JPS5933295A - Antihuman fibronectin antibody - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody, reactive specifically with all the human fibronectins, and capable of linking to a domain, obtained by cleaving a human fibronectin with a thermolysin, and having 150,000-140,000 or 40,000 molecular weight [measured by the electrophoresis with sodium dodecyl sulfate(SDS)/ polyacrylamide gel]. USE:For purifying and measuring human fibronectins. PROCESS:A human fibronectin and a complete Freund adjuvant are administered to a mammal, e.g. a mouse, subcutanesouly four times at an interval of 10days in the same amount to immunize the mammal. Three days after the final immunization, the spleen is extirpated to give the splenic cell, which is then mixed with a myelomatous cell of a mouse, etc. A polyethylene glycol solution is dropped to the resultant mixture to carry out the plasmogamy and prepare a hybridoma, which is then cloned and cultivated in a culture medium to separate the aimed monoclonal antibody.

Description

Detailed Description of the Invention The present invention is directed to a novel human fibronectin (hereinafter abbreviated as 1-FNJ) monoclonal antibody.
lant, 1 body).

FN is a sticky glycoprotein that exists on cell surfaces, delaminants, body fluids, etc., and is a polymeric substance with approximately 400,000 or more subunits α and β linked by two disulfide bonds. It exhibits binding properties to fibrin, promotes cell adhesion and proliferation, and also has properties that are involved in creating an environment for immune response [Molecular
nd Celural Biochem.

29, 103-128 (198'0); Oo
llagenRes, 1, 95-128 (1
981); Nature275, 179-184
(1978); Blood.

56.145-158 (1980)).

In recent years, FN has attracted attention due to its relationship with cancer, but there are still many unknowns regarding its structure, dynamics,
Many studies have been conducted on in vivo activity. For this research, it is advantageous to use specific antibodies as well as general physiologically active substances, but FN obtained by conventional methods is advantageous.
-Antibodies (polyclonal antibodies) have various recognition sites, react with FN of other species, and [
It could not be used in the above research because it was non-specific in that it neutralized most of the various physiological activities of FN.

Under these circumstances, as a result of intensive research over a long period of time, the present inventors were able to obtain a monoclonal antibody that specifically reacts with FH and has a clear reaction site and ability to neutralize the physiological activity of FN. Successful.

That is, the present invention provides a vibrator 1) produced by the fusion of myeloma cells with immune cells of a mammal immunized with human fibronectin, (a) essentially specific to all human fibronectin; reacts to (
b) Molecular weight of human fibronectin cleaved with Thermoregi/(SDS/po1) Alumidodel electrophoresis method) 1 s 0.006 and 140.00.0 or 4
0. The present invention provides a monoclonal antibody that specifically binds to the P main of OGO.

Such anti-human fibronectin antibodies of the present invention are human FN
In addition to being useful for research, it can also be used for general purposes of antibodies, such as purification, measurement, etc.

The anti-human fibronectin antibody of the present invention is produced by fusing immune cells of a mammal immunized with human FN with bone marrow hepatocytes to form Vibe II P-ma, and separating the monoclonal antibody from this. .

In the present invention, according to the present invention, 1) IS-Ma's key 41, methods known per se, e.g. Nature, 256, 49
5-497 (1975).

As the antigen FN, those derived from plasma, body fluids such as amniotic fluid, cell surfaces, etc. should be used; especially human plasma FN.
N is preferred. These human FN&main columns, hepa-11 column, etc. can be used to purify the single meat, or commercially available 11-oderecivitate (blood coagulation factor preparation) can be used.
This can be obtained by moving the distance further by m.

The mammal to be immunized with human FN ('!, although not particularly limited, is preferably selected in consideration of compatibility with the myeloma cells used for cell fusion, and generally mice, rats, etc. are used). Immunization is also carried out by a general method, for example, human FN is diluted with physiological saline or the like to an appropriate concentration, made into a suspension with Freund's auxiliary solution, etc., and administered to the animal by sterile injection or the like.

Administration is carried out several times every 1 to 2 weeks, with a total dose of 75 to 10
It is preferable to set the amount to about 0 ■/mouse. As the immune cells, it is preferable to use lung cells extracted about 6 days after the final immunization.

cells, e.g. MS-1, P3, P3 in mice
- Ul, MPO-11, sp2, X 63.6.5.
3. Y3, Ag1,2,3 in rats
etc. are used. The fusion reaction is carried out using a known cell fusion method, for example, by incubation in a medium in the presence of a fusion promoter.

As the fusion promoter, for example, poly-11 ethylene glycol (PEG), Sendai virus (H'T'J), etc. are used, and if desired, an adjuvant such as dimethyl sulfoxide may be added to increase the fusion effect. I can do it. The ratio of immune cells to bone marrow lung cells used is the same as in general methods; for example, the number of immune cells used is about 1 to 10 times that of myeloma cells. MFiM medium used for cell line proliferation, Gerpelaco's modified medium, ppMx-1640 medium, and various other usual media used for cell culture of this type can be used, and serum supplements such as Fe2 are usually used. It is better to remove it.

For fusion, the above-mentioned immune cells and myeloma cells are mixed well in a manual medium, centrifuged, the supernatant is removed, and a PEG solution preheated to about 37°C, for example, with an average molecular weight of 1. [10
About 60 to 6 n-1,011 [1] in normal medium
This is done by adding at a #degree of 0 w/v% and mixing. Thereafter, by repeating the steps of sequentially adding an appropriate medium, centrifuging, and removing the supernatant, one idelidoma is formed.

Isolation of the desired hyperprime r-ma is carried out by culturing the cells after the cell fusion described above in a usual culture medium for selection of Nov. 1j doma. The myeloma cell line described above has hypoxanthine phospho-11 bosyltransferase () (C).
PET) and therefore cannot grow in HAT medium (medium containing hyboxanthin, aminobutylene, and thymidine). Obey. The cells may be cultured in the HAT medium for a sufficient period of time, usually from several days to several weeks, to kill the cells (unfused cells, etc.) of the desired hybridoma.

This Vibe 11 doma may be cultured for about 1 to 2 weeks in an HT medium containing hyboxanthin and thymidine, and then cultured in a normal medium.

The hybridoma obtained by sniffing is subjected to a conventional limiting dilution method to search for a strain producing the desired antibody and to reject the clone. The thus obtained Vibe 1j doma producing the monoclonal antibody of the present invention can be subcultured in a normal culture medium and can be easily stored for a long period of time in liquid nitrogen. The present inventor maintains D5 and H5, which are representative of this hybridoma, in a state in which they can be separated by themselves in the Examples described later.

To find out whether the hybridoma obtained in this way produces the desired monoclonal antibody, for example, KL Engineering S.
A method CE11g'Vall, L; M8th,
Finzymol.

70.419-439 (1980)]x. That is, by adding hybridoma culture supernatant to a microtiter well in which a specific domain of human y'N has been immobilized as an antigen and incubating it, the antibody in the supernatant and the antigen fixed on the well surface ( h) FN
specific domain). After that, anti-mouse antibody labeled with various enzymes (if the immune cells are mouse), anti-rat antibody (if the immune cells are rat), or the same protein A are added and incubated, and after thorough washing, the enzymes in each well are incubated. This is done by checking the activity.

C The Jour
nal of Biologj, cal O
bemistry, Val, 25
6.

Bian 12. DT)-<')452 6462 (1981)
:] Sunawachihito F N k Thermolysin
(Protease.

type
aemrnlj , U, K, , Natu
re 227.680-6F35 (see 197[])], the molecular product is 150,000 and 140. On
(hereinafter referred to as domain A) and 40,000 (hereinafter referred to as group main B) are used. This domain is purified and isolated based on its physicochemical activity and physiological activity, for example, by column chromatography, dialysis, etc. The physiological activities of the domains are shown in Table 1.

Table 1 also shows that anti-mouse antibodies, anti-rat antibodies, or protein A labeled with various enzymes may be commercially available or prepared by conventional methods. Historically, this search method is not limited to the above-mentioned ELJEA method, but also general pressure, which is used for detecting antibodies such as the plaque method, spot method, agglutination reaction method, Ouchterlony method, and RIA method, which are commonly used in this type of field. Various methods can be adopted [[Vibe 11 Doma method and monoclonal antibodies]
Reference: ■R&D Planning, June 5, 1957].

In order to obtain the anti-human fibronectin antibody of the present invention from the specific Vibe 11 doma obtained as described above, there is a method of tissue culturing the Hydelyma according to a conventional method and separating it from the culture supernatant, or a method such as Vibe I) doma. A method is adopted in which the drug is administered to compatible mammals, grown, and isolated from the ascites fluid.

The former method is good for obtaining high-purity products, and the latter method is excellent for producing large trenches and is rated A.

As shown above, the novel anti-human fibronectin antibody of the present invention, which specifically binds to domain A from Vibe 11V-ma H5 and to domain B from hybridoma D5, can be obtained. Conventionally known monoclonal antibodies [5cand, J, Immunol, 12+3
61-637 (1980) i Ce1l Jolo
gy international sports,
4, 734 (1980); Ce1l, 25
, 133-141 (1981); Ce1l,
26, 259-267 (1981)) are not known to specifically bind to domain A or genome B.

Next, reference examples and examples will be given and explained.

Reference Example 1 Human plasma fibronectin Fibronectin-rich supernatant [supernatant after fraction Knee 1(r)/l'+J Schiff precipitation, which is a by-product during the purification of human blood coagulation factor ■Dr. K. Fujikawa (University of Washington)] was prepared using a conventional method.
In, type engineering; Sigma Corporation], Cephalo-C4B
(Pharmacia); Methods ljnzy
mol.

22.345-378 (1971)] 3~
4Wr Latin/medel; Vera H+ $11um approx. 40 ml), 0.5mM ITA, 150'
25mM Tris buffer with mM Na11, 1mM phenylmethylnulfonyl fluoride CpH=7.6)
After thorough washing with the above buffer solution containing 1M urine, the plate is further washed with the above buffer solution. Elute fipronectin with the above buffer containing 4M urea, 0.5mM EDTA, 5QmM
10 mM Naol * (pH = 7.
6) at 4°C for 6 days. Human Derasma phipronectin obtained at 75\ (preserved at -80'0).

Reference example 2) Production of Eve++ S-Ma■ Ba1b/
c human plasma fibronectin 3.5' obtained in Reference Example 1 to mice? Inject 75-100 μl/tnl of Freund's complete adjuvant subcutaneously per mouse. 10
The same amount was administered 4 times at daily intervals, the lungs were removed 3 days after the final immunization, and the snails were inoculated with RPM Knee 164N medium for 6 days.
Wash twice.

Mau-' (f% filltm cell line NS/l (Nat
ure Val, 25YS.

August 7 (1975)) after washing in the same way.
This Is/1 1 X I 07 pieces and the above tile boat cavity 4
Place the X i O' solids in a 50-centrifuge tube and mix. 2'
After centrifugation for 2.5 minutes at 00x, remove the supernatant with a Pasteur pipette.

The temperature was kept at 37°C. +3? +1 ethylene chloride 011 call 1500 (Ffastman *engineering nc,) 50w
/v% RPM knee 1640 solution 4 solution 1°Id was added dropwise,
Mix slowly for 2 minutes.

37℃ VC retention WALjc 15% yos, 1
mm%! ' Add 1 ml of Rupete's RPMI-1640 (hereinafter referred to as "Complete RPM Knee 1640J") for 1 minute.
Add the same amount of Complete RPM: r-1640 for 1 minute, then dropwise add 8-Complete RPMI and stir gently for 2 minutes. After centrifugation at 200 x 2.5 minutes, remove the supernatant and
Suspend cells in complete RPMI-1640 incubated at 10°C to a density of 107 cells/Ie, and place 100μ in microtest plate (Falcon). inoculated at 37°
Culture C15 carbonate in a spin incubator. 24
After 1.0 x 10-4 M Hyboxanthin, 4.
OX, 10-7M aminodeten-11, C6x 10-6
The above complete RPMI-16411 containing M-thymidine (
Add 100 μl (hereinafter referred to as “HAT medium”) to each well. Thereafter, half of the supernatant was replaced with fresh HAT medium on days 2.3.5.8 and 11, respectively, and half of the supernatant was replaced with fresh HAT medium on day 14. Change to complete RPM Nee 1640 (hereinafter referred to as (-'HT medium)) containing OX 10-'M hypoxanthin, C6X 10-5M thymidine.

Similarly, half of the supernatant was replaced with HT medium on days 18.22.25 and 26, and half of the supernatant was replaced with complete Rk on day 28.
Replace with MI-1640. Thereafter, the cells are grown and maintained using this complete IRPM knee 1640. The hybridoma obtained by sniffing was cloned by the limiting dilution method.

That is, 2.5 x 10 hyperdomas/ml, B
a1b/c-r mouse thymocytes 4 x 106 cells/m7!
iIMIM was inoculated into a complete RPM knee 1640 so that the iIMIM was cultured in a 2F)0 well plate at a density of 5 hyperprimers/well. The proliferating hybridomas were further cloned in the same manner at 0.25 hybridomas/well. The culture supernatant of each clone, domain A or domain B obtained in the same manner as in Test Example (3) described later, was added to a U-shaped microtiter plate insolubilized, and after standing at room temperature for 5 hours, the culture supernatant was removed. , Zo1/
- The cells were washed four times with PBS. 50, ul of peroxidase-labeled protein A (LY.

(manufactured by Laborad 11-Inc.) was added, the anti-cheat was removed at room temperature for 2 hours, and the mixture was washed 4 times with PBS. 0.03 in each well
0.2 M citrate-phosphate buffer (pH
= 5.0 ) , [1,5- and 4 "It' /
Add 1.0 - of me-orthophenylenediamine in saline and incubate for 10 minutes at room temperature. 1N Hot
The reaction is stopped by adding an aqueous solution and a clone producing the desired antibody is searched for by absorbance, thus clone f
Single clonal strains of Jo, H5 and D5 were obtained.

The monoclonals produced by both cell lines are identified by antibodies, and have the characteristics shown in the test examples below. Both cell lines are each stably stored in liquid nitrogen.

Example 1 11) Complete RPMI of the above H5 or D5 as usual
-1640 at 37°C in a 5% carbon dioxide gas incubator for 48 hours, followed by centrifugation (3n00 rpm).
.

10 minutes) and H5-producing monoclonal antibody (antibody A).
Alternatively, culture supernatants containing the D5-producing monoclonal antibody (antibody B) were obtained.

(21Ba1b/c? 1 of the above H5 or D5
×10'/mouse in RPux-1640 medium 0.5
m/[Suspend and administer intraperitoneally. After 2-3 weeks, the accumulated ascitic fluid was collected I7 and 2-5π7! / Obtain mouse ascites fluid containing antibody A or antibody B.

The antibody concentration was approximately 0.2 to 1/- in each case.

Test example (1) Rabbit antibodies against mouse immunoglobulin classes for each immunoglobulin class (
Litton, Bionetico, Inc.
, Kensington.

(MD20795) and 125-labeled protein A following the method of Yeh et al. (Ming-Ya
ngYeh etal Proc, Natl, Ac
ad, Sci, USA, VOl, 7<S.

No, 6. pp, 2927-2931. (1979))
.

The results are shown in Figure 1.

(2) Specificity test A polystyrene microtiter plate containing 1 μf/rnl of H. deranuma fipronectin at 137 mM Na0
t, 2.7 mM KCl, 8.1 mM Na2
After 2 hours of incubation with a phosphate buffer solution (hereinafter referred to as PBB) containing HPO4, 1.5mM KH, PO,
Incubate for an additional 2 hours in PBS containing BSA.

Plate y7 The above Example 1-(
Incubate in the same manner as antibody A obtained in 2). After washing the plate with PBS, rabbit anti-mouse immunoglobulin antibody was incubated in the same manner as 12-)I,--y° lotiin A, and the specificity of antibody A was tested by RIA using solid-phase competition. In the above, the plate was added with 0.1 μL//d PI3 of Human Derasma fipronectin.
8 Incubating with antibody B instead of antibody A
The specificity of antibody B was tested using the same pressures except that . The results are shown in Figure 2.

In the figure, the horizontal axis represents the concentration of 4 fibronectins, and the vertical axis represents the binding rate of 125 protein A (the number of binding counts when no fibronectin is added is taken as a factor of 100).
are shown respectively.

The marks in the diagram represent the following.

○; human plasma fibronectin; human cell fibronectin Δ;, bovine fetal plasma fibronectin; neonatal bovine plasma fibronectin; guinea pig deplasm fibronectin ■; chicken cell fibronectin; mouse plasma fibronectin Delonectin Each plasma hydronectin was purified by affinity chromatography using Delatin-Sepharose in the same manner as in Reference Example 1 from plasma or serum obtained from each animal in a conventional manner. Human cell fibronectin was isolated in the same manner from the culture solution of Noki-38 and used.

Chicken cell fibronectin was obtained according to the method of Yamaaa et al. [Yamada, K., M.,
Biochemistry.

16:5552-5559 (1977)), from the figure,
It can be seen that antibodies A and B of the present invention are antibodies specific to human fibronectin.

(3) Specificity test Human Deras maphie fronectin obtained in the same manner as in Reference Example 1 was treated with trypsin (Worthin) at room temperature.
gton company; 111? /me, 3 Q minutes) or thermolysin treatment (protease, type X
, 'y Gua company; 5μ? /rne, 4 hours) to obtain human fibronectin-main, respectively. [8ekig
uchi, K. et al.

J, Biol, Ohem, 256, 6452-
6462 (1981)), each of the above I-main and untreated human derasma phipronectin is subjected to 5DS-polyacrylamicidal electrophoresis. The first group was stained with common blue. [Panel (1)] The other two groups received antibody A after adsorption to nitrocellulose [Panel (2)] The father received antibody B (Panel (3)
] and then rabbit anti-mouse immunoglodelin antibody followed by 1251-Protein A. CI (arry Towbin et al.
, Proc, Natl, Acad, Sci.
USA Vol, 76, A9 T)l), 4350
-4354, (1979)], the results are shown in Figure 3.

In the figure, v-ya c shows untreated human derasma fibronectin; lane b shows human derasma fibronectin treated with trypsin; lane C shows human derasma fibronectin treated with thermo-IJ thin.

From the figure reading, Antibody A is 150.000 and 141 of thermolysin-treated human derasma hydronectin], [)
00 domain (domain A), and antibody B specifically binds to the same 40.000 domain (domain B).
It was found that it specifically binds to.

Incidentally, the 200.000 and 180.00 (]gemains of to11decine-treated human derasma phipronectin include both the domains A and B described above.

(4) Incubate the polystyrene microtiter plate with 5μ2/- of human delasma phipronectin in PBS for 2 hours, then incubate for an additional 2 hours with 5 Lt)BSA in PBS, and wash the plate 3 times with PBS. This was serially diluted with PBS in Example 1-(
Incubate in the same manner as with 50 μE of antibody A or antibody B obtained in step 21, wash the plate with PBS, and incubate in the same manner with 1011 cV ml of PBS'5Qμ). Wash the plate with PBS, cut out the latter wells, and Its radioactivity (1) was measured.

Setting the radioactivity when the antibody concentration is O as 10, a proposal for suppressing the fibronectin-rlatin binding of antibody A or antibody B was proposed using the following formula.

Upper O The results are shown in Figure 4.

In the figure, the vertical axis shows the inhibition rate, the horizontal axis shows the dilution factor of the antibody, . . . indicates antibody A, and α indicates antibody B.

1y shows that antibody B suppresses the binding of rlatin to fibronectin.

[Brief explanation of the drawing]

FIG. 1 is a drawing showing the immunoglobulin class of the antibody of the present invention, FIG. 2 is a drawing showing the specificity of the antibody of the present invention,
FIG. 3 is a drawing showing the human fibronectin domain to which the antibody of the present invention binds, and FIG. 4 is a drawing showing the inhibition of human fibronectin-delatin binding by the antibody of the present invention. Above yI: 1 figure 0 5 1 (
1. Radiant bipolarity, cpm
t'+, body A1 几い本l) full 4QK-□

Claims (1)

[Claims] (1) Hybridomas formed by the fusion of immune cells of a mammal immunized with human fibronectin and bone marrow lung cells (a) react specifically with essentially all human fibronectin2 , (b) Molecular weight of human fibronectin cleaved with thermo-11cin (S
DS/Polyacrylamide electrophoresis method) 150
．． Monoclonal antibodies that specifically bind to domains of 000 and 140.00 CJ or 4[1,000.