JPH0610140B2 - Method for producing stabilized antibody complex - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION A complex in which an antibody having a function of specifically binding to an antigen and various compounds are covalently bonded is used in various fields. For example, a substance having a γ-ray emitting radioisotope (hereinafter referred to as RI) bound to an antibody having specificity to a specific tissue in a living body, for example, a malignant tumor, or a chelating agent is bound to the antibody first. Then, the RI-labeled antibody obtained by chelating the obtained complex with a γ-ray emitting metal nuclide accumulates radioactivity in the tumor when administered in vivo, and thus can be used for diagnostic imaging of cancer. . In addition, a RI-labeled antibody obtained by binding a radioactive substance bound with β-ray-emitting RI to an antibody or chelating a β-emitting metal nuclide with an antibody-chelating agent complex is selective for tumors. It becomes a cancer treatment drug that works.

Furthermore, a complex in which a bioactive substance is bound to an antibody also becomes a drug with high selectivity. For example, a complex in which an antitumor antibody is bound to an anticancer drug or other cytotoxic substance becomes a cancer therapeutic drug that also selectively acts on tumor. The antibody is a complex in which a fluorescent substance or an enzyme is bound, and is used, for example, as a diagnostic agent or an immuno / biochemical reagent. Also, newer complexes will be devised for future needs.

The method often used to bind a substance to an antibody is
It is a method of binding a substance to an antibody by an acylation reaction.
For example, in the preparation of RI-labeled antibodies, a method of reacting a radioactive substance, Bolton-Hunter reagent, with an antibody at its active ester group is often used to obtain ¹²⁵ I-labeled antibodies (AEBolton and WMHunter, Biochem.J. 13
3, 529-539 (1973)). Further, in order to obtain the ¹¹¹ In-labeled antibody, for example, after the diethylenetriaminepentaacetic acetate tic acid (abbreviated as DTPA) and mixed acid anhydride, bound to the antibody by acylation reaction, the resulting complex ¹¹¹ A method of chelating In is used (BAKhaw, JTFallon, HWStrauss, E.Haber, Scienc
e, 209, 295).

Also, in the production of a complex of an antibody and a bioactive substance,
For example, the method of binding the anticancer drug methotrexate (PNKulkar
ni, AHBlair, TIGhose, Cancer Research, 41, 2700‐27
06 (see 1981)), a carboxyl group contained in a biologically active substance is made into an active ester or a mixed acid anhydride, and then bound to an antibody by an acylation reaction.

Further, in order to bind the A chain of cytotoxic ricin to an antibody, first, an active disulfide group is introduced using a cross-linking agent, for example, N-succinimidyl-3- (2-pyridyldithio) propionate, and thus the introduced active disulfide is introduced. A thiol group contained in the lysine A chain is reacted with the group to form a new disulfide bond (KA
Krolick, C. Villemez, P. Isakson, JWUhr. And ESVite
tta, Proc. Natl. Acad. Sci. USA., 77, 5419-5423 (1980)).

However, the conventional method for producing a complex in which various substances are bound to an antibody or a fragment thereof by an acylation reaction has a problem common to them. That is, the acylation reaction of the antibody by the carboxyl group and its derivative group does not necessarily react with the amino group of the antibody as well as other functional groups such as a hydroxyl group, and in addition to a stable amide bond,
It is also to generate an unstable bond such as an ester bond which is relatively easily cleaved. Therefore, the substance bound to the antibody is released from the antibody complex, causing various problems.
For example, first, there arises a problem that the complex decomposes and deteriorates during storage. In the case of a RI-labeled antibody in which a γ-ray or β-ray emitting nuclide is bound to an antibody / chelating agent complex, when administered to humans, RIs apart from the antibody give non-specific γ-ray images, Alternatively, it causes a decrease in the selectivity of cell killing action and a decrease in the antitumor effect. Further, even a complex of an antibody with an anticancer drug or the like causes a problem of reduced tumor selectivity and / or reduced antitumor effect.

The present inventors have earnestly studied to develop a method for producing a stable antibody-substance complex that does not cause such inconvenience that is unavoidable by the conventional method, and as a result, an antibody or a fragment thereof is desired to undergo desired reaction by an acylation reaction. When the substance is introduced, the substance reacts with a functional group such as a hydroxyl group other than the amino group of the antibody to give an unstable complex in which a part of the bound substance is released from the antibody. Many of the bonds can be cleaved by treating the product with a water-soluble amine, and it was found that the antibody conjugate treated with such an amine has excellent stability, and thus the present invention was reached.

That is, the present invention provides the production of a stable complex of an antibody or a fragment thereof with a substance having an acylating functional group, which is characterized in that the antibody or a fragment thereof is acylated, and then the product is treated with a water-soluble amine. Is the way.

In the present invention, the antibody that binds a substance by an acylation reaction may be an antibody against any antigen or hapten with respect to its specificity. That is, for example, cancer, bacteria,
Antibodies against viruses, molds, mycoplasma, parasites, pathogenic substances, tumor-related substances, tumor-related antigens, cell differentiation antigens, histocompatibility antigens, antibodies against other cell membrane antigens, toxins, enzymes, allergens, hormones, Antibodies against drugs and other biologically active substances can be used. Further, in the present invention, the antibody may be, for example, a polyclonal antibody prepared by immunizing an animal with an antigen or a hapten, or a monoclonal antibody prepared by cell fusion or a method of transforming antibody-producing cells using EB virus. Good. Monoclonal antibodies have the advantage that a large amount of highly pure antibodies with clear specificity for various antigens can be obtained by the cell fusion method or virus transformation method.

The method of the present invention can be applied to antibodies of any class and subclass. That is, IgG, I
The classes of gA, IgM, IgD and IgE are known, and IgG has IgG1, IgG2a, IgG2b and IgG3 subclasses of I
IgA1 and IgA2 subclasses for gA, IgM1 and IgM2 for IgM
Although any of these classes and subclasses can be used as the antibody of the present invention. The antibody may use the whole molecule, but a fragment containing the antigen-binding portion thereof may be used. Such fragments include, for example, monomeric IgMs and IgM of IgM antibody.
gA monomer, divalent fragment F (ab ') ₂ obtained by degrading antibody molecule with pepsin, monovalent fragment Fab obtained by degrading with papain
Etc. can be mentioned.

The water-soluble amine preferably used in the present invention can be selected from a wide range of amines, and particularly preferable amines are ammonia, hydroxylamine, lower alkylamine, lower alkyldiamine and the like. Examples thereof include methylamine, ethylamine, n-propylamine, N-ethylmethylamine and the like.

In the method of the present invention, the substance having an acylating functional group may be any substance as long as it is a substance that needs to be stably introduced into an antibody or a fragment thereof and has an acylating functional group. Examples thereof include radioactive substances, bioactive substances, chelating agents, fluorescent substances, and crosslinking agents. These substances may be substances originally having an acylating functional group or substances which are chemically converted to have an acylating functional group.

The bioactive substance as a substance having an acylating functional group in the method of the present invention includes cytotoxic substances, antitumor substances, antibacterial substances, antiviral substances, antifungal substances, antimycoplasma substances, antiparasitics. Contains sex substances, hormones, enzymes, etc.

The acylating functional group in the substance having such an acylating functional group is a carboxyl group or an activation inducing group thereof, and therefore, the activation inducing group of the carboxyl group is represented by the general formula [I], There are active ester groups, active amide groups, acid azide groups, acid anhydride groups, acid halides, etc. That is, Z in the formula [I] is an alkoxy group forming an active ester group, an acyloxy group or an alkoxycarbonyloxy group forming an active amide group, a halo group forming an acid halide group, and the like. 2,4-dinitrophenoxy group 4-nitrophenoxy N-succinimidyloxy group N- (5-norbornene-2,3-dicarboxyimidyl) oxy group Alkoxy group such as N-imidazolyl group Amino group, etc., isovaleryloxy group Pivalyloxy group Acyloxy group, ethoxycarbonyloxy group Isobutoxycarbonyloxy group Examples thereof include alkoxycarbonyloxy groups and the like. Z forming the acid halide group represented by the formula [I] is a halogen atom such as a chlorine atom or a bromine atom.

Specific examples of the substance having an acylated functional group that is preferably used in the method of the present invention include radioactive substances such as Bolton Hunter reagent. As a chelating agent, diethylenetriaminepentaacetic acid The mixed acid anhydride And intramolecular anhydride 2,6-dioxo-N- (carboxymethyl) -morpholine , Etc., as the bioactive substance, a nitrosourea derivative In the cross-linking agent, N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP, N-succinimidyl 4- (N-maleimido) butyrate N-succinimidyl m- (N-maleimido) benzoate Etc. can be mentioned.

The method for producing a stable complex of an antibody or a fragment thereof with a substance having an acylating functional group according to the present invention comprises two reactions. The first reaction is a step of reacting an antibody or a fragment thereof with an acylating functional group. The second reaction is a reaction of acylating with a substance having a second reaction, in which the product of the first reaction is treated with a water-soluble amine, and the substance having an acylating functional group reacts with a hydroxyl group other than the amino group of the antibody. Is a reaction that cleaves a relatively unstable bond generated by The second reaction may be performed after the product of the first reaction is once subjected to a purification operation such as dialysis or gel filtration, but in many cases, the product of the first reaction is not purified at all. The second reaction can subsequently be carried out.

First, in the first acylation reaction, it is preferable that the pH is selected from the range of 4 to 10 and the reaction temperature is selected from the range of -4 to 40 ° C, and the reaction is carried out in a buffer solution. The amount of the substance having an acylating functional group used in the reaction is 1 to 10 of the antibody or its fragment in a molar ratio.
It is appropriately selected from 00 times depending on the properties of the acylating agent and the purpose of acylation. The product of the acylation reaction is, if necessary, carried out by a method such as dialysis, gel permeation, ammonium sulfate precipitation method, ethanol precipitation method, ion exchange chromatography and isoelectric focusing method.

The second reaction in which a substance having an acylated functional group reacts with a hydroxyl group other than the amino group of an antibody to cleave a relatively unstable bond formed is, for example, 0 to 40 ° C. and a pH of 6 to 10 It can be carried out by adding a water-soluble amine to a concentration of 0.001 to 5 M, and the required time is 1 hour to 5 days. In addition,
Particularly preferred pH and water-soluble amineno concentration are pH 7-10,0.
01 to 1M.

Purification of the antibody complex produced as described above can be carried out by appropriately selecting from methods such as dialysis, gel permeation, ammonium sulfate precipitation method, ethanol precipitation method, ion exchange chromatography, and isoelectric focusing method.

Next, examples will be described to explain the present invention in more detail, but the present invention is not limited to these examples.

Reference Example 1 Dextran, poly-L-lysine (PLL), and succinylated immunoglobulin and methotrexate (MTX)
Reaction with N-succinimidyl ester (MTX-OSu) and stability of the bond between these macromolecules and MTX in the resulting complex 40 times molar MTX-OSu in dextran, 0.1M boric acid React in buffer solution (pH 8.6) at 25 ℃ for 20 hours to react with MTX /
Dextran Dextran-MTX with a molar binding ratio of 7.7
A complex was obtained. On the other hand, PLL has 20 times mole of MTX-OSu.
Was reacted in phosphate buffered saline (pH 7.4) at 25 ° C. for 4.5 hours to give a PLL-containing MTX / PLL molar binding ratio of 14.3.
An MTX complex was obtained. That is, MTX-OSu is more reactive towards amino groups than hydroxyl groups, but is also significantly reactive towards hydroxyl groups. (When an antibody is acylated, it is shown that the acyl group is introduced into the amino group more than into the hydroxyl group, but is also introduced into the hydroxyl group.) The above dextran-MTX and PLL-MTX complexes are mixed with 0.1M boric acid. Incubate with 0.1 M hydroxylamine in a buffer solution (pH 9.0) and add phosphate buffered saline (pH
After dialysis against 7.2) (PBS) for 24 hours, MTX remaining in the complex was quantified by the absorbance at 372 nm. The ester bond between dextran and MTX is easily cleaved, and
After time incubation, 97% of MTX was released from dextran and removed. (Release in the absence of hydroxylamine is 52% after incubation at 37 ° C for 24 hours.
Stayed in. ) On the other hand, the amide bond between PLL and MTX is stable, and after incubating at 25 ° C for 24 hours,
Only 9% of MTX was released. (It is shown that, among the acyl groups introduced into the antibody by the acylation of the antibody, the acyl group introduced into the hydroxyl group while leaving the acyl group introduced into the amino group can be selectively removed by the hydroxylamine treatment. Reference Example 2 Reaction between succinylated immunoglobulin and MTX-OSu, and immunoglobulin and MT in the formed complex
Stability of bond between X Rabbit γ-globulin (RGG) was reacted with 994 times mol of succinic anhydride in 0.1M borate buffer (pH 8.6) at 20 ° C for 1 hour, and then hydroxylamine was added to 0.1%. in addition to the M concentration for 24 hours at 37 ° C., and dialyzed against PBS, 93% of the amino groups have been succinylated {Satake et al [K.Satake e
t al ., J. Biochem., Tokyo , 47, 654-660 (1960)] and Gold
farb et al [ARGold farb, et al., Biochem . 5, 2570-2574
(1966)] to quantify free amino groups} RGG
Got

The succinylated RGG thus obtained was mixed with 50-fold molar amount of MTX-
OSU was reacted in 0.1 M borate buffer (pH 8.0) at 4 ° C. for 4 hours and then dialyzed against PBS to give MTX / RGG.
A complex with a molar binding ratio of 17.9 was obtained. Quantification of MTX is 372n
The absorbance at m was measured. The quantification of globulin was 2.52 times the absorbance at 372 nm than the absorbance at 280 nm of the complex (absorbance at 280 nm derived from bound MTX).
Was carried out by subtracting from the absorbance at 280 nm derived from globulin.

The above succinylated RGG-MTX complex was mixed with 0.1M hydroxylamine and 4 ° C. in 0.1M borate buffer (pH 9.0).
Incubation for 3 minutes. 86% of MTX was released during this time, and the MTX / RGG molar binding ratio dropped to 2.5. (Although functional groups such as hydroxyl groups other than amino groups of the antibody also undergo acylation reaction, it is shown that the acyl group introduced by such reaction can be smoothly removed by hydroxylamine treatment.) Example 1 Mouse against mouse breast cancer MM46 A complex (MTX / IgG molar binding ratio 4.6) produced by reacting an IgG2a monoclonal antibody (anti-MM46 antibody) with MTX-OSu was mixed with 0.1M hydroxylamine in a 0.1M borate buffer (pH 9.0). After incubation at ° C, it was dialyzed against PBS.
After incubation for 24 hours, 41% of MTX was released from the complex, but only 3% of MTX was released even after incubation for another 2 days. This result indicates that MTX bound to the antibody was not removed by hydroxylamine treatment (MTX bound to the amino group by an amide bond) and M was removed.
TX (MTX having an ester bond or the like bonded to a water distribution group). Further, it is shown that once the hydroxylamine treatment is carried out, a stable complex with less release of MTX can be obtained thereafter.

Example 2 A mouse monoclonal antibody against human melanoma (anti-melanoma antibody) was reacted with 8-fold molar MTX-OSu in a 70 mM borate buffer (pH 8.4) at 4 ° C. for 4 hours to prepare MT.
After 30 times mol of glycine was added to X-OSu to stop the reaction, the reaction solution was divided into two and one was dialyzed against PBS to obtain an anti-melanoma antibody-MTX complex. In addition, the other reaction solution was added with hydroxylamine to a concentration of 0.1 M, incubated at 25 ° C. for 24 hours, and then dialyzed against PBS to remove MTX bound to a hydroxyl group or the like by an unstable bond. , Stabilized anti-melanoma antibody-M
A TX complex was made.

Anti-melanoma antibody-M not treated with hydroxylamine
The TX complex showed non-specific cytotoxicity against cultured mouse breast cancer MM46 cells based on extracellular release of MTX (2.5 × 10 ⁴ cells / ml of MM46 cells were treated with the complex). Five.
When added with 0 μM and cultured for 3 days, the number of viable cells was 10% of that without the complex), hydroxylamine-treated, stabilized anti-melanoma antibody-
The MTX complex did not show non-specific cytotoxicity on cultured mouse breast cancer MM46 cells.

Example 3 An anti-human melanoma antibody was added with a 0.3-fold molar amount of a Bolton-Hunter reagent (N-succinimidyl 3- [p-iodo ( ^125I ) phenyl] propionate) in a 0.1 molar borate buffer solution (pH 8.5) at 0 ° C. After reacting for 20 minutes and stopping the reaction by adding glycine, 0.1M NaCl and 0.25%
Gelation with Sephadex G-25 was performed using 50 mM phosphate buffer (pH 7.5) containing gelatin to obtain ¹²⁵ I-labeled anti-human melanoma antibody.

Bolton Hunter reagent against anti-human melanoma antibody at 0.
Using a 5 times molar amount, hydroxylamine was added to a concentration of 0.1 M before the reaction mixture was passed through Sephadex G-25 and gelled.
After adjusting the pH, ¹²⁵ I-labeled anti-human melanoma antibody was prepared in the same manner as above except that incubation was carried out at 25 ° C. for 24 hours.

A part of both ¹²⁵ I-labeled anti-human melanoma antibodies was incubated at 37 ° C. for 2 days and then subjected to gel filtration with Sephadex G-25. Due to gel filtration, 21% of the radioactivity was transferred to the low molecular weight fraction in the labeled antibody not treated with hydroxylamine, but the radioactivity was not transferred to the low molecular weight fraction in the labeled antibody treated with hydroxylamine. It was only 2%. That is, a labeled antibody with less release of radioactivity could be produced by the treatment with hydroxylamine.

Example 4 A 50-fold molar amount of an intramolecular cyclic acid anhydride of diethylenetriaminepentaacetic acid (DTPA) represented by the following formula was added to an anti-human melanoma antibody with 0.25M phosphoric acid. Allow to react for 5 minutes at room temperature in buffer (pH 7.5), then
Hydroxylamine dissolved in 0.1M borate buffer (pH 9.0) was added to a concentration of 0.1M and incubated at 25 ° C for 24 hours, and then Sephadex G50 in 0.5M acetate buffer (p 6.0). Gel filtration, and unreacted DT
PA was removed. Then, it was dissolved in 0.5 M acetate buffer (pH 6.0). The thus prepared DTPA-conjugated antibody (1 mg solution of 7 mg protein / ml) was added with 2 M acetate buffer (pH 6.0) to a final concentration of 1 M acetate buffer, and the carrier-free [ ¹¹¹ In] indium chloride was added. It was added to the solution (5 mCi) and reacted at 37 ° C. for 15 minutes. From the reaction solution, in 75 mM acetate buffer (pH 5.0), Chelex 100 column [New York Bio-Rad (Bio-Rad Laboratori
es) to remove free ¹¹¹ In and inactive 3.6 mCi / mg
A ¹¹¹ In-labeled antibody of the protein was obtained.

A ¹¹¹ In-labeled antibody of inactive 4 mCi / mg protein was obtained in the same manner as above except that the reaction solution was not treated with hydroxylamine.

The two ¹¹¹ In-labeled antibodies thus prepared were incubated at 37 ° C. for 48 hours and then subjected to gel filtration with Sephadex G50 in PBS. ¹¹¹ I without hydroxylamine treatment at the antibody-DTPA complex stage
With the n-labeled antibody, 20% of the radioactivity at the time of measurement was transferred to the low molecular weight fraction, but hydroxylamine treatment was performed.
^{With the 111} In-labeled antibody, the transfer of radioactivity to the low molecular weight fraction was only 3%.

Example 5 RGG dissolved in PBS was added to the crosslinker N-succinimidyl 3- (2-pyridyldithio) propionate (SPD).
P) was added in a 12-fold molar amount and reacted at 23 ° C. for 1 hour.

The reaction solution was divided into two, and one of them was subjected to gel filtration with Sephadex G-25 in PBS to remove a low molecular weight fraction. On the other hand, adjust the pH by adding 1/5 volume of 0.4 M borate buffer (pH 9.0), add hydroxylamine to a concentration of 0.1 M, incubate at 25 ° C for 24 hours, and then perform gel filtration in the same manner. I went. Thus, in the former, one molecule
7.6 3- (2-pyridyldithio) propyl groups (PD
PGG) -bound RGG was obtained, and the hydroxylamine-treated latter gave RGG with 4.9 PDP groups attached per molecule. The PDP group was quantified by adding dithiothreitol (final concentration 5 mM) to release 2-
It was performed by measuring the absorbance at 343 nm derived from mercaptopyridine [T. Stuchbury et al., Biochem. J., 151, 417-432,
(1975)]. In addition, the quantitative determination of RGG was performed by measuring the absorbance at 280 nm with PD.
The calibration was carried out by correcting the amount of P group.

The two PDP group-introduced RGG thus produced were oversterilized and incubated in PBS at 37 ° C. for 48 hours,
Gel filtration with Sephadex G-25 was performed in PBS.

When RGG and PDP groups were quantified as described above, the PDP group per RGG molecule was reduced to 6.4 in the one without hydroxylamine treatment, while the RDP and PDP group per hydroxylamine treatment was reduced in one group. PDP
The group was almost unchanged at 4.7.

That is, this indicates that stable PDP group-introduced γ-globulin can be obtained by the treatment with hydroxylamine.

Claims (11)

[Claims] 1. Preparation of a stable complex of an antibody or a fragment thereof with a substance having an acylating functional group, which comprises acylating the antibody or a fragment thereof and then treating the product with a water-soluble amine. Method. 2. The method for producing an antibody complex according to claim 1, wherein the antibody is a monoclonal antibody. 3. The method for producing an antibody complex according to claim 1 or 2, wherein the water-soluble amine is ammonia, hydroxylamine, lower alkylamine or lower alkylenediamine. 4. The method for producing an antibody complex according to claim 1, 2, or 3, wherein the substance having an acylating functional group is a bioactive substance or a derivative thereof. 5. The method for producing an antibody complex according to claim 4, wherein the bioactive substance is a substance exhibiting a cell-killing effect. 6. The method for producing an antibody complex according to claim 5, wherein the substance exhibiting a cell-killing effect is an antitumor substance. 7. The method for producing an antibody complex according to claim 1, 2, or 3, wherein the substance having an acylating functional group is a radioactive substance. 8. The method for producing an antibody complex according to claim 1, 2, or 3, wherein the substance having an acylating functional group is a chelating agent. 9. The method for producing an antibody complex according to claim 1, 2, or 3, wherein the substance having an acylating functional group is a crosslinking agent. 10. The first and second claims, wherein the acylating functional group is a carboxyl group or its activation-inducing group.
Item 9. A method for producing the antibody complex according to Item 3, Item 3, Item 4, Item 7, Item 8, or Item 9. 11. An antibody or a fragment thereof is acylated, and the product is then added in an aqueous solution of pH 7-10 at 0.01-.
The method for producing an antibody complex according to claim 1, which comprises treating with 1 M of a water-soluble amine.