CA1202897A - Selective antitumor agent - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
This invention relates to a selective antitumor agent consisting of a special carrier portion and a voluntary antitumor ingredient. The carrier portion is a monoclonal antibody (MoAb) which is produced by a hybri-doma formed by cell fusion of two kinds of animal cells, one of which is an animal cell sensitized by human tumor cell such as spleen cell of BALB/c mouse and another of which is another animal cell such as mouse myeloma cell.
The antibody is isolated from the hybridoma and chemical-ly bound with the voluntary antitumor ingredient such as an alkylating agent, an antimetabolic agent, a cytotoxic antibiotic protein or polypeptide, toxin, or alkaloid.
The resultant medicine-MoAb conjugate selectively binds with specific tumor cells or tissue having corresponding antigens and the medicine selectively attacks said cells or tissue as its target.

Description

~%02~,~7 SELECTIVE ANTITUMOR AGENT

FIELD OF THE INVENTION
This invention relates to a selective antitumor agent, more particularly, to a selective antitumor agent having a specific affinity with human tumor cells wherein a monoclonal antibody (hereinafter referred to as "MoAb") to human tumor antigen produced by hybridoma is being chemically conjugated with an antitumor active substance.
That is, a special antitumor agent in which a monoclonal antibody to specific antigen of human tumor is being conjugated with a voluntary antitumor substance, and, therefore, the conjugate (the agent of the present inven-tion) selectively attacks special tumor tissues or tumor cells as its attacking target when it is administered into tumor patients.
BACKGROUND OF THE INVENTION
Heretofore, various cytotoxic substances have been studied and applied for clinical use as a treating agent for a malignant tumor such as cancer. However, these antitumor agents are not specific for the tumor cells and such chemotherapeutic agents are toxic to normal cells as well as tumor cells. Therefore, these chemotherapeutic agents have comparatively low reliabil-ity and it is impossible to administer sufficient amount 120Z~97 for treatment of the malignant tumor to the patients.
Accordingly, in the conventional treatment, the chemo-therapy lacks in a reliability, and the early diagnosis and surgical treatment have been main current.
Recently, considerable efforts have been done to develop the application of an antibody to tumor antigen in accordance with the method, which comprises conjugating the cytotoxic antitumor active substance with a special carrier and then selectively transport the antitumor agent to objective tumor cells. Originally, the same or different antitumor antibody to the same subject does not always exhibit the effect of treating the tumor cells but they have a common property in conjugating with the tumor cells at a high probability.
The developments carried out using the above properties have been carried out as described in Japanese Patent Application (OPI) No. 144723/76 (the term "OPI" as used herein refers to a "published unexamined Japanese patent application"), J. Natl. Cancer Inst., Vol. 55, pp. 473 to 477 (1975) and Nature, Vol. 271, pp. 752 to 754 (1978).
In Japanese Patent Application (OPI) No.
144723/76, it is described that the antitumor agent such as daunomycin, etc., is covalently bonded with Fab' dimers of antitumor immunoglobulin.

~20Z~97 In J. Natl. Cancer Inst., it is described that the diphtheria toxin is cross-linked with the rabbit Sv40 antibody by glutaraldehyde to prepare a complex.
In Nature, the diphtheria toxin is conjugated antilumph cell antibody by chlorambucil.
Further, the published unexamined Japanese Application Nos. 136235/80, 49321/80, 16418/81 and 16417/81 have been published in addition to the above publications.
However, since each antibody used in the above literatures is not only single antibody but also the antibody against the normal antigens to normal cells, it is not specific antibody to the tumor antigen. There-fore, they are insufficient in selectivity and possibly injure normal cells in high rate.
SUM~IARY OF THE INVENTION
As a result of the extensive investigations to prepare the antitumor agents capable of selectively act-ing on the tumor cells, it has been found that the mono-clonal antibody (MoAb) which selectively connects tospecial antigen of human tumor cell can be prepared by the purification of the proteins produced from the propagates of a fused cell (hybridoma) in accordance with a cell-fusion method and the selective antitumor agent having a specificity for human tumor cells can be !

1~021!397 obtained by chemically conjugating the MoAb l~it]l an anti-tumor active substance or a cytotoxic substance.
BRIEF DESCRIPTION OF TIIE DRAWINGS
Figure 1 is a graph elution curve showing fractionation of an SP-H-antibody conjugation-reaction product through Sephadex G-200.
Figure 2 is a graph showing selective absorp-tion property of SP-H~antibody conjugate to Colo 38 cells.
Figure 3 and Figure 4 are a graph showing inhibition effect of SP-H-MoAb conjugate against Colo 3 cells ~melanoma cells) and Raji cells ~normal cells).
Figure 5 and Figure 6 are a graph sho~ing thymidine-intake inhibition effect of SP-H-MoAb conjugate against Colo 38 cells and Raji cells.
Figure 7 is a graph showing cytotoxic effect of Adreamycin~MoAb conjutate against Colo 38 cells.
Figure 8 and Figure 9 is a graph showing survival effect of SP-H ~oAb conjugate to cancerized mice.
Throughout Figure 2 and Figure 6, straight line sho~s the effect to Colo 38 and dotted line shows the effect to Raji cells, and symbols A, O, ~ and ~
sho-~ ~loAb alone, conjugate I (F-l in Figure 1), conjugate II ~F-II in Figure 1), Fraction III ~F-III in Figure 1) and SPH-alone (Substance No. SUN 9102), respectively.

* Trade Mark 4 Z~,9'7 Symbols O and ~9 in Figure 7 show adreamycin MoAb conjugate, Adreamycin alone and MoAb alone, respectively.
Symbols ~ , O , ~3 and ~in Figure 8 and Figure 9 show SP-H MoAb conjugate, MoAb alone, non-treatment and SP-H
alone, respectively.
DETAILED DESCRIPTION OF THE INVENTION
The invented antitumor agent is substantially constituted rom a monoclonal antibody human tumor antigen, that is a carrier ingredient and a voluntary antitumor active ingredient burdened by the former.
That is, the pharmaceuticals of the present invention comprise the active components having the properties capable of preventing the growth of the tumor cells or resulting in the extinction thereof, and the carrier components supporting the active components to be trans-ported to the tumor cells. The active component is existing or not existing but to be discovered or developed in the future voluntary antitumor substances, however, it is preferable that these substances has strong cytotoxic property as possible. For example, many alkylating agent~ssuch as endoxane, nitromin, sarcolysine, myleran, cyclophosphamide, etc.; anti-metabolic substance such as ametoputerin, 8-azaguanine, 5-fluorourasil, cytosine arabinoside, 6-mercapto purin, etc.; antibiotics such as mitomycin C, actinomycin D, 1202~3~

adreamycin, daullomycin, sarcomycin, etc.; alkaloids such as vinblastine; basic polypeptides of barley or wheat such as SPl, SP2, SP-H, etc.; and toxic proteins or peptides such as diphtheria toxin, botulinus toxin, tetanus toxin, ricin, etc.
The carrier components (specific antibody) used in the present invention is a single antibody which is main structural element of the present invention.
The carrier components can be prepared from the propagates of a fused cell ~hybridoma) caused by a cell-fusion between an animal cell (e.g., a spleen cell from BALB/c mouse) sensitized by human tumor cell (such as malignant melanoma cell) and another animal cell (e.g., a myeloma cell of mouse), the above propagates are subjected to cloning whereby special hybridoma capable of producing monoclonal antibody (MoAb) which selectively connects to special antigen of human tumor is selected and then desired MoAb is isolated from SUC}I special hybridomas.
The fused cells (hybridoma) propagated accord-ing to the culture method or the fused cells propagated in vivo are collected and then the antibody is isolated according to a suitable manner. Under the purpose of the present invention, the tumor antibody must be substantially in a high pure form. Therefore, it is ~202~97 preferred that the antibody is purified by an electro-phoresis until it becomes a single substance.
The purified antibody is then chemically conjugated with the desirable antitumor active substance or cytotoxic substance.
The conjugation between such antitumor active substance and the above-mentioned monoclonal antibody (~oAb) is carried out by chemically combine the free amino or carboxyl radical of the antibody with amino, carboxyl or aldehyde radical, etc., of the antitumor active substance as follows:
(i) In case that the antitumor substances are basic protein or polypeptide: In this case, it is important problems how to avoid the formation of by-product such as antibody-antibody, medicine-medicine, antibody-medicine-medicine in the conjugation. Further, it is essential that the conjugated substances have a specificity as an antibody. However, according to the present invention, the major parts of amino radicals in medicines are guanizylated and remaining free amino radicals are then condensated with the carboxyl radicals of acidic amino acid moiety among the antibody using carbodiimide reagent.
This method commonly gives good result to basic 1~)2~7 polypeptides of barley or wheat (SPl, SP2, SP-H), mitomycin, daunomycin, adreamycin, ACNU, carbocan, merfalan, chlorambucil, etc.
(ii) In case that the medicines (antitumor substances) contain sugars as their constituent: The -C-C-bond between adjacent hydroxyl radicals (one hydroxyl radical may be replaced with an amino radical) in the sugar moiety of the substance is cut with sodium metaperiodate so as to form aldehyde radical ~ollowed by the reaction with the antibody so as to form a Schiff's base between basic amino radical of the antibody; then the Schiff's base is reduced with a selective reducing agent such as sodium boron hydride so as to form corresponding aminomethyl compound.
This method is applicable for daunomycin, adrea-mycin, FT-207, sitauvin, thioinosine, etc.
(iii) In case that the pharmaceutical substances have amino radical: The both amino radicals among the substances and antibody are cross-linked with two valent aldehyde such as glutaraldehyde. The conjugation is reduced to corresponding saturated compound if need be.
The present invention will now be described in greater detail by reference to the following examples which are given here for illustrative purposes only and 1;~021397 are by no means intended to limit the scope of the inven-tion.
EXAMPLE
Cultivation of llybridoma (225. 28s) and Purification of MoAb Cells (each 5 x 106) of hybridoma~225~ 28s) preliminary cultivated in Dulbecco's Modified Eagle medium (D-MEM) containing 10% calf serum were inoculated intrapenetreally into BALB/c mice cells (~ , aged 8-12 weeks) into which 0.3 mQ of pristein (2, 6, 10, 14-tetramethyl pentadecane)has been injected intrapenetreal-ly before one week of the inoculation.
The penetreal water of the mice was collected during their living until to be elapsed from two weeks after the inoculation of the hybridoma. The collected penetreal water was then centrifuged at 3,000 rpm for 10 minutes in order to remove erythrocytes and pristeine.
To the supernatant, there was added saturated ammonium sulfate solution up to final concentration reaches 33.3%
and stand over one hour at 4C. The precipitate formed was collected centrifugally ~10,000 rpm, 10 minutes, 4C).
The precipitate was then dissolved in a small amount of phosphate buffer saline (Ca and Mg were not existing) (PBS) and dialysed for 24 hours for PBS with a dialysing tube (Cellophane Tubing-Seamless, Union Carbide, U.S.A.).

~ ~02~7 The outer liquid was replaced at least 4 times during the dialyses and finally dialyzed for M/100 tris-ll5Q
buffer (pH 8.0) and then loaded on DE-52 column ~2.5 x 16.5 cm) bufferized with the equal buffer solution. This column was eluted with the same buffer but so that the NaCQ concentration in the buffer was linearly changed from 0 to 0.5 M under the addition of NaCQ. MoAb activity in the eluted peak fraction ~at 280 nm) was measured with 125I-protein A by the amount of binding to human malignant melanoma. The active fraction was concentrated at 4C and then was passed through Sephadex G-200 column (1.5 x 90 cm). Thus, the fraction was purified up to unitary peak at 280 nm.
Ihis peak fraction was unitary even by an electro-phoresis and this can be stored through freeze-dry or under freeze condition. Besides, the aforegoing cultiva-tion, purification and isolation techni~ues are applica-ble for other MoAb produced by the hybridoma to human malignant melanoma, for example, 376.74, 376.96, 465.14, 473.54, 653.25 ~hybridoma and MoAb produced thereby).
Each antibody specifically combined with each correspond-ing antigen of the melanoma, but the bonding portion is somewhat differs depend on the kind of each antibody.
Thus, this fact conjugates suggest a possibility for a cocktail sherapy by conjugates between several different MoAb and antitumor agent.

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ExAr~PLE 2 Preparation of Medicine-MoAb Conjugate, Part 1

2.582 g (0.5 mol) of O-methylisourea was dissolved into 25 mQ of water. The solution was adjusted to pH 3.2 with 10 N NaOH. 300 mg of SP-H (which is a basic polypeptide isolated from barley by the present inventors, constituted from 45 amino acids and cross-linked by 4 "-S-S-" linkages as described in Japanese Patent Application (OPI) No. 49342/81~ was added to the above solution and dissolved. The solution thus-formed was further adjusted to pH 9.2 with further addition of NaOH solution and then allowed to stand for 48 hours at 4C. The reaction mixture was then subjected to gel-filtration through Sephadex G-10, and the eluate was freeze-dried so as to give 299 mg of guanidilated SP-H.
In this sample, about 4 free amino radicals among 5-e-lysins in original SP-H was guanidilated but cytotoxic acitivty against tumor cells was almost not changed.
35 mg of above guanidilated SP-H and 10 mg of freeze-dried antibody obtained by the Example 1 were dissolvedin 1.5 mR of PBS. To the solution 60 ~Q of water solution of water-soluble carbodiimide were added and adjusted to pH 8.5 with 6N HCQ and stirred for 4 hours at room temperature. The reaction mixture was then immediately subjected to a gel-filtration with Sephadex ~2021~97 G-200 column (1.5 x 90 cm). The column is eluted with PBS in the velocity of 24 mQ/hour and the eluate is fractioned into each 4 mQ of fractions. As shown by the Figure 1, the eluate is fractioned into 3 fractions (F-I, F-II and F-III) under the parameter of absorption at 280 nm. As after mentioned, the fraction F-I (tube No. 9-10) and F II (tube No. 12-15) were desired SP-H
MoAb conjugates. Contrary to this, the fraction F-III
(tube No. 26-30) was perhaps unreacted SP-H.

Preparation of Medicine-MoAb Conjugate, Part 2 40 mg of adreamycin was dissolved in 1 mQ of PBS. To this solution, a somewhat excessive amount of sodium metaperiodate (NaIO4) was added followed by stand-ing in the dark for one hour. To the reactant solutionobtained, there was added 1 M glycerol water up to the final concentration of 0.05 M in order to decompose excess sodium metaperiodate. 20 mg of antibody of Bxample 1 dissolved in 1 mQ of 0.15 M potassium carbonate solution (pH 9.5) was added into above medicine solution and reacted for one hour at room temperature. To the reactant solution, sodium boron hydride is added in the ratio of 0.3 mg per 1 mQ of the former solution, allowed to stand for 2 hours at 37~C
and gel-filtered so as to give desired adreamycin-MoAb conjugate.

~0%8~7 Preparation of Medicine.MoAb Conjugate, Part 3 30 mg of the antibody obtained by Example 1 and 5 mg of daunomycin were dissolved into 3 mQ of PBS.
To the solution, glutaraldehyde was added up to 0.1~ and allowed to stand for 15 minutes. The reactant solution is then purified through a gel-filtration as in the former example so as to aimed daunomycin MoAb conjugate.
Immunological Properties of the Conjugates of the Present Invention The medicine-MoAb conjugates thus-obtained are selectively combined with target tumor cells as follows.
Material Cells: Colo 38 cells (human malignant melanoma cells) and Raji cells (human lymphoid cells derived from B-cells) Culture Media:
For Colo 38: D-MEM ~hereinbefore described) For Raji: RMPI 1640 with 10% calf serum 0 Medicine: SP-H.MoAb conjugate according to Example 2.
The medicine is applied as PBS solution and further diluted respective culture medium for the use.
Experimental Method Colo 38 and Raji cells are preincubated for

3 days. The pr~pagated cells are washed 5 times with respective culture medium and poured into plastic plate ~OZ~7 so as to contain 105 cells in the respective plate. Toeach plate, 0-100 ~g/mQ ~final concentration) of the medicine (the conjugate) is added and allowed to stand for one hour under stirring at 4C. Then, the cells are washed fifth similarly, and then an appropriate amount of protein A (which is derived from Staphylococcus aureus sp.) marked with 125I is added to the cells and allowed to stand for one hour. Then, the cells are further washed fifth and the adhered amount of 125I to the cells is measured with Gamma counter (by Beckmann, U.S.A.). Since the protein A combines to Fc portion of the conjugate, the protein A combines the cells providing that the conjugates combine the cell. There-fore, the combining state can be evaluated by measuring the radioactivity.
Result In Figure 2, Colo 38 having a specific antigen for tumor is represented by a solid line and the Raji having no antigen is represented by a dotted line. As shown by the Figure 2, Colo 38 cells ~epend on the concentration of ~loAb ~225. 28s), conjugate I (F-I) and conjugate II ~F-II), but Raji cells having no antigen never combine with any antigen (225, 28s), conjugate I
(F-I) or conjugate II (F-II). Fraction F-III (presum-ably unreacted SP-H) and SP-H alone naturally do not combine with either Colo 38 or Raji cells.

~2(~

Cytotoxic Effect of the Conjugate of the Present Inven-tion, Part 1 The medicine-MoAb conjugates thus obtained have selectivity for the antigen cells as follows.
Material As same as the above.
Method Each 2 x 105 per mQ of cells are dispersed into each plate and 0-100 ~Q of the conjugate per 1 mQ
of the culture medium is added into the plate followed by incubation at 37C for 48 hours. Then the cells are sampled from each plate and stained with tripane blue of the final concentration of 0.05%. The live and death of the cell are judged by the stainability (the stained:
death, the stainless: live) and counted by Tuerk-Buerker counting disk.
Result As shown by Figures 3 and ~, conjugates I and II show cyto~oxic activity against Colo 38 cells having melanoma antigen (cf. Figure 3), but they are almost inactive against Raji (cf. Figure ~).
On the contrary, MoAb (225, 28sl does not effect on any cells bu~ medicine (SP-H) alone and frac-tion F-[II exert strong toxicity to all cells.

1~02t~97 Cytotoxic Effect of the Conjugate of the Present Inven-tion, Part 2 The same experiment as the above Part 1 was repeated using the adreamycin-MoAb conjugate produced in Example 3. The results are shown in Figure 7. As shown in the figure, the antibody alone is not effective to Colo 38 cells. The adreamycin and the medicine of the present invention both show fatal effect against said Colo 38 cells. The latter is stronger than the former.
Antitumor Effect of the Conjugate on Experimental Animals, Part 1 In the above éxperiments, since it has been confirmed that the conjugates of the present invention provide a selective cytotoxic effect against target tumor cells in vitro. Consequently, the experiment evaluating the availability in vivo has been carried out in accordance with the method which comprises inoculating the target cel:Ls (human melamona cells, Colo 38~ into nude mice and then administrating the conjugates of the present invention into the mice. As a result, the availability was confirmed. These experiments are as follows:
Materials Cells: Colo 38 (human malignant melanoma cells) Culture Medium: RMPI 1640 having 10% calf serum Medicine: SP-H-MoAb conjugate obtained according to Example 2 ~202~3g7 Animals: BALB/cA (-nu/JCR) nude mice, female, aged 6 weeks Method The human melanoma cells were inoculated in the amount of 3.6 x 106 to each mouse by hypodermic injection. The medicine and controls ~antibody alone and SP-H alone) are intrapenetreally administered into the animals in the amount of 0.1 mg/mouse, 1 mg/mouse and 0.5 mg/kg at l, 3, 5 and 20 days after the inocula-tion.
Result The result is shown by the attached Figure 8.
The mice of con~rol group (each constituted from 6 mice) died within 30 days. Namely, SP-H alone or antibody alone could not prolong the life of the tested mice.
On the contrary, the medicine-administered group (each constituted from 6 mice) were clearly survived up to 37 days after the inoculation.
Antitumor Effect of the Conjugate on Experimental Animals, Part 2 In the foregoing Part 1 experiment, the target cells were changed to penetreal water type Colo 38. The result is shown by the attached Figure 9. All mice in the control groups died within 20 days but the mice of the medicine-treated groups were survived at least for 25 days including a mouse which was living even after 60 days.

1~02F,~97 Cell Physiological Effect of the Conjugate of the Present Invention The reason that the conjugates of the present invention exhibit inhibition effects for the target tumor cells seems to be same as case of using a medicine alone. In order to verify the above facts, the following experiments were carried out regarding the conjugates in which SP-H known as a substance having an effect of inhibiting the incorporation of thymidine into the cells is conjugated with an antibody.
Material As same as the above.
Experimental Method Each 2 x 105 per mQ of cells are dispersed into each plate and 0-200 ~g of the conjugates and the objective substances per 1 mQ of the culture medium is added into the plate followed by incubation at 37C for 8 hours. Then, 1 ~Ci of 3H-thymidine is added into each plate. After 16 hours, the amount of incorporation of thymidine into each cell is measured.
Result As shown by Figure 5, it can be seen that the conjugates I and II exhibit the incorporation of thymidine into Colo 38 cells as a target tumor cell in accordance with the concentration thereof added. On the ~o~7 other hand, there is no influence with respect to the incorporation of the thymidine into the Raji cells having no antigen as indicated in Figure 6. In addition, when the antigen and SP-H are used alone, respectively, the former does not inhibit the incorporation of thymidine and the latter strongly inhibits such incorporation.
As described above, the medicine-~loAb conjugates exert strong affinity to specific tumor cells having corresponding antigen and kill said cells or inhibit the growth of said cells, but it does not affect to normal cells. Therefore, it can be expected that the new conjugate contribute on expectful chemotherapy of many malignant tumors or cancers.
The conjugate substances of the present inven-tion is preferably administrated by the intravascularinjection or intramuscular injection or preferably applied directly to the tumor tissues. As shown in the above experimental results, the conjugates are combined with the tumor antibody of the target tumor cell and they are incorporated by the phagocytosis and pinocytosis of the tumor cell. As a result, it can be expected that the conjugates kill said cells or inhibit the growth of said cells. In this case, the conjugates do not affect normal cells because they are not combined with the other normal cells having no antigen.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one ski.lled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

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Claims (6)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A selective antitumor agent wherein a mono-clonal antibody to human tumor antigen produced by hybri-doma is chemically conjugated with an antitumor active substance which is cytotoxic basic polypeptides derived from barley or wheat.

2. An antitumor agent according to Claim 1, wherein the hybridoma is fused cell between animal (BALB/c mouse) spleen cell sensitized by human malignant melanoma and animal myeloma cell.

3. An antitumor agent according to Claim 1, wherein free carboxyl radical of acidic amino acid among the antibody and amino radical of E-lysine in the anti-tumor active substance are being combined by forming acid amide.

4. An antitumor agent according to Claims 1 or 2, wherein free amino radical of basic amino acid in the antibody and free carboxyl radical in the antitumor active substance are being combined by forming acid amide.

5. An antitumor agent according to Claims 1 or 2, wherein free amino radical of basic amino acid in the antibody and nuclear carbon atom in the antitumor active substance including sugar as the elemental constituent are being combined.

6. An antitumor agent according to Claims 1 or 2, wherein free amino radical of basic amino acid in the antibody is being cross-linked with the antitumor active substance by a saturated divalent aldehyde.