular weight, accounting for 76.2%; the content of monomer is 18.4%.

[0119] Table 2 Liquid chromatography condition settings

[0120] Time / min B％ 0.00 5 2.00 5 3.00 35 10.00 95 14.00 95 14.10 5 15.00 5

[0121] Table 3 Liquid chromatography tandem mass spectrometry identification results

[0122]

[0123] The above results show that the antibody Fab fragment C H1 The modification method of introducing (CP)3 at the tail end can effectively form an antibody dimer structure, and the dimer formation ratio is significantly higher than the existing technology.

[0124] Example 4: Flow cytometry comparison of Anti-CD19Fab-(CP)3, Anti-CD19Fab-(CPP)3, Anti- Binding ability of CD19Fab to the hCD19 high-expressing cell line Raji

[0125] 1. Direct binding test of Anti-CD19Fab-(CPP)3, Anti-CD19Fab-(CP)3, Anti-CD19Fab and hCD19 high expression cell line Raji: The antibodies were respectively mixed with 5x10 5Raji cells were co-incubated at room temperature for 30 minutes, centrifuged at 1500 rpm for 8 minutes, the supernatant was discarded, and the cells were washed twice with PBS; the cells were resuspended in 100 μl PBS, and PE / CY5-labeled mouse anti-human IgG antibody (abacm, clone number 4A11, catalog number ab239260) was used as the secondary antibody, 0.8 μl per tube, incubated at room temperature in the dark for 30 minutes, centrifuged at 1500 rpm for 8 minutes, the supernatant was discarded, and the cells were washed twice with PBS, and the cells were resuspended in 400 μl PBS. At the same time, blank control tubes and secondary antibody control tubes were set up, and the fluorescence intensity was measured by FACS, and the Median value was calculated. The results are shown in Figure 3A , Figure 3B , Figure 3C and Figure 3D As shown: the binding ability of Anti-CD19Fab-(CPP)3, Anti-CD19Fab-(CP)3, and Anti-CD19Fab to Raji cells increased with the increase of antibody concentration in a dose-dependent manner, and at the same concentration, the affinity of Anti-CD19Fab-(CP)3 was significantly stronger than that of Anti-CD19Fab-(CPP)3 and Anti-CD19Fab.

[0126] 2. Anti-CD19Fab-(CP)3, Anti-CD19Fab-(CPP)3, Anti-CD19Fab and CD19 parent mouse whole antibody competition binding test: Take the antibody to make the final concentration of 9μg / ml, 27μg / ml, 81μg / ml, then add CD19 parent mouse whole antibody (Kuangbo Bio, clone HI19a, catalog number A6361) with a final concentration of 15μg / ml, and at the same time add PD-1 mouse whole antibody (Kuangbo Bio, clone HI2E7, catalog number A7541) with the same final concentration as the antibody to deduct the non-specific binding background of CD19 parent mouse whole antibody Fc end and cells, and then add 5x10 5Raji cells were incubated at room temperature for 30 minutes, centrifuged at 1500 rpm for 8 minutes, the supernatant was discarded, and the cells were washed twice with PBS; 200 μl PBS was used to resuspend the cells, 1 μl of APC-labeled anti-mouse IgG Fab2 antibody (CST, catalog number 4410S) was added, incubated at room temperature in the dark for 30 minutes, centrifuged at 1500 rpm for 8 minutes, the supernatant was discarded, and the cells were washed twice with PBS. The cells were resuspended in 400 μl PBS, and blank control tubes, positive control tubes (uncompeted), secondary antibody control tubes, and nonspecific isotype control tubes (PD-1-APC, Kuangbo Bio, catalog number 2022060201) were set up at the same time. For details, see Table 4. FACS was used to measure the fluorescence intensity, and the Median value was calculated. The values ​​of all test tubes and uncompeted control tubes after deducting the secondary antibody control tube were taken as the fluorescence intensity values, and the competition percentage was calculated. The calculation formula was: (uncompeted control tube-test tube) / (positive control tube-secondary antibody control tube)X100%, and the competition percentage of the antibody was plotted. Results are shown in Figure 4A , Figure 4B , Figure 4C and Figure 4D As shown: Anti-CD19Fab-(CPP)3, Anti-CD19Fab-(CP)3 and Anti-CD19Fab gradually increase their competition with the parent CD19 antibody as the concentration increases. At medium concentration (27μg / ml), the competition rate of Anti-CD19Fab-(CP)3 with the parent antibody reached 57.8%, and at high concentration (81μg / ml), the competition rate of Anti-CD19Fab-(CP)3 with the parent antibody reached 78.1%, while the competition rates of Anti-CD19Fab-(CPP)3 with the parent antibody were 49.6% and 74.3%, respectively, and those of Anti-CD19Fab were 14.4% and 54.2%, respectively. This result proves that the affinity of the modified Anti-CD19Fab-(CP)3 with the target CD19 is stronger than that of Anti-CD19Fab-(CPP)3 and Anti-CD19Fab.

[0127] Table 5 Competition tube design table

[0128]

[0129] Examples 1-4 demonstrate that the present invention provides antibody Fab fragment C H1The modification method of introducing (CP) 3 at the tail end can effectively make the exemplary anti-CD19-Fab form dimers, and the dimer ratio and affinity of the obtained product to the CD19 target are significantly stronger than the existing modification technology of introducing (CPP) 3. To prove the versatility of this scheme in different antibody sequences, Examples 5-8 and Examples 9-12 respectively apply this modification method to the exemplary anti-CD20-Fab and the exemplary anti-BCMA-Fab, and compare the ability of the modified products to form dimers and the affinity to the corresponding targets.

[0130] Example 5: Construction of Anti-CD20Fab-(CP)3 expression plasmid

[0131] The construction method is the same as in Example 1. Figure 1B As shown, based on the original variable region sequence of the Anti-CD20 monoclonal antibody of the applicant, the light and heavy chain variable regions are connected to the N-terminus of the human antibody constant region CL and CH1 to form Anti-CD20Fab. The (CP)3 gene sequence is introduced into the C-terminus of the Anti-CD20Fab heavy chain CH1 to form Anti-CD20Fab-(CP)3 or the (CPP)3 sequence is introduced to form Anti-CD20Fab-(CPP)3 (such as Figure 1A The combined fragments were connected to the prokaryotic expression vector PAYZ by gene synthesis to obtain the reconstructed expression plasmids PAYZ-Anti-CD20Fab-(CP)3 and PAYZ-Anti-CD20Fab-(CP)3, and the monomer control plasmid was PAYZ-Anti-CD20Fab.

[0132] SEQ ID No.3: Anti-CD20Fab(VL+CL)

[0133] DIELTQSPAILSASPGEKVTMTCRANSSVSYMLWYQQKPGSSPKPWIYATSHLASGVPTRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGAGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0134] SEQ ID No.4: Anti-CD20Fab(VH+CH1)

[0135] QVKLQQSGAELVKPGASVKMSCKASGYTFISYNMHWVKQTPGQGLEWIGGIYPGNGDTSYNQKFKGKATLTADKSSSAAYMQLSSLTSEDSAVYYCARWNYGNFGGGTMDYWGQG ITVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

[0136] Example 6: Expression and purification of Anti-CD20Fab-(CP)3, Anti-CD20Fab-(CPP)3, and Anti-CD20Fab

[0137] The expression plasmids PAYZ-Anti-CD20Fab-(CPP)3, PAYZ-Anti-CD20Fab-(CP)3, and PAYZ-Anti-CD20Fab constructed in Example 5 were expressed and purified according to the method of Example 2. TM After quantification by BCA protein analysis kit (Thermo catalog number 23225), the yields were 1.7 mg / L for Anti-CD20Fab-(CPP)3, 1.9 mg / L for Anti-CD20Fab-(CP)3, and 1.4 mg / L for Anti-CD20Fab.

[0138] Example 7: Identification of Anti-CD20Fab-(CPP)3 and Anti-CD20Fab-(CP)3 dimerization

[0139] Coomassie brilliant blue staining was used to identify protein components.

[0140] The purified proteins Anti-CD20Fab-(CPP)3, Anti-CD20Fab-(CP)3, and Anti-CD20Fab were electrophoresed according to the method of Example 3. The results are shown in Figure 5A and Figure 5B As shown: In the electrophoresis diagram under non-reducing conditions, the position corresponding to ① is the dimer band, and the position corresponding to ② is the monomer band. It can be seen from the figure that Anti-CD20Fab-(CPP)3 and Anti-CD20Fab-(CP)3 both form dimers. The non-reducing electrophoresis bands were scanned and analyzed using Image J image analysis software. The dimer component of Anti-CD20Fab-(CP)3 accounted for 20.35%, while the dimer component of Anti-CD20Fab-(CPP)3 accounted for 10.76%. This result shows that anti-CD20Fab introduced with (CP)3 structure is more likely to form dimers.

[0141] Example 8: Flow cytometry comparison of Anti-CD20Fab-(CP)3, Anti-CD20Fab-(CPP)3, Anti- Binding ability of CD20Fab to the hCD20 high-expressing cell line Raji

[0142] 1. Direct binding test of Anti-CD20Fab-(CPP)3, Anti-CD20Fab-(CP)3, Anti-CD20Fab and hCD20 high expression cell line Raji: The method is the same as Example 4(1). The results are shown in Fig. 6A , Figure 6B , Figure 6C and Fig.6D :The binding ability of Anti-CD20Fab-(CPP)3, Anti-CD20Fab-(CP)3, and Anti-CD20Fab to Raji cells increased with the antibody concentration in a dose-dependent manner. At the same concentration, the affinity of Anti-CD20Fab-(CP)3 for CD20 high-expressing cells Raji was significantly stronger than that of Anti-CD20Fab-(CPP)3 and Anti-CD20Fab.

[0143] 2. Anti-CD20Fab-(CP)3, Anti-CD20Fab-(CPP)3, Anti-CD20Fab and CD20 parent mouse whole antibody competition binding test: The method is the same as Example 4 (2). The antibody to be competed is CD20 parent mouse whole antibody (Kuangbo Bio, clone number HI20a, item number A6381); when incubating the secondary antibody, resuspend it in 100μl PBS. The results are shown in Fig. 7A , Figure 7B , Figure 7C and Fig.7D :It can be seen that the competition of Anti-CD20Fab-(CPP)3, Anti-CD20Fab-(CP)3 and Anti-CD20Fab for the parent CD20 antibody gradually increases with the increase of the concentration used. At low concentration (9μg / ml), the competition rate of Anti-CD20Fab-(CP)3 for the parent antibody reached 58.5%, while the competition rate of Anti-CD19Fab-(CPP)3 for the parent antibody was 41.2%, and Anti-CD20Fab was only 25.3%. When the antibody concentration reached a high concentration, the competition result tended to be stable. This result proves that the affinity of the modified Anti-CD20Fab-(CP)3 for the target CD20 is stronger than that of Anti-CD20Fab-(CPP)3 and Anti-CD20Fab.

[0144] Example 9: Construction of Anti-BCMAFab-(CP)3 expression plasmid

[0145] The construction method is the same as in Example 1: Figure 1BAs shown in the figure, based on the original variable region sequence of the Anti-BCMA monoclonal antibody in our laboratory, the light and heavy chain variable regions were connected to the N-terminus of the human antibody constant region CL and CH1 to form Anti-BCMA Fab. The (CP)3 gene sequence was introduced into the C-terminus of the Anti-BCMA Fab heavy chain CH1 to form Anti-BCMA Fab-(CP)3 or the (CPP)3 sequence was introduced to form Anti-BCMA Fab-(CPP)3 (as shown in the figure). Figure 1A The combined fragments were connected to the prokaryotic expression vector PAYZ by gene synthesis to obtain the reconstructed expression plasmids PAYZ-Anti-BCMA Fab-(CPP)3 and PAYZ-Anti-BCMA Fab-(CP)3, and the control plasmid was PAYZ-Anti-BCMAFab.

[0146] SEQ ID No.5: Anti-BCMAFab(VL+CL)

[0147] DIVLTQSPASLTVSLGQRATISCRASKSVSTSGYSYMHWYQQKPRQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRDLPYTFGGG TKLEIKRAVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC

[0148] SEQ ID No.6: Anti-BCMAFab(VH+CH1)

[0149] QVQLQQSGAEVVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGPEWIGVITPGRGDTKYNAKFAGKATLTADKSSSTAYMQLSSLTFDDSAVYFCARGTTAWFPYWGQGTL VTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT

[0150] Example 10: Expression of Anti-BCMAFab-(CP)3, Anti-BCMAFab-(CPP)3, Anti-BCMA Fab change

[0151] The expression plasmids PAYZ-Anti-BCMAFab-(CPP)3, PAYZ-Anti-BCMAFab-(CP)3, and PAYZ-Anti-BCMA Fab constructed in Example 9 were expressed and purified according to the method of Example 2. TM After quantification by BCA protein analysis kit (Thermo catalog number 23225), the yields were 2.1 mg / L for Anti-BCMA Fab-(CPP)3, 3.0 mg / L for Anti-BCMAFab-(CP)3, and 2.2 mg / L for Anti-BCMAFab.

[0152] Example 11: Identification of Anti-BCMA Fab-(CP)3 and Anti-BCMA Fab-(CPP)3 Dimerization

[0153] Coomassie brilliant blue staining was used to identify protein components.

[0154] The purified proteins Anti-BCMAFab-(CPP)3, Anti-BCMAFab-(CP)3, and Anti-BCMA Fab were electrophoresed according to the method of Example 3. The results are shown in Fig. 8A and Figure 8B : In the electrophoresis diagram under non-reducing conditions, the position corresponding to ① is the dimer band, and the position corresponding to ② is the monomer band. It can be seen from the figure that Anti-BCMAFab-(CPP)3 and Anti-BCMAFab-(CP)3 both formed dimers. The non-reducing electrophoresis bands were scanned and analyzed using Image J image analysis software. The dimer component of Anti-BCMAFab-(CP)3 accounted for 43.64%, while the dimer component of Anti-BCMAFab-(CPP)3 accounted for only 9.67%. This result shows that the ability of anti-BCMAFab introduced with (CP)3 structure to form a dimer structure is significantly stronger than the modification method of introducing (CPP)3.

[0155] Example 12: Flow cytometry comparison of Anti-BCMAFab-(CPP)3, Anti-BCMAFab-(CP)3, Anti- Binding ability of BCMAFab to the hBCMA high-expressing cell line H929

[0156] 1. Direct binding test of Anti-BCMAFab-(CPP)3, Anti-BCMAFab-(CP)3, Anti-BCMAFab and hBCMA high-expressing cell line H929: The method is the same as Example 4(1). The results are shown in Fig. 9A , Fig. 9B , Fig. 9C and Fig.9D:The binding ability of Anti-BCMAFab-(CPP)3, Anti-BCMAFab-(CP)3, and Anti-BCMA Fab to H929 cells increased with the increase of antibody concentration in a dose-dependent manner, and at the same concentration, the affinity of Anti-BCMAFab-(CP)3 was significantly stronger than that of Anti-BCMAFab-(CPP)3 and Anti-BCMA Fab.

[0157] 2. Anti-BCMAFab-(CPP)3, Anti-BCMAFab-(CP)3, Anti-BCMAFab and BCMA parent mouse whole antibody competition binding test: The method is the same as Example 4 (2). The antibody being competed is BCMA parent mouse whole antibody (Kuangbo Bio, clone number HIBCMA, catalog number A7561); when incubating the secondary antibody, resuspend it in 100μl PBS. The results are shown in Fig. 10A , Fig. 10B , Fig. 10C and Fig. 10D :The competition of Anti-BCMA Fab-(CPP)3, Anti-BCMAFab-(CP)3 and Anti-BCMA Fab for the parental BCMA antibody gradually increased with the increase of the concentration used. At low concentrations, the competitive ability of Anti-BCMA Fab-(CP)3 for the parental antibody was significantly stronger than that of Anti-BCMAFab-(CPP)3 and Anti-BCMAFab. This result proves that the affinity of the modified Anti-BCMAFab-(CP)3 for the target BCMA is stronger than that of Anti-BCMAFab-(CPP)3 and Anti-BCMAFab.

[0158] Examples 1-12 demonstrate that the present invention provides antibody Fab fragment C H1 The modification method of introducing (CP)3 at the tail end can effectively form homodimers in different antibody Fab fragments. The dimer ratio and affinity of the resulting products to the corresponding targets are significantly stronger than the existing modification technology of introducing (CPP)3 and the monomer Fab form.

[0159] Antibody-drug conjugates are an important branch of antibody drug research, and the location of the conjugate drugs is often set at the CH1 tail end. In order to verify the application prospects of the present invention in the dimerization of antibody-drug conjugates, Examples 13-16 constructed a protein portion of an antibody-drug conjugate, that is, on the basis of the (CP)3 connected to the CH1 tail end of Anti-CD19Fab, the lidamycin cofactor protein LDP was further connected to verify whether this modification method can still effectively promote the antibody protein to form a dimer, and the affinity of its product to the target.

[0160] Example 13: Construction of Anti-CD19Fab-(CP)3-G4S-LDP expression plasmid

[0161] like Figure 1A As shown, based on the constructed Anti-CD19Fab-(CP)3 and Anti-CD19Fab-(CPP)3, the flexible peptide G4S sequence and the lidamycin cofactor protein LDP sequence were introduced at the end of (CP)3 to form Anti-CD19Fab-(CP)3-G4S-LDP (as shown in Figure 1C or introduce the flexible peptide G4S sequence and the lidamycin cofactor protein LDP sequence at the end of (CPP) 3 to form Anti-CD19Fab-(CPP) 3-G4S-LDP (as shown in Figure 1D As shown). The combined fragments were connected to the prokaryotic expression vector PAYZ by gene synthesis to obtain the reconstructed expression plasmids PAYZ-Anti-CD19Fab-(CPP)3-G4S-LDP and Anti-CD19Fab-(CP)3-G4S-LDP. The control plasmid was PAYZ-Anti-CD19Fab-G4S-LDP (as shown). Figure 1E shown).

[0162] SEQ ID No.7: Anti-CD19Fab-(CPP)3-G4S-LDP(VL+CL)

[0163] DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0164] SEQ ID No.8: Anti-CD19Fab-(CPP)3-G4S-LDP(VH+CH1+cpcpcp+G4S+LDP)

[0165] QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPCPCPGGGGSAPAFSVSPASGLSDGQSVSVSVSGAAAGETYYIAQCAPVGGQDACNPATATSFTTDASGAASFSFVVRKSYTGSTPEGTPVGSVDCATAACNLGAGNSGLDLGHVALTFG

[0166] Example 14: Anti-CD19Fab-(CP)3-G4S-LDP, Anti-CD19Fab-(CPP)3-G4S-LDP, Anti- Expression and purification of CD19Fab-G4S-LDP

[0167] The expression plasmids Anti-CD19Fab-(CPP)3-G4S-LDP, Anti-CD19Fab-(CP)3-G4S-LDP, and Anti-CD19Fab-G4S-LDP constructed in Example 13 were expressed and purified according to the method of Example 2, and Pierce TM After quantification by BCA protein analysis kit (Thermo catalog number 23225), the yields were 3.6 mg / L for Anti-CD19Fab-(CPP)3-G4S-LDP, 3.2 mg / L for Anti-CD19Fab-(CP)3-G4S-LDP, and 7.3 mg / L for Anti-CD19Fab-G4S-LDP.

[0168] Example 15: Dimerization of Anti-CD19Fab-(CP)3-G4S-LDP and Anti-CD19Fab-(CPP)3-G4S-LDP Identification

[0169] Coomassie brilliant blue staining was used to identify protein components.

[0170] The purified proteins Anti-CD19Fab-(CPP)3-G4S-LDP, Anti-CD19Fab-(CP)3-G4S-LDP, and Anti-CD19Fab-G4S-LDP were electrophoresed according to the method of Example 3. The results are as follows Fig.11A and Fig. 11BAs shown: In the electrophoresis diagram under non-reducing conditions, the position corresponding to ① is the dimer band, and the position corresponding to ② is the monomer band. It can be seen from the figure that Anti-CD19Fab-(CPP)3-G4S-LDP and Anti-CD19Fab-(CP)3-G4S-LDP have formed dimers. The non-reducing electrophoresis bands were scanned and analyzed using Image J image analysis software. The dimer component of Anti-CD19Fab-(CP)3-G4S-LDP accounted for 22.64%, while the dimer component of Anti-CD19Fab-(CPP)3-G4S-LDP accounted for 11.39%. Anti-CD19Fab-G4S-LDP exists in the form of a monomer. This result shows that the modification of (CP)3 can form a higher proportion of dimer structure than (CPP)3.

[0171] Example 16: Flow cytometry comparison of Anti-CD19Fab-(CPP)3-G4S-LDP, Anti-CD19Fab-(CP)3- Binding ability of G4S-LDP and Anti-CD19Fab-G4S-LDP to the hCD19 high-expressing cell line Raji

[0172] 1. Direct binding test of Anti-CD19Fab-(CPP)3-G4S-LDP, Anti-CD19Fab-(CP)3-G4S-LDP, and Anti-CD19Fab-G4S-LDP with the hCD19 high-expressing cell line Raji: The method was the same as in Example 4(1), wherein the final concentration gradient of the antibody was set to 0.165μg / ml, 0.825μg / ml, and 1.65μg / ml. The results are shown in Fig. 12A , Fig. 12B , Fig. 12C and Fig.12D : The binding ability of Anti-CD19Fab-(CPP)3-G4S-LDP, Anti-CD19Fab-(CP)3-G4S-LDP and Anti-CD19Fab-G4S-LDP to Raji cells increased with the increase of antibody concentration in a dose-dependent manner, and at the same concentration, the binding ability of Anti-CD19Fab-(CP)3-G4S-LDP to Raji cells was significantly stronger than that of Anti-CD19Fab-(CPP)3-G4S-LDP and Anti-CD19Fab-G4S-LDP.

[0173] 2. Competition binding test between Anti-CD19Fab-(CPP)3-G4S-LDP, Anti-CD19Fab-(CP)3-G4S-LDP, Anti-CD19Fab-G4S-LDP and CD19 parent mouse whole antibody: The method was the same as in Example 4 (2), wherein the final concentration gradient of the antibody was set to 11 μg / ml, 33 μg / ml, and 99 μg / ml, and the concentration of PD-1 mouse whole antibody (Kuangbo Bio, clone number HI2E7, item number A7541) was the same as the antibody concentration (specific setting type is the same as Table 5). The results are shown in Fig.13A , Fig. 13B , Fig. 13C and Fig.13D :As the concentration of Anti-CD19Fab-(CPP)3-G4S-LDP, Anti-CD19Fab-(CP)3-G4S-LDP and Anti-CD19Fab-G4S-LDP increases, the competition for the parental CD19 antibody gradually increases. And at the same concentration, especially at low and medium concentrations, the competitive ability of Anti-CD19Fab-(CP)3-G4S-LDP for the parental antibody is significantly stronger than that of Anti-CD19Fab-(CPP)3-G4S-LDP and Anti-CD19Fab-G4S-LDP. This result proves that the modified Anti-CD19Fab-(CP)3-G4S-LDP has a stronger affinity for the target CD19 than Anti-CD19Fab-(CPP)3-G4S-LDP and Anti-CD19Fab-G4S-LDP.

[0174] Example 17: Construction of eukaryotic expression plasmids Anti-CD19Fab-(CP)3-TRAIL, Anti-CD19Fab-TRAIL

[0175] The schematic diagram of the expression frame of the eukaryotic expression vector used in this example is as follows Fig.14A and Fig. 14B Based on the variable region sequence of the original Anti-CD19 monoclonal antibody with internalization ability of the applicant, the light and heavy chain variable regions were connected to the N-termini of the human antibody constant regions CL and CH1 to form Anti-CD19Fab (shown in SEQ ID No. 1), (CP)3 was introduced at the end of the Fab segment CH1 to form Anti-CD19Fab-(CP)3, and the TRAIL protein (amino acids at positions 114-281) was further coupled to the end of (CP)3 through the flexible peptide G4S to form Anti-CD19Fab-(CP)3-TRAIL (shown in SEQ ID No. 1). Fig.14A As shown), the control plasmid is Anti-CD19Fab-TRAIL without the introduction of (CP)3 structure (as shown Fig. 14B The above gene sequence was cloned into the lentiviral vector pCDH-CMV-MCS-EF1-copGFP by gene synthesis, and the light and heavy chains were connected by T2A.

[0176] Example 18: Expression and purification of Anti-CD19Fab-(CP)3-TRAIL and Anti-CD19Fab-TRAIL

[0177] The expression plasmids Anti-CD19Fab-(CP)3-TRAIL and Anti-CD19Fab-TRAIL constructed in Example 17 were transformed into DH5α competent cells by heat shock method, plated, cultured overnight at 37°C, and single clones were picked and placed in 2xYT medium containing ampicillin. The culture was maintained at 37°C and 200 rpm with shaking for 12-16 h. The plasmid was extracted using an endotoxin-free plasmid mini-extraction kit the next day.

[0178] One day before transfection, 6x10 293T cells in logarithmic growth phase were collected. 6 The cells were plated in a 10 cm dish using DMEM (Gibco, catalog number C11995500BT) + 10% FBS (Gibco, catalog number 10099141) in an 8 ml volume. After 16 hours of plating, the 293T cells were replaced with fresh culture medium. During transfection, first dilute 8 μg of the target plasmid to a volume of 800 μL with serum-free culture medium OPTI-MEM (Gibco, catalog number 31985070), mix gently, add 24 μL of Roche transfection reagent X-tremeGENE HP DNA Transfection Reagent (Roche, catalog number 06366236001) at a ratio of 1:3 of plasmid (μg): transfection reagent (μL), mix gently, let stand at room temperature for 15 min, evenly add dropwise to the plate containing 293T cells, gently shake the plate, and place it in a 37°C, 5% CO2 incubator. Collect the 293T cell culture supernatant 48 h and 72 h after transfection, centrifuge at 4000 rpm, 4°C for 15 min, and filter the supernatant through a 0.45 μm filter to remove cell debris. The obtained samples were purified using AKTAprime purification instrument and 1mL His tag purification column (Cyiva, Catalog No. 17371205) according to the GE purification manual, and the obtained antibody was concentrated and replaced to 1mL in a 10K ultrafiltration concentration tube with PBS solution. The His Tag ELISA Detection Kit (GenScript, Catalog No. 2209K019) was used for quantification, and the yield (protein amount per 1L cell culture supernatant) was 6.4μg / L for Anti-CD19Fab-(CP)3-TRAIL and 7.1μg / L for Anti-CD19Fab-TRAIL.

[0179] Example 19: Identification of Anti-CD19Fab-(CP)3-TRAIL and Anti-CD19Fab-TRAIL

[0180] This example uses Western Blot identification.

[0181] 3 μg of purified protein Anti-CD19Fab-(CP)3-TRAIL and Anti-CD19Fab-TRAIL were mixed with loading buffer (5x loading buffer) and placed in a 95-100°C metal bath for 10 min. The reducing sample was added with 2% mercaptoethanol at a final concentration. After 4%-20% gradient gel SDS-PAGE, Coomassie Brilliant Blue R250 staining for 5 min, and decolorization with decolorizing solution for more than 16 h, clear bands were displayed. SDS-PAGE results are shown in Figure 2A . Western Blot identification was performed simultaneously. After separation by electrophoresis gel, the membrane was transferred and blocked with 5% skim milk powder. The primary antibody of rabbit anti-TRAIL (abacm, product number ab2056) was added at a ratio of 1:5000, incubated overnight at 4°C, washed 3 times with PBST for 10 minutes each time, and HRP-goat anti-rabbit antibody (Zhongshan Jinqiao, product number ZB2301) was added at a ratio of 1:3000 and incubated at room temperature for 2 hours. The cells were washed 3 times with PBST for 10 minutes each time, and ECL developer was added in the dark room for color development for 1 minute. The images were observed and saved on a chemiluminescence imager. Results are shown in Fig.15 .

[0182] The theoretical molecular weight of Anti-CD19Fab-(CP)3-TRAIL and Anti-CD19Fab-TRAIL monomers is approximately 68KD. Fig.15 It can be seen that at about 150KD of the marker, which corresponds to the position of the fusion protein dimer, Anti-CD19Fab-(CP)3-TRAIL obviously forms a higher proportion of dimer protein compared with Anti-CD19Fab-TRAIL, and at about 250KD, which is the position of the multimeric fusion protein, Anti-CD19Fab-(CP)3-TRAIL also expresses the target protein. It can be seen that the introduction of the (CP)3 structure can effectively form dimers and even multimers.

[0183] Example 20: Flow cytometry detection of Anti-CD19Fab-(CP)3-TRAIL and Anti-CD19Fab-TRAIL Binding ability of hCD19 high-expressing cell line Raji

[0184] Flow cytometry was used to detect the binding ability of the target protein to the hCD19 high-expressing cell line Raji. Equal amounts (1 μg) of Anti-CD19Fab-(CP)3-TRAIL and Anti-CD19Fab-TRAIL were taken and mixed with 5x10 5Raji cells were incubated at room temperature for 30 minutes, centrifuged at 1500 rpm for 8 minutes, the supernatant was discarded, and the cells were washed twice with PBS; 100 μL PBS was used to resuspend the cells, and PE / CY5-labeled mouse anti-human IgG antibody (abacm, clone number 4A11, catalog number ab239260) was used as the secondary antibody, 0.8 μL per tube, incubated at room temperature in the dark for 30 minutes, centrifuged at 1500 rpm for 8 minutes, the supernatant was discarded, and the cells were washed twice with PBS, and the cells were resuspended in 400 μl PBS. At the same time, an isotype control tube was set up, and the fluorescence intensity was measured by FACS and plotted. The results are shown in Fig.16 The left picture is the isotype control tube, the right picture a is Anti-CD19Fab-TRAIL, and b is Anti-CD19Fab-(CP)3-TRAIL. Fig.16 It can be seen that at the same concentration, the binding affinity of Anti-CD19Fab-(CP)3-TRAIL to target cells is higher than that of Anti-CD19Fab-TRAIL.

[0185] Example 21: CCK8 method to detect the in vitro killing ability of Anti-CD19Fab-(CP)3-TRAIL on target cells

[0186] 1. In vitro killing experiment on CD19 positive cell line Raji:

[0187] Take 8x10 Raji cells (CD19 positive, TRAIL resistant cell line) in logarithmic growth phase 3 / 100μL was spread in a 96-well flat-bottom plate and mixed. Four antibody proteins, Anti-CD19Fab-(CP)3-TRAIL, Anti-CD19Fab-TRAIL, Anti-CD19Fab-(CP)3, Anti-CD19Fab, and TRAIL protein control (abacm, item number ab9960) were added respectively. Eight concentration gradients (double dilution) were set for each protein, and 3 replicates were set for each concentration. The concentration gradients of the five proteins are shown in Table 6. 37°C, 5% CO2 incubator culture for 72h, 10μLCK8 reagent (DOJINDO, item number TU738) was added to each well, mixed, and continued to be cultured in the incubator for 2h. The OD450 absorbance value was detected by an enzyme reader. At the same time, blank wells (culture medium without cells, plus PBS) and control wells (culture medium with cells, plus PBS) were set. The absorbance value measured in each well minus the blank absorbance mean was taken as the OD450 value of each well in this experiment, and the cell viability was calculated as follows: OD450 of each well minus the blank absorbance mean / (control well-blank value) x 100%. GraphPad was used to draw a graph and calculate the IC50 value of each protein.

[0188] The results are as follows Fig.17As shown. First, Anti-CD19Fab-(CP)3 and Anti-CD19Fab that are not coupled to TRAIL protein have almost no killing effect on Raji. Since Raji cells express low levels of TRAIL-related receptors and have a strong tolerance to TRAIL protein, the killing rate of TRAIL protein on Raji cells at high doses is only 22%. The antibody-coupled drugs Anti-CD19Fab-(CP)3-TRAIL and Anti-CD19Fab-TRAIL can effectively kill Raji cells, and the killing effect is dose-dependent. The IC50 value was calculated using GraphPad, and the killing effect of Anti-CD19Fab-(CP)3-TRAIL on cells was significantly stronger than that of Anti-CD19Fab-TRAIL (P<0.05). The IC50 of Anti-CD19Fab-(CP)3-TRAIL is 0.049nM, and the IC50 value of Anti-CD19Fab-TRAIL is 0.25nM.

[0189] Table 6 Five protein concentration gradient settings

[0190]

[0191] 2. In vitro killing of CD19 negative cell line Jurkat cells:

[0192] Jurkat cells that are sensitive to Trail and negative for CD19 expression were selected to detect the target specificity of Anti-CD19Fab-(CP)3-TRAIL and Anti-CD19Fab-TRAIL. 8x10 Jurkat cells in the logarithmic growth phase were taken. 3 / 100 μL was spread in a 96-well flat-bottom plate and mixed. Three proteins, Anti-CD19Fab-(CP)3-TRAIL, Anti-CD19Fab-TRAIL, and TRAIL control, were added respectively. Eight concentration gradients (double dilutions) were set for each protein, and 3 replicates were set for each concentration. The specific concentration settings are shown in Table 7). The culture conditions and time after drug addition were the same as those in the operation of (1) in this embodiment. The results are shown in Fig.18 As shown, the killing of Jurkat cells by the TRAIL control group gradually increased with the increase of protein concentration, and was dose-dependent. The IC50 value of the TRAIL control group was calculated by GraphPad to be 3.09nM. As the protein concentration increased, the survival rate of Jurkat cells in the Anti-CD19Fab-(CP)3-TRAIL and Anti-CD19Fab-TRAIL groups decreased slightly until it stabilized. The survival rates of both groups were stable at around 80%.

[0193] Table 7 Concentration settings

[0194]

[0195]

[0196] The results of this embodiment show that compared with the TRAIL control, the antibody-TRAIL drug after TRAIL and Anti-CD19-Fab coupling can effectively mediate the killing of CD19 positive cells, significantly increase the sensitivity of target cells to TRAIL protein, and reduce the toxic side effects of TRAIL protein on CD19 negative cells Jurkat. Compared with Anti-CD19Fab-TRAIL, the killing ability of Anti-CD19Fab-(CP)3-TRAIL with the introduction of (CP)3 structure is significantly enhanced, and the IC50 value for the lymphoma cell line Raji is reduced to one fifth of the latter.

[0197] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An antibody-drug conjugate (ADC), comprising a dimer of an antibody Fab fragment, wherein the antibody Fab fragment comprises V L 、V H , C L and C H1 part, the dimer comprises two monomeric Fab fragments, characterized in that The C H1 The C-terminus of the part is directly connected to the amino acid sequence (CP) n , or the C of the monomeric Fab fragment H1 The C-terminus of the moiety is indirectly connected to the amino acid sequence (CP) via at least one linker n ; The amino acid sequence (CP) n The last proline residue of the amino acid sequence (CP) is also directly linked to the effector molecule. n The last proline residue of is also indirectly connected to the effector molecule via at least one linker; Wherein, n=3, and the effector molecule is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

2. The antibody-drug conjugate according to claim 1, characterized in that: The dimer is a homodimer or a heterodimer.

3. The antibody-drug conjugate according to claim 1, characterized in that: The monomeric Fab fragments are connected via disulfide bonds.

4. The antibody-drug conjugate according to claim 1, characterized in that: The V L and / or V H Partially replaced with other protein fragments that specifically bind to the target protein.

5. The antibody-drug conjugate according to claim 4, characterized in that: The protein fragment is a ligand, a receptor, a scFv, a Fv or a dAb that specifically binds to a target protein, or a multivalent body of the ligand, the receptor, the scFv, the Fv or the dAb.

6. The antibody-drug conjugate according to claim 1, characterized in that: The antibody Fab fragment specifically binds to one or two of the following protein molecules: CD19, CD20, BCMA, CD56, CD17, CD123, CD52, CD22, HER2, EGFR, VEGFR1, VEGFR2, FLT3, CD38, FGFR3, CD7, PD-1, PD-L1, CTLA4, PTK7, Notch3, FGF2, TNF-α, IL-1, IL-2, IL-6, IL-7, IL-17A, IL-12, IL-23, IL-36, TSLP, TSLPR, GRP78, integrin, adhesion protein-4, SDC1, AGS-16 or MUC1.

7. The antibody-drug conjugate according to claim 1, characterized in that: The antibody Fab fragment also carries a fluorescent marker, a radioactive marker or an enzyme marker.

8. The antibody-drug conjugate according to claim 1 or 6, characterized in that: The antibody-drug conjugate comprises Fab of anti-CD19 antibody, amino acid sequence (CP) 3 and TRAIL molecule.

9. Use of the antibody-drug conjugate according to any one of claims 1 to 8 in the preparation of a drug for treating tumors.

10. The use according to claim 9, characterized in that The tumor uses the following molecules as therapeutic targets: CD19, CD20, BCMA, CD56, CD17, CD123, CD52, CD22, HER2, EGFR, VEGFR1, VEGFR2, FLT3, CD38, FGFR3, CD7, PD-1, PD-L1, CTLA4, PTK7, Notch3, FGF2, TNF-α, IL-1, IL-2, IL-6, IL-7, IL-17A, IL-12, IL-23, IL-36, TSLP, TSLPR, GRP78, integrin, adhesion protein-4, SDC1, AGS-16 or MUC1.

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