A recombinant protein for improving the efficacy of ADC drugs and its applications

Abstract

This application relates to the field of biotechnology, and specifically discloses a recombinant protein for improving the efficacy of ADC drugs and its application. The recombinant protein includes a tumor cell surface targeting structure, a cell-penetrating structure, and a toxin molecule; the toxin molecule is linked and fused with the tumor cell surface targeting structure and / or the cell-penetrating structure; the tumor cell surface targeting structure is a protein structure capable of specifically binding to a target on the surface of tumor cells; the cell-penetrating structure is a protein structure that mediates the penetration of the recombinant protein through the cell membrane into the cell interior. This application uses the tumor cell surface targeting structure to specifically recognize the target on the surface of target cells and exert the killing activity of conventional antibodies, and then brings the toxin molecule into the tumor cells through the cell-penetrating structure, and the toxin is released to play a role in killing tumors, improving the efficacy of ADC drugs. Among them, the cell-penetrating efficiency of the cell-penetrating structure in this application can be increased to 50-90%, thereby improving the therapeutic window of traditional ADC drugs.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular, to a recombinant protein for improving the efficacy of ADC drugs and its applications. Background Art

[0002] Led by monoclonal antibody cancer drugs, antibody-drug conjugates (ADCs), which have developed rapidly in Europe and the United States in recent years, have become one of the most cutting-edge pharmaceutical technologies. Antibody-drug conjugates generally consist of three parts: 1. A monoclonal antibody that specifically binds to a target; 2. A small molecule chemical drug with cytotoxicity; 3. A linker that links the small molecule drug and the monoclonal antibody, also called a linker.

[0003] Antibody-drug conjugates utilize the target specificity of monoclonal antibodies and the cytotoxicity of chemical drugs to kill tumor cells. Its mechanism of action is as follows: (1) The antibody-drug conjugate specifically binds to the target antigen on the tumor cells using the monoclonal antibody; (2) The complex of the antibody-drug conjugate and the target antigen is endocytosed into the cell through target antigen-mediated endocytosis; (3) The antibody-drug conjugate degrades inside the cell, releasing the cytotoxic chemical drug; (4) The cytotoxic chemical drug kills the tumor cells.

[0004] The core of whether an antibody-drug conjugate can become a safe and effective drug is the therapeutic window. Since the connected chemical drug (such as a toxin) is highly toxic, high dosing concentrations will cause side effects, and low dosing concentrations will have no effect. Finding the therapeutic window is the key to drug development. The key to the therapeutic window is the efficiency of the antibody entering the tumor cells. Currently, the efficiency of natural antibodies entering tumor cells is relatively low, resulting in many targets being unable to be developed into drugs. Summary of the Invention

[0005] The purpose of this application is to overcome the deficiencies of the above-mentioned prior art and provide a recombinant protein for improving the efficacy of ADC drugs and its applications. This application first specifically recognizes the target on the surface of the target cell through the tumor cell surface targeting structure, exerts the killing activity of the conventional antibody, and then brings the ADC drug into the interior of the tumor cell through the cell-penetrating structure. The toxin of the ADC drug is released, resulting in the death of the tumor cell. The transmembrane efficiency of the recombinant protein of this application is 50-90%, thereby greatly enhancing the therapeutic window of traditional ADC drugs.

[0006] To achieve the above purpose, the technical solutions adopted in this application are as follows:

[0007] In the first aspect, this application provides a recombinant protein for improving the efficacy of ADC drugs, and the recombinant protein includes a tumor cell surface targeting structure, a cell-penetrating structure, and a toxin molecule;

[0008] The toxin molecule is linked and fused with a tumor cell surface targeting structure and / or a cell-penetrating structure;

[0009] The tumor cell surface targeting structure is a protein structure that can specifically bind to a target on the surface of tumor cells;

[0010] The cell-penetrating structure is a protein structure that mediates the penetration of the recombinant protein through the cell membrane and into the cell interior.

[0011] In this application, first, the tumor cell surface targeting structure in the recombinant protein specifically recognizes the target on the surface of the target cell, exerting the killing activity of a conventional antibody. Then, the toxin molecule is brought into the tumor cell through the cell-penetrating structure, and the toxin is released to exert the effect of killing tumors, thereby improving the efficacy of the ADC drug.

[0012] The transmembrane efficiency of the cell-penetrating structure adopting the structure of this application is 50-90%, thus greatly enhancing the therapeutic window of traditional ADC drugs.

[0013] As a preferred embodiment of the recombinant protein described in this application, the cell-penetrating structure includes at least one of Fab antibody-1, single-chain antibody-1, and cell-penetrating peptide;

[0014] The Fab antibody-1 contains a heavy chain variable region VH1-a and a light chain variable region VL1-a; the heavy chain variable region VH1-a contains an amino acid sequence having at least 95% identity with at least one of the sequences SEQ ID NO: 1-4; the light chain variable region VL1-a contains an amino acid sequence having at least 95% identity with at least one of the sequences SEQ ID NO: 5-8;

[0015] The single-chain antibody-1 contains an amino acid sequence having at least 95% identity with at least one of the sequences SEQ ID NO: 9-13.

[0016] The single-chain antibody-1 includes a single domain or scfv.

[0017] More preferably, the single domain contains an amino acid sequence having at least 95% identity with at least one of the sequences SEQ ID NO: 9-12; scfv contains an amino acid sequence having at least 95% identity with the sequence SEQ ID NO: 13.

[0018] The amino acid sequences of the heavy chain variable region VH1-a, the light chain variable region VL1-a, and the single-chain antibody-1 are shown in Table 1 below.

[0019] Table 1

[0020]

[0021]

[0022] More preferably, the Fab antibody-1 contains a heavy chain variable region VH1-a and a light chain variable region VL1-a; the heavy chain variable region VH1-a comprises an amino acid sequence having at least 95% identity with the sequence SEQ ID NO:1 or SEQ ID NO:2; the light chain variable region VL1-a comprises an amino acid sequence having at least 95% identity with at least one of the sequences SEQ ID NO:5 or SEQ ID NO:6;

[0023] The single-chain antibody-1 comprises an amino acid sequence having at least 95% identity with the sequence SEQ ID NO:9.

[0024] In some specific embodiments, in the Fab antibody-1, the heavy chain variable region VH1-a is an amino acid sequence as shown in the sequence SEQ ID NO:1 or SEQ ID NO:2;

[0025] In the Fab antibody-1, the light chain variable region VL1-a is an amino acid sequence as shown in the sequence SEQ ID NO:5 or SEQ ID NO:6;

[0026] The single-chain antibody-1 is an amino acid sequence as shown in the sequence SEQ ID NO:9.

[0027] The present application optimizes the cell-penetrating structure. By using the cell-penetrating structure of the above structure, the ADC drug can be brought into the interior of tumor cells (the penetration efficiency can be increased to 50-90%). The release of the toxin of the ADC drug causes the death of tumor cells, greatly improving the therapeutic window of traditional ADC drugs. Furthermore, it can also enable targets that cannot be made into drugs to be made into drugs.

[0028] As a preferred embodiment of the recombinant protein of the present application, the cell-penetrating peptide includes at least one of the cell-penetrating peptide HIV-1 TAT, the cell-penetrating peptide M918, the cell-penetrating peptide YTA2, the cell-penetrating peptide YTA4, the cell-penetrating peptide Pep-1, the cell-penetrating peptide MAP, the cell-penetrating peptide CADY, the cell-penetrating peptide pVEC, the cell-penetrating peptide MPG, the RVG-9R peptide, the rabies virus peptide RVG, the Peptide C105Y, the cell-penetrating peptide TP10, the cell-penetrating peptide NLS, the KALA amphiphilic peptide, melittin, polyarginine (Arg)n (n≥3), and the cell-penetrating peptide with the amino acid sequence as shown in any one of SEQ ID NOs:14-24.

[0029] The amino acid sequences of the above cell-penetrating peptides are shown in Table 2.

[0030] Table 2

[0031]

[0032] As a preferred embodiment of the recombinant protein described in the present application, the tumor cell surface targeting structure contains at least one protein structure capable of specifically binding to a cell surface target, and the at least one protein structure capable of specifically binding to a cell surface target can bind to the same or different cell surface targets.

[0033] As a preferred embodiment of the recombinant protein described in the present application, the tumor cell surface targets include at least one of EGFR, CEACAM5, Trop2, MSLN, CLDN6, ROR1, Her2, Her3, Her4, FLT-3, IGF-1R, c-MET, EphA2, IL13Rα2, RON, VEGFR, ADORA2A, FGFR, PDGFR, CD19, BCMA, CD22, CD20, CD70, CD123, CD38, CEACAM6, CD25, CD46, CD138, PSMA, MUC1, MUC16, GD2, Mesothelin, GPC3, PRAME, CDH6, Globo H, FOLR1, IDO, GITRL, CD30, CD2 / SLAM family; PD-L1, CD47, CD73, CD33, TNFR2, WNT / βcatenin, TOLI-like receptor, B7-H3, chemokine CCR, CXCR, FRα, nectin-4, ROR2, Claudin18.2, CD133, CD44, EPCAM.

[0034] The cell surface targets involved in the present application are not limited to the above selections, but also include common cell surface targets in the art. The cell surface targets can also be, for example, signal transduction proteins, inflammatory mediators, ligands, cell surface receptors or fragments thereof.

[0035] As a preferred embodiment of the recombinant protein described in the present application, the tumor cell surface target is MSLN, CEACAM5, CLDN6, ROR1 or Trop2; the tumor cell surface targeting structure is one of Fab antibody-2 or scFV domain capable of specifically binding to the cell surface target;

[0036] The Fab antibody-2 contains a heavy chain variable region VH-b and a light chain variable region VL-b;

[0037] The heavy chain variable region VH-b contains an amino acid sequence having at least 95% identity with at least one of the sequences SEQ ID NO: 25-29; the light chain variable region VL-b contains an amino acid sequence having at least 95% identity with at least one of the sequences SEQ ID NO: 30-34.

[0038] As a preferred embodiment of the recombinant protein described in the present application, the heavy chain variable region VH-b comprises at least one amino acid sequence among SEQ ID NO: 25 to 29;

[0039] and / or, the light chain variable region VL-b comprises at least one amino acid sequence among SEQ ID NO: 30 to 34.

[0040] In the present application, only the above cell surface targets are exemplified, and the cell targeting structures corresponding to MSLN, CEACAM5, CLDN6, ROR1, and Trop2 are used. When other types of cell surface targets are used, the cell targeting structures with different sequences can be correspondingly replaced. The cell targeting structures of the present application can specifically recognize the target cell surface targets and accurately exert the killing activity of conventional antibodies.

[0041] In the technical solution of the present application, the amino acid sequences of the heavy chain variable region VH-b, the light chain variable region VL-b, and the scFV domain are shown in Table 3.

[0042] Table 3

[0043]

[0044] In some specific embodiments, the cell surface targeting structure is Fab antibody-2 containing at least one heavy chain variable region VH-b and at least one light chain variable region VL-b.

[0045] As a preferred embodiment of the recombinant protein described in the present application,

[0046] the tumor cell surface targeting structure is Fab antibody-2;

[0047] the cell penetrating structure is single-chain antibody-1;

[0048] and the cell penetrating structure is connected to the N-terminus or C-terminus of the tumor cell surface targeting structure.

[0049] As a preferred embodiment of the recombinant protein described in the present application,

[0050] the tumor cell surface targeting structure is Fab antibody-2;

[0051] the cell penetrating structure is a cell-penetrating peptide;

[0052] and the cell penetrating structure is connected to the C-terminus of the tumor cell surface targeting structure.

[0053] As a preferred embodiment of the recombinant protein described in the present application,

[0054] the tumor cell surface targeting structure is the scFV domain;

[0055] The cell-penetrating structure is single-chain antibody-1;

[0056] And the cell-penetrating structure is connected to the C-terminus of the tumor cell surface targeting structure.

[0057] As a preferred embodiment of the recombinant protein described in the present application, the tumor cell surface targeting structure and the cell-penetrating structure are connected by a linker peptide.

[0058] As a preferred embodiment of the recombinant protein described in the present application, the linker peptide includes at least one of GGGGS, GGGGSGS, GGGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, AKTTPKLEEGEFSEAR, AKTTPKLEEGEFSEARV, AKTTPKLGG, SAKTTPKLGG, SAKTTP, RADAAP, RADAAPTVS, RADAAAAGGPGS, RADAAAA(G4S)4, SAKTTPKLEEGEFSEARV, ADAAP, ADAAPTVSIFPP, TVAAP, TVAAPSVFIFPP, QPKAAP, QPKAAPSVTLFPP, AKTTPP, AKTTPPSVTPLAP, AKTTAPSVYPLAP, ASTKGP, ASTKGPSVFPLAP, GENKVEYAPALMALS, GPAKELTPLKEAKVS, GHEAAAVMQVQYPAS, AKTTAP.

[0059] The linker peptides involved in the present application are not limited to the above-listed ones. Linker peptides also include peptide linker peptides that can be cleaved in vivo, protease (such as MMP)-sensitive linker peptides, disulfide bond-based linker peptides that can be cleaved by reduction, etc., or any cleavable linker peptides known in the art, and are all within the protection scope of the present application.

[0060] As a preferred embodiment of the recombinant protein described in the present application, the toxin molecule includes at least one of radioactive isotopes, antimetabolites, DNA alkylating agents, DNA cross-linking agents, DNA intercalating agents, topoisomerase inhibitors, taxanes, vinca alkaloids, maytansine alkaloids, colchicine, podophyllotoxin, griseofulvin, SPDB, SMCC, SN38.

[0061] The types of toxin molecules mentioned in the present application are not limited to the above examples, and also include toxin molecules commonly used in the art, which are all within the protection scope of the toxin molecules of the present application.

[0062] In some specific embodiments, the toxin molecule is linked to the tumor cell surface targeting structure and / or the cell-penetrating structure through a linker.

[0063] In a second aspect, the present application provides a nucleic acid molecule encoding the above-mentioned recombinant protein.

[0064] In a third aspect, the present application provides an expression vector comprising the above-mentioned nucleic acid molecule.

[0065] In some specific embodiments, the expression vector includes pKS001.

[0066] The expression vectors involved in the present application are not limited to the above-listed vectors, but also include expression vectors commonly used in the art for cloning and expressing the above-mentioned nucleic acid molecule, all of which are within the protection scope of the present application.

[0067] In a fourth aspect, the present application provides a host cell comprising the above-mentioned nucleic acid molecule or expression vector.

[0068] As a preferred embodiment of the host cell described in the present application, the host cell is eukaryotic. Preferably, the host cell is a mammalian cell or other cells suitable for preparing antibodies or fusion proteins.

[0069] In some specific embodiments, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies.

[0070] In a fifth aspect, the present application provides an antibody-drug conjugate, which comprises the above-mentioned recombinant protein and a drug for treating tumors.

[0071] In a sixth aspect, the present application provides a pharmaceutical composition, which comprises the above-mentioned antibody-drug conjugate and a pharmaceutically acceptable pharmaceutical excipient.

[0072] In a seventh aspect, the present application provides the use of the above-mentioned antibody-drug conjugate or pharmaceutical composition in the preparation of a drug or combination product for preventing or treating tumors.

[0073] As a preferred embodiment of the use described in the present application, the drug or combination product is administered in combination with one or more other therapies.

[0074] As a preferred embodiment of the use described in the present application, the tumors include one of lung cancer, liver cancer, breast cancer, ovarian cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, melanoma, bone cancer, skin cancer, prostate cancer, kidney cancer, bladder cancer, anal cancer, malignant blood diseases, gallbladder cancer, cholangiocarcinoma, head and neck cancer, glioma, nasopharyngeal cancer, laryngeal cancer, cervical cancer, uterine body cancer, osteosarcoma, esophageal cancer, small intestine cancer, head and neck squamous cell carcinoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, myeloma, lymphoma.

[0075] This application also covers any combination of any of the embodiments described herein. Any of the embodiments described herein or any combination thereof is applicable to any and all recombinant proteins or fragments thereof or pharmaceutical compositions or combination products or kits, methods, and uses described herein.

[0076] Compared with the prior art, this application has the following beneficial effects:

[0077] This application provides a recombinant protein for improving the efficacy of ADC drugs and its application. First, this application uses the tumor cell surface targeting structure in the recombinant protein to specifically recognize the target on the surface of target cells and exert the killing activity of a conventional antibody. Then, the toxin molecule is brought into the tumor cell through the cell-penetrating structure, and the toxin is released to play a role in killing tumors, thereby improving the efficacy of ADC drugs. Among them, the cell-penetrating efficiency of the cell-penetrating structure adopting the structure of this application can be increased to 50-90%, thereby improving the therapeutic window of traditional ADC drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Schematic diagram of the structure of the recombinant proteins (BY501-1, BY501-2, BY501-3, BY501-4, BY501-5) in Example 1;

[0079] Figure 2 Schematic diagram of the structure of the recombinant proteins (BY502-1, BY502-2, BY502-3) in Example 2;

[0080] Figure 3 Schematic diagram of the structure of the recombinant proteins (BY503-1, BY503-2, BY503-3) in Example 3;

[0081] Figure 4 Schematic diagram of the structure of the recombinant proteins (BY504-1, BY504-2, BY504-3) in Example 4;

[0082] Figure 5 Schematic diagram of the structure of the recombinant proteins (BY505-1, BY505-2, BY505-3, BY505-4, BY505-5) in Example 5;

[0083] Figure 6 Graph showing the measurement results of the antigen-binding affinity and biological activity of the recombinant proteins in Example 1;

[0084] Figure 7 Graph showing the measurement results of the antigen-binding affinity and biological activity of the recombinant proteins in Example 2;

[0085] Figure 8 Graph showing the measurement results of the antigen-binding affinity and biological activity of the recombinant proteins in Example 3;

[0086] Figure 9 Graph showing the determination results of the antigen-binding affinity and biological activity of the recombinant protein of Example 4;

[0087] Figure 10 Graph showing the determination results of the antigen-binding affinity and biological activity of the recombinant protein of Example 5;

[0088] Figure 11 Graph showing the in vivo activity results of the recombinant protein obtained in Example 1;

[0089] Figure 12 Graph showing the in vivo activity results of the recombinant protein obtained in Example 5;

[0090] Figures 13 - 14 Schematic diagram of a recombinant protein formed by combining a tumor cell surface targeting structure (denoted as D1) and a cell-penetrating structure (denoted as D2) with different structures. Detailed implementation manners

[0091] To better illustrate the purpose, technical solution, and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0092] In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0093] In the following examples, Fc refers to the antibody hinge region + CH2 + CH3 (CH: constant region of the antibody heavy chain);

[0094] VH refers to the variable region of the antibody heavy chain; CH refers to the constant region of the antibody heavy chain;

[0095] VL refers to the variable region of the antibody light chain; CL refers to the constant region of the antibody light chain;

[0096] In one embodiment, CH1 and Fc are from the antibody heavy chain, or derivatives thereof.

[0097] In one embodiment, the "CH1Fc" of the polypeptide chain in the following examples is in the form of IgG, such as in the form of IgG1, IgG2, or IgG4. In one embodiment, it is in the form of IgG1. It will be understood that the Fc in the constant domain can be mutated to achieve the function of stabilizing the antibody or enhancing the effector function. For example, in one specific embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0098] In one embodiment, there is a disulfide bond between CH1 and CL.

[0099] Unless otherwise indicated, the abbreviations in this specification have the following meanings:

[0100] ADCC: antibody-dependent cell-mediated cytotoxicity;

[0101] Ig: immunoglobulin;

[0102] As used herein, when the term "comprising" or "including" is used, unless otherwise specified, the situation consisting of the recited elements, integers or steps is also covered. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover an antibody variable region consisting of that specific sequence.

[0103] The term "antibody" is used herein in the broadest sense to refer to a protein containing an antigen-binding site, covering natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, intact antibodies, and antibody fragments.

[0104] "Antibody fragment" refers to a molecule different from an intact antibody, which contains a part of the intact antibody and binds to the antigen bound by the intact antibody. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, F(ab’)2; diabodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent or bispecific antibodies or their fragments; camelid antibodies; and bispecific or multispecific antibodies formed from antibody fragments.

[0105] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that participates in antibody-antigen binding. The variable domains of the heavy and light chains of a natural antibody usually have a similar structure, in which each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, VH or VL domains from an antibody that binds a specific antigen can be used to isolate antibodies that bind the antigen, to screen libraries of complementary VL or VH domains, respectively.

[0106] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parental cell, but may contain mutations. Mutant progeny having the same function or biological activity as selected or screened in the originally transformed cell are included herein. A host cell is any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells; and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells, and also include cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0107] The term "pharmaceutical composition" refers to a composition that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0108] The term "pharmaceutical excipient" refers to diluents, adjuvants (such as Freund's adjuvant (complete and incomplete)), carriers, excipients, or stabilizers, etc. that are administered together with the active substance.

[0109] As used herein, "treatment" refers to slowing, interrupting, arresting, alleviating, halting, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis. In some embodiments, the antibody molecules of the present invention are used to retard the development of a disease or to slow the progression of a disease.

[0110] As used herein, "prevention" includes the inhibition of the occurrence or development of a disease or disorder or the symptoms of a particular disease or disorder. In some embodiments, a subject with a family history of cancer is a candidate for a preventive regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in subjects at risk of cancer.

[0111] The term "combination product" refers to a fixed combination or non-fixed combination in the form of a dosage unit or a kit of parts for combined administration, wherein two or more therapeutic agents can be administered independently simultaneously at the same time or separately at certain time intervals, especially when these time intervals allow the combination partners to exhibit cooperation, for example, a synergistic effect. The term "fixed combination" means that the antibody of the present invention and the combination partner (such as other therapeutic agents) are administered to a patient simultaneously in the form of a single entity or dose. The term "non-fixed combination" means that the antibody of the present invention and the combination partner (such as other therapeutic agents) are administered to a patient simultaneously, in parallel or sequentially as separate entities, without a specific time limit, wherein such administration provides a therapeutically effective level of the two therapeutic agents in the patient. The latter also applies to combination therapies, such as the administration of three or more therapeutic agents. In a preferred embodiment, the drug combination is a non-fixed combination.

[0112] The term "combination therapy" or "combined therapy" refers to the administration of two or more therapeutic agents to treat cancer or an infection as described in the present disclosure. Such administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administering or separately administering or sequentially administering the individual active ingredients in multiple or separate containers (such as tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In some embodiments, the administration also includes using each type of therapeutic agent at approximately the same time, or in a sequential manner at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating the disorders or conditions described herein.

[0113] This application provides a recombinant protein for improving the efficacy of an ADC drug, the recombinant protein comprising a tumor cell surface targeting structure, a cell-penetrating structure, and a toxin molecule;

[0114] The toxin molecule is linked and fused to the tumor cell surface targeting structure and / or the cell-penetrating structure;

[0115] The tumor cell surface targeting structure is a protein structure capable of specifically binding to a tumor cell surface target;

[0116] The cell-penetrating structure is a protein structure that mediates the penetration of the recombinant protein through the cell membrane into the cell interior.

[0117] In some specific embodiments, the toxin molecule is linked to the tumor cell surface targeting structure and / or the cell-penetrating structure through a linker.

[0118] Examples are given below of the tumor cell surface targeting structure specifically binding to different cell surface targets and bringing the toxin molecule into the tumor cell through cell-penetrating structures of different structural forms.

[0119] Example 1. A recombinant protein against MSLN

[0120] This example provides a recombinant protein against MSLN, including a tumor cell surface targeting structure, a cell-penetrating structure, and a toxin molecule;

[0121] Among them, the tumor cell surface targeting structure specifically binds to the human MSLN antigen. The toxin molecule uses DM4, and the toxin molecule is linked to the tumor cell surface targeting structure through a linker. The cell-penetrating structure uses the cell-penetrating peptide TAT, or the cell-penetrating structure uses single-chain antibody-1, and single-chain antibody-1 is the amino acid sequence shown in SEQ ID NO:10;

[0122] The tumor cell surface targeting structure is Fab antibody-2 containing two heavy chain variable regions VH-b and two light chain variable regions VL-b;

[0123] The heavy chain variable region VH-b contains the amino acid sequence shown in SEQ ID NO:25;

[0124] The light chain variable region VL-b contains the amino acid sequence shown in SEQ ID NO:30;

[0125] Moreover, the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure through a linker (SPDB) to form a recombinant protein (BY501-1);

[0126] Moreover, the cell-penetrating structure (containing two single-chain antibodies-1) is respectively connected to the N-terminus of VH-b of the tumor cell surface targeting structure through a linker peptide, and then the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure through a linker (SPDB) to form a recombinant protein (BY501-2);

[0127] Moreover, the cell-penetrating structure (containing two single-chain antibodies-1) is respectively connected to the N-terminus of VL-b of the tumor cell surface targeting structure through a linker peptide, and then the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure through a linker (SPDB) to form a recombinant protein (BY501-3);

[0128] Moreover, the cell-penetrating structure (containing two cell-penetrating peptides TAT) is respectively connected to the N-terminus of VH-b of the tumor cell surface targeting structure through a linker peptide, and then the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure through a linker (SPDB) to form a recombinant protein (BY501-4);

[0129] Moreover, the cell-penetrating structure (containing two cell-penetrating peptides TAT) is respectively connected to the N-terminus of VL-b of the tumor cell surface targeting structure through a linker peptide, and then the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure through a linker (SPDB) to form a recombinant protein (BY501-5);

[0130] The schematic structural diagrams of BY501-1, BY501-2, BY501-3, BY501-4, and BY501-5 are as Figure 1 shown.

[0131] The specific polypeptide chains formed by BY501-1, BY501-2, BY501-3, BY501-4, and BY501-5 are shown in Table 4-5.

[0132] Table 4

[0133]

[0134] Note: VH MSLN : the variable region of the heavy chain of the anti-MSLN antibody; VL MSLN : the variable region of the light chain of the anti-NSLN antibody; V D2 : the variable region of the cell-penetrating structure; V TAT : the cell-penetrating peptide TAT.

[0135] Table 5

[0136]

[0137]

[0138] The amino acid sequences of BY501-1, BY501-2, BY501-3, BY501-4, and BY501-5 obtained above were sent to Nanjing Genscript for synthesis, and the synthesized genes were respectively cloned into the pKS001 vector to construct BY501-1, BY501-2, BY501-3, BY501-4, and BY501-5 expression vectors.

[0139] Example 2. A recombinant protein against CEACAM5

[0140] This example provides a recombinant protein against CEACAM5, including a tumor cell surface targeting structure, a cell-penetrating structure, and a toxin molecule;

[0141] Among them, the tumor cell surface targeting structure specifically binds to the human CEACAM5 antigen. The toxin molecule is SN38, and the toxin molecule is linked to the tumor cell surface targeting structure or the cell-penetrating structure through a linker. The cell-penetrating structure is the cell-penetrating peptide TAT, or the cell-penetrating structure is a Fab antibody-1 formed by a heavy chain variable region VH1-a (SEQ ID NO:3) and a light chain variable region VL-a (SEQ ID NO:7);

[0142] The tumor cell surface targeting structure is a Fab antibody-2 containing a heavy chain variable region VH-b and a light chain variable region VL-b;

[0143] The heavy chain variable region VH-b contains the amino acid sequence shown in SEQ ID NO:26;

[0144] The light chain variable region VL-b contains the amino acid sequence shown in SEQ ID NO:31;

[0145] Moreover, the toxin molecule is respectively linked to the CH1 and CL of the tumor cell surface targeting structure through a linker (CL2) to form a recombinant protein (BY502-1);

[0146] Moreover, the cell-penetrating structure (Fab antibody-1) and the tumor cell surface targeting structure are connected and fused, and then the toxin molecule is respectively linked to the CH1 and CL of the tumor cell surface targeting structure and the CH1 and CL of the cell-penetrating structure (Fab antibody-1) through a linker (SPDB) to form a recombinant protein (BY502-2);

[0147] Moreover, the cell-penetrating structure (cell-penetrating peptide TAT) and the tumor cell surface targeting structure are connected and fused, and then the toxin molecule is respectively linked to the CH1 and CL of the tumor cell surface targeting structure and the CH1 and CL of the cell-penetrating structure (cell-penetrating peptide TAT) through a linker (SPDB) to form a recombinant protein (BY502-3);

[0148] The schematic diagrams of the structures of BY502-1, BY502-2, and BY502-3 are as Figure 2 shown.

[0149] The specific polypeptide chains formed by BY502-1, BY502-2, and BY502-3 are shown in Table 6-7.

[0150] Table 6

[0151]

[0152] Note: VH CEACAM5 : Heavy chain variable region of anti-CEACAM5 antibody; VL CEACAM5: the light chain variable region of the anti-CEACAM5 antibody;

[0153] VH D2 : the heavy chain variable region of the antibody with a cell-penetrating structure; VL D2 : the light chain variable region of the antibody with a cell-penetrating structure; V TAT : the cell-penetrating peptide TAT.

[0154] Table 7

[0155]

[0156]

[0157] The amino acid sequences of BY502-1, BY502-2, and BY502-3 obtained above were sent to GenScript for synthesis. The synthesized genes were respectively cloned into the pKS001 vector, thereby constructing the BY502-1, BY502-2, and BY502-3 expression vectors.

[0158] Example 3. A recombinant protein against CLDN6

[0159] This example provides a recombinant protein against CLDN6, including a tumor cell surface targeting structure, a cell-penetrating structure, and a toxin molecule;

[0160] Among them, the tumor cell surface targeting structure specifically binds to the human CLDN6 antigen. The toxin molecule uses DM1, and the toxin molecule is linked to the tumor cell surface targeting structure cell-penetrating structure through a linker. The cell-penetrating structure uses the cell-penetrating peptide TAT, or the cell-penetrating structure uses single-chain antibody-1, and single-chain antibody-1 is the amino acid sequence shown in SEQ ID NO:11;

[0161] The tumor cell surface targeting structure is a Fab antibody-2 containing the heavy chain variable region VH-b and the light chain variable region VL-b;

[0162] The heavy chain variable region VH-b contains the amino acid sequence shown in SEQ ID NO:27;

[0163] The light chain variable region VL-b contains the amino acid sequence shown in SEQ ID NO:32;

[0164] Moreover, the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure through a linker (SMCC) to form a recombinant protein (BY503-1);

[0165] Moreover, the cell-penetrating structure (single-chain antibody-1) is connected and fused with the tumor cell surface targeting structure, and then the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure and the cell-penetrating structure (single-chain antibody-1) through a linker (SPDB) to form a recombinant protein (BY503-2);

[0166] Moreover, the cell-penetrating structure (cell-penetrating peptide TAT) is connected and fused with the tumor cell surface targeting structure, and then the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure and the cell-penetrating structure (cell-penetrating peptide TAT) through a linker (SPDB) to form a recombinant protein (BY503-3).

[0167] The schematic diagrams of the structures of BY503-1, BY503-2, and BY503-3 are as Figure 3 shown.

[0168] The specific polypeptide chains formed by BY503-1, BY503-2, and BY503-3 are shown in Table 8-9.

[0169] Table 8

[0170]

[0171] Note: VH CLDN6 : anti- CLDN6 heavy chain variable region of the antibody; VL CLDN6 : anti- CLDN6 light chain variable region of the antibody;

[0172] V D2 : variable region of the cell-penetrating structure; V TAA : cell-penetrating peptide TAA.

[0173] Table 9

[0174]

[0175] The amino acid sequences of BY503-1, BY503-2, and BY503-3 obtained above were sent to Nanjing Genscript for synthesis, and the synthesized genes were respectively cloned into the pKS001 vector to construct the BY503-1, BY503-2, and BY503-3 expression vectors.

[0176] Example 4. A recombinant protein against ROR1

[0177] This example provides a recombinant protein against ROR1, including a tumor cell surface targeting structure, a cell-penetrating structure, and a toxin molecule;

[0178] Among them, the tumor cell surface targeting structure specifically binds to the human ROR1 antigen. The toxin molecule is MMAE. The toxin molecule is linked to the tumor cell surface targeting structure or the cell-penetrating structure through a linker. The cell-penetrating structure is the cell-penetrating peptide TAA, or the cell-penetrating structure is scfv, and scfv contains the amino acid sequence shown in SEQ ID NO: 13;

[0179] The tumor cell surface targeting structure is Fab antibody-2 containing the heavy chain variable region VH-b and the light chain variable region VL-b;

[0180] The heavy chain variable region VH-b contains the amino acid sequence shown in SEQ ID NO: 28;

[0181] The light chain variable region VL-b contains the amino acid sequence shown in SEQ ID NO: 33;

[0182] Moreover, the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure through a linker (mc-vc-PAB) to form a recombinant protein (BY504-1);

[0183] Moreover, the cell-penetrating structure (scfv) and the tumor cell surface targeting structure are connected and fused, and then the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure and the cell-penetrating structure (scfv) through a linker (mc-vc-PAB) to form a recombinant protein (BY504-2);

[0184] Moreover, the cell-penetrating structure (cell-penetrating peptide TAT) and the tumor cell surface targeting structure are connected and fused, and then the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure and the cell-penetrating structure (cell-penetrating peptide TAT) through a linker (mc-vc-PAB) to form a recombinant protein (BY504-3);

[0185] The schematic diagrams of the structures of BY504-1, BY504-2, and BY504-3 are as Figure 4 shown.

[0186] The specific polypeptide chains formed by BY504-1, BY504-2, and BY504-3 are shown in Table 10-11.

[0187] Table 10

[0188]

[0189] Note: VH ROR1 : heavy chain variable region of anti-ROR1 antibody; VL ROR1 : light chain variable region of anti-ROR1 antibody;

[0190] V D2 : The variable region of the antibody of the cell-penetrating structure; V TAT : The cell-penetrating peptide TAT.

[0191] Table 11

[0192]

[0193] The amino acid sequences of BY504-1, BY504-2, and BY504-3 obtained above were sent to Nanjing GenScript for synthesis. The synthesized genes were respectively cloned into the pKS001 vector, thereby constructing BY504-1, BY504-2, and BY504-3 expression vectors.

[0194] Example 5. A recombinant protein against TROP2

[0195] This example provides a recombinant protein against TROP2, including a tumor cell surface targeting structure, a cell-penetrating structure, and a toxin molecule;

[0196] Among them, the tumor cell surface targeting structure specifically binds to the human TROP2 antigen. The toxin molecule is SN38, and the toxin molecule is linked to the tumor cell surface targeting structure or the cell-penetrating structure through a linker. The cell-penetrating structure uses the cell-penetrating peptide TAT, or the cell-penetrating structure uses single-chain antibody-1, and single-chain antibody-1 is the amino acid sequence shown in SEQ ID NO:9;

[0197] The tumor cell surface targeting structure is Fab antibody-2 containing two heavy chain variable regions VH-b and two light chain variable regions VL-b;

[0198] The heavy chain variable region VH-b contains the amino acid sequence shown in SEQ ID NO:29;

[0199] The light chain variable region VL-b contains the amino acid sequence shown in SEQ ID NO:34;

[0200] Moreover, the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure through a linker (CL2A) to form a recombinant protein (BY505-1);

[0201] Moreover, the cell-penetrating structure (single-chain antibody-1) is respectively connected to the Fc end of the tumor cell surface targeting structure through a connecting peptide, and then the toxin molecule is respectively linked to CH1 and CL of the tumor cell surface targeting structure through a linker (CL2A) to form a recombinant protein (BY505-2);

[0202] Moreover, the cell-penetrating structure (single-chain antibody-1) is respectively connected to the CL of the tumor cell surface targeting structure through a linker peptide, and then the toxin molecule is respectively linked to the CH1 and CL of the tumor cell surface targeting structure through a linker (CL2A) to form a recombinant protein (BY505-3);

[0203] Moreover, the cell-penetrating structure (cell-penetrating peptide TAT) is respectively connected to the Fc end of the tumor cell surface targeting structure through a linker peptide, and then the toxin molecule is respectively linked to the CH1 and CL of the tumor cell surface targeting structure through a linker (CL2A) to form a recombinant protein (BY505-4);

[0204] Moreover, the cell-penetrating structure (cell-penetrating peptide TAT) is respectively connected to the CL of the tumor cell surface targeting structure through a linker peptide, and then the toxin molecule is respectively linked to the CH1 and CL of the tumor cell surface targeting structure through a linker (CL2A) to form a recombinant protein (BY505-5);

[0205] The schematic diagrams of the structures of BY505-1, BY505-2, BY505-3, BY505-4, and BY505-5 are as Figure 5 shown.

[0206] The specific polypeptide chains formed by BY505-1, BY505-2, BY505-3, BY505-4, and BY505-5 are shown in Table 12-13.

[0207] Table 12

[0208] BY505 molecule Polypeptide chain #1 Linker Polypeptide chain #2 Connector Toxin BY505-1 <![CDATA[VH TROP2 -CH1-Fc]]> <![CDATA[VL TROP2 -CL]]> CL2A SN38 BY505-2 <![CDATA[VH TROP2 -CH1-Fc-linker-V D2 > GGGGSGGGGSGGGGS <![CDATA[VL TROP2 -CL]]> CL2A SN38 BY505-3 <![CDATA[VH TROP2 -CH1-Fc]]> GGGGSGGGGSGGGGS <![CDATA[VL TROP2 -CL-Joint-V D2 > CL2A SN38 BY505-4 <![CDATA[VH TROP2 -CH1-Fc-linker-V TAT > GGGGSGGGGSGGGGS <![CDATA[VL TROP2 -CL]]> CL2A SN38 BY505-5 <![CDATA[VH TROP2 -CH1-Fc]]> GGGGSGGGGSGGGGS <![CDATA[VL TROP2 -CL-Joint-V TAT > CL2A SN38

[0209] Note: VH TROP2 : the variable region of the heavy chain of the anti-TROP2 antibody; VL TROP2 : the variable region of the light chain of the anti-TROP2 antibody;

[0210] V D2 : the antibody variable region of the cell-penetrating structure; V TAT : the cell-penetrating peptide TAT.

[0211] Table 13

[0212]

[0213]

[0214] The amino acid sequences of BY505-1, BY505-2, BY505-3, BY505-4, and BY505-5 obtained above were sent to GenScript Nanjing for synthesis. The synthesized genes were respectively cloned into the pKS001 vector, thereby constructing the expression vectors of BY505-1, BY505-2, BY505-3, BY505-4, and BY505-5.

[0215] Example 6. Expression, Purification, and Analysis Methods of Recombinant Proteins

[0216] This example provides the expression, purification, and analysis methods of the above recombinant proteins, including the following steps:

[0217] (1) Protein expression:

[0218] Pre-prepare CHO cell medium (CD CHO Medium + 8 mM GlutaMAX; Gibco, 10743-029; Gibco, 35050-061) in a T75 flask and preheat it at 37 °C. Prepare CHO-K1 cells in the logarithmic growth phase. Mix 1×10 7 cells with 30 μg of the expression vectors obtained in Examples 1-5 above in an electroporation cuvette respectively, and perform electroporation according to the electroporation program: 300 V, pulse width 15 ms, pulse interval 1 s, 2 pulses. Transfer the electroporated cell suspension to a T75 flask, and place the T75 flask in an incubator at 37 °C and 8% CO2 for static culture. After 24-48 h, take samples for counting. Within 48 h, replace the medium (CHO CD04 Medium + 25 μM MSX; Quacell, A11004; Sigma, M5379) for pressure screening until the cell viability recovers to more than 90% for large-scale Fedbatch culture, and harvest the cell culture supernatant.

[0219] (2) Protein purification:

[0220] Antibody purification mainly includes affinity chromatography and ion exchange chromatography, as follows:

[0221] ① Affinity chromatography (PrismA, GE):

[0222] a) Equilibration: Equilibrate the chromatography column with binding buffer (25 mM tris(hydroxymethyl)aminomethane, pH 7.0-7.4) until the UV detector and conductivity values remain stable or at the baseline, and equilibrate for at least 5 column volumes;

[0223] b) Loading: Load the filtered supernatant at a flow rate of 5 ml / min;

[0224] c) Wash equilibration: Wash with binding buffer for 5 column volumes;

[0225] d) Elution: Elute the sample with elution buffer (50 mM acetic acid = sodium acetate, pH 3.6 ± 0.1) at a flow rate of 5 ml / min for 5 column volumes, and collect the elution peak.

[0226] e) Neutralization: Neutralize the eluate with 1 M Tris (tris(hydroxymethyl)aminomethane) pH 8.0, and adjust the pH of the sample to 5.0 - 5.5.

[0227] ② Anion exchange chromatography (CaptoQ, GE)

[0228] a) Sample preparation: After microfiltration, dilute the chromatography sample with ultrapure water to a conductivity of 3.0 - 7.0 mS / cm, and then adjust the pH to 7.6 - 8.2.

[0229] b) Equilibration and loading: First, rinse with pre-equilibration buffer (50 mM Tris-HCl + 1 M NaCl, conductivity 80 - 100 mS / cm, pH 7.8 - 8.2) for 2 CV; then equilibrate with equilibration buffer (50 mM Tris-HCl, conductivity 2.0 - 5.0 mS / cm, pH 7.8 - 8.2) for 2 CV until the conductivity, pH, and UV baseline are stable; then load the sample at a flow rate of 1 ml / min.

[0230] c) Start collecting the flow-through peak when the UV280 reading rises to 0.1 AU; after loading, switch to the equilibration buffer to rinse the chromatography column; stop collecting the flow-through peak when the UV280 drops to 0.1 AU.

[0231] d) Add 10% acetic acid dropwise to the collected sample to adjust the pH to 6.0 - 6.4; detect the sample.

[0232] ③ Cation exchange chromatography (CaptoS ImpAct, GE):

[0233] a) Sample preparation: After microfiltration, dilute the affinity chromatography sample with ultrapure water to a conductivity less than 5 mS / cm, and then adjust the pH to 6.0 ± 0.1.

[0234] b) Equilibration and loading: First, equilibrate with 5 column volumes of buffer B (50 mM acetic acid - sodium acetate + 1 M sodium chloride, conductivity should be 80 - 90 mS / cm, pH 5.5 ± 0.1), and then equilibrate the chromatography column with at least 5 column volumes of buffer A (50 mM acetic acid - sodium acetate, conductivity should be less than 5 mS / cm, pH 5.5 ± 0.1). After the conductivity, pH, and UV baseline are stable, load the sample at a flow rate of 1 ml / min.

[0235] c) Wash and equilibrate: Wash with 5 column volumes of buffer A at a flow rate of 1 ml / min.

[0236] d) Elution: 0 - 30% buffer B, 20 column volumes; 100% buffer B, 10 column volumes. The flow rate is 1 ml / min throughout the process. The eluate is collected in fractions and detected.

[0237] (3) Protein detection:

[0238] The purified protein is dialyzed and exchanged into PBS (pH 7.0). After SDS-PAGE and HPLC-SEC analysis, it is ensured that the protein purity is greater than 98%.

[0239] Detection of the antigen-binding affinity of the recombinant proteins obtained in Example 7 and Examples 1 - 5

[0240] 1) Detection of the antigen-binding affinity of BY501-1, BY501-2, BY501-3, BY501-4, BY501-5:

[0241] The target-binding activity is detected by the ELISA method:

[0242] Analyze the target-binding activity of MSLN. The specific steps are as follows: Dissolve human MSLN antigen in the coating buffer and dilute it to 0.5 μg / mL; then add it to a 96-well ELISA plate, 100 μL per well, and place the ELISA plate in a 4°C refrigerator overnight. During detection, wash it 3 times with the washing solution (TBS, 0.05% Tween-20, pH 7.4); first block it at 37°C for 1.5 h with the blocking solution (washing solution, 2% BSA), and wash it 3 times with the washing solution; then add the diluted experimental group antibodies (set amatuximab monoclonal antibody as the positive control and IgG1 as the negative control), incubate at 37°C for 1 h, and wash it 3 times with the washing solution; then add the goat antibody (Jakson Immuno Research, 109-035-098) labeled with horseradish peroxidase (HRP) against the human IgG Fc fragment, and incubate at room temperature for 1 h; after washing 6 times, add the substrate of HRP, develop color in the dark for 10 - 15 min, then add the termination solution (1 M H2SO4) to terminate the reaction, and measure the optical density at a wavelength of 450 nm.

[0243] 2) Similarly, detect the target-binding activity of BY502-1, BY502-2, BY502-3 to CEACAM5, and the detection method is the same as the above method for detecting the target-binding activity of MSLN.

[0244] 3) Similarly, detect the target-binding activity of BY503-1, BY503-2, BY503-3 to CLDN6, and the detection method is the same as the above method for detecting the target-binding activity of MSLN.

[0245] 4) Similarly, detect the target-binding activities of BY504-1, BY504-2, and BY504-3 to ROR1. The detection method is the same as that for detecting the target-binding activity of MSLN described above.

[0246] 5) Similarly, detect the target-binding activities of BY505-1, BY505-2, BY505-3, BY505-4, and BY505-5 to TROP2. The detection method is the same as that for detecting the target-binding activity of MSLN described above.

[0247] The detection results of the antigen-binding affinities of the recombinant proteins obtained in Examples 1 to 5 are respectively referred to Figures 6 - 10 .

[0248] Analysis: The recombinant proteins (BY501-1, BY501-2, BY501-3, BY501-4, BY501-5) of Example 1 of this application have relatively high affinities for targeting MSLN.

[0249] The recombinant proteins (BY502-1, BY502-2, BY502-3) of Example 2 of this application have relatively high affinities for targeting CEACAM5.

[0250] The recombinant proteins (BY503-1, BY503-2, BY503-3) of Example 3 of this application have relatively high affinities for targeting CLDN6.

[0251] The recombinant proteins (BY504-1, BY504-2, BY504-3) of Example 4 of this application have relatively high affinities for targeting ROR1.

[0252] The recombinant proteins (BY505-1, BY505-2, BY505-3, BY505-4, BY505-5) of Example 5 of this application have relatively high affinities for targeting TROP2.

[0253] Determination of the biological activities of the recombinant proteins obtained in Examples 8, 1 to 5

[0254] (1) Use a cytotoxicity detection experiment to measure the biological activities of the recombinant proteins obtained in Examples 1 to 5 above to inhibit the proliferation of tumor cells.

[0255] Briefly, according to the growth rate of tumor cells, adherent tumor cells in the logarithmic growth phase were inoculated into 96-well culture plates at 100 μL / well, and drugs were added 24 hours after adherent growth. Three replicates were set for each concentration; a blank group (culture medium, detection solution) and a control group (culture medium, cells, detection solution) were also set. The tumor cells were cultured at 37 °C and 5% CO2 for 48 hours. The culture plates were taken out, 10 μL of CCK-8 detection reagent was added to each well, incubated at 37 °C and 5% CO2 for 4 hours, and detected with an enzyme-linked immunosorbent assay (ELISA) at 450 nm. The tumor cell growth inhibition rate was calculated according to the formula:

[0256] Inhibition rate = [A(control) - A(experiment)] / [A(control) - A(blank)] × 100%

[0257] (2) The endocytic activity of the recombinant proteins obtained in Examples 1 to 5 above was determined by immunofluorescence assay.

[0258] The coverslips were placed in 24-well plates, and an appropriate amount of cells were added to 0.5 ml of medium containing 10% fetal bovine serum (FBS) and cultured at 5% CO2 and 37 °C for 12 hours. When the cells were stabilized, each well was treated with 10 μM antibody (the recombinant proteins and control antibody in Examples 1-6 above) in 0.5 ml of fresh medium and incubated at 37 °C and 5% CO2 for 6 hours. Then the medium was removed and each well was washed with phosphate-buffered saline (PBS). The cells were treated with a weak acidic solution (200 mM glycine, 150 mM NaCl, pH 2.5) to remove cell surface proteins. Then each well was washed with PBS, fixed with 4% paraformaldehyde at 25 °C for 10 minutes; after washing with PBS, each well was treated with a permeabilization solution and permeabilized at 25 °C for 5 minutes; after washing with PBS, each well was blocked with PBS buffer containing 2% bovine serum albumin (BSA) at 25 °C for 1 hour to eliminate non-specific binding; then each well was treated with a fluorescein isothiocyanate (FITC)-labeled fluorescent secondary antibody specifically recognizing human IgG Fc, incubated at 25 °C in the dark for 1 hour, the cells were collected, and the FITC-labeled fluorescence was detected by flow cytometry to measure the transmembrane efficiency of the recombinant proteins obtained in Examples 1 to 5 above.

[0259] The measurement results of the biological activities of the recombinant proteins obtained in Examples 1 to 5 are referred to Figures 6 - 10 .

[0260] Analysis: The recombinant proteins (BY501-1, BY501-2, BY501-3, BY501-4, BY501-5) obtained in Example 1 could better inhibit the proliferation of ASPC-1 cells, and both antibody-dependent cell-mediated cytotoxicity (ADCC) activity and endocytic activity were relatively high. Among them, BY501-2, BY501-3, BY501-4, and BY501-5 had better inhibitory effects on the proliferation of ASPC-1 cells, and their ADCC activity and endocytic activity were superior to BY501-1.

[0261] The recombinant proteins (BY502-1, BY502-2, BY502-3) obtained in Example 2 could inhibit the proliferation of MKN-45 cells well, and both the ADCC activity and the endocytosis activity were relatively high. Among them, BY502-2 and BY502-3 showed better inhibitory effects on the proliferation of MKN-45 cells, and their ADCC activity and endocytosis activity were superior to those of BY502-1.

[0262] The recombinant proteins (BY503-1, BY503-2, BY503-3) obtained in Example 3 could inhibit the proliferation of OVCAR-3 cells well, and both the ADCC activity and the endocytosis activity were relatively high. Among them, BY503-2 and BY503-3 showed better inhibitory effects on the proliferation of OVCAR-3 cells, and their ADCC activity and endocytosis activity were superior to those of BY503-1.

[0263] The recombinant proteins (BY504-1, BY504-2, BY504-3) obtained in Example 4 could inhibit the proliferation of AGS cells well, and both the ADCC activity and the endocytosis activity were relatively high. Among them, BY504-2 and BY504-3 showed better inhibitory effects on the proliferation of AGS cells, and their ADCC activity and endocytosis activity were superior to those of BY504-1.

[0264] The recombinant proteins (BY505-1, BY505-2, BY505-3, BY505-4, BY505-5) obtained in Example 5 could inhibit the proliferation of BxPC-3 cells well, and both the ADCC activity and the endocytosis activity were relatively high. Among them, BY505-2, BY505-3, BY505-4, and BY505-5 showed better inhibitory effects on the proliferation of BxPC-3 cells, and their ADCC activity and endocytosis activity were superior to those of BY505-1.

[0265] It was demonstrated that adding the cell-penetrating structure of the present application was helpful to improve the cell-penetrating efficiency of the antibody, and the cell-penetrating efficiency could be increased to 20 - 50%. The toxin molecule was brought into the interior of the tumor cell through the cell-penetrating structure, and the toxin was released to play a role in killing the tumor, thereby increasing the therapeutic window of traditional ADC drugs by 5 - 10 times.

[0266] In vivo activity evaluation of the recombinant proteins obtained in Example 9, Example 1, and Example 5

[0267] 1. Using the ASPC-1 cell subcutaneous tumor model, the in vivo anti-tumor activity of the recombinant protein obtained in Example 1 was studied. 35 nude mice were injected subcutaneously at the tail with ASPC-1 cells, and each mouse was injected with 3×10 6 cells, and the tumor volume was 100 mm 3Random grouping was performed at that time. The first group was intraperitoneally injected with PBS, the second group was injected with BY501-1 (30 mg / kg), the third group was injected with BY501-2 (10 mg / kg), the fourth group was injected with BY501-3 (10 mg / kg), the fifth group was injected with BY501-4 (10 mg / kg), the sixth group was injected with BY501-5 (10 mg / kg), and the seventh group was intraperitoneally injected with anti-MSLN monoclonal antibody (30 mg / kg). The drug was administered twice a week for 4 consecutive weeks. The condition of the mice was observed and the body weight was measured every day.

[0268] 2. Using the subcutaneous tumor model of BxPC-3 cells, the in vivo antitumor activity of the recombinant protein obtained in Example 5 was studied. 35 nude mice were subcutaneously injected with BxPC-3 cells at the tail, and each mouse was injected with 3×10 6 cells, and the tumor volume was 100 mm 3 Random grouping was performed at that time. The first group was intraperitoneally injected with PBS, the second group was injected with BY505-1 (30 mg / kg), the third group was injected with BY505-2 (10 mg / kg), the fourth group was injected with BY505-3 (10 mg / kg), the fifth group was injected with BY505-4 (10 mg / kg), the sixth group was injected with BY505-5 (10 mg / kg), and the seventh group was intraperitoneally injected with anti-TROP2 monoclonal antibody (30 mg / kg). The drug was administered twice a week for 4 consecutive weeks. The condition of the mice was observed and the body weight was measured every day.

[0269] The in vivo activity results of the recombinant proteins obtained in Examples 1 and 5 are referred to Figures 11 - 12 .

[0270] Analysis: The in vivo activity results of the recombinant proteins obtained in Examples 1 and 5 are both better.

[0271] Example 10. Preparation of Antibody-Drug Conjugate

[0272] This example provides an antibody-drug conjugate and its preparation method, including the following steps:

[0273] (1) Prepare the reduction buffer: Dissolve TCEP (Tris-2-carboxyethyl-phosphine) and DTPA (diethylenetriaminepentaacetic acid) in PBS. The concentrations of the two substances in the reduction buffer are 0.26 mM and 2 mM respectively;

[0274] (2) Antibody reduction: Mix 20 mg / mL mAb (in PBS buffer) with the reduction buffer at a volume ratio of 1:1, and stir and react at 25 °C for 2 h;

[0275] (3) Preparation of small molecule drug solution: Dissolve the small molecule toxin DM4 in DMSO (dimethyl sulfoxide) to a final concentration of 10 mM;

[0276] (4) Conjugation: Add 25% DMSO to the reduced protein, and then slowly add the small molecule drug solution according to a molar ratio of 4.4 between the small molecule drug and the recombinant proteins of Examples 1-5 for conjugation. Stir and react at 25 °C for 1 h. Finally, dialyze the conjugated protein in PBS to remove the unconjugated small molecule drug to obtain ADC (mAb-SPDB-DM4) for use.

[0277] Detection of antibody-drug conjugate: Analyze the drug conjugation ratio (DAR) of ADC by hydrophobic interaction chromatography (HIC-HPLC).

[0278] Analyze the anti-MSLN antibody (mAb) and ADC (mAb-SPDB-DM4) using a 1260bio high performance liquid chromatograph (purchased from Agilent) and a TSK-GEL Butyl-NPR chromatographic column (4.6 mm × 35 mm, TOSOH). Mobile phase A: 75% (v / v) 20 mM phosphate buffer (pH = 7.0), 25% (v / v) isopropanol; Mobile phase B: 20 mM phosphate buffer (pH = 7.0), 1.5 M ammonium sulfate. Detection wavelength: 280 nm; Column temperature: 25 °C; Flow rate: 1.0 ml / min; Injection volume: approximately 10 μg.

[0279] This application also provides recombinant proteins formed by combining cell surface targeting structures (denoted as D1) and cell-penetrating structures (denoted as D2) in different forms and structures. Schematic diagrams of the recombinant proteins formed by the above different combinations are as Figures 13 - 14 shown.

[0280] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of this application rather than to limit the protection scope of this application. Although this application has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of this application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of this application.

Claims

1. A recombinant protein for improving the efficacy of ADC drugs, characterized in that: The recombinant protein includes a tumor cell surface targeting structure, a cell membrane-penetrating structure and a toxin molecule; The toxin molecule is connected and fused with the tumor cell surface targeting structure and / or the cell-penetrating structure; The tumor cell surface targeting structure is a protein structure that can specifically bind to a target point on the surface of a tumor cell; The cell-penetrating structure is a protein structure that mediates the recombinant protein to penetrate the cell membrane and enter the cell interior; The amino acid sequence of the cell-penetrating structure is shown in SEQ ID NOs: 9 to 12; The tumor cell surface target is Trop2 or MSLN; The tumor cell surface targeting structure is a Fab antibody-2 that specifically binds to a cell surface target, and the Fab antibody-2 is connected to Fc; The Fab antibody-2 contains a heavy chain variable region VH-b and a light chain variable region VL-b; Wherein, VH-b comprises the amino acid sequence shown in SEQ ID NO: 25; VL-b comprises the amino acid sequence shown in SEQ ID NO: 30; and the cell-penetrating structure is connected to the N-terminus of VH-b or VL-b of the tumor cell surface targeting structure; Alternatively, VH-b comprises the amino acid sequence shown in SEQ ID NO: 29; VL-b comprises the amino acid sequence shown in SEQ ID NO: 34; and the cell-penetrating structure is connected to the Fc terminus or CL of the tumor cell surface targeting structure.

2. The recombinant protein according to claim 1, characterized in that The tumor cell surface targeting structure and the cell-penetrating structure are connected via a connecting peptide; The connecting peptide includes at least one of GGGGSGS, GGGGSGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, AKTTPKLEEGEFSEAR, AKTTPKLEEGEFSEARV, RADAAAAGGPGS, ADAAPTVSIFPP, AKTTPPSVTPLAP, and GHEAAAVMQVQYPAS.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the recombinant protein according to claim 1 or 2 and a pharmaceutically acceptable excipient.

4. Use of the pharmaceutical composition according to claim 3 in the preparation of a drug for treating tumors or a combination product, characterized in that: The tumor cell surface target is MSLN, and the tumor is selected from one of lung cancer, breast cancer, ovarian cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, bile duct cancer, cervical cancer, and esophageal cancer; or, The target on the tumor cell surface is Trop2, and the tumor is selected from one of lung cancer, liver cancer, breast cancer, ovarian cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, bladder cancer, bile duct cancer, nasopharyngeal cancer, cervical cancer, and esophageal cancer.

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