CS263087B1 - Mouse lymphocyte hybridoma producing anti-human keratin 18 antibody - Google Patents

Abstract

The invention does not concern a mouse lymphocyte hybridoma producing antibodies against human keratin 18, stored in the hybridoma collection of the Research Institute of Clinical and Experimental Oncology in Brno under the designation RICEO-DA-7. The monoclonal antibody of the hybridoma DA-7 itself is suitable for use in enzyme-immunological and radioimmunological analysis of tested tissues, cells and body fluids, as well as for visualization of keratin in cells at the optical and electron microscope level and immunohistochemical detection of keratin 18 on tissue sections.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, prepared by a biotechnology process, i.e., by cell fusion of the mouse myeloma line P3-X63-Ag8,653 and mouse splenic lymphoid cells producing an antibody against human keratin 18.

Until now, antibodies have generally been produced by immunizing animals by repeatedly administering the antigen, usually together with an adjuvant, to experimental animals, most often rabbits, guinea pigs, pigs or goats. The serum of animals immunized in this way is collected after a certain period of exposure to the antigen and serves as a source of antibodies that are used for the diagnosis and treatment of a number of diseases, but also for studying the occurrence and function of these antigens in cellular pathophysiology. This procedure, called conventional immunization, has several disadvantages. The serum of immunized animals contains a heterogeneous mixture of antibodies, the spectrum of which is different and unrepeatable in each individual organism. The organism usually produces, in addition to antibodies against the desired antigen, antibodies against impurities in the antigen preparation, which must be removed from the serum in a complex manner called saturation. In the case of antisera against only one of the 19 known keratins of the human cytoskeleton (Moll 2., Franke W.1V,, Schiller D.L., Geiger 5,, Krepler R.: The cstalog of human cytokefatin polypeptides: patterns of expression of specific cytokeratins in normal epithelio, tumors and cultured cells. Cell 31: 11 - 24, 1982) the situation is further complicated by the considerable mutual similarity of the individual keratins, so that preparing a conventional antiserum against a single keratin by saturation is extremely laborious and only rarely leads to obtaining the desired monospecific antibody. Production batches of conventional antisera can therefore be standardized with great difficulty and come out of production with a wide range of quality.

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For the production of each batch, it is necessary to prepare a large amount of pure immunizing antigen and additional antigens to saturate ballast antibodies against impurities or against similar molecules, such as vimentin, desmin or lamins.

The above-mentioned shortcomings of the previously used procedure will be eliminated if a hybridoma producing a completely homogeneous antibody, the so-called monoclonal antibody against any class of 19 known human keratins, in our case against keratin 18, is available. Such a hybridoma is stored in the hybridoma collection of the Research Institute of Clinical and Experimental Oncology in Brno, Žlutý kopec 7, under the designation RICEO-DA-7,

The hybridoma was obtained by a method known from the literature (Kohler G., Milstein C,: Continuous cultures of fused cells secreting antibody of predefined specificity.

Nature 256: 495- , 1975, Galfré G.. Howe S.C., Milstein C,, Butcher G.W,, Howard 3.C.: Antibodies to major histocompatibility antigen produced by hybrid cell line. Nature 266:

550- , 1977, Fazekas de St. Groth S., Scheidigger D.; Production of monoclonal antibodies: Strategies and tactics. □.Immunol.Meth. 35: 1-21, 1980) by cloning a set of hybrid cells, resulting from the fusion of cells of the mouse myeloma line P3-X63-Ag8.653 and cells obtained from the spleen of BALB/c mice, immunized with an isolated cytoskeletal preparation of human epithelial cells.

Hybridoma DA-7 can be cultured in vitro in media suitable for growing animal cells or in vivo in the peritoneal cavity of BALB/c mice. Hybridoma DA-7 is suitable for long-term storage in cans stored in liquid nitrogen. Production of monoclonal antibody can be initiated after thawing of the cell can without the need for additional antigen. The monoclonal antibody produced by hybridoma DA-7 is specific only for human keratin with a molecular weight of approximately 45,000 - designated keratin 18, and does not react with any other intermediate filament protein or with other cellular proteins, as determined by immunoblotting, enzyme-linked immunosorbent assay, indirect immunoperoxidase and indirect immunofluorescence methods. Evidence was obtained

263 087 after electrophoretic transfer of a wide range of cell and tissue lysates and preparations of isolated cytoskeleton of various human and animal tissues and cell lines, after separation of protein components in polyacrylamide gel in sodium dodecyl sulfate environment under reducing conditions. To determine the isoelectric point characteristic of the target keratin recognized by the DA-7 antibody, the proteins of the above-mentioned lysates and cytoskeletal isolates were first subjected to isoelectric focusing, followed by the already mentioned electrophoresis in polyacrylamide gel. After transfer of the separated proteins to a nitrocellulose membrane, keratin 18 was specifically visualized (detected) using the DA-7 antibody and subsequently rabbit antiserum against mouse immunoglobulins conjugated with horseradish peroxidase. The result was also confirmed by an enzyme-linked immunosorbent assay, which demonstrated binding of the DA-7 antibody to keratin 10, but not to vimentin, serum proteins, or keratins of other classes.

Using immunoperoxidase reaction on tissue sections (confirmed by the results of indirect immunofluorescence method - see below) of human and animal tissues, reactivity with epithelial tissues containing keratin 18 and non-reactivity with other tissues was found for the DA-7 antibody. In indirect immunofluorescence using DA-7 antibodies and subsequent incubation with rabbit or pig antisera against mouse immunoglobulins labeled with fluorescein-isothiocyanate, binding occurred only to intermediate filaments of the keratin type in the tested cell types in vitro. Microtubules or microfilaments distinguishable from keratins after the treatment with colcemid and cytochalasin B did not react with the DA-7 antibody, and other structures of the tested cells, with the exception of keratin filaments, were also negative.

The advantage of the DA-7 lymphocyte hybridoma is that it produces an antibody that reacts only with human keratin 18. The advantage of this narrow specificity is the possibility of identifying and studying human carcinomas growing as transplants in athymic mice (nu/nu strain) or other immunosuppressed animals, when antibodies that react with keratins of other species cannot be used.

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263,087 animal species, e.g. antibody RICE0-C-04,

Another advantage is that it binds to the target structure of keratin 18, which is stable even during relatively drastic fixation of human material with formaldehyde and subsequent embedding in paraffin, which is the routinely used fixation of histological material in all histological laboratories. Known monoclonal antibodies react only on frozen tissue sections, or require a more gentle special fixation with metacarn.

Table 1 shows the strength of the antibody reaction when using different fixation methods.

Table 1

Intensity of the DA-7 antibody reaction on tissue sections fixed in three different ways (weak reaction +, moderate reaction ++, strong reaction + + +)

Fixation used: methanol/acetone metacarb formaldehyde

Frozen paraffin-embedded sections

Reaction + + -)· + + τ- +

This antibody can be used in all pathology departments for routine differential diagnostics!< differentiating carcinomas from other types of tumors, e.g. sarcomas, melanomas or lymphomas, but also for differentiating and denocrating carcinomas from carcinomas from stratified squamous epithelium. To our knowledge, a hybrid with these properties is not commercially available in any country, nor has a hybrid with similar properties been described in the professional literature.

Example 1

In order to propagate RICEO-DA-7 hybridoma cells in vitro, 1 x 10"' hybridoma cells were inoculated into a Legroux culture flask containing 20 ml of DMEM culture medium with 10/. fetal calf serum and antibiotics (see below). After four days of hybridoma cell culture growth, the culture medium enriched with the monoclonal antibody DAg ¹ ? released into the medium by the hybridoma cells was obtained from the flask.

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cells. The specificity of the obtained antibody was tested on a panel of cells from tissue cultures, both in indirect immunofluorescence using a pig or rabbit antibody against mouse immunoglobulin, labeled with fluorescein-iso-thiocyanate, and after electrophoretic transfer of cell lysates* and cytoskeleton preparations to a nitrocellulose membrane with subsequent detection by an indirect immunoperoxidase test (see above). The results are summarized in Table 2.

Table 2

Reactivity of the DA-7 antibody with cells of various human lineages (+ strong positivity, - negativity,

XF immunofluorescence, IB immunoblotting, V vimentin, l< keratins labeled with numerical indices of the keratin catalog, G glial acidic protein, D desmin)

Type Tissue origin, cell type Line name Medium filament type Reaction IF IB Man Rhabdomyosis of the hand RD D + V - - Glioma At 118 MG G + V - - Fibroblasts Primoculture 3 V - T-lymphoblstoid line MOLT-3 In - - Breast cancer MCF-7 ^To 8,18,19 + + Urinary bladder cancer T 24 ^To 7,8,18,19 + + Intestinal cancer HT-29 ^To 8,18,19 4 4 Squamous cell carcinoma A 431 ^For 5,6,7,8/13, 14,15,16.17,18 4 4 Cervical Carcinoma Hela ^K 7,8.17,18 4 4 Cow Mammary gland epithelium BMGE-H ^To 7,8,18,19 - Rat Morris hepatoma MH ^To 8.18 - - Mouse Firoblast 3T3 In - - From results, listed in Table No. it is clear that the anti-

DA-7 reacts exclusively with human keratin 18. The narrow specificity for keratin 18 was clarified in immunoblotting, where a strong reaction was found with keratin 18, while other keratins present were negative (see also Example 2: tissue

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Example 2\

Culture medium enriched with monoclonal antibody DA-7 was obtained by culturing hybridoma cells RICEO-DA-7 as in Example 1. The tissue specificity of the antibody reaction was tested by indirect immunofluorescence test and confirmed also by indirect immunoperoxidase reaction, in both cases on frozen and paraffin-embedded tissue sections. Tissue samples listed in Table 5 were taken from surgical or autopsy material. Frozen tissue sections were fixed after air drying with a methanol-acetone mixture (1:1) pre-cooled to -15°C and repeatedly washed with phosphate buffered saline at pH 7.4 (PBS). Paraffin sections were deparaffinized and rehydrated sequentially with xylene, alcohol and distilled water. Now the procedure was common for frozen and paraffin-embedded sections. The sections were coated with monoclonal antibody DA-7, which was removed from the section after 1 hour of incubation by repeated washing (3 x 5 minutes each) in PBS, and rabbit or pig antibody with fluorescein-isothiocyanate was applied to the section. Incubation (40 - 60 minutes) was completed by washing three times in PBS for 5 minutes each, the sections were left at room temperature until dry, and the preparations were mounted, for example, in a medium with glycerin. The results of the evaluation of the reaction of the DA-7 antibody with selected tissues are shown in Table 3; no differences were found between the results obtained by indirect immunofluorescence and indirect immunoperoxidase reaction, therefore these methods are not presented separately in Table 3.

Table 3

Reactivity of the DA-7 antibody with normal tissues (+ strong positivity, - negativity, V vimentin,

D desmin, G glial acidic protein, N neurofilament, l< keratin with the appropriate numerical index)

Type Tissue Man Connective tissue Sva 1 Brain Vessels □Atra - hepatocytes - bile ducts Urinary bladder - epithelium Endometrium Intestinal epithelium Respiratory tract epithelium Mesothelioma Mammary gland epithelium Gallbladder epithelium Epidermis (incl. spid.pla Cow Connective tissue Sva 1 Vessels Epidermis Datrc - hepatocytes - bile ducts Intestinal epithelium Mammary gland epithelium Urinary bladder epithelium Calcaneal gland epithelium E o i t o 1 z a n k r a t u

Type of filament Reaction

IN

D

N + G

V+D ^K 8,18 ^{+ !<} 7,8,18,19 ⁺ ^4,5,7,8,13,18,19 ^{+ K} 7,8,1.8,19 ⁺ ^8,18,19 ^{+ K} 7,8,13,19 ^7,8,18,19 ^{+ K} 5,7,8,14,16,17,18,19 ^{+ !<} 7,8,18,19 ⁺

Λ Ώ r! 1 C ' jf ^K 1 ,2,5,5,9,10.11. 14,15,16'

IN

D

D+V

K (ala not Κ^θ) ^K 8,18 '<7,8,18,19 '<8,18,19

K, ívČ5tn ^? · K _1n ) o

_ i< - ~

AND

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Table 3 - continued

Type Tissue

Hamster Ligament Sva 1 Vessels

Lymphoid tissue Epidermis □ atra - hepatocytes

- bile ducts Intestinal epithelium

Rat Connective tissue Sva 1 Vessels □ atra - hepatocytes

- bile ducts Intestinal epithelium Mammary gland epithelium Urinary bladder epithelium

Hys Connective tissue Sva 1 Vessels □atra - hepatocytes

- bile ducts Mammary gland epithelium

Type of filament

Reaction

IN

D

D+V

IN

K (but not Κ-^θ) K including K _n K -

"K-"

IN

D

D + V

Including<

To -

To -

K - ” « - ”

IN

D

D + V '18

K including Κ^θ K - K - From the spectrum of tissues (see Table 3) reacting with the DA-7 antibody, it is clear that all tissues containing keratin 18 are positive, while all tissues lacking this keratin are negative. It is also evident from the table that the antibody does not react with keratins of the tested animal species, which can be considered its advantage, especially from the point of view of clinical use. Immunoblotting analysis after electrophoretic separation of proteins of tissue lysates or cytoskeletal preparations confirmed that the only keratin recognized by

263,087 antibody DA-7 in epithelial tissues containing multiple keratins is keratin 18.

Example 3/

With regard to the possibility of practical use in the differential diagnosis of tumors, the reactivity of the DA-7 antibody was tested on tissue sections prepared from various human tumors by immunofluorescence and immunoperoxidase methods (see description of the method in Example 2). The results obtained are summarized in Table No.-4.Table 4

Reactivity of the DA-7 antibody on tissue sections of human tumors (+ positive reaction, - negativity, V vimentin, D desmin, G glial acidic protein.

To keratin with additives

Tumor type

Sarcomas

Melanomas

Lymphomas

Gliomas

Carcinomas of the intestine, breast, lung, stomach, ovary, bladder

Hepa t omy ear numerical index)

Type of medium filaments Reaction

V (or V+D)

IN

VG ^K 8,18,19 ^{+ K} 7,8.18,19 ^{+ K} 7,8,18,19 .. ^{+ K} 8,18,19 ' ^{+ K} 7,8,18,19 ^{+ K} 4,7,8,13,17,18,19 ^{+ K} 8,18 ⁺

The results of determining the reactivity of the DA-7 antibody with various human tumors confirm the specificity of this antibody for tumors of epithelial origin that contain keratin 18. Together with the negative results in other tumor types, this indicates the clinical value of this antibody for the diagnosis of primary carcinomas and their metastases.

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RICEO-DA-7 hybridoma cells have a morphological appearance of typical myeloma cells. Under in vitro conditions they grow in a similar semi-suspension culture. The basic culture medium is Dulbecco's modification of minimal essential medium (DMEM) supplemented with L-glutamine (3mM), penicillin, streptomycin and inactivated fetal calf serum (10%) from JZD Sokolnice u Brna. The hybridoma is cultured at 37°C, the mean generation time is 18 hours and the modal chromosome number 6 months after the construction of the hybridomas was 72. The produced antibody is a mouse monoclonal immunoglobulin of the IgG1 class, specifically binding the epitope of human keratin 18.

The monoclonal antibody produced by the RICEO-DA-7 hybridoma reacts with human keratin 18 in formaldehyde-fixed paraffin-embedded histological sections and is therefore usable in all histological laboratories using various tests (immunohistochemical, enzyme-immunological, radioimmunological, immunofluorescence, immunoblotting). In addition to medical practice, due to its property of specifically reacting only with human keratin, it is useful in the field of theoretical research of human carcinomas on xenotransplants, where it is impossible to use an antibody reacting with a target structure also present on animal keratins, such as the RICE0-C-04 antibody.

Claims (1)

Mouse lymphocyte hybridoma RICEO-DA-7 producing a monoclonal antibody class IgG1 against human keratin 18.