JPS6359898A - Human/human hybridoma producing human immunogloblin specific to cancer-relating antigen and antibody produced thereby - Google Patents

Abstract

PURPOSE:To separate an antigen-specific human immunogloblin, by preparing a human/human fused cell strain of a human B-cell of a human colon carcinoma patient and a subclone of a human lymphoblast cell strain and culturing the human/human fused cell strain. CONSTITUTION:A human/human hybridoma CoLNE10 (human/human fused cell strain) having biochemical properties to link with a human established cell CaCo-2 (established cell originated from human colon carcinoma) and a human established cell A549 (established cell originated from human pulmonary carcinoma) is produced by using a human immunogloblin A (IgA). An antigen- specific human immunogloblin can be produced from a human/human fused cell strain (human/human hybridoma CoLNE10) of a human B-cell of a human colon carcinoma patient and a subclone of a human lymphoblast cell strain.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides human B-cell immunity that has immunoglobulin-producing ability and human B-cell immunity that is substantially deficient in globulin-producing ability.
- A new type of human/human fusion cell clones with cells, antigen-specific human immunoglobulins produced by them, and a method for producing the human immunoglobulins.

More specifically, the present invention relates to a human/human hybridoma that is a human/human fusion cell line of human B-cells of a human colon cancer patient and a subclone of a human lymphoblastoid cell line, and has the ability to produce antigen-specific human immunoglobulin. Regarding. The invention also relates to antigen-specific human immunoglobulins produced by the human/human fusion cell line.

Furthermore, the present invention also relates to a patented method for producing human immunoglobulin, which comprises culturing the human/human fusion cell line in a medium and collecting antigen-specific human immunoglobulin from the culture.

The antigen-specific immunoglobulin can be used in the medical and pharmaceutical fields, such as prevention, treatment, and diagnosis of human colon cancer, lung cancer, and gastric cancer, and in the pharmacological field, such as biochemical reagents and purification reagents for biopolymers. It is useful in a wide range of fields such as research in biochemical fields such as the purification of cancer antigens.

Conventionally, fusion cells of mouse B cells sensitized with cancer or other antigens and mouse B cells from myeloid leukemia (my61oIIa) mice are formed outside the mouse body, and mouse immunoglobulin production based on t=N- to the above antigens is performed. have the ability,
In addition, there are reports of the formation of mouse/mux fusion cells with self-propagating properties [for example, Kayla No, Milstein S., Nature (Kijl+Ier G.
+MilsteL+C. , Nature).

256, 495 (1975); Diebold E.N., Lloyd K.O.L.T.C.
Ikeg H, Etgen H Eng 7.

Old L.G., Proceedings of the National Academy of Sciences of the United States of America (Dippo
lcLE. G. +LIoyd+K, o
．． , Li.

L, T, C. +I keda+H. yoeL
tgcnlI{, F”, &Old, I-, J.
, Pore. Nat. Acad. Sci. U.

S. Δ． ), 7 7.6 1 1 4 (1 9 8 0
); etc. 1.

In addition, 8 synergistic cells of human B-cells sensitized by the antigen and mouse B-cells from myeloid leukemia Mawas were formed in vitro, and these cells had the ability to produce human immunoglobulin against the above-mentioned antigen and had self-propagating properties. There are also reports of the formation of human/mouse fusion cells with [e.g. Shlom Noei,
Schlom J., Wunde Proceedings of the National Academy of Sciences of the United States of America
rricb P, , and Ter
amoto Y, A, , Proc.
.

Nat. Acad. Sci. , U, S, A, )
,? ? C1 1 )+6841 (1980) J.

However, in the latter attempt to obtain fused cells capable of producing human immunoglobulin, the ability to produce human immunoglobulin is lost over time due to chromosome deletion, and the ability to produce immunoglobulin is extremely reduced. It has a fatal flaw of instability.

In an attempt to form human/human fusion cells with the ability to produce human immunoglobulin, human B-cells sensitized with the antigen Deca virus or Hapten [2,4-nonitrophenyl 1] were used. is used as a donor, and a cell line (Hi) B-cell, which has the ability to produce human immunoglobulin from a myeloid leukemia patient, is used as a donor! a
There are reports of the formation of human/human fusion cells with the human immunoglobulin production ability against the above antigens and self-propagation properties by forming fusion cells with the above-mentioned antigens [e.g. -C.M., Leinenbatzha.A., Hole.Gbru, Stepleski.Z., Kobrodsky.H., Nature (Croee).

C, M, , Linnenbach+A, +Hall
, W, +5tepl.

viski+Z & Koprowski+H,,
Nature, 288w488 (1980); Cabran H. Nis, Proceedings
of the United States of America (01)
93on, L & Kaplan, H, S+tPr
oc+Nat, Acad, Sci, U, S, A,
), 7(9), 5429 (1980); et al. 1° However, in mice, the above-mentioned method is a method for producing mouse immunoglobulins, and in mouse/human hybridomas, mouse-derived products are produced in 1apA culture. It is inevitable that it will be generated during the performance. Loss of human immunoglobulin production may also occur due to deletion of human chromosomes. This is why it is desirable to produce human immunoglobulin obtained from human/human fusion cells. However, the reports on human immunoglobulin produced from the human/human hybridomas described above do not provide any demonstration of binding to cancer cells. In particular, there is no description of binding to colon cancer, gastric cancer, or lung cancer cells.

One of the present inventors has developed a human/human fusion cell line of human B-cells from a human uterine cancer patient and a subclone of a human lymphoblastoid cell line, which has the ability to produce antigen-specific human immunoglobulin. dormer is fr4'll
(Japanese Patent Application Laid-Open No. 59-B7497). And normal #! for the cervical squamous epithelial am blemish cell and established cell lines Hela and Ca5ki derived from the cell. & Fibroblast (WI-38)
It was reported that the binding force was stronger than that for

However, there is no mention of binding to human lung cancer, gastric cancer, colon cancer cells, etc.

Furthermore, Cote et, at, +Proc, Na
t, Acad.

Sci, USA, Val, 80. pp2026-
2B30 (1983) reports on a human/human fusion cell line of B-cells from human cancer patients and a human lymphoblastoid cell line, and the human immunoglobulin produced by the line. According to this report, LICR-2 or 5KO-007 was used as a human lymphoblastoid cell line, and a human/human fusion cell line with B-cells from human breast cancer patients, human cancer patients, and human kidney cancer patients was used. It has been shown that some of the fused cell lines formed produce IgA.

However, this report does not include human B-
Human cells and subclones of human lymphoblastoid cell lines/
No human fusion cell lines are shown. Furthermore, specific examples of binding to cancer cells include human colon cancer-derived cell lines 5W-1083, 1116, and -122.
2. No binding to Hi”-29 (nonr
eacLive).

Furthermore, JP-A-61-44900 describes a monoclonal antibody that specifically reacts with y4 to pancreatic cancer, colon cancer, and liver & lfj-related antigens, and the fusion cells that produce it are antibody-producing cells. and bone marrow cells, but it is stated that they are preferably derived from an allogeneic animal. However, what is specifically disclosed in this proposal is only a mouse/mouse fusion cell line.

The present inventors have been conducting research on the creation of human/human hybridomas. As a result, we found that a human/human hybridoma, which is a human/human fusion cell line of human colon cancer patient's human B-cell and a subclone of a human lymphoblastoid cell line, has the ability to produce antigen-specific human immunoglobulin. successfully created.

Furthermore, the immunoglobulin produced by the human/human hybridoma of the present invention has binding ability not only to colon cancer, especially colon cancer, but also to lung cancer cells of different origins, such as human cell lines! It was found that it has binding power to α-cell A549 (lung cancer).

The human/human hybridoma of the present invention is a human large HvI
Human/human hybridomas of cancer patients have the immunological characteristics of B-cells and can stably maintain such characteristics, and the human/human hybridoma has the ability to produce human immunoglobulin A (IbiA). And these produced antibodies are human lung cancer cells, I''
They discovered that it is an antigen-specific monoclonal antibody that shows binding to J cancer cells and colon cancer cells, and succeeded in developing such an antigen-specific human immunoglobulin, 91y:L.

Furthermore, the above-mentioned new type of human/human hybridoma is derived from human cell populations containing human B-cells of human colorectal cancer patients and subclones of human lymphoblastoid cell lines [human B-cells].
- fusing a group of human cells containing Cell J outside the human body to produce a fused cell of H) B-cell and H) B-cell;
We have discovered that it is possible to produce fused cell clones by culturing them in a conventional HAT medium, and by such a method, we can produce fused cell clones (e.g., C.
.

We succeeded in producing 111 LNEIO and 'l' OCo/A 1 ).

Therefore, the purpose of the present invention is to develop subclones of human I3-cells and human lymphoblast J12 cell lines from human colorectal cancer patients.
- To provide a human/human hybridoma, which is a human/human fusion cell line with Cell J and has the ability to produce antigen-specific human immunoglobulin.

Another object of the present invention is to produce cancer-related antigen-specific human immunoglobulins (monoclonal antibodies) that exhibit binding properties to human colon cancer cells, lung cancer cells, v4 cancer cells, etc. using the human/human hybridoma. A method and the human immunoglobulin are provided.

For the above objects and many more objects of the present invention, I!
7. will become clearer from the description below.

The human/human hybridoma of the present invention is a human B-cell of a human colorectal cancer patient, for example, a human B-cell and a human lymphoblastoid cell line that can be obtained from the patient's blood lymph nodes, viscera, bone marrow, etc. Subcloned human I3-cell 1, for example UC729-6 strain (ATCC CRL8061:@
Engineering 1iN deposit tLl knowledge IF notification number, 57 @ deposit rjS
Human/human fusion cell lines such as HIH/To1 strain (No. 664) and HIH/To1 strain (Notice of Refusal of Fiber Technology Deposit No. 60 Micro-Yorobun No. 1198), human colon cancer cells, lung cancer cells,
) 4 It has the ability to produce antigen-specific human immunoglobulin (monoclonal antibody production ability) that shows binding properties to cancer cells, etc.

From research 1 of the present inventors, human B-
A subclone of the human IJ lymphoblastoid cell line that is substantially deficient in immunoglobulin production ability [
H) B-Cell] and fused outside the human body, large! A@i
For the first time, a human/human hybridoma was produced that has the ability to produce human immunoglobulin that specifically binds to cancer-related antigens such as cancer, gastric cancer, and lung cancer.

The fusion operation itself to produce this fused cell may be performed by any known method. For example, the fusion operation can be carried out by bringing human B-cells of a human colon cancer patient into contact with a subclone of a human lymphoblastoid cell line [human B-cell J] in a liquid medium in the presence of a fusion promoter. Can be done. An example of such a fusion method is Sendai Virus (HVJ).
Examples include a method using a fusion accelerator such as polyethylene glycol, and a method of electrically fusing at high voltage.

For example, in an aqueous medium, in the presence of a fusion accelerator as exemplified above, if desired, gently stir 4'1! to homogenize the 'ζ system, and then incubate for a few minutes, for example, to produce a fused cell consisting of one of the above B-cells and 1"F of the subclone (B-cells). Desired fused cells can be produced by using the device.Examples of the liquid medium include water, physiological saline, 5% aqueous solution of trimethyl sulfoxide, and aqueous 596 glycerol solution.

The system in which the desired fused cells have been produced is, for example, centrifuged to collect the cell group, and then transferred to an appropriate medium again, for example, at 10%
MI-1640PO (Rf containing calf serum)
In L-body medium 1: HA T d l in Oeta medium,
The collected cell group is dispersed, for example, the dispersed cells are injected in a fixed amount into the holes of a micro titer plate, and cultured at 37° C. in the presence of, for example, 5% CO2. It can be carried out. In place of the above RPMI-1640 medium, Ri) MI-1640:DME:F-
12=2:1:1 mixed medium (RDF) can also be used. Refresh the culture solution in each hole, for example, every 3 days!
After continuing to culture for, for example, 2 weeks, the presence of fused cells can be determined using visualization. The culture fluid of a sample in which colonies are observed can be collected, and the presence or absence of human immunoglobulin can be detected by, for example, Lanao φ immunoassay using +zs■ or enzyme antibody method.

In this manner, a colony that has been found to be producing human immunoglobulin is transferred to a new culture medium and cultured to proliferate the fused cells, thereby making it possible to obtain a fused #II cell wolfberry clone. Furthermore, if necessary, sub-cloning can be performed to obtain a human immune gummy purine that specifically binds to a desired cancer-related antigen such as human colon cancer, gastric cancer, or lung cancer.
You can get the following information.

In implementing this aspect, methods known, for example, from the following documents can be used. [Glassy M.C., Handley H.H. Shizuku Hagiwara H.,
and Royston Eye, Proceedings Op.
National Academy of Sciences
United States of America (GIassy)
+M, C,, Handle)+, I1. H,rHa
giwara+H,, arld Roy! 3ton+
1, ,Proc, Nat, Acad, Sci,
U, S, A, ).

80.6327. (1983)J.

For example, lymphoid cells such as lymphoblasts and lymphocytes of myeloid leukemia patients are taken from a healthy human lung by biopsy (B 1opsy) using the method described in the above-mentioned literature, and immunoglobulin production therein is investigated. B cell lymphocytes that have substantially no ability are sorted and separated, for example, 6
- It is possible to obtain human B-cells that are resistant to thioguanine and are susceptible to death in 1-+AT medium. It can be grown by culturing in a thioguanine-containing medium.

By the above-described method, subclones of human colon cancer patient's B-cell and human lymphoblastoid cell line [B-1] were obtained.
Uc729-6 strain (A'l'CCCR1-8061)l
Power to refuse entrustment of Huiko cultivation
Antigen-specific immunoglobulin-producing human/human hybridoma produced from No. 64 No. 1 (IIuIIIan
t(ybridoma)Col-4N E I
+l lmicroI', research deposit entrustment 71 杏notification notification number 26
1 Hui Yong Wen No. 7534 1 and H 1 Yuki LIC729-6 strain (7)') l: m, l (I II/''01 strain Sentence No. 1198 1
The antigen-specific immunoglobulin-producing human/human hybridoma i'OCo was used as a fusion partner.
/ A 11 The cell biochemical properties of the 11 Microtechnical Research Institute Deposit Rejection Notification Notification Number = 61 Requisition Letter No. 1075 1 are shown below.

Human human hybridoma Cor, N F: J O
and i'('ICo/Al (1) Human immunoglobulin A (TgA) secretion (production), (2) Doubling time (Gubbling time) approximately 20
time, (3) DNA content is normal) +7 lymphocytes approximately 2
(4) The IgA in (1) above is derived from human cell line CaCo.
2 (colon cancer) and human cell line A 549 (Iklf
(5) It is capable of dividing and proliferating in a HAT-containing medium (medium containing hypoxanthine, amethopterin, and thyminone).

Furthermore, the IgA in (1) above also shows binding to human cell line LoVo (colon cancer-derived cell line; ATCCCCL229).

In addition, relative DNA content +i (normal human) 17 member bulb I)
The ratio to the NA content is determined by staining the hybridoma with probicuium iodide and then staining it with flourcy.
It was determined by the method of minute g1 analysis with to+aeLer.

The antigen-specific human immunoglobulin of the present invention is a human/human fusion cell line of a human colon cancer patient's B-cell and a subclone of a human lymphoblastoid cell line [B-cell]. #l with y4-like human immunoglobulin production ability
It can be produced by culturing the Il cell strain in a medium and collecting antigen-specific human immunoglobulin from the culture.

For example, human/human hybridomas, which can be produced as described above, are cultured in a suitable medium, such as RP containing 10% calf seedlings.
By culturing in MI-1640 medium and collecting 11 liquids of the medium, an immunoglobulin-containing substance that specifically binds to cancer-related antigens such as human colon cancer, vJ cancer, and lung cancer can be obtained. When culturing the human/human hybridoma, a serum-free medium can also be used, which is advantageous because it facilitates the purification of the obtained immunoglobulin. An example of such a serum-free medium is the serum-free medium proposed in Japanese Patent Application No. 1988-192145 filed by the same applicant. Furthermore, if desired, it may be purified to obtain purified immunoglobulin.

Purification can be carried out by appropriately using M production methods similar to those available for collecting and purifying immunoglobulins from biological fluids, such as ammonium sulfate fractionation, affinity chromatography, del filtration, ion exchange chromatography, and electrophoresis. can be used.

According to the present invention, when obtaining a human immunoglobulin-containing substance or the immunoglobulin that specifically binds to cancer-related antigens such as human colon cancer, gastric cancer, and cancer from the above-mentioned fused cell clone or its culture solution, Antigenic groups (e.g., cancer tissues) are often exposed to living organisms that lack or have an extremely weak ability to produce immune substances, such as nude mice.
mouse)h! ? After maintaining a well-established organization, you can use it for cultivation! ! It is advantageous to seek out and isolate human immunoglobulins (antibodies) that are reactive against the tissue returned to the system.

Other examples of media that can be used to culture the human/human hybridoma include RPMI containing 10% fetal eagle serum.
A three-way mixed medium (mixing ratio 2:1:1) of 1640 medium, Dulbeco medium, and HΔM medium was used with 71RDF medium, and the addition of transferrin, insulin, sodium selenite, ethanolamine, and β-merptoethanol. Examples include RDF medium.

In addition, as culture conditions, for example, in the presence of 5% CO□,
An example is the condition of 37°C.

As an example of the cancer-related antibody of the present invention which can be obtained as described above (specific human immunoglobulin), the above-mentioned human/human hybridoma CoLNE10 or the above-mentioned human/human hybridoma T.

The globulin of the present invention obtained using Co/Al can be exemplified. Its characteristics are shown below. Human/human bilidoma CoLNE10 or human/human hybridoma T O
Cancer-related antigen-specific human immunoglobulin produced by Co/A 1: (a) Human immunoglobulin A (IgA), which is (
b) It has binding strength to human cell line CaCo-2 (colon cancer) and human cell line A549 (lung cancer). Furthermore, human cell line I, 0■0 (colon cancer) and K A TO
-H[j (heavy cbai) with a molecular weight of approximately 60,000;
n), Lli (light chai) with a molecular weight of approximately 20,000
n) and J[((J chain
) contains subunits of

The human cell line A549 (1M! cancer) is ATC
CCCLl, 85 further includes human cell line CaC.

-2 is ATCCHTB37, human cell line LoV
o1. Human cell line K as tATCCCCCl229
ATO-111 is ATCCHTB103, ATC
C (American Type Culture Flexion)
You can freely provide labor from

The antigen-specific human immunoglobulin of the present invention suppresses the growth of cancer cells and kills cancer cells by acting as an antibody or itself on human cancers such as human colon cancer, particularly colon cancer, lung cancer, and gastric cancer. In addition, these cancer tissue-recognizing antibodies mass-produced outside the human body can be used to inhibit cancer cell proliferation and kill cancer cells with the help of complement or T-lymphocytes. Furthermore, cancer-specific antibodies produced outside the human body can be used as carriers to produce, for example, chemotherapeutic agent-conjugated human monoclonal antibodies, interferon-conjugated human monoclonal antibodies, polymer toxin-conjugated human monoclonal antibodies, and drug-containing antibodies. Ribosome binding - cancer #I in the form of human monoclonal antibodies, etc.
It is useful for inhibiting cell proliferation and killing cells. In addition, the human monoclonal antibody obtained by the method of the present invention is used as a carrier, and a radiation-sensitive substance is conjugated to the antibody and administered to the patient.The affected area is detected by utilizing its property of selectively gathering on cancer cells, and radiotherapy is performed. It can be used for. When using this type of cancer,
A complete antibody may be used as a human minoclonal antibody, or an antibody may be chemically cut into smaller molecules containing a specific antigen recognition site, or such small molecules or specific A more effective I1#1 human monoclonal antibody can also be created and used by a chemical method by combining only the antigen recognition site with a non-specific antigen recognition portion of another antibody.

Hereinafter, one embodiment of the method of the present invention will be described in more detail with reference to Examples.

Example 1 Lymph nodes around cancer tissue were obtained from colon cancer patients during surgery. Cut the lymph node into small pieces with scissors, and remove the lymphocytes inside in medium RDF (RP M I 1640:DM
E:F-12=2:1:1), followed by filtration using double gauze to remove the fat layer, and then centrifuging the cells (lymphocytes>80%) in the solution. collected by.

After that, the lymph acid content derived from this cancer patient i1! ! I (donor B cells) were frozen (-B0°C) in RDF medium containing 20% tallow serum and 10% dimethyl sulfoxide (DMSO) and stored until the day of cell fusion.

Donor B cells and 17 fusion partners (UC729-6) that are substantially deficient in immunopropurine production ability were mixed, 1.3 x 10' and V2.3 x 17 cells, at 35%
Fusion was completed with an aqueous solution containing polyethylene glycol 1540. Thereafter, polyethylene glycol was removed by centrifugation at 1100X for 10 minutes, and RD I'' medium containing 10% tallow serum and hypoxanthine, amethopterin, and thymidine (HAT) was added. 150 μl of the culture solution containing this cell group ( This includes 1,2X10'
cells) were dispensed into each well of three microplates with 96 wells, and incubated at 37°C and 5% CO2 for about 3 weeks.
Cultured in an incubator under the following conditions.

During this time, the RDF medium containing HAT and 10% beef tallow blood was replaced approximately once every three days. After 3 weeks of culture, 5 hybridoma subclones were found in 288 wells of 3 microplates. Among these, one hybridoma subclone was further cloned in RDF medium containing HAT and 10% tallow serum.
It was named.

To further confirm that this CoLNElo is a hybridoma, the intracellular DNA content was examined. The method will be explained below.

A cultured cell solution (CoL, NEiO) is placed in a centrifuge tube, and the supernatant is removed by centrifugation. 70% ethanol:I
Fix with 8 solutions for 30 minutes or more. The supernatant is removed by centrifugation, an RNA degrading enzyme (RNasc) solution is added, and the mixture is gently shaken to degrade the RNA. At 37℃! After reacting for 30 minutes, wash twice with #C distilled water.

Add Providium Ioguide (PI) and
After staining for a minute, wash twice with double distilled water. As a result of diluting with double-distilled water and analyzing using a flow cytometer, CoLNElo was found to have approximately twice the DNA content of donor B cells.

From the above results, it was confirmed that CoLNElo is a hybridoma.

This CoLNElo is human immunoglobulin (HuIg)
The production of the following was confirmed using an enzyme-antibody method.6 The method will be explained below.

Drop anti-human immunopropurine antibody (50 μN) into a microtiter plate and incubate the plate at 37°C for 1 hour;
let a. 10mM PBS containing 0.3% gelatin (
Gelachi 7ajffla) r Washed 3 times L? , after n, 5%
Add bovine serum albumin solution dropwise (200 μN) L, 37°C
Let it absorb for 1 hour. Wash 3 times with gelatin buffer fL,
Remove unadsorbed material. Next, a test solution (culture supernatant) was added dropwise (50 μk), and after reacting for 1 hour at 37° C., the plate was washed three times with gelatin buffer.

Veroxyguse-conjugated goat anti-human immunoglobulin antibody was added dropwise (50 μl) and reacted at 37° C. for 30 minutes.
Binds with human immunoglobulin in the test solution. After washing three times with gelatin buffer, a substrate solution containing hydrogen peroxide and o-phenylenediamine is added and reacted for 10 minutes in the dark. 5N
Add H2SO, (50 μm2) to stop the reaction.

490nm in proportion to the amount of human immunoglobulin in the test solution.
A yellow substrate reactant with absorption is produced. By measuring this amount using an absorption photometer and comparing it with the absorbance of human immunoglobulin whose concentration is known in advance, the human immunoglobulin concentration in the test solution can be determined.

As a result of using the above measurement method, it was found that CoLNE10 produced human shrimp A (immunoglobulin with the same characteristics as donor B cells).

One month later, CoLNElo was transplanted into a microplate with 24 holes (2 ml/well), and culture was continued for another week. That cultivation 1! The supernatant of the liquid was collected and various cell lines were used as target cells to obtain I produced from CoLNElo.
We investigated the specificity of H. The method will be explained below.

Various cultured human cell lines were cultured in DF medium (DME:
The cells are cultured in a medium containing F-12=1:1) and 10% tallow serum. When the number of cells reaches 5xlO' to 1xio', trypsin is used to peel the cells from the bottom of the Petri dish, the cells are collected using a centrifugation method, and the cells are thoroughly washed with a medium. A fixed number of cells (10'/100 μm) are dispensed into each hole of a microtiter plate having 96 holes and allowed to adsorb to the plate overnight at 37°C. Next, a 3% glugulaldehyde solution (50 μl) was added dropwise and the cells were fixed at 37"C for 30 minutes. The cells were further fixed by centrifugation (200 gtlO minutes) and washed three times with gelatin buffer. 5
% bovine serum albumin solution (200μj2) L, 3
Adsorb onto the plate for 1 hour at 7°C. gelatin itll
Wash three times with Li solution to remove unadsorbed substances.

Then drop the test solution (culture supernatant) onto the cells (50μλ)
Then, react at 37°C for 1 hour, and wash three times with gelatin buffer. This was followed by peroxyguse-conjugated anti-human 1. Add antibody dropwise (50μ2) and react at 37°C for 3θ minutes.

After washing three times with gelatin buffer, H)I in the culture medium is bound to the cells by the method described in the enzyme antibody method above.
Measure the amount of g.

As a result of investigating the target cell specificity of IgA produced by CoLNElo using the above method, it was found that IgA in the culture medium of CoLNElo () was derived from human colon cancer-derived cell line CaCo.
2 (A'r'CCHTB37), LoV.

(ATCCCCCl229) Human lung cancer-derived cell line A5
49 (ATCCCCCL185), was found to have binding ability to the t11% mountain rice cell line KATO-1 (ATCCHTB103).

In other words, since donor B cells are collected from patients with colon cancer in their bodies, self-propagating hybridoma clones created by cell fusion method have the same characteristics as donor B cells and specific antigen binding. This shows that a monoclonal (single) antibody with a specific site is produced. It was found that it reacts not only with cancer but also with other types of cancer.

After about 4 weeks, HA'r was removed from the hybridoma culture medium and cultured at 37°C in RI) F medium containing insulin, transferrin, sodium selenite, ethanolamine, β-merca (7') ethanol, and serum albumin.
The cells were cultured under 5% CO2 conditions and continued to produce IgA at a rate of 2.2 μg/IgA with a growth rate of 1×10 6 cells/1. The doubling time in the above culture was about 20 hours.

A large scale culture of Vibrido-ma was precipitated with 70% ammonium sulfate to obtain a crude shrimp fraction. The obtained precipitate was dissolved in physiological saline and goat anti-1. IgA was purified by affinity chromatography using Sepharose bound to antibody A.

IgA 0 purified from CoLNElo culture solution 12
.

7 mg was obtained. This purified IgArj product was
8- As a result of electrophoretic analysis, the molecular weight is approximately 60,000, 20,000,
It was confirmed that fgA containing 15,000 subunits was produced.

Example 2 LJ C used as a fusion partner in Example 1
Instead of the 7296 strain, the HI H/T O1 strain was used as a fusion partner, and R.
R containing ouabain instead of D F medium
A) Hybridoma subclones were obtained in the same manner as in Example 1, except that DF medium was used. This hybridoma was cloned in the same manner as in Example 1, and i'OC
It was named o/A1 and was confirmed to be a hybridoma in the same way as IL.

In the same manner as in Example 1, it was found that the 'OCo/A I produced human shrimp A. Furthermore, in the same manner as in Example 1, it was found that the 'OCo/A I produced 1 As a result of examining the target cell specificity of The same procedure as in Example 1 was carried out to obtain purified 1 g Al and OIllg containing the same subunits.

Procedural amendment July 16, 1986

Claims (1)

[Scope of Claims] 1. Antigen-specific human immunoglobulin produced by a human/human fusion cell line of a human B-cell of a human colon cancer patient and a subclone of a human lymphoblastoid cell line. 2. The antigen-specific human immunoglobulin according to claim 1, wherein the human/human fusion cell line is human/human hybridoma CoLNE10. 3. The antigen-specific human immunoglobulin according to claim 1, wherein the human/human fusion cell line is the human/human hydridoma TOCo/A1. 4. The human immunoglobulin is (a) human immunoglobulin A (IgA), and (b) human cell line CaCo-2 (human colon cancer-derived cell line) and human cell line A549 (human cell line). Human/human hybridoma Co
The antigen-specific human immunoglobulin according to claim 1, which is produced by LNE10. 5. The human immunoglobulin is (a) human immunoglobulin A (IgA), and (b) human cell line CaCo-2 (human colon cancer-derived cell line) and human cell line A549 (human cell line). Human/human hybridoma TO
The antigen-specific human immunoglobulin according to claim 1, which is produced by Co/A1. 6. A human/human fusion cell line of a human B-cell of a human colon cancer patient and a subclone of a human lymphoblastoid cell line, which has the ability to produce antigen-specific human immunoglobulin.
human hybridoma. 7. The human/human hybridoma according to claim 6, which is human/human hybridoma CoLNE10. 8. The human/human hybridoma according to claim 6, which is the human/human hybridoma TOCo/A1. 9. The following (1) to (5) (1) Human immunoglobulin A (IgA) secretion (production),
(2) Doubling time: approximately 20 hours, (
3) DNA content is approximately twice that of normal human lymphocytes, (4) IgA in (1) above shows binding to human cell line CaCo-2 (colon cancer) and human cell line A549 (lung cancer). (5) capable of dividing and proliferating in a HAT-containing medium (hypoxanthine, amethopterin, and thymidine-containing medium); Human/human hybridoma.