WO2004065418A1 - 抗pci中和抗体 - Google Patents
抗pci中和抗体 Download PDFInfo
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- WO2004065418A1 WO2004065418A1 PCT/JP2004/000429 JP2004000429W WO2004065418A1 WO 2004065418 A1 WO2004065418 A1 WO 2004065418A1 JP 2004000429 W JP2004000429 W JP 2004000429W WO 2004065418 A1 WO2004065418 A1 WO 2004065418A1
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- antibody
- pci
- apc
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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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
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
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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
-
- 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
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- 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
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the present invention relates to a neutralizing antibody for protein C inhibitor (PCI).
- PCI protein C inhibitor
- Venous thrombosis frequently occurs after lower limb joint replacement or open surgery.
- the treatment is mainly performed prophylactically with small molecule heparin and opfarin.
- small molecule heparin requires daily subcutaneous administration, and perfurin can be administered orally, but its protein binding rate is so high that interaction with other drugs has become a problem.
- all drugs show a bleeding tendency.
- DIG dissected intravascular coagulation
- causes include solid tumors, infectious diseases and obstetric diseases.
- Heparin intravenous or subcutaneous
- the drug itself has a bleeding-promoting effect, and a sufficient effect cannot be obtained due to a decrease in antithrombin III concentration.
- There are drawbacks such as. Synthetic protease inhibitors are also prescribed, but their efficacy is not always clear.
- Anticoagulants such as heparin ⁇ synthetic antithrombin are prescribed for other conditions involving the blood coagulation system such as coronary artery syndrome and peripheral circulatory insufficiency. (Quality of Life) is low. Therefore, the effect is long (Several weeks) Antithrombotic drugs that are persistent and do not show a bleeding tendency can prevent thrombosis and improve the patient's QOL.
- Blood clots are formed by the activation of platelets and the blood coagulation system, and it is generally believed that platelets work in arterial thrombus and coagulation system works in venous thrombus.
- the thrombin generated by the activation of the blood coagulation system produces fipurin, which is the main component of the thrombus network, and changes its properties by binding to thrombomodulin present on the vascular endothelium.
- Activated PC (aPC), which uses protein S as a coenzyme, acts to inactivate Factor Va and illa and to suppress the rotation of the coagulation system.
- fibrinolytic inhibitors such as 1 (Plasminogen Activator Inhibitor-l) and TAFI (Thrombin Activated Fibrinolysis Inhibitor), thus promoting the fibrinolytic system.
- 1 Pulsminogen Activator Inhibitor-l
- TAFI Thrombin Activated Fibrinolysis Inhibitor
- aPC when aPC itself is used as a drug, its half-life in blood is extremely short, 20 to 30 minutes, so continuous intravenous administration or continuous administration over a long period of time is necessary.From the viewpoint of medical economics and Q0L of patients, Is also inconvenient.
- the reason for the short half-life is that aPC is irreversibly inactivated by an inactivating substance in the living body, such as PCI or ⁇ -antitrypsin (AAT).
- PCI Fibrinolysis & Proteolysis 2000; 14: 133-145
- a PC / PCI complex in blood On symptoms such as syndrome, DIC, and deep vein thrombosis Is reported to be elevated (Blood Coagul. Fibrinolysis 2001; 12: 503-5 10; Am. J. Hematol. 2000; 65: 35-40; Thromb. Haemost. 2001; 86: 1400-1408)
- PCI irreversibly inhibits enzyme activity by forming an acyl complex with aPC (J. Biochem. 1984; 95: 187-195). In addition, it inhibits the aPC-forming enzyme thrombin / tombo mombomodulin (Thr / TM) complex and suppresses aPC production (Blood 1998; 91: 1542-1547). That is, PCI suppresses the action of aPC by inhibiting both the production and activity of aPC. Therefore, by inhibiting the action of PCI, the action of endogenously generated aPC or exogenously administered aPC can be enhanced, and an effective anticoagulant action can be obtained. Disclosure of the invention
- An object of the present invention is to provide a use of an anti-PCI antibody having a neutralizing effect on PCI that inhibits enzyme activity of producing aPC.
- PCI (Suzuki, K. et al., J. Biol. Chem. 1983, 258: 163-168; Suzuki, ⁇ ⁇ , Fibrinolysis Proteolysis 2000, 14: 133-145) is soluble in blood. "It is a raw protein with a half-life of 23 hours. So, if a neutralizing antibody against this protein is produced and administered in a sufficient amount, it will be a long-lasting anticoagulant.
- PCn3 ⁇ 4 (1) has an inhibitory action on aPC generation by the Thr / TM complex (that is, an inhibitory action on PC activation), and (2) an inhibitory action on aPC activity. Therefore, the present inventors prepared a hybridoma that produces a monoclonal antibody against PCI, and screened each of the two actions of PCI for an inhibitory action. As a result, the inventors succeeded in isolating antibodies having an inhibitory effect on each of (1) and (2), and some of them inhibited both effects.
- the antibody of the present invention suppresses the action of the blood coagulation system by enhancing the action of aPC, and is therefore extremely useful for the treatment and prevention of thrombosis.
- the antibody of the present invention can be used in combination with aPC in the treatment of sepsis with aPC, etc. Thus, it can be used as a drug that enhances the action of aPC by suppressing the inactivation of aPC in blood.
- the present invention relates to the use of an anti-PCI antibody having a neutralizing effect on PCI which inhibits aPC generation and enzymatic activity, and more specifically, to the invention described in each claim.
- the present invention relates to inventions consisting of one or more (or all) of the inventions described in the claims, particularly to the same independent claim (inventions not included in the inventions described in other claims).
- the present invention also relates to an invention comprising a desired combination of one or more (or all) of the inventions described in the paragraphs (dependent claims) citing (a).
- the invention described in each independent claim also contemplates an invention composed of any combination of the dependent claims. That is, the present invention includes the following inventions.
- an anti-PCI antibody which comprises (a) an activity of inhibiting the inhibitory effect of a protein C inhibitor (PCI) on activated protein C (aPC) activity, or (b) a thrombin / thrompomodulin (Thr / TM) an activity of inhibiting the inhibitory effect of protein C inhibitor (PCI) on the production of activated protein C (aPC) by the complex;
- an anti-PCI antibody comprising: (a) an activity for inhibiting the inhibitory effect of a protein C inhibitor (PCI) on the activity of protein C (aPC); and (b) a thrombin / thrombomodulin (Thr / TM) antibodies that both inhibit the inhibitory effect of protein C inhibitor (PCI) on the production of active protein C (aPC) by the complex,
- an antibody containing a variable region of an antibody selected from the group consisting of PC19G8, PC23A7, PC23D8, PC30G1, PC31E2, PC31F1, and PC39C6, and an anti-sense site compete with the antibody, (1) or (2). Described antibodies,
- the complementarity-determining region comprises the amino acid sequence of any one of the following (a) to (f) or a functionally identical region:
- An antibody having a complementarity determining region such as (a) the amino acid sequence according to SEQ ID NOs: 49, 50 and 51,
- the antibody according to any one of [1] to [4], wherein the antibody is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, an antibody fragment, a single-chain antibody, and a diapody; Antibodies,
- composition comprising the antibody according to any one of (1) to (5) and a pharmaceutically acceptable carrier,
- composition according to (6) further comprising protein C and / or active protein C,
- composition according to [6] or [7] which is a pharmaceutical composition used for prevention or treatment of a disease that develops and / or develops due to a decrease or deficiency in the activity of Activated Protein C.
- composition according to (8), wherein the disease is a disease caused by enhanced blood coagulation reaction and enhanced Z or inflammatory response.
- a disease caused by enhanced blood coagulation reaction Z or an increased inflammatory response is selected from the group consisting of sepsis, disseminated intravascular coagulation syndrome, arterial thrombosis, and venous thrombosis;
- a method for preventing or treating a disease that develops or develops due to a decrease or deficiency in the activity of active protein D comprising: (a) protein C and no or active protein C; and (b) The method according to any one of (1) to (5), comprising:
- Onset due to decreased or insufficient activity of Activated Protein C Is a method for preventing or treating a developing disease, comprising: (1) administering the antibody according to any of (5),
- kits for use in the prevention or treatment of a disease that develops and / or develops due to a decrease or deficiency in the activity of Actividai Protein C comprising: (a) a kit comprising: A kit comprising the antibody of any one of the above, and (b) protein C, activated protein II, or both;
- a kit for use in the prevention or treatment of a disease that develops and / or develops Z or develops due to a decrease or lack of the activity of activated protein D comprising (a) protein ⁇ , activated protein C, and (1) to (5), and (b) (i) a therapeutically effective amount of protein C and Z or activated protein C, and (ii) the antibody, A recording medium comprising a link to the description.
- the present invention provides an anti-PCI antibody having a neutralizing effect on PCI that inhibits aPC generation and / or enzymatic activity, and use thereof.
- PCI has (1) an inhibitory action on aPC production (ie, activation of PC) by the Thr / TM complex, and (2) an inhibitory action on aPC activity.
- the antibody of the present invention It has a function of significantly inhibiting either the activity of (1) or (2), more preferably the activity of both (1) and (2). The inhibition of these activities can be measured, for example, by the method described in the Examples or other methods.
- the activity of PCI for inhibiting the activity of aPC is determined by incubating PCI with an anti-PCI antibody, adding it to an aPC solution, incubating the solution, and measuring the activity of aPC. Based on the aPC activity values when no PCI was added (aPC activity without PCI inhibition) and when no anti-PCI antibody was added (aPC activity with PCI inhibition), The rate at which the PCI ⁇ C body inhibited the inhibitory effect of PCI on aPC activity was determined. If incubation of the antibody with PCI reduces the degree of inhibition of PCI on aPC activity as compared to the absence of the antibody, this antibody is considered to have the activity of inhibiting the inhibitory effect of PCI on aPC activity. You.
- the activity of aPC is anticoagulant
- it can be quantified by measuring APTT (activated partial thromboplastin time) by a known method.
- APTT activated partial thromboplastin time
- the aPC activity can be assayed using a low-molecular compound such as the chromogenic substrate pyroGlu-Pro-Arg-pNA ⁇ HC1 (S-2366) (see Examples).
- PCI for example, PCI, an anti-PCI antibody, thrombin (Thr), and thrompomodulin (TM) are incubated. Do. Thereafter, the amount of generated aPC is measured by measuring the aPC activity. Based on the value of aPC activity when PCI was not added (aPC generation under conditions without inhibition by PCI) and without anti-PCI antibody (aPC generation when inhibited by PCI), the PCI Thr Of the anti-PCI antibody in the “inhibition of aPC generation by the / TM complex”.
- this antibody will inhibit the activity of PCI to inhibit the Thr / TM complex's inhibitory effect on aPC generation. It is determined to have.
- the activity of aPC can be quantified in the same manner as described above.
- the present invention also relates to a method for screening an anti-PCI antibody having a neutralizing effect on PCI, comprising the steps of: i) a step of measuring an activity of inhibiting aPC activity of PCI to which an anti-PCI antibody is bound or not; And ii) a step of selecting an antibody that inhibits the inhibitory effect of PCI bound with the antibody as compared to PCI not bound with the antibody.
- the present invention also relates to a method for screening an anti-PCI antibody having a neutralizing effect on PCI, comprising the steps of: i) using a Thr / TM complex of PCI with or without an anti-PCI antibody bound thereto.
- the present invention also provides antibodies obtainable by these screening methods.
- the antibody of the present invention can enhance the production and / or life span of aPC in vivo by preventing the production and / or activity of aPC in blood, thereby enhancing the activity.
- the anti-PCI antibody of the present invention may be a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, or a variant of those antibodies. Monoclonal antibodies are preferred because homogeneous antibodies can be stably produced.
- the “monoclonal antibody” in the present invention refers to a substantially homogeneous population of antibodies, that is, a population of antibodies in which the individual antibodies constituting the population are uniform except for a small amount of a naturally occurring variant that can occur in nature.
- the antibody obtained from Monoclonal antibodies are highly specific, acting against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single antigenic determinant on the antigen, as compared to a conventional polyclonal antibody preparation, which typically contains different antibodies directed against different antigenic determinants (epitopes).
- monoclonal antibodies are advantageous in that they are synthesized by hybridoma cultures that are not contaminated by other immunopurines.
- the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and does not limit that the antibody is produced by any particular method.
- the monoclonal antibody used in the present invention is produced by, for example, the hybridoma method (Kohler and Milstein, Nature 256: 95 (1975)) or the recombinant method (US Pat. No. 4,816,567). May be.
- the monoclonal antibodies used in the present invention may also be isolated from phage antibody libraries (Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1991)).
- a part of the heavy chain (H chain) and / or the light chain (L chain) is derived from a specific species or a specific antibody class or subclass, and the remaining part of the chain is Included are "chimeric" antibodies (immunoglobulins), antibody variants, and fragments of antibodies that are derived from another species or another antibody class or subclass (US Patent No. 4,816,567; Morrison et al., Pro Natl. Acad. Sci. USA 81: 6851-6855 (1984)).
- an “antibody variant” is one in which one or more amino acid residues are modified.
- the amino acid sequence variant of the antibody can be modified to improve the biological properties of the antibody, such as binding to the antigen.
- modifications can be made by methods such as site-specific mutation (see Kunkel, Proc. Natl. Acad. Sci. USA 82: 488 (1985)), PCR mutation, cassette mutation and the like.
- Such variants may have at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more, the amino acid sequence of the variable region of the heavy or light chain of the antibody.
- it has at least 90%, and most preferably at least 95%, amino acid sequence identity.
- the sequence identity is the same as that of the amino acid sequence of the original antibody after aligning the sequences as necessary so that the sequence identity is maximized, introducing appropriate gaps, and so on. Is defined as a percentage of
- the identity between the nucleotide sequence and the amino acid sequence can be determined by the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). Based on this algorithm, a program called BLASTN or BLASTX has been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990).
- BLASTN BLASTTN
- BLASTX BLASTX
- Polyclonal and monoclonal antibodies can be prepared by methods well known to those skilled in the art. For example, it can be manufactured by the following method.
- the PCI used for animal immunization is PCI prepared by the recombinant DNA method or chemical synthesis. And all or part of the amino acid sequence of The amino acid sequences of human and other mammalian PCIs are known (Suzuki, K. et al., J. Biol. Chem. 1987, 262: 611-616).
- a mammalian PCI for example, a mouse, rat, or mouse PCI can be used, but is not limited thereto (Zechmeister-Machhart, M., et. Al., Gene, 186, (1), 61-66, 1997; Wakita, T., et.
- the recombinant PCI protein can be prepared, for example, as described in Examples.
- an antigen PCI or its partial peptide itself can be used, or immunization can be performed by binding to a carrier protein.
- the antigen PCI is used as the carrier protein.
- an adjuvant After binding to (eg, thyroglobulin), an adjuvant is added.
- the adjuvant include Freund's complete adjuvant, Freund's incomplete adjuvant, and the like, and any of these may be mixed.
- the antigen obtained as described above is administered to mammals, for example, mammals such as mice, rats, hamsters, guinea pigs, pomas, monkeys, peas, goats, and sheep.
- Immunization can be performed by any existing method, but is mainly performed by intravenous injection, subcutaneous injection, intraperitoneal injection, or the like.
- the interval of immunization is not particularly limited, and immunization is performed at intervals of several days to several weeks, preferably at intervals of 4 to 21 days.
- the amount of the antigen protein to be immunized at a time can be, for example, 10 to: ⁇ ⁇ g (for example, 20 to 60 ⁇ g) per mouse. ⁇
- a flocculant such as pan-meal is preferably used.
- the selected mammalian antibody usually has a sufficiently strong binding affinity for the antigen.
- Antibody affinity can be determined by saturation binding, enzyme-linked immunosorbent assay (ELISA), and competition analysis (eg, radioimmunoassay).
- ELISA enzyme-linked immunosorbent assay
- competition analysis eg, radioimmunoassay.
- a screening method for polyclonal nano-antibodies a conventional cross-linking analysis as described in Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratoriey Harlow and David Lane edit. (1988)) can be performed.
- epitope mapping (Champe et al., J. Biol. Chem. 270: 1388-1394 (1995)) may be performed.
- a preferred method for measuring the efficacy of a polypeptide or antibody is a method using quantification of antibody binding affinity, but in other embodiments, in addition to that, or instead of binding affinity measurement And methods for assessing one or more biological properties of the antibody.
- Such assays are particularly useful because they indicate the therapeutic efficacy of the antibody.
- antibodies that exhibit improved properties in such assays also have increased binding affinity.
- the preparation of a hybridoma in the preparation of a monoclonal antibody can be performed, for example, according to the method of Milstin et al. (Kohler, G., and Milstein, C., Methods Enzyraol. 1981, 73, 3-46).
- myeloma (bone myeloma) cells to be fused with the antibody-producing cells cell lines derived from various animals such as mice, rats, and humans and commonly available to those skilled in the art are used.
- a cell line to be used a cell line which has drug resistance, has a property that it cannot survive in a selection medium (for example, HAT medium) in an unfused state, and can survive only in a fused state is used.
- a selection medium for example, HAT medium
- 8-azaguanine-resistant strains are used, which are deficient in hypoxanthine-guanine-phosphoribosyltransferase and cannot grow on hypoxanthine. Aminopterin. Thymidine (HAT) medium. .
- Myeloma cells include various cell lines already known, for example, P3x63Ag8.653 (J. Immunol. (1979) 123: 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81: 1- 7), NS-1 (Kohler,
- Antibody-producing cells are collected, for example, from animals sacrificed 2-3 days after the last immunization.
- Examples of the antibody-producing cells include spleen cells, lymph node cells, and peripheral blood cells, and spleen cells are generally used. Specifically, the spleen, lymph nodes, etc. are removed or collected from the various animals, and these tissues are crushed. The obtained crushed product is suspended in a medium or buffer such as PBS, DMEM, or RPMI1640, filtered through a stainless mesh or the like, and then subjected to centrifugal separation to prepare a desired antibody-producing cell.
- a medium or buffer such as PBS, DMEM, or RPMI1640
- the myeloma cells are fused with the antibody-producing cells.
- Cell fusion is performed by mixing myeloma cells and antibody-producing cells at a mixing ratio of 1 ::! To 1:20 in a medium for animal cell culture such as MEM, DMEM, and RPMI-1640 medium in the presence of a fusion promoter. This is done by contacting at 30-37 ° C for 1-15 minutes.
- a commercially available cell fusion device using a fusion promoter such as polyethylene glycol, polyvinyl alcohol or Sendai virus having an average molecular weight of 1,000 to 6,000 (Da) or a fusion virus is used.
- Antibody-producing cells and myeloma cells can also be fused.
- Select the desired hybridoma from the cells after cell fusion treatment examples include a method utilizing selective growth of cells in a selection medium. In other words, after diluting the cell suspension with an appropriate medium, spread it on a microtiter plate, select each well; add ground (such as HAT medium); Do. As a result, cells that grow can be obtained as hybridomas.
- McCafferty et al. (Nature 348: 552-554 (1990)).
- An antibody or an antibody fragment can be isolated from an antibody phage library produced using the techniques described above. Clackson et al. (Nature 352: 624-628 (1991)) and O3 ⁇ 4larks et al. (J. Mol. Biol.
- the anti-PCI neutralizing antibody of the present invention can be suitably selected by the following screening.
- the binding specificity of the antibody can be determined by known techniques such as EIA (enzyme immunoassay), RIA (radioimmunoassay), ELISA (enzyme linked immunosorbent assay), HTR F (homogenous time-resolved) Measure the fluorescence; or the fluorescence immunity method (Antibodies A Laboratory Manual. Ed Harlow, David Lane, old Spring harbor Laboratory, 1988), and select the one that binds to PCI.
- EIA enzyme immunoassay
- RIA radioimmunoassay
- ELISA enzyme linked immunosorbent assay
- HTR F homogenous time-resolved
- the aPC / PCI assay is an assay of the aPC activity inhibitory action of PCI as described above, and the Thr / TM / PCI assay is an aPC generation by the Thr / TM complex of PCI (PC activity ).
- the degree of inhibition of these PCI actions is measured, and an antibody having a relatively high degree of inhibition is selected. For example, if an antibody is obtained using an antibody obtained from an antibody-producing cell such as a hybridoma, an antibody having a desired activity is produced.
- the clones are subcloned by limiting dilution and grown by standard methods (Goding, Monoclonal Antibodies: Principals an Practice, pp. 59-103, Academic Press, 1986).
- As the culture medium for example, D-MEM or RPIM-1640 medium can be used.
- D-MEM or RPIM-1640 medium can be used as the culture medium.
- the relative value of PCI activity inhibition in the culture supernatant (1/5 (vol / vol)) is preferably 45 or more, more preferably 48 or more, and more preferably 50 or more. More preferably, the present invention provides a hybridoma having 60 or more, more preferably 70 or more, more preferably 80 or more, and more preferably 90 or more.
- the aPC / PCI assay of the hybridoma culture supernatant is performed, for example, according to the method described in Examples.
- Antibodies can be purified from the hybridoma culture supernatant by a conventional method.
- the antibody of the present invention has a 50% inhibitory concentration of preferably 100 g / ml or less, more preferably 100 g / ml in an aPC / PC I assay where the value when no antibody is added is 100% and the value when no PCI is added is 0%.
- 80 ig / ml or less more preferably 60 // g / ml or less, more preferably 50 ⁇ g / ml or less, more preferably 40 ⁇ / ⁇ 1 or less, more preferably 25 It is more preferably at most 15 ⁇ g / ml, more preferably at most 12.5 / ig / ml.
- the relative value of PCI inhibition at an antibody concentration of 25 ⁇ g / ml is preferably 40% or more, more preferably 50% or more, and more preferably 60% in the aPC / PCI assay. Above, more preferably It is at least 70%, more preferably at least 80%.
- the aPC / PCI assay of the purified antibody is performed, for example, according to the method described in Examples. In order to determine the 50% inhibition concentration, the assay may be performed by changing the antibody concentration, and a graph may be prepared to determine the antibody concentration corresponding to 50% inhibition.
- the antibody of the present invention preferably has an inhibitory activity on Thr / TM / PCI.
- the antibody of the present invention has a 50% inhibitory concentration of preferably 200 ⁇ g /% in the Thr / TM / PCI assay where the value when no antibody was added was 100% and the value when no PCI was added was 0%.
- ml or less more preferably 150 ⁇ g / ml or less, more preferably 100 ⁇ g / ml or less, more preferably 80 ⁇ g / ml or less, more preferably 50 ⁇ g / ml or less, more preferably 30 ⁇ g / ml or less.
- the relative value of PCI inhibition at an antibody concentration of 25 tg / ml is preferably 10% or more, more preferably 20% or more, more preferably 30% in the Thr / TM / PCI assay. It is above, more preferably 40% or more, and more preferably 50% or more.
- Thr / TM / PCI assay of the purified antibody is performed, for example, according to the method described in Examples. To determine the 50% inhibition concentration, perform an assay with different antibody concentrations, create a graph, and determine the antibody concentration corresponding to 50% inhibition! / ,.
- the antibody of the present invention may have either the PCI inhibitory activity on PC / PCI assay or the PCI inhibitory activity on Thr / TM / PCI assay 1 ", more preferably aPC / PCI.
- the antibody of the present invention has both a PCI inhibitory activity in the aPC / PCI assay and a PCI inhibitory activity in the Thr / TM / PCI assay.
- the inhibitory concentration is preferably 100 and 200 ⁇ g / ml or less, more preferably 80 and 150 g / ml or less, more preferably 60 and 100 ⁇ g / ml or less, more preferably 50 and 80 ⁇ g / ml, respectively.
- ⁇ g / ml or less more preferably 40 and 50 g / ral or less, more preferably 25 and 30 ⁇ g / ml or less, more preferably 15 and 25 ⁇ g / ml or less, more preferably 12.5 and Antibodies up to 25 g / ml are included.
- the antibodies of the present invention are considered to be more preferred if they have a stronger binding force to PCI.
- the antibody of the present invention has a dissociation constant (KD) of 50 nM or less, more preferably 20 nM or less, more preferably 10 nM or less, more preferably 5 nM or less, more preferably 3 nM or less.
- KD dissociation constant
- it is more preferably 1 nM or less, more preferably 0.8 nM or less, more preferably 0.6 nM or less, more preferably 0.4 nM or less, more preferably 0.2 nM or less.
- Kinetic parameters such as dissociation constant, dissociation rate constant (ka), dissociation rate constant (kd) and maximum binding amount (Rmax) can be determined by surface plasmon resonance analysis such as BIAC0RE. .
- the antibody of the present invention preferably has an activity of suppressing inactivation of aPC by blood.
- the antibody of the present invention suppresses inactivation of aPC by blood, preferably at least 10%, more preferably at least 15%, more preferably at least 20%, more preferably at least 25%, more preferably at least 30%.
- Antibody Such an inhibition level is defined as an inactivation inhibition rate (%), and the aPC activity at the same level as the aPC activity when inactivated by blood is 0%, and the aPC activity at the same level when not inactivated. It is expressed as a relative value with the aPC activity taken as 100%.
- the inactivation inhibition rate may be measured under optimal conditions while appropriately changing the dose of the antibody, and can be specifically determined by the following procedure. 10 / ig / mL aPC (SIGMA, # P-2200) solution ⁇ and antibody solution (for hybridoma screening, for example, hybridoma culture supernatant) or solution without antibody as control
- the culture supernatant of myeoma cells or HAT medium (For example, the culture supernatant of myeoma cells or HAT medium), and incubate at room temperature for a certain time (for example, 60 minutes).
- 50 zL of plasma eg, human standard plasma
- 50 AP TT reagent eg DADE BEHRING, GAA-200A
- Blood clotting time of aPC is measured by adding it to the plasma immediately before adding the APTT reagent.
- the antibody of the present invention may be an antibody that binds to a PCI site involved in, for example, interaction with aPC or Thrombin or selectivity.
- the site where PCI reacts with Thrombin and aPC is Arg 354 -Ser 355 (Suzuki, K. et ah, J. Biol. Chem. 1987, 262: 611-616) in human PCI after signal peptide cleavage. is there.
- sites involved in the selection of the PCI of Thrombin is Sites involved in selectivity to Phe 353, PCI of aPC is Thr 35 2 (Cooper, ST et al, Biochemistry 1995, 34:.
- the antibodies of the present invention include, for example, antibodies that bind to any of these amino acids in PCI or in the vicinity thereof (for example, a site within 10 amino acids).
- an oligopeptide containing the desired PCI moiety is synthesized, and the resulting antibody is used as an antigen to immunize an animal to produce the antibody.
- a method for collecting a monoclonal antibody from the obtained hybridoma includes a normal cell culture method, an ascites formation method, and the like.
- the hybridoma is cultured in an animal cell culture medium such as RPMI-1640 medium, MEM medium, or serum-free medium containing 10 to 20% fetal serum at normal culture conditions (eg, 37 ° C, 5 ° C). % C0. 2 to 2 concentration); cultured for 14 days, to obtain the culture supernatant antibody.
- an animal cell culture medium such as RPMI-1640 medium, MEM medium, or serum-free medium containing 10 to 20% fetal serum at normal culture conditions (eg, 37 ° C, 5 ° C). % C0. 2 to 2 concentration); cultured for 14 days, to obtain the culture supernatant antibody.
- the ascites formation method a hybridoma is administered intraperitoneally to an animal of the same species as a mammal derived from Mye chinoma cells, and the hybridoma is grown
- Antibodies used in the present invention may be prepared by known methods such as protein A-sepharose, protein G-sepharose, hydroxyapatite chromatography, sulfur salting out, ion exchange chromatography, and affinity chromatography. Purification can be achieved by selecting or combining these.
- the gene of the antibody obtained according to the above method is cloned from a hybridoma, inserted into an appropriate vector, introduced into a host, and produced using a gene recombination technique.
- Recombinant antibodies can be used (see, eg, Carl, AK Borrebaeck, James, W. Larrick, THERAPEUTIC M0N0CL ONAL ANTIBODIES, Published in the United Kingdom by MCMILLAN PUBLISHERS LTD, 1990).
- cDNA for the variable region (V region) of the antibody is synthesized from the mRNA of the hybridoma using reverse transcriptase.
- DNA encoding the V region of the desired antibody is obtained, it is ligated to the DNA encoding the desired antibody constant region (C region) and inserted into an expression vector.
- DNA encoding the V region of the antibody may be incorporated into an expression vector containing the DNA of the C region of the antibody.
- Expression control regions such as enhancers and promoters, are assembled into an expression vector to control expression. Put in.
- host cells can be transformed with this expression vector to express the antibody.
- the present invention provides a cell that expresses the antibody of the present invention.
- Cells expressing the antibody of the present invention include cells transformed with such antibody genes, and hybridomas.
- the antibody binding site overlaps with the antibody containing the variable region of any of the monoclonal antibodies (PC19G8, PC23A7, PC23D8, PC30G1, PC31E2, PC31F1, or PC39C6) isolated in the Examples. (Or the same) antibody.
- such an antibody is referred to as an antibody that binds to a site substantially the same as the PCI binding site of the monoclonal antibody (PC19G8, PC23A7, PC23D8, PC30G1, PC31E2, PC31F1, or PC39C6).
- Whether two antibodies bind to the same site on the antigen protein can be determined, for example, by competition experiments.
- the first antibody and the second antibody bind to the same antigen site Is determined.
- the epitope of the above monoclonal antibody is analyzed by a known epitope mapping method using a partial peptide of PCI, etc., and an antibody containing the identified epitope is used as an antigen to prepare an antibody that binds to the epitope.
- an antibody that binds to substantially the same site as the PCI binding site of the monoclonal antibody is expected to exert the same inhibitory effect on aPC generation and / or aPC activity as the antibodies isolated in the Examples.
- an antibody that binds to a site substantially the same as the PCI binding site of the antibody isolated in the Examples, and has an activity of inhibiting the inhibitory effect of PCI on aPC activity and / or Thr / TM For aPC generation by complex Antibodies having an activity to inhibit the inhibitory action of PCI are included in the present invention.
- the antibody of the present invention includes the capture determining region (CDRs) of any of the monoclonal antibodies (PC19G8, PC23A7, PC23D8, PC30G1, PC31E2, PC31F1, or PC39C6) isolated in the Examples or a functional region thereof. And antibodies containing a complementarity-determining region equivalent to the above. Functional equivalent means having an amino acid sequence similar to the amino acid sequence of CDRs of any of the monoclonal antibodies isolated in the Examples, and inhibiting the function of binding to PCI and inhibiting its function. Say you have. CDRs are regions in the variable region (also called V region) of an antibody that determine the specificity of binding to an antigen.
- CDRs are regions in the variable region (also called V region) of an antibody that determine the specificity of binding to an antigen.
- the CDRs are present in three places in each of the H chain and L chain, and are each located from the N-terminal side. Named CDR1, CDR2, CDR3. Four regions with high conservativeness of the amino acid sequence called a framework are interposed so as to sandwich the CDR. CDRs can be transplanted to other antibodies, and recombinant antibodies can be produced by combining the CDRs with a framework of a desired antibody. It is also possible to modify one or several CDR amino acids while maintaining antigen binding. For example, one or several amino acids in a CDR can be substituted, deleted, and / or added.
- amino acid residue to be mutated is desirably mutated to another amino acid that preserves the properties of the amino acid side chain.
- amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), Amino acids with aliphatic side chains (G, A, V, Les I, P), Amino acids with hydroxyl side chains (S, ⁇ , ⁇ ), Sulfur atom-containing side Amino acids with chains
- the number of amino acids to be mutated is not particularly limited, but is usually within 40%, preferably within 35%, and more preferably within 30% (eg, within 25%) of the amino acid of each CDR.
- the identity of the amino acid sequence may be determined as described herein.
- Examples of the antibodies included in the present invention include: ) (T / Y) (F / Y) (M / I) H (SEQ ID NO: 49), RID (Y / L) (V / E) (N / K) (G / V) N (T / I) (K / I) YDP (K / N) FQ (G / D) (SEQ ID NO: 50) and GGYDV (R / P) (E / S) FAY (SEQ ID NO: 51) (separated by slash Represents an amino acid of any of the above), or an antibody having CDRs functionally equivalent thereto. Each amino acid sequence corresponds to CDR1, CDR2, and CDR3 of the antibody heavy chain.
- the PCI neutralizing antibody of the present invention can be prepared. More specifically, preferred amino acid sequences for the H chain CDR of such an antibody are as follows: DTFMH (SEQ ID NO: 22) or DYYIH (SEQ ID NO: 23) as CDR1, and RIDYVN GNTKYDPKFQG (SEQ ID NO: 26), RIDLVNVNTKYDPNFQD (SEQ ID NO: 27), or RIDLEKGNIIYDPKFQG (SEQ ID NO: 28), and GGYDVREFAY (SEQ ID NO: 32) or GGYDVPSFAY (SEQ ID NO: 33) as CDR3.
- DTFMH SEQ ID NO: 22
- DYYIH SEQ ID NO: 23
- RIDYVN GNTKYDPKFQG SEQ ID NO: 26
- RIDLVNVNTKYDPNFQD SEQ ID NO: 27
- RIDLEKGNIIYDPKFQG SEQ ID NO: 28
- the amino acid may be appropriately modified by substitution, etc.
- conservative substitution of the amino acid of each CDR is included in the scope of the present invention. (See Figure 5.)
- These antibodies can be used to bind the H chain CDR1, 2, and 3 of PC23D8, PC19G8, PC23A7, or PC39C6 (see Figure 5). Expected to have activity It is.
- Antibodies containing the above H chain CDRs can be used in combination with antibody L chain variable regions as appropriate.
- L chain CDRs include amino acids of SA (T / S) SS (L / V) (I / S) YMH (SEQ ID NO: 55), STSNLASGVPA (SEQ ID NO: 56), and RSSYPFT (SEQ ID NO: 57) It is preferable to combine CDRs consisting of a sequence or CDRs functionally equivalent thereto. Each amino acid sequence corresponds to antibody CDR1, CDR2, and CDR3. Further, these L chain CDRs may be used independently of the above H chain. These CDRs are inserted at positions corresponding to CDR1, CDR2, and CDR3 between the frameworks of the desired L chain variable region.
- preferred amino acid sequences for the L chain CDR of such an antibody are: SATSSLIYMH (SEQ ID NO: 37) or SASSSVSYMH (SEQ ID NO: 38) as CDR1, and STSNLASGVPA (SEQ ID NO: 42) as CDR2. ), RS SYPFT (SEQ ID NO: 46) is used as CDR3, but is not limited thereto.
- the antibody of the present invention includes an antibody having the following H chain complementarity-determining region, and has an activity of inhibiting the inhibitory effect of PCI on aPC production by a Thr / TM complex and / or an activity of aPC Antibodies having an activity of inhibiting the inhibitory effect of PCI on the activity are included.
- a complementarity determining region consisting of an amino acid sequence having 70% or more identity to SEQ ID NOS: 49, 50, and 51, respectively.
- the number of amino acid modifications in (c) is preferably within 2 and more preferably 1 in SEQ ID NO: 49. Also preferably, within 7 of SEQ ID NO: 50, more preferably within 6, more preferably within 5, more preferably within 4, more preferably Or no more than three. Further, the number is preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and still more preferably 1 of SEQ ID NO: 51.
- the identity in (d) is preferably 75% or more, more preferably 80% or more, further preferably 90% or more, and further preferably 95% or more.
- the antibody of the present invention includes an antibody having the following L chain complementarity determining region, wherein the activity of inhibiting the inhibitory effect of PCI on aPC production by the Thr / TM complex and / or the inhibitory effect of PCI on aPC activity: Antibodies that have the activity of inhibiting A.
- up to 5 amino acids of SEQ ID NO: 55, up to 5 amino acids of SEQ ID NO: 56, and up to 4 amino acids of SEQ ID NO: 57 consist of substituted, deleted and / or modified amino acid sequences Complementarity determining region.
- a complementarity determining region consisting of an amino acid sequence having 70% or more identity to SEQ ID NOs: 55, 56, and 57, respectively.
- the number of amino acid modifications in (c) is preferably no more than 4, more preferably no more than 3, more preferably no more than 2, and even more preferably 1, in SEQ ID NO: 55.
- the number of SEQ ID NO: 56 is 4 or less, more preferably 3 or less, further preferably 2 or less, and more preferably 1 or less.
- the number of SEQ ID NO: 57 is 3 or less, more preferably 2 or less, and still more preferably 1.
- the identity in (d) is preferably at least 75%, more preferably at least 80%, further preferably at least 90%, further preferably at least 95%.
- an antibody having both the above-described H chain complementarity determining region and L chain complementarity determining region is suitable as the antibody of the present invention.
- the antibodies of the present invention include RYWMS (SEQ ID NO: 52), EINPDSSTI (N / T) YT (P / S) S LKD (SEQ ID NO: 53), and amino acids of (F / FYYGTPDY (SEQ ID NO: 54)) Array Or an antibody having a CDR functionally equivalent thereto. As above, each amino acid sequence corresponds to CDR1, CDR2, and CDR3 of the antibody H chain.
- preferred amino acid sequences for the H chain CDR of such an antibody are as follows: RYWMS (SEQ ID NO: 24) as CDR1, EI PDSSTINYTPSLKD (SEQ ID NO: 29) or EINPDSSTITYTSSLKD as CDR2 (SEQ ID NO: 30), CDR3 includes FFYY GTPDY (SEQ ID NO: 34) or LFYYGTPDY (SEQ ID NO: 35).
- a combination of CDR1, 2, and 3 of the H chain of the monoclonal antibody PC30G1 or PC31F1 can be used. These antibodies are expected to have a PCI neutralizing activity equivalent to PC30G1 or PC31F1.
- L chain CDRs for example, the amino acid sequence of KASQDVI (V / K) AVA (SEQ ID NO: 58), S (A / T) SYRYTGVPD (SEQ ID NO: 59), and 3 ⁇ 4YSSPPWT (SEQ ID NO: 60) Or a CDR functionally equivalent thereto.
- Each amino acid sequence corresponds to CDR1, CDR2, and CDR3 of the antibody light chain.
- these L chain CDRs may be used independently of the above H chain.
- CDR 1 is KASQDVIVAVA (SEQ ID NO: 39) or KASQDVIKAVA (SEQ ID NO: 40);
- CDR2 is SASYRYTGVPD (SEQ ID NO: 43) or STSYRYTGVPD ( SEQ ID NO: 44), and HYSSPPWT (SEQ ID NO: 47) is used as CDR3, but is not limited to these sequences.
- the antibody of the present invention includes an antibody having the following complementarity-determining region, which has an activity of inhibiting the inhibitory effect of PCI on aPC production by the Thr / TM complex and / or an activity of inhibiting the aPC activity.
- Antibodies having an activity of inhibiting the inhibitory action of PCI are included.
- SEQ ID NO: within 3 of 52, SEQ ID NO: within 8 of 53, and SEQ ID NO: 5 of 54 A complementarity determining region consisting of an amino acid sequence in which no more than one amino acid has been substituted, deleted and / or added.
- the number of amino acid modifications in (c) is preferably within 2 and more preferably 1 in SEQ ID NO: 52. It is also preferable that the number of SEQ ID NO: 53 is 7 or less, more preferably 6 or less, further preferably 5 or less, more preferably 4 or less, and still more preferably 3 or less. Also preferably, the number of SEQ ID NO: 54 is 4 or less, more preferably 3 or less, further preferably 2 or less, and more preferably 1 or less.
- the identity in (d) is preferably 75% or more, more preferably 80% or more, further preferably 90% or more, and further preferably 95% or more.
- the antibody of the present invention includes an antibody having the following L chain complementarity determining region, wherein the activity of inhibiting the inhibitory effect of PCI on aPC production by the Thr / TM complex and / or the inhibitory effect of PCI on aPC activity: Antibodies that have the activity of inhibiting A.
- up to 5 amino acids of SEQ ID NO: 58, up to 5 amino acids of SEQ ID NO: 59, and up to 4 amino acids of SEQ ID NO: 60 are composed of 'substituted, deleted and / or added amino acid sequences Complementarity determining region.
- the number of amino acids modified in (c) is preferably within 4 in SEQ ID NO: 58, more preferably within 3, more preferably within 2, and even more preferably 1.
- the number of SEQ ID NO: 59 is 4 or less, more preferably 3 or less, even more preferably. Or less than two, more preferably one.
- the number of SEQ ID NO: 60 is 3 or less, more preferably 2 or less, and still more preferably 1.
- the identity in (d) is preferably at least 75%, more preferably at least 80%, further preferably at least 90%, and further preferably at least 95%.
- an antibody having both the above-described H chain complementarity determining region and L chain complementarity determining region is suitable as the antibody of the present invention.
- the antibodies of the present invention include CDRs comprising the amino acid sequences of TYPIE (SEQ ID NO: 25), KFHPDNDDTNYNEKFKG (SEQ ID NO: 31), and GHDYDYGMDY (SEQ ID NO: 36), or CDRs functionally equivalent thereto.
- CDRs comprising the amino acid sequences of TYPIE (SEQ ID NO: 25), KFHPDNDDTNYNEKFKG (SEQ ID NO: 31), and GHDYDYGMDY (SEQ ID NO: 36), or CDRs functionally equivalent thereto.
- CDRs consisting of the amino acid sequence of KASQSVDYDGDSYLN (SEQ ID NO: 41), GASNLESGTPA (SEQ ID NO: 45), and SNEDPPT (SEQ ID NO: 48) or CDRs functionally equivalent thereto are used as L chain CDRs. Is preferably combined. Each amino acid sequence corresponds to the antibody L chain CDR1, CDR2, and CDR3.
- the antibody of the present invention includes an antibody having the following H chain complementarity-determining region, and has an activity of inhibiting the inhibitory effect of PCI on aPC production by a Thr / TM complex and / or an activity of aPC Antibodies having an activity of inhibiting the inhibitory effect of PCI on the activity are included.
- (d) A complementarity determining region consisting of an amino acid sequence having 70% or more identity to SEQ ID NOs: 25, 31, and 36, respectively.
- the number of amino acid modifications in (c) is preferably within 2 and more preferably 1 in SEQ ID NO: 25.
- the number of SEQ ID NO: 31 is 7 or less, more preferably 6 or less, further preferably 5 or less, more preferably 4 or less, and still more preferably 3 or less.
- it is within 4 of SEQ ID NO: 36, more preferably within 3, more preferably within 2, and still more preferably 1.
- the identity in (d) is preferably 75% or more, more preferably 80% or more, further preferably 90% or more, and further preferably 95% or more.
- the antibody of the present invention includes an antibody having the following L chain complementarity determining region, wherein the activity of inhibiting the inhibitory effect of PCI on aPC production by the Thr / TM complex and / or the inhibitory effect of PCI on aPC activity: Antibodies that have the activity of inhibiting A.
- a complementarity-determining region consisting of an amino acid sequence having 70% or more identity to SEQ ID NOs: 41, 45, and 48, respectively.
- the number of amino acid modifications in (c) is preferably within 4 in SEQ ID NO: 41, more preferably within 3, more preferably within 2, and even more preferably 1.
- the number of SEQ ID NO: 45 is 4 or less, more preferably 3 or less, further preferably 2 or less, more preferably 1 or less.
- the number of SEQ ID NO: 48 is 3 or less, more preferably 2 or less, and still more preferably 1.
- the identity in (d) is preferably at least 75%, more preferably at least 80%, further preferably at least 90%, and further preferably at least 95%.
- the H chain complementarity determination An antibody having both a region and an L chain complementarity determining region is suitable as the antibody of the present invention.
- an oligonucleotide encoding a variable region containing a CDR in which the amino acid sequence has been modified is synthesized, and based on these, a nucleic acid encoding the variable region is generated by PCR.
- An antibody having a desired CDR can be obtained by incorporating this into an appropriate expression vector for expression. For example, bases are mixed during the synthesis of an oligonucleotide to prepare a DNA library encoding an antibody having various amino acids at specific positions in the CDR. From the library, the antibody of the present invention can be obtained by selecting a clone encoding an antibody that binds to PCI and suppresses its function.
- the present invention relates to a nucleic acid encoding the antibody of the present invention, a vector containing the nucleic acid, and a host cell containing the nucleic acid or the vector.
- the nucleic acid may be DNA or RNA.
- the vector may be a known desired vector such as a plasmid, a phage or a virus vector.
- Host cells include pacteria, yeast, insect, plant cells, and mammalian cells.
- a recombinant antibody artificially modified for the purpose of, for example, reducing the antigenicity against humans such as a chimeric antibody or a humanized antibody
- modified antibodies can be produced using known methods.
- Chimeric antibodies include antibodies in which the variable region and the constant region of the antibody are different from each other.For example, mammals other than humans, such as the heavy chain of a mouse antibody, the variable region of a light chain and the heavy chain of a human antibody, Antibodies consisting of the constant region of the light chain.
- Such an antibody can be obtained by ligating DNA encoding the variable region of a mouse antibody to DNA encoding the constant region of a human antibody, incorporating the DNA into an expression vector, introducing the resulting DNA into a host, and producing the antibody. .
- a humanized antibody is also called a reshaped human antibody.
- the complementarity determining region (CDR) of the heavy or light chain of a non-human mammal such as a non-human antibody such as a mouse antibody is replaced with a human. It is replaced by the phase capture individual determination region of the antibody, and general gene recombination techniques are also known (for example, Jones e etal., Nature 321: 522-525 (1986); Reichmann et al., Nature 332: 323-329 (1988); Presta Curr. Op. Struct. Biol. 2: 593-596 (1992)).
- CDR complementarity determining region
- the obtained DNA is ligated to DNA encoding the constant region of a human antibody, then inserted into an expression vector, and introduced into a host to produce it (European Patent Application Publication No. EP 23 9400, International Patent Application Publication Number W0 96/02576).
- the human antibody FR linked via CDR is selected so that the complementarity determining region forms a favorable antigen-binding site.
- the humanized antibody may contain amino acid residues that are not included in either the CDR or the framework sequences introduced into the recipient antibody. Usually, such introduction of an amino acid residue is performed in order to more accurately optimize the antigen recognition / binding ability of an antibody.
- amino acids in the framework region of the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen-binding site (Sato, K. et al. , Cancer Res. (1993) 53, 851-856).
- a method for obtaining a human antibody is known.
- a human lymphocyte is sensitized in vitro with a desired antigen or a cell expressing the desired antigen, and the sensitized lymphocyte is fused with a human myeloma cell such as U266 to obtain a desired human having an antigen-binding activity.
- Antibodies can also be obtained (see Japanese Patent Publication No. 1-59878).
- a desired human antibody can be obtained by immunizing a transgenic (Tg) animal having part or all of the repertoire of the human antibody gene with an antigen (Nature Genetics 7:13).
- Tg animals are disrupted in the locus of the endogenous immunoglobulin heavy chain and light chain of a non-human mammal, and instead use yeast artificial chromosome (YAC) vectors or the like.
- YAC yeast artificial chromosome
- Functional inactivation of the immunoglobulin heavy chain locus can be achieved, for example, by introducing a disorder in a portion of the J region or C region (e.g., the C / region), and by immunoglobulin light chain (e.g., The functional inactivation of the ( ⁇ chain) can be achieved, for example, by introducing a disorder into the ⁇ region of the J region or C region, or a region including a region extending between the J region and the C region.
- a eukaryotic cell is transformed with a cDNA encoding each of the heavy chain and light chain of such a humanized antibody, preferably a vector containing the cDNA, by a genetic recombination technique.
- This antibody can also be obtained from the culture supernatant by culturing the transformed cells that produce E. coli.
- the host is, for example, a desired eukaryotic cell, preferably a CH0 cell, or a mammalian cell such as a lymphocyte or myeoma.
- a technique for obtaining a human antibody by Panjung using a human antibody library is also known.
- a phage that binds to an antigen can be selected by expressing the variable region of a human antibody as a single-chain antibody (scFv) on the surface of a phage by a phage display method.
- scFv single-chain antibody
- the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined.
- a human antibody can be obtained by preparing an appropriate expression vector having the sequence and introducing it into an appropriate host for expression.
- animal cells include (1) mammalian cells, for example, CHO, COS, myeloma, BHK (ba by hamster kidney), HeLa, Vero, (2) amphibian cells, for example, African omega oocytes, or (3) insects Cells include, for example, individuals such as Sf9, Sf21, Tn5, and the like.
- Known plant cells include cells derived from the genus Nicotiana, for example, Nicotiana tabacum, which may be callus cultured.
- Fungal cells include yeast, for example, the genus Saccharomyces, for example, Saccharomyce s serevisiae, filamentous fungi, for example, the genus Aspergillus, for example, Aspergillus niger. It has been known. When using prokaryotic cells, some production systems use bacterial cells. Escherichia coli (E. coli) and Bacillus subtilis are known as bacterial cells. An antibody can be obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro.
- yeast for example, the genus Saccharomyces, for example, Saccharomyce s serevisiae
- filamentous fungi for example, the genus Aspergillus, for example, Aspergillus niger. It has been known.
- prokaryotic cells some production systems use bacterial cells. Escherichia coli (E. coli) and Bacillus subtilis are known as bacterial cells
- the isotype of the antibody of the present invention is not limited, and includes, for example, IgG (igGl, IgG2, IgG3, IgG4), IgM, IgA (IgAl, IgA2), IgD or IgE, and preferably IgG or IgM. is there.
- the antibody of the present invention may be an antibody fragment having an antigen-binding portion of the antibody or a modified product thereof.
- An “antibody fragment” refers to a portion of a full-length antibody and generally refers to a fragment that contains an antigen-binding or variable region.
- antibody fragments include Fab, F (ab ') 2 , Fv, or single-chain Fv (scFv), diabody (diabodies), Examples include linear antibodies, and multispecific antibodies formed from antibody fragments.
- antibody fragments have been produced by digestion of natural antibodies with proteases. At present, however, methods for expressing them as recombinant antibodies by genetic engineering are also known (Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); Brennan et al., Science 229: 81 (1985); Co, MS et al., J. Immunol., 1994, 152, 2968-2976; Better, M.
- an “Fv” fragment is the smallest antibody fragment and contains a complete antigen recognition and binding site.
- This region is a dimer in which the variable regions of one heavy and light chain are tightly linked by non-covalent bonds ( VH - VL dimer).
- the three complementarity determining regions (CDRs) of each variable region interact to form an antigen-binding site on the surface of the VH -dimer. That is, six CDRs, including the heavy chain and the light chain, function as the antigen-binding site of the antibody.
- CDRs complementarity determining regions
- the antibody fragment in the present invention is preferably an Fv fragment, but is not limited thereto.
- Polypeptides including antibody fragments having heavy chain or light chain CDRs conserved and capable of recognizing and binding to antigens are conserved. It may be a peptide.
- the Fab fragment (also called F (ab)) further contains a light chain constant region and a heavy chain constant region (CH1).
- Fab fragment an antigen-binding fragment called the Fab fragment, which contains the variable regions of the heavy and light chains forming one antigen-binding site, and ⁇ Fc '' for easy crystallization.
- the resulting fragment is called.
- Fab 'fragments differ from Fab fragments by the addition of a few residues derived from the carboxy terminus of the heavy chain CH1 region including one or more cysteines in the hinge region of the antibody, but have a single antigen binding It is structurally equivalent to Fab in that it is an antigen-binding fragment containing the variable regions of the heavy and light chains that form the site.
- Fab-like antibody An antigen-binding fragment containing the variable regions of the heavy and light chains forming one antigen binding site, equivalent to that obtained by pine digestion, is called a Fab-like antibody.
- Fab'-SH is a protein in which one or more cysteine residues in the constant region are free 1 shows Fab ′ having a thiol group.
- F (ab ') 2 fragments are produced by cleavage of disulfide bonds in cysteines at the hinge of F (ab') 2 .
- Other chemically ligated antibody fragments are known to those of skill in the art.
- F (ab ') 2 fragment Digestion of the antibody with pepsin yields a F (ab ') 2 fragment that has two antigen-binding sites and is capable of cross-linking antigen, and another fragment, referred to as pFc'.
- an antibody fragment having two antigen-binding sites and capable of cross-linking an antigen, which is equivalent to that obtained by pepsin digestion is referred to as an F (ab ') 2- like antibody.
- these antibody fragments can be produced by recombinant techniques.
- an antibody fragment can be isolated from the antibody phage library described above.
- the F (ab,) 2 -SH fragment can be directly recovered from a host such as Escherichia coli and chemically bound to the form of the F (ab ') 2 fragment (Carter et al., Bio / Technology 10: 163). -167 (1992)).
- F (ab,) 2 fragments can be isolated directly from recombinant host cultures.
- the antibodies used in the present invention may be multispecific antibodies.
- Multispecific antibodies are antibodies that have specificity for at least two different antigens. Usually such molecules bind two antigens (i.e., bispecific antibodies), but "multispecific antibodies" in the present invention include more (e.g., 3 ) Antigens.
- a multispecific antibody can be a full-length antibody, or a fragment of such an antibody (eg, a F (ab ') 2 bispecific antibody).
- Bispecific antibodies can be produced by combining the heavy chain and light chain (HL pair) of two types of antibodies, or by combining hybridomas that produce different monoclonal antibodies to produce bispecific antibodies.
- Fusion cells can also be produced and obtained (Millstein et al., Nature 305: 537-539 (1983)).
- bispecific antibodies can be produced by genetic engineering techniques.
- the variable region of the antibody with binding specificity is fused to the constant domain sequence of an immunoglobulin.
- the constant domain sequence preferably contains at least a part of hinge, CH2 and CH3 regions in the constant region of the heavy chain of immunoglobulin. Preferably, it is necessary for further binding to the light chain. Contains the CHI region of the required heavy chain.
- the DNA encoding the immunoglobulin heavy chain fusion and, if desired, the DNA encoding the immunoglobulin light chain are inserted into separate expression vectors, and transformed into a suitable host organism.
- each gene By introducing each gene into a separate expression vector, it is convenient to control the expression ratio of each chain if the abundance ratio of each chain is not the same, if the yield of the obtained antibody increases.
- genes encoding a plurality of chains can be inserted into one vector and used.
- Diapod (Db) refers to a bivalent antibody fragment constructed by gene fusion (P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-644 8 (1993)). , EP404, 097, W093 / 11161, etc.).
- a diabody is a dimer composed of two polypeptide chains, each of which is in a position where the light chain variable region () and heavy chain variable region (V H ) cannot bind to each other in the same chain. It is linked by a short, for example, about 5 residue linker.
- V L and V H in the same polypeptide chain, to form a dimer can not be form a single chain V region fragments for the linker one short therebetween, Daiabodi binding two antigens Site.
- two different antigens (a, b) V
- Db may be used as the antibody of the present invention.
- a single-chain antibody (also referred to as scFv) can be obtained by linking the heavy chain V region and the light chain V region of the antibody.
- scFv single-chain antibody
- Methods for making single chain antibodies are well known in the art (eg, US Pat. No. 4,946,778, US Pat. No. 5,260,203, US Pat. No. 5,091,513). And US Pat. No. 5,455,030).
- the heavy and light chain V regions are linked via a linker, preferably a polypeptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. US A, 1988). , 85, 587 9-5883).
- the heavy chain V region and light chain V region in the scFv may be derived from the same antibody or from different antibodies.
- As the peptide linker connecting the V regions for example, any single-chain peptide consisting of 12 to 19 residues is used.
- the DNA encoding the scFv may be any of the DNA encoding the heavy chain or heavy chain V region and the DNA encoding the light chain or light chain V region of the antibody, and all or a desired amino acid sequence thereof.
- the DNA sequence encoding the acid sequence was type III, amplified by the PCR method using a pair of primers defining both ends, and then the DNA encoding the peptide linker portion, and both ends were separated from the heavy and light chains, respectively. It is obtained by combining and amplifying a primer pair that is defined to be ligated.
- an expression vector containing them and a host transformed with the expression vector can be obtained according to a conventional method.
- scFv can be obtained according to a conventional method.
- These antibody fragments can be obtained and expressed in the same manner as described above, and can be produced by a host.
- an antibody bound to various molecules such as polyethylene glycol (PEG) can also be used. Methods for modifying antibodies are well established in the art.
- the “antibody” in the present invention also includes these antibodies.
- the obtained antibody can be purified to homogeneity.
- the separation and purification of the antibody may be performed by the separation and purification methods used for ordinary proteins. For example, if you select and combine chromatographic columns, filters, ultrafiltration, salting out, dialysis, polyacrylamide gel electrophoresis for preparation, isoelectric focusing, etc. Saru (Strategies for Protein Purification and Charcterization: A Laboratoy Course Manual, Daniel R. Marshak et al. Eds., Cold Spring
- Chromatography includes affinity chromatography, ion exchange chromatography, hydrophobic 1 "raw chromatography, and gel filtration. And reversed-phase chromatography, adsorption chromatography and the like. These chromatographys can be performed using liquid phase chromatography such as HPLC and FPLC.
- Columns used for affinity chromatography include a protein tin A column and a protein G column. For example, columns using a protein A column include Hyper D, POROS, Sepharose FF (Pharmacia) and the like. It is also possible to purify the antibody by using the carrier having the antigen immobilized thereon and utilizing the binding property to the antigen.
- the present invention provides an inhibitor of the activity of PCI, comprising the antibody of the present invention.
- the present invention also relates to the use of the antibody of the present invention for inhibiting the activity of PCI.
- the present invention also relates to a method for inhibiting the activity of PCI, comprising the step of contacting the antibody of the present invention with PCI. By contacting the antibody of the present invention with PCI, it is possible to suppress the inhibition of aPC production by PCI and / or the inhibition of aPC activity by PCI.
- the present invention also provides an enhancer for the production or activity of aPC, comprising the antibody of the present invention.
- the present invention also relates to the use of the antibody of the present invention for generating aPC and / or enhancing the activity.
- the step of contacting the antibody of the present invention with PCI it is possible to suppress a decrease in intrinsic or foreign PC activity and / or aPC activity through inhibition of PCI.
- the antibody can be administered alone or in combination with PC and / or aPC.
- aPC is known to have the effect of suppressing blood coagulation and inflammation, and the step of administering the PCI-neutralizing antibody of the present invention makes it possible to enhance the effect of aPC that suppresses blood coagulation or inflammation.
- the present invention relates to a method for suppressing blood coagulation or inflammation, comprising a step of administering the antibody of the present invention.
- the method may further include the step of administering PC and / or aPC.
- PC and / or aPC and the antibody of the present invention may be mixed in advance and administered, or may be administered separately.
- a pharmaceutical preparation containing the antibody of the present invention as an active ingredient, the effect of aPC in treatment with aPC (eg, prevention and treatment of thrombosis and sepsis) can be improved. It is possible to increase.
- “containing the antibody of the present invention as an active ingredient” means that the antibody of the present invention is contained as at least one of the active ingredients, and does not limit the content of the antibody of the present invention.
- the antibody of the present invention is useful for the prevention or treatment of a disease that develops and / or progresses due to a decrease or deficiency in the activity of activated protein C. Among them, the blood coagulation reaction and / or the inflammatory reaction are enhanced.
- the present invention also provides a kit comprising (a) the antibody of the present invention, and (b) PC and / or aPC. It can be used for the prevention or treatment of a disease that develops and / or progresses due to a decrease or deficiency in the activity of the protein.
- Ishi is a kit used for prevention or treatment of a disease that develops and / or progresses due to deficiency, wherein (a) at least one selected from the group consisting of PC, aPC, and the antibody of the present invention; And (b) a recording medium containing a therapeutically effective amount of PC and / or aPC in combination with the antibody or a recording medium containing a link to the description.
- the disease include a disease caused by an enhanced blood coagulation reaction and / or an increased inflammatory response as described above, and specifically include arterial thrombosis, venous thrombosis, DIC, and sepsis.
- the kit is useful for relatively increasing the in vivo activity of endogenous aPC or PC or aPC administered from outside the living body, thereby enabling prevention and treatment of the above-mentioned diseases.
- Desirable recording media such as printing media such as paper and plastic, flexible discs (FD), compact discs (CD), digital video discs (DVD), and computer-readable recording media such as semiconductor memory are exemplified.
- FD flexible discs
- CD compact discs
- DVD digital video discs
- computer-readable recording media such as semiconductor memory
- the link does not directly describe the use of the antibody in combination with a therapeutically effective amount of PC and / or aPC, but refers to the fact that it is linked to the description by a mark or the like. This is the case when you can get to ⁇ .
- the instruction sheet contains a description that indicates or suggests that a reference is made to a separate sheet or a URL.
- Such links are preferably reached up to the description with up to three references (with a link depth of less than 3), more preferably with two references (with a link depth of 2), and more preferably with one reference. (The link depth is 1).
- the antibody of the present invention can be administered either orally or parenterally, but is preferably administered parenterally.
- injection, nasal, pulmonary, transdermal Dosing type is an example.
- injection forms include systemic or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like.
- the administration method can be appropriately selected depending on the age and symptoms of the patient.
- the dose can be selected, for example, in the range of 0.0001 mg to 1000 mg / kg body weight at a time. Alternatively, for example, the dose can be selected within the range of 0.001 to 100000 mg / body per patient.
- the antibody of the present invention is not limited to these doses.
- the antibody of the present invention can be formulated according to a conventional method (for example, Remington's Pharmaceutical Science, latest edition, Mark PuDiishing Company, Easton, U.S.A.), a pharmaceutically acceptable carrier and / or Alternatively, it may contain an additive.
- a pharmaceutically acceptable carrier for example, a pharmaceutically acceptable carrier and / or Alternatively, it may contain an additive.
- an additive for example, surface active agents (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), coloring agents, flavoring agents, preservatives, stabilizers, buffers (phosphoric acid, quinic acid, etc.) Acids, chelating agents (EDTA, etc.), suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity enhancers, flavoring agents, etc., but are not limited thereto.
- aqueous solution for injection for example, physiological saline, isotonic solution containing glucose and other adjuvants, for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc.
- a solubilizing agent for example, an alcohol (eg, ethanol), a polyalcohol (eg, propylene glycol, PEG), or a nonionic surfactant (eg, polysorbate 80, HC0-50).
- the antibody of the present invention may be encapsulated in microcapsules (microcapsules of hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid], etc.), or a colloid drug delivery system (ribosome, albumin microsphere).
- micro Emar Ji may be a nanoparticle ⁇ Pi nanocapsules) ( "Remington s pharmaceutical Science 16 th edition, Oslo Ed. (1980) see the like).
- a method for the drug and drug sustained-release are also known and can be applied to the antibodies of the invention (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981); Langer, Chem. Tech. 12: 98-105 (1982).
- Gene encoding antibody of the present invention In addition to direct administration with naked plasmid, it can be packaged in ribosomes, retroviral vectors, adenovirus vectors, vaccinia virus vectors, Various virus vectors such as box virus vector, adeno-associated virus vector and HVJ vector (See Adolph "Viral Genome Method", CRC Press, Florid (1996)), or coated on a bead carrier such as colloidal gold particles (W093 / 17706 etc.) and administered.
- the antibody may be administered by any method as long as the antibody is expressed in vivo while exerting its action.
- injection, infusion, or gas-induced particle bombardment intravenous, intraperitoneal, subcutaneous, intradermal, adipose tissue, adipose tissue, mammary tissue, inhalation or intramuscular routes
- a sufficient amount is administered by an electron gun or the like, or via a mucosal route such as a nasal drug.
- the antibody of the present invention can be administered to cells ex vivo using liposome transfection, particle bombardment (U.S. Pat. No. 4,945,050), or viral infection, and reintroducing the cells into an animal. May be administered.
- FIG. 1 is a diagram showing the base sequence (without tag) of PCI cDNA.
- the nucleotide sequence of the full-length PCI gene is shown.
- EcoRI and BaraHI recognition sequences (underlined) at the 5 'end and 3' end for the purpose of insertion between EcoRI and BamHI of an animal cell expression vector (pCHOI), and a start codon to further increase transcription efficiency Kozak sequence is added before.
- the nucleotide sequence was SEQ ID NO: 4, and the amino acid sequence was SEQ ID NO: 5.
- FIG. 2 is a diagram showing a base sequence (with a FLAG tag) of a PCI cDNA.
- the nucleotide sequence of the PCI gene with a Flag tag is shown.
- a Flag sequence By inserting cDNA encoding full-length PCI between EcoR I-BamHI of an animal cell expression vector (pCH02-FLAG), a Flag sequence (wavy line) was added to the 3 ′ side of the full-length PCI gene.
- the nucleotide sequence was SEQ ID NO: 6, and the amino acid sequence was SEQ ID NO: 7.
- FIG. 3 is a photograph showing the analysis results of PCI-Flag: ⁇ and PCI by SDS-PAGE and Western plotting.
- A Separate PCI-Flag (lanes 1 and 2) or
- B untagged PCI (lanes 3 and 4) by SDS-PAGE and use coomassie-pull staining (lanes 1 and 3) or anti-PCI antibody Western blotting (lanes 2 and 4) ). .
- FIG. 4 is a diagram showing a comparison of neutralizing activities of each anti-PCI antibody in aPC / PCI and Thr / TM / PCI assays.
- the white circles represent the results of aPC / PCI Atssay, and the black circles represent the results of Thr / TM / PCI Atsushi.
- the values are shown as relative values when 100% when no PCI was added and 0% when no antibody was added when PCI was added.
- FIG. 5 is a view showing the amino acid sequence of the H chain of each anti-PCI neutralizing antibody. CDRs 1, 2, and 3 are boxed. The amino acid sequences in the figure are SEQ ID NOS: 8 to 14 in order from the top.
- FIG. 6 is a diagram showing the amino acid sequence of the L chain of each anti-PCI neutralizing antibody. CDRs 1, 2, and 3 are boxed. The amino acid sequences in the figures are SEQ ID NOs: 15 to 21 in order from the top.
- PCI-low 5 "-CTG GAT CCT CAG GGG CGG TTC ACT TTG C (SEQ ID NO: 2)
- human kidney marathon ready cDNA (Clontech) as type PCR
- the digested DNA fragment was digested with EcoRI and BamHI, and inserted into the EcoRI and BamHI cleavage sites of pCHOI, an animal cell expression vector.
- the nucleotide sequence of the PCI gene in the vector was analyzed, and the plasmid having the correct sequence was selected to complete the construction of the pCHOI-PCI vector ( Figure 1).
- PCI-Flag PCI-Flag expression vector with a Flag tag was performed as follows. Mirror the pCHOI-PCI vector and perform PCR using PCI-up and PCI-low2 primers! / Amplification of the PCI gene was performed.
- PCI-low2 5, TTG GAT CCG GGG TTC ACT TTG CCA AG (SEQ ID NO: 3)
- This DNA fragment is digested with EcoRI and BamHI, and a Flag tag is added immediately after the cloning site. And inserted into the BamHI cleavage site. After confirming the base sequence, the construction of pCHOII-PCI-Flag was completed. ( Figure 2 )
- pCHOI-PCI and pCHOII-PCI-Flag were each cut with Pvul to perform linearization.
- 30 g of this DNA was introduced into CH0 cells (DXB11 strain) by electoporation. Then, the cells were cultured in ⁇ ; (-) MEM (containing no nucleic acid) (GIBCO BRL CAT # 12561-056) supplemented with 5% FBS (GIBCO BRL CAT # 10099-141), and then PCI or PCI-Flag. Producing strains were selected.
- the cell line obtained at this stage was cultured in the same medium supplemented with MTX to a final concentration of 50 nM to establish a high-producing cell line.
- the expression of PCI and PCI-Flag was confirmed using an anti-PCI antibody (Affinity Biologicals CAT # GAPCI-IG).
- the PCI-Flag high expression CH0 strain was cultured in a (-) MEM (nucleic acid-free) medium supplemented with 5% FBS using a roller bottle (1700 cm 2 ). After culturing until cells became confluent (37 ° C, 0.5 rpm), the medium was removed and washed with PBS, and CH0-S-SFMII medium (GIBCO BRL CAT # 12052_098) was added, followed by culturing for 72 hours. . The obtained culture supernatant was subjected to centrifugation to remove cell debris, and then filtered through a 0.45 ⁇ filter for use in the following purification.
- the culture supernatant was prepared from the high PCI expressing CH0 strain using a roller bottle (1700 cm 2 ) in the same manner as described above. 50 mM Tris buffer containing culture supernatant 0. 05% Tween20 (P H7. 0) at and ⁇ Ka ⁇ equilibrated with CM sepharose Fast Flow, washed, and eluted with the same buffer solution containing 400 mM NaCl . Next, the fraction containing PCI was added to a HiTrap Heparin HP (Amersham CAT # 17-0407-01) column equilibrated with 10 mM phosphate buffer (PH 7.0) containing 0.05% Tween20.
- HiTrap Heparin HP Amersham CAT # 17-0407-01
- PCI-Flag as an immunizing antigen, five Balb mice (female, 13 weeks old, Charles River Japan) were immunized according to a standard method.
- the antigen was prepared to be lOO / zg / head and emulsified with FCA ⁇ Freund's complete adjuvant (H37Ra), Difco (3113-60) Beta ton Dickinson (cat # 231131) ⁇
- FCA ⁇ Freund's complete adjuvant H37Ra
- Difco (3113-60) Beta ton Dickinson cat # 231131
- the mixture was adjusted to 50 / z g / head and emulsified with FIA ⁇ mouth incomplete adjuvant, Difco (0639-60), Betta ton Dickinson (cat # 263910) ⁇ Were administered subcutaneously.
- mice were subcutaneously subjected to final immunization, and mouse myeloma cells P3U1 and mouse spleen cells were mixed according to a standard method, and cell fusion was performed with PEG1500 (Roche Diagnostics, cat # 783 641).
- PEG1500 Roche Diagnostics, cat # 783 641.
- Each mouse-derived hybrid was cultured on 16 96-well culture plates. From the day after the fusion, the selection was opened with HAT medium ⁇ 10% FBS I RPMI1640 / 1 x HAT media supplement (SIGMA CAW H-0226) I 4% BM-Con dimed HI Hybridoma cloning supplement (Roche CAT # 1088947) ⁇ and fusion was started. Ten days later, the culture supernatant was collected and subjected to ELISA screening. ELISA screening was performed using an immunoplate coated with 0.5 ⁇ g / ml of PCI and 100 1 / well in the same manner as in the antibody titer measurement
- the positive wells were selected by ELISA screening using PCI.
- the selected positive wells were expanded to a 24-well plate and then cloned by limiting dilution (100 cells per positive well were seeded on a 96-well plate).
- the cloned hybridoma was expanded and cultured, and the antibody was purified from the culture supernatant.
- 111 were selected from the higher 0D value by primary screening and cloned by the P-field dilution method.
- the culture supernatant was collected from the 179 cells that had not been subjected to limiting dilution, and only the cells were stored. Finally, 81 clones that stably produce antibodies were established from 111 limiting dilution tubes.
- the method for measuring the neutralizing activity against PCI by aPC / PCI assay is as follows: Reaction solution (50 mM Tris-HC1 (pH 8.0), 150 mM NaCl, 2 mM CaCl 2 , 0.1% BSA, 5 U / ral Heparin) Then, the culture supernatant of the hybridoma or the purified antibody, and ⁇ g / ml PCI were added thereto, and the mixture was heated at 37 ° C for 30 minutes. 0.25 tg / ml aPC was added, and the mixture was further heated at 37 ° C for 30 minutes.
- Spectrozyme aPC American Diagnostica Inc.
- colorimetry was performed at 405 nm. (All display densities are final densities).
- the aPC / PCI assay was performed on 81 single-cloned hybridoma culture supernatants, and clones in which aPC activity was recovered by neutralizing PCI activity were selected. 16 clones were selected in order from the clones having strong neutralizing activity against PCI, and the antibody was purified from the culture supernatant using a Protein G column. When aPC / PCI assay was performed using the purified antibody, strong neutralizing activity was confirmed in 8 out of 16 clones depending on the antibody dose.
- FIG. 4 shows the dose-dependent curves of seven of these clones.
- the method for measuring the neutralizing activity against PCI by Thrombin (Thr) / Thrombomodulin (TM) / PCI assay is as follows: Reaction solution (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM CaCl 2 , 0.1% BSA, 5 U / ml Heparin) was added with purified antibody, 5 zg / ml PCI, 2 nM Thr, 10 nM TM, and heated at 37 ° C for 30 minutes. After adding 0.73 / ig / ml PC and further heating at 37 ° C for 50 minutes, the reaction was stopped by adding 0.775 ⁇ g / ml argathroban. After adding 0.4 mM Spectrozyme aPC and reacting at room temperature for 2 hours, colorimetry was performed at 405 nm. (All display densities are final densities). ' ⁇
- Thr / TM / PCI assay was performed on 7 clones whose neutralizing activity was confirmed by the purified antibody in aPC / PCI assay.
- 3 out of 7 clones PC31E2, PC31F1, and PC30G1 were confirmed to have a strong dose-dependent neutralization activity of 1 "(FIG. 4).
- Antibodies whose isotypes were IgGl, IgG2a, and IgG2b were obtained by equilibrating the hybridoma culture supernatant with 20 mM phosphate buffer (pH 7.0) for Hi Trap Protein G HP (Am ersham CAT # 17-0404-01), washed, and purified by elution with 0.1 M glycine buffer (pH 2.7). The eluted fraction was immediately neutralized with 1 M Tris buffer (pH 9.0). After pooling the fractions containing the antibody, dialysis was carried out with PBS containing 0.05% Tween 20 all day and night, and after solvent replacement, NaN 3 was added to a concentration of 0.02%.
- Isotyping of anti-PCI antibody was performed using I MarunoPure Monoclonal Antibody Isotyping Kit II (PIERCE CAT # 37502), and the method was in accordance with the attached manual. Isotype analysis of the established 81 anti-PCI antibody clones revealed that IgGl was 70 clones, IgG2a was 6 clones, IgG2b was 4 clones, and IgM was 1 clone.
- Each antibody R easy than High Priestess dormer about IX 10 7 cells that produce Plant Mini Ki ts were extracted total RNA using.
- CDNA was synthesized from total RNA using SMART RACE cDNA Amplification Kit (Clontech, Cat. No. K1811-1).
- PC19G8, PC30G1, PC31E2, PC31F1, and PC39C6 were used for primers specific to the constant region of IgGl, and for PC23A7 and PC23D8, primers specific for the constant region of IgG2a were used.
- the H chain and the L chain were amplified.
- the amplified H chain and L chain DNA fragments were cloned using pGEM-T easy vector (Promega, Cat. No. A1360), and the nucleotide sequences were determined.
- the amino acid sequences of the variable regions of the H chain and the L chain were as shown in FIGS. 5 and 6, respectively. Since the amino acid sequences of PC19G8 and PC23D8 were identical, it was predicted that the antibodies were derived from the same clone. However, for Isoty pe, PC19G8 was IgGl and PC23D8 was IgG2a, so it was considered that class switching occurred. PC23A7 and PC39C6 have sequences similar to the above two clones. Therefore, it was predicted that the epitopes of the antibodies of these four clones were nearby.
- PC30G1 and PC31F1 had low similarity to the antibodies of the four clones described above, these two clones had similar sequences, and thus it was predicted that their epitopes were close to each other. It was revealed that the PC31E2 sequence had low similarity to the other 6 clones.
- PC19G8, PC23A7, PC23D8, and PC39C6 suppress only aPC-PCI system, and PC30G1, PC31E2, and PC31F1 suppress both aPC-PCI system and Thr-TM-PCI system. It was presumed that this was almost reflected in the mode of neutralization activity against.
- an anti-PCI antibody having a neutralizing effect on PCI has been provided.
- the antibody of the present invention maintains the activity of aPC by acting to inhibit the activity of PCI, which inhibits the production and enzymatic activity of aPC, thereby suppressing the activity of the blood coagulation system or suppressing the anti-inflammatory activity. It has the function of maintaining the effect of the physiological activity of aPC.
- the antibody of the present invention can be used for the prevention or treatment of diseases or injuries that develop and / or progress due to a decrease or deficiency in the activity of activated protein C, and in particular, prevention of aPC such as thrombosis and sepsis. It is very useful for treatment.
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Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2005508090A JPWO2004065418A1 (ja) | 2003-01-20 | 2004-01-20 | 抗pci中和抗体 |
EP04703509A EP1598368A4 (en) | 2003-01-20 | 2004-01-20 | ANTI-PCI neutralizing ANTIBODY |
US10/542,682 US20060167230A1 (en) | 2003-01-20 | 2004-01-20 | Anti-pci neutralizing antibody |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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JP2003011529 | 2003-01-20 | ||
JP2003-011529 | 2003-01-20 |
Publications (1)
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WO2004065418A1 true WO2004065418A1 (ja) | 2004-08-05 |
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ID=32767283
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PCT/JP2004/000429 WO2004065418A1 (ja) | 2003-01-20 | 2004-01-20 | 抗pci中和抗体 |
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US (1) | US20060167230A1 (ja) |
EP (1) | EP1598368A4 (ja) |
JP (1) | JPWO2004065418A1 (ja) |
WO (1) | WO2004065418A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006041048A1 (ja) | 2004-10-15 | 2006-04-20 | Mie University | 肝再生・修復制御剤 |
US8106002B2 (en) | 2005-04-18 | 2012-01-31 | Chugai Seiyaku Kabushiki Kaisha | Anti-cancer agent comprising protein C inhibitor |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
TW200407335A (en) * | 2002-07-22 | 2004-05-16 | Chugai Pharmaceutical Co Ltd | Non-neutralizing antibody to inhibit the inactivation of activated protein C |
IN2015DN02826A (ja) | 2009-09-03 | 2015-09-11 | Merck Sharp & Dohme | |
US8765432B2 (en) | 2009-12-18 | 2014-07-01 | Oligasis, Llc | Targeted drug phosphorylcholine polymer conjugates |
EP2646466B1 (en) * | 2010-12-02 | 2017-03-29 | AIMM Therapeutics B.V. | Means and methods for producing high affinity antibodies |
UA114108C2 (uk) * | 2012-07-10 | 2017-04-25 | Борд Оф Ріджентс, Дзе Юніверсіті Оф Техас Сістем | Моноклональне антитіло для застосування в діагностиці і терапії злоякісних пухлин і аутоімунного захворювання |
ES2819217T3 (es) | 2013-09-08 | 2021-04-15 | Kodiak Sciences Inc | Conjugados de polímeros iónicos dipolares y factor VIII |
WO2015120187A1 (en) * | 2014-02-05 | 2015-08-13 | The University Of Chicago | Chimeric antigen receptors recognizing cancer-spevific tn glycopeptide variants |
US9840553B2 (en) | 2014-06-28 | 2017-12-12 | Kodiak Sciences Inc. | Dual PDGF/VEGF antagonists |
EP3207128B1 (en) | 2014-10-17 | 2022-07-27 | Kodiak Sciences Inc. | Butyrylcholinesterase zwitterionic polymer conjugates |
RU2744860C2 (ru) | 2015-12-30 | 2021-03-16 | Кодиак Сайенсиз Инк. | Антитела и их конъюгаты |
CA3059938A1 (en) | 2017-04-14 | 2018-10-18 | Kodiak Sciences Inc. | Complement factor d antagonist antibodies and conjugates thereof |
AU2020364071A1 (en) | 2019-10-10 | 2022-05-26 | Kodiak Sciences Inc. | Methods of treating an eye disorder |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH11124399A (ja) * | 1997-10-21 | 1999-05-11 | Eisai Co Ltd | プロテインcインヒビターとプロテアーゼの複合体測定用モノクローナル抗体 |
WO2000047626A1 (en) * | 1999-02-09 | 2000-08-17 | Protease Ab | Monoclonal antibody |
EP1222929A2 (en) * | 2001-01-11 | 2002-07-17 | D. Collen Research Foundation vzw | Method and pharmaceutical composition for preventing and/or treating systemic inflammatory response syndrome |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
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US5279956A (en) * | 1991-06-24 | 1994-01-18 | The Scripps Research Institute | Activated protein C polypeptides and anti-peptide antibodies, diagnostic methods and systems for inhibiting activated protein C |
JPH1160500A (ja) * | 1997-08-11 | 1999-03-02 | Nippon Chem Res Kk | 血液凝固異常亢進抑制剤 |
HUP0001237A3 (en) * | 1997-10-20 | 2002-01-28 | Lilly Co Eli | Methods for treating vascular disorders |
-
2004
- 2004-01-20 US US10/542,682 patent/US20060167230A1/en not_active Abandoned
- 2004-01-20 WO PCT/JP2004/000429 patent/WO2004065418A1/ja active Application Filing
- 2004-01-20 JP JP2005508090A patent/JPWO2004065418A1/ja not_active Withdrawn
- 2004-01-20 EP EP04703509A patent/EP1598368A4/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH11124399A (ja) * | 1997-10-21 | 1999-05-11 | Eisai Co Ltd | プロテインcインヒビターとプロテアーゼの複合体測定用モノクローナル抗体 |
WO2000047626A1 (en) * | 1999-02-09 | 2000-08-17 | Protease Ab | Monoclonal antibody |
EP1222929A2 (en) * | 2001-01-11 | 2002-07-17 | D. Collen Research Foundation vzw | Method and pharmaceutical composition for preventing and/or treating systemic inflammatory response syndrome |
Non-Patent Citations (2)
Title |
---|
See also references of EP1598368A4 * |
WARE J. ET AL: "Localization of a factor VIII-inhibiting antibody epitope to a region between residues 338 and 362 of factor VIII heavy chain", PNAS USA, vol. 85, 1988, pages 3165 - 3169, XP002928101 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006041048A1 (ja) | 2004-10-15 | 2006-04-20 | Mie University | 肝再生・修復制御剤 |
US8106002B2 (en) | 2005-04-18 | 2012-01-31 | Chugai Seiyaku Kabushiki Kaisha | Anti-cancer agent comprising protein C inhibitor |
Also Published As
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JPWO2004065418A1 (ja) | 2006-06-29 |
US20060167230A1 (en) | 2006-07-27 |
EP1598368A1 (en) | 2005-11-23 |
EP1598368A4 (en) | 2007-07-25 |
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