WO1988005537A1

WO1988005537A1 - Conjugate of biological molecules and a chelating agent

Info

Publication number: WO1988005537A1
Application number: PCT/FI1987/000163
Authority: WO
Inventors: Pirkko Vihko; Marja SÖDERVALL; Reijo Vihko
Original assignee: Innofarm Oy
Priority date: 1987-01-14
Filing date: 1987-12-08
Publication date: 1988-07-28
Also published as: FI80801C; FI870122A0; FI80801B; FI870122A

Abstract

The conjugate of a biological molecule, especially a protein, such as a monoclonal antibody or its fragment and of mono- or bicyclic DTPA, which conjugate is labeled with a fluorescent agent. For the preparation of the labeled conjugate the protein and the chelating agent are first combined, whereafter the conjugate is labeled with the fluorescent agent. The invention also concerns a method for the determination of the degree of conjugation of conjugates.

Description

Conjugate of biological molecules and a cheiating agent.

The present invention concerns conjugates of biological molecules labeled with a fluorescent agent, especially labeled antibody conjugates.

Immunological methods have become standard methods in clinical chemistry, especially for the determination of biologically active compounds in low concentrations. Radioimmunoassays are the most generally used assay methods. Research has been carried out in order to find substitutes for the radioactive labels due to their detrimental effect on health and also to their often short storage time. One alternative is a fluorescent label. Some of the lanthanide ions, specifically Eu³⁺ and Tb³⁺, form strongly fluorescing chelates with suitable organic, multiteeth ligands, e.g. ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) .

In the patent publication WO 84/03698 Eu³⁺ cheiating DTPA derivatives have been described, which can be coupled to antibodies for in vitro diagnostic purposes. The cheiating compounds, for example N¹- and N²-(p- isothiocyanatobenzyl)diethylenetriaminetetraacetic acid, are synthesized by means of a laborious four stage process, wherein use is made of reagents which from the point of view of safety of the working environment are irritating and toxic to the working environment (bromoacetic acid and thiophosgene). In the second stage of the synthesis a mixture of isomers is formed. The isomers are separated by means of HPLC and the synthesis sequence is continued using the pure isomers. Eu3⁺ is chelated in the third stage of the synthesis series (I. Hemmilä et al., Anal. Biochem. 197 (1984) 335), also in a multistage process. The Eu³⁺-chelate of the DTTA isocyanate derivative is reacted with the antibody. The reaction is slow (overnight, 4°C) and the degree of conjugation low, about 10%, wherefore the reagent has to be used in a large excess (chelate:antibody = 60:1). The unbound chelate thus has to be removed by gel filtration. At this ratio the conjugate contains 7 Eu³⁺/lgG.

The claims of the US patent 4,454,106 comprise coupling of DTPA to an antibody using the process of mixed anhydrides and the labeling of the conjugate obtained i.a. with a fluorescent metal, e.g. Eu³⁺. in the method according to the patent appr. 1.5 DTPA/IgG is obtained. This binding ratio has been obtained by testing with a 1¹¹in-label. The patent does not, however, contain an example describing the use of Eu³⁺ as a label. Also the literature contains no example of Eu³⁺-labeling with DTPA anhydrides, and thus the labeling cannot be considered to have been carried out with any prior described method. For example the TR-FIA sensitivity is not sufficient enough with the ratio 1.5 Eu³⁺/igG, but the withinassay and betweenassay variations are large.

DTPA has been coupled to proteins using also the cyclic anhydride of DTPA (cDTPAA) (I) of the formula

CD

(e.g. Hnatowich, US 4,479,930 and Hnatowich et al.. Int. J. Appl. Radiat. Isot. 33 (1982) 327). The use of cDTPAA has several advantages. It is a substance that is easily synthesized in a single step. It is also available commercially (Sigma, Aldrich). It is an unam- biguous, under dry conditions stable substance as opposed to the mixed anhydride (US-patent 4,454,106), which always has to be prepared shortly before use. The re- producibility of the process is because of the unstability poor, and there is no reliable analytical method for the anhydride formed.

On the other hand, cDTPAA is poorly soluble substance. Hnatowich has used for the administration of small amounts of cDTPAA a chloroform suspension. With this method, administration from an unhomogenous mixture is not very precise.

According to the present invention the DTPA-conjugate of the antibody is prepared with the mono- or bicyclic anhydride of DTPA and the conjugate obtained is labeled for diagnostic use, for example with Eu³⁺ (TR-FIA).

In time-resolved fluoroimmunoassay use is made of the difference between the duration of the fluorescence of a specific signal and that of a non-specific background, whereby the signal-noise ratio is improved. Almost in all published methods use is made of Eu³⁺- labeled antibody, wherefrom the bound Eu³+ is determined quantitatively subsequent to dissociative fluorescence development. Wallac-LKB has patented DEL- FIA (dissociative Eu fluorescence enhancement FIA) (EP 64484 (1984)).

This invention simplifies the method described in the patent publication WO 84/03698, according to which the Eu3⁺-DTTA-chelate obtained in several synthesis stages is coupled to the antibody using a reaction time of 12 hours at +4°C. According to the present invention, initially a DTPA-conjugate is formed from the protein with the mono- or bicyclic anhydride. The bicyclic an- hydride is either a commercially available product or prepared according to Eckelman (Eckelman et al., J. Pharm. Sci. 64 (1975) 704). The monocyclic anhydride is prepared according to Halpern (Radioimmunoimaging and Radioimmunotherapy, ed . S W Burchiel & B A Rhodes, Elsevier 1983, p. 198-9). Typically when preparing the derivative, the mono- or bicyclic anhydride is dissolved in dry DMSO, as opposed to Hnatowich, whereby due to complete dissolution, the pipetting accuracy is good. From this homogenous solution a suitable amount is pipetted into a bicarbonate solution of the antibody. The reaction time is 1 hour at room temperature. The antibody DTPA-derivative thus obtained may be used for different diagnostic purposes in vitro and in vivo.

In TR-FIA, the Eu³⁺-chelate has to be sufficiently stable but easily dissociated in acid environment. The more teeth the complex former has, the more stable a complex it forms. Also the dissociation from such complexes is slow. It has, however, been discovered (I Hemmila, Lanthanides as Probes for Time-resolved Fluorometric Immunoassays, Thesis 1986) that from the unsymmetrical N'-benzyl-DTTA containing four cheiating acid groups, Eu³⁺ dissociates faster (1 min) than from the corresponding symmetrical N²-benzyl-DTTA (20 min).

When using a cyclic anhydride of DTPA for the preparation of the antibody DTPA-derivative, an unsymmetrical conjugate (II) is formed containing four cheiating acid groups, and having a structure corresponding to that of the N'-benzyl-DTTA-conjugate (III). Only the group of DTTA and the bond connecting to the protein are different. ( III )

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Thus the antibody DTPA-derivative prepared with a cyclic anhydride is comparable to the N-benzyl-DTTA- derivative for use in TR-FIA. It is easier and much faster to work with the cyclic anhydride. The commercial DTPA-anhydride or the DTPA-anhydride prepared in one simple synthesis step is reacted with the antibody for 1 hour at room temperature.

Excess DTPA is removed by dialyzing while simultaneously exchanging the buffer to one suitable for labeling. The cheiating substance is added in the form of the commercially easily available chloride. There is no need to use an excess of EuCl₃, but a tested suitable amount, wherefore no gel filtration is needed. On the other hand, the reagent disclosed in the patent publication WO 84/03698 is laboriously prepared in four synthesis steps. In the synthesis two isomers are formed of which only one forms a rapidly dissociating Eu- chelate. From this follows that the yield of usable chelate after the separation of the isomers is poor. In addition, as the degree of conjugation is low, the consumption of a laboriously prepared reagent is high. Also, as the cheiating metal according to the present invention is added in the last stage, the same antibody DTPA-conjugate may be used for different diagnostic purposes, both in vitro and in vivo, by choosing a suitable metal for each purpose.

For the preparation of the derivatives, the purified F(ab')₂-fragment of PAP (Prostatic Acid Phosphatase) monoclonal antibody and purified whole antibody as well as SHBG (Sex Hormone Binding Globulin) purified monoclonal antibody was used. The bicyclic DTPA is manufactured by Sigma. The mono-cDTPA we have manufactured ourselves. The dimethylsulphoxide (DMSO) is manufactured by SNEA or Aldrich.

Example 1

Coupling of DTPA to a monoclonal antibody

1) Treatment of the protein

Purified monoclonal PAP-antibody is used as such or is split with pepsin to F(ab')₂, which is purified using protein-A-Sepharose-4B-affinity chromatography. The F(ab')₂-fraction obtained (0.02 M PBS buffer, pH 8.0) or the whole monoclonal antibody (IgG) is concentrated in an Amicon concentration device and dialyzed into a NaHCO₃-buffer, pH 8.0. The dialyzed sample is lyo- philized and deep frozen. F(ab')₂ is dissolved in water shortly before use so that the protein concentration is 10 mg/ml and the concentration of NaHCO₃ is 0.1 M. The SHBG antibody is used as whole (IgG).

2) Preparation of the derivative

A. cDTPA/F(ab')₂-derivative/IgG-derivative

For the conjugation reaction cDTPAA is dissolved in dry DMSO, from which a desired amount is pipetted into the protein solution. The solution is mixed for 30 s to 1 min and incubated at room temperature for one hour. The pH of the solution is checked. The sample is dialyzed into a 0.1 M Na-citrate buffer, pH 5, to remove free DTPA. For example when cDTPA/F(ab')₂/IgG = 5 the cDTPA- DMSO-solution (17.85 mg/ml), 5 /ul, is pipetted into the protein solution, wherein 5 mg F(ab')2 or IgG/0.5 ml.

When adding DMSO to the protein solution one has to take into account that the amount of DMSO does not exceed 5% of the volume of the protein solution.

B. mono cDTPA/F(ab')₂-derivative/IgG-derivative

Preparation in the same way as for the cyclic derivative. Mono-cDTPAA is added in a DMSO-solution to the protein solution.

Example 2

Labeling of the DTPA-conjugate of the PAP-antibody or its F(ab')₂-fragment with EuCl₃

The labeling was carried out using 10 M Eu with respect to the antibody-DTPA-conjugate. When labeling is carried out with the Eu-DTPA-derivative chelate as described in the patent WO 84/03698, it is used in an excess of 160 M with respect to the antibody.

EuCl₃ (Fluka 46130) was dissolved in 0.1 M sodium citrate buffer, pH 5 to 6, 2.75 mg EuCl₃/mI, 15 minutes before use. The DPTA-conjugate of the antibody or its fragment dissolved in 0.1 M sodium citrate buffer (10 mg conjugate/ml, pH 5 to 6) was mixed with a suitable amount of EuCl₃ - sodium citrate solution and incubated for one hour at room temperature. The percentage of labeling is 98. Example 3

Labeling of the DTPA-conjugate of a SHBG-antibody with EuCl₃

The antibody DTPA-conjugate was prepared using a ratio of DTPA/IgG of 20:1. EuCl₃ (Fluka 46130) was dissolved in 0.1 M sodium citrate buffer at pH 6. Labeling was carried out using Eu at a ratio of approximately 10 M with respect to the IgG-DTPA-derivative at room temperature for one hour.

Example 4

For reason of comparison the SHBG-antibody was labeled with the Eu-chelate described in the patent specification WO 84/03698. Thereby the Eu-chelate was used in an excess of 160 M with respect to the antibody. The remaining labeling conditions were: 50 mM K₂HPO₄, pH 9.4, incubation over night at +4°C. Separation of the labeled antibody was carried out using gel filtration in order to remove excess chelate.

After incubation the reaction mixture was poured into a gel filtration column (Sepharose 6B 1.5 cm x 30 cm, at the top Sephadex 6-25 1.5 cm x 5 cm). Elution was carried out using 0.05 M tris-HCl, 0.9% NaCl, 0.1% NaN₃ (pH 7.75, 25°C) as elution buffer.

The fluorescence was measured by adding a sample of 10/ul into 200/ul Delfia Enhancement-solution. After shaking the fluorescence was measured with an Arcus fluorimeter. The Eu-concentration was calculated using as the reference value 0.2 pmol Eu = 106 _cps. Comparison results:

Using the EuCl₃-label, the following numbers of Eu- molecules attached to antibodies were obtained:

Eu-molecules/protein molecules cDTPA/F(ab')₂ (5:1) 1.9-2.0 cDTPA/F(ab')₂ (10:1) 2.7 cDTPA/IgG (10:1) 2.4 cDTPA/IgG (20:1) 3.7 (Ex. 3) cDTPA/IgG (50:1) 6.9 m-cDTPA/IgG (300:1) 11.0

In the Example of the patent publication W0 84/03698 when labeling using an excess of 60 M of Eu-chelate 7 Eu-molecules/IgG is obtained and when raising the ratio to 160- 500 M excess Eu-chelate the ratio does not exceed 10 Eu-molecules/IgG. With the ratio 160 M of Eu- chelate in excess, 9.3 Eu-molecules per one antibody molecule were obtained (Example 4).

The labels according to the Examples 3 and 4 were tested with the SHBG Delfia kit-method using approximately the same protein concentration in the dilutions of both labels. This means in brief that the wells of the micro- titer plates were coated with polyclonal anti-SHBG- antibodies. The comparison solutions and the labels were pipetted into the wells and incubated, washed and measured using Delfia Enhancement-solution. SHBG-comparison solns EuCl₃-label Eu-chelate label nmol/1 (Example 3) cps (Example 4) cps

0 3197 4281

6.25 43535 81940

12.5 77255 147983

25 146026 263334

50 238831 442793

100 389177 841566

200 787518 1579440

TR-FIA of SHBG functioned in the same way with both labels and their distinction with respect to the different standards was as good.

According to one embodiment the invention also concerns a method for the determination of the degree of conjugation between a cheiating agent and a biological molecule. By the degree of conjugation is meant the molar ratio cheiating agent/biological molecule. The degree of conjugation is usually determined by coupling to the cheiating groups in the conjugate a radioactive isotope. This mode of determining is, however, cumbersome and unprecise due to the short half-life of the isotopes. When, however, as in this invention, Eu is coupled to the cheiating groups and the fluorescence is measured, the degree of conjugation may be determined very precisely and in a simple manner.

Claims

Claims:

1. Conjugate of a biological molecule and a cheiating agent and labeled with a fluorescent agent, c h a r a c t e r i z e d in that the cheiating agent is mono- or bicyclic DTPA.

2. Conjugate according to the Claim 1, c h a r a c t e r i z e d in that the biological molecule is a poly- or monoclonal antibody or a fragment thereof, a pepti- de, nucleic acid, hapten, enzyme, synthetic probe or cDNA.

3. Conjugate according to the Claim 1, c h a r a c t e r i z e d in that the biological molecule is a monoclonal antibody or a fragment thereof.

4. Conjugate according to the Claim 3, c h a r a c t e r i z e d in that the monoclonal antibody or its fragment is the antibody of SHBG-antigen or a fragment thereof.

5. Conjugate according to the Claim 3, c h a r a c t e r i z e d in that the monoclonal antibody or its fragment is the antibody of PAP-antigen or a fragment thereof.

6. Conjugate according to the Claims 1-5, c h a r a c t e r i z e d in that the fluorescent agent is Eu.

7. Process for the preparation of a conjugate according to the Claims 1-6, c h a r a c t e r i z e d in that the biological molecule and the cheiating agent are first combined, whereafter the labeling of the conjugate is carried out with the fluorescent agent.

8. In vitro-diagnostic immunological method, c h a r a ct e r i z e d in that a conjugate according to the claims 1-6 is used for determining the antigen of an analyte.

9. Method for the determination of the degree of conjugation between a cheiating agent and a biological molecule, c h a r a c t e r i z e d in that the chelate mixed in a solution buffered to pH 5-6 and a EuCl₃- buffer solution are incubated, whereafter the fluorescence is determined in a manner known.