JPS6067434A - Antitumor agent - Google Patents

Abstract

PURPOSE:To provide an antitumor agent capable of selectively transferring the toxin and carcinostatic agent to the cancer cells by the missile therapy, and having high remedying effect, by bonding a monoclonal antibody recognizing alpha- fetoprotein with the toxin or carcinostatic agent effective to tumor cells. CONSTITUTION:The objective antitumor agent is produced by bonding (A) a monoclonal antibody capable of recognizing the alpha-fetoprotein(AFP) especially preferably existing at the surface of a cell membrane with (B) a toxin or a carcinostatic agent effective to tumor cells. The monoclonal antibody can be prepared by producing a hybridoma from the AFP originated from human placenta by conventional method, dyeing the hepatocarcinoma cerulein with the monoclonal antibody separated from the supernatant liquid of the hybridoma, and selecting the positive antibody. The toxin is bonded with the antibody protein by crosslinking method. The carcinostatic agent is preferably sealed in liposome, and the monoclonal antibody is bonded to the surface of the liposome.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to antitumor agents.・As a cancer marker, α-fetoprotein (A
F P J and fetal antigens (CF, A) are well known. CEA is said to be present on the membrane surface of cancer cells in the digestive system. On the other hand, AFP is known to be present on the membrane surface of cancer cells in the digestive system. Although it has been recognized that it is produced in cell lines, it has only been confirmed that it exists in the cytoplasm, and it is not necessarily clear whether it exists on the membrane surface or not.

The present inventors bind anticancer drugs and toxins to monoclonal antibodies. In order to find a so-called "missile therapy," we conducted seven various studies, discovered AFP present on the membrane surface and a monoclonal antibody that recognizes it, and arrived at the present invention.

That is, the gist of the present invention is AFP'? An antitumor agent is a monoclonal antibody that recognizes tumor cells combined with a toxin or anticancer agent.

The present invention will be explained in detail below.

The monoclonal antibody used in the present invention is AF
Any monoclonal antibody that recognizes P'%l- may be used, but a monoclonal antibody that recognizes AFPg present on the cell membrane surface is particularly preferably used. Such monoclonal antibodies can be obtained by the following method.

In clonal antibodies.

The present invention will be explained in detail below.

The monoclonal antibody according to the present invention can be obtained by the following method.

That is, first, AFP77° derived from human placenta is fused with mouse myeloma cells, such as P3-Tl, which have been immunized with BALB/C mice, to obtain hybridomas by a conventional method. Monoclonal antibodies are then obtained from the hybridoma supernatant.

Next, using these antibodies obtained by conventional methods, liver cancer cell lines (for example, PLOlKN-NuE
) Y immunohistochemical staining and select antibodies showing positive. This detection was carried out using an abiocin: biotinylated horseradish peroxidase complex (ABC) kit.
Through the enzyme activity of horseradish peroxidase.

It is carried out using diaminobenzdine as a substrate.

To obtain a positive amount of antibodies, select the selected antibody-producing hybridomas by BALB/C! This can be done by intraperitoneally injecting mice, allowing them to grow, and collecting ascites fluid.

Alternatively, it is also possible to use method f, in which hybridomas are cultivated in large quantities in a culture tank.

The monoclonal antibody thus obtained has the following properties Z.

i) Using seven +40-labeled antibodies, we examined whether placenta-derived AFF inhibited the binding of PLO to liver and cancer cell line membranes. It can be seen that it inhibits

1i) When PLO cell line is labeled with l-14C-leucine, its membrane components are solubilized with "Triton

In addition, binding is also confirmed to secreted proteins in the culture supernatant.1ii) If it is a solubilized membrane component of the PLc cell line, is υζ an antibody reactant in the culture supernatant? , O'Farre
l) When performing two-dimensional electrophoresis according to the method of et al.
It can be seen that both membrane-derived and culture supernatant-derived bound substances have the properties of AFP (isoelectric point, molecular weight).

When the membrane fraction of the 1VJPLc cell line is digested with proteolytic enzymes such as trypsin, grotease, and thymotrypsin, binding to the antibody [i] decreases or tends to decrease.

In addition, it shows a tendency to decrease with ")IJt/X" treatment, and its binding property increases with lipase treatment.

The antitumor agent according to the present invention is a monoclonal antibody that recognizes AFP, preferably the above-mentioned antibody that recognizes AFP present on the membrane surface, and a toxin for tumor cells or an anticancer agent combined with this 7J. Examples include plant seed toxins such as ricin and abrin, and bacterial toxins such as diphtheria toxin.

These blue-purple colors are the B chain that participates in cell binding and the intracellular V chain.
The A chain, which is toxic to humans, is linked by disulfide (S-S) bonds. This S-S bond is reduced,
The A chain having -8H group is separated and used.

In addition, Cobra 4i, which is known as a complement activator
) can also be used.

bound to proteins.

Examples of the crosslinking method include MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester),
A conventional method using 5PDP (N-succinimidyl 3-(2-pyridyldithio)propionate) or the like can be used.

In addition, the above-mentioned anticancer drug is not particularly limited, but for example, adriamycin
, adriamycin (acttnomyctn), daunomycin, noga'i'Rycin, bleomycin, chromomycin, mithramycin 7 (
mitLxramycin) and the like.

Binding with monoclonal antibodies involves reactions such as diazotization and carbodiimidation depending on the type of anticancer drug and its functional group. A method using Dextra/, etc. is adopted.

When using an anticancer drug, it is preferable to encapsulate (incorporate) it in liposomes (lipid membrane vesicles) from the viewpoints of drug uptake into cells, sustained release, and reduced toxicity.

Liposomes consisting of double lipid layers mainly composed of phospholipids include (1) multilayers, (11) small unilamellar membranes,
(111) A large single film, both of which can be used.

Lipids include phosphatidylcholine (lecithin), lysolecithin, phosphatidylethane-/l/7 mino,
Cholesterol etc. are used.

The preparation is carried out according to a conventional method, and the size of (a) is usually obtained in a range of about 1 nm to several μm.

In this case, the anticancer drug is usually incorporated into the drug solution at the time of liposome preparation. As a result, hydrophilic substances are encapsulated within the liposome, and hydrophobic substances are encapsulated in the liposome membrane.

Encapsulation (incorporation) i'ii' can be determined as appropriate depending on the type of drug, but can be higher in concentration than in normal administration methods.

When binding a monoclonal antibody to the surface of this liposome, methods such as attaching a hydrophobic substance to the antibody and inserting it into the liposome, or crosslinking with phosphatidyl ethanolamine M, or Kurtal are used. However, preferably the following method is used.
11. Ru.

a) A reagent that reacts with phosphatidylethanolamine [-EIH group is covalently bonded and incorporated into liposomes. Is this an antibody '9SH modified or an S-8 bond between antibodies? Remove and combine the S) 1 parts.

5PDP, MBS, etc. are used for the above-mentioned co-opt combination. Or b) Incorporate glycolipids into liposomes and react the aldehyde groups generated by periodic acid treatment with antibodies. Using glycolipids and gangliosides, etc., the liposome surface is converted to aldehyde by treatment with periodate. Then Na
Antibodies are liposome bound in the presence of BH3CN.

The antitumor agent according to the present invention can be administered by conventional methods.

Since the antitumor agent according to the present invention can selectively transport toxins or anticancer agents to cancer cells, high therapeutic effects can be obtained.

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

Reference example (]) Production of mouse monoclonal antibody: h) AFP
Is it purified from placenta, has a purity of 9q% or more, and does not immunochemically react with human albumin (IISA) (made by Morinaga Institute of Biosciences)? Using.

This human AFP was administered to BALB/C mice.
01Lji was sensitized 91 times with Freund's complete adjuvant, the final immunization was administered intravenously, the spleen was removed 3 days later, P3-TJ/mouse mini-Romado fusion was performed using polyethylene glycol + llθoy7, and hyperimmunization was carried out using the conventional method. Dormer was created. Cloning was performed using the limiting dilution method over 79 times. Note that a large amount of antibody was collected from the ascites system of BAL B/C mice.

About qoq clones of hybridomas producing anti-AFP antibodies were selected through six fusions. Among them, l
/Culture of clones' double antibody titer IgG state? Shown in Table 1 (measured after double concentration 10 times).

Table 7 *“hybritec”: Anti-AFP manufactured by Hybritec
Monoclonal Antibodies (2) Selection of Antibodies l1l) Cell line is more than -weeks, Eagle Mr';
After culturing in M'y-based medium, washing with phosphate buffer several times and immediately reacting with iJ hybridoma culture supernatant; A method was used in which endogenous enzymes were inactivated with a H2O2 solution and reacted with antibodies. The antibody was detected using the ABC kit from Vectastain.
Due to the enzymatic activity of horseradish peroxidase, diaminobenzidine was used as the substrate.

b. Cell lines and maintenance H1- Cell lines for liver cancer include KN, P TJ.
C5 Also, cancer cell line NuEy derived from fetal liver cells, and human gastric moth/cultured line KATO as a control.
, M K N 4'ri.

Each cell line of colon cancer C-no was used. In addition.

The cultured cells were RP M'l:lA containing 20% tallow serum.
37. l/, O culture solution. θ℃, 5%CO2゜qs%A
The cells were maintained and grown under IR conditions.

C) Culture supernatant containing the anti-AFP monoclonal antibody of (1) above and its dilution (up to 28 times) for dandruff liver cancer cell lines PLO, KN, which were immunohistochemically reacted with human fetal liver cells NuE; A strong reaction was observed for l?111'l. This reaction was the same for unfixed specimens, fixation, methanol treatment, and H2O2 treatment.

In addition, as a control, a commercially available anti-AFP monoclonal antibody C) (manufactured by Ibritec) was used as an antibody titer of /
0" [Th, but other) Ibrid-
Like McCrone, there were no positive tests.

As shown in Table 1, 15F/2 does not have a particularly strong antibody titer compared to 5, nor does it have a large amount of Ig(), but
As mentioned above, unlike the others, it was positive.

However, /9F/2 did not react with cell lines MKNGS, KATOll'J, and C/ in which AFP production was not observed.

Example 1 (1) Method and materials (a) Cell line: ((2)b) PLc, N u E% described in the above reference example
C-/and KATO1] 'J and Lic (human liver cancer)''
I used it.

(1)) Culture method.

According to the method of the above reference example.

(C) Monoclonal antibody: Antibody obtained in the above reference example (/9F/, 2)? Using.

(eL) Incorporation of 3H-thymidine = 96 wells (cells were cultured in a Well J culture plate.

In the culture solution, YuμCi/Well (anticancer drug)
Or 3H- of s7+ 017me (when toxin)
After adding thymidine and culturing, the cells were washed and the radioactivity in the cells was measured using a liquid scintillation counter.

Ce) Toxin: Using ricin haf, its purification and binding with antibody (Bio ch
emie try 'Vol/2, A/A
+7973) Da 2,11! ;7-11411. /
By qg Cobb.

(f) Anticancer agent d Adriamycin was used.

(g) Liposome: The preparation and antibody binding are performed by the following method.

(iJ anti-human AFP mouse monoclonal antibody (lq
Ftx)tme and S P D P' (0,, LmM)'
f React at room temperature for 30 minutes, then add acetate buffer (pH
11,3)'l using.

The protein fraction was eluted using Sephadex 0-50'' and dithiothreitol (DTT) 30mMf.
Use ty-.

The reaction was carried out for 1 minute at room temperature to cleave the S-S bond, and the protein fraction/
get me

(11) On the other hand, as liposomes, egg yolk phosphatidylcholine qμmole, cholesterol/IzJμmole
, D T P - D P P 'K O,001-μ
Wash with adriamycin using mole y and suspend in acetate buffer ime.

(iii) Next, the products of (1) and (11) above are mixed, pHLOK is prepared, and ink coated for 2 hours to obtain the desired adriamycin-encapsulated posome-monoclonal antibody conjugate.

(2) F T of ricin A-monoclonal antibody conjugate
, Effect on Cy (in VitrOJ) The whole ricin molecule showed strong cytotoxicity even at 10-9M, but ricin A
As for the chain, its toxicity (suppression of 3.1-thymidine incorporation) was hardly observed at the order of 10-8M.

On the other hand, monoclonal J/antibody-bound or syn-A is 1
Even on the order of 0-8M, Iff incorporation of 3H-thymidine was inhibited and depended on the concentration.

In addition, synA bound to the anti-H8A monoclonal antibody used as a control was 5X/0-7M
The uptake of 3H-thymidine was suppressed, but a lower concentration of 10-8 M had no effect at all.

(3) Effect of adriamycin-encapsulated liposome-monoclonal antibody conjugate (in vitro) on cells 72
After being brought into contact with the drug for a period of time, it was washed, and the 3H-thymidine incorporation described above was carried out in step 7.

The results are shown in Figure/-3.

It is a natural result that the anticancer drug adriamycin leads to in vitro inhibition, suppression of tumor growth, and reduction of 3H-thymidine uptake, but what is the difference between adriamycin-encapsulated liposomes and a monoclonal antibody further conjugated to them? In comparison, PLO (Figure 7), Lie' (Figure Yun,
In the AFP-producing tumor liver cancer cell line of NuE (FIG. 3), the monoclonal binding type always showed a strong antitumor effect, and in %NIIK, a stronger effect than adriamycin alone appeared.

On the other hand, no difference was observed in tumors other than liver cancer, C-/ and KATO■1, regardless of the presence or absence of monoclonal antibody binding.

(4) Nude mouse transplantable liver cancer cell line Li7
According to the protocol of Barachil Columbus Laboratories, the major axis (LJ and minor axis CW) was measured once a week using a sliding caliper (S'L). (W2
The estimated tumor weight was calculated from x L ) / , 2, and the tumor weight was ioo ~ 300 m! Treatment started at the age of 7,
This was done three times on each day.

The results for adriamycin-encapsulated liposomes, monoclonal antibody-bound adriamycin-encapsulated liposomes, and adriamycin alone are shown in FIG.

In other words, adriamycin alone was clearly more effective than the control group (no treatment), and the liposono-conjugate was more effective than the control group (no treatment).
iVO has produced therapeutic effects.

[Brief explanation of drawings]

Figure/=5 shows the in vitro results of the antitumor agent according to the present invention. Figure 6 shows the in vivo test results. Applicant Mitsubishi Chemical Industries, Ltd. Agent Patent Attorney Hase - 1 other person0ii'jノi'+':! F (F'J'ef: No change) Nagi l 7F' Adriamycin concentration diagram (Figure 2) 3 Adriamycin concentration diagram 3 Adriamycin concentration diagram 5 Adriamycin concentration. Continuing from page 1 of the number of days 100 square meters of the old town inside the Ken Mortuary Mitsubishi Chemical Industries, Ltd.゛1Special M'F Application No. 176773 OJ2 Name of Butomei Anti-tumor agent S3 Relationship with person who writes 7 mountains j1 Kushii'1 41h i'1 Applicant (!+9
) Mitsubishi Kasei Kogyo Co., Ltd. (2, Shikikai Ne) / 1 Agent 〒100 Tokyo R-dai 1711 Marunouchi 2-chome 5) No. 2〉] Mitsubishi Kasei Kogyo Co., Ltd. Ne1! ”+ sleeve l-l',, fi) Q ノI'lf'l
Showa E) 9 'L January 3'3 'I 8 (Qm
11) 1i'l'l, Specification i↓) and drawing cleanup procedure Neichi Masae 1 (Voluntary) 1 Indication of matter A'1 Koma Application No. 176773 of 1981 2 Name of the invention Anti-tumor agent 3. Relationship with the case of the person making the amendment Patent W To applicant (59(i) Mitsubishi Kasei Koukai Co., Ltd. 4 Agent Address: 1 o. Chiyo, Tokyo [5-5 Mitsubishi Kasei Kogyo Co., Ltd., 2-5-28 Marunouchi, 0-ku) [Amendment] Column 6 of “Illustrated!Ill. 1-Detailed description of the invention” Contents of amendment

Claims (1)

[Claims] (1) An antitumor agent comprising a monoclonal antibody that recognizes α-fetoprotein (AFPJ) combined with a toxin against tumor cells or an anticancer agent.