JPS5942323A - Conjugated protein killing selectively cell and its preparation - Google Patents

Abstract

PURPOSE:To obtain the titled conjugated protein having improved efficiency as a carcinostatic agent, by bonding an immunoglobulin capable of being selectively linked to a specific antigen of the target cell or its fragment to neocarcinostatin through a specified bifunctional organic group. CONSTITUTION:A thiol group is produced in an immunoglobulin which is capable of being selectively linked to a specific antigen possessed by a cell to be killed (the target cell for short) or its fragment, or the thiol group is introduced to the immunoglobulin or its fragment, while a maleimide group is introduced to neocarcinostatin, a carcinostatic agent. Both of them are reacted to give the desired conjugated protein wherein they are bonded through a bifunctional organic group shown by the formula [X1 and X2 are bifunctional organic group; m is 0, or 1; with the proviso that the immunoglobulin or its fragment is bonded in the (X1)m side, and neocarcinostatin is bonded in the X2 side]. Since the toxic part of the cell and a carrier part are bonded by a safe chemical bond, the toxicity of the cell on the target cell can be manifested selectively with improved efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel selective cell-killing protein complex and a method for producing the same. More specifically, a component consisting of an immunoglobulin that can selectively bind to a specific antigen of cells to be killed (hereinafter referred to as target cells) or a fragment containing its antigen-binding site, and an anticancer drug neocarcinostasis/
The present invention relates to a novel selective cell-killing protein complex having a component consisting of the following, and a method for producing the same.

For the purpose of selectively killing only certain types of cells, attempts have been made to combine various anticancer drugs with immunoglobulins that can selectively bind to the target cells (for example, T.

Ghose et al., Journal of the National Cancer Institute (J, Natl.

Cancer In5t, ) + Volume 61, No. 657
~676 pages.

(see 1978). Because neocarzinostatin has strong cytotoxicity, it is thought to be suitable as an anticancer agent for such purposes, and a conjugate with immunoglobulin has been investigated. For example, wood is 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (hereinafter wsc
r) as a dehydration condensation agent to bind immunoglobulin and neocarzinostane, and that the resulting complex possesses antibody activity as well as neocarzinostatin activity, and that it is more active than neocarzinostatin alone. It has been reported that it effectively inhibited DNA synthesis in target cells (see Cancer and Chemotherapy, Vol. 6, Special Edition I, pp. 75-81, 1979). In addition, Ji Jung et al. created a complex by forming a disulfide bond between the sulfur atoms introduced into immunoglobulin and neocarzinostatin using a crosslinking agent, and the complex was used as neocarzinostatin. It has been reported that nostatin activity was maintained (Biochemical and Biophysical Research Communications).
Biophysical Research
101, pp. 599-606.

(see 1981). However, when preparing the complex,
In the method using sar as a condensing agent, wsor is a reagent that causes dehydration condensation between an amino group and a carboxyl group to form an amide bond, and in addition to crosslinking between immunoglobulin and neocarzinostatin-5, Cross-linking within one molecule, cross-linking between immunoglobulins, or cross-linking between neocarzinostatins also occurs, making it difficult to obtain desired products. In addition, since immunoglobulin and neocarzinostatin have multiple amino groups and carboxyl groups, a considerable amount of polymeric substances in which multiple immunoglobulins and neocarzinostatin are bonded to each other is produced, and W80I is used as Depending on the crosslinking method, it is extremely difficult to obtain a complex that can actually be used for therapy. In addition, in the above-mentioned complex of Jung et al., immunoglobulin and neocarzinostatin are combined with disulfide (-8-
8-) Since the non-natural disulfide bonds are cleaved in the blood, the efficacy of such complexes is reduced before they reach the target cells.

The present inventors have overcome such drawbacks of the prior art,
For example, as a result of intensive research to develop a selective cell-killing protein complex that selectively exerts strong toxicity on cells to be killed, such as cancer cells, the present invention was arrived at.

That is, the present invention combines an immunoglobulin or a fragment thereof capable of selectively binding to an antigen possessed by cells to be killed and neocarzinostatin via a divalent organic group represented by the following formula [1]. It is a selective cell-killing protein complex consisting of Among such protein complexes, a protein complex represented by the following formula %[1113] is particularly preferred in view of its biological activity.

In the present invention, immunoglobulin refers to an antibody that recognizes a target cell to be destroyed or a globulin containing such an antibody. Such immunoglobulins are called anti-tumor immunoglobulins when the target cells are cancer cells. Anti-tumor immunoglobulins can be obtained, for example, from the serum of cancer patients or from monkey tumor cells or tumor-specific or tumor-associated antigens.

Cohn's ethanol fractionation method, ammonium sulfate fractionation method,
Protein IX (immunoglobulin) having antibody activity, which can be prepared by known means such as ion-exchange chromatography and further subjected to absorption and adsorption operations using various cells, if necessary, or cancer Antibody-producing lymphocytes obtained by immunizing an animal with cells or cancer antigens are fused with, for example, bone marrow tumor cells to obtain culturable antibody-producing fused cells (hybridomas), which are then incubated in vitro. Immunoglobulin with highly specific antibody activity is produced from the culture fluid, serum, or peritoneal fluid by culturing it in vitro) or transplanting it into animals and culturing it in vivo. included. The immunoglobulin of the present invention also includes immunoglobulin having antibody activity, which is prepared by releasing anti-tumor antibodies from tumor tissue using a denaturing agent such as a surfactant, and then preparing the anti-tumor antibodies by the same means as described above.

Immunoglobulins include IriQ, IyA, IyM, and I
Riri.

It is known that there are five classes of IfE, and its basic structure, as schematically shown in Figure 1, consists of two L chains shown in the figure and an H chain shown in the figure. The two chains are connected by at least three disulfide bonds (-S-S- bonds), and as shown in Figure 1, the Fab part has antigen-binding activity and the Fc part has effector activity. They are in agreement in that they are the same. However, IBM exists as a pentamer and IPA exists as a partial dimer, but from the perspective of tissue permeability of the cell-killing protein complex, these should be reduced with mercaptan to form a monomer and then the complex. It is preferable to use it for the guiding part.

An immunoglobulin or a fragment thereof is used as one component of the selective cell-killing protein complex of the present invention. A fragment containing the site is used. Such fragments include in particular Fab,
P(ab)4, which is a dimer of Pab' (a portion consisting of a Fab portion and a so-called hinge portion (the shaded portion in FIG. 1)) and Fab′, is desirable. Any of these 7 immunoglobulin-derived fragments can be used as the derivative of the protein complex of the present invention, regardless of the class of immunoglobulin used as the raw material.

Neocarzinostatin used in the present invention is S
lreptomyces 0arzinostatic
Acute leukemia with a molecular weight of approximately 11,000 obtained from culture fluid of US.

It is a protein antibiotic that exhibits anti-malignant activity against pre-cancer, bladder cancer, liver cancer, etc., and has two free amine groups in its molecule. In addition, neocarzinostatin has a non-protein part, that is, a chromophore, and this chromophore is responsible for the biological activity of neocarzinostatin, and the protein part is involved in the stabilization, transport, activation, etc. of chromo7oa. (Edo 2 Ishida, History of Medicine.

120, pp. 79-80, 1982), such molecular structural features are advantageously used in the present invention.

In the above formulas [:I), (II:] and [■], m=
In the case of 0, S is a sulfur atom derived from the immunoglobulin or a fragment thereof (t), and in the case of m=1, it is a sulfur atom introduced by a crosslinking agent.

X is, for example, a crosslinking agent represented by the following formula [111) % formula %), the following formula (V) Q-0-x3-s-y -...
)1 H-HR, a crosslinking agent represented by the following formula [■], a crosslinking agent (2-iminothiolactone) represented by the following formula [■], a crosslinking agent represented by the following formula [■] (N-acetylhomocystine) K
It is a divalent organic group derived from

Specific examples of the monovalent organic group represented by Y that can form an active disulfide group together with the bonded sulfur atom include 2-pyridylthio group (Q-s-), 4-bi')
? O group (1'J3-8-).

The divalent organic group represented by 13-X3 is not particularly limited as long as it is chemically inert, but is generally appropriately selected from alkylene groups with or without branches, phenylene groups, etc. Active 14-enoxy group, 2,4-dinitrophenoxy group represented by 2, 284.
Examples include a 5-toIJ chlorophenoxy group and a pe/tachlorophenoxy group. Specific examples of the alcohol residue of the imidoester represented by Q include methoxy and ethoxy groups. Specific examples of the halogen atom represented by chlorine include chlorine, bromine, and iodine. X2 is a divalent organic group derived from a crosslinking agent represented by the following formula [■]. X
The divalent organic group represented by 4 is not particularly limited as long as it is chemically inert, but is generally appropriately selected from alkylene groups, phenylene groups, etc. with or without a monomer. 2
Specific examples of the alcohol residue of the active ester represented by are the same as those of the above formula ('W) and CVI'J.

Specific examples of the crosslinking agent include N-succinimidyl 3-(2-pyridyldithio)gropionate, N-succinimidyl a as a crosslinking agent represented by the formula CM)
-(2,4-dinitrophenoxy)butyrate with the formula [
methyl 3-(2-pyridyldithio)propionimidate hydrochloride as a crosslinking agent represented by the formula [■]
As a crosslinking agent represented by N-succinimidyl 3-
Bromopropionate is used as a crosslinking agent represented by the formula [■], metamaleimidobenzoic acid N-hydroxysuccinimide ester, ortho-rV imidobenzoic acid-hydroxysuccinimide ester, bramaleimidobenzoic acid N-hydroxysuccinimide ester, maleimidoacetic acid N-hydroxysuccinimide ester.

Maleimidopropionic acid N-hydroxy-5-norbornene-2,3-diorposiimide ester. Maleimidobutyric acid 2,4-dinitrophenol ester and maleimidovaleric acid pentachlorophenol ester can be mentioned.

The selective cell-killing protein complex of the present invention is produced by generating or introducing a thiol group into an immunoglobulin or a fragment thereof, introducing a maleimide group into neocarzinostatin, and reacting the two. can do.

That is, an immunoglobulin or its 7 lagmate is reacted with a crosslinking agent represented by the formula [F/] or [v], and the product is reduced with, for example, 2-mercaptoethanol or diteothreitol. [Reaction (su,
(2) ) or [x] 17- An immunoglobulin or a fragment thereof of the formula (W),
React with a crosslinking agent represented by (■] or [■] [Reaction (3), (4), (5)), (XI) (XI-t) Cx-1) Formula Cx), CM), ( XI-1) and [X-1:l
Obtain an immunoglobulin into which a thiol group represented by the formula (1) to (5) above (in the above formulas (1) to (5), p is 1 to 2 when Ab is an intact rfM).
An integer of 0, or 1 to 5 if not an intact IpM.
). On the other hand, neocarzinostatin is reacted, for example, with a crosslinking agent represented by the formula [■] to obtain neocarzinostatin into which a maleimide group represented by the following formula [expletive] 18- has been introduced. and the thus obtained immunoglobulin or fragment thereof into which a thiol group has been introduced,
The selective cell-killing protein complex of the present invention can be produced by reacting neocarzinostatin into which a maleimide group has been introduced.

One precursor of the selective cell-killing protein complex of the present invention is an immunoglobulin or a fragment thereof having a thiol group; In addition, if the immunoglobulin or its fragment itself originally has a thiol group, or if it has a cystine-based disulfide bond, the disulfide group can be produced by, for example, reduction. It may also be a thiol group that can.

In the production of the selective cell-killing protein complex of the present invention, when reacting immunoglobulin or its fragment with a crosslinking agent, immunoglobulin or its fragment 1
It is preferable to use 1 to 100 moles of the crosslinking agent per mole. The reaction is an immunoglobulin or its fragment p
The protein concentration in the buffer of H5-8 is 0.5-to Ow/
ld (more preferably 1 to 20 q/-), add a small amount of the crosslinking agent to an aqueous solution of the crosslinking agent while stirring at 0 to 40°C, or if the crosslinking agent is not soluble in water. An organic solvent such as N.

N-dimethylformamide, dimethyl sulfoxide, 1
, 2-dimethoxyethane, methanol.

This is done by adding a solution dissolved in ethanol, acetone, etc. Reaction time is the reaction scale.

Depending on the reaction conditions, it is generally within 2 days.

After the reaction is completed, unreacted crosslinking agents and low-molecular reaction products are removed by dialysis or molecular sieve force chromatography. When a disulfide group is introduced by a crosslinking agent, the disulfide group is reduced to a thiol group, but such a reaction uses an excess of a thiol reagent (e.g., 2-mercaptoethal, diteothreitol), and the above reaction is performed. The reaction can be carried out by applying a temperature and two reaction times, and the reaction product can be purified by the same method as described above. Furthermore, the reaction conditions for introducing a maleimide group into neocarzinostatin using a crosslinking agent are the same as those for reacting the crosslinking agent with the above-mentioned immunoglobulin or its fragment.

The reaction between an immunoglobulin having a thiol group or a fragment thereof having a thiol group generated or introduced therein and neocarzinostatin having a maleimide group introduced can be carried out by mixing the two (the preferred pH of the reaction solution is 5 to 8, The preferred protein concentration is 1-20 cap/-), 2-20 at 0-40°C.
It will be held for 24 hours. Separation of the complex of immunoglobulin or its fragment obtained by the above method and Neocalte=21-nostatin from the reaction mixture.

Purification can be carried out by commonly used operations, such as molecular sieve column chromatography.

The selective cell-killing protein complex of the present invention has a component consisting of neocarzinostatin that is toxic to target cells such as cancer cells, and a component that selectively recognizes target cells and targets neocarzinostatin. It has a constituent part made of immunoglobulin or its fragment, which is a carrier that selectively reaches cells, and the constituent parts are linked by chemically safe bonds, so it selectively suppresses cytotoxicity to target cells. Moreover, it has the characteristic of being able to perform effectively. Further, such a complex can be produced with high purity by the method of the present invention.

Hereinafter, the present invention will be explained in detail with reference to Examples.

22- Example 1 (a) Preparation of anti-mouse mammary cancer MM46 monoclonal antibody Anti-MM46 ISIG2b antibody-producing hybridoma obtained by cell fusion method (Kata Seto et al., Journal Op Immunology (J, Immunol), No. 128
Vol., pp. 201-205, 1982) was inoculated into the peritoneal cavity of 15 nude mice at 2×10' mice per mouse.
Ascites fluid was collected after 0 days, and 5 tons of the obtained ascites fluid 50-
The mixture was thoroughly dialyzed against 0.1 M phosphate buffer (PHs, o ). The dialysis solution was thoroughly equilibrated with the same buffer using a Protein A/Sepharose column (column size 1.5
12.5 on) to sufficiently wash out the protein that passed through, elute the impure protein with 0.1M citrate buffer (pH 5,0), and then add 0.1M citrate buffer (pH 5.0) to elute the impure protein.
The adsorbed IfG2b was eluted with 2MT Hot buffer (pH 8, 2), and the pH of the eluate was returned to neutral with 2MT Hot buffer (pH 8, 2), and then 5 tons of 20mM phosphate buffer (pH 8, 2) was added to the eluate.
It was thoroughly dialyzed to I-t7.5) and 1 osq (t7.7-)
An anti-MM46 monoclonal antibody (IfG2b) was obtained.

In addition, non-immune ryo2b 25 W (7.0 ml) was obtained from normal mouse serum 50-ml in the same manner as above.

(b) Preparation of rpo2b antibody having a thiol group (
Anti-MM46 monoquine antibody 1fG2b obtained in b)
Add 36.4 μt (4 times equivalent of IpG2b) of a 20 mM ethanol solution of N-succinimidyl 3-(2-pyridyldithio) gropionate (hereinafter abbreviated as 5PDP) to 27.3 cape (4,6, t, 0.182 μmole). After reacting at room temperature for 30 minutes, 2MTr was added to the reaction solution.
1/1 volume of is-HO4 buffer (pH 7,5),
2M2-ME 10. lJl, (30 for SPDP
double equivalent amount) and incubated at 37°C for 1 hour.

Then, the reaction solution was diluted with 20mM J acidic acid solution (pH 7.5).
Approximately 20M9 (4,76 mj
) anti-M M 4619G2b into which a thiol group was introduced
Obtained antibodies. Similarly, for non-immune Iy2b, 2
IfG2b into which about 20 capes (7J+d) of thiol groups were introduced was obtained from 5m9 of IpG2b.

(c) Preparation of m-maleimidobenzoyl neocarzinostatin Neocarzinostatin (hereinafter abbreviated as NO8) in the dark
N-succinimisyl m-
Maleimidehen/-1--to (hereinafter abbreviated as 8MB) N
, N'-dimethylformamide solution (173mM) 0.
15- was added and reacted at 23°C for 30 minutes. The reaction solution was centrifuged and the supernatant was transferred to a Sephadex G-25 column (1cr) equilibrated with 50mM phosphate buffer (pH 6.2).
R x 36 crn) to remove unreacted low molecular weight substances such as SMB, m-maleimidobenzoyl neocarcinostate/(hereinafter abbreviated as NO8-MB) 9.9 capes (4, s-)
I got it.

25- In addition, 10 times the equivalent of dinitrophenyl-cysteine (hereinafter abbreviated as DNPN sheetin) was added to 100 μt of a portion of the product, incubated at 23°C for 1 hour, equilibrated with physiological saline, and transferred to Sephadex G-25. Through,
After removing unreacted DNP-cysteine, 2 of the obtained sample
When absorbance was measured at 78 nm and 361 nm (
NO88% 1cPn+278nm'15tDN P -V , r, Te/(7B%l, , I, 361 n
m = 17”o), on average NC! 0.0 per 81 pieces.
It was found that 32 m-maleimidobenzoyl groups were introduced. ○) Preparation of a complex of anti-M M 46 IfG2b antibody and NO8 The thiol group obtained as above (I:I) was introduced. Anti-M M 46 IpG2b antibody 20v (4,y6
-), add NO8-MB obtained as in (c) above to 4
．． Add 73 Misaki (2,15-) and adjust the pH with sodium hydroxide.
7.0 and allowed to react overnight at 4°C. After the completion of the reaction, the reaction solution was applied to a Sephadex G-150 Super Fine column (1,5X s 9 cm) that had been equilibrated with physiological saline, and both the diagonally shaded portions in Figure 2 were collected and concentrated. 20.8 mg (s, xwIt) of product containing the conjugate of the invention was obtained. When the obtained product was analyzed by sodium lauryl sulfate polyacrylamide gel electrophoresis (hereinafter abbreviated as 5DS-PAGE), a band pattern as shown in disk 2 of Fig. 3 was obtained, and this product unreacted anti-M
M 461fG2b antibody (molecular weight approximately 15,5000) and I
fG2b antibody bound to one NO8 (molecular weight approximately 1
70,000) and an IyG2b antibody bound to two NO8s (
The molecular weight was approximately 180,000).

Non-immune If! Using G2b antibody, NO
When the reaction with 8 was carried out, the product 19DS-
The PAGE analysis results were as shown in Disc 3 of FIG. 3, and were similar to those for the anti-MM46IpG2b antibody.

Note that disk 1 in FIG. 1 is a band pattern of anti-MM46IfG2b antibody.

(c) Therapeutic experiment in mice A product containing a complex of anti-MM46 G2b antibody and NO8 prepared as described above (hereinafter simply referred to as anti-MM4
6 IfG2b antibody and NO8 complex), MM
The therapeutic effect on mice transplanted with 46 tumors was investigated.

That is, 3×1G5 MM4 were administered to each group of 5 03H mice.
6 cells were transplanted intraperitoneally, and 24 hours after transplantation, the samples (complex of anti-MM46IpG2b antibody and NO8, complex of non-immune IfG2b antibody and NO8, anti-MM46
A 1=1 mixture of IpG2b antibody and NO8 (50 μt or 500 μ2) was administered, and the lifespan of mice in each group was compared to examine the antitumor activity of the complex of anti-MM 46 IpG2b antibody and NO8.

The results are shown in Figure 4. Non-immune rfGRb antibody and N
The O8 complex did not show antitumor activity even when administered with SOOμV, and the lifespan of the mice was almost the same as that of the saline-administered control group, with all mice dying by 15 days after tumor implantation and fc (fc). Figure 4(b)).

Anti-M M 46 IyG 2 b antibody and NO8 1:1
Although some antitumor effects were seen in the mixture when administered at 500 and 50μ2, only -1 mice survived for a long period of time (Figure 4(C)).

In contrast, anti-MM46 IfG2b antibody and NO8
In the complex administration group, 3 mice survived for a long period of time when 500μ2 was administered, indicating that the complex of the present invention has strong antitumor properties (Figure 4(a)).

Example 2 (a) Preparation of anti-mouse leukemia L1210I, G 1X10' mouse leukemia L1210 cells were made into an emulsion with Freund's complete adjuvant and intravenously injected into rabbits. On subsequent pages, three times at one-week intervals, each about I
10' L1210 cells were injected subcutaneously with an adjuvant, and blood was collected 8 days after the final injection. The obtained blood was pooled, the serum was separated, and the 29-serum was inactivated by heating at 56°C for 30 minutes.

To 200+d of the anti-L1210 serum thus obtained, 200-d of a saturated aqueous solution of ammonium sulfate was added, and the resulting precipitate was dispersed by centrifugation. This precipitate was dissolved in 0.01 M phosphate buffer (pH 7,6) 50- and then thoroughly dialyzed against the same buffer. This dialyzed solution was subjected to DEAE cellulose column chromatography (column size 3tyRX94m) equilibrated with the same buffer solution to obtain a solution containing anti-L1210IfG as an unadsorbed fraction.

(b) Immunoglobulin! l) F'(ab')z
Isolation of fragments 1 of anti-L1210 rtG obtained as in 0) above.
．． 2f was dissolved in 0.1M acetate buffer (pH 4, s) at 4°, and 24 pepsin was added to decompose it at 37°C for about 18 hours. By column chromatography (column size 3.52 x 140 am), the molecular weight was approximately 1.
Pure F (a b')2 as a protein flowing out at 30-00000
7 fragments were obtained.

(c) Preparation of Fab' fragment F(ab')2 fragment obtained as in (b) above) 0.01 M lithium hydrochloric acid-0.14 M sodium chloride-2mM BDTA solution containing 18.4 capes (
pH 8,3) 0.02 wt of 150 mM 2-mercaptoethanol aqueous solution was added to 2.0-, and the mixture was heated to 1 at 37°C.
Time was saved. After the reaction, the solution was diluted with 5mM acetate buffer.
0,14M sodium chloride-1mM EDTAll solution (
Sephadex 025 column chromatography (pH 5.5) (hereinafter abbreviated as ANE buffer) equilibrated with
1,03 x 20 car) to remove 2-mercaptoethanol and prepare Fab' having one thiol group.
7 fragments were obtained.

) Anti-mouse leukemia L 1210 TpG protein)
Preparation of a complex of Fab' and NO8 To the anti-L 1210 I, G Fab' 7 fragment sq+ (2w) obtained as in e9 above, was added the complex obtained as in Example 1 (c). 3 NO8-MB
5 Misaki (1,6+d) plus 0.5M phosphate buffer (+)
1,710 volumes of H7,0) were added, and the mixture was reacted overnight at 4°C. After the reaction was completed, the complex was purified using a Sephadex G-150 superfine column (1,5 x s96n) equilibrated with physiological saline, and the anti-L1210I
Complex of pG Fab' fragment and NO8 ewq
(4+d) was obtained. The resulting complex contains 5DS-p
When analyzed by AGFi, the molecular weight was approximately 50,000F a
b' and a protein (complex) with a molecular weight of approximately 60,000.

[Brief explanation of the drawing]

FIG. 1(a) is a schematic diagram showing the basic structure of immunoglobulin, and ←) is a schematic diagram showing the structure of human immunoglobulin IpG1. FIG. 2 shows the protein efflux pattern of the reaction product obtained in Example 1) in Sephadex G-150 Superfine Φ column chromatography. FIG. 3 shows the pattern of 8D8-PAGE, disk 1 is anti-MM 46 IfG2b antibody, and disk 2 is anti-MM 46 IfG2 obtained in Example 1.
b Complex of antibody and NO8, 6 disks 3 enlarged <The complex of non-immune IyG2b and NO8 obtained in Example 1 was
This is a band pattern obtained by DS-PAGE. Figure 4 line shows the results of examining the antitumor properties of the complex of anti-MM46 antibody and NO8 in Example 1■ by evaluating the therapeutic effect on mice transplanted with MM46 tumors. It is a diagram shown against the number of days after transplantation. (a) In the case of a complex of U anti-M M 46 IpG2b and NO8, -〇- is physiological saline, -〇- is 500
μ2, -Δ- indicates the survival rate when 50 μV of the complex was administered. (b) is the case of a complex of non-immune IpG2b and NO8, -・- is physiological saline, -〇- is SOOμ
V, -Δ- indicates the survival rate when 50 μt of the complex was administered. (C) Anti-MM46 IfG2b and NO8
In the case of a mixture with a ratio of 1:1, -・- is physiological saline, -〇- is 500 μ? , 33-1Δ- indicates the survival rate when 50μ of the mixture was administered. Patent applicant Teijin Ltd. 34- Figure 2 0 50 Now Figure 3

Claims (1)

[Claims] L An immunoglobulin or a fragment thereof capable of selectively binding to a specific antigen possessed by turtle cells to be killed and neocarzinostatin are combined with the following formula [! ] A selective cell-killing protein complex bonded via a divalent organic group represented by: 2. The selective cell-killing protein complex according to claim 1, which is represented by the following formula [1]. & The selective cell-killing protein complex according to claim 1, which is represented by the following formula. 4. The immunoglobulin or its fragment having a generated or introduced thiol group or its fragment and neocarzinostatin having an introduced maleimide group are reacted with the immunoglobulin or its fragment and neocarzinostatin, A method for producing a selective cell-killing protein complex bound via a divalent organic group represented by the following formula [■].