AU658403B2 - Technetium-99m labeling of proteins - Google Patents

Description

OPI DATE 06/10/92 AOJP DATE 12/11/92

INTEI

(51) International Patent Classification 5 A61K 49/02 APPLN. ID 14576 92 PCT NUMBER PCT/US92/01577 ION TREATY (PCT) (11) International Publication Number: WO 92/15333 A (43) International Publication Date: 17 September 1992 (17.09.92) (21) International Application Number: PCT/US92/01577 (22) International Filing Date: 27 February 1992 (27.02.92) Priority data: 661,793 27 February 1991 (27.02.91) US (71) Applicant: AKZO N.V. [NL/NL]; Velperweg 76, P.O. Box 186, NL-6800 LS Arnhem (NL).

(71)(72) Applicant and Inventor: SUBRAMANIAN, Ramaswamy [US/US]; 352 Catoctin Avenue, Frederick, MD 21701 (US).

(74)Agent: BLACKSTONE, William, Akzo Pharma, 1330-A Piccard Drive, Rockville, MD 20850 (US).

(81) Designated States: AT (European patent), AU, BE (European patent), CA, CH (European patent), DE (European patent), DK (European patent), ES (European patent), FI, FR (European patent), GB (European patent), GR (European patent), IT (European patent), JP, KR, LU (European patent), MC (European patent), NL (European patent), SE (European patent).

Published With international search report.

658403 (54)Title: TECHNETIUM-99m LABELING OF PROTEINS (57) Abstract A method for attaching technetium-99m to proteins using reducing metal reagents to achieve binding to high affinity binding sites and high specific activity. The reagents play a dual role under the given experimental conditions by reducing disulfide bonds in the proteins to sulfhydral groups suitable for binding to technetium, and reducing pertechnetate from Tc(VII) to Tc(III) or Tc(V). Reduction of disulfide on the protein is conducted initially with an excess of reducing metal reagent, a pertechnetate reagent is added at the end of the protein reduction reaction and allowed to continue to reduce the technetium. Thereafter a chelator scavenger is added to remove poorly bound or unbound technetium.

WO 92/15333 PC/US92/01577 1 TECHNETIUM-99m LABELING OF PROTEINS DESCP'PTION OF THE INVENTION This invention relates to a procedure for attaching technetium-99m to antibodies using reducing metal reagents.

These reagents play a dual role in the labeling reaction under the specified conditions. The method of this invention overcomes two problems with prior art methods, which are low specific activity and binding of Tc-99m to low affinity binding sites.

BACKGROUND OF THE INVENTION Prior art methods for labeling antibodies with technetium- 99m used stannous chloride as a reducing agent to generate sulfhydral groups on antibodies. At the same time the antibodies were contacted with technetium and a chelator, typically DTPA, to achieve binding of the technetium to the antibodies, while scavenging unbound technetium with the DPTA present in the reaction medium.

Paik et al. reported that carrying out technetium-99m labeling in presence of excess DTPA (MoAb:DTPA 1:10) one could selectively attach technetium-99m to high affinity sites.

Stannous chloride was present in 10-fold excess over the protein. Their typical reaction conditions (Paik et al.) are as follows: [MoAb] [SnClz] 100 pum [DTPA] 100 uim Selective binding to high affinity sites, however, was obtained only under experimental conditions where both DTPA and antibody were competing for the reduced technetium ion. Paik et al.

reported that about 10 times molar excess of DTPA was required to avoid technetium-99m binding to low affinity sites.

Unfortunately the presence of excess DTPA resulted in reduced specific activity (~mCi/mg). Following their procedures with antibody 88BV59, an IgG 3 the yield was only 0.01-0.5mCi/mg.

Summary of the Invention This invention relates to a procedure for attaching technetium-99m to proteins such as monoclonal antibodies using reducing metal reagents such as tin and zinc according to which 99 mTc binds is to high affinity binding sites and high specific activity is maintained. The reagents play a dual role under the given experimental conditions by reducing disulfide bonds in the proteins to sulfhydryl groups suitable for binding to technetium, and reducing pertechnetate from Tc(VII) to Tc (III) or Tc(V).

According to a broad form of the present invention there is provided a method for labelling proteins containing cysteine with technetium-99m, comprising reacting a protein with a reducing metal bound to a ligand by a covalent or coordinate bond to reduce disulfide groups in the protein to sulfhydryl groups in reaction mixture, adding pertechnetate to the reaction mixture and incubating to reduce technetium in the pertechnetate from Tc(VII) to Tc(II) or Tc(V) and to react the reduced technetium with sulfhydryl groups on the protein, thereby binding to the protein, and adding a chelator to the reaction mixture to react with unbound technetium, thereby quenching the reaction and binding any free or loosely bound technetium.

By the preferred method of this invention, reduction of the disulfide groups on the protein is conducted initially with an excess of tin or zinc reagent, a pertechnetate reagent 20 is added at the end of the protein reduction reaction and allowed to continue to reduce the Stechnetium. Thereafter a chelator scavenger is added to remove poorly bound or unbound 99mTc Brief Description of the Drawings Figure 1 illustrates stability studies of 99 mTc bound to IgG 3 antibody 88BV59 in S 25 saline solution with excess DTPA in a ratio of IgG:DTPA of 1:1000 at 37°C.

Figure 2 shows HPLC radiochromatographs of 99 mTc-88BV59 in the reaction medium after preparation according to the method of the invention. Figure 2a shows the peak of technetium antibody conjugate as the major peak. The minor peak is technetium bound to DTPA. Figure 2b shows the technetium antibody conjugate purified with all 30 measurable chelator bound technetium removed.

Figure 3 illustrates the immunoreactivity of the antibody technetium conjugate prepared according to the invention compared with the immunoreactivity of the antibody alone. Immunoreactivity was determined by indirect ELISA on specific antigen coated wells. The reactivity of the radiolabelled antibody (Tc-antibody conjugate) was determined by comparison with the reactivity of native (unbound) antibody by their ability to bind conjugate antigen for which the antibody (88BV59) has specificity.

Figure 4a illustrates the retention of antibody technetium conjugate by tumor 7 xenografts in 6 to 8 week old athymic Balb/c [G:\WPUSER\LIBVV]00383:TCW WO 92/15333 PCr/US92/01 577 3 mice. The xenografts were developed using enzymatically dissociated human tumor cells containing antigens recognizable by 88BV59. Ten micrograms (1-2 uCi/ug) of labeled antibodies were injected into the veins of the mice for biodistribution studies. A comparison is made between conjugates with intact antibodies and conjugates with F(ab')2.

Figure 4b illustrates serum retention of 88BV59 technetium conjugates in mice having human colon tumor xenografts.

Figure 4c illustrates the tumor retention of antibody and technetium conjugates in the mice.

Figure 4d illustrates kidney retention of and intact antibody technetium conjugates in the mice.

Figure 4e illustrates liver retention of F(ab') 2 and intact antibody technetium conjugates.

Figure 5 shows a coronal view of the liver SPECT scan of a human patient who has received 15 mCi/10mg 99 'Tc-88BV59 at 4 to 5 hours after administration. Large numbers of lesions in the liver of a size less than or equal to 0.5 cm can be seen.

These results were later confirmed by CT scan.

DETAILED DESCRIPTION OF THE EMBODIMENTS This procedure describes the protocol for attaching technetium-99m 99 "Tc) to proteins using reagents containing reducing metals such as tin and zinc. These reagents play a dual role under the given experimental conditions.

Binding to the protein is through a sulfhydral group (SH) obtained by reduction of disulfide in the protein. Thus, cysteines must be present in the protein for conjugation.

The reagents contain well known reducing metals bound to ligands through covalent or coordination bonds. They are sufficiently powerful enough to reduce disulfide bonds present in the protein molecule, creating sulfhydryl groups suitable for attachment to technetium, but not so powerful as to form metal hydroxide colloids. Examples of the preferred metals are Sn, Zn, Rn and Co. They are bound to ligands such as oligosaccharides, polysaccharides and other sugar derivatives by covalent or coordinate bonds.

WO 92/15333 PCF/US92/01577 4 The reagents also reduce pertechnetate for attachment to the protein. Tc(VII) is reduced to either Tc(III) or Tc(V) and concomitantly coupled to the sulfhydryl group on the protein.

Any loosely bound technetium is chelated with DTPA, EDTA, iminodiacetate, cysteine, diaminedithiol or other chelators, which are added to the reaction mixture after reduction and binding of Tc to the protein to quench the reaction by scavenging unbound and loosely bound Tc. The ratio of MoAb to quencher is preferably from about 1:1 to 1:5, and should not to exceed about 1:8. The chelators may be attached to an immobile surface, or may be removed by gel filtration chromatography.

Our imaging experiments with Tc-antibody conjugates clearly show that the presence of small amounts of Tc-DTPA does not affect the quality of imaging because Tc-DTPA is rapidly cleared from circulation by renal filtration. Thus, it is not always necessary to remove chelator bound 99 "Tc from the preparation before administration.

This process of making the radiolabeled antibody is unique. In the preferred embodiment tin or zinc saccharate or glucarate is used to produce sulfhydryl groups and to reduce technetium for conjugation to sulfhydryls in the antibody.

Also, the process is unique in using chelators as quenchers, rather than competing for reduced technetium in the reaction mixture by adding them earlier. Our reducing reagent is preferably tin saccharate prepared by adding saccharic acid 20 mg/ml, deaerated) solution to tin chloride solution 5 mg/ml in 0.02M HCl). Tin saccharate may also be prepared by treating tin chloride with excess saccharic acid, removing the precipitated tin saccharate and storing the precipitate in dry nitrogen. It is also possible to combine the metal chloride and the acid together and add that reaction mixture to the protein combining stannous chloride and glucaric acid).

The antibody (10 mg/ml or lyophilized powder) in a buffer solution, or alternatively in a reducing buffer solution is added to the tin saccharate solution and incubated at about to 60 C for 5 to 60 minutes. This incubation leads to formation of sulfhydryl groups. The period of incubation varies inversely with temperature. Reaction temperature is WO 92/15333 PC/US92/01577 limited by the stability of the protein. A temperature of incubation cannot be used that will denature the protein.

Preferred reaction conditions are about 15 minutes to minutes at about 20° to 37 0 C. Under experimental conditions 1 to 3 SH groups are generated per antibody molecule. This method of labeling has proved to be particularly suitable for antibodies such as an IgG's. Under the same reaction conditions use of tin chloride alone, not as a saccharic acid salt, leads to formation of a colloidal solution not suitable for further use. Thus the reducing metal must be bound to a ligand for the method to work.

Reduction of the antibody is followed by addition of pertechnetate. Incubation to reduce Tc(VII) to Tc(III) or Tc(V) and to conjugate with the sulfhydrals on the antibody is carried out at about 200 to 37 0 C for about two minutes to one hour. Preferably, labeling is accomplished by incubation at about 230 37 0 C for about 30 to 60 minutes. Thereafter, a chelator is added DTPA) to quench the reaction and to scavenge unbound Tc by conversion to Tc-DTPA. This resulting pharmaceutical preparation is purified before administering or, alternatively, directly administered to cancer patients without removing excess Tc-DTPA. As a general rule, at least 90% of the Tc should be bound to the antibody. Otherwise it should be purified. Within 1-2 hour after administration non-antibody conjugated Tc in the original preparation in the form of Tc- DTPA will be removed by the kidneys. Patient studies with radiolabeled antibody preparations containing Tc-DTPA have shown good tumor localization. If the composition is to be purified before administration, excess Tc-DTPA is removed by gel filtration column chromatography, leaving pure radiolabeled antibody.

Tc labeled antibodies prepared according to this invention are very stable. Results obtained with cancer patients using such preparations have clearly shown that even 4 hours after administration the technetium-99m is firmly bound to the antibody. Excellent localization of the radiolabeled antibody was also observed in th.se cases making it possible to obtain good radioimmunoscintigraphs. Loosely bound Tc, if any, would bind to human serum albumin. HPLC analysis of the serum from WO 92/15333 PCr/US92/01577 6 a patient treated with Tc-99m labeled 88BV59 did not show any transfer to human serum albumin even 4 hours after administration.

Another advantage of this method is its ability to label relatively difficult systems, such as Reductive labeling with technetium of frequently results in formation of 99 "Tc labeled F(ab). In fact many researchers use the reductive method to obtain s 99 Tc labeled Fab fragment from F(ab') 2 In this invention, using appropriate concentrations and reaction conditions, particularly reacting at room temperature (20 0 one can mildly introduce technetium in F(ab') 2 without alteration.

We radiolabeled the fragment of 88BV59, an IgG 3 using this method and about 10 mg/10 mCi of the radioimmunoconjugate was administered to cancer patients.

Planar and SPECT images showed localization of the radiolabeled antibody in lesions. Also HPLC analysis of serum from patients showed that 99 "Tc was firmly bound to the antibody. The immunoreactivity of radiolabeled antibody was not affected by this procedure.

Example 1 We found out that by treating a concentrated antibody solution (10-50 m solution) with 30 to 50 molar equivalents of a stannous salt solution (in particular stannous glucarate) for a short period of time at elevated temperatures (4 0 -60 0 one could generate large numbers of -SH groups (2-3 per molecule, as determined by DTNB tests using Elmans reagent suitable for Tc-binding). This method is specifically suitable for -SH rich proteins. Sodium pertechnetate was added at the end of the reaction. The reaction was allowed to continue for additional 20-30 minutes in an inert atmosphere (vacuum or nitrogen).

Scavenging solutions containing chelators such as DTPA, ETDA, cysteine or diaminidithiol chelators were added at the end of the reaction and incubated at room temperature for about 5 to 10 minutes. This converted any remaining TcO 4 unbound to MoAb, to Tc-DTPA.

7 Experimental conditions were as follows: Stannous Glucarate 1-2mm Reaction at 37 0 C, for 15-30 min (alternate condition are room temperature for min. or 45 0 C, 3-6 min.) in an evacuated vial.

TcO 4 (50-100mCi) was added and reacted to 37 0 C for 15 min. (alternatively 23 0 C for 30 min.).

DTPA was then added (1-100pm solution). DTPA to MoAb ratio was 0.1:1 to 5:1.

Reaction yields of 10-15mCi/pg of protein was easily achieved.

If radiolabelling yields were less than 90%, the radiolabelled antibody would be purified by gel filtration chromatography. In general, yields were >90% (with 88BV59).

Results of purification are illustrated in Figure 2.

In vivo biodistribution data in mice showed that: the radiolabelled antibody was retained in serum and tumor; uptake in normal tissues as liver, bone, spleen, muscle and intestine were low and, depending on the nature of the antibody, kidney uptakes were low to moderate.

Early studies in colon cancer patients shows that the radiolabelled antibody localised to tumor metastases (Figure Example 2: Three vial kit Vial one (component A) contains 200-300ptg of stannous chloride and 400-600tg of 20 glucaric acid as stannous glucarate in the form of lyophilised powder. This vial may also S' contain excipients or additives such as lactose, fructose, mannose etc. w/v, preferred) to enhance the stability of the product. Vial two (component B) contains lyophilised protein (0.1-10mg of protein preferred for single i.v. dose preparation).

Excipients such as lactose, fructose and/or sucrose can also be present.

Vial three (component C) contains DTPA solution (preferred 0.1-0.2ml of 0.2mM DTPA solution in NaHCO 3 pH 8 buffer) and 3-10ml of injectable saline solution. The radiolabelling procedure consists of adding 1.0 to 2.0ml of water for injection to vial one containing the stannous glucarate. The contents of this vial are then added to vial two containing the lyophilised protein. The reaction mixture is incubated at temperatures 4-37 0 C for a period of time (5 min 24 hours). The higher the temperature, the less time has to be involved.

Temperature Time 37 0 C pref. 2 hours 20-25 0 C pref. /4 6 hours 4 0 C pref. 1 24 hours In a radiopharmacy or hospital, temperatures above 20 0 C are preferred because of the smaller amount of time. The above step is followed by addition of sodium pertechnetate 99 mTcO 4 and further incubation for about 5-15 minutes at temperatures [G:\WPUSER\LIBVV]00383:TCW 7A ranging from 20-37°C. The final step involves addition of DTPA solution containing saline (vial three).

99 mTc labelled antibody prepared in this way 99 mTc-88BV59) requires no purification radiochemical purity) and is stable in human serum as well as in saline solution over a period of 24 hours (see Figures 2A and 2B).

Example 3: Two vial kit.

Vial one contains component A and component B described above. This vial hence will contain stannous glucarate (prepared from 100-300p[g of stannous chloride and 200- 600.g of glucaric acid) and protein -10mg, or, in the case of human monoclonal antibody 88BV59, 10-12mg in 0.04M PBS solution, pH 7.2) and excipients (lactose 5-6% w/v) if needed.

This mixture is lyophilised to enhance the stability of the product.

Prior to lyophilisation, the mixture containing components A and B can be allowed to sit at temperatures ranging from 4 0 C-37 0 C over a period of time ranging from 5 min 24 hours (see above). All the above conditions will be acceptable, as Vial one used in the 2 vial kit can be prepared in a commercial vialing facility. Vials can be stored at appropriate temperatures until ready for use in the clinic, or radiopharmacy.

Vial two containing DTPA/saline solution is prepared as described for Vial three of the 3 vial kit. The radiolabeliing reaction is performed as follows: 20 reconstitute the contents of vial one with injectable 99 mTcO 4 (in saline oxidant free solution the volume of 99 mTc can be 0.5 to 5ml or more. Activity can be 20-200 mCi depending on the amount of protein in the vial.

allow the labelling reaction to occur at temperatures ranging from 40 to 37C° for a period of time (2 min 2 hours).

Add at the end an aliquot of DTPA/saline solution and perform ITLC and HPLC analysis to determine the percentage of 99 mTc bound to the protein.

Example 4: Two vial kit, preferred reaction conditions i**I Lyophilised stannous glucarate powder prepared from stannous chloride (275 pg) and saccharic acid (550pug) was reconstituted with water for injection. This solution was S 30 added to a vial containing lyophilised monoclonal antibody 88BV59 (alternately one could add a solution of MoAb 88BV59 to a vial containing lyophilised stannous glucarate). The reaction was allowed to occur at 37 0 C for about 75 min. The vial was stoppered, frozen and lyophilised.

To the lyophilised vial containing stannous glucarate and MoAb 88BV59, 54.6mCi of 99 mTcO 4 in 1.3ml of saline solution was added. The radiolabelling reaction was allowed to happen at room temperature for about 30 minutes. At the end a scavenger solution containing DTPA (0.1ml of 0.2mM DTPA solution in pH 8 NaHCO 3 buffer), 2 [G:\WPUSER\LIBVV]00383:TCW 7B minutes at room temperature followed by the addition of 3ml of 0.9% saline solution was added.

The percentage 99 mTc bound to 88BV59 was determined using an ITLC system, in which Gilman ITLC-SG strips and a buffer system consisting of 50/50 mixture of 0.6M acetate solution, pH 5.0 and 0.06M citrate solution, pH 5.0 were used. The results showed that 98.3% of 99 mTc was bound to 88BV59.

HPLC analysis using a TSK 3000 SW gel filtration column and radioactivity analyser showed a single peak corresponding to 99 mTc-labelled antibody 88BV59 (Figure 3A).

Immunoreactivity of 99 mTc-88BV59 was determined using affinity column containing sephadex bead resins with antigen for 88BV59 covalently attached to the beads. The extent of binding by 99 mTc 88BV59 in a given amount of radioactivity gave the reactive fraction values. The immunoreactivity or reactive fraction value determined by this assay for 99 mTc-88BV59 prepared using the 2 vial kit was 84.1%.

Stability: 99 mTc-88BV59 prepared using the above method was incubated in saline solution as well as in human serum solution. As shown in the enclosed figures (3B and 3C), HPLC analysis indicates that more than 80% of 99 mTc remained bound to 88BV59 even after incubation in serum at 37 0 C, and also in saline solution at 4 0

C.

4.

4*4 4 4* 4 4 xs [G:\WPUSER\LIBVV]00383:TCW WO 92/15333PC/S2O57 PCT/US92/01577 References Fritzberg, Abramns, Beaumier, P2.

Proceedings of National Academiy of services, 4025-4029 (1988).

et al. USA, 8 Paik, Pham, Hong, Suharni, Heald, S.C., Reba, R. C. Steiginan, and Eckelman, W. C. International Journal of Nuclear Medicine and Biology, 12:3-8 (1985).

Paik, Eckelman, W.C. and Reba, Nuc. Med. Biol., 131:359-362 (1986).

Rhode, Torvestad, Breslow, Burchiel, Reed, andAustior, R.W. In: S.W. Burchiel and B.A. Rhodes, "Tumor Imaging", p. 111, New York, Masson Publishing, USA, 1982.

Claims (4)

1. A method for labelling proteins containing cysteine with technetium-99m, comprising reacting a protein with a reducing metal bound to a ligand by a covalent or coordinate bond to reduce disulfide groups in the protein to sulfhydryl groups in reaction mixture, adding pertechnetate to the reaction mixture and incubating to reduce technetium in the pertechnetate from Tc(VII) to Tc(Il) or Tc(V) and to react the reduced technetium with sulfhydryl groups on the protein, thereby binding to the protein, and adding a chelator to the reaction mixture to react with unbound technetium, thereby quenching the reaction and binding any free or loosely bound technetium.

2. 'tUe method of claim 1 wherein the reducing metal is selected from the group consisting of tin, zinc, ruthenium and cobalt.

3. The method of claim 1 wherein the ligand is a sugar derivative.

4. The method of claim 1 wherein the reducing metal bound to a ligand is selected from the group consisting of stannous saccharate, stannous glucarate, zinc saccharate and zinc glucarate. A method for labelling proteins containing cysteine with technetium-99m which method is substantially as herein described with reference to Example 1. S 20 Dated 19 January, 1995 S""Raraswamy Subramanian -Akzo N.V. Patent Attorneys for the Applicants/Nominated Persons SPRUSON FERGUSON [G :\WPUSER\LIBVV]00383:TCW a