CN113769058B - Pharmaceutical composition and application thereof - Google Patents

Abstract

The invention discloses a pharmaceutical composition and application thereof. The pharmaceutical combination comprises: 1) A polypeptide capable of reducing blood IgG levels, the polypeptide being capable of specifically recognizing human FcRn protein; and 2) viral vector drugs. The virus vector medicine is selected from oncolytic viruses, virus vector vaccines and gene therapy viruses; and the pharmaceutical combination allows for the administration of the viral vector drug and agent alone. The polypeptide specifically recognizing human FcRn protein has low immunogenicity and can be continuously and repeatedly administered. The polypeptide capable of specifically recognizing human FcRn protein is combined with oncolytic virus and gene therapy virus, so that the interference of oncolytic virus and gene therapy virus neutralizing antibodies is avoided, side effects of oncolytic virus, gene therapy virus intravenous injection mediated cytokine storm and the like are eliminated, intravenous injection of oncolytic virus and gene therapy virus is not an application obstacle, treatment of oncolytic virus in migration type tumor is possible, and vein scavenging obstacle for gene therapy is realized.

Description

Pharmaceutical composition and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a medicine combination and application thereof, and particularly relates to a combination and application of a reagent for reducing blood IgG level and a virus vector medicine.

Background

Viruses are now commonly used therapeutic vectors, such as oncolytic viruses, viral vector vaccines, gene therapy viruses. Common viruses include HSV-1, adenoviruses, and the like. A major problem with the use of these viruses in therapy is that most of the human beings are infected and immunogenic, and neutralizing antibodies are widely present in the human population. The positive rate of the neutralizing antibody pre-stored in HSV-1 is considerable. These neutralizing antibodies block the effect of the virus and are one of the major limitations of viral vector therapy. In addition, once the viral vector therapy is completed, the titer of neutralizing antibodies increases and the administration of neutralizing antibodies is not possible within one year, which is another major obstacle to the viral vector therapy. Immunosuppressants are mainly used at present to solve the problem, but the treatment effect of the method is poor.

Oncolytic viruses (Oncolytic virus) are a class of viruses that preferentially infect and kill tumor cells. Initially, part of the tumor cells are specifically infected and destroyed by the oncolytic virus. Subsequently, oncolytic viruses replicate and proliferate in tumor cells, releasing new infectious viral particles to infect and destroy other tumor cells. Oncolytic viruses exert oncolytic efficacy by either directly lysing tumor cells or stimulating the host to produce an anti-tumor immune response.

Oncolytic viruses are administered by both intratumoral and intravenous injection. Wherein, the intratumoral injection is the current mainstream administration mode because of the characteristics of uneasy drug resistance, excitation of tumor microenvironment immune response, uneasy generation of cytokine storm and the like. However, in advanced tumors which have metastasized to the whole body, the effect of the intratumoral injection administration is limited, and the intratumoral injection cannot achieve the effect of eliminating the metastasized tumor. Therefore, research and development of intravenous injection of oncolytic viruses are more and more compared with research and development of intratumoral injection, but the problems of neutralizing antibodies and safety brought by systemic injection are the main obstacle .(Russell,S.,Peng,K.&Bell,J.Oncolytic virotherapy.Nat Biotechnol 30,658–670,2012.https://doi.org/10.1038/nbt.2287). of intravenous injection of oncolytic viruses at present, and in addition, the pre-existing neutralizing antibodies in human bodies are one of the treatment key obstacles encountered by viral vector vaccines. There is therefore a need for a therapeutic agent that can slow or eliminate the effects of neutralizing antibodies on oncolytic virus and viral vector vaccine intravenous injection.

WO2020102740A2 discloses a method of gene therapy, which is a treatment method using IdeS or EndoS for the enzymatic cleavage of IgG or sugar chains contained in IgG in serum of an AAV subject. But IdeS, endoS and the like are bacterial source proteins, on the one hand, most antibodies of the proteins naturally exist in human bodies, and the use of IdeS has the risk of generating cytokine storm and the like; on the other hand, bacterial source proteins have strong immunogenicity, can only be administered once, and the effect and efficacy of the bacterial source proteins are greatly reduced when the bacterial source proteins are administered for the second time within a short period (within 1 year), and on the other hand, the high-titer human blood pre-stores neutralizing antibodies, so that the risk of re-administration is also improved.

Therefore, there is a need for a safe and effective agent for reducing the level of viral vector neutralizing antibodies in serum of a subject treated with a viral vector as a drug.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a drug combination of a reagent and a viral vector drug, which can safely and effectively reduce the level of a viral vector neutralizing antibody in serum of a treatment object taking the viral vector as the drug, and application thereof. The main obstacle of intravenous administration of oncolytic viruses and gene therapy viruses is cleared by pre-clearing neutralizing antibodies related to viral vector medicaments in human bodies and/or clearing neutralizing antibodies generated after viral vector administration. The virus vector vaccine is subjected to pre-elimination of virus vector drug neutralizing antibodies, and the influence of the neutralizing antibodies is eliminated.

The invention provides a reagent which can effectively reduce the level of immunoglobulin in blood, has low immunogenicity, can be repeatedly administered and is safe to use. In order to solve the technical problems, the following technical scheme is as follows:

In a first aspect, there is provided a pharmaceutical combination, wherein the pharmaceutical combination comprises: 1) A polypeptide capable of reducing blood IgG levels, wherein the polypeptide is capable of specifically recognizing human FcRn protein; and 2) a viral vector drug, wherein the viral vector drug is selected from the group consisting of oncolytic viruses, gene therapy viruses, viral vector vaccines; and wherein the pharmaceutical combination allows for separate administration of the antibody and the agent. Preferably, the pharmaceutical combination comprises a therapeutically effective amount of the agent that reduces blood IgG levels and a viral vector drug. Preferably, the pharmaceutical composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier or diluent.

Preferably, a pharmaceutical combination as described above, wherein the polypeptide is capable of blocking the binding of blood IgG and FcRn protein.

Preferably, a pharmaceutical combination as described above, wherein the polypeptide has a higher affinity for human FcRn protein than for blood IgG and human FcRn protein; the IgG is selected from IgG1, igG2, igG3, and IgG4.

Preferably, a pharmaceutical combination as described above, wherein the polypeptide comprises an antibody Fc fragment variant comprising a mutation, preferably YTE, YTEKF, LS, NHS, capable of increasing Fc and FcRn affinity, for example Efgartigimod. The variants may be monomers, dimers, multimers. Mutations such as YTE, YTEKF, LS, NHS that can be used in the present invention are described by Dall' Acqua et al (WF, D.A., et al (2002). Journal of immunology (Baltimore, md.: 1950) 169 (9): 5171-5180), lee et al (Lee, C.H., et al (2019). Nat Commun 10 (1): 5031), respectively. The mutant subject is selected from human IgG selected from IgG1, igG2, igG3, igG4.

Other Fc fragment variants useful in the present invention include, but are not limited to, mutations described by Dall' Acqua et al (WF, D.A.), et al (2002), journal of immunology (Baltimore, md.:1950) 169 (9), shan et al (Shan, L., et al (2016), PLoS One 11 (8): e 0160345), lee et al (Lee, C.H., et al (2019), nat Commun 10 (1): 5031), mackness et al (Mackness, B.C., et al (2019), MAbs 11 (7): 1276-1288), christophe et al (Dumet Chusto, pottier J emy, gouilleux-Gruart Val, 20124. rie et, MAbs 1-13411).

Preferably, a pharmaceutical combination as described above, wherein the polypeptide comprises an antibody Fc fragment variant comprising a mutation capable of increasing Fc and fcγr affinity, preferably the variant is a S239D/I322E, S D/I322E/a330L, K W/E333S, R K mutation; the variant is preferably free of fucose modification. The variants may be monomers, dimers, multimers. Other Fc fragment variants useful in the present invention include variants including, but not limited to, mutations described by Wang et al (Wang Xinhua.,Mathieu Mary.,Brezski Randall J.(2018).Protein Cell,9(1),63-73.doi:10.1007/s13238-017-0473-8)

Preferably, the pharmaceutical combination as described above, wherein the variant comprising a polypeptide capable of increasing the affinity of Fc and FcRn comprises a mutation capable of increasing the affinity of Fc and fcγr. The variants may be monomers, dimers, multimers.

Preferably, the pharmaceutical combination as described above, wherein the polypeptide is selected from anti-FcRn antibodies, e.g. Nipocalimab, rozanolixizumab, RVT-1401, HBM9161, ALXN1830, SYNT001, nirsevimab.

Preferably, a pharmaceutical combination as described above, wherein the polypeptide is selected from small peptide fragments capable of specifically binding FcRn, said small peptide fragments being 10 to 70 amino acids in length; such as ABY-039.

Preferably, a pharmaceutical combination as described above, wherein the polypeptide is selected from Fc multimers capable of specifically binding FcRn, e.g., GL-2045, M230, PRIM, hexaGardTM, CSL777, hexavalent molecules by UCB.

Preferably, a pharmaceutical combination as described above, wherein the polypeptide comprises, but is not limited to, a polypeptide fragment as described by Sockolosky et al (Sockolosky Jonathan T, szoka Francis c.adv. Drug deliv. Rev.,2015, 91:109-24.).

Preferably, the combination as described above, wherein, in the viral vector medicament, the virus used in the viral vector medicament is selected from ssDNA-like virus, dsDNA-like virus, ssRNA-like virus or dsRNA-like virus; and/or the virus used by the virus vector drug is selected from wild type virus strain or natural attenuated strain, genetically engineered selective attenuated strain, genetically loaded virus strain and genetically transcription targeted virus strain.

Preferably, the pharmaceutical combination as described above, wherein the wild-type strain or naturally attenuated strain is selected from newcastle disease virus, reovirus, mumps virus, west nile virus, adenovirus, vaccinia virus, and the like.

Preferably, a pharmaceutical combination as described above, wherein the genetically engineered selectively attenuated strain achieves tumor selectivity of viral replication by deleting key genes artificially, e.g. genetically engineered human herpes simplex virus I (HSV-1) knocked out by thymidine kinase (THYMIDINEKINASE, TK), e.g. ONYX-015, G207 or dl1520.ONYX-015 deleted 827bp in E1B region and point mutation was performed on the gene for E1B55K protein to terminate its expressed gene prematurely, failing to express E1B55K protein. The gamma 34.5 gene was deleted from G207, which is a neurotoxicity determinant of HSV-1.

Preferably, a pharmaceutical combination as described above, wherein the gene-loaded virus strain is loaded with an exogenous gene, such as granulocyte macrophage colony-stimulating factor (GM-CSF), such as JX-594 or T-VEC.

Preferably, a pharmaceutical combination as described above, wherein the gene transcription targeted virus strain, e.g. G92A, is inserted into a tissue or tumor specific promoter prior to the viral essential genes to control replication of the oncolytic virus in tumor cells.

Preferably, a pharmaceutical combination as described above, wherein said ssDNA viroid is selected from the group consisting of parvoviruses (parvovirus), preferably said parvoviruses are H-1PV viruses.

Preferably, a pharmaceutical combination as described above, wherein the dsDNA-like virus is selected from the group consisting of herpes simplex virus (herpes simplex virus), adenovirus (adeno virus), poxvirus; preferably, the herpes simplex virus is preferably herpes simplex virus type I HSV-1; preferably, the adenovirus is selected from Enadenotucirev、DNX-2401、C-REV、NG-348、ProsAtak、CG0070、ADV-TK、EDS01、KH901、H101、H103、VCN-01、Telomelysin(OBP-301), the herpes simplex virus is selected from R3616, T-VEC, HF10, G207, NV1020, orienX010, and the poxvirus is selected from Pexa-Vec (vaccinia viruse), JX-594 (vaccinia viruse), GL-ONC1, myxoma.

Preferably, a pharmaceutical combination as described above, wherein said ssRNA viroid is selected from Picornavirus, alphavirus, retroviruses, paramyxoviruses, rhabdoviruses; preferably, said Picornavirus is selected from the group consisting of CAVATAK, PVS-RIPO, CVA21 (enterhead), RIGVIR, said alphavirus is selected from the group consisting of M1, sindbis AR339, semliki Forest virus, said Retroviruses is selected from the group consisting of Toca511, said Paramyxoviruses is selected from the group consisting of MV-NIS, PV701 (NEWCASTLE DISEASE viruses), said Rhabdoviruses is selected from the group consisting of VSV-IFN beta, MG1-MAGEA3, VSV-GP.

Preferably, the pharmaceutical combination as described above, wherein the dsRNA virus is selected from the group consisting of reovirus, vaccinia virus, mumps virus, human immunodeficiency virus (human immunodeficiency virus, HIV), coxsackie virus (coxsackievirus), polio virus (polio virus), swine celecoxib virus (SENECA VALLEY virus), measles virus (measles virus), newcastle disease virus (NEWCASTLE DISEASE virus), vesicular stomatitis virus (vesicular stomatitis virus, VSV), influenza virus; preferably, said Reoviruses is selected from Pelareorep, reolysin.

Preferably, in the pharmaceutical combination as described above, the viral vector used for the viral vector medicament comprises a lentiviral vector, an adenoviral vector or an adeno-associated viral (AAV) vector. Wherein the AAV vector preferably comprises an antibody-bound capsid protein, preferably VP1, VP2 and/or VP3 capsid protein. The lentivirus is preferably selected from the group consisting of lentivirus, which may be human immunodeficiency-1 (HIV-1), human immunodeficiency 2 (HIV-2), simian Immunodeficiency Virus (SIV), feline Immunodeficiency Virus (FIV), bovine Immunodeficiency Virus (BIV), jembrana Disease Virus (JDV), equine Infectious Anemia Virus (EIAV) or Caprine Arthritis Encephalitis Virus (CAEV). More preferably, the lentiviral vector comprises an antibody-bound envelope protein.

Preferably, a pharmaceutical combination as described above, wherein the oncolytic virus expresses an exogenous gene, preferably a bispecific T cell binding molecule (Bispecific T CELL ENGAGERS, biTE), scFv fragment, cytokine, chemokine. The BiTE can be combined with molecules such as CD3 and the like for activating T cells, and can be combined with antigen targets on the surfaces of cancer cells; the scFv targets an immune checkpoint; the immune checkpoints include CTLA-4, PD-1, TIM-3, LAG3, siglec15, 4-1BB, GITR, OX, CD40L, CD, TIGIT, VISTA. Such as GM-CSF, interleukin-2 (IL-2), interleukin-12 (IL-12), interferon (IFN), tumor Necrosis Factor (TNF), soluble CD80, CCL3.

Preferably, a pharmaceutical combination as described above, wherein the gene therapy virus expresses an exogenous gene encoding a protein required for a gene-deficient disease, the protein is selected from acid alpha-glucosidase, copper-transporting ATPase2, alpha-galactosidase, arginine succinate synthase, beta-glucocerebrosidase, beta-hexosaminidase A, cl protease inhibitor or Cl esterase inhibitor, glucose 6 phosphatase, insulin, glucagon, growth hormone, parathyroid hormone, growth hormone releasing factor, insulin, glucose 6 phosphatase, insulin, glucose-receptor follicle stimulating hormone, luteinizing hormone, human chorionic gonadotrophin, vascular endothelial growth factor, angiogenin, angiostatin, granulocyte colony stimulating factor, erythropoietin, connective tissue growth factor, basic fibroblast growth factor, acidic fibroblast growth factor, epidermal growth factor transforming growth factor a, platelet-derived growth factor, insulin growth factors I and II, TGF, bone morphogenic protein, nerve growth factor, brain-derived neurotrophic factor, neurotrophin NT-3 and NT4/5, ciliary neurotrophic factor, glial cell line-derived neurotrophic factor, neurotrophin, lectin, netrin-1 and netrin-2, hepatocyte growth factor, ephrins, tyrosine hydroxylase, thrombopoietin, interleukins (IL-1 to IL-36, etc.), monocyte chemotactic protein, leukemia inhibitory factor, granulocyte macrophage protein colony stimulating factor, fas ligand, tumor necrosis factors a and b, interferon a/b/g, stem cell factor, flk-2/flt3 ligand, igM, igA, igD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, MHC class I and II molecules, cystic fibrosis transmembrane regulator, coagulation factor (factor XIII, factor IX, factor VIII, factor X, factor VII, factor VIIa, protein C, etc.), retinal pigment epithelium-specific 65kDa proteins, LDL receptors, lipoprotein lipase, ornithine transcarbamylase, β -globulin, α -globulin, shadow proteins, α -antitrypsin adenosine deaminase, metal transporter (ATP 7A or ATP 7), sulfonamide enzymes, enzymes involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, b-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched ketoacid dehydrogenase.

Preferably, the pharmaceutical combination as described above, wherein the gene therapy virus carries an exogenous gene encoding an inhibitory nucleic acid selected from the group consisting of siRNA, antisense molecule, miRNA, RNAi, ribozyme and shRNA. The inhibitory nucleic acid binds to a gene associated with a polynucleotide repeat disease, a transcript of the gene, or a polynucleotide repeat of a transcript of the gene. The disease gene encodes a related protein selected from Huntingtin (HTT), androgen receptor on the spinal cord amyotrophic X chromosome, human Ataxin-1/-2/-3/-7, cav2.1p/Q voltage-dependent calcium channel (CACNA 1A), TATA binding protein, ataxin8 reverse chain (ATXN 80S), serine/threonine protein phosphatase 2a55kDa subtype B subtype of spinocerebellar ataxia (type 1,2, 3, 6, 7, 8, 1217), FMR1 (fragile 1 of fragile X syndrome), FMR1 of fragile X-related tremor/ataxia syndrome (fragile X dysnoesia 1), fragile XE mental retardation FMR1 (fragile X dysnoesia 2), or AF4/FMR2 family member 2; troponin kinase (MT-PK), frataxin in myotonic dystrophy. The disease genes are selected from mutants of superoxide dismutase 1 (SOD 1) gene, genes involved in the pathogenesis of parkinson's disease and/or alzheimer's disease, apolipoprotein B (APOB), PCSK9, HIV infection associated genes (HIVTat, TAR, HIVTAR, CCR), influenza a genome/gene sequences in influenza infection, severe Acute Respiratory Syndrome (SARS) coronavirus genome/gene sequences in SARS infection, respiratory syncytial virus genome/gene sequences in respiratory syncytial virus infection, ebola virus genome/gene sequences in ebola virus infection, genome/gene sequences of hepatitis B and c virus in hepatitis B and c virus, herpes Simplex Virus (HSV) genome/gene sequences of HSV infection, coxsackievirus B3 genome/gene sequences of coxsackievirus B3 infection, pathogenic alleles (allele specific silencing) of genes in silent primary dystonia such as torsinA, specific class I and dominant inheritance in HLA, and the genetic mutation of the retina.

Preferably, the pharmaceutical combination according to any one of the preceding claims, wherein the pharmaceutical combination further comprises a targeting drug selected from the group consisting of an epigenetic drug, an inhibitor targeting PI3K/Akt/mTOR signaling pathway, and a tyrosine kinase inhibitor, or a chemotherapeutic drug selected from the group consisting of an immunosuppressant, a proteasome inhibitor, a cytotoxic drug, and a cell cycle non-specific drug, or an immune checkpoint blocker selected from the group consisting of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-TIM-3 antibody, an anti-LAG 3 antibody, an anti-Siglec 15 antibody, an anti-4-1 BB antibody, an anti-GITR antibody, an anti-OX 40 antibody, an anti-CD 40L antibody, an anti-CD 28 antibody, an anti-TIGIT antibody, an anti-VISTA antibody; the epigenetic drug is, for example, a histone deacetylase inhibitor, the inhibitor targeting the PI3K/Akt/mTOR signaling pathway is, for example Tricibine, the tyrosine kinase inhibitor is, for example, sunitinib, the immunosuppressant is, for example, cyclophosphamide, the proteasome inhibitor is, for example, bortezomib, the immunosuppressant is, for example, thalidomide, pomalidomide, the cytotoxic drug is, for example, gemcitabine, temozolomide, and the cell cycle non-specific drug is, for example, mitoxantrone.

In a second aspect, there is provided the use of a pharmaceutical combination according to any one of the preceding claims for the manufacture of a medicament for the treatment or prophylaxis of a disease, wherein the agent is administered to a subject by intravenous infusion or subcutaneous injection. Preferably, the disease is a cancer, a viral infection, a bacterial infection or a fungal infection, a gene-deficient disease. The cancer or infectious disease vaccine may be a veterinary vaccine, or may be a human vaccine, for example as shown in the following table.

Such gene defect related diseases, including but not limited to protein overexpression, protein expression loss, heterologous protein expression caused by viral infection; preferably, the gene therapy agent is for the treatment of gene overexpression or gene underexpression or gene defect or infectious disease; the disease is selected from pulmonary diseases (e.g., cystic fibrosis), hemorrhagic diseases (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, blood diseases (e.g., anemia), alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic Lateral Sclerosis (ALS), epilepsy, lysosomal storage diseases (e.g., aspartyl-glucose diabetes, betay disease, late infant neuronal lipofuscinosis type 2 (CLN 2), cystine disease, fabry disease, gaucher type I, II and III, glycogen storage disease type II (Pompe disease), type I GM 2-gangliopathies (Tay Sachs disease), GM 2-gangliosis type II (Sandhoff disease), type I mucoseborrheic diseases (type I and II sialacidosis, type II (I cell disease), type III (pseudo Hurler disease) and type IV, mucopolysaccharidoses (Hurler disease and variants, hunter, sanfilippo A, type B), C, D, morquio A and type B, maroteaux-Lamy and Sly disease), niemann-Pick disease A/B, C1 and C2 and Schindler disease type I and II), hereditary Angioedema (HAE), copper or iron accumulation disorders (e.g., wilson's disease or Menkes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative diseases, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage disease), solid organs (e.g., brain, liver, kidney, heart) or infectious viruses (e.g., type B and C hepatitis, HIV, etc.), bacterial or fungal diseases; coagulation disorders.

Preferably, wherein the subject has hemophilia a, hemophilia a B with inhibitory antibodies, hemophilia B with inhibitory antibodies, any clotting factor: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor or FV/FVIII co-deficiency, thalassemia, vitamin K cyclooxygenase Cl deficiency or gamma-carboxylase deficiency.

Preferably, wherein the disease caused by the gene deficiency is anemia, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated Intravascular Coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e., FXa inhibitors) or platelet diseases (e.g., bernard Soulier syndrome, glanzmann blood deficiency or reservoir deficiencies).

Preferably, the agent is administered prior to administration of the viral vector drug or the agent is administered after administration of the viral vector drug.

In the use of the invention, the agent and the viral vector agent are present as a combined preparation for simultaneous, separate or sequential use.

In some embodiments, the method comprises the steps of: 1) Administering the agent to a subject; subsequently, 2) administering the viral vector drug to the subject. Preferably, the agent is a polypeptide capable of specifically recognizing human FcRn protein, the polypeptide capable of specifically recognizing human FcRn protein and the administration of the viral vector drug being at time intervals.

In some embodiments, the method comprises the steps of: 1) Administering the viral vector drug to the subject; subsequently, 2) administering the agent to the subject. Preferably, the agent is a polypeptide capable of specifically recognizing human FcRn protein, the polypeptide capable of specifically recognizing human FcRn protein and the administration of the viral vector drug being at time intervals.

Preferably, the agent is administered in an amount and for a time interval sufficient to reduce the level of immunoglobulin in the subject to 60% of the initial level. More preferably, the agent is administered in an amount and for a time interval sufficient to reduce immunoglobulin level binding in the subject to less than 50%, 40%, 30%, 20% or 10% of the initial level in the subject. The agent may be administered at a single point in time or within a set period of time.

Preferably, wherein the agent is administered by intravenous infusion or subcutaneous injection and/or the amount of agent administered is 1mg/kg body weight to 250mg/kg body weight, 1mg/kg body weight to 120mg/kg body weight, 1mg/kg body weight to 80mg/kg body weight, 1mg/kg body weight to 60mg/kg body weight, 2 to 40mg/kg body weight, 5mg/kg body weight to 35mg/kg body weight, 10mg/kg body weight to 30mg/kg body weight or 10mg/kg body weight to 25mg/kg body weight.

Preferably, wherein the agent is administered a plurality of consecutive repetitions, the consecutive repetition being at time intervals of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days or at least 8 days, respectively;

Preferably, wherein the first administration of the agent and the viral vector agent is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, or at least 8 days apart; and at most 35 days, at most 28 days, at most 21 days, at most 18 days, at most 14 days, at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days. Preferably, the agent and the oncolytic virus or the gene therapy virus may be administered simultaneously or at intervals, and it is clear that the agent is administered in an amount and for a time interval sufficient to reduce immunoglobulin level binding in the subject to less than 50%, 40%, 30%, 20% or 10% of the initial level in the subject during the subsequent administration.

Preferably, wherein the first dosing interval of the agent and the viral vector drug is 0 to 35 days, 1 to 35 days, 3 to 35 days, 5 to 35 days, 7 to 35 days, 9 to 35 days, 11 to 35 days, 13 to 35 days, 15 to 35 days, 17 to 35 days, 19 to 35 days, 21 to 35 days, 23 to 35 days, 25 to 35 days, 27 to 35 days, 29 to 35 days, 31 to 35 days, 33 to 35 days. Preferably, the agent and the oncolytic virus or the gene therapy virus may be administered simultaneously or at intervals, and it is clear that the agent is administered in an amount and for a time interval sufficient to reduce immunoglobulin level binding in the subject to less than 50%, 40%, 30%, 20% or 10% of the initial level in the subject during the subsequent administration.

Preferably, wherein the agent and the viral vector vaccine are administered at a time interval of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, or at least 8 days; and at most 35 days, at most 28 days, at most 21 days, at most 18 days, at most 14 days, at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days. More preferably, the administration of the agent is performed first and then the administration of the viral vector vaccine is performed.

Preferably, wherein the agent and the viral vector vaccine are administered at intervals of 0 to 35 days, 1 to 35 days, 3 to 35 days, 5 to 35 days, 7 to 35 days, 9 to 35 days, 11 to 35 days, 13 to 35 days, 15 to 35 days, 17 to 35 days, 19 to 35 days, 21 to 35 days, 23 to 35 days, 25 to 35 days, 27 to 35 days, 29 to 35 days, 31 to 35 days, 33 to 35 days. More preferably, the administration of the agent is performed first and then the administration of the viral vector vaccine is performed.

In a preferred embodiment, the agent is preferably an agent that reduces blood IgG levels with a viral vector drug for the treatment of cancer.

In a preferred embodiment, the agent is preferably an agent that reduces blood IgG levels with a viral vector drug for the prevention of cancer.

In a preferred embodiment, the agent is preferably an agent that reduces blood IgG levels with a viral vector drug for preventing viral infection.

In a preferred embodiment, the agent is preferably an agent that reduces blood IgG levels with a viral vector drug for the treatment of viral infections.

In a preferred embodiment, the agent is preferably an agent that reduces blood IgG levels with a viral vector drug for preventing bacterial infection.

In a preferred embodiment, the agent is preferably an agent that reduces blood IgG levels with a viral vector drug for the treatment of bacterial infections.

In a preferred embodiment, the agent is preferably an agent that reduces blood IgG levels with a viral vector drug for preventing fungal infection.

In a preferred embodiment, the agent is preferably an agent that reduces blood IgG levels with a viral vector drug for the treatment of fungal infections.

In a preferred embodiment, the agent is preferably an agent that reduces blood IgG levels and a viral vector drug for preventing a gene-deficiency related disorder.

In a preferred embodiment, the agent is preferably an agent that reduces blood IgG levels and a viral vector drug for the treatment of a gene deficiency related disorder.

In a third aspect, the invention also provides the use of an agent that reduces the binding of an Fc receptor to an endogenous serum antibody in the manufacture of a medicament for the treatment or prophylaxis of cancer or an infection or a gene-deficient disease, wherein the agent comprises a polypeptide capable of specifically recognizing human FcRn protein, for use in reducing the level of blood virus neutralising antibodies. Preferably, the medicament is for use in the treatment of cancer, prevention of infection, treatment of infection-related diseases, treatment of diseases related to gene deficiency. Preferably, the medicament is for the treatment of cancer. Preferably, the medicament is for the treatment and prophylaxis of infections. Preferably, the medicament is for the treatment of a disease associated with a virus. Preferably, the medicament is for the treatment of gene-deficient disorders.

Preferably, a pharmaceutical combination as described above, wherein the polypeptide is capable of blocking the binding of blood IgG and FcRn protein.

Preferably, a pharmaceutical combination as described above, wherein the polypeptide has a higher affinity for human FcRn protein than for blood IgG and human FcRn protein; the IgG is selected from IgG1, igG2, igG3, and IgG4.

Preferably, a pharmaceutical combination as described above, wherein the polypeptide comprises an antibody Fc fragment variant comprising a mutation, preferably YTE, YTEKF, LS, NHS, capable of increasing Fc and FcRn affinity, for example Efgartigimod. The variants may be monomers, dimers, multimers. Mutations such as YTE, YTEKF, LS, NHS that can be used in the present invention are described by Dall' Acqua et al (WF, D.A., et al (2002). Journal of immunology (Baltimore, md.: 1950) 169 (9): 5171-5180), lee et al (Lee, C.H., et al (2019). Nat Commun 10 (1): 5031), respectively. The mutant subject is selected from human IgG selected from IgG1, igG2, igG3, igG4.

Other Fc fragment variants useful in the present invention include those that include, but are not limited to, the mutations described by Dall' Acqua et al (WF, D.A., et al (2002), journal of immunology (Baltimore, md.: 1950) 169 (9): 5171-5180.), the mutations described by Shan et al (Shan, L., et al (2016), PLoS One 11 (8): e 0160345), the mutations described by Lee et al (Lee, C.H., et al (2019), nat Commun 10 (1): 5031), mackness et al (Mackness,B.C.,et al.(2019)."Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life."MAbs 11(7):1276-1288.)

Preferably, a pharmaceutical combination as described above, wherein the polypeptide comprises an antibody Fc fragment variant comprising a mutation capable of increasing Fc and fcγr affinity, preferably the variant is a S239D/I322E, S D/I322E/a330L, K W/E333S, R K mutation; the variant is preferably free of fucose modification. The variants may be monomers, dimers, multimers. Other Fc fragment variants useful in the present invention include variants including, but not limited to, mutations described by Wang et al (Wang Xinhua.,Mathieu Mary.,Brezski Randall J.(2018).Protein Cell,9(1),63-73.doi:10.1007/s13238-017-0473-8)

Preferably, the pharmaceutical combination as described above, wherein the variant comprising a polypeptide capable of increasing the affinity of Fc and FcRn comprises a mutation capable of increasing the affinity of Fc and fcγr. The variants may be monomers, dimers, multimers.

Preferably, the pharmaceutical combination as described above, wherein the polypeptide is selected from anti-FcRn antibodies, e.g. Nipocalimab, rozanolixizumab, RVT-1401, HBM9161, ALXN1830, SYNT001, nirsevimab.

Preferably, a pharmaceutical combination as described above, wherein the polypeptide is selected from small peptide fragments capable of specifically binding FcRn, said small peptide fragments being 10 to 70 amino acids in length; such as ABY-039.

Preferably, a pharmaceutical combination as described above, wherein the polypeptide is selected from Fc multimers capable of specifically binding FcRn, e.g., GL-2045, M230, PRIM, hexaGard ^TM, CSL777, hexavalent molecules by UCB.

Preferably, a pharmaceutical combination as described above, wherein the polypeptide comprises, but is not limited to, a polypeptide fragment as described by Sockolosky et al (Sockolosky Jonathan T, szoka Francis c.adv. Drug deliv. Rev.,2015, 91:109-24.).

In a fourth aspect, the invention also provides a method of treating or preventing a disease or cancer or infection associated with a gene defect wherein the method results in a 20-50%,50-75%,75-90%,90-95% or more reduction of the viral vector binding antibodies by the subject. Preferably, the medicament is for use in a method of treating a disease associated with a gene defect. Preferably, the medicament is for use in a method of treating cancer, preventing infection. The infection is preferably a viral infection, a bacterial infection or a fungal infection. Preferably, the medicament is for use in a method of treatment of cancer.

Preferably, wherein the viral vector drug is an oncolytic virus; more preferably, the cancer is selected from the group consisting of prostate cancer, breast cancer, bladder cancer, colon cancer, rectal cancer, pancreatic cancer, ovarian cancer, lung cancer, cervical cancer, endometrial cancer, renal (renal cell) cancer, esophageal cancer, thyroid cancer, lymphoma, skin cancer, melanoma, and leukemia.

Preferably, wherein the viral vector drug is a viral vector vaccine; more preferably, the viral vector vaccine is used for targeting or treating diseases caused by prostate cancer, breast cancer, bladder cancer, colon cancer, rectal cancer, pancreatic cancer, ovarian cancer, lung cancer, cervical cancer, endometrial cancer, renal (renal cell) cancer, esophageal cancer, thyroid cancer, lymphoma, skin cancer, melanoma, leukemia or coronavirus, novel coronavirus.

Preferably, wherein the viral vector drug is a gene therapy virus; more preferably, the gene therapy virus is used to treat gene overexpression or gene underexpression or gene deficiency or infectious disease; the disease is selected from pulmonary diseases (e.g., cystic fibrosis), hemorrhagic diseases (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, blood diseases (e.g., anemia), alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic Lateral Sclerosis (ALS), epilepsy, lysosomal storage diseases (e.g., aspartyl-glucose diabetes, betay disease, late infant neuronal lipofuscinosis type 2 (CLN 2), cystine disease, fabry disease, gaucher type I, II and III, glycogen storage disease type II (Pompe disease), type I GM 2-gangliopathies (Tay Sachs disease), GM 2-gangliosis type II (Sandhoff disease), type I mucoseborrheic diseases (type I and II sialacidosis, type II (I cell disease), type III (pseudo Hurler disease) and type IV, mucopolysaccharidoses (Hurler disease and variants, hunter, sanfilippo A, type B), C, D, morquio A and type B, maroteaux-Lamy and Sly disease), niemann-Pick disease A/B, C1 and C2 and Schindler disease type I and II), hereditary Angioedema (HAE), copper or iron accumulation disorders (e.g., wilson's disease or Menkes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative diseases, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage disease), solid organs (e.g., brain, liver, kidney, heart) or infectious viruses (e.g., type B and C hepatitis, HIV, etc.), bacterial or fungal diseases; coagulation disorders.

Preferably, wherein the components of the pharmaceutical combination are administered separately or the components of the pharmaceutical combination are administered simultaneously.

In a fifth aspect, the invention also provides the use of an agent for reducing blood IgG levels comprising a polypeptide that specifically recognizes FcRn and a viral vector medicament in a method of enhancing the efficacy of a viral vector medicament.

In a sixth aspect, the invention also provides a product comprising an agent that reduces blood IgG levels, including a polypeptide that specifically recognizes FcRn and a viral vector medicament, as a combined preparation for simultaneous, separate or sequential use in the treatment of a gene-deficiency related disorder, in the treatment of cancer, in the prevention of cancer and/or infection. Preferably, the combined preparation is for use in a method of treating cancer. Preferably, the combined preparation is used in a method of treatment of a disease associated with a gene defect.

In some embodiments, the cancer is selected from the group consisting of: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal carcinoma, appendicular carcinoma, astrocytoma, childhood cerebellum or brain carcinoma, basal cell carcinoma, extrahepatic cholangiocarcinoma, bladder carcinoma, bone carcinoma, osteosarcoma/malignant fibrous histiocytoma, brain stem glioma, brain carcinoma, brain tumor-cerebellar astrocytoma, brain tumor-brain astrocytoma/glioblastoma, brain tumor-ependymoma, brain tumor-medulloblastoma, brain tumor-supratentorial primitive neuroectodermal tumor, brain tumor-visual pathway and hypothalamic glioma, Breast cancer, bronchial adenoma/carcinoid, burkitt lymphoma, carcinoid tumor, gastrointestinal carcinoid tumor, unknown primary carcinoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, cervical cancer, chronic lymphocytic leukemia, chronic myeloproliferative leukemia chronic myeloproliferative disease, colon cancer, cutaneous T cell lymphoma, connective tissue proliferative small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, ewing's sarcoma in the ewing tumor family, extracranial germ cell tumor, childhood extragonadal germ cell tumor, extrahepatic biliary tract cancer, ocular cancer-intraocular melanoma, Eye cancer-retinoblastoma, gall bladder cancer, stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), extracranial, extragonadal, or ovarian germ cell tumor, gestational trophoblastoma, brain stem glioma, childhood brain astrocytoma glioma, childhood visual pathway and hypothalamic glioma, gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, hodgkin lymphoma, hypopharynx cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell carcinoma (endocrine pancreas), kaposi sarcoma, renal carcinoma (renal cell carcinoma), laryngeal carcinoma, leukemia, acute lymphoblastic leukemia (also known as acute lymphoblastic leukemia), and the like, Acute myeloid leukemia (also known as acute myelogenous leukemia), chronic lymphocytic leukemia (also known as chronic lymphocytic leukemia), chronic myelogenous leukemia (also known as chronic myelogenous leukemia), hairy cell leukemia, lip and mouth cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, lymphoma, AIDS-related lymphoma, burkitt's lymphoma, cutaneous T-cell lymphoma, hodgkin's lymphoma, non-Hodgkin's lymphoma (old classification: all lymphomas except Hodgkin's lymphoma), primary central nervous system lymphoma, macroglobulinemia, Osteomalignant fibrous histiocytoma/osteosarcoma, medulloblastoma, melanoma, intraocular (ocular) melanoma, merkel cell carcinoma, mesothelioma, adult malignant mesothelioma, primary recessive metastatic squamous neck carcinoma, oral cancer, multiple endocrine adenoma syndrome, multiple myeloma/plasmacytoid granuloma, myelodysplastic syndrome, myelodysplastic/myeloproliferative disorders, chronic myelogenous leukemia, adult acute myelogenous leukemia, pediatric acute myelogenous leukemia, multiple myeloma (myelocarcinoma), myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-hodgkin lymphoma, Non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma/bone malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer (surface epithelium-stroma), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic islet cell pancreatic cancer, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal gland germ cell tumor, pineal gland blastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasmacytoma/multiple myeloma, pleural pneumoblastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal pelvis and ureteral renal cell carcinoma (renal cancer), Transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, ewing family tumor sarcoma, kaposi' S sarcoma, soft tissue sarcoma, uterine sarcoma, szebra (Sze zary) syndrome, skin carcinoma (non-melanoma), skin carcinoma (melanoma), merkel cell skin carcinoma, small cell lung carcinoma, small intestine carcinoma, soft tissue sarcoma, squamous cell carcinoma, primary recessive squamous neck carcinoma, metastatic gastric cancer, supratentorial primitive neuroectodermal tumor, cutaneous T-cell lymphoma (see mycosis fungoides and Szechurian syndrome), testicular carcinoma, throat carcinoma, thymoma and thymus carcinoma, thyroid carcinoma, Transitional cell carcinoma of the renal pelvis and ureter, ureter and renal pelvis trophoblastoma, transitional cell carcinoma of the urinary tract, endometrial carcinoma, uterine sarcoma, vaginal carcinoma, visual pathway and hypothalamic glioma, vulval carcinoma, macroglobulinemia and nephroblastoma (renal carcinoma).

In some embodiments, the gene defect related disease such as a disease caused by over-expression or under-expression of a gene or a gene defect or an infectious disease; the disease is selected from pulmonary diseases (e.g., cystic fibrosis), hemorrhagic diseases (e.g., hemophilia A or hemophilia B with or without inhibitors), thalassemia, blood diseases (e.g., anemia), alzheimer's disease, parkinson's disease, huntington's disease, amyotrophic Lateral Sclerosis (ALS), epilepsy, lysosomal storage diseases (e.g., aspartyl-glucose diabetes, betay disease, late infant neuronal lipofuscinosis type 2 (CLN 2), cystine disease, fabry disease, gaucher type I, II and III, glycogen storage disease type II (Pompe disease), type I GM 2-gangliopathies (Tay Sachs disease), GM 2-gangliosis type II (Sandhoff disease), type I mucoseborrheic diseases (type I and II sialacidosis, type II (I cell disease), type III (pseudo Hurler disease) and type IV, mucopolysaccharidoses (Hurler disease and variants, hunter, sanfilippo A, type B), C, D, morquio A and type B, maroteaux-Lamy and Sly disease), niemann-Pick disease A/B, C1 and C2 and Schindler disease type I and II), hereditary Angioedema (HAE), copper or iron accumulation disorders (e.g., wilson's disease or Menkes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative diseases, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage disease), solid organs (e.g., brain, liver, kidney, heart) or infectious viruses (e.g., type B and C hepatitis, HIV, etc.), bacterial or fungal diseases; coagulation disorders.

Preferably, wherein the subject has hemophilia a, hemophilia a B with inhibitory antibodies, hemophilia B with inhibitory antibodies, any clotting factor: VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor or FV/FVIII co-deficiency, thalassemia, vitamin K cyclooxygenase Cl deficiency or gamma-carboxylase deficiency.

Preferably, wherein the disease caused by the gene deficiency is anemia, bleeding associated with trauma, injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated Intravascular Coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotics (i.e., FXa inhibitors) or platelet diseases (e.g., bernard Soulier syndrome, glanzmann blood deficiency or reservoir deficiencies).

In a seventh aspect, the present invention also provides a pharmaceutical composition or therapeutic agent for use in a method of treatment of a gene defect related disorder, treatment of cancer, prevention of cancer and/or infection, the pharmaceutical composition or therapeutic agent comprising: 1) A therapeutically effective amount of an agent that reduces blood IgG levels includes a polypeptide that specifically recognizes FcRn; and 2) a therapeutically effective amount of a viral vector drug, preferably an oncolytic virus, preferably a gene therapy vector virus, preferably a viral vector vaccine; and 3) a pharmaceutically acceptable carrier or diluent.

In an eighth aspect, the present invention also provides a kit or kit of parts for preventing or treating a gene-deficiency related disease, cancer or infection, the kit comprising: 1) A therapeutically effective amount of a drug that reduces blood immunoglobulin levels; and 2) a therapeutically effective amount of a viral vector drug. The kit may further comprise 3) a targeting drug or a chemotherapeutic drug or an immune checkpoint blocker. The targeting drug is selected from epigenetic drugs, inhibitors targeting PI3K/Akt/mTOR signaling pathway, and tyrosine kinase inhibitors, the chemotherapeutic drug is selected from immunosuppressants, proteasome inhibitors, cytotoxic drugs, and cell cycle non-specific drugs, and the immune checkpoint blocker is selected from anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-TIM-3 antibodies, anti-LAG 3 antibodies, anti-Siglec 15 antibodies, anti-4-1 BB antibodies, anti-GITR antibodies, anti-OX 40 antibodies, anti-CD 40L antibodies, anti-CD 28 antibodies, anti-TIGIT antibodies, anti-VISTA antibodies; the epigenetic drug is, for example, a histone deacetylase inhibitor, the inhibitor targeting the PI3K/Akt/mTOR signaling pathway is, for example Tricibine, the tyrosine kinase inhibitor is, for example, sunitinib, the immunosuppressant is, for example, cyclophosphamide, the proteasome inhibitor is, for example, bortezomib, the immunosuppressant is, for example, thalidomide, pomalidomide, the cytotoxic drug is, for example, gemcitabine, temozolomide, and the cell cycle non-specific drug is, for example, mitoxantrone.

The kit of parts comprises a kit a comprising a therapeutically effective amount of a drug that reduces blood immunoglobulin levels comprising a polypeptide that specifically recognizes FcRn and a kit B comprising a therapeutically effective amount of a viral vector drug. The kit of parts may also comprise a kit C. The kit C comprises a targeted drug or a chemotherapeutic drug or an immune checkpoint blocker. The targeting drug is selected from epigenetic drugs, inhibitors targeting PI3K/Akt/mTOR signaling pathway, and tyrosine kinase inhibitors, the chemotherapeutic drug is selected from immunosuppressants, proteasome inhibitors, cytotoxic drugs, and cell cycle non-specific drugs, and the immune checkpoint blocker is selected from anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-TIM-3 antibodies, anti-LAG 3 antibodies, anti-Siglec 15 antibodies, anti-4-1 BB antibodies, anti-GITR antibodies, anti-OX 40 antibodies, anti-CD 40L antibodies, anti-CD 28 antibodies, anti-TIGIT antibodies, anti-VISTA antibodies; the epigenetic drug is, for example, a histone deacetylase inhibitor, the inhibitor targeting the PI3K/Akt/mTOR signaling pathway is, for example Tricibine, the tyrosine kinase inhibitor is, for example, sunitinib, the immunosuppressant is, for example, cyclophosphamide, the proteasome inhibitor is, for example, bortezomib, the immunosuppressant is, for example, thalidomide, pomalidomide, the cytotoxic drug is, for example, gemcitabine, temozolomide, and the cell cycle non-specific drug is, for example, mitoxantrone.

The kit may comprise instructions for administration (e.g., dose information, dosing interval information) of a therapeutically effective amount of the agent that reduces blood immunoglobulin levels and a therapeutically effective amount of the viral vector agent.

In any aspect of the invention, the administration of the agent that reduces blood immunoglobulin levels and the viral vector agent may be simultaneous, separate or sequential, e.g., for the treatment of a gene deficiency related disease, the treatment of cancer, the prevention of cancer and/or infection. The agent that reduces blood immunoglobulin levels and the viral vector agent may be provided as separate formulations or as a combined formulation.

The term "agent (or reagent) that reduces blood immunoglobulin levels" as used herein preferably refers to an agent or reagent that reduces blood immunoglobulin levels to 60% or less of the original level. Preferably, the agent or reagent reduces the blood immunoglobulin to at most 60% of original level, at most 50% of original level, at most 40% of original level, at most 30% of original level, at most 20% of original level, at most 10% of original level, or at most 0% of original level. More preferably, the agent or reagent reduces the blood immunoglobulin to at most 20% of the original level, at most 10% of the original level, or at most 0% of the original level.

The mature expression system can be used to manufacture viral vector pharmaceuticals. Some examples of methods include the use of mammalian cell expression systems to produce viral particles, such as adenovirus-like viral vector drugs using HEK293 cells (Freedman Joshua D,Duffy Margaret R,Lei-Rossmann Janet et al.An Oncolytic Virus Expressing a T-cell Engager Simultaneously Targets Cancer and Immunosuppressive Stromal Cells.[J].Cancer Res.,2018,78:6852-6865.).

The pharmaceutical carrier may be a liquid and the pharmaceutical composition may be in the form of a solution. Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient may be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, an organic solvent, a mixture of both, or a pharmaceutically acceptable oil or fat.

Pharmaceutical compositions for parenteral administration are sterile, substantially isotonic, pyrogen-free, and prepared according to the FDA or GMP of a similar agency. The viral vector drug may 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 may be a sterile liquid such as water, oil, saline, glycerol or ethanol). In addition, auxiliary substances such as wetting or emulsifying agents, surfactants, and pH buffering substances, among others, 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. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions. The viral vector drug may be administered in depot injection or implant formulations which can be formulated to allow sustained release of the active ingredient. Typically, the compositions are prepared as injectables, either as liquid solutions or suspensions; it may also be prepared in solid form suitable for dissolution or suspension in a liquid carrier prior to injection.

The pharmaceutical compositions of the present invention may be used in combination with targeted drugs. The targeting agent is selected from the group consisting of epigenetic agents, inhibitors targeting PI3K/Akt/mTOR signaling pathway, and receptor tyrosine kinase inhibitors.

Histone deacetylase inhibitors (HDACi) are epigenetic drugs that are widely studied, and HDACi can not only promote differentiation and apoptosis of tumor cells by inhibiting proliferation of tumor cells and inducing cell cycle arrest, but also reduce antiviral immune response of the body by inhibiting interferon signaling pathway. Histone deacetylase inhibitor HDAC6 is proved to be capable of remarkably improving the replication level of HSV-1 oncolytic virus in glioma cells, and can be used for killing tumors in a synergistic way with HSV-1.

The PI3K/Akt signal pathway is an important signal pathway for regulating cell proliferation and apoptosis under a stress condition, and the Akt inhibitor Tricibine can cooperate with oncolytic virus MG18L to induce glioma cells to undergo apoptosis, so that the curative effect of the combination of the two for treating the glioma of the mice is obviously better than that of single-drug treatment. Rapamycin is an inhibitor of the mTOR signaling pathway and can synergistically kill both adenovirus and HSV-1 in tumor cells that are not susceptible.

Protein Tyrosine Kinase (PTKs) inhibitors have multiple effects of inhibiting tumor angiogenesis and anti-tumor cell growth. Sunitinib is a small-molecule receptor tyrosine kinase inhibitor, can improve the replication of VSV oncolytic viruses in tumor cells by inhibiting the activity of intracellular PTK, and can enhance the infection capacity of oncolytic viruses in tumors by inhibiting VEGFR signal channels to destroy tumor angiogenesis, thereby remarkably enhancing the treatment effect of oncolytic viruses.

The pharmaceutical compositions of the present invention may be used in combination with chemotherapeutic agents. The combination is capable of immunogenic cell death, enhancing tumor cell antigenicity or susceptibility to immune cells, inhibiting Treg cells with negative regulation, and Myeloid Derived Suppressor Cells (MDSCs). The chemotherapeutic agent is selected from the group consisting of immunosuppressants such as cyclophosphamide, proteasome inhibitors such as bortezomib, immunosuppressants such as thalidomide, pomalidomide, cytotoxic agents such as gemcitabine, temozolomide, and cell cycle non-specific agents such as mitoxantrone. Preferably, the combination is an oncolytic reovirus and bortezomib combination.

The pharmaceutical compositions of the invention may be used in combination with immune checkpoint blockers. The immune checkpoint is selected from CTLA-4, PD-1, TIM-3, LAG3, siglec15, 4-1BB, GITR, OX, CD40L, CD, TIGIT, VISTA; the immune checkpoint blocker is selected from anti-CTLA-4 antibody, anti-PD-1 antibody, anti-TIM-3 antibody, anti-LAG 3 antibody, anti-Siglec 15 antibody, anti-4-1 BB antibody, anti-GITR antibody, anti-OX 40 antibody, anti-CD 40L antibody, anti-CD 28 antibody, anti-TIGIT antibody, anti-VISTA antibody.

The agent and oncolytic virus or viral vector vaccine or gene therapy virus may be administered by any suitable route. Preferably, both the agent and oncolytic virus are administered intravenously (i.v.). Alternatively, both the agent and the viral vector vaccine are administered intravenously (i.v.). Preferably, the agent is administered intravenously (i.v.), and the viral vector vaccine is administered intramuscularly.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

By combining polypeptide capable of specifically recognizing human FcRn protein with oncolytic virus and gene therapy virus, on one hand, interference of neutralizing antibodies of oncolytic virus and gene therapy virus is avoided, and on the other hand, side effects of cytokine storm and the like mediated by intravenous injection of oncolytic virus and gene therapy virus are also eliminated, so that intravenous injection of oncolytic virus and gene therapy virus is not an obstacle for application of oncolytic virus and gene therapy virus, treatment of oncolytic virus in migration type tumor is possible, and obstacle is cleared for vein of gene therapy. By combining polypeptide specifically recognizing human FcRn protein with virus vector vaccine, the interference of virus neutralizing antibody is reduced, and side effects such as cytokine storm mediated by virus vector vaccine injection are eliminated, so that the safety of virus vector vaccine is greatly improved.

Drawings

FIG. 1 is a map of KJ-V01 expression vector.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

The technical scheme of the invention is further described by the following specific embodiments. It will be appreciated by persons skilled in the art that the implementations described are merely to aid in the understanding of the invention and are not to be construed as limiting the invention in any way.

The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 sample preparation

The entrusted gene synthesis company performs codon optimization for the provided amino acid sequence KJ037 (SEQ ID No.1, amino acid sequence identical Efgartigimod) and total gene synthesizes a DNA fragment encoding KJ037, cloned into the KJ-V01 expression vector (fig. 1) constructed by the inventors, said expression vector consisting of:

1) The glutamine synthetase gene, as a screening marker,

2) The origin of replication, ori,

3) An origin of replication from the vector pUC18, which allows this plasmid to replicate in E.coli,

4) A beta-lactamase gene which confers ampicillin resistance in E.coli,

5) Immediate early enhancers and promoters from human cytomegalovirus,

6) Human 1-immunoglobulin polyadenylation ("poly A") signal sequences.

As described above, the KJ037 sequence was synthesized by total gene synthesis, and cloned into the same digested KJ-V01 vector after digestion with EcoRI and HindIII, and KJ037/KJ-V01 was finally constructed.

E.coli TOP10 is transformed by the constructed KJ037/KJ-V01 expression vector, and positive clones are selected and inoculated into 100ml LB culture medium for amplification. Transfection-grade DNA was prepared using the Qiagen Plasmid purification kit (Plasmid Plus MIDI KIT CAT:12943). The plasmid obtained was linearized by single cleavage with ScaI, and the plasmid after cleavage was purified by phenol-chloroform extraction and ethanol precipitation (method from third edition of the Experimental guidelines for molecular cloning). Plasmid DNA was transfected into CHO-K1 (Chinese hamster ovary cells, available from ATCC) using Gibco's liposome method kit, and the procedure was followed according to the manufacturer's instructions.

Cells are inoculated into a 96-well plate at a certain cell density 24-48 hours after transfection, a cell culture medium is a screening culture medium containing screening drugs, and clones are grown successively after stationary culture for 3-4 weeks. And selecting larger monoclonal to perform liquid exchange and blowing, and taking the supernatant after 24 hours to perform E/KJ-V01 detection. The anti-gamma antibody is coated on ImmunoX ELISA plates, supernatant is diluted 2000 times by PBS-T and is sampled, anti-gamma-HRP antibody is adopted for detection and color development, clones with higher response values are selected to be amplified to 24 pore plates, T25 culture flasks, T75 culture flasks and 125mL shake flasks in sequence, well-grown clones are selected for amplifying the recovery liquid, and after the recovery liquid, the supernatant is purified by a ProA column or a nickel column and endotoxin is removed. The purified protein was stored in PBS, pH 7.4.

EXAMPLE 2 KJ037 decreases IgG level, virus neutralizing antibody level

And detecting the level of the AAV8 neutralizing antibody in the blood of the cynomolgus monkey, and selecting 1 cynomolgus monkey with the neutralizing antibody detection negative, and 2 cynomolgus monkeys with the neutralizing antibody level of 1:64 for the experiment. 1 cynomolgus monkey with neutralizing antibody was given KJ037 at 25mg/kg, once every 4 days, 3 times in total. The other 2 cynomolgus monkeys were given normal saline and a tracer antibody, wherein the tracer antibody was an anti-human CD38 antibody, and did not cross-react with monkey CD 38. The tracer antibody was administered only once at D0, 1mg/kg. On the day after the third administration, AAV8 was given by intravenous infusion of 5X 10 ¹² vg/kg. D0, D4, D8, D12, D16 were bled prior to dosing. Animals were sacrificed 28 days after AAV8 administration and tissues were reserved for detection of viral transduction efficiency.

The tracer antibody detection method comprises the following steps: the content of tracer antibody in monkey blood was detected by conventional ELISA method. Specifically, a CD38 antigen 2 mug/ml plate was used, and the tracer antibody was diluted at 9 spots at a 2-fold ratio starting at 100 ng/ml. Blood was diluted in four proportions 1:10, 1:100, 1:1000, 1:10000. The measurement results were converted into a ratio to the non-administered sample.

The method for detecting the total IgG of the monkey comprises the following steps: the IgG content in the monkey blood was detected by conventional ELISA. Specifically, monkey IgG gamma chain-specific antibodies were used to coat 2. Mu.g/ml, and blood was diluted in four proportions, 1:10,000, 1:100,000, 1:1,000,000, 1:10,000,000. Detection of murine anti-monkey HRP antibody. The measurement results were converted into a ratio to the non-administered sample.

AAV8 neutralizing antibody titer assay: HEK293 cells were seeded in 96-well plates 2×10 ⁴ each at 37 ℃ with 5% co ₂ for approximately 7hr. Monkey serum was diluted and mixed with AAV8 virus (1X 10 ⁵ vg/cell) in equal volumes at a 2-fold ratio for 10 gradients, and incubated for 2 hours at room temperature. Each dilution was 3 duplicate wells and negative controls were not serum or virus added. After the incubation was completed, the cells were cultured in a cell culture plate at 37℃for 24hr with 5% CO ₂. After the completion of the incubation, a Bio-Bright ^TM One-Step firefly luciferase assay kit reagent was added and chemiluminescent signals were detected according to the protocol.

Liver transduction efficiency: the tissue was weighed, diced into tubes, 300 μl of reporter gene-specific lysate (Rhinobio) was added, homogenized at low speed for 30 seconds each time with a homogenizer, and ice-bath was performed for 1 minute each time at intervals until the tissue was completely lysed. The lysate was centrifuged at 14,000Xg for 15 minutes in a pre-chilled centrifuge. The supernatant is immediately transferred into a new centrifuge tube and stored for later use. Mu.l of the supernatant of the tissue fluid was added to 60. Mu.l of the luciferase substrate solution (Rhinobio), and the luminescence value was measured within 5 minutes.

Neutralizing antibody titer assay experiments showed that KJ037 administration reduced the titer of AAV8 neutralizing antibodies, the level of tracer antibodies, and the level of total monkey IgG in the blood (table 1). The KJ037 group of tracer antibodies was significantly lower in content than the other two groups on day 4, by more than 95% and about 70% reduction in total IgG (table 1). Liver fluorescence detection showed that KJ037 can significantly increase viral gene expression in the liver by reducing circulating antibodies (table 1).

TABLE 1 serum antibody level and viral transduction efficiency detection

The invention has various embodiments, and all technical schemes formed by equivalent transformation or equivalent transformation fall within the protection scope of the invention.

SEQUENCE LISTING

<110> Shanghai Baoji pharmaceutical Co., ltd

<120> A pharmaceutical composition and use thereof

<130> P21015299C

<150> CN202010525070.4

<151> 2020-06-10

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 227

<212> PRT

<213> Artificial Sequence

<220>

<223> Efgartigimod amino acid sequence

<400> 1

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr

20 25 30

Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu Lys Phe His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

Claims (5)

1. A pharmaceutical composition, characterized in that, the pharmaceutical combination comprises: 1) A polypeptide capable of reducing blood IgG levels, wherein the polypeptide is capable of specifically recognizing human FcRn protein, and the sequence of the polypeptide is shown in SEQ ID No. 1; and 2) an AAV8 viral vector agent, wherein the pharmaceutical combination allows for separate administration of the AAV8 viral vector agent and the polypeptide.

2. The pharmaceutical combination of claim 1, wherein the AAV8 viral vector drug carries a foreign gene; the exogenous gene is selected from the group consisting of an inhibitory nucleic acid of siRNA, miRNA, ribozyme and shRNA.

3. Use of a pharmaceutical combination according to claim 1 or 2 for the preparation of a medicament for gene therapy.

4. The use of claim 3, wherein the components of the pharmaceutical combination are administered separately.

5. The use of claim 3, wherein the components of the pharmaceutical combination are administered simultaneously.