EP0573577A1 - TECHNETIUM-99m LABELING OF PROTEINS - Google Patents

TECHNETIUM-99m LABELING OF PROTEINS

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Publication number
EP0573577A1
EP0573577A1 EP92907824A EP92907824A EP0573577A1 EP 0573577 A1 EP0573577 A1 EP 0573577A1 EP 92907824 A EP92907824 A EP 92907824A EP 92907824 A EP92907824 A EP 92907824A EP 0573577 A1 EP0573577 A1 EP 0573577A1
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EP
European Patent Office
Prior art keywords
technetium
protein
antibody
reduction
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92907824A
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German (de)
French (fr)
Other versions
EP0573577A4 (en
Inventor
Ramaswamy Subramanian
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Per Immune Holdings Inc
Original Assignee
Akzo NV
Akzo Nobel NV
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Filing date
Publication date
Application filed by Akzo NV, Akzo Nobel NV filed Critical Akzo NV
Publication of EP0573577A1 publication Critical patent/EP0573577A1/en
Publication of EP0573577A4 publication Critical patent/EP0573577A4/en
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/008—Peptides; Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • 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.
  • 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 Tc 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 sulfhydral groups suitable for binding to technetium, and reducing pertechnetate from Tc(VII) to Tc ⁇ III) or Tc(V) .
  • reduction of the disulfide groups on the protein is conducted initially with an excess of tin or zinc 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 99m Tc.
  • Figure 1 illustrates stability studies of 99l TMTc bound to IgG 3 antibody 88BV59 in saline solution with excess DTPA in a ratio of IgG:DTPA of 1:1000 at 37°C.
  • Figure 2 shows HPLC radiochromatographs of 99m Tc-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 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 radiolabeled antibody (Tc-antibody conjugate) was determined by comparison with the reactivity of native (unbound) antibody by their ability to bind cognate antigen for which the antibody (88BV59) has specificity.
  • Figure 4a illustrates the retention of antibody technetium conjugate by tumor xenografts in 6 to 8 week old athymic Balb/c mice.
  • the xenografts were developed using enzymatically dissociated human tumor cells containing antigens recognizable by 88BV59.
  • 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 F(ab * )_ 2 technetium conjugates in the mice.
  • Figure 4d illustrates kidney retention of F(ab') 2 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/lOmg 99m 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.
  • This procedure describes the protocol for attaching technetium-99m ( S9m 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.
  • SH sulfhydral group
  • 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. 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 ⁇ n Tc from the preparation before administration.
  • tin or zinc saccharate or glucarate is used to produce sulfhydryl groups and to reduce technetium for conjugation to sulfhydryls in the antibody.
  • 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 (e.g., 20 mg/ml, deaerated) solution to tin chloride solution (e.g., 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 (e.g., 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 4° 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 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 60 minutes at about 20° to 37°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.
  • 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 20° to 37°C for about two minutes to one hour.
  • labeling is accomplished by incubation at about 23° - 37°C for about 30 to 60 minutes.
  • a chelator is added (e.g., 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.
  • Tc should be bound to the antibody. Otherwise it should be purified.
  • 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 these 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 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 F(ab * ) 2 .
  • Reductive labeling with technetium of F(ab') 2 frequently results in formation of ""Tc labeled F(ab) .
  • many researchers use the reductive method to obtain ""Tc labeled Fab fragment from F(ab') 2 -
  • using appropriate concentrations and reaction conditions, particularly reacting at room temperature (20°-25°C) one can mildly introduce technetium in F(ab' ) 2 without alteration.
  • Tc0 4 (50-100 mCi) was added and reacted at 37°C for 15 min. (alternatively 23°-25°C for 30 min.). DTPA was then added (1-100 ⁇ m solution). DTPA to MoAb ratio was 0.1:1 to 5:1. Reaction yields of 10-15 mCi/ ⁇ g of protein was easily achieved.
  • radiolabeling yields were less than 90%, the radiolabeled antibody would be purified by gel filtration chromatography. In general, yields were always >90% (with 88BV59). Results of purification are illustrated in Figure 2.

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Abstract

Procédé de fixation de technétium-99m à des protéines au moyen de réactifs de métal afin d'obtenir une liaison à des sites de liaisons à haute affinité et une activité spécifique élevée. Les réactifs jouent un double rôle dans les conditions expérimentales données par réduction de liaison de disulfure dans les protéines en groupes sulfhydriques permettant une liaison au technétium, et réduction de pertechnétate de Tc(VII) en Tc(III) ou Tc(V). La réduction du disulfure sur la protéine est effectuée initialement avec un excédent de réactif de métal de réduction, un réactif de pertechnétate étant ajouté à la fin de la réaction de réduction des protéines et continuant à réduire le technétium. Ensuite, on ajoute un intercepteur chélateur afin d'éliminer le technétium faiblement lié ou non lié.A method of attaching technetium-99m to proteins using metal reagents to obtain binding to binding sites with high affinity and high specific activity. The reagents play a double role under the experimental conditions given by reduction of disulfide bond in proteins in hydrogen sulfide groups allowing a binding to technetium, and reduction of pertechnetate from Tc (VII) to Tc (III) or Tc (V). The reduction of the disulfide on the protein is carried out initially with an excess of reduction metal reagent, a pertechnetate reagent being added at the end of the protein reduction reaction and continuing to reduce technetium. Then, a chelating interceptor is added in order to eliminate the weakly bound or unbound technetium.

Description

TECHNETIUM-99m LABELING OF PROTEINS
DESCRIPTION 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.
Pai 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] = lOμm [SnCl2] = 100 μm [DTPA] = 100 μm
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 IgG3, 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 Tc 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 sulfhydral groups suitable for binding to technetium, and reducing pertechnetate from Tc(VII) to Tc{III) or Tc(V) . 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 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 99mTc.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates stability studies of 99l™Tc bound to IgG3 antibody 88BV59 in saline solution with excess DTPA in a ratio of IgG:DTPA of 1:1000 at 37°C.
Figure 2 shows HPLC radiochromatographs of 99mTc-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 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 radiolabeled antibody (Tc-antibody conjugate) was determined by comparison with the reactivity of native (unbound) antibody by their ability to bind cognate antigen for which the antibody (88BV59) has specificity.
Figure 4a illustrates the retention of antibody technetium conjugate by tumor xenografts in 6 to 8 week old athymic Balb/c mice. The xenografts were developed using enzymatically dissociated human tumor cells containing antigens recognizable by 88BV59. Ten micrograms (1-2 μCi/μg) of labeled antibodies were injected into the veins of the mice (n=6) 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 F(ab*)_2 technetium conjugates in the mice.
Figure 4d illustrates kidney retention of F(ab')2 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/lOmg 99mTc-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 (S9mTc) 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. 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 99ιnTc 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 (e.g., 20 mg/ml, deaerated) solution to tin chloride solution (e.g., 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 (e.g., 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 4° 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 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 60 minutes at about 20° to 37°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 20° to 37°C for about two minutes to one hour. Preferably, labeling is accomplished by incubation at about 23° - 37°C for about 30 to 60 minutes. Thereafter, a chelator is added (e.g., 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 these 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 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 F(ab*)2. Reductive labeling with technetium of F(ab')2 frequently results in formation of ""Tc labeled F(ab) . In fact many researchers use the reductive method to obtain ""Tc labeled Fab fragment from F(ab')2- In this invention, using appropriate concentrations and reaction conditions, particularly reacting at room temperature (20°-25°C), one can mildly introduce technetium in F(ab' )2 without alteration.
We radiolabeled the F(ab*)2 fragment of 88BV59, an IgG3, 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 ""Tc was firmly bound to the antibody. The immunoreactivity of radiolabeled antibody was not affected by this procedure. Example
We found out that by treating a concentrated antibody solution (10-50 urn 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°-60°C); 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 Tc04 unbound to MoAb, to Tc-DTPA. Experimental conditions were as follows: Stannous Glucarate : l-2mm
Reaction at 37°C. for 15-30 min (alternate condition are room temperature for 60 min. or 45°C, 3-6 min.) in a evacuated vial.
Tc04 (50-100 mCi) was added and reacted at 37°C for 15 min. (alternatively 23°-25°C for 30 min.). DTPA was then added (1-100 μm solution). DTPA to MoAb ratio was 0.1:1 to 5:1. Reaction yields of 10-15 mCi/μg of protein was easily achieved.
If radiolabeling yields were less than 90%, the radiolabeled antibody would be purified by gel filtration chromatography. In general, yields were always >90% (with 88BV59). Results of purification are illustrated in Figure 2.
In vivo biodistribution data in mice showed that: the radiolabeled antibody was retained in serum and tumor? uptakes in normal tissues such as liver, bone, spleen, muscle and intestine were low (<3% I.D./g); and, depending on the nature of the antibody, kidney uptakes were low to moderate.
Early studies in colon cancer patients showed that the radiolabeled antibody localized to tumor metastases (Figure 5) .
References
Fritzberg, A.R. , Abrams, P.G., Beaumier, P.L. et al., Proceedings of National Academy of Services, USA, 85: 4025-4029 (1988).
Paik, C.H. , Pham, L. , Hong, J.J., Suhami, M.S., Heald, S.C., Reba, R.C., Steigman, J. and Eckelman, W.C., International Journal of Nuclear Medicine and Biology, 12:3-8 (1985).
Paik, C.H. , Eckelman, W.C. and Reba, R.C., Nuc. Med. Biol., 13:359-362 (1986). Rhode, .A., Torvestad, D.A., Breslow, K. , Burchiel, S.W., Reed, K.A. , and Austior, R.W. In: S.W. Burchiel and B.A. Rhodes, "Tumor Imaging", p. Ill, New York, Masson Publishing, USA,

Claims

WE CLAIM:
1. A method for labeling 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 sulfhydral groups in a reaction mixture, adding pertechnetate to the reaction mixture and incubating to reduce technetium in the pertechnetate and to react the reduced technetium with sulfhydral 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. The 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.
EP92907824A 1991-02-27 1992-02-27 TECHNETIUM-99m LABELING OF PROTEINS Withdrawn EP0573577A1 (en)

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GB9324807D0 (en) * 1993-12-03 1994-01-19 Cancer Res Campaign Tech Tumour antibody
US7232888B2 (en) 2002-07-01 2007-06-19 Massachusetts Institute Of Technology Antibodies against tumor surface antigens
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EP0248506A1 (en) * 1986-03-05 1987-12-09 Mallinckrodt, Inc. (a Delaware corporation) Metal ion labeling of carrier molecules
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KR100238558B1 (en) 2000-02-01
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CA2104943A1 (en) 1992-08-28
EP0573577A4 (en) 1994-03-02
JPH06505990A (en) 1994-07-07
AU658403B2 (en) 1995-04-13
WO1992015333A1 (en) 1992-09-17
AU1457692A (en) 1992-10-06

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