17 results about "Blood coagulation factor VIII" patented technology

The invention provides a dual-signal peptide, the dual-signal peptide comprises a first signal peptide and a second signal peptide, the first signal peptide comprises an exogenous signal peptide, the second signal peptide comprises an endogenous signal peptide of a blood coagulation factor VIII, and the first signal peptide and the second signal peptide are connected in series. According to the dual-signal peptide, the secretion effect and the blood coagulation activity of the blood coagulation factor VIII are substantially improved, the production of FVIII can be greatly improved, the industrial production efficiency of FVIII is improved, the application of FVIII in hemophilia treatment drugs is further promoted, and the treatment cost is reduced.

Provided is a dual-signal peptide. The dual-signal peptide comprises a first signal peptide and a second signal peptide, wherein the first signal peptide comprises an exogenous signal peptide, the second signal peptide comprises an endogenous signal peptide of blood coagulation factor VIII, and the first signal peptide and the second signal peptide are linked in tandem. The dual-signal peptide of the present invention enables substantial improvements in both the secretion effect and coagulation activity of blood coagulation factor VIII, so that the yield of FVIII is greatly increased, and the industrial production efficiency thereof is improved.

Various bispecific antibodies that specifically bind to both blood coagulation factor IX / activated blood coagulation factor IX and blood coagulation factor X and functionally substitute for the cofactor function of blood coagulation factor VIII, that is, the function to promote activation of blood coagulation factor X by activated blood coagulation factor IX, were produced. From these antibodies, multispecific antigen-binding molecules having a high activity of functionally substituting for blood coagulation factor VIII were successfully discovered.

The invention provides a recombinant blood coagulation factor VIII preparation and a preparation method and application thereof, and belongs to the field of biological medicine. The recombinant blood coagulation factor VIII preparation provided by the invention is prepared from the following components: 500 to 1500 IU / mL of recombinant blood coagulation factor VIII, 20 to 100 mM of glycine, 50 to 100 mM of raffinose, 2 to 10 mM of divalent calcium salt, 0.5 to 5 percent of dextran-40 and 0.05 to 0.15 percent of tween-80, the preparation is simple in component, so that the recombinant blood coagulation factor VIII is long in preservation time and stable in activity, and can be used for preparing drugs for treating hemophilia.

Bispecific antibodies whose FIX activation-inhibiting activity is not elevated and whose FVIII cofactor function-substituting activity is elevated have been successfully discovered.

The invention discloses a blood coagulation factor VIII activity determination kit and a determination method. The reagent comprises a reagent R1, a reagent R2, a reagent R3 and a sample diluent, the reagent R1 comprises the following components: a bovine X factor, a fibrin polymerization inhibitor, salt ions, a stabilizer, a preservative and a buffer solution; the R2 reagent comprises the following components: a bovine IXa factor, bovine thrombin, phospholipid, calcium chloride, a stabilizer, a preservative and a buffer solution; the R3 reagent comprises the following components: an FXa chromophoric substrate, a thrombin inhibitor, salt ions, a stabilizer, a preservative and a buffer solution; the sample diluent comprises the following components: a heparin antagonist, salt ions, a stabilizer, a buffer solution and a preservative; the activity of the blood coagulation factor VIII is quantitatively detected based on the principle of a chromophoric substrate method, the detection operation is simple, the sensitivity is high, the result is reliable, and the reagent stability is good.

The present invention addresses the problem of providing a novel cell for ex vivo gene therapy that produces an FVIII-modified body. A cell for isolated gene therapy according to the present invention, which is a means for solving the problem, produces a human FVIII modified body comprising the amino acid sequence at positions 20to 2351st in the amino acid sequence represented by SEQ ID NO: 1 in which the B domain (the amino acid sequence at positions 760 to 1667th) is modified, the number of Asn residues modified by an N-type sugar chain is 4 to 6 from the N-terminal side, and the number of Asn residues modified by the N-type sugar chain is 4 to 6 from the N-terminal side, and the number of Asn residues modified by the N-type sugar chain is 4 to 6 from the N-terminal side. And a modified FVIII having a defect in the function of the furin recognition site.

The invention discloses a composition for gene editing and application thereof. The composition comprises i) a CRISPR / Cas gene editing element and a delivery vector thereof, and ii) a recombinant adeno-associated virus (AAV) vector carrying a target gene, and the target gene comprises at least one selected from the following genes: a blood coagulation factor VIII (F8) gene, an LDLR gene, a TTR gene, a PAH gene, an F9 gene, a PCSK9 gene or an SERPINC1 gene. The composition for gene editing disclosed by the invention can realize efficient gene integration and expression, reduce immune response and increase safety, and is suitable for patients of all ages, so that effective treatment choices are provided for different people.

The present application belongs to the technical field of blood product preparation, and particularly relates to a method for separating blood coagulation factor VIII and vWF factor from blood plasma. The method comprises the following steps in sequence: after cellulose is balanced, the cellulose is mixed with raw material blood plasma, filtered after stirring, and cellulose precipitate is obtained; after the cellulose precipitate is eluted, the cellulose precipitate is soaked in precipitate dissolving solution, and filtrate is obtained by filtering; the filtrate is subjected to three times of ion exchange chromatography, and factor VIII and / or vWF factor product is obtained. The technical scheme solves the technical problems of the prior art, such as complicated process steps for purifying and enriching human blood coagulation factor VIII and vWF from blood plasma cold precipitate, and unsatisfactory production efficiency and product quality. The cold precipitate protein solution prepared by the method can separate factor VIII, vWF factor and fibrinogen product at the same time through chromatography, improve the utilization rate of plasma protein, and has an ideal application prospect.

The present inventors examined the procoagulant activity of a multispecific antigen-binding molecule that functionally substitutes for FVIII using blood and plasma derived from FIX disorder patients. The result showed that multispecific antigen-binding molecules that functionally substitute for FVIII can be used not only as methods for preventing and / or treating bleeding in hemophilia A, acquired hemophilia A, von Willebrand disease, and hemophilia C, which are caused by FVIII dysfunction, but also as methods for preventing and / or treating bleeding in FIX disorders, because of their procoagulant activity. Furthermore, the effect of a FIX formulation could be enhanced by using it in combination with a multispecific antigen-binding molecule that functionally substitutes for FVIII, and it was shown that the combined use is promising as a combination therapy that shows stable hemostatic effects.

The invention discloses a preparation of a recombinant blood coagulation factor VIII, such as a pharmaceutical preparation and a freeze-drying preparation, particularly, the recombinant blood coagulation factor VIII is preferably a long-acting blood coagulation factor VIII, and the preparation has good stability and is suitable for long-term storage and clinical application.

The invention belongs to the technical field of blood product preparation, and particularly relates to a process for preparing cryoprecipitate from plasma, which is used for preparing a blood coagulation factor VIII or fibrinogen from plasma based on cellulose adsorption. The method comprises the following steps: balancing cellulose, mixing with raw material plasma, adsorbing, and filtering to obtain cellulose precipitate; after leaching the cellulose precipitate, soaking the cellulose precipitate in a precipitate dissolving solution; and filtering and taking filtrate to obtain a protein solution rich in the blood coagulation factor VIII and the human fibrinogen. According to the technical scheme, cellulose is added into low-temperature plasma, and the cellulose precipitate adsorbed with the plasma cryoprecipitate is separated in a filtering or filter-pressing mode and the like. And dissolving the cellulose precipitate, and purifying a conventional VIII factor and the like to obtain a high-purity blood coagulation factor VIII and fibrinogen product. According to the technical scheme, the technical problems that an existing blood coagulation factor VIII preparation method is high in process cost, not prone to expanding production and low in yield are solved, and ideal application and popularization value is achieved.

To provide a multi-specific antigen-binding molecule having an alternative function to the function of blood coagulation factor VIII.SOLUTION: The present invention provides a multi-specific antigen-binding molecule with high activity substituting for the function of blood coagulation factor VIII. The molecule is found out among various prepared bispecific antibodies each specifically binding to both the blood coagulation factor IX / activated blood coagulation factor IX and the blood coagulation factor X and having an alternative function to the cofactor function of the blood coagulation factor VIII, i.e., the function of promoting the activation of the blood coagulation factor X by the activated blood coagulation factor IX.SELECTED DRAWING: None

The inventors discovered that by administering a pharmaceutical composition comprising a bispecific antigen-binding molecule that recognizes blood coagulation factor IX and / or activated blood coagulation factor IX and blood coagulation factor X and / or activated blood coagulation factor X according to a given dosage regimen, diseases that develop and / or progress due to a decrease or deficiency in the activity of blood coagulation factor VIII and / or activated blood coagulation factor VIII can be prevented and / or treated more effectively.

The present inventors produced a variety of bispecific antibodies that specifically bind to both F. IX / F. IXa and F. X, and functionally substitute for F. VIIIa, i.e., have a cofactor function to promote F. X activation via F. IXa. Among these antibodies, the antibody A44 / B26 reduced coagulation time by 50 seconds or more as compared to that observed when the antibody was not added. The present inventors produced a commonly shared L chain antibody from this antibody using L chains of A44, and showed that A44L can be used as commonly shared L chains, although the activity of the resulting antibody is reduced compared to the original antibody (A44HL-B26HL). Further, with appropriate CDR shuffling, the present inventors successfully produced highly active multispecific antibodies that functionally substitute for coagulation factor VIII.

The invention discloses a prediction method for pharmacokinetic parameters of a blood coagulation factor VIII and a training method of a corresponding model. The training method comprises the following steps: acquiring historical clinical data of a child patient with hemophilia A; determining a truth value label of a pharmacokinetic parameter corresponding to the historical clinical data, and storing the historical clinical data and the truth value label as a first training data set in a table form; inputting the first training data set into a predetermined pharmacokinetic parameter prediction model for the blood coagulation factor VIII to obtain a predicted value of a corresponding pharmacokinetic parameter; and according to the truth value label and the predicted value, adjusting a preset model parameter of a pharmacokinetic parameter prediction model for the blood coagulation factor VIII to obtain a trained pharmacokinetic parameter prediction model for the blood coagulation factor VIII. Under the condition of greatly reducing the blood sampling frequency demand of the patient, the accurate prediction of the important PK parameter of the blood coagulation factor VIII is realized.

The present inventors produced a variety of bispecific antibodies that specifically bind to both F. IX / F. IXa and F. X, and functionally substitute for F. VIIIa, i.e., have a cofactor function to promote F. X activation via F. IXa. Among these antibodies, the antibody A44 / B26 reduced coagulation time by 50 seconds or more as compared to that observed when the antibody was not added. The present inventors produced a commonly shared L chain antibody from this antibody using L chains of A44, and showed that A44L can be used as commonly shared L chains, although the activity of the resulting antibody is reduced compared to the original antibody (A44HL-B26HL). Further, with appropriate CDR shuffling, the present inventors successfully produced highly active multispecific antibodies that functionally substitute for coagulation factor VIII.