WO2022057942A1 - 一种药物组合及其应用 - Google Patents
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Classifications
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
Definitions
- the invention relates to the field of biomedicine, in particular to a drug combination and application thereof, in particular to the application in the preparation of a therapeutic drug for diseases with abnormally elevated blood IgG levels.
- Immunoglobulins include IgA, IgD, IgE, IgM and IgG, of which gamma immunoglobulin IgG excess is the most common.
- M protein M-Protein
- M-Protein is a large amount of abnormal immunoglobulin produced by the monoclonal malignant proliferation of plasma cells or B lymphocytes. Its essence is an immunoglobulin or immunoglobulin fragment, also known as monoclonal protein, monoclonal immunoglobulin, myeloma protein, or M-spike.
- M protein positivity is seen in: malignant monoclonal gammopathy, such as multiple myeloma, heavy chain disease, malignant lymphoma, chronic lymphocytic leukemia, macroglobulinemia, etc.; secondary monoclonal gammopathy , such as non-lymphoreticular tumors, monocytic leukemia, cryoglobulinemia, etc.; benign M proteinemia, the incidence of elderly people over 70 years old is about 3%.
- the purpose of the present invention is to reduce the influence of globulin on treatment by combining immunoglobulin degrading enzymes with Fc agents for hyperglobulinemia, so that Fc agents can play a better role, and at the same time
- the clinical dose of some Fc agents can also be reduced, which can reduce the side effects of the therapeutic agent on the one hand, and reduce the economic burden of medication on the other hand.
- “Comprising at least" the function of the immunoglobulin degrading enzyme IdeE means that the mutant may contain other functions on the basis of retaining the immunoglobulin degrading enzyme IdeE, or that the mutant is more efficient in cleaving human IgG than IdeS , or the mutant was as effective as IdeS in cleaving human IgG.
- the IgG degrading enzyme comprises the amino acid sequence shown in Table 1 or a variant thereof having the same function or is a protein consisting of the amino acid sequence or a fragment thereof.
- the IgG degrading enzyme comprises the amino acid sequence shown in Table 1, and may further comprise: 1) amino acid sequences SEQ ID NOs: 1-42, 59, 60, 61-95; 2) variants thereof, the The variants are at least 50% identical to the amino acid sequences SEQ ID NOs: 1-42, 59, 60, 61-95 and possess an IgG degrading enzyme; or fragments of 1) and 2) having IgG degrading enzyme activity.
- the mutant comprises any amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the sequence shown in SEQ ID NO: 5 or consists of any such amino acid sequence. Sequence composition and has IgG cysteine protease activity.
- Modified immunoglobulin degrading enzymes modified by a modification means selected from the group consisting of glycosylation, sialylation, albuminylation, farnesylation, carboxylation, hydroxylation, phosphorylation and conjugation to polymers.
- a modified immunoglobulin degrading enzyme wherein the modified polypeptide is modified by conjugation to dextran, PEG, a multimerization domain, a toxin, a detectable label, or a drug.
- Modified immunoglobulin degrading enzymes which are HSA fusion proteins or Fc fusion proteins.
- Fc agent that is, an Fc-containing protein or preparation, the following is an example of an Fc-containing protein
- the pharmaceutical combination as described above, wherein the Fc-containing protein is selected from one of antibodies, immunoglobulins, immunoadhesins, therapeutic agents, diagnostic agents, imaging agents and antibody drug conjugates or variety.
- the pharmaceutical combination as described above wherein the Fc-containing protein is directed against the group consisting of IL1 ⁇ , IL2, IL2R, IL4, IL6, IL10, IL11, IL12, IL23, C5, BAFF, BLyS, BCMA, CXCR-4 , ICAM-1, SLAMF7/CS1, TNF ⁇ , IgE, B7.1/B7.2 antibodies.
- An antibody that specifically recognizes CD38 has binding specificity to human effector cells, and/or the antibody is capable of killing by apoptosis, and/or antibody-dependent cell-mediated cytotoxicity, and/or complement-dependent cytotoxicity cells expressing CD38.
- the immunoconjugate of the CD38 antibody wherein the antibody is a monomeric IgM antibody linked to a cytotoxic agent, radioisotope or drug.
- the CD38 antibody has binding specificity to CD3, CD4, CD138, IL-15R, membrane-bound or receptor-bound TNF- ⁇ , human Fc receptor, or membrane-bound or receptor-bound IL-15.
- the CD38 antibody is characterized in that the antibody comprises at least one heavy chain and at least one light chain, wherein the light chain comprises the amino acid sequences of SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45.
- the light chain comprises the amino acid sequence shown in SEQ ID NO.55 or 57
- the heavy chain comprises the amino acid sequence shown in SEQ ID NO.56 or 58;
- amino acid sequence of the light chain is shown in SEQ ID NO.55, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.56; or the amino acid sequence of the light chain is shown in SEQ ID NO.56. 57, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.58.
- compositions described above also include chemotherapeutic agents, hormonal agents, and targeted small molecule agents.
- the combined use is capable of immunogenic cell death, enhancement of tumor cell antigenicity or susceptibility to immune cells, suppression of Treg cells with negative regulation and myeloid derived suppressor cells (MDSCs).
- MDSCs myeloid derived suppressor cells
- the chemotherapeutic drugs are selected from immunosuppressants, proteasome inhibitors, cytotoxic drugs and cell cycle non-specific drugs; the immunosuppressants such as cyclophosphamide, thalidomide, pomalidomide, lenalidomide; The proteasome inhibitor such as bortezomib; the cytotoxic drugs such as gemcitabine, temozolomide, cisplatin; the cell cycle non-specific drugs such as milphalan, mitoxantrone, vincristine, doxorubicin.
- immunosuppressants such as cyclophosphamide, thalidomide, pomalidomide, lenalidomide
- the proteasome inhibitor such as bortezomib
- the cytotoxic drugs such as gemcitabine, temozolomide, cisplatin
- the cell cycle non-specific drugs such as milphalan, mitoxantrone, vincristine, doxorubicin.
- Such hormonal agents are eg dexamethasone, prednisone.
- the targeted small molecule preparation is selected from epigenetic drugs, inhibitors targeting PI3K/Akt/mTOR signaling pathway and tyrosine kinase inhibitors.
- Inhibitors of tyrosine kinases have multiple effects of inhibiting tumor angiogenesis and anti-tumor cell growth.
- gefitinib is an oral epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor. Inhibition of EGFR-TK hinders tumor growth, metastasis, and angiogenesis, and increases tumor cell apoptosis.
- Also included are other therapeutic agents including pamidronate, zoledronic acid, clodronate, risedronate, ibandronate, etidronate, alendronate, tiludronate Phosphonates, arsenic trioxide, filgrastim, pegylated filgrastim, sagrastim, suberoylanilide hydroxamic acid and SCIO-469, all-trans retinoic acid (ATRA), arabinoside Cytidine, chlorambucil, vascular endothelial growth factor inhibitor, CD40-binding immunosuppressive antibody, TOR inhibitor, IL-6-binding immunosuppressive antibody, IGF-IR inhibitor.
- ATRA all-trans retinoic acid
- therapeutic agents include analgesics, anti-inflammatory agents, antimicrobial agents, anti-amebic agents, trichomicides, anti-Parkinsonian agents, anti-dysentery agents, anti-convulsants, anti-depressants, anti- Rheumatic, antifungal, antihypertensive, antipyretic, antiparasitic, antihistamine, alpha-adrenergic agonist, alpha blocker, anesthetic, bronchodilator, biocide, bactericide , bacteriostatic agents, beta-adrenergic blockers, calcium channel blockers, cardiovascular agents, contraceptives, decongestants, diuretics, sedatives, diagnostic reagents, electrolyte reagents, hypnotics, hormones, blood sugar-promoting agents, Muscle relaxants, muscle contractions, ophthalmic agents, parasympathomimetic agents, psychostimulants, tranquilizers, sympathomimetic agents, tranquilizers, urinar
- compositions for parenteral administration are sterile, substantially isotonic, pyrogen-free, and are prepared in accordance with FDA or similar agency GMP.
- the viral vector drug can be administered as an injectable dosage form of a solution or suspension of the substance in a physiologically acceptable diluent and a pharmaceutical carrier (which can be a sterile liquid such as water, oil, saline, glycerol, etc.) or ethanol).
- auxiliary substances such as wetting or emulsifying agents, surfactants, pH buffering substances, and the like may be present in the composition.
- Other components of the pharmaceutical composition are those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil and mineral oil.
- glycols such as propylene glycol or polyethylene glycol are the preferred liquid carriers, especially for injectable solutions.
- the Fc-containing protein can be administered in the form of a depot injection or implant formulation that can be formulated to allow sustained release of the active ingredient.
- compositions are prepared as injectables, either liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
- the present invention also provides a product containing reagents for reducing blood IgG levels, including immunoglobulin degrading enzymes and Fc-containing proteins, as a combined preparation for simultaneous, separate or sequential use in the treatment and/or treatment of hyperglobulinemia or infection prevention.
- the combined formulation is used in a method of treating hyperglobulinemia.
- the present invention also provides a kit or kit for preventing or treating hyperglobulinemia, the kit comprising: 1) a therapeutically effective amount of an immunoglobulin degrading enzyme; and 2) a therapeutically effective amount of Fc-containing protein.
- the kit may also include other active ingredients in addition to 1) and 2).
- the kit may contain instructions relating to the administration of a therapeutically effective amount of a drug for reducing blood immunoglobulin levels and a therapeutically effective amount of a viral vector drug (eg, dosage information, dosing interval information).
- the present invention also provides a composition such as, but not limited to, a package and a kit, a kit or a kit for preventing or treating globulinemia, the kit comprising: 1) an immunoglobulin degrading enzyme; and 2) a therapeutically effective amount of a pharmaceutically active agent comprising a CD38 antibody; and 3) a label with instructions for performing the methods disclosed herein.
- 1) and 2) are in separate or the same container.
- any one of the modified immunoglobulin degrading enzymes in combination with an Fc-containing protein for the manufacture of a medicament for the treatment of hyperglobulinemia.
- the use of the modified immunoglobulin degrading enzyme of any one and the Fc-containing protein combination for the manufacture of a medicament for the treatment of multiple myeloma.
- Hyperglobulinemia abnormally elevated IgG is characterized by higher than normal levels of immunoglobulins in patients, including IgA, IgD, IgE, IgM and IgG, of which gamma immunoglobulin IgG is the most common excess.
- Excessive immunoglobulins are produced by leukocytes selected from B cells, abnormal B cells, IgM-producing cells, IgE-producing cells, and IgA-producing cells; the globulins include gamma globulin; the hyperglobulin Proteinemia, including malignant monoclonal gamma globulinemia (such as multiple myeloma, heavy chain disease, malignant lymphoma, chronic lymphocytic leukemia, macroglobulinemia, etc.), primary monoclonal gamma globulinemia disease, secondary monoclonal gammopathy (such as non-lymphoreticular tumors, monocytic leukemia, cryoglobulinemia, etc.), benign M proteinemia, monoclonal gammopathy of undetermined significance ( MGUS), Waldenström macroglobulinemia, AL amyloidosis, solitary plasmacytoma (osseous or extraosseous), POMES syndrome, reactive plasmacytosis, osteolytic lesions of meta
- connective tissue diseases such as systemic lupus erythematosus, rheumatoid arthritis, scleroderma, Sjögren's syndrome, etc.; liver disease, chronic viral active hepatitis, cryptic cirrhosis, lupus-like hepatitis, etc.; infectious diseases, Tuberculosis, leprosy, kala-azar, infectious mononucleosis, venereal disease, lymphogranuloma, actinomycosis, malaria, trypanosomiasis, etc.; others, sarcoidosis, myasthenia gravis, Hodgkin's disease, monocytic Leukemia, Behcet's disease, nephritis, allergic purpura, immune (or spontaneous) thrombocytopenia, etc.
- connective tissue diseases such as systemic lupus erythematosus, rheumatoid arthritis, scleroderma,
- CD38 is a therapeutic target for multiple myeloma and other hyperglobulinemic hematological tumors.
- CD38-related cancers or autoimmune diseases also include leukemias and lymphomas, such as B-cell chronic lymphocytic leukemia, T- and B-cell acute lymphoblastic leukemia, Waldenstrom's macroglobulinemia, primary systemic amyloidosis, mantle cells Lymphoma, prolymphocytic/myeloid leukemia, acute myeloid leukemia, chronic myeloid leukemia, follicular lymphoma, Burkitt lymphoma, large granular lymphocyte (LGL) leukemia, NK cell leukemia and plasma cell leukemia, Diffuse Large B-Cell Lymphoma (DLBCL), Follicular Lymphoma (FL), Mantle-Cell Lymphoma (MCL), Chronic Lymphocytic Leukemia Leukemia, CLL), acute myeloid leukemia (Acute My
- Multiple myeloma is a blood tumor that results from the canceration of plasma cells in the bone marrow.
- Fc agents targeting CD38 including but not limited to daratumumab, Isatuximab, MOR202, and the like.
- a pharmaceutical combination in the manufacture of a medicament for a method for inhibiting the growth and/or proliferation of cells expressing CD38, the method comprising administering the pharmaceutical composition, thereby inhibiting the growth and/or proliferation of the cells.
- a pharmaceutical combination in the manufacture of a medicament for a method for treating a disease or condition in a patient involving CD38-expressing cells, the method comprising administering the pharmaceutical composition to a patient in need of treatment.
- a pharmaceutical combination in the manufacture of a medicament for a method for preventing a disease or condition involving CD38-expressing cells in a patient, the method comprising administering the pharmaceutical composition to a patient in need of treatment.
- SLE systemic lupus erythematosus
- the hyperglobulinemia is selected from the following conditions or the group consisting of: including malignant monoclonal agammaglobulinemia (such as multiple myeloma, heavy chain disease, malignant lymphoma, chronic lymphocytic leukemia, macroglobulinemia blood, etc.), secondary monoclonal gamma globulinemia (such as non-lymphoreticular tumors, monocytic leukemia, cryoglobulinemia, etc.), benign M proteinemia, monoclonal gamma globulin of undetermined significance Proteinopathy (MGUS), Waldenström macroglobulinemia, AL amyloidosis, solitary plasmacytoma (osseous or extraosseous), POMES syndrome, reactive plasmacytosis, osteolytic lesions of metastatic carcinoma , plasmablastic lymphoma, monoclonal immunoglobulin-related renal damage (MGRS) and so on.
- the disorder is preferably multiple myeloma.
- the immunoglobulin degrading enzyme When the immunoglobulin degrading enzyme is administered before the administration of the Fc agent, before administration of the immunoglobulin degrading enzyme, after administration of the agent and before administration of the Fc agent, administration of the Fc agent Afterwards, the level of immunoglobulin, especially M protein, in the blood of the subject is quantitatively detected.
- the immunoglobulin degrading enzyme When the immunoglobulin degrading enzyme is administered after the administration of the Fc agent, before administration of the Fc agent, after administration of the Fc agent and before administration of the immunoglobulin degrading enzyme, the immunoglobulin After administration of the proteolytic enzyme, the level of immunoglobulins, particularly M protein, in the blood of the subject is quantitatively detected.
- the immunoglobulin degrading enzyme is present in the Fc agent as a combined preparation for simultaneous, separate or sequential use.
- the method comprises the steps of: 1) administering the immunoglobulin degrading enzyme to a subject; subsequently, 2) administering the Fc agent to the subject.
- the Fc agent Preferably, there is a time interval between the administration of the immunoglobulin degrading enzyme and the Fc agent.
- the method comprises the steps of: 1) administering the Fc agent to the subject; subsequently, 2) administering the immunoglobulin degrading enzyme to the subject.
- the method comprises the steps of: 1) administering the Fc agent to the subject; subsequently, 2) administering the immunoglobulin degrading enzyme to the subject.
- the immunoglobulin degrading enzyme is administered in an amount and at intervals sufficient to reduce the immunoglobulin level in the subject to 60% of the starting level. More preferably, the agent is administered in an amount and at a time interval sufficient to reduce binding of the immunoglobulin level in the subject to 50%, 40%, 30%, 20% or 10% of the starting level in the patient.
- the agent can be administered at a single point in time or over a set period of time.
- the agent and oncolytic virus or viral vaccine can be administered by any suitable route.
- the immunoglobulin degrading enzyme is administered by intravenous infusion or subcutaneous injection, and/or the amount of the agent administered is 0.01 mg/kg body weight to 2 mg/kg body weight, 0.04 to 2 mg/kg body weight, 0.12 mg/kg body weight to 2 mg/kg body weight, 0.24 mg/kg body weight to 2 mg/kg body weight or 1 mg/kg body weight to 2 mg/kg body weight.
- the administration time interval of the immunoglobulin degrading enzyme and the Fc agent is 30 minutes to 1 hour, 30 minutes to 2 hours, 30 minutes to 3 hours, 30 minutes to 4 hours, 30 minutes to 30 minutes 5 hours, 30 minutes to 6 hours, 1 to 2 hours, 1 to 3 hours, 1 to 4 hours, 1 to 5 hours, 1 to 6 hours, 2 to 3 hours, 2 to 4 hours, 2 to 5 hours, 2 to 6 hours, 3 to 4 hours, 3 to 5 hours, 3 to 6 hours, 4 to 5 hours, 4 to 6 hours, or 5 to 6 hours.
- the method comprises the steps of: 1) ex vivo processing blood from the subject with the immunoglobulin degrading enzyme; 2) returning the blood to the subject; 3 ) administering the Fc agent to the subject.
- the method comprises the steps of: 1) administering the Fc agent to the subject; 2) subjecting blood from the subject ex vivo with the immunoglobulin degrading enzyme processing; 3) returning the blood to the subject.
- the immunoglobulin degrading enzyme and Fc agent are used to treat hyperglobulinemia.
- the immunoglobulin degrading enzyme and Fc agent are used to prevent hyperglobulinemia.
- both the immunoglobulin degrading enzyme and the Fc agent administration can be administered simultaneously, separately or sequentially for the treatment and prevention of hyperglobulinemia.
- the immunoglobulin degrading enzyme and the Fc agent can be provided as separate formulations or as a combined formulation.
- Figure 8 is an SDS-PAGE gel electrophoresis image of cleavage products produced by cleavage of human IgG1 by E97D_del18 mutant and IdeS at different concentrations.
- Figure 9 is an SDS-PAGE gel electrophoresis image of cleavage products produced by E97D_del18 mutant and IdeZ cleavage of human IgG1 at different concentrations.
- Figure 12 is an SDS-PAGE gel electrophoresis image of cleavage products produced by the E97D_del18 mutant in mouse and human serum.
- Figure 13 is an SDS-PAGE gel electrophoresis image of the cleavage products produced by E97D_del18 cleaving human IVIg at different times in mice.
- Figure 14 shows the inhibition of antibody-suppressed tumor by immunoglobulin-degrading enzyme-reducing IgG in the Daudi cell tumor model.
- Daratumumab is harvested from cell culture fluids using immobilized Protein A affinity chromatography, cation exchange chromatography (e.g. SP Sepharose FF), a filtration step to remove viral contamination, followed by hydrophobic chromatography (e.g. Butyl Sepharose HP) and ultrafiltration exchange steps for purification, to prepare pharmaceutical compositions according to the examples, daratumumab was provided at a concentration of about 100 mg/mL in 20 mM histidine buffer (pH about 6.0). Isatuximab was prepared in the same way.
- Entrusted Nanjing GenScript Biotechnology Co., Ltd. to synthesize the polynucleotide sequences encoding SEQ ID NO: 1 to SEQ ID NO: 5 after codon optimization by Escherichia coli, and added N-terminal methionine and C-terminal 6 ⁇
- the histidine tag was inserted into the pET32a expression vector after the sequence was synthesized, and the recombinant plasmid for polypeptide expression was obtained after the sequence was correct.
- the prepared recombinant plasmids were electrotransformed into Escherichia coli BL21 Star (DE3) and plated on LB agarose plates containing 100 ⁇ g/mL ampicillin. Incubate overnight at 37°C until colonies grow.
- the supernatant was collected by centrifugation, and the supernatant was purified by IDA-Ni agarose magnetic beads. After purification, the eluted protein was changed into a PBS buffer system with an ultrafiltration centrifuge tube. SDS-PAGE was used to assess the purity of each protein.
- IdeE T8D (SEQ ID NO: 63), T8W (SEQ ID NO: 65), T24A (SEQ ID NO: 69), A59L (SEQ ID NO: 73), A59V (SEQ ID NO: 69) were synthesized according to the method of Example 1 74), E97D (SEQ ID NO: 75) and R280H (SEQ ID NO: 76), these 7 single point mutants cleavage the activity of human IgG1.
- the cleavage effect of 7 single-point mutants at 0.001mg/ml concentration of IgG1 is not worse than that of 0.002mg/ml wild-type IdeE, indicating that the cleavage activity of 7 single-point mutants is not lower than that of wild-type IdeE. times.
- the mutant polynucleotide sequences in Table 3 were synthesized according to the method of Example 1, the mutant expression recombinant plasmid was constructed, E. coli BL21 Star (DE3) was transformed, and the mutant purified protein was prepared.
- the purified mutant or wild-type IdeE were diluted to 0.002 mg/mL, respectively. 50 ⁇ l of the diluted mutant or wild-type IdeE was taken and added to 50 ⁇ l of the reaction system containing 2 mg/ml trastuzumab to start the cleavage reaction, and the reaction system was placed at 37° C. for 30 minutes. The samples were mixed with the same volume of 2 ⁇ SDS loading buffer, and then water bathed at 75°C for 5 minutes, and the cleavage products were detected by SDS-PAGE.
- the cleavage activity of the five truncation mutants was not significantly different from that of the wild-type IdeE.
- mutant polynucleotide sequences in Table 4 were synthesized according to the method of Example 1, the mutant expression recombinant plasmid was constructed, transformed into Escherichia coli BL21 Star (DE3), and the mutant purified protein was prepared.
- the purified mutant or wild-type IdeE were diluted to 0.002 mg/mL, respectively. 50 ⁇ l of the diluted mutant or wild-type IdeE was taken and added to 50 ⁇ l of the reaction system containing 2 mg/ml trastuzumab to start the cleavage reaction, and the reaction system was placed at 37° C. for 30 minutes. The samples were mixed with the same volume of 2 ⁇ SDS loading buffer, and then water bathed at 75°C for 5 minutes, and the cleavage products were detected by SDS-PAGE.
- the cleavage activity of the two truncation mutants was more than 2 times higher than that of the wild-type IdeE.
- the mutant polynucleotide sequence in Table 5 was synthesized according to the method of Example 1, the mutant expression recombinant plasmid was constructed, E. coli BL21 Star (DE3) was transformed, and the mutant purified protein was prepared.
- Figure 13 shows electropherograms of cleavage products produced by E97D_del18 cleaving human IVIg at different times in mice. The results showed that E97D_del18 had a significant effect on cleaving IVIg in mice, and the cleavage was basically reached within 15 minutes.
- a single intraperitoneal injection of 1 g/kg IVIg was administered on the day of cell inoculation.
- a single intravenous injection of 5 mg/kg immunoglobulin degrading enzyme E97D_del18 was administered 24 h later.
- 10 days after IVIg administration 10 mg/kg anti-CD38 monoclonal antibody was administered intraperitoneally, once a week, for a total of 4 administrations. After CD38 administration was started, tumor volume was measured twice a week for a total of 4 weeks.
- the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented.
- Those skilled in the art should understand that any improvement to the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
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Abstract
提供一种药物组合及其应用。所述的药物组合用于制备治疗高球蛋白血症的药物,其包含免疫球蛋白降解酶和含有Fc的蛋白,所述药物组合通过将免疫球蛋白降解酶与Fc剂联合使用于高球蛋白血症,降低球蛋白对治疗的影响,使得Fc剂可更好的发挥作用,同时也可降低部分Fc剂的临床使用剂量,一方面可以减轻治疗剂的副作用,另一方面也减轻用药经济负担。
Description
本发明涉及生物医药领域,具体涉及一种药物组合及其应用,特别是在制备血液IgG水平异常升高疾病的治疗药物制备中的应用。
高球蛋白血症表现为患者体内具有高于正常水平的免疫球蛋白。免疫球蛋白包括IgA、IgD、IgE、IgM和IgG,其中γ免疫球蛋白IgG过量最为常见。M蛋白(M-Protein)是浆细胞或B淋巴细胞单克隆恶性增殖所产生的一种大量的异常免疫球蛋白,其本质是一种免疫球蛋白或免疫球蛋白的片段,又叫做monoclonal Protein、monoclonal immunoglobulin、myeloma protein,或者M-spike。
因其多见于多发性骨髓瘤(multiple myeloma)、巨球蛋白血症(macroglobulinemia)及恶性淋巴瘤(malignant lymphoma),都是以M开头的疾病,故称为“M蛋白”。M蛋白阳性见于:恶性单克隆丙种球蛋白血症,如多发性骨髓瘤、重链病、恶性淋巴瘤、慢性淋巴细胞白血病、巨球蛋白血症等;继发性单克隆丙种球蛋白血症,如非淋巴网状系统肿瘤、单核细胞白血病、冷球蛋白血症等;良性M蛋白血症,70岁以上的老年人发生率约为3%。
多发性骨髓瘤是由于骨髓中的浆细胞癌变而导致的血液肿瘤。异常浆细胞在骨髓中聚集,在身体多处骨骼产生肿瘤。这些细胞不但不能行使正常功能,它们还会导致骨髓无法生成健康的血细胞。骨髓浆细胞异常增生伴有无功能的单克隆免疫球蛋白或轻链(M蛋白)过度生成,这一异常免疫球蛋白在多数患者体内水平可高达30g/L或更高(正常人血液IgG水平在10g/L左右),大约50%患者的M-蛋白呈IgG亚型。目前治疗多发性骨髓瘤的抗体包括达雷妥尤单抗、伊沙妥昔单抗(isatuximab)。本发明人意外的发现,通过免疫球蛋白降解酶预先清除病理性免疫球蛋白,可以降低单抗治疗量的同时增强抗体治疗效果。
发明内容
针对现有技术的不足,本发明的目的在于通过将免疫球蛋白降解酶与Fc剂联合使用于高球蛋白血症,降低球蛋白对治疗的影响,使得Fc剂可更好地发挥作用,同时也可降低部分Fc剂的临床使用剂量,一方面可以减轻治疗剂的副作用,另一方面也减轻用药经济负担。
本发明的目的,将通过以下技术方案得以实现:
1.药物组合
第一方面,提供一种药物组合,所述药物组合可用于治疗高球蛋白血症药物的制备,其中,所述药物组合包含:1)免疫球蛋白降解酶,和2)含有Fc的蛋白。其中,其中所述药物组合允许所述降解酶和所述免疫球蛋白的单独给药。优选地,所述药物组合包含治疗有效量的所述免疫球蛋白降解酶和免疫球蛋白。优选地,所述药物组合是药物组合物,并且还包含药学上可接受的载剂或稀释剂。其为冻干粉末或为液体。
1.1免疫球蛋白降解酶
本文所用术语“免疫球蛋白降解酶”优选是指将血液免疫球蛋白水平降至原水平60%或以下的蛋白酶药物或试剂,其氨基酸序列通常来源于细菌,或者是包含部分细菌来源序列。优选的是,该药物或试剂将血液免疫球蛋白降到至多原水平60%、至多原水平50%、至多原水平40%、至多原水平30%、至多原水平20%、至多原水平10%或者至多原水平0%。更优选的是,该药物或试剂将血液免疫球蛋白降到至多原水平20%、至多原水平10%或至多原水平0%。
该试剂通常为蛋白质,且具有IgG半胱氨酸蛋白酶活性的蛋白质,优选酶切免疫球蛋白分子的铰链区。这种蛋白质的一个例子是IdeE(化脓性链球菌的免疫球蛋白G降解酶)。IdeE是具有独特特异性程度的链球菌蛋白酶;它酶切免疫球蛋白G(IgG)抗体,但不将其任何其他底物(包括IgA、IgD、IgE和IgM)。IdeE在铰链区的COOH末端的特定位点将人IgG酶切为F(ab’)
2片段和Fc片段。IdeE的成熟序列见SEQ ID NO:5。该试剂可为包含SEQ ID NO:5的氨基酸序列的蛋白质或由该序列组成的蛋白质,或可为来自替代性细菌的其同源物。
优选地,如上所述的药物组合,其中,所述免疫球蛋白降解酶为IgG降 解酶,所述IgG降解酶选自化脓性链球菌的IgG半胱氨酸蛋白酶或其变体或片段或者人来源MMP蛋白酶或其变体或片段,所述变体或片段保持了酶切IgG的活性;优选地,所述IgG降解酶选自IdeS、MAC2、IdeZ、IdeZ2、IdeE、IdeE2、IdeP、MMP。
IdeE蛋白质的变体可包含这样的氨基酸序列或由该序列组成:该氨基酸序列相对SEQ ID NO:5氨基酸序列可具有1、2、3、4、5、10、20、30或更多个氨基酸取代、插入或缺失,条件是该变体具有IgG半胱氨酸蛋白酶活性。所有氨基酸取代优选是保守性的。保守性取代将氨基酸替换为其他具有相似化学结构、相似化学性质或相似侧链体积的氨基酸。所引入的氨基酸可与被取代的氨基酸具有相似极性、亲水性、疏水性、碱性、酸性、中性或电荷。或者,保守性取代可在原先存在的芳香族或脂肪族氨基酸位置处引入另一个芳香族或脂肪族氨基酸。保守性氨基酸改变在本领域是熟知的。
IdeE蛋白质的变体,至少对免疫球蛋白降解酶IdeE所述氨基酸序列的位置8、10、24、59、97和280中的一位或者多位进行替换后得到所述突变体;所述的突变体的功能至少包含所述免疫球蛋白降解酶IdeE的功能。
“至少包含”免疫球蛋白降解酶IdeE的功能指的是所述突变体在保留免疫球蛋白降解酶IdeE的基础上还可含有其他功能,或者所述突变体在切割人IgG上比IdeS更有效,或者所述突变体在切割人IgG上与IdeS一样有效。
或者免疫球蛋白降解酶可为包含SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3的片段的蛋白质或由该片段组成,并且具有IgG半胱氨酸蛋白酶活性,优选地,其中所述片段的长度为100至300个氨基酸、150至300个氨基酸或200至300个氨基酸。该片段可通过缺失SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3的氨基酸序列的一个或多个氨基酸残基来产生。可缺失至多1、2、3、4、5、10、20、30、40或50个残基,或更多个残基。所缺失的残基可彼此连续。
优选地,所述IgG降解酶包含如表1的氨基酸序列或其具有相同功能的变体或为由所述氨基酸序列组成的蛋白质或其片段。在一个实施方式中,所述IgG降解酶包含如表1的氨基酸序列,还可包括:1)氨基酸序列SEQ ID NO:1~42、59、60、61~95;2)其变体,该变体与氨基酸序列SEQ ID NO:1~42、59、60、61~95具有至少50%的同一性并具有IgG降解酶;或1)和2)的具有IgG降解酶活性的片段。
表1.免疫球蛋白降解酶序列
本发明所述的突变体优选通过基因工程重组方式生产得到。
较佳地,所述突变体包含与SEQ ID NO:5所示序列至少有70%、75%、80%、85%、90%、95%或99%同一性的任何氨基酸序列或由任何该序列组 成,并且具有IgG半胱氨酸蛋白酶活性。
修饰的免疫球蛋白降解酶,其通过选自糖基化、唾液酸化、白蛋白化、法尼基化、羧化、羟基化、磷酸化和缀合至聚合物的修饰方式而修饰。
修饰的免疫球蛋白降解酶,其中所述修饰多肽通过与葡聚糖、PEG、多聚化结构域、毒素、可检测标记或药物缀合而修饰。
修饰的免疫球蛋白降解酶,其是HSA融合蛋白或Fc融合蛋白。
1.2 Fc剂(即为含Fc的蛋白或者制剂,下面以含有Fc的蛋白为例进行说明)
优选地,如上所述的药物组合,其中,所述含有Fc的蛋白选自抗体、免疫球蛋白、免疫粘附素、治疗剂、诊断剂、成像剂和抗体药物缀合物中的一种或多种。
优选地,如上所述的药物组合还包含白细胞消耗剂。其中的白细胞优选B细胞。所述的白细胞消耗剂优选与CD4、CD10、CD19、CD20、CD21、CD22、CD23、CD24、CD37、CD53、CD69、CD70、CD72、CD74、CD75、CD77、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、CD85、CD86、HLA-DRA、TNFRSF5、TNFRSF6、TNFSF5、TNFSF6、BLR1、HDAC4、HDAC5、HDAC7A、HDAC9、ICOSL、IGBP1、MS4A1、RGS1、SLA2、CD81、IFNB1、IL10、TNFRSF5、TNFRSF7、TNFSF5、AICDA、BLNK、GALNAC4S-6ST、HDAC4、HDAC5、HDAC7A、HDAC9、IL4、INHA、INHBA、KLF6、TNFRSF7、DPP4、FCER2、IL2RA、TNFRSF8、TNFSF7、CR2、IL1R2、ITGA2、ITGA3、MS4A1、ST6GAL1、CD1C、CHST10、HLA-A、HLA-DRA或者NT5E特异性结合的抗体。
优选地,如上所述的药物组合,其中所述含有Fc的蛋白是针对选自CD3、CD11a、CD14、CD25、CD28、CD30、CD33、CD36、CD38、CD40、CD44、CD52、CD55、CD59、CD56、CD103、CD134、CD137、CD138、CD152、CD3、IL-1β、IL-2R、IL-6、IL-12、IL-23、C5、BAFF、BLyS、BCMA、CXCR-4、ICAM-1、SLAMF7/CS1、TNFα和IgE的抗体。
优选地,如上所述的药物组合,其中所述含有Fc的蛋白是针对选自IL1β、IL2、IL2R、IL4、IL6、IL10、IL11、IL12、IL23、C5、BAFF、BLyS、BCMA、CXCR-4、ICAM-1、SLAMF7/CS1、TNFα、IgE、B7.1/B7.2的抗体。
优选地,如上所述的药物组合,其中所述含有Fc的蛋白为利妥昔单抗 (CD20)、达克珠单抗(IL2R)、巴利昔单抗(IL2R)、莫罗单抗-CD3、英夫利昔单抗(TNFα)、阿达木单抗(TNFα)、奥马珠单抗(IgE)、依法利珠单抗(CD11a)、那他珠单抗(Integrinα4)、托珠单抗(IL6)、依库珠单抗(C5)、戈利木单抗(TNFα)、卡那单抗(IL1β)、优特克单抗(IL12/IL23)、贝利木单抗(BLyS)、西妥昔单抗、贝伐单抗、Elotuzumab(SLAMF7),或其组合。
特异性识别CD38的抗体与人效应细胞有结合特异性,和/或所述抗体能够通过凋亡、和/或抗体依赖性细胞介导的细胞毒性、和/或补体依赖性细胞毒性来杀死表达CD38的细胞。
所述的CD38抗体的免疫连接物,其中所述抗体是与细胞毒素药剂、放射性同位素或药物连接的单体IgM抗体。
所述的CD38抗体,与CD3、CD4、CD138、IL-15R、膜结合或受体结合的TNF-α、人Fc受体、或膜结合或受体结合的IL-15有结合特异性。
所述的CD38抗体,选自达雷妥优单抗、isatuximab、MOR202、TAK-079、TAK-573、SAR442085、TAK-169、T-007、AMG424、GBR1324、Hexabody-CD38、TSK011010、STI-5171、anti-CD38/IGF-1R bsAb、Anti-CD38SIFbody、Actinium-225 dara、STI-6129、Anti-CD38/anti-CD3、CD38 TCE和Y-150。
所述的CD38抗体,其特征在于所述抗体包含至少一条重链和至少一条轻链,其中所述轻链包含SEQ ID NO:43、SEQ ID NO:44和SEQ ID NO:45的氨基酸序列所示的三个连续互补性决定区,或者如SEQ ID NO:49~51的氨基酸序列所示的三个连续互补性决定区,其中所述重链包含SEQ ID NO:46~48的氨基酸序列所示的三个连续互补性决定区,或者如SEQ ID NO:52、SEQ ID NO:53和SEQ ID NO:54的氨基酸序列所示的三个连续互补性决定区;其中所述表位结合片段为Fab、Fab’和F(ab’)
2、scFv或二硫化物连接的Fv片段。
较佳地,所述的轻链包含如SEQ ID NO.55或57所示的氨基酸序列,所述重链包含如SEQ ID NO.56或58所示的氨基酸序列;
更佳地,所述轻链的氨基酸序列如SEQ ID NO.55所示,且所述重链的氨基酸序列如SEQ ID NO.56所示;或者所述轻链的氨基酸序列如SEQ ID NO.57所示,且所述重链的氨基酸序列如SEQ ID NO.58所示。
表2.抗体相关序列
所述含Fc的蛋白还包含结合选自以下的一个或多个免疫检查点靶标的抗体。所述免疫检查点选自CTLA-4、PD-1、TIM-3、LAG3、Siglec15、4-1BB、GITR、OX40、CD40L、CD28、TIGIT、VISTA;所述免疫检查点抗体选自抗CTLA-4抗体、抗PD-1抗体、抗TIM-3抗体、抗LAG3抗体、抗Siglec15抗体、抗4-1BB抗体、抗GITR抗体、抗OX40抗体、抗CD40L抗体、抗CD28抗体、抗TIGIT抗体、抗VISTA抗体。
1.3还包括的其他试剂
如上所述的药物组合物还包含化疗剂、激素试剂、靶向小分子制剂。所述联合使用能免疫原性细胞死亡、增强肿瘤细胞抗原性或对免疫细胞的易感 性、抑制具有负调控的Treg细胞和髓样来源的抑制细胞(MDSC)。
所述化疗药物选自免疫抑制剂、蛋白酶体抑制剂、细胞毒药物和细胞周期非特异性药剂;所述免疫抑制剂例如环磷酰胺、沙利度胺、泊马度胺、来那度胺;所述蛋白酶体抑制剂例如硼替佐米;所述细胞毒药物例如吉西他滨、替莫唑胺、顺铂;所述细胞周期非特异性药物例如米尔法兰、米托蒽醌、长春新碱、多柔比星。
所述激素试剂例如地塞米松、泼尼松。
所述靶向小分子制剂选自表观遗传学药物、靶向PI3K/Akt/mTOR信号通路的抑制剂和酪氨酸激酶抑制剂。酪氨酸激酶(PTKs)抑制剂具有抑制肿瘤血管生成和抗肿瘤细胞生长的多重作用。举例如吉非替尼是一种口服表皮生长因子受体酪氨酸激酶(EGFR-TK)抑制剂。对EGFR-TK的抑制可阻碍肿瘤的生长、转移和血管生成,并增加肿瘤细胞的凋亡。
还包含其它治疗性药剂,包括帕米膦酸盐、唑来膦酸、氯膦酸盐、利塞膦酸盐、伊班膦酸盐、依替膦酸盐、阿仑膦酸盐、替鲁膦酸盐、三氧化二砷、非格司亭、聚乙二醇化非格司亭、沙格司亭、辛二酰苯胺异羟肟酸和SCIO-469、全反式视黄酸(ATRA)、阿糖胞苷、苯丙氨酸氮芥、血管内皮生长因子抑制剂、与CD40结合的免疫抑制抗体、TOR抑制剂、与IL-6结合的免疫抑制抗体、IGF-IR抑制剂。
还包含其它治疗性药剂,包括表皮生长因子(EGF)、成纤维细胞生长因子(FGF)、肝细胞生长因子(HGF)、组织因子(TF)、蛋白质C、蛋白质S、血小板衍生生长因子(PDGF)、Heregulin、巨噬细胞-刺激蛋白(MSP)或血管内皮生长因子(VEGF)的拮抗剂,或者表皮生长因子(EGF)、成纤维细胞生长因子(FGF)、肝细胞生长因子(HGF)、组织因子(TF)、蛋白质C、蛋白质S、血小板衍生生长因子(PDGF)、Heregulin、巨噬细胞-刺激蛋白(MSP)或血管内皮生长因子(VEGF)的受体的拮抗剂。
还包含其它治疗性药剂,包括止痛剂、抗炎剂、抗微生物剂、抗阿米巴虫剂、杀毛滴虫剂、抗帕金森剂、抗痢疾剂、抗痉挛剂、抗抑郁剂、抗风湿剂、抗真菌剂、抗高血压剂、退热剂、抗寄生虫剂、抗组胺剂、α-肾上腺素激动剂、α封阻剂、麻醉剂、支气管扩张剂、杀生物剂、杀菌剂、抑菌剂、β肾上腺素封阻剂、钙通道封阻剂、心血管药剂、避孕药剂、解充血药剂、利尿剂、镇静剂、诊断试剂、电解质试剂、催眠药剂、激素、促血糖增高药 剂、肌松弛剂、肌收缩剂、眼用药剂、拟副交感神经药、心理兴奋剂、镇定剂、拟交感神经药、安定剂、泌尿剂、阴道用药剂、杀病毒剂、维生素药剂、非类固醇类抗炎药剂、血管紧张素转化酶抑制剂、多肽、蛋白质、核酸、药物、有机分子和促睡眠剂。
1.4组合物的载体或赋形剂
药物载剂可以是液体,并且药物组合物可以为溶液形式。液体载剂用于制备溶液、悬浮液、乳液、糖浆、酏剂和加压组合物。活性成分可以溶解或悬浮在药学上可接受的液体载剂中,例如水、有机溶剂、二者的混合物或药学上可接受的油或脂肪。
用于肠胃外施用的药物组合物是无菌的、基本上等渗的、无热原的,并根据FDA或类似机构的GMP来制备。病毒载体药物可以作为该物质的溶液或悬浮液的可注射剂型施用,其中,该物质处在生理上可接受的稀释剂和药物载剂(可以是无菌液体,例如水、油、盐水、甘油或乙醇)中。另外,组合物中可存在辅助物质,例如润湿剂或乳化剂、表面活性剂和pH缓冲物质等。药物组合物的其他组分有石油、动物、植物或合成来源的组分,例如花生油、大豆油和矿物油。通常,例如丙二醇或聚乙二醇等二醇是优选的液体载剂,对于可注射溶液尤其如此。含Fc的蛋白可以以积存注射剂或植入制剂的形式施用,这些形式能够被配制成允许活性成分持续释放。通常,将组合物制备成可注射物,即液体溶液或悬浮液;也可以制备成适合于在注射前溶解或悬浮在液体载质中的固体形式。
组合产品,其中所述免疫球蛋白固定于一个表面,所述药物活性试剂游离于溶液中。
1.5组合物的制备所得产品的应用
本发明还提供了一种产品,所述产品含有降低血液IgG水平的试剂包括免疫球蛋白降解酶和含Fc的蛋白,作为联合制剂同时、分开或依次应用于高球蛋白血症的治疗和/或感染的预防中。优选的是,所述联合制剂应用于治疗高球蛋白血症的方法中。
1.6试剂盒或套装药盒,保护除了免疫球蛋白降解酶、含Fc的蛋白之外的第 三种治疗用成分
本发明还提供了一种用于预防或治疗高球蛋白血症的试剂盒或套装药盒,所述试剂盒包含:1)治疗有效量的免疫球蛋白降解酶;和2)治疗有效量的含Fc的蛋白。所述试剂盒还可以包括1)和2)之外的其他活性成分。
所述套装药盒包括药盒A和药盒B,所述药盒A包括治疗有效量的降低血液免疫球蛋白水平的药物包括IgG降解酶,所述药盒B包括治疗有效量的病毒载体药物。所述套装药盒还可以包括药盒C。所述药盒C还可以包括1)和2)之外的其他活性成分。
该试剂盒可以包含涉及治疗有效量的降低血液免疫球蛋白水平的药物和治疗有效量的病毒载体药物施用(例如,剂量信息、给药时间间隔信息)的说明书。
本发明还提供了一种例如但不限于包装和试剂盒的组合物,用于预防或治疗球蛋白血症的试剂盒或套装药盒,所述试剂盒包含:1)免疫球蛋白降解酶;和2)治疗有效量的药物活性试剂,其包含CD38抗体;和3)带有用于执行本文公开的方法的指令的标签。在某些实施方案中,1)和2)在分开的或相同的容器中。
2.组合在制备治疗高球蛋白血症中的用途
在优选的实施方式中,任一项的修饰的免疫球蛋白降解酶与含Fc的蛋白组合用于制备治疗高球蛋白血症药物中的用途。
在优选的实施方式中,任一项的修饰的免疫球蛋白降解酶和含Fc的蛋白组合用于制备用于治疗多发性骨髓瘤的药物中的用途。
在优选的实施方式中,任一项的试剂盒或套装药盒用于制备治疗高球蛋白血症药物中的用途。
2.1高球蛋白血症
高球蛋白(IgG异常升高)血症表现为患者体内具有高于正常水平的免疫球蛋白,免疫球蛋白包括IgA、IgD、IgE、IgM和IgG,其中γ免疫球蛋白IgG过量最为常见。过量的免疫球蛋白由白细胞产生,所述白细胞选自B细胞、异常B细胞、产生IgM的细胞、产生IgE的细胞、产生IgA的细胞;所述球蛋白包括γ球蛋白;所述的高球蛋白血症,包含恶性单克隆丙种球蛋白血症(如多发性骨髓瘤、重链病、恶性淋巴瘤、慢性淋巴细胞白血病、巨 球蛋白血症等)、原发性单克隆丙种球蛋白血症、继发性单克隆丙种球蛋白血症(如非淋巴网状系统肿瘤、单核细胞白血病、冷球蛋白血症等)、良性M蛋白血症、意义未明的单克隆丙种球蛋白病(MGUS)、华氏巨球蛋白血症、AL型淀粉样变性、孤立性浆细胞瘤(骨或骨外)、POMES综合征、反应性浆细胞增多症、转移性癌的溶骨性病变、浆母细胞性淋巴瘤、单克隆免疫球蛋白相关肾损害(MGRS)等,其中MGRS是由于单克隆免疫球蛋白或其片段导致的肾脏损害,其血液学改变更接近MGUS。还包含结缔组织病,如系统性红斑狼疮、类风湿性关节炎、硬皮病、干燥综合征等;肝脏病,慢性病毒性活动性肝炎、隐慝性肝硬化、狼疮样肝炎等;传染病,结核、麻风、黑热病、传染性单核细胞增多症、性病、淋巴肉芽肿、放射线菌病、疟疾、锥虫病等;其他,类肉瘤病、重症肌无力、霍奇金病、单核细胞性白血病、白塞(Behcet)病、肾炎、过敏性紫癜、免疫性(或自发性)血小板减少症等。
2.2 CD38相关癌症或自体免疫疾病
CD38是多发性骨髓瘤等多种高球蛋白血症类血液肿瘤的治疗靶点。CD38相关癌症或自体免疫疾病还包括白血病和淋巴瘤,诸如B细胞慢性淋巴细胞白血病、T细胞和B细胞急性淋巴细胞白血病、华氏巨球蛋白血症、原发性系统性淀粉样变性、套细胞淋巴瘤、幼淋巴细胞/髓细胞白血病、急性髓性白血病、慢性髓性白血病、滤泡性淋巴瘤、伯基特淋巴瘤、大颗粒淋巴细胞(LGL)白血病、NK细胞白血病和浆细胞白血病、弥漫性大B细胞淋巴癌(Diffuse Large B-Cell Lymphoma,DLBCL)、滤泡性淋巴瘤(Follicular Lymphoma,FL)、套细胞淋巴瘤(Mantle-Cell Lymphoma,MCL)、慢性淋巴细胞白血病(Chronic Lymphocytic Leukemia,CLL)、急性髓性白血病(Acute Myeloid Leukemia,AML)。多发性骨髓瘤是由于骨髓中的浆细胞癌变而导致的血液肿瘤。目前已有多种靶向CD38的Fc剂,包括但不限于达雷妥尤单抗、Isatuximab、MOR202等。
药物组合在制备用于抑制表达CD38的细胞的生长和/或增殖的方法的药物中的用途,所述方法包括给药所述的药物组合物,从而抑制所述细胞的生长和/或增殖。
药物组合在制备用于治疗患者因表达CD38的细胞参与的疾病或症状的方法的药物中的应用,所述方法包括对所需要治疗的患者给药所述的药物组合物。
药物组合在制备用于预防患者由表达CD38的细胞参与的疾病或症状的方法的药物中的应用,所述方法包括对所需要治疗的患者给药所述的药物组合物。
2.3全身性红斑狼疮
全身性红斑狼疮(SLE)的一个免疫致病性标志是多克隆B细胞活化,其导致高球蛋白血症、自体抗体的产生及免疫复合物形成。主要的异常看来是由于泛化的T细胞失调导致了T细胞不能抑制这些被禁止的B细胞克隆。此外,引发第二信号的若干细胞因子(如IL10)以及共刺激分子(如CD40和CD40L、B7与CD28和CTLA-4)也促进B细胞与T细胞的相互作用。这些相互作用,连同削弱的对免疫复合物和细胞凋亡物质的吞噬细胞清除作用一起,使免疫反应得以保持和由此引起组织损伤。本发明的组合将增强针对SLE的治疗功效(参见Sfikakis PP等,2005 Curr Opin Rheumatol 17:550-7;Peng SL(2004)Methods Mol Med.;102:227-72)。
3.组合治疗高球蛋白血症的方法
在优选的实施方式中,所述免疫球蛋白降解酶与Fc剂用于预防和或治疗高球蛋白血症。
所述高球蛋白血症选自由以下病症或以下病症组成的组:包含恶性单克隆丙种球蛋白血症(如多发性骨髓瘤、重链病、恶性淋巴瘤、慢性淋巴细胞白血病、巨球蛋白血症等)、继发性单克隆丙种球蛋白血症(如非淋巴网状系统肿瘤、单核细胞白血病、冷球蛋白血症等)、良性M蛋白血症、意义未明的单克隆丙种球蛋白病(MGUS)、华氏巨球蛋白血症、AL型淀粉样变性、孤立性浆细胞瘤(骨或骨外)、POMES综合征、反应性浆细胞增多症、转移性癌的溶骨性病变、浆母细胞性淋巴瘤、单克隆免疫球蛋白相关肾损害(MGRS)等。所述病症优选多发性骨髓瘤。
所述免疫球蛋白降解酶在所述Fc剂给药前施用时,所述免疫球蛋白降解酶给药前、所述试剂给药后且所述Fc剂给药前、所述Fc剂给药后,定量检测所述受试者血液中免疫球蛋白的水平,特别是M蛋白。
所述免疫球蛋白降解酶在所述Fc剂给药后施用时,所述述Fc剂给药前、所述Fc剂给药后且所述免疫球蛋白降解酶给药前、所述免疫球蛋白降解酶给 药后,定量检测所述受试者血液中免疫球蛋白的水平,特别是M蛋白。
优选地,所述免疫球蛋白降解酶在所述Fc剂给药前施用,或者所述免疫球蛋白降解酶在所述Fc剂给药后施用。
在本发明的应用中,所述免疫球蛋白降解酶在所述Fc剂作为用于同时、分开或依次使用的联合制剂存在。
在一些实施方式中,所述方法包括以下步骤:1)向受试者施用所述免疫球蛋白降解酶;随后,2)向所述受试者施用所述Fc剂。优选地,所述免疫球蛋白降解酶和所述Fc剂的施用存在时间间隔。
在一些实施方式中,所述方法包括以下步骤:1)向所述受试者施用所述Fc剂;随后,2)向受试者施用所述免疫球蛋白降解酶。优选地,所述免疫球蛋白降解酶和所述Fc剂的施用存在时间间隔。
优选的是,所述免疫球蛋白降解酶的施用量和时间间隔足以将受试者体内的免疫球蛋白水平降至起始水平的60%。更优选的是,试剂施用量和时间间隔足以将受试者体内的免疫球蛋白水平结合降低至低于该患者体内起始水平的50%、40%、30%、20%或10%。该试剂可以在一个单一时间点施用或在设定的时间内施用。
所述试剂和溶瘤病毒或者病毒疫苗可以通过任何适合的途径施用。优选的是,其中所述免疫球蛋白降解酶通过静脉输液或者皮下注射施用,并且/或者所施用的所述试剂的量为0.01mg/kg体重至2mg/kg体重、0.04至2mg/kg体重、0.12mg/kg体重至2mg/kg体重、0.24mg/kg体重至2mg/kg体重或1mg/kg体重至2mg/kg体重。
优选的是,其中所述免疫球蛋白降解酶和所述Fc剂的给药时间间隔至少30分钟、至少1小时、至少2小时、至少3小时、至少4小时、至少4小时、至少5小时或至少6小时;并且最多35天、最多28天、最多21天、最多18天、最多14天、最多13天、最多12天、最多11天、最多10天、最多9天、最多8天、最多7天、最多6天、最多5天、最多4天、最多3天、最多2天、最多24小时、最多18小时、最多12小时、最多10小时、最多8小时、最多7小时或最多6小时。
优选的是,其中所述免疫球蛋白降解酶和所述Fc剂的给药时间间隔为30分钟至1小时、30分钟至2小时、30分钟至3小时、30分钟至4小时、30分钟至5小时、30分钟至6小时、1至2小时、1至3小时、1至4小时、1至5 小时、1至6小时、2至3小时、2至4小时、2至5小时、2至6小时、3至4小时、3至5小时、3至6小时、4至5小时、4至6小时或5至6小时。
在又一实施方式中,所述方法包括以下步骤:1)用所述免疫球蛋白降解酶对来自所述受试者的血液进行离体处理;2)将血液返回所述受试者;3)向所述受试者施用所述Fc剂。
在又一实施方式中,所述方法包括以下步骤:1)向所述受试者施用所述Fc剂;2)用所述免疫球蛋白降解酶对来自所述受试者的血液进行离体处理;3)将血液返回所述受试者。
在优选的实施方式中,所述免疫球蛋白降解酶与Fc剂用于治疗高球蛋白血症。
在优选的实施方式中,所述免疫球蛋白降解酶与Fc剂用于预防高球蛋白血症。
在本发明的任何方面,免疫球蛋白降解酶和Fc剂施用均可以同时、分开或依次施用,用于高球蛋白血症的治疗和预防。免疫球蛋白降解酶和Fc剂可以作为分开的制剂或作为联合制剂提供。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果为:通过将免疫球蛋白降解酶与Fc剂联合使用于高球蛋白血症,降低球蛋白对治疗的影响,使得Fc剂可更好的发挥作用,同时也可降低部分Fc剂的临床使用剂量,一方面可以减轻治疗剂的副作用,另一方面也减轻用药经济负担。
图1为7个单点突变体及野生型IdeE切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:1000)。
图2为7个单点突变体及野生型IdeE切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:2000)。
图3为5个N端截短突变体切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:1000)。
图4为50℃条件下保温1小时后5个N端截短突变体及野生型IdeE切 割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:1000)。
图5为2个C截短突变体切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:1000)。
图6为5个组合突变体切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:2000)。
图7为50℃条件下保温1小时后5个组合突变体切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:2000)。
图8为不同浓度的E97D_del18突变体和IdeS切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图。
图9为不同浓度的E97D_del18突变体和IdeZ切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图。
图10A和10B为具有不同突变组合的突变体和IdeE切割人IgG1产生的切割产物SDS-PAGE凝胶电泳图(酶:底物=1:2000)。
图11为E97D_del18突变体在小鼠血清和血浆中切割人IVIg产生的切割产物SDS-PAGE凝胶电泳图。
图12为E97D_del18突变体在小鼠和人血清中产生的切割产物SDS-PAGE凝胶电泳图。
图13为E97D_del18在小鼠体内不同时间内切割人IVIg产生的切割产物SDS-PAGE凝胶电泳图。
图14为在Daudi细胞肿瘤模型中,免疫球蛋白降解酶降低IgG对抗体抑制肿瘤的抑制。
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例1.蛋白制备
1)CD38单抗液体制剂的制备
自细胞培养物流体收获达雷木单抗,使用固定化的蛋白A亲和层析、阳离子交换层析(如SP Sepharose FF)、除去病毒污染的过滤步骤,接着进行疏水层析(如Butyl Sepharose HP)和超滤换液步骤来纯化,为了制备依照实 施例的药物组合物,在20mM组氨酸缓冲液(pH约6.0)中以约100mg/mL的浓度提供达雷木单抗。伊沙妥昔单抗(Isatuximab)采用相同的方法制备。
2)IdeS和IdeZ的制备
委托南京金斯瑞生物科技有限公司经大肠杆菌偏好的密码子优化后合成编码SEQ ID NO:1至SEQ ID NO:5的多核苷酸序列,并加入N末端甲硫氨酸和C末端6×组氨酸标签,序列合成后插入到pET32a表达载体中,测序正确后获得用于多肽表达的重组质粒。将制备的重组质粒电转化到大肠杆菌BL21 Star(DE3)中,并接种在含100μg/mL氨苄青霉素的LB琼脂糖平板上。37℃培养过夜至菌落长出。挑取单个菌落,接于3ml含100μg/mL氨苄青霉素的LB培养基中,37℃、250rpm培养过夜。过夜培养物接于50mL含100μg/mL氨苄青霉素的LB培养基中,37℃培养至OD600达到0.4~0.6,加入0.1mM IPTG,20℃继续培养过夜。过夜培养物通过离心收集菌体沉淀,用PBS重悬至100g/L后通过超声破胞处理。破胞后通过离心收集上清,上清再用IDA-Ni琼脂糖磁珠纯化,纯化后,洗脱的蛋白用超滤离心管再换液至PBS缓冲体系中。使用SDS-PAGE来评价每种蛋白质的纯度。
实施例2.单点突变体切割人IgG1活性的比较
按照实施例1的方法合成IdeE T8D(SEQ ID NO:63)、T8W(SEQ ID NO:65)、T24A(SEQ ID NO:69)、A59L(SEQ ID NO:73)、A59V(SEQ ID NO:74)、E97D(SEQ ID NO:75)和R280H(SEQ ID NO:76)这7个单点突变体切割人IgG1的活性。
通过SDS-PAGE上显现有每种突变体的不同浓度对人IgG1所产生的切割产物来进一步评价不同突变体相对于野生型IdeE的切割人IgG1活性的高低。将纯化后的突变体或野生型IdeE分别稀释至0.002mg/mL和0.001mg/mL。分别取50μl不同浓度的突变体或野生型IdeE加入50μl含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30分钟。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5分钟,SDS-PAGE检测切割产物。
图1展示了7个单点突变体及野生型IdeE切割人IgG1产生的切割产物电泳图(酶:底物=1:1000)。图2展示了7个单点突变体及野生型IdeE切割人IgG1产生的切割产物电泳图(酶:底物=1:2000)。7个单点突变体在0.001mg/ml浓度下切割IgG1的效果都不差于0.002mg/ml野生型IdeE,说明 7个单点突变体的切割人IgG1的活性不低于野生型IdeE的2倍。
实施例3.N端截短截短突变体切割人IgG1活性和热稳定性的比较
在野生型IdeE基础上分别删除N端前15个(D1-V15)、前16个(D1-P16)、前17个(D1-H17)、前18个(D1-Q18)和前19个氨基酸(D1-I19),构建5个N端截短突变体(见表3)。
表3.截短突变体序列设计
突变体 | 相对于野生型或突变体序列的修饰 | SEQ ID NO: |
WT_del15 | 删除SEQ ID NO:62前15个氨基酸 | 78 |
WT_del16 | 删除SEQ ID NO:62前16个氨基酸 | 79 |
WT_del17 | 删除SEQ ID NO:62前17个氨基酸 | 80 |
WT_del18 | 删除SEQ ID NO:62前18个氨基酸 | 81 |
WT_del19 | 删除SEQ ID NO:62前19个氨基酸 | 82 |
1、突变体的表达和纯化
按照实施例1的方法合成表3中的突变体多核苷酸序列,构建突变体表达重组质粒,转化大肠杆菌BL21 Star(DE3),并制备突变体纯化蛋白。
2、突变体切割人IgG1活性的比较
将纯化后的突变体或野生型IdeE分别稀释至0.002mg/mL。分别取50μl稀释后的突变体或野生型IdeE加入50μl含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30分钟。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5分钟,SDS-PAGE检测切割产物。
图3展示了5个截短突变体切割人IgG1产生的切割产物电泳图(酶:底物=1:1000)。5个截短突变体切割活性都与野生型IdeE没有明显差别。
3、突变体热稳定性的比较
将纯化后的突变体或野生型IdeE分别稀释至0.1mg/ml,50℃条件下保温1小时,保温后再稀释至0.002mg/mL。分别取50μl稀释后的突变体或野生型IdeE加入50μl含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30分钟。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5分钟,SDS-PAGE检测切割产物。
图4展示了50℃条件下保温1小时后5个截短突变体及野生型IdeE切割人IgG1产生的切割产物电泳图(酶:底物=1:1000)。50℃热处理后5个截短突变体的残余活性明显高于野生型,说明5个截短突变体热稳定性相对于 野生型都有明显提高。
实施例4.C端截短截短突变体切割人IgG1活性的比较
在野生型IdeE基础上分别删除C端最后5个(W311-S315)和最后10个氨基酸(S306-S315),构建2个C端截短突变体(见表4)。
表4.截短突变体序列设计
突变体 | 相对于野生型或突变体序列的修饰 | SEQ ID NO: |
WT_delC5 | 删除SEQ ID NO:62后5个氨基酸 | 83 |
WT_delC10 | 删除SEQ ID NO:62后10个氨基酸 | 84 |
1、突变体的表达和纯化
按照实施例1的方法合成表4中的突变体多核苷酸序列,构建突变体表达重组质粒,转化大肠杆菌BL21 Star(DE3),并制备突变体纯化蛋白。
2、突变体切割人IgG1活性的比较
将纯化后的突变体或野生型IdeE分别稀释至0.002mg/mL。分别取50μl稀释后的突变体或野生型IdeE加入50μl含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30分钟。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5分钟,SDS-PAGE检测切割产物。
图5展示了2个C端截短突变体切割人IgG1产生的切割产物电泳图(酶:底物=1:1000)。2个截短突变体切割活性都比野生型IdeE高出2倍以上。
实施例5.组合突变体切割人IgG1活性和热稳定性的比较
在T24A、A59L、A59V、E97D和R280H 5个单点突变体的基础上分别删除前18个(D1-Q18)氨基酸,构建5个组合突变体(见表5)。
表5.组合突变体序列设计
突变体 | 相对于野生型或突变体序列的修饰 | SEQ ID NO: |
T24A_del18 | 删除SEQ ID NO:69前18个氨基酸 | 85 |
A59L_del18 | 删除SEQ ID NO:73前18个氨基酸 | 86 |
A59V_del18 | 删除SEQ ID NO:74前18个氨基酸 | 87 |
E97D_del18 | 删除SEQ ID NO:75前18个氨基酸 | 88 |
R280H_del18 | 删除SEQ ID NO:76前18个氨基酸 | 89 |
1、突变体的表达和纯化
按照实施例1的方法合成表5中的突变体多核苷酸序列,构建突变体表达重组质粒,转化大肠杆菌BL21 Star(DE3),并制备突变体纯化蛋白。
2、突变体切割人IgG1活性的比较
将纯化后的突变体分别稀释至0.001mg/mL。分别取50μl稀释后的突变体或野生型IdeE加入50μl含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30分钟。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5分钟,SDS-PAGE检测切割产物。
图6展示了5个组合突变体切割人IgG1产生的切割产物电泳图(酶:底物=1:2000)。比较图6和图2的切割效果,5个截短突变体和单点组合突变体切割活性没有明显差别,说明组合突变体切割人IgG1的活性同样不低于野生型IdeE的2倍。
3、突变体热稳定性的比较
将纯化后的突变体分别稀释至0.1mg/ml,50℃条件下保温1小时,保温后再稀释至0.001mg/mL。分别取50μl稀释后的突变体或野生型IdeE加入50μl含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30分钟。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5分钟,SDS-PAGE检测切割产物。
图7展示了50℃条件下保温1小时后5个组合突变体切割人IgG1产生的切割产物电泳图(酶:底物=1:2000)。比较图7和图2的切割效果,5个组合突变体50℃热处理后活性只是略有下降,说明5个组合突变体相对于野生型热稳定性也都有明显提高。
实施例6.E97D_del18突变体与IdeS和IdeZ活性比较
将实施例5中纯化的E97D_del18突变体依次稀释至20μg/mL,10μg/mL,5μg/mL,2.5μg/mL和1.25μg/ml。将IdeS(
货号:A0-FRI-020,Genovis)按照其标识分别稀释至2U/μl,1U/μl,0.5U/μl,0.25U/μl和0.125U/μl。将IdeZ(
货号:A0-FRZ-020,Genovis)分别稀释至0.4U/μl,0.2U/μl,0.1U/μl,0.05U/μl和0.025U/μl。分别取50μl不同浓度的突变体、IdeS或IdeZ加入50μl含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30分钟。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5分钟,SDS-PAGE检测切割产物。
图8展示了不同浓度E97D_del18突变体和IdeS切割人IgG1产生的切割产物电泳图。从电泳图上的酶蛋白条带可以判断1号泳道IdeS酶浓度介 于7号和8号泳道E97D_del18突变体酶浓度之间,由此递推3号泳道IdeS酶浓度介于9号和10号泳道E97D_del18突变体酶浓度,而3号泳道的IgG1的酶切效果介于10号和11号泳道,由此可以推断E97D_del18突变体切割人IgG1的活性接近IdeS的2倍。
图9展示了不同浓度E97D_del18突变体和IdeZ切割人IgG1产生的切割产物电泳图。从电泳图上的酶蛋白条带可以判断1号泳道IdeZ酶浓度高于7号泳道E97D_del18突变体酶浓度,由此递推3号泳道IdeZ酶浓度高于9号泳道E97D_del18突变体酶浓度,即高于11号泳道E97D_del18突变体酶浓度的4倍,而3号泳道的IgG1的酶切效果接近11号泳道,由此可以推断E97D_del18突变体切割人IgG1的活性高于IdeZ的4倍。
实施例7.组合突变体切割人IgG1活性的比较
在E97D_del18突变体的基础上删除C端最后5个(W311-S315)和最后10个氨基酸(S306-S315),构建2个C端截短突变体以及一种三突变组合(见表6)。
表6.组合突变体序列设计
突变体 | 相对于野生型或突变体序列的修饰 | SEQ ID NO: |
E97D_del18_delC5 | 删除SEQ ID NO:88后5个氨基酸 | 90 |
E97D_del18_delC10 | 删除SEQ ID NO:88后10个氨基酸 | 91 |
A59V_del18_delC5 | 删除SEQ ID NO:87后5个氨基酸 | 92 |
A59L_del18_delC5 | 删除SEQ ID NO:86后5个氨基酸 | 93 |
R280H_del18_delC5 | 删除SEQ ID NO:89后5个氨基酸 | 94 |
E97D_A59V_R280H | E97D、A59V、R280H三突变组合 | 95 |
1、突变体的表达和纯化
按照实施例1的方法合成表6中的突变体多核苷酸序列,构建突变体表达重组质粒,转化大肠杆菌BL21 Star(DE3),并制备突变体纯化蛋白。
2、突变体切割人IgG1活性的比较
将纯化后的突变体分别稀释至0.001mg/mL。分别取50μl稀释后的突变体或野生型IdeE加入50μl含有2mg/ml曲妥珠单抗的反应体系中开始切割反应,反应体系置于37℃反应30分钟。将样品与同等体积的2×SDS上样缓冲液混合后75℃水浴5分钟,SDS-PAGE检测切割产物。
图10A和10B展示了6个组合突变体切割人IgG1产生的切割产物电泳 图(酶:底物=1:2000)。
实施例7.E97D_del18突变体切割人IgG1活性的体外检测
通过检测加入E97D_del18突变体与人IVIg处理过的小鼠血清或血浆中完整或单切割IVIg的量来评价E97D_del18突变体对人IgG1的体外切割活性。按照表7配制不同组别的小鼠血清或血浆酶切体系。
表7.小鼠血清或血浆酶切体系
碘乙酸处理组中碘乙酸的作用是抑制IgG降解酶的活性。
将体系置于37℃反应30min。取20μl样品与同等体积的2×SDS非还原上样缓冲液混合后75℃水浴5min,SDS-PAGE检测切割产物。
图11展示了E97D_del18突变体在小鼠血清和血浆中切割人IVIg产生的切割产物电泳图。结果显示E97D_del18在小鼠血清和血浆中都可以有效切割人IVIg。
通过检测加入E97D_del18突变体处理过的小鼠或人血清来评价E97D_del18突变体是否具有对人IgG1的体外切割活性。按照表8配制不同组别的小鼠或人血清酶切体系。
表8.小鼠或人血清酶切体系
将体系置于37℃反应24小时。取20μl样品与同等体积的2×SDS还原 性上样缓冲液混合,再用1×SDS还原性上样缓冲液稀释20倍,75℃水浴5分钟,SDS-PAGE检测切割产物。
图12展示了E97D_del18突变体在小鼠和人血清中产生的切割产物电泳图。结果显示E97D_del18在人血清中切割产生明显可见的25kD的Fc片段,而在小鼠血清中则没有该片段,说明E97D_del18能有效特异性切割人血清中IgG1,而对小鼠血清中的IgG1切割活性很低或无切割活性。
实施例8.人体内针对E97D_del18突变体的预存抗体低
该测定是基于E97D_del18突变体与IdeS之间对于与抗E97D_del18/IdeS抗体结合的竞争。测试酶和人血清的预温育将使得抗E97D_del18/IdeS抗体与E97D_del18突变体与IdeS能够结合。
将E97D_del18突变体与IdeS在孔板上包被过夜,然后用PBST洗涤并在2%BSA封闭液中封闭1小时,用逐步稀释的待测突变体与IdeS和人血清制备混合板,将混合版在室温下振荡温育1小时,PBST洗涤之后加入生物素标记的E97D_del18突变体与IdeS,再加入SA-HRP,用TMB显色并读数。平行比对获得约80个人血样本中E97D_del18和IdeS预存抗体的情况。
结果如表9所示,IdeS在正常人血清中预存抗体的比例高达约90%,而E97D_del18突变体仅为约20%。E97D_del18突变体在体内的预存抗体明显少于IdeS,证明E97D_del18突变体的免疫原性更低,更有利于体内用药。
表9.E97D_del18突变体和IdeS在人血样本中预存抗体比对情况
E97D_del18突变体 | IdeS | |
人血样本总数(例) | 76 | 76 |
预存抗体阳性样本比例(%) | 18.4 | 89.5 |
实施例9.E97D_del18突变体切割人IgG1活性的体内检测
在无菌条件下用人IVIg(静注人免疫球蛋白)腹腔注射2只小鼠(两只小鼠为平行实验,小鼠编号1号和2号),注射剂量为1g/kg。注射人IVIg 24h后,再将IgG降解酶(E97D_del18)以5mg/kg的剂量静脉注射小鼠,2只小鼠都分别在注射E97D_del18后0h、15min、2h、6h和24h采血并收集血清样品。取20μl血清样品与同等体积的2×SDS非还原上样缓冲液混合后,再用1×SDS非还原性上样缓冲液稀释20倍,75℃水浴5min,SDS-PAGE检测。
图13展示了E97D_del18在小鼠体内不同时间内切割人IVIg产生的切割产物电泳图。结果显示,E97D_del18在小鼠体内切割IVIg效果显著,15min基本已经达到完全酶切。
实施例10.免疫球蛋白降解酶有效排除高免疫球蛋白对单抗抗肿瘤效
果的负面影响
采用Daudi小鼠模型评价KJ103对抗CD38单抗。实验设计为4组:1)溶剂对照组;2)抗CD38单抗;3)抗CD38单抗+IVIg;4)抗CD38单抗+免疫球蛋白降解酶+IVIg。具体的,取对数生长期细胞,小鼠皮下接种,每只CB-17SCID小鼠无菌条件下右侧腋窝皮下接种5×10
6cells的Daudi细胞,接种量均为0.1mL/只。细胞接种当天单次腹腔注射给予1g/kg IVIg。24h后给予单次静脉注射5mg/kg免疫球蛋白降解酶E97D_del18。IVIg给药后10天腹腔注射给予10mg/kg抗CD38单抗,每周给药1次,共给药4次。CD38开始给药后,每周测量2次肿瘤体积,共检测4周。
实验结果显示(图14),IgG(IVIg)的存在会抑制抗CD38单抗治疗肿瘤的疗效。免疫球蛋白降解酶可降低IgG对抗体疗效的抑制,增强治疗性抗体的治疗效果。
申请人声明,本发明通过上述实施例来说明本发明的详细方法,但本发明并不局限于上述详细方法,即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了,对本发明的任何改进,对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等,均落在本发明的保护范围和公开范围之内。
Claims (20)
- 一种药物组合,其包含免疫球蛋白降解酶和含有Fc的蛋白,所述药物组合用于制备治疗高球蛋白血症的药物。
- 如权利要求1所述的药物组合,其特征在于,所述免疫球蛋白降解酶选自化脓性链球菌的IgG半胱氨酸蛋白酶或其变体以及人来源MMP蛋白酶或其变体,所述变体至少具有酶切IgG的活性;优选地,所述免疫球蛋白降解酶选自IdeS、MAC2、IdeZ、IdeZ2、IdeE、IdeE2、IdeP以及MMP中的一种或多种。
- 如权利要求1或2所述的药物组合,其特征在于,所述免疫球蛋白降解酶包含如SEQ ID NO:1~42、59、60或者61~95所示的氨基酸序列或由所述氨基酸序列组成。
- 如权利要求1-3中任一项所述的药物组合,其特征在于,所述含有Fc的蛋白选自抗体、免疫球蛋白、免疫粘附素、治疗剂、诊断剂、成像剂和抗体药物缀合物中的一种或多种。
- 如权利要求1~4中任一项所述的药物组合,其特征在于,其还包含化疗剂、激素试剂、蛋白酶体抑制剂、靶向小分子制剂、B细胞消耗剂以及免疫抑制剂中的一种或多种。
- 如权利要求5所述的药物组合,其特征在于,所述B细胞消耗剂为与CD10、CD19、CD20、CD21、CD22、CD23、CD24、CD37、CD53、CD70、CD72、CD74、CD75、CD77、CD79a、CD79b、CD80、CD81、CD82、CD83、CD84、CD85或CD86特异性结合的抗体。
- 如权利要求1~6中任一项所述的药物组合,其中所述含有Fc的蛋白是针对选自CD3、CD11a、CD14、CD25、CD28、CD30、CD33、CD36、CD38、CD40、CD44、CD52、CD55、CD59、CD56、CD103、CD134、CD137、CD138、CD152、CD3、IL-1β、IL-2R、IL-6、IL-12、IL-23、C5、BAFF、BLyS、BCMA、CXCR-4、ICAM-1、SLAMF7/CS1、TNFα、IgE的抗体。
- 如权利要求7所述的药物组合,其包含特异性识别CD38的抗体,所述抗体与人效应细胞有结合特异性,和/或所述抗体能够通过凋亡、和/或抗体依赖性细胞介导的细胞毒性、和/或补体依赖性细胞毒性来杀死表达CD38的细胞。
- 如权利要求8所述的药物组合,其特征在于,所述的CD38抗体是与细胞毒素药剂、放射性同位素或药物连接的抗体。
- 如权利要求8所述的药物组合,其特征在于,所述的CD38抗体与CD3、CD4、CD138、IL-15R、膜结合或受体结合的TNF-α、人Fc受体、或膜结合或受体结合的IL-15有结合特异性。
- 如权利要求8所述的药物组合,其特征在于,所述的CD38抗体包含至少一条重链和至少一条轻链,其中所述轻链包含SEQ ID NO:43、SEQ ID NO:44和SEQ ID NO:45的氨基酸序列所示的三个连续互补性决定区,或者如SEQ ID NO:49、SEQ ID NO:50和SEQ ID NO:51的氨基酸序列所示的三个连续互补性决定区,其中所述重链包含SEQ ID NO:46、47和48的氨基酸序列所示的三个连续互补性决定区,或者如SEQ ID NO:52、SEQ ID NO:53和SEQ ID NO:54的氨基酸序列所示的三个连续互补性决定区;其中表位结合片段为Fab、Fab’和F(ab’) 2、scFv或二硫化物连接的Fv片段;较佳地,所述的轻链包含如SEQ ID NO.55或57所示的氨基酸序列,所述重链包含如SEQ ID NO.56或58所示的氨基酸序列;更佳地,所述轻链的氨基酸序列如SEQ ID NO.55所示,且所述重链的氨基酸序列如SEQ ID NO.56所示;或者所述轻链的氨基酸序列如SEQ ID NO.57所示,且所述重链的氨基酸序列如SEQ ID NO.58所示。
- 药物组合物,其包含如权利要求1-11中任一项所述的药物组合,以及药学上可接受的载剂或稀释剂。
- 如权利要求12所述的药物组合物,其为冻干粉末或为液体。
- 一种包含如权利要求1-11中任一项所述的药物组合或者权利要求12或13所述的药物组合物的产品,其中所述免疫球蛋白降解酶固定于一个表面。
- 如权利要求1-11中任一项所述的药物组合、如权利要求12或13所述的药物组合物在制备用于治疗高球蛋白血症的药物中的用途;较佳地,所述的高球蛋白由白细胞产生,所述白细胞选自B细胞、异常B细胞、产生IgM的细胞、产生IgE的细胞、产生IgA的细胞;所述球蛋白包括γ球蛋白;所述的高球蛋白血症包括原发性单克隆丙种球蛋白血症、恶性单克隆丙种球蛋白血症、继发性单克隆丙种球蛋白血症、白血病、淋巴瘤、结缔组织病、肝脏病、传染病、类肉瘤病、重症肌无力、霍奇金病、单核细胞性白血病、 白塞(Behcet)病、肾炎、过敏性紫癜、免疫性(或自发性)血小板减少症。
- 如权利要求1~11中任一项所述的药物组合、如权利要求12或13所述的药物组合物在制备药物中的用途,所述药物用于治疗CD38相关性癌症或自体免疫疾病,所述CD38相关性癌症是多发性骨髓瘤,所述CD38相关性自体免疫疾病为系统性红斑狼疮。
- 如权利要求1~11中任一项所述的药物组合、如权利要求12或13所述的药物组合物在制备用于抑制表达CD38的细胞的生长和/或增殖的药物中的用途。
- 如权利要求1~11中任一项所述的药物组合、如权利要求12或13所述的药物组合物在制备用于治疗患者中表达CD38的细胞参与的疾病或病症的药物中的用途。
- 如权利要求1~11中任一项所述的药物组合或者如权利要求12或13所述的药物组合物在制备用于预防患者中表达CD38的细胞参与的疾病或病症的药物中的用途。
- 一种试剂盒,其包含如权利要求1~11中任一项所述的药物组合或者如权利要求12或13所述的药物组合物。
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US20230374083A1 (en) | 2023-11-23 |
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