DE3222084A1 - MONOCLONAL ANTIBODIES - Google Patents

Description

BAYER AKTIENGESELLSCHAFT 5090 Leverkusen, Bayerwerk

Central area no ι ·

Patents, trademarks and licenses Dn / by-c * »* ♦ June Monoclonal Antibodies

The present invention generally describes new hybridoma cell lines and in the special hybridoma cell lines for Production of monoclonal antibodies against human ■ borrowed plasminogen activator of tissue type (PA II), antibodies, which are produced in this way, preparative, therapeutic and diagnostic uses as well pharmaceutical preparations containing these antibodies.

The fusion of mouse myeloma cells with spleen cells from immunized mice (Kohler and Milstein, Nature 256 , 495-497 (1975)) was the first indication of the possibility of obtaining continuous cell lines which produce uniform (so-called "monoclonal") antibodies . - Since then, numerous attempts have been made to produce various hybrid cells (so-called "hybridomas") and to use the antibodies that are formed by them for various scientific investigations (eg: Current Topics in Microbiology and Immunology , Vol 81 - "Lymphocyte Hybridomas ", F. MeIchers, et al, Springer-Verlag (1978), as well as

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References in it; CJ. Barnstable et al., Cell 14: 9-20 (1978); P. Parham, WF Bodmer, Nature 276: 397-399 (1978); Handbook of Experimental Immunology , 3rd ed., Vol. 2, DM Wier, Editor: Backwell, 1978, Chapter 25, Chem. Eng. News, 15-17 (1979)).

This work describes the basic technology for the production of monoclonal antibodies by hybridomas.

Monoclonal antibodies against histocompatibility antigens, against haptens, protein and enzymes have already been generated. However, nothing is known about yet somatic cell hybrid antibody production against human tissue-type plasminogen activator.

The tissue-type plasminogen activator is an enzyme of the fibrinolytic system that converts plasminogen to plasmin converts, which in turn can break down fibrin. Thus it has a thrombolytic effect. The three proteins mentioned (Plasminogen, plasmin, fibrin) occur in the blood. Plasminogen activator can therefore be used in the therapy of hyper-coagulation conditions, especially in thromboembolic Diseases, be of use.

Currently, urokinase, obtained from human urine or from kidney cell cultures, and streptokinase, those from culture filtrates ß-hemolyzing Streptococcus isolated is used as a fibrinolylic agent. However, both are in their therapeutic uses not unproblematic.

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Plasminogen activator of the tissue type can be distinguished from urokinase and streptokinase. The tissue type plasminogen activator was found to have a molecular weight of about 72,000. It is produced by human melanoma cells and can be isolated from the corresponding culture hypersensitivities by the method of Rijken and Collen (JBC 256 , 7035-7041 (1981)).

Monoclonal antibodies against the plasminogen activator tissue type are of interest in several respects. For example, the previous three-stage cleaning process for the plasminogen activator can be converted to a single-stage one can be simplified by using such antibodies in immunoadsorption chromatography.

The present invention provides hybridoma cell lines Available that synthesize highly specific monoclonal antibodies against tissue-type plasminogen activator and secrete. These hybridomas are produced by the Köhler and Milstein method mentioned at the beginning. After immunization of mice with tissue-type plasminogen activator, their spleens become Mice fused with cells of a mouse myeloma cell line; The resulting hybridomas were systematically examined for antibodies that were selectively produced with the Plasmino tissue type gene activator attached to microtiter plates react. In this way they became hybridomas that produce antibodies against the plasminogen activator. Some of these antibodies react exclusively with tissue-type plasminogen activator,

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while others cross-react with urokinase. The antibodies produced in this way are a further subject of the invention. They are monospecific for one certain antigenic determinants in the plasminogen activator molecule. The present invention further describes Methods for making these hybridomas.

The antibodies according to the invention can be used, for example, to determine of plasminogen activator in normal and malignant cells. They are thus for diagnostic and prognostic purposes, e.g. to check tumor growth or the functional status of normal cells, applicable.

The preparation of the hybridomas generally comprises the following steps:

A. Immunization of mice with the human plasminogen activator. Female Balb / c mice have been shown to be useful for this purpose, but others can also Mouse strains can be used. The immunization regimen and the concentration of plasminogen activator

20 should be chosen so that sufficient amounts of antigen-stimulated lymphocytes are formed. Three immunizations 14 days apart with 100 μg / protein / mouse / injection with phosphate-buffered physiological saline solution

25 were found to be effective.

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B. Obtaining the spleens of the immunized mice and Preparation of a spleen cell suspension in a suitable medium. Approximately 1 ml of medium per spleen •is sufficient. The experimental techniques

5 are known.

C. Fusion of the suspended spleen cells with mouse myeloma, cells of a suitable cell line (e.g. P 3 χ 68 Ag 8) by adding a suitable fusion promoter (e.g. Polyethylene glycol) is used. Preferred fusion promoters are medium molecular weight polyethylene glycols between 1,000 and 4,000 (e.g. commercially available PEG 1,000 etc.). However, you can other known fusion promoters can also be used. A ratio of about 10 microcells is preferred per myeloma cell. A total volume of about

8 0/5 - 1.0 ml fusion medium is sufficient for 10 Spleen cells. There are many mouse myeloma cell lines known and available, e.g. from scientific institutes or from cell deposit institutions such as the SaIk Institute Cell Distribution Center, La Jolla, CA. The cell line used should be preferably of the so-called "drug resistant" type, so that unfused myeloma cells in a selection medium die while hybrids survive.

Cell lines resistant to 8-azaguanine, which contain the enzyme hypoxanthine-guanine-phosphoribosyl transferase, are most common is missing, and is therefore in a HAT medium (hypoxanthine, aminopterin, Thymidine) are not capable of growth.

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Preferably the myeloma cell line used should also be of the "non-secreting" type so that they do not have antibodies or H or L chains of immunoglobulins themselves forms. In certain cases, however, secreting myeloma cells can be beneficial.

D. Dilution and cultivation in individual vessels of the

unfused spleen cells, the unfused myeloma cells and the fused cells in one Selective medium in which the unfused myeloma

10 cells do not multiply, so that they did not fuse Cells die off (approx. 1-2 weeks). The individual fused cells are isolated by adding the Volume of the diluent is adjusted so that a certain number of cells (about 1-4) in the

15 individual vessels (e.g. each well of a microtiter plate). The medium (e.g. HAT medium) prevents the resistant (e.g. against 8-azaguanine) unfused myeloma cell line grows out; it dies with it. The non-merged MiIz-

cells have only a limited number of cycles of division; therefore these cells also die afterwards a certain time (about 1-2 weeks). The fused cells, however, continue to multiply, since it is the permanent growth of the parental myeloma cells and of the parental spleen cell the ability to use the enzyme hypoxanthine guanine phosphoribosyl transferase to synthesize, have inherited and thus can survive in the selection medium.

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E. Check for the presence of antibodies to plasminogen activator in each tube.

F. Selection (e.g. by limited dilution) and cloning of the hybridomas that have the desired anti-

5 bodies produce.

Once the desired hybridoma has been selected and has been cloned, the antibody can be produced in two different ways. Monoclonal antibodies of very high purity is obtained if the hybridomas cultivated in a suitable medium for a certain time and the antibody from the supernatant wins. A suitable medium and the optimal culture time are easy to determine. This in vitro technique delivers monoclonal antibodies with only low levels

15 quantities of proteins from the heterologous serum (e.g. fetal calf serum) are contaminated.

To have a much higher concentration of monoclonal The selected hybridoma of a mouse can be used to produce antibodies with only slightly lower purity

20 ~ preferably a syngeneic or semi-syngeneic -

inject. After a certain incubation time, this leads to the formation of a tumor in the mouse, which has high antibody concentrations (5 - 20 mg / ml) in the blood and in the
free peritoneal exudate (ascites) of the host animal

25 places. Even if these mice have normal antibodies in the blood and ascites, these only come in

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a concentration of approx. 5% of the monoclonal antibodies before. The monoclonal antibody thus obtained has

3 has a high titer (it is active in dilutions of 10 or below), and the ratio between specific and non-specific immunoglobulin is about 20: 1.

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example 1 Production of the antigen used for immunization

Plasminogen activator was produced from melanoma cell culture supernatants by the method of Rijken and Colien (JBC 256, 7035-7041 (1981)).

Immunization of Balb / c Mice

Female Balb / c mice were administered subcutaneously with 100 μg of plasminogen activator in complete friend's view adjuvant immunized. The animals were administered subcutaneously with 100 μg of plasminogen activator boosted after 14, 28 and 42 days. The spleens were removed for cell fusion on day 53. Four days before the spleens were removed, the animals received a further 30 μg of plasminogen activator i.p. daily.

Preparation of a spleen cell suspension

Single cell suspensions were prepared from the removed spleens by passing the organs through a stainless steel sieve Steel were pressed. The cells were in Dulbecco's Minimal Essential Medium (DMEM), which was prepared with 4.5 g / l glucose, 1000 units / ml penicillin and 100 μg / ml Streptomycin was supplemented, transferred. The cells were washed three times with DMEM and then in desired Concentration resuspended in the same medium.

8th
Generally about 10 cells were obtained per spleen.

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Preparation of the myeloma cells

The myeloma cell line X 6 3-Ag 8-653 used here is a Subclone of the mouse myeloma cell line P 3-X 63-Ag 8. The cell line does not express heavy or light chains of

5 immunoglobulins ( J. Immunol. 123 , 1548-1550 (1980)).
It was made by Dr. Lemke, Institute for Genetics at the University of Cologne. The cells are
resistant to 20 μg / ml 8-azaguanine in a medium containing hypoxanthine, aminopterin, thymidine (HAT).

They were in DMEM containing 4.5 g / l glucose, 20 mM glutamine, 1,000 units / ml penicillin, 100 ug / ml streptomycin and 10% fetal calf serum was added (complete medium). The myeloma cells were at the time of fusion in the logarithmic phase of cell proliferation.

Zeil merger

The spleen cell suspensions were added to the myeloma cells in DMEM without serum in a ratio of 10: 1 and centrifuged for 10 minutes at 200 g in round-bottom beakers. After the sediment had settled down, the supernatant medium was carefully decanted. ^{The sediments were incubated at 37 °} C. for 1 minute with 2 ml of a solution of polyethylene glycol of MW 2000 (diluted to 30% w / w with DMEM, pH 7.6). Then 20 ml of DMEM were added and the cells were carefully resuspended for a few minutes. They were then centrifuged again for 5 minutes at 200 g

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and now resuspended in complete medium so that a concentration of 10 cells / ml was obtained, whereupon, they were distributed in 1 ml portions on Costar plates.

Selection and cultivation of the hybridomas

After the cell fusion, the cells were placed in HAT medium

(Hypoxanthine, aminopterin, thymidine) ^{cultivated at 37 0} C with 5% CO ₂ in a humid atmosphere. After a few weeks, 100 μΐ of the supernatants from the hybridoma cell cultures were activated for anti-plasminogen activator

^ q did checked.

Those hybridoma cell lines which showed positive results in the anti-plasminogen activator test were used for the Clone selected. The hybridomas were subjected to a double-dilution cloning technique, at

15 of the 96 micron wells in one calculated At a dilution of 0.5 cells / well, 10 mouse thymocytes were used as "feeder cells". The cells still producing antibodies after this cloning process were multiplied, frozen and

in liquid nitrogen in the complete medium, the 25% fetal calf serum and 7.5% dimethyl sulfoxide contains ie11, kept.

Determination of antiplasmin activity

100 μl culture supernatant were removed from the cell cultures 25 and placed in microtiter wells to the plasmino-

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gene activator (5 μg / ml) for 3 hours at room temperature

had been fixed / and which was then

1 25 had been scooped up. J (goat) anti

4 mouse immunoglobulin (8-10 χ 10 cpm) was added added; then the plates were incubated for 3 hours at room temperature. After washing again with distilled water, the samples were counted in an LKB gamma counter. Table 1 shows some representative ones Examples of hybridomas secreting antibodies to human tissue-type plasminogen activator.

The same hybridomas were given to mice with 0.5 ml Pristane were pretreated, injected intrape.ritoneal to ascites fluid containing antibodies 10-15 days 15 to win after that. The antibody activity was determined as described above. As shown in Table 1, the antibody titers of the ascites fluids range over a range of 10 to 3 χ 10

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¹ Table 1

Titer of the activities of antibodies in hybridoma supernatants against human tissue plasnujnogenic

125 "^ activator as measured in (J) anti-mouse IgG binding test.

(12SJ) -Anti ^ Ig (cpm) bound by the culture supernatant number 10 ^J

7th C. 6th 4.03 22nd D. 12th 4.22 31 C. 10 4.76 33 B. 12th 8.38 34 C. 4th 9.24 46 D. 10 4.39 50 F. 8th 1.98 57 F. 11th 2.07 71 C. 12th 3.97 73 E. 10 5.42 77 G 7th 2.16 86 E. 6 6 1.83 90 C. 10 3.55 100 E. 11th 2.80

Anti-ascites
body titre
(reciprocal) 10 ⁵ number B 11 From the culture supernatant
bound (125J) -
Anti ^ Ig (cpm)
χ 10 Anti-ascites
body titre
(reciprocal) X 10 ⁵ 1 χ 10 ⁵ 131 C 11 5.47 3 X 10 ⁵ 1 χ 10 ⁵ 135 G 7 3.18 1 X 10 ⁴ 3 χ 10 ⁶ 137 G 6 1.72 1 X 10 ⁴ 1 χ 10 ⁶ 164 E 9 2.04 1 X 10 ⁴ 1 χ 10 ⁵ 167 C 8 2.56 1 X 10 ³ 1 χ 10 ³ 180 G 5 1.47 1 X 10 ⁵ 1 x 10 ³ 206 E 8 5.21 3 X 10 ⁶ 1 χ 10 ⁵ 239 P 8 13.01 3 X 10 ⁶ 1 x 10 ⁴ 243 D 8 10.04 3 X 10 ⁴ 3 χ 10 ³ 245 G 9 2.97 3 X 10 ⁶ 1 χ 10 ³ 251 D 12 10.53 3 X TO ⁵ 1 χ 10 ⁵ 260 E 9 3.82 1 X 10 ⁵ 3 χ 10 ⁴ 265 A 7 2.95 1 X 10 ⁵ 1 X 280 4.69 3

Table 1 (port setting)

Titer of the activities of antibodies in eybridoma supernatants against human tissue plasminogen

125 activator, measured in (J) -AntirMau3-IgG binding test.

(125J) -Anti-Ig bound by the culture supernatant Nuircter (cpm)

Ascites antibody titer (reciprocal)

Nuniner from the culture supernatant
bound. (125J) anti-Ig (cpm)

Ascites antibody titer (reciprocal)

108 G, 12

109 F 6
124 D 8

5.32
3.34
9.76

1 χ ΙΟΙ χ 10 1 χ 10 ⁽

282 B. 8th 9.68 286 G 8th 2.46 299 G 12th 12.65

1 χ 10 3 χ 10 ⁴ 3 χ ΙΟ ⁶

125 3

cpm of (J) goat anti - = - mouse IgG χ 10, that of plasminogen activator fixed on microtiter plates

is bound. The initial volumes of the samples were 100 μΐ. ι t j

* · ■ cc «« *

Characterization of the immunoglobulins produced by the cloned hybridomas

The classes and subclasses of antibodies produced by the hybrid clones were analyzed. The respective Antibody was enriched from the culture supernatants by precipitation with ammonium sulfate (40% saturation) and partially cleaned. Using class-specific anti-mouse immunoglobulin antisera, the respective immunoglobulin classes in the Ouchterlony gel diffusion test certainly. As shown in Table 2, most of the antibodies against the plasminogen activator belong to IgM or IgG class; only one is from

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ro table 2

> (Characterization of itonoclonal antibodies against human plasminogen activator of the tissue type ^ by isotype analysis

IgG ₁ ¹ ^ b ^{IgM K} cell line IgG ₁ I ^ 2b ^{IgM K}

+ + 160 P 9 +

+ + 164 G 6 + +

+ + 167 E 9 +

+180 G 8 ++

+ 191 B. 12 + +

+198 +,

+ 206 G 5 + + do

+ + 211 + +

+ 219 C 10 +

+ + 239 E 8 +

+ 243 D 8 + ^!

+ + 245 D 8 + +

++ 251 G 9 ++

+ 260 D 12 + '

+ 265 E 9 + +

+ + 267 +

Ul
- ¹ cell line C. 6th 7th D. 9 8th D. 12th 22nd C. 10 31 B. 12th 33 C. 4th 34 C. 5 36 D. 10 46 F. 8th 50 H 11th 57 D. 10 64 C. 12th 71 E. 10 • 7.3 G 7th 77 E. 6th 86 C. 10 90

Table 2 (continued) Characterization of inonoclonal antibodies against human tissue-type plasminogen activator by isotype analysis

Cell line

^{IgM K}

^{IgM K}

100 E.11 106 E 9

108 G 12

109 F 6

110 D 7 124 D 8 131 B 11 135 C 11 137 G 7

268 A. 8th + 275 + 280 A. 7th 282 B. 8th 285 A. 10 + 286 G 8th 297 C. 6th 299 G 12th

Example 2

Inhibition of plasminogen activator activity by antibodies against plasminogen activator.

Plasminogen activator is a protease called plasminogen converted into plasmin. The activity of plasminogen activators can be measured with the pibrin plate technique. Plasminogen, which is incorporated into fibrin, is thereby used is activated by the plasminogen activator. The effect of anti-plasminogen activator antibodies - either individually or in various combinations - was determined in the fibrin plate test. Table 3 shows that 6 antibodies of 34 inhibit the fibrinolytic response in this test system. The six active antibodies differentiate quantitatively in their inhibitory properties. An additive inhibition in the fibrin plate test is observed when two antibodies are mixed. This is an indication that the two antibodies are directed against different antigenic determinants.

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t * Table 3

Φ

jp Measurement of plasminogen activator of the tissue type in the fibrin plate test by mixtures of antibodies

to against plasminogen activator

- * ■.

131 B 11 282 B 8 206 G 5 33 B 12 137 G 7 299 G 12 73 E.

13.1 B 11 65% 100% 100% 91% 86% 79 % 72%

282 B 8 100% 97% 100% 80% 65% 52% 45%

206 G 5 100% 100% 65% 62% 65% 65% 55%

33 B 12 91% 80% 62% 30% 45% 30% 30%

137 G 7 86% 65% 65% 45% 22% 30% 20%

299 G 12 79% 52% 65% 30% 30% 14% 14%

73 E 10 72% 45% 55% 30% 20% 14% 0%

Dilution of the antibodies: 10 " ²

Example 3

Cross reaction of the antibodies against the human plasminogen activator vom .Gewebetyp with urokinase

Urokinase, a protease / derived from either human

(R)
Urine (e.g. Ukidan from Serono) or from tissue culture

(R)

supernatants (e.g. Abbokinase from Abbott) obtained

is capable of plasminogen in a similar way to plasminogen activator. convert from tissue type to plasmin. For this reason, the antibodies were tested for their cross-reactivity Investigated urokinase of the two types mentioned above. In Table 4 it is shown that 5 out of 35 antibodies are directed against tissue-type plasminogen activator alone, while the other 30 antibodies

(Ό)

also react with · Ukidan ^v ', and below that 10 with Abbokinase 15.

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Table 4

Cross reactions of the antibodies against plasminogen activator with urokinase

Line

Reactivity of antibodies against plasminogen activator with:

(R)

Plasminogen

7 C 6 +

22 D 12 +

31 C 10 +

33 B 12 +

34 C 4 + 46 D 10 + 50 F 8 + 57 H 11 + 71 C 12 + 73 E 10 + 77 G 7 + 86 E 6 +

MG = molecular weight

Ukidan (urokinase MG 54000

Abbokinase (urokinase

MG 33000

Line

0 0

0 0 0

0 0 Reactivity of antibodies against plasminogen activator with:

(R)
Plasminogen Ukidan Abbokinase

(Uro- (urokinase kinase

MG MG

54000 33000

135 C 137 G 164 G 167 E 180 G 206 G 239 E 243 D 245 D 251 G 260 D 12 + 265 E 9 +

0 0 0 0 0 +

* Tv *

tr ¹ table 4 (continued)

Cross reactions of the antibodies against plasminogen activator with urokinase

Ükidan ^iR) Abbokinase ^(R)

Reactivity of antibodies against plasminogen activator with:

Zeil- plasmidnogen-

line (Oro- (urokinase

kinase

MG MG 54000 33000

E. 9 Reactivity of antibodies against
Plasminogen activator with: + A. 7th Plasminogen Ukidan Abbokinase
(Uro- (urokinase
kinase
MG MG
54000 33000 + Cell
line B. 8th + 0 265 A. 10 + + 0 280 G 8th + '0 0 282 G 12th + + ₊ 285 + '0 286 + + 299

86 E 6
90 C 10
100 E 11

108 G 12

109 F 6
124 D 8
131 B 11

0 0 0 0 0 0 0

MG = molecular weight

CO K) N) N) O OO

This cross-reactivity of the antibodies could be due to common Structural elements between the tissue-type plasminogen activator and urokinase can be attributed, however also for a contamination of this protein with uro-

kinase. To decide which of the two interpretations the more likely one was. plasminogen activator of the tissue type prepared by immunosorbent chromatography, a monoclonal Antibody used exclusively with

10 this protein reacts. The thus purified plasminogen activator was then used as the antigen for the test system. The binding test showed that this plasminogen activator too reacted with all antibodies. This experiment makes it clear that between plasminogen

Tissue-type activator and urokinase cross-reactions appear; thus both enzymes share antigenic determinants.

Example 4

Purification and concentration of plasminogen activator tissue type by immunoadsorption chromatography.

Those antibodies that react exclusively with human tissue-type plasminogen activator - viz 131, 206 and 282 - were covalently attached to Sepharose

(R)
4 B * in a concentration of 10 mg protein per ml gel according to the method of SC March, J. Parikh, P. Cuatrecasas, Anal., Bichem. 60, 149 (1974) bound.

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The binding capacity of the column for plasminogen activator was determined to be 0.5 to 1/5 mg / ml gel. The enzymatic The activity of the material thus purified was concentrated more than 1,000 times that of the starting solution. The electrophoresis pattern of the plasminogen activator purified in this way was identical to that of material / that has been cleaned in a conventional manner. The yield of plasminogen activator at the immunoadsorption chromatography was up to 90% compared to approx. 40% for the conventional method.

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Claims (7)

Claims 1. Monoclonal antibodies against plasminogen activator of tissue type. 2. Process for the production of monoclonal antibodies against plasminogen activator of the tissue type, characterized in that one myeloma cells with Fused cells obtained from mammals immunized with tissue-type plasminogen activator and the resulting cells Hybridomas selected those that produce the antibodies. 3. The method according to claim 2, characterized in that mouse cells are used. 4. Hybridoma cells that raise monoclonal antibodies against Produce tissue-type plasminogen activator. 5. Hybridoma cells according to claim 4, characterized in that they are produced by fusion of spleen cells immunized mice with mouse myeloma cells became. 6.. Use of monoclonal antibodies against Plasminogen activator of the tissue type z.ur. cleaning of plasminogen activator using immunoadsorption chromatography. Le A 21 751 ': V3222Q84 7. Use of tissue-type monoclonal antibodies against plasminogen activator for detection of plasminogen activator in normal or malignant cells and tissues. Le A 21 751