JPS62106027A - Production of l-asparginase-mammarian protein binding compound - Google Patents

Abstract

PURPOSE:The reaction between a specific mammarian protein and L-asparginase is effected in an aqueous solution in the presence of transglutaminase to give the titled binding compound which is used as a remedy for acute leukemia and malignant lymphoma. CONSTITUTION:The reaction of a mammarian protein which can become an acyl-donating substrate for transglutaminase such as casein, fibronectin or plasmin hydrolyzate of fibrinogen, with L-asparginase, from e.g., Escherichea coli, is effected in an aqueous solution, in the presence of transglutaminase, further glycerol and glutathione as stabilizers at a pH of 6.5-9.2 over 55 deg.C for several hours. The reaction is stopped with ammonium sulfate, fractionated and purified to isolate L-asparginase-mammarian protein binding compound. EFFECT:The process according to the present invention has high reaction specificity almost without side-reactions. The binding compound does not residue in living bodies.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-asparaginase-binding protein, which is useful as a therapeutic agent for acute leukemia and malignant lymphoma.

L-asparakinase, which is used as a treatment for acute leukemia and malignant lymphoma, produces antibodies against L-asparaginase immediately after administration, and if treatment is continued, fever, cloudiness, and chills may occur. There are many side effects caused by the drug, such as vomiting. In addition, the half-life in living trees is extremely short, so in order to expect sufficient effects, it is necessary to administer varaginase frequently, but this is difficult due to the side effects described below. be.

Fire Intestinal Relaxation - Asparaginase's - Detailed Disadvantages)
Experiments to modify L-asparakinase (L-7 or balaginase combined with a non-immunogenic (or weak) protein)
For example, L-asparaginase combined with polyDL-alanine (Cancer Rpserch3
9, +927.1979) and those bound with serum albumin (Ibid. 42, 1020.1982) have been confirmed to exhibit benign properties in some cases. However, since the bond between L-asparakinase and protein is conventionally produced by a synthetic method, abnormal protein Abnormal protein release bonds or protein E in which the constituent amino acids cannot be formed or cleaved at the base of the protein.
It had the disadvantage that intraplasmic bonds were formed.

The invention tries to solve l'! 18 The present invention does not rely on the conventional methods for producing L-asparaginase-protein conjugates using organic synthesis methods, which have the drawbacks mentioned above, but instead produces L-asparaginase-protein conjugates by pure enzymatic reactions! ! The aim is to provide a method for creating

Means for Solving Skin Problems To first give an overview of the present invention, the present invention takes a mammalian protein that can be an acyl donor substrate for transglutaminase and reacts it with asparaginase in an aqueous solution state in the presence of transglutaminase. It is characterized by this.

Transglutaminase catalyzes the acyl transfer reaction between the γ-carboxyamide group of glutamine residues and the ε-amino group of phosphorus residues in proteins, resulting in the transfer of ε between or within protein molecules. -(γ-glutamyl)-lysine cross-linking occurs. The production method of the present invention utilizes this action of transglutaminase to bind the above-mentioned milk protein to the ε-7 amino group of the lysine residue in L-asparaginase.

Hereinafter, the method for producing the L-asparaginase-mammary protein conjugate according to the present invention will be described in detail.

In the production method of the present invention, the transglutaminase may be a weeping mammalian tissue-type transglutaminase activated by blood coagulation factor
Either force can be used.

Examples of mammalian proteins that can serve as acyl donor substrates in the enzyme reaction of the present invention include casein, fibronectin, plasmin-degraded products of fibrinogen, plasmin-degraded products of fibrin, and N,N-dimethylated products thereof. In addition, N,N-dimethylated products of mammalian proteins were obtained by the method of Kuba Lin et al. (J, Biol, C.
hem.
It can be prepared by adding sodium borohydride to the acyl donor protein and adding formaldehyde in a reduced state. Therefore, it also acts as an acyl acceptor substrate, and polymerization between the acyl donor proteins occurs.Therefore, the ε~ruamitsu of the lysine residue is pre-N.

N-dimethylation is advantageous because polymerization of acyl donors with each other can be prevented. ).

As L-asparaginase, commercially available Escherichia coli L
- Asparaginase can be used as is. Asparakinase of the origin of ponds, such as Erwinia
Carotovora L-asparaginase can also be used in exactly the same way as E. coli L-lua barraginase.

The reaction between L-asparaginase and a milk protein is typically carried out as follows. First, an aqueous solution of transglutaminase is prepared, and stabilizers such as glycerin and glutathione are added to it. Furthermore, if transglutaminase is a blood coagulation factor Xll+, thrombin and Ca are added to activate it. Next, L-asparaginase and milk protein are added to this solution and reacted for several hours at a temperature below 2.55°C. At an appropriate time, ammonium sulfate (hereinafter referred to as ammonium sulfate) is added to stop the transglutaminase reaction, and QL-asparaginase, which has not reacted with the milk protein conjugate, is separated from mono-asparaginase by a fractionation method such as ammonium sulfate fractionation. When solubilized casein is used, casein binds to unreacted solubilized casein and -asparaginase r, +48.
6, unreacted L-asparakinase precipitates only at ammonium sulfate saturations of 70% or higher. Furthermore, when a plasmin-degraded product of fibrinogen is used, L-asparaginase bound to the plasmin-degraded product of fibrinogen precipitates at ammonium sulfate saturation of 65% or less. When solubilized casein is used as the acyl donor protein, in addition to ammonium sulfate fractionation, a calcium chloride solution is added to precipitate unreacted solubilized casein and casein-bound -asparaginase. Asparaginase and asparaginase can be separated.

The thus obtained asparakinase is obtained by dissolving the precipitate containing the milk protein conjugate in an appropriate buffer solution, and dissolving the unreacted 1. =-It is possible to obtain an L-asparaginase-mammalian protein conjugate solution that does not contain asparaginase.

Thereafter, the L-asparaginase can be isolated as a milk protein conjugate by further purification using separation means such as gel filtration and ion exchange chromatography.

As mentioned above, the production method of the present invention, in which transglutaminase is used to bind L-asparaginase to 7L-class proteins, is an enzymatic reaction, so it is less reaction-specific than conventional synthetic methods. It has the characteristics of high compatibility and almost no side reactions.

Furthermore, the action of transglutaminase (double +1)
L-Akubaragi'nase is known because cross-links formed with mammalian proteins are broken down by γ-glutamylamine cyclotransferase present in the mammalian body (Proc, Natl, Acad, Sci.

It is also an advantage of the process of the present invention that the resulting L-asparaginase-mammalian protein conjugate is not expected to exhibit biopersistence.

The present invention will now be described with reference to Examples. The unit of each enzyme activity shown in the examples is a value defined as follows.

L-asparaginase activity: One unit is an enzyme layer that releases 1 nmole of ammonia from asparagine in 1 minute at pH 8, 6, and 37°C.

Transglutaminase activity of blood coagulation Xll+ factor
When bovine milk casein was used as the niacyl donor substrate and monogunsylcadaverine was used as the acyl acceptor substrate, lnmo per minute at pH 7, 5, and 37 °C was obtained.
The amount of enzyme that causes bovine milk casein to incorporate fluorescence corresponding to the fluorescence intensity at a wavelength of 525 nm (excitation light wavelength 355 nm) of le monogunsylcagberin is defined as one unit.

Thrombin activity: INIH unit is the amount of enzyme that solidifies 1+nl of standard fibrinogen (Cohn fraction I) at 25°C for 115 seconds.

Plasmin activity: pt(?, -
The amount of enzyme that liberates acid-insoluble tyrosine equivalent to 4501+8 thousand rosins per hour at 1.37°C is defined as 1 casein l'l-position.

Example 1 Bovine blood FJta fixed Xll + factor solution (transglutaminase activity 318.2 units/ml; 0.05M) Rimata-hydrochloric acid buffer; containing 1 mN4 EDTA; p
H7,5) 50% glycerol solution (0,
Contains 1M glutathione; pH 8,6) 0. Lnl
0.2 ml of bovine thrombin solution (containing 20 mM calcium chloride and 25% glycerol; pH 8, G) of 125 NII+ units/ml was added and reacted for 20 minutes at 37 °C to activate blood coagulation factor X111. Na. This one has 1013 units/ml of E. coli. Hairsparaginase solution ((1,05M) Lis-HCl buffer; L
TH8,6) 0.5n+l and 1% solubilized human milk casein solution (0,05\4 Tris-HCl buffer; containing 0.4M NaCl, 4.5mM Calcium Chloride and 10% Glycerol; p ) (8,6) and add 1 ml, 37
The reaction was allowed to proceed for 3 hours at °C. Next, 100% saturated ammonium sulfate solution was added to make the human casein E. coli L solution 65% saturated.
- Asparakinase conjugate and unreacted human casein were precipitated.

Collect the precipitate by centrifugation and add 65% saturated ammonium sulfate solution (
After repeating washing with 5 ml of pH 8,6 twice,
0.05M) Lis-HCl buffer (pH 8,6; 1m
(Including M's E D T A) 4m1: This? 8 to obtain a human casein-E. coli L. asparaginase conjugate. The apparent recovery rate of L-asparaginase activity is 25
．． It was 7%.

Example 2 2% human fibril/dens solution (containing 40mM sodium citrate and 177mM glucose; pH 6,8
) 1 (-)ml was added with 10 zein units of human plasmin and reacted at 37°C for 3 hours. This was passed through a Linone-Sepharose IB (manufactured by Pharmacia) column to remove free plasmin, and then ammonium sulfate was added to give 50% saturation at pH 6.8, followed by centrifugation. The precipitated fraction was dissolved in a 0.1M saline solution, dialyzed against the same solution, and then lyophilized to obtain a plasmin-degraded product of human fibril-7-gen (hereinafter referred to as FEDP).

Another blood sample was prepared in the same manner as in Example 1;
Activate the factor and add 557.6 units/ml of E. coli L-asparaginase solution (0,05\1 Tris-
Hydrochloric acid buffer; p) L3.6)]+01 was added, and the above 1% solution of FgDP (0°t) 5 M Tris-HCl fiii; Contains glycerol;
1) H8, 6) 1. ml was added and reacted at 37°C for 3 hours. Next, a 100% saturated ammonium sulfate solution was added to the reaction system to precipitate the asparaginase conjugate of F, DP and unreacted F, DP to 65% saturation.

Washing with 5 ml of 65% saturated ammonium sulfate solution (pH 8,6) was repeated twice, and then washed with O.
! (3, G: l"includes 0 and 1 E D T A)
The mixture was dissolved in 4 ml of 8 volumes to obtain a FgDP-coliform-asparakinase conjugate. The apparent recovery rate of L-asparakinase activity was 19.7%.

Add 1 ml of this melted fL to Toyo Soda TSK Del G-300.
(l SW column, f), 0', > N
i) Lis-HCl buffer (p)l7,5:0.2
The solution was drawn out at a flow rate of 0.8 ml/min, and separated into two 1.6 ml aliquots. Honkyu ζ
The L-asparakinase activity of the eyes is determined by the unreacted E. coli L.
- Asparaginase and unreacted F-DP (F):; Asparaginase and unreacted F-DP (F):;
Escherichia coli L-7 human barraginase conjugate (to is unreacted E. coli L)
- It was confirmed that crystal molecules were broken from asparaginase and unreacted F and DP.

Example 3 Blood fL coagulation Xll+ factor was activated in the same manner as in Example. However, the X111 factor solution used had a transglutaminase activity of 71.0 units/ml. After activation, 224 units/ml of E. coli L-
Amatabaraginase solution (0.05M) Lis-HCl buffer; I]88.6) 0.5 ml and 2% N,N-dimethyl bovine milk casein solution (0.05M) Lis-HCl buffer: +'), Contains 4 M NaCl, 4.5+oM Calcium Chloride and 10% Glycerol: p
H8,6) was added and reacted at 37°C for 4 hours to form N,N-dimethyl bovine milk casein-E. coli l-1.
-Asparaginase conjugate (hereinafter referred to as CB A) was produced. Then 200mM calcium chloride solution (
p) Add 48.6) to 2 [o1, and at 37°C
BA and unreacted N,N-dimethyl bovine milk casein were precipitated. Collect the precipitate by centrifugation, and
After repeating washing with 5 ml of OmM calcium chloride solution (p) (8,6) three times, 50n+M) lith-hydrochloric acid-
5ml of 20+nM EDT A solution (pH 8,6)
A solution of I[LCBl\ was obtained. The apparent recovery of L-asparaginase activity was 20.5%.

Example 4 After producing CBA in the same manner as in Example 3, the reaction mixture was placed in a column of 5ephacryl S-300 and subjected to gel filtration without performing precipitation treatment with calcium chloride. Collect the minutes. Then, calcium chloride solution was added to precipitate CBA, the precipitate was collected and dissolved with EDTA solution, and again 5 epha
Gel filtration was performed using a cryl column. In this purification, both CBA fractions in the second gel filtration were divided into two sections: a high molecular side elution section (hereinafter referred to as 7-lactation A) and a low-molecule side elution section (hereinafter referred to as 7-lactation B). Table 1 shows the apparent recovery of asparaginase activity at each stage of the reaction and purification described above.

Table 1 - Recovery rate (raw material L-asparaginase 100 after 1st gel filtration)
34.1 After Ca2+ precipitation
1296 Fraction A after the second gel filtration 4.47 Fraction 8 5 , 0 Table 2 shows the fraction obtained after the second gel filtration.
This is a summary of the results obtained by comparing the enzymatic chemical properties of BA and the raw material Escherichia coli L-asparaginase. From the molecular weight value, it can be seen that E. coli L-asparaginase and N,N-trimethylbovine milk casein were combined to produce an extremely high molecular weight substance having asparaginase activity.

Claims (2)

[Claims] (1) A method for producing an L-asparaginase-mammalian protein conjugate, which comprises reacting a mammalian protein that can serve as an acyl donor substrate for transglutaminase with L-asparaginase in an aqueous solution in the presence of transglutaminase. (2) Claim 1 uses casein, fibronectin, a plasmin-degraded product of fibrinogen, a plasmin-degraded product of fibrin, or an N,N-dimethylated product thereof as a mammalian protein that can serve as an acyl donor substrate for transglutaminase. manufacturing method.