JPH07119240B2 - Antigen-specific human immunoglobulin - Google Patents

Description

Detailed Description of the Invention

The present invention relates to two types of human B-cells having different immunoglobulin-producing ability, in particular, human B-cell (A) having immunoglobulin-producing ability and substantially lacking immunoglobulin-producing ability. that it is produced by the new type of fused cells clone of human B- cell (B) which are related to antigen-specific human immunoglobulins. The human immunoglobulin, for example, the prevention of human antigenic, treat, medical and pharmaceutical fields and biochemical reagents, such as diagnosis, pharmacological fields such as purification reagents of biopolymers, such as wide such biochemistry It is useful in the field.

More particularly, the present invention relates to cancer cells.
Production of immunoglobulins produced and specific for cancer antigens
Human B-cells of human lymph node lymphocytes with function and human
Human / human fusion cells with subclones of lymphoblastoid cell lines
Human immunoglobulin produced by a cell line of (a) cervical cancer-derived cell lines Hela and Ca
Has binding ability to Ski, (b) Cell line A431 (vulvar cancer), lymphoid tumor
And WI · 38 (normal human fibroblasts)
It relates to an antigen-specific human immunoglobulin that does not have it.
The human immunoglobulin of the present invention is particularly (c) human immunoglobulin G (IgG), and (d) an antigen-specific molecule having a molecular weight of about 150,000 (monomer).
Human immunoglobulin, or (c) human immunoglobulin M (IgM), (d) an antigen-specific molecular weight of about 180,000 (monomer)
Human immunoglobulin is preferred. Conventionally, mouse B-cells sensitized with antigen and myeloid leukemia (myelo)
It is known that ma) fused cells with mouse B-cells from mice were formed outside the mouse body to form mouse / mouse fused cells having the ability to produce mouse immunoglobulin against the above-mentioned antigen and having self-proliferation. (Eg Na
pure, Vol. 256, 1975, 495-49
Page 7: Proc. Natl. Acad. Sci. US
A, Vol. 75, no. 7, 1978, 3405
3409, etc.). Also, human B sensitized with the antigen
-A fusion cell of a cell and a mouse B-cell from a myeloid leukemia mouse was formed in vitro to form a human / mouse fusion cell having human immunoglobulin-producing ability and self-proliferating ability against the above-mentioned antigen. Known (eg,
Proc. Natl. Acad. Sci. USA, Vo
l. 77, No. 11, 1980, 6841-684
Page 5: The journal of Immunol
ology, Vol. 125, No. 3, 1980, 10
37-1043, etc.).

However, in the latter attempt to obtain fused cells having human immunoglobulin-producing ability, the human immunoglobulin-producing ability is lost over time, resulting in the loss of human immunoglobulin producing ability. It has a fatal defect that its productivity is extremely unstable.

Human having human immunoglobulin production ability /
In an attempt to form human fused cells, human B-cells sensitized by Hashika virus or hapten [2,4-dinitrophenyl] as antigen were used as donors and this Cell line capable of producing human immunoglobulin (human B-
It has been known that human / human fused cells having the ability to produce human immunoglobulin against the above-mentioned antigen and having self-proliferation ability were formed by forming fused cells with cells) (parent strains) outside the human body (Nature, Vol.288, 1980
483-483: Proc. Natl. Aca
d. Sci. USA, Vol. 77, No. 9, 198
0, 5429-5431).

However, according to the method of this report,
Since the latter parent cell line (human B-cell) has a human immunoglobulin-producing ability different from that of the former donor human B-cell, the obtained human / human fused cells are obtained from both human B-cells. It is a mixture of cells with immunoglobulin formation in the cell. For this reason, immunoglobulins collected from such human / human fusion cells become a mixture of immunoglobulins of several traits, and immunoglobulins of the same trait as lymphocytes (A) maintain a specific trait and It becomes impossible to be produced as a single antibody.

As a result, for example, even if an antigen-specific human immunoglobulin having the same trait as lymphocyte (A) is used,
Interfering with immunoglobulins of other traits makes it difficult to carry out the desired treatment, prevention, test, purification, and the like.

Furthermore, the cell line (human B-cell), which is the parent strain used in the above-mentioned report, has reversible drug sensitivity and frequently loses sensitivity. When it occurs, it becomes difficult to separate and collect the desired fused cells from the human B-cell used as the donor and parent strains. Further, there is a drawback that aggregates are easily formed during the culture of the parent cell line, and as a result, the probability of producing fused cells is low.

The present inventor can produce an antigen-specific human immunoglobulin having the same trait as B-cell [hereinafter also referred to as B-cell (A)] of human lymphocytes used as a donor while maintaining a certain trait. Studies were conducted with the purpose of producing human / human fusion cells and from them producing single trait immunoglobulins.

As a result, cancer cells are sensitized and the cancer
A human with the ability to produce an immunoglobulin specific to an antigen
A novel human / human fused cell is prepared by fusing a B-cell (A) of a lymphoid lymphocyte to a human B-cell (B) that is substantially deficient in immunoglobulin-producing ability outside the human body. It has been discovered that it is possible to produce and that the human / human fusion cells produced have the immunological traits of the human B-cell (A) and are capable of stably sustaining such traits. Furthermore, a unique human / human fusion that facilitates efficient production of this new type of human / human fusion cell, has a high probability of forming fused cells, and enables mass production of human immunoglobulin in vitro. I knew it was a cell.

As a result of further research based on the above new findings, according to the present invention, (1) sensitization with cancer cells
And immunoglobulin-specific ability to produce cancer antigens
Human B-cells (A) of human lymph node lymphocytes having
A human cell group containing, (2) (b) has a self-proliferating in a suitable medium, (ii) substantially to growth arrest or death in the absence of presence or specific ingredients in the specified reagent Has an irreversible sensitivity, and (c) is substantially deficient in immunoglobulin-producing ability,
And (d) Single cells (single cells) under appropriate medium and culture conditions.
Cell- like proliferative, bone marrow-like cells with permanent division potential
(3) A human cell group containing human B-cell (B) of human lymphocytes is fused outside the human body to produce a fused cell of human B-cell (A) and human B-cell (B). Then, the obtained fused cells are cultured in a medium in which the human cell group of (1) above and the human cell group of (2) above stop or die but the fused cells can grow to obtain a fused cell clone. (4) From the fused cell clone, the B-cell (A)
It was discovered that by collecting an antigen-specific immunoglobulin-containing substance or immunoglobulin having the same trait, human immunoglobulin having a desired and constant immune trait can be reproducibly obtained.

[0011] Thus, antibody was produced according to the present invention with reference to the lymphocytes taken from one patient whose principal (Au
It has been experimentally found that the antigen specifically reacts with the antigen of the same species and a human antigen having the same trait.

[0012] As long as the present inventors know, one of the (1)
Human B-cells of lymph node lymphocytes were used as a donor, and fused with a parent strain substantially lacking immunoglobulin-producing ability like the human B-cells of (2) above, in vitro. Up to now, there have been no known examples of successful production of human / human fusion cells. In addition, it has not yet been reported that the human immunoglobulin produced from this fused cell specifically reacts with the person (patient) and an antigen (tissue) having the same trait as that of the person other than the person.

However, the present inventor used the appropriate human B-cell of the above (1) as a donor, and using this as a donor, the human B-cell substantially deficient in the immunoglobulin-producing ability of the above (2) (parent strain). And that the fused cells can be produced by fusing in vitro outside the human body, and the produced fused cells have different sensitivities to reagents or medium components from the parent B-cell, and the donor B-cell is It is possible to separate it from these B-cells due to its poor growth ability, and when many of the separated fused cells are cultured in another medium, many of them stop growing or die. It has been discovered that some of them stably grow to form clones, and that the fused cells forming this clone are capable of long-term passage expansion.

Accordingly, it is an object of the present invention to provide an antigen-specific human immunoglobulin produced by using a new type of clone of fused cells.

In the present invention, the human B-cell (A) capable of producing an immunoglobulin used for producing a fused cell is a cancer cell.
Human B- of lymph node lymphocytes sensitized by cells and capable of producing immunoglobulin specific for cancer antigen
It can be a cell. Such human B-cell (A) preferably has substantially no passage self-propagation outside the human body.

In the present invention, human B-cell (B) is a human B having substantially no immunoglobulin production ability.
-The cell (B) is used, and the human B-cell (A) having an immunoglobulin-producing ability is used as the human B-cell (A). Therefore, the above human B-cell (A) is obtained from the clone of the fused cell produced from this. There is an advantage that a desired antigen-specific human immunoglobulin can be produced by appropriately selecting the trait of the human immunoglobulin produced by.

In the present invention, tumor (particularly cancer) cells are used.
Sensitized and specific for cancer antigens
It may be a B-cell of human human lymph node lymphocytes capable of producing , or it may also be B of collected human lymphocytes.
-Human B having enhanced immunoglobulin-producing ability, which is obtained by using a technique such as sensitizing cells outside the human body, for example, by proliferating with a growth factor and / or with an antigenic substance
-May be a cell. Examples of such growth factors include lipopolysaccharides and lectins.
You can do it .

The above-mentioned operation for increasing the productivity is known, for example, from the following documents and can be used in the present invention. Proc. Natl. Acad. Sci. U. S.
A, Vol. 77, pp 1139-1143 (198)
0) Michael Hoffman; “Natur
e ", Vol. 282, (1979), Robin.
E. Callard. According to a preferred embodiment, the human B-cell (A) separated and collected from the above-mentioned collection source is cultured in a liquid medium containing a growth factor such as RPMI-1640 (containing 10% calf serum) as described above. Then, the medium may be further cultured by adding an antigenic substance, or may be cultured in a liquid medium containing both. Examples of the culture conditions include, for example, conditions at 37 ° C. for about 5 to 7 days.

The human B-cell (B) used in the present invention is a human B-cell which satisfies the following requirements (a) to (d).
Any cell may be used, and it can be appropriately selected and used.

(A) Self-propagating in a suitable medium , preferably
Kudou doubling time within 48 hours
It has a self-proliferative property , (b) has a susceptibility to stop or die in the presence of a specific reagent or in the absence of a specific component, and (c) is substantially deficient in immunoglobulin-producing ability. Cage,
And (d) Single cells (single cells) under appropriate medium and culture conditions.
Cell) .

The above-mentioned human B-cell (B) is a suitable source such as human lymph node, human lymph gland, human spleen,
It can be collected from lymphocytes such as human blood, human bone marrow, etc., or can be selected from those grown or proliferated and differentiated outside the human body. The human B-cell (B) satisfying the above requirements (a), (b) , (c) and (d) can be directly selected, but from the operational point of view, From the requirements of (a), (b) , (c), and (d) , until the human B-cell that satisfies only one or two requirements is first collected and other requirements that are lacking are satisfied. It is preferable to perform a treatment such as proliferation, differentiation or the like.

For example, B-cells having (a) self-proliferation property in an appropriate medium and (c) substantially lacking immunoglobulin-producing ability, but not having the property (b) are collected. ,
It can be endowed with the susceptibility to growth arrest or death in the presence of a specific reagent or in the absence of a specific component outside the human body. In carrying out this aspect, for example, a method known from the following documents can be used. H
ybridoma in Cancer Diagnostic
sis and Treatment ed. M. S.
Mitchell and H.M. F. Oettgen
RavenPress New York (198
2), pp125-132.

For example, utilizing the method described in the above-mentioned document, a healthy human spleen can be biopsied.
, Lymphoid cells such as lymphoblasts are taken, and B cell lymphocytes having substantially no immunoglobulin-producing ability therein are sorted and separated. For example, 6-thioguanine, 8
- azaguanine, when cultured in such enzyme-mutagenic containing medium bromo Deo shea uridine, has self-proliferating in Sento medium, susceptible to killing in且one HAT medium not produce immunoglobulin Human B-cell (B) can be obtained. For example, the human B cell UC729-6 used in Examples described later can be obtained by culturing the above-mentioned lymphoblasts in a medium containing 6-thioguanine.

The above-mentioned human B cell UC729-6 has received the deposit refusal notice issued by the Director of Institute for Microbial Technology, Institute of Industrial Science and Technology.

Further, for example, (a) human B having self-reproducing property in a suitable medium and not having the properties of (b) and (c).
-Collecting cells, imparting the property (b) thereto by the same method as described above, culturing and proliferating the cells, and screening the proliferated product by a sub-cloning method known per se (c) Human B-cells that satisfy the properties can be selected.

The self-proliferative property of the requirement (a) of human B-cell (B) can be obtained because it is a trait inherited by a fused cell of human B-cell (A) and human B-cell (B). It is preferred to select human B-cells (B) which have self-renewal properties for as long as possible, preferably substantially permanent self-renewal properties. Industrially, it is preferable to select one that repeats self-propagation at least 100 times, more preferably at least 200 times, and especially at least 500 times.

Furthermore, the doubling time of self-reproduction is about 4 in relation to the self-reproduction property of the requirement (a).
It is preferable to use human B-cell (B) having a self-proliferative property such that it is 8 hours or less, and further about 20 hours or less. This short doubling time is also a trait that is inherited by the fused cells of human B-cell (A) and human B-cell (B), so that the desired human immunity produced by the clone of the fused cells is produced. An increase in globulin production can be achieved. Therefore, it is possible to provide a particularly excellent method for producing human immunoglobulin by selecting the human B-cell (B) that maintains the self-proliferation property for a long period of time and has a short doubling time.

The susceptibility to growth arrest or death in the presence of the specific reagent of the requirement (b) or in the absence of the specific component enables screening for obtaining a desired fused cell clone described in detail later. This is the property required for Examples of the specific reagent include aminopterin, alancin, waabain and the like.

The above-mentioned sensitivity reversibly returns to a state in which the requirement (b) is not satisfied in the fused cells produced from human B-cell (A) and human B-cell (B).
Since a "mutation" (back mutation) may occur, in the present invention, human B having irreversible susceptibility to growth arrest or death in the presence of a specific reagent or in the absence of a specific component is used. It is advantageous to choose cell (B).

The above-mentioned human B-cell (B) used for producing a fused cell (hybridoma) in the present invention can grow as a single cell under appropriate medium and culture conditions, that is, form a cell aggregate. It is desirable not to.
Examples of the human B-cell (B) medium include serum-containing RPMI-1640 medium such as calf serum, human serum, newborn calf serum and horse serum. The culture conditions are, for example, 37 ° C. in the presence of 5% CO _2.
The conditions of can be illustrated.

[0031] is in the present onset bright, and human cell group containing the human B- cell (A) having such immunoglobulin-producing ability of the above, such as the above-mentioned (a), (b), (c) and
A human cell group containing human B-cell (B) satisfying (d) is fused outside the human body to produce a fused cell of human B-cell (A) and human B-cell (B). To do.

The fusion operation for producing this fused cell may be any known method. The fusion operation can be performed by bringing human B-cell (A) and human B-cell (B) into contact with each other in a liquid medium in the presence of a fusion accelerator. Examples of such a fusion accelerator include Sendai virus (HVJ), polyethylene glycol and the like. For example, in an aqueous medium, in the presence of the fusion promoter as exemplified above,
If necessary, gentle agitation is added to homogenize the system, and then the time for producing a fused cell consisting of one human B-cell (A) and one human B-cell (B), for example, several minutes. The desired fused cells can be produced by allowing the cells to stand for an order. Examples of the liquid medium include water, physiological saline, 5% dimethylsulfoxide aqueous solution, 5% glycerol aqueous solution and the like.

The system in which the desired fused cells are produced is subjected to, for example, centrifugation to collect a cell group, which is again added to an appropriate medium.
For example, the above-exemplified reagent is added to the serum-containing RPMI-1640 liquid medium as described above to disperse the collected cell groups, and this dispersion liquid is dispensed into a well of, for example, a microtiter plate in a fixed amount. While preparatively injecting, culture is performed at 37 ° C. in the presence of 5% CO ₂ , for example. The culture solution in each well is replaced with a new culture solution every 3 days, for example, after culturing for 2 weeks, the presence or absence of fused cells can be inspected under a microscope to collect a culture solution of a sample in which colonies are recognized. The presence or absence of human immunoglobulin can be detected by a radio immuno assay using ¹²⁵ I, for example.

As described above, a fused cell clone can be obtained by transferring a colony in which human immunoglobulin production has been observed to a new culture medium and culturing the same to grow the fused cell. In addition, if necessary,
By cloning, an antigen-specific human immunoglobulin-producing clone having the same trait as the desired human B-cell (A) can be obtained.

[0035] According to the onset bright, appropriate medium a fusion cell clones obtained as described above, for example, were cultured in 10% serum-containing RPMI-1640 medium, human B- cell by collecting the culture solution ( An antigen-specific immunoglobulin-containing substance having the same trait as in A) can be obtained. Furthermore, if desired, the immunoglobulin can be purified to give a purified immunoglobulin. For the purification, for example, ammonium sulfate fractionation method, affinity chromatography, gel filtration, ion exchange chromatography, electrophoresis method, and the like, which are the same as those used when collecting and purifying immunoglobulin from a biological fluid, are used. be able to.

According to the present invention, when an antigen-specific human immunoglobulin-containing substance having the same trait as human B-cell (A) or the immunoglobulin is obtained from the above-mentioned fused cell clone or its culture, the antigen is separated. If possible, find a human immunoglobulin (antibody) that reacts with the antigen,
When the antigen cannot be separated, an antigenic tissue (for example, cancer tissue) is once used as a living body that lacks or has an extremely weak immunogen-producing ability, such as a nude mouse.
It is advantageous to search for a human immunoglobulin (antibody) that reacts with the tissue or the tissue returned to the culture system after isolating the tissue, and then separating it.

The human B-cell (A) used in the present invention does not substantially exhibit self-propagation outside the human body, and for example, it is preferable that it undergoes division several times at most.

As an example of a fused cell (hybridoma) produced by fusing the human B-cell (A) and the human B-cell (B) used in the present invention outside the human body, a human described later in Examples. / Human hybridoma (Humanhybr
The cytological properties of the Idoma) CLN / SUZH5 strain and human / human hybridoma CLN / SUZ H11 strain are shown below.

Human / Human hybridoma CLN / SUZ
H5:-(1) Chromosome number 92 (2) Secretion (production) of human immunoglobulin M (IgM) (3) Double time (doubling time) 37 hours (4) Lymphocyte single cell human / human hybridoma CLN / SUZ H11:-(1) Chromosome number 92 (2) Human immunoglobulin G (IgG) secretion (production) (3) Doubling time 37 hours (4) Lymphocyte single cell The above human hybridomas are microorganisms of the Institute of Industrial Science and Technology. I received a deposit refusal notice issued by the Director of AIST.
However, after that, the human hybridoma became ATCC.
The human / human hybridoma CLN / S described above has been deposited.
UZ H5 is humanized as ATCC HB8206
/ Human hybridoma CLN / SUZH11 is ATCC
Commissioned as HB8307.

According to the present onset bright, clinical and basic medicine the beginning pharmaceutical and pharmacological field, a human immunoglobulin having utility should unique and attention in the field of biochemistry other broad fields, especially desired and An antigen-specific human immunoglobulin having a certain trait can be industrially advantageously produced outside the body.

[0041] Thus, therefore to the onset Akira, a human / human
A single antibody (monoclonal antibody) produced from a hybridoma covers a wide range of pharmaceutical, medical, pharmacological, biochemical and other fields including treatment and treatment of difficult-to-treat human diseases such as cancer. It has novel and notable utility in the field.

For example, in the mouse / mouse monoclonal antibody, the mouse antibody is administered to humans,
Naturally, there is a risk of allergic symptoms and shock symptoms. In addition to the same risks as above, human / mouse monoclonal antibodies have a drawback in the stability of hybridomas. It cannot be produced over a long period of time. Further, even in the case of human / human monoclonal antibody, if the B-cell corresponding to the human B-cell (B), which is the parent strain used for cell fusion, has antibody-producing ability, such antibody Is preferably removed sufficiently.
However, since it is a human / human monoclonal antibody,
Even when administered to another person, its antigenicity is low and there is no serious risk as in the above mouse / mouse monoclonal antibody.

The most desired characteristic is that an antigen-specific antibody (immunoglobulin) having the same trait as the antibody produced by the patient's lymphatic system is mass-produced outside the human body, and again without any fear of side effects within the person himself. It is possible to put it back. Then, the onset Ming Thus for the first time, became such antigen-specific immunoglobulin and can be industrially produced in human body.

[0044] According to the onset bright, for example, a cancer antigen specific human immunoglobulin available in wide range of fields as illustrated below can be produced in human body.

[0045] For example, an antibody against cells that have been changed in the self within the body, such as cancer (altered self) Ru can be produced in the human body.

More specifically, examples of cancer will be described in more detail. The action of the cancer-specific antibody itself mass-produced outside the human body suppresses the growth of cancer cells and causes the death of cancer cells.
Complement or T to the cancer tissue recognition antibody mass-produced outside the human body
-With the help of lymphocytes, it is possible to suppress the growth of cancer cells and act to kill cancer cells. Furthermore,
Using a cancer-specific antibody mass-produced outside the human body as a carrier, for example, chemotherapeutic agent binding-human monoclonal antibody, interferon binding-human monoclonal antibody,
It is useful for inhibiting the growth or killing of cancer cells in the form of macromolecule toxin binding-human monoclonal antibody, drug-containing liposome binding-human monoclonal antibody, or the like. In addition, the human monoclonal antibody of the present invention is used as a carrier, and a radiosensitizer is bound to the carrier to administer to a patient, and the affected area is detected by utilizing the property of selectively collecting in cancer cells, and is used for radiotherapy. can do. In the use for such cancer, a complete antibody may be used as a human monoclonal antibody, or the antibody may be cleaved into smaller molecules containing a specific antigen recognition site by a chemical method, or Can bind such small molecule or specific antigen recognition site to non-specific antigen recognition part of other antibody to create more effective modified human monoclonal antibody by chemical method. .

Furthermore, the human immunoglobulin of the present invention can be used for diagnosis and prevention of human cancer antigenic diseases. According to this utilization mode, a specific antibody against a biopolymer that appears in a living body with cancer is produced by the above-described method, and the obtained antibody is bound with, for example, an isotope or a similar tracing substance (sensitive substance), The presence of the antibody should be made detectable, and by injecting this antibody into the body and detecting the binding site, the lesion or pathogen in the body can be detected, or the antigen present in the extract of the tissue outside the body can be detected. It can be used for diagnosis and prevention of diseases by the method of detecting and quantifying. By such a method, for example, the state of metastasis after surgery of a cancer patient can be monitored, it can be used for early detection of cancer, or the degree of healing of cancer can be confirmed.

In addition, for tissue-cultured human cells,
By investigating the action of the human / human single antibody obtainable by the present invention, basic knowledge useful for the medical application of the antibody, for example, elucidation of factors affecting cell proliferation and differentiation, cell It is useful for elucidation of basic knowledge of cell biology such as qualitative and quantitative determination of constituents, and further, broad biochemistry such as purification of enzymes, proteins and nucleic acids, analysis of constitution and function of biopolymers, extraction of DNA and RNA. It can also be used in the area.

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

[0050]

Example 1 Cancer tissue and lymph nodes were obtained from a patient with squamous cell carcinoma of the cervix during surgery to remove the entire uterine body and lymph nodes. The latter lymph node is cut into small pieces with scissors, the internal lymphocytes are dispersed in the culture medium (RPMI-1640), and then, filtration is performed using double gauze,
Cells during filtration after removing the fat layer (lymphocytes> 80%)
Are collected by centrifugation [human lymphocyte fraction containing human B-Cell donor (donor B-cell)]. This lymphocyte fraction was then frozen (-70% in RPMI-1640 medium containing 10% fetal calf serum and 10% glycerol).
C.) and stored until the day of cell fusion.

A parent human B-cell (UC729-, which is substantially deficient in donor lymphocyte and immunoglobulin producing ability)
Mix 1 x 10 ⁷ and 5 x 10 ⁶ of each of 6) and complete the fusion in RPMI-1640 medium with 10% fetal calf serum in the presence of 35% polyethylene glycol. Then, centrifugation is performed at 800 G for 10 minutes to remove polyethylene glycol, and a medium containing RPMI-1640 containing 10% fetal calf serum and hypoxanthine / aminopterin / thymidine (HAT) is newly added. 200 μl of a culture solution containing this cell group (including 1.5 × 10 ⁵ cells) was dispensed into a microplate having 96 holes, and the culture was performed at 37 ° C. and 5% C for about 2 weeks.
It was cultured in an O ₂ incubator. During this period, 10% fetal calf serum-RPMI-1640 medium containing HAT was used for 1 day on 3 days.
It changed times. The parent B-cell (UC729-6) cannot live in the presence of aminobuterin because it lacks hypoxanthine phosphoriboseyltransferase. In addition, lymphocytes (A) can be used in a normal medium such as RPMI
In -1640 + 10% fetal calf serum, it grows permanently and cannot survive. Therefore, cells that have proliferated permanently in the medium containing HAT are lymphocytes (A) and B-Cells.
(B) The fused cells. After culturing for 2 weeks, clones of hybridoma were found in 6 of 96 microplates. Two of these 6 hybridomas (CLN / SUZ H5 and CLN, respectively)
/ SUZ H11) is a human immunoglobulin (Hu
The production of Ig) was confirmed using an immobilized radioimmunoassay and an immobilized enzyme antibody method. The method will be described below.

The culture medium of the hybridoma is dropped (50 ul) on the glass filter and dried to immobilize the protein in the culture medium. Then, ¹²⁵ I-binding anti-human immunoglobulin serum (radioimmunoassay method) or peroxidase-binding anti-human immunoglobulin serum is dropped (50 ul) (enzyme antibody method) to bind to human Ig that should be in the culture solution. After reacting at room temperature for 30 minutes and thoroughly washing with physiological saline, in the case of radioimmunoassay, the radioactivity of ¹²⁵ I remaining on the glass filter was quantified with a γ-counter to be contained in the hybridoma culture solution. Know the amount of human Ig. On the other hand, in the case of the enzyme antibody method, a substrate solution containing hydrogen peroxide and O-phenylenediamine is further added, and the reaction is performed for 30 minutes in a dark room. If the peroxidase-conjugated anti-human Ig serum remains on the glass filter, that is, if human Ig or anti-human Ig serum reacts on the glass filter, a yellow substrate reaction product detected at an absorbance of 490 nm is produced. To be done. This amount is measured by using an absorptiometer to know the amount of human Ig contained in the hybridoma culture solution. If no human Ig is present in the hybridoma culture, anti-human Ig serum is washed through the glass filter at the washing stage.

As a result of using the above measuring method, CLN / S
UZ H5 produces human IgM, and CLN / SU
ZH11 was found to produce human IgG (Ig syngeneic with donor B-cell). 2 weeks later, CL
Each of N / SUZ H5 and CLN / SUZ H11 was subcultured in a microplate having 24 holes (2 ml / well), and the culture was continued for another week. Three weeks after the fusion, the supernatant of the culture solution was collected to examine the specificity of Ig produced from human / hybridoma using various cell lines as target cells (eg, Ig having a certain trait or specific traits). With Ig).

The method will be described below.

Various human cell line cultures (these are AT
(Available from CC) are cultured in a synthetic medium of DME: F-12 = 1: 1 with 10% fetal calf serum. When the number of cells reached 5 × 10 ⁶ to 1 × 10 ⁷ , the cells were peeled from the bottom of the dish without using trypsin, and a fixed number (using a 96-well microtiter plate having a glass filter at the bottom ( Approximately 5 × 10 ⁴ ) is placed on a glass filter and dried to fix the cells on the glass filter. After that, CLN / SUZ H5 and CLN /
For each SUZ H11, the culture supernatant (50 u
l) was dropped on the cells and reacted at room temperature, and then ¹²⁵ I
-Bonded human Ig serum or peroxidase-conjugated human Ig serum is added dropwise and reacted at room temperature. After thorough washing, the amount of human Ig in the culture solution bound to the cells is measured by the methods described in the radioimmunoassay method and the enzyme antibody method described above.

CLN / SUZ H5 is prepared by the following method.
Cell specificity of IgM and CLN / SUZ H1
As a result of examining the target cell specificity of IgG of 1 respectively, C
IgM in culture medium of LN / SUZ H5, and CLN
IgG in the culture solution of / SUZ H11 each has a binding force to the cell lines Hela and Caski derived from cervical cancer, and according to the study of the present inventor, other than cervical cancer It did not have a binding force with the cell line of A., A431 (vulvar cancer), lymphoid tumor, W1.38 (normal human fibroblast), and the like.

That is, since the donor B-cell was collected from a patient with cervical cancer in the body, the donor B-cell was selected from the self-proliferating hybridoma clone produced by the cell fusion method. It is shown that a monoclonal (single) antibody having the same trait as the cell and having a specific antigenic determinant site is produced. In general, it is shown that the cancer tissue in the body of a patient with cancer acts as an antigen and is capable of producing a cancer-specific antibody by the immunosurveillance mechanism of the patient.

From the same patient, CLN / SUZ H5 was prepared by using cells obtained by transferring the collected cancer tissue into culture as target cells.
As a result of examining the specificity of CLN / SUZ H11 and CLN / SUZ H11 by the same method as above, it was found that they have binding force.

Approximately 3 weeks later, HAT was removed from the culture medium of the hybridoma, and the cells were cultured in RPMI-1640 + 10% calf serum. The doubling time was 37 hours, and CLN / SUZ H5 was 2.4.
μg IgM (HuMoIgM) / 10 ⁶ cells / ml /
human IgM has been continuously produced in the amount of day, and CLN /
SUZ H11 is 2.6 μg of IgG (HuMoIgG)
Human IgG continues to be produced in an amount of / 10 ⁶ cells / ml / day.

The hybridoma mass culture was precipitated with 50% ammonium sulfate and the crude Ig fractions were collected. The obtained precipitate was dissolved in physiological saline, IgG was used as IgG in sheep anti-human IgG serum, and IgM was used as affinity chromatography using Sepharose to which IgG in sheep anti-human IgM serum was bound. Purified by the technique. Yields are from 1 liter of CLN / SUZ H5 culture to 2.
2 mg of IgM, CLN / SUZ H11 culture medium 1 liter
Gave 3.0 mg of IgG. As a result of SDS-electrophoresis analysis of the Ig standard purified by using these affinity chromatography, IgG (CLN / SUZ H1) having the same trait as human Ig and a molecular weight of about 150,000 was used.
1) and IgM (CLN with a molecular weight of about 180,000 (monomer))
/ SUZ H5) was confirmed to be produced. In addition, both human hybridomas can be grown in the peritoneal cavity of Nudemouse, and 3 hybrids in 5 ml of ascites.
It is possible to produce ~ 10 mg of human Ig.

Example 2 A lymph node was obtained from a patient with epithelial adenocarcinoma of the cervix during surgery, and lymphocytes were prepared by the same method as that used in Example 1. In this example, lymphocytes were stored in RPMI-1640 + 10% calf serum without cryopreservation.
After daily culture, cell fusion was carried out in the same manner as in Example 1 using the parent B-cell (UC729-6). As a result, hybridoma clones were found in 16 out of 96 wells, and 5 out of 16 produced human IgM by the detection method using radioimmunoassay.
It was confirmed that 2 out of 6 produced human IgG.

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Claims (3)

[Claims] 1. A sensitized cancer cell and against a cancer antigen.
Human lymph node with specific immunoglobulin-producing ability
Human B-cells of lymphocytes and sub-lines of human lymphoblast cell lines
Human immunity produced by human / human fusion cell line with clone
A globulin, (b) cell lines Hela and Ca from cervical cancer
Has binding ability to Ski , (b) Cell line A431 (vulvar cancer), lymphoid tumor
And WI · 38 avidity and (normal human fibroblasts)
Antigen-specific human immunoglobulin having no. 2. The human immunoglobulin is (c) human immunoglobulin G (IgG), and (d) the molecular weight is about 150,000 (monomer).
The above antigen-specific human immunoglobulins. 3. The human immunoglobulin is (c) human immunoglobulin M (IgM), and (d) the molecular weight is about 180,000 (monomer).
The above antigen-specific human immunoglobulins.