JPH03164192A - Bi-specific antibody - Google Patents

Abstract

NEW MATERIAL:A bi-specific antibody having different antigenic specificities, i.e., activated platelet antigen specificity and antithrombogenic substance specificity, on one antibody molecule. USE:An antithrombogenic agent and a remedy for thrombosis. PREPARATION:The objective bi-specific antibody can be produced e.g. by using an activated platelet specific antibody (e.g. anti-GMP-140 monoclonal antibody) and an antithrombogenic substance specific antibody (e.g. anti-tissue plasminogen activator antibody) as raw materials, digesting the materials with a protease such as pepsin to form F(ab')2 fragments, reducing a disulfide bond bonding H chains of the F(ab')2 fragments to split the F(ab')2 into two half molecules Fab', mixing the Fab' originated from each antibody, oxidizing again under a bonding condition to produce a bi-specific antibody and purifying the antibody with an affinity column containing each antigen in immobilized state.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a bispecific antibody having different antigen specificities on one antibody molecule, a method for producing the same, an application thereof, and a drug containing the antibody.

[Conventional technology]

Fibrinolytic therapy has traditionally been used to treat thrombosis. This mainly dissolves fibrin, which is the main stimulant in blood clots, and uses blasminogen 7 activator, which produces plasmin, a fibrinolytic enzyme.

Recently, it has been discovered that some plasminogen activators have a high affinity for fibrin, while others have a low affinity for fibrin, and tissue-type plasminogen activators and prouronase, which have a high affinity for fibrin, have come to be used. Ta. Furthermore, to increase fibrin affinity,
A complex has been created in which plasminogen 7 cutipator is chemically and covalently bonded to a fifurin-specific antibody (Runge M.S. et al.
c. Nsh A+-J P-! t+
+ H +- - On the other hand, activation of platelets is an important trigger for thrombus formation, and a state in which platelets are activated can be considered to be in a stage of preparation for thrombus or a tendency to thrombus. Recently, molecular weight 1, which specifically appears on the membrane surface of activated platelets and does not appear on resting platelets, has been reported.
40,000 glycoproteins were identified, and the possibility of image diagnosis of thrombus sites using their specific antibodies was demonstrated (Balagrica T.
．． K (Parabrica T. K) et al.
oc. Nat1, Acad. Sci. U. S
．． A. 8 61036 (1989)).

[Problem to be solved by the invention]

Generally, blood clots that form in arteries are mainly platelets,
It is said that intravenous thrombi are mainly composed of platelets and fibrin. Therefore, in conventional fibrinolytic therapy that simply targets fibrin, no matter how much the affinity of the thrombolytic agent for fibrin is increased, it only increases the ability of the formed thrombus to dissolve fibrin.
It was difficult to hope for an inhibitory effect on the very early stages of thrombus formation, and an effect on preventing thrombus formation. Furthermore, when a thrombolytic agent and an antibody as described above are chemically bonded, the complex lacks stability, and there is a risk that the activity of the thrombolytic agent itself may be reduced due to the chemical bonding process.

The present invention has been made in order to overcome the problems of the prior art thrombolytic agents, and specifically and stably delivers an antithrombotic substance to the site of thrombus formation, and further to the site in the preparation stage of thrombus formation. Another object of the present invention is to provide a compound that is transported and trapped in an activated state.

[Means to solve the problem]

To summarize the present invention, the first invention of the present invention is a bispecific antibody characterized by having different antigen specificities of activated platelet antigen specificity and antithrombotic substance specificity on one antibody molecule. Regarding this, the second invention of the present invention uses an activated platelet antigen-specific antibody and an antithrombotic substance-specific antibody as raw materials, enzymatically digests these, reduces the disulfide bonds between the H chains of each antibody, and then produces both antibodies. Regarding the method for producing the above-mentioned bispecific antibody, which is characterized by including a step of reoxidizing the H chains of each of the fragments, the third invention of the present invention provides the method for producing the above-mentioned bispecific antibody alone or in advance with an antithrombotic antibody. A fourth aspect of the present invention relates to a method for transporting an antithrombotic substance to a thrombotic site, which is characterized by mixing the antithrombotic substance with an antithrombotic substance and injecting the substance into the blood circulatory system. Alternatively, the present invention relates to an antithrombotic drug characterized by containing a mixture of a bispecific antibody and an antithrombotic substance as an active ingredient.

The above problem is solved by a bispecific antibody that has different antigen specificities: activated platelet antigen specificity and antithrombotic substance specificity. Bispecific antibodies are produced by Nisonov (Niso
Various known methods have been reported, such as the one prepared by chemical methods from two types of rabbit antibodies by Nisonoff et al. (Nisonoff A.,
Arch. Biocherw Biophys. ,−
Dan-2 460 (1961). Hammer rig U(H)
ammerling U), J. Exp. M
ed. , 1 2 8 1461 (l968),
Laso V. (Rago V.), Fed. P
roc. Brennan K), Science,
2 2 9 8 1 (1985)).

That is, the basic steps of these methods are as follows. Antibodies to each of the two antigens are generated by methods well known in the art. Each antibody is then digested with a suitable protease, such as pevcin, to generate F(ab') fragments. Each F(ab') fragment is then digested by reducing the disulfide bonds connecting the chains. By subjecting F(ab') to two half molecules (Fab'),
cleaved into. Fab' derived from one antibody is then mixed with Fab' derived from the other antibody and reoxidized under conditions that allow binding, creating a bispecific antibody. This bispecific antibody is then separated from the mixture. One separation method involves contacting the mixture with an affinity matrix containing an antigen that can specifically bind to either half of the bispecific antibody, and then eluting the bound fraction, which is combined with the other half of the bispecific antibody. The second step is to contact the child with an affinity matrix containing an antigen to which it can specifically bind. This second one L n +'+ h −v rw is seven
M l J- m o Lk A! Death H Jk
J−+ −f′ 1 . 17-specific antibody.

The activated platelet-specific antibody is preferably an antibody that recognizes an antigen that specifically appears on the membrane surface of activated platelets and does not appear on resting platelets.
The monoclonal antibody PL7-6 which recognizes MP-140 antigen is mentioned. Anti-GMP-1 4
0 monoclonal antibody was prepared by K8hler (K8hler).
) et al.'s method (Naeure, 25
6, 4 7 6 (1 9 7 5))
It can be prepared using For example, B a l b/
c Mice are intraperitoneally immunized with GNP-140 or washed platelets, booster immunized 4 weeks later, and spleens are removed from the mice 3 days later. Spleen cells and mouse myeloma cells are fused by the action of polyethylene glycol and cultured in a 96-well plate by a conventional method. The supernatant of each culture was collected, cells producing specific antibodies were selected by ELISA, and further cloned by limiting dilution method to obtain anti-GMP-140.
Obtain monoclonal antibody producing cells (hyperidoma).

The obtained hybridoma is, for example, Hybridoma PL7-6 (Hybridoma PL7-6).
6 (FERMP-11073)), this hybridoma secretes anti-GMP-140 monoclonal antibody PL7-6 into the culture medium, and the supernatant is cultured using a commercially available serum-free medium. Antibody fractions can be obtained by operations such as ammonium sulfate precipitation. Alternatively, a hybridoma can be cultured in a syngeneic mouse and an antibody fraction can be obtained from the animal's body fluid by the same procedure.

On the other hand, antithrombotic substance-specific antibodies are antibodies against compounds used for the prevention and treatment of thrombosis and platelet activation and aggregation, such as anti-tPA monoclonal antibodies, among which there is a report by Yamamoto et al. (Thrombosis Re
search, 50, 637-646
(1988)) monoclonal antibody C9-5,
K8-2 and the like are antibodies that increase the activity of ePA by binding to tPA, and the use of antibodies with the ability to activate such antithrombotic substances is more suitable.

Although polyclonal antibodies derived from various animals can of course be used as these antibodies, monoclonal antibodies are more suitable. Especially in vivo
o), the use of chimeric antibodies is optimal, and the production of chimeric antibodies is described by Winter G. (Wint
erG. ) et al ( Nature 332323 (
1988)) can be used.

Furthermore, the bispecific antibody according to the present invention can be used as an antithrombotic substance that can dissolve thrombus sites and suppress platelet activation and aggregation by reacting the corresponding antithrombotic substance with the antibody in advance. can be used to specifically direct blood to the site of thrombus formation. As such antithrombotic substances, for example, tPA, urokinase, strebtokinase, prourokinase, antiplatelet agents, etc. can be used. In addition, by injecting the present antibody itself into the blood circulatory system, biologically derived antithrombotic substances can be transported to the thrombotic site and the antithrombotic drug containing an effective amount of the bispecific antibody of the present invention can be concentrated. When administered, it is administered by oral or parenteral route. Its dosage forms include tablets, sugar-coated tablets, pills, capsules, powders, troches, solutions, suppositories, injections, etc., which may contain pharmaceutically acceptable excipients, disintegrants, binders, spreading agents, etc. It is manufactured by blending agents, lubricants, pigments, coating agents, and diluents. For example, 1-5 x 10-10 of this bispecific antibody per 1-5 M of distilled water for injection or buffer solution.
Prepared by dissolving 1 g, and adding as necessary, for example, human serum anorepmin, lactose, glucose, sutucarose,
Saccharides such as dextrin, mannitol, inosit, and cyclodextrin, and amino acids such as glutamine, gnoretamic acid, asparagine, aspartic acid, glycine, and alanine are contained at least 0.1%, preferably at least 0.1%. It can be added at a concentration of 2 to 10%, prepared by ordinary drying means such as freeze drying or vacuum drying, and formulated as an injection to be dissolved before use. In addition, the above-mentioned anti-blood serum may be mixed with the bispecific antibody at the time of use, or may be mixed in advance and then prepared in the form of an injection. At this time, it is preferable to use the antithrombotic substance in an amount equal to or more than the amount of the bispecific antibody used.

The human dosage of the antithrombotic agent containing the bispecific antibody of the present invention is usually 1 to 500 ml of bispecific antibody per day.
The dose is 0Tn9, but the dose may be increased or decreased as appropriate depending on the severity of symptoms, gender, age, etc.

[Effect]

Next, the physiological activity of the bispecific antibody of the present invention will be illustrated by experimental examples.

Experimental Example 1 Antithrombotic effect in rabbit jugular vein model Collen et al. (J. Clin. I
nvest. 7 1369 (1983)).

A rabbit (2.4±0.26k9) is anesthetized with 7cetobrotamine and ketamine. A midline incision is made in the neck through which the jugular vein is exposed and isolated.

After the external jugular vein is cut and bleeding has subsided, the jugular vein is closed with a vascular ramp. Approximately 50000 in the jugular vein
0 cpm of 1'-labeled human fibrinogen 10
μl1 concentrated human red blood cells 100 μl1 fresh human platelet-rich plasma 1 0 0 μ110. 5M CaCL*10
Inject 10 μl of μj1 trompin (8 NIB units). After 30 minutes, remove the clamp and resume blood flow. A mixture of tPA and bispecific antibody at various concentrations or a control drug solution is then injected into the peripheral ear vein for 4 hours using a Harvard infusion pump. After 4 hours, the external jugular vein was isolated and removed, and the radioactivity incorporated into the thrombus formed in the jugular vein was measured.

Thrombolysis rate was calculated using the following formula.

Thrombolytic rate (%) = UNIT × 100D Here, D represents the radioactivity incorporated into the thrombus when physiological saline was added, and C represents the radioactivity incorporated into the thrombus when the drug was added. Shows radioactivity.

The results are shown in Table 1.

When the mixture of tPA and bispecific antibody was used, the rate of thrombolysis was clearly higher than that of tPA alone or a mixture of normal mouse IgG and LPA as a control drug.

Table 1 Bispecific antibody (200ln9) tPA (100,000 units) 58 bispecific antibody (
20019) 42 Mouse IgG (200!n9) Ren PA (100,000 units) 18 Ren PA (100,000 units) 20 Experimental example 2 Acute toxicity test Rabbits (2.4 ± 0.26 k9) were administered to 5 animals per group. P
A saline solution of the A-bispecific antibody mixture was intravenously administered at 100,000 units/mouse as tPA activity and 200 rn9/mouse as bispecific antibody amount, and the weekly progress was observed. All rabbits survived and no abnormalities were observed.

As is clear from the above experiments, the drug of the present invention is useful.

〔Example〕

Next, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

Example 1 Production of bispecific antibodies Monoclonal antibody PL7-6 against GMP-140 and monoclonal antibody K against jPA
8-2 each with ImmunoPu
re) (registered trademark) F(ab')t Preparation Kit (manufactured by Pierce) and treated with vepsin to create an F(ab')t fragment. Next, two types of F (a b'). The fragments were reduced and reoxidized to generate bispecific antibodies as described below. First, each F (a b') of 20m9. Mix the pieces and add 0. 37 by OIM mercabutethylamine
C. and pH 5 for 1 hour to cleave these fragments into half molecules Fab' without disrupting the bond between the H and L chains.

This mixture was pH-tested in boric saline buffer.

Example 2 Purification of bispecific antibodies 700gl5 from blood re-collected with citrate from a normal person
After obtaining the platelet fraction by centrifugation for 1 minute, RCD solution (36mM citric acid, 5mM glucose, 1mM
MgCL, 5mM KCL, 103m
MNaCl, 20 ng/1714! Gamma platelets were centrifugally washed twice with prostaglandin E,
Washed using the umin density gradient method and CAT+-free Hepes Tyrode (HEP)
Washed platelets were obtained by resuspending them in ES-Tyrode buffer. Next, Triton
(Uhiinin) Passed through an immobilized agarose column, and eluted both fractions adsorbed on the column with 0.1glycine-HCI buffer (pH 2.2), and purified! l! cMp-14o
was prepared. This GMP-140 was converted into CNBr-activated Sepharose (Sepharose)4B.
(manufactured by Pharmacia), GNP-1
A 407 affinity carrier was prepared. On the other hand, epA (manufactured by Cosmo Bio) t CNBr-activated Sepharose 4
B was covalently attached to the tPA affinity carrier. GNP-1 was prepared by equilibrating the mixture of Example 1 with 0.1M Tris}ICL buffer (pH 7.5).
40 Sepharose 4B column, then 0.1
Washed with M Tris-11Cl (pH 7.5) and further washed with 0.
The anti-GMP specificity-containing fraction was eluted with 1M glycine (pH 2.5) and then neutralized with Tris. Furthermore, this was passed through an equilibrated tPA-Sepharose 4B column and O
, 1M Tris-HCI, then O. The anti-tPA specificity-containing fraction was eluted with 1M glycine (pH 2.5),
Neutralized with Tris. As described above, anti-GMP-
A bispecific antibody having 140 specificity and anti-tPA specificity on the same antibody molecule was obtained.

Example 3: Targeting of tPA to GMP-140 antigen GMP-140 antigen was immobilized on a polystyrene microplate as a carrier, blocked with 1% BSA, washed, and then treated with tPA obtained in Example 2. bispecific antibody (2
A pre-mixed solution of 0 nM) and tPA (200 nM) is added and incubated.

Next, after washing, 0.0%, which is a coloring substrate for tPA, 8mM
S-2288 (manufactured by Daiichi Kagaku Co., Ltd.) was added, and color development at 405 nm was measured. For comparison, the same procedure as above was performed when normal mouse IgG (K8-2, PL7-6) was used instead of the bispecific antibody or when the GNP-140 antigen was not immobilized. . 405nm
Comparison of color development shows that the bispecific antibody according to the invention targets tPA specifically to the GMP-140 antigen that specifically appears on activated platelet membranes, whereas the control antibody Alternatively, the control antigen showed no targeting of tPA at all.

Example 4 Bispecific antibody preparation Physiological saline was added to 40 parts by weight of bispecific antibody (hereinafter abbreviated as "parts"), 20 parts of human albumin, and 20 parts of sucrose, and the total amount was sterilized using 2000 parts by iip. Filter.

10.2g of this filtrate. The mixture was placed in a nl vial and lyophilized to obtain a lyophilized injection containing 40% l of the bispecific antibody in a l vial.

〔Effect of the invention〕

As explained in detail above, the present invention provides a bispecific antibody that has activated platelet antigen specificity and antithrombotic substance specificity on one antibody molecule, and uses this to target the site of thrombus formation. Methods for the delivery (targeting) of antithrombotic substances and antithrombotic agents have been provided.

Claims (1)

[Scope of Claims] 1. A bispecific antibody characterized by having different antigen specificities, namely activated platelet antigen specificity and antithrombotic substance specificity, on one antibody molecule. 2. Using an activated platelet antigen-specific antibody and an antithrombotic substance-specific antibody as raw materials, they are enzymatically digested to reduce the disulfide bonds between the H chains of each antibody, and then each H chain of both antibody fragments is
2. The method for producing a bispecific antibody according to claim 1, which comprises a step of reoxidizing the chains. 3. Transport of an antithrombotic substance to a site of thrombus formation, characterized by injecting the bispecific antibody according to claim 1 alone or in advance with an antithrombotic substance and injecting the mixture into the blood circulatory system. Method. 4. An antithrombotic drug comprising the bispecific antibody according to claim 1 or a mixture of the bispecific antibody and an antithrombotic substance as an active ingredient.