WO2000021573A1 - Site-specific labeling of disulfide-containing targeting vectors - Google Patents
Site-specific labeling of disulfide-containing targeting vectors Download PDFInfo
- Publication number
- WO2000021573A1 WO2000021573A1 PCT/US1999/023614 US9923614W WO0021573A1 WO 2000021573 A1 WO2000021573 A1 WO 2000021573A1 US 9923614 W US9923614 W US 9923614W WO 0021573 A1 WO0021573 A1 WO 0021573A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- peptide
- thiol
- protein
- diagnostic
- hydrazone
- Prior art date
Links
Classifications
-
- 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
- A61K51/1093—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 conjugates with carriers being antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
- A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
-
- 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
- A61K51/083—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being octreotide or a somatostatin-receptor-binding peptide
-
- 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
- A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
-
- 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
- A61K51/1027—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 against receptors, cell-surface antigens or cell-surface determinants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- This invention relates to methods for introducing thiol-containing linkers onto disease targeting agents that contain disulfide bonds, the radiolabeled targeting agents and drug conjugates produced using these methods, and use of the radiolabeled or drug-bearing targeting agents for diagnosis and treatment.
- Free thiols offer a unique chemical handle for the attachment of numerous species to specific targeting agents, because of the specificity of the thiol group for reactive groups such as haloacetates, maleimides, and activated sulfonyl groups, and for reduced metal species such as reducedgnacchenetate and perrhenate, and certain other thiophilic metals such as zinc, copper, mercury, cadmium, platinum, palladium, lead and bismuth.
- reactive groups such as haloacetates, maleimides, and activated sulfonyl groups
- reduced metal species such as reducedgnacchenetate and perrhenate
- certain other thiophilic metals such as zinc, copper, mercury, cadmium, platinum, palladium, lead and bismuth.
- a complicating issue with many proteins, polypeptides and peptides is the presence of disulfide bonds that are critical to their structural integrity. The inherent reactivity of the thiol group can lead to breakage of such disulf
- Antibody fragments as well as sub-Fab' fragments, single-chain antibodies, diabodies, polypeptides and peptides, offer advantages for in-vivo targeting of radioimaging and radio- therapeutic isotopes and drugs because the smaller fragments will target and clear faster than an intact IgG or larger protein.
- a divalent antibody fragment such as a F(ab') 2 fragment should have increased total targeting compared to a Fab' fragment because the divalent binding region will increase the affinity of the protein for the antigen.
- F(ab')2 fragments are made up of two Fab' fragments joined by one or more disulfide bonds, which are sensitive to reduction by free thiols both during and after the conjugation of a thiol-containing moiety.
- conjugate may be bound to or near the antigen-binding region of an antibody or the receptor-binding region of a peptide/polypeptide, which can reduce or eliminate the binding affinity of the antibody or peptide for the antigen or receptor.
- the conjugation of haptens to periodate-oxidized carbohydrate sites is one method of site- specifically forming conjugates .
- the carbohydrate regions can be genetically engineered into specific sites on proteins or peptides so, for example, it is possible to place a carbohydrate at a site on a F(ab')2 fragment that will not interfere with the binding of the antibody fragment to the antigen.
- carbohydrate residues on the light chains of certain IgGs has been established. Such residues remain on F(ab')2S and F(ab)2S after pepsin or papain digestion, respectively. As such, they represent a masked potential site-specific chemical handle for haptenic attachment.
- certain murine antibodies have been reengineered to produce humanized complementarity determining region versions of the same antibodies, while simultaneously engineering glycosylation sites at positions remote to the antibody's antigen- binding site. This enables the insertion of carbohydrate at desired positions within the protein, including insertion of carbohydrate in the CHi domain and the variable region on either light or heavy chains.
- One object of the present invention is to provide conjugates of disulfide-containing targeting proteins, polypeptides and peptides, e.g., divalent antibody fragments and (sv)2S, with thiol-containing ligands without cleaving the disulfide bonds of the targeting proteins.
- Another object of the invention is to use the substituted thiol group attached to the disulfide-containing proteins or peptides as a specific chemical handle to further attach certain radioisotopes or chemotherapy agents.
- Another object of the invention is to provide radiolabeled proteins that are stable in vitro and in vivo.
- Yet another object is to provide methods for the use of stably substituted disulfide- containing proteins, polypeptides and peptides for radiodiagnosis, radiotherapy and chemotherapy of disease.
- a method of producing a diagnostic or therapeutic conjugate of a protein, polypeptide or peptide containing at least one disulfide bond which is necessary to maintain its biological activity, and bearing at least one thiol- containing moiety linked thereto through a hydrazone or hydrazine linkage comprising contacting the protein, polypeptide or peptide with a thiol-reactive diagnostic or therapeutic agent, either preformed or generated in situ, to form a stable diagnostic or therapeutic conjugate of the protein, polypeptide or peptide without substantial cleavage of the disulfide bond.
- the thiol-containing moiety linked to the protein, polypeptide or peptide through a hydrazone or hydrazine linkage is joined by reacting the disulfide bond- containing protein, polypeptide or peptide which also contains an aldehyde or ketone group with a thiol-hydrazine of the formula HS-Q-NHNH2 , wherein Q is a linking moiety selected from the group consisting of alkyl groups, aryl groups, cycloalkyl groups, peptides, and combinations thereof; and optionally reducing the resultant hydrazone to a hydrazine.
- the diagnostic or therapeutic agent in the conjugate can be a thiol-binding cationic radioisotope or a drug derivative comprising a thiol-binding linker.
- the protein is a glycosylated divalent antibody fragment whose partially oxidized carbohydrate portion is joined through the hydrazone or hydrazine linkage to the thiol-containing moiety.
- the present inventors have developed a method for conjugating a thiol-containing peptide linker or ligand to a disulfide-containing protein or peptide, e.g., a F(ab')2, through a carbonyl function, e.g., a periodate oxidized carbohydrate portion of the protein, without reducing disulfide bonds that maintain structure and/or conformation related to activity, e.g., reduction of a F(ab')2 fragment to Fab' during the conjugation or labeling process.
- a carbonyl function e.g., a periodate oxidized carbohydrate portion of the protein
- Conjugates have been produced in which the attachment of the linker or ligand to the disulfide bond- containing protein is stable and the attachment of a radiolabel, e.g., Tc-99m, to the ligand is stable in vitro and in vivo.
- a radiolabel e.g., Tc-99m
- preferred embodiments of Tc-99m-labeled peptide chelators delivered a higher percentage of the injected dose to a tumor than I-125-labeled F(ab')2or Tc-99m-labeled Fab'.
- acyl hydrazides commonly used for the conjugation of drug and chelate-nuclide molecules to antibodies through oxidized carbohydrate moieties are very unstable, even in-vitro. This was shown in stability experiments with the Tc-99m radiolabeled conjugates IMP 126-LL2-F(ab')2 and IMP 140-LL2- F(ab')2.
- LL2 is an anti-CD-22 monoclonal antibody (mab) that is described in U.S. Patent 5,789,554.
- the Tc-99m labeled peptide would dissociate from the protein when stored in solution over time.
- the in-vitro loss of the labeled peptide was monitored by size exclusion HPLC and reverse phase HPLC.
- the stability of the acyl hydrazide connection could be controlled to some extent by changing the amino acids adjacent to the acyl hydrazide.
- the peptide IMP 126 formed a more stable (though still unstable) connection to the LL2 F(ab than IMP- 140: possibly the aspartic acid residue catalyzed the dissociation of the labeled peptide.
- the antibody conjugate could be formulated into a single vial kit and labeled with Tc- 99m at room temperature.
- the Tc-99m labeled conjugate was labeled site-specifically on the peptide attached to the oxidized carbohydrate. This was shown first in control experiments in which the LL2 F(ab')2 was put through the conjugation process except that no periodate was added during the oxidation step.
- the control was a treated with Tc-99m-glucoheptonate and formed only 6% Tc-99m-labeled protein as measured by ITLC whereas the IMP 155-LL2 F(ab labeled under the same conditions afforded substantial quantities of the labeled antibody fragment (70-80 %).
- the other proof that the Tc-99m is attached to the peptide is that the labeled acyl- hydrazide peptides, which used the same Tc-99m ligand as IMP 155, had the activity dissociated from the protein as the Tc-99m labeled peptide, as shown by size exclusion and reverse phase HPLC analysis.
- the proof that the peptide is attached to the oxidized carbohydrate is that the conjugation with the periodate oxidized LL2 F(ab')2 produces a protein which contains free thiols (2-3 thiols/LL2 F(ab')2 measured by UV) and conjugation with the unoxidized antibody produces a protein which contains no free thiols.
- Tc-99m labeled IMP 155-LL2 F(ab conjugate was stable in-vitro, and in-vivo.
- the Tc-99m labeled antibody showed tumor targeting at 24 hr in Ramos tumor- bearing mice (see Example 3 and 4 below).
- the divalent antibody fragment delivered a higher dose to the tumor than the iodinated LL2-F(ab')2 or the Tc-99m-Fab'.
- the free thiol conjugates that have been made by the present method can be used to form conjugates to other moieties such as drugs, antibodies, antibody fragments, proteins, glycoproteins, DNA, RNA, PNA, metal complexes, radiolabeled species (imaging and therapy), enzymes, toxins and sugars.
- moieties such as drugs, antibodies, antibody fragments, proteins, glycoproteins, DNA, RNA, PNA, metal complexes, radiolabeled species (imaging and therapy), enzymes, toxins and sugars.
- Such fragment-present carbohydrate can be used to attach haptens such as miol-containing chelators which can be radiolabeled subsequently with thiol-binding radiometals.
- Carbohydrate containing vicinal diols can be oxidized to produce aldehyde and ketone functions with an agent such as periodate, and mixed with a thiol-containing hydrazine-containing hapten, generally represented as HS-Q-NHNH2, to effect conjugation.
- the formed hydrazones linking the hapten to the protein carbohydrate can be reduced with a reductant such as sodium cyanoborohydride to produce a thiol-hapten conjugate without compromising hinge-region disulfide bonds.
- the thiol-containing hydrazine-containing moiety HS-Q-NHNH2 can have a wide range of structures.
- the group, Q can include: alkylene groups, including straight or branched chain C2-30 alkylene groups; Cs-s cycloalkylene groups; CMO fused or linked aryl groups, optionally incorporating from one to eight heteroatoms in one or more of the aromatic rings, including but not limited to phenylene, naphthylene, furylene, benzofurylene, pyridylene, purinylene, piperidylene, and the like; peptides and/or peptidyl mimetics of one to 20 amino acids or amino acid analogs in length, preferably wherein one or more of the amino acids is cysteine, for its thiol function, and a terminal serine or threonine, oxidized to an aldehyde, reacted with hydrazine and reduced to form the hydrazinyl substituent.
- Combinations of the foregoing structural components also can be used to construct the group, Q.
- the foregoing components can bear one or more substituents that do not interfere with the conjugation reactions, including but not limited to halogens, hydroxyl or alkoxyl groups, including protected hydroxyl groups, carboxyls and carboxylic ester groups, alkyl groups, cyano groups, primary, secondary and tertiary amino groups, including protected amino groups, amides, urethanes, ureas, nitro groups, and the like.
- the peptides disclosed herein are examples of peptides suitable for this purpose.
- the structures represented by Q can be readily synthesized by conventional methods. Many aliphatic and aromatic single, multiple or fused ring compounds are commercially available, with substituents suitable or adaptable for further elaboration. Ring compounds bearing one or two carbonyl compounds, e.g., aldehydes, ketones, carboxylic acids or esters, and amides can be found in reagent catalogues. Other substituents such as hydroxyls, haloalkyl groups, hydroxyls, amines, cyano groups, isocyanates, and the like can be used as such or transformed into handles for further elaboration.
- Small linker synthons such as glyoxyl esters, sugar derivatives, alpha-halo acyl compounds, e.g., alpha-bromoacetyl esters, acid chlorides, are useful for introducing free or masked carbonyl groups for reaction with hydrazine, followed by reduction with, e.g., sodium cyanoborohydride, to produce the hydrazine function of HS-Q- NHNH2 or for introducing alkyl halide groups for reaction with sodium sulfide or hydrosulfide to produce the thiol function of HS-Q-NHNH2.
- Peptides can introduce the thiol function by incorporation of cysteines.
- nascent aldehyde and ketone residues into targeting vectors using standard methods of molecular biology may also be used.
- a polypeptide can be constructed with an N-terminal serine or threonine moiety, which can then be specifically oxidized to generate N-terminal carbonyl groups.
- Such derivatives then constitute specific chemical 'handles' for the attachment of tMol-containing haptens.
- Preferred ligand-bearing peptides such as IMP 155, are advantageous because they contain a hydrazine, several hydrophilic D-amino acids and a metal binding ligand.
- the hydrazine is used to form a hydrazone linkage to aldehydes or ketones on the oxidized portion of the carbohydrate groups on a glycosylated antibody or antibody fragment.
- the ligand forms a stable Tc(V) oxo complex with the diagnostic imaging isotope Tc-99m.
- hydrophilic amino acids make the peptide sufficiently hydrophilic so that disulfide interchange or mixed disulfide formation is minimized during the conjugation of the free thiol-containing peptide to the antibody.
- the hydrophilic nature of the peptide should keep the conjugated peptide at the surface of the protein where it can react with the Tc-99m when it is added.
- D- amino acids are used to minimize metabolism of the metal-complexed peptide after injection. This is done so that, in the event the protein is degraded, the hydrophilic metal-containing peptide will not be metabolized and, because it is hydrophilic, any labeled peptide which escapes the cell will be rapidly renally excreted.
- a 500 mL Parr bottle was charged with 18.00 g (1.96x10 " ' mol) glyoxylic acid monohydrate, 25.84 g (1.95x10 " ' mol) t-butylcarbazate, 0.72 g 10 % palladium on carbon, 100 mL methanol, 50 mL dioxane and then placed on a Parr hydrogenation apparatus under an atmosphere of hydrogen (50 PSI). The reaction mixture was shaken at room temperature and the hydrogen pressure was adjusted several times to maintain the pressure at 50 PSI. A precipitate formed as the reaction proceeded.
- Peptide IMP 155 (H 2 NHN-CH2-CO-D-Asp-D-Lys(TscG-Cys-)-D-Asp-D-Lys-NH 2 ) was synthesized by Fmoc based solid phase synthesis on an Advanced ChemTech 348 multiple peptide synthesizer. The peptides were synthesized using Rink amide resin on a 0.05 mmol/well scale (48 well synthesis block). The repetitive coupling process is as follows: Fmoc Cleavage.
- the resin is vortex mixed for 4 min with 1.5 mL of 25 % piperidine in DMF.
- the block is then drained and the resin is vortex mixed for 15 min with 1.5 mL of 25 % piperidine in DMF.
- the resin is washed with 1.5 mL portions of NMP, isopropanol, NMP, isopropanol, and 4 NMP washes.
- the resin is vortex mixed with each of the washing solutions for at least one miute before the liquid is drained.
- the protected amino acid is dissolved in NMP (0.5 M) which contains 0.5 M HOBt.
- NMP 300 ⁇ L
- diisopropylcarbodiimide solution 600 ⁇ L, 0.5 M in NMP, 6 equivalents
- 600 ⁇ L of the amino acid solution 6 equivalents relative to the resin.
- the mixture is vortex mixed for 1 hr at room temperature and then washed as described above. The entire process is repeated for the addition of each amino acid.
- the carbohydrate portion of murine LL2 F(ab')2 (4 mg/mL) was oxidized with 15 mM NaTC at 0°C for 1 hr in the dark at pH 5.3.
- a glycerol/water solution (1:1) was then added (50 ⁇ L for 2.5 mL antibody solution) and the solution was incubated for 15 mm at 0°C in the dark.
- the oxidized antibody was then purified through a Sephadex G 50-80 spin column in pH 5.3 acetate buffered saline (ABS, 50 mM acetate).
- the peptide, IMP 155 was conjugated to the periodate oxidized LL2 F(ab')2 fragment (4 mg/mL, LL2 F(ab')2) in a molar ratio of 100: 1 peptide/antibody at pH 5.3 in acetate buffered saline for 2 hr at room temperature by adding the antibody solution to the appropriate amount of peptide in a septum sealed evacuated vial.
- the conjugated antibody fragment was purified twice through Sephadex G 50-80 gel spin columns (pH 5.3 ABS) to remove the unbound excess peptide at the end of the conjugation time. Analysis of the conjugate by MALDI mass spectroscopy showed that an average of four peptides were conjugated per antibody fragment.
- a buffer solution was prepared by making a solution which contained 200 mM ⁇ -D-glucoheptonic acid sodium salt and 21 mM sodium acetate at pH 5.3. This solution was diluted in half with water to give a 100/10.5 mM (glucoheptonate/acetate) solution used for the kit formulation.
- SnCb A bulk SnCh solution was prepared by dissolving tin metal in 6 M HC1 at a concentration of 200 mg/mL. A 2 mg/mL solution of stannous was prepared by diluting an aliquot of the 200 mg/mL stannous solution 100 fold in the glucoheptonate buffer.
- Sucrose Solution
- a I M sucrose solution was prepared for addition to the kits.
- the purified conjugate solution was passed through a pH 7.3, 0.1 M phosphate buffered Sephadex G 50-80 gel spin column (A mg/mL LL2 F(ab')2.
- the conjugate, 1 mg, (250 ⁇ L) was mixed with 12.5 ⁇ g SnCk (6.25 ⁇ L), 446 ⁇ g ⁇ -D-glucoheptonic acid sodium salt, (11.7 ⁇ L of glucoheptonate buffer + the buffer in the stannous solution), and 19 mg sucrose (55.6 ⁇ L) in a total volume of approximately 0.3 mL.
- the mixture was frozen immediately, lyophilized and septum sealed under vacuum.
- kits were dissolved in 0.4 mL saline and the solution was allowed to stand for 5 min before the addition of Na99m-TcO4 in saline (0.4 mL).
- the kit solution was incubated for 30 mm at room temperature before use.
- HPLC analysis on a Bio-Sil size exclusion column indicated that 96 % of the activity was attached to the protein and 4 % of the activity was present as small molecular weight material.
- the LL2-F(ab')2 conjugate was labeled at room temperature by exchange with Tc-99m glucoheptonate as follows:
- a GlucoscanTM (DuPont) glucoheptonate labeling kit was labeled with 30 mCi Tc-99m in 2 mL.
- the Tc-99m glucoheptonate 0.6 mL was added to the IMP 155-LL2-F(ab')2 conjugate kit vial (containing 1 mg conjugate, and sucrose) prepared as described in Example 1.
- the vial was incubated at room temperature for 30 min.
- the labeled material was then purified through two successive 3-mL Sephadex G 50-80 spin columns (pH 7.3, 0.1 M PBS). The product was diluted with saline to 5 mCi/mL in an empty sterile vial.
- mice Nine BALB/c mice were injected with 100 ⁇ L (500 ⁇ Ci) of the purified Tc-99m-LL2 F(ab')2 conjugate. The animals were anesthetized and sacrificed, 3 animals per time point, at 1 hr, 4 hr, and 24 hr. Serum for analysis was also collected at 1 hr, 4 hr and 24 hr. The serum samples were frozen after isolation and thawed just before HPLC analysis on the size exclusion HPLC column (Bio-Sil SEC 250, Gel-filtration HPLC Column, 300mm x 7.8 mm). The following tissues and organs were collected and counted: Blood, Liver, Kidneys, Spleen, Lungs, Stomach, Small Intestine, Large Intestine, Muscle, Urine.
- the antibody fragment LL2-F(ab')2, 23.3 ⁇ L (5.15 mg/mL) was mixed with 50 ⁇ L 0.5 M, pH 7.5, phosphate buffer and added to a vial containing 2.5 mCi 1-125.
- a solution of chloramine-T, 12 ⁇ L (0.0034 g in 2 mL PBS) was added and the reaction was allowed to proceed for 3 min at room temperature before it was quenched with 20 ⁇ L (0.0254g in 10 mL) of a solution of sodium metabisulfite.
- the 1-125 LL2-F(ab')2 was in a solution which contained 1.6 mCi in " 3 mL.
- the I- 125 LL2-F(ab')2 and the Tc-99m LL2 F(ab') 2 were mixed in a ratio of 5 ⁇ Ci 1-125 to 25 ⁇ Ci Tc-99m.
- the final mixed solution contained 220 ⁇ Ci 1-125 LL2-F(ab')2and 1.04 mCi Tc-99m IMP 155-LL2-F(ab')2in 2.0 mL of solution.
- a group of five Ramos tumor-bearing nude mice with similar sized tumors were used per time-point. Each animal was injected with 50 ⁇ L of the premixed solution (5 ⁇ Ci 1-125 and 25 ⁇ Ci Tc-99m). The animals were anesthetized and sacrificed by cervical dislocation at 1 hr, 4 hr, and 24 hr. The following tissues and organs were collected and counted: Tumor, Blood, Muscle, Liver, Kidneys, Spleen, Stomach.
- LL2-Fab' kit (LymphoscanTM - Immunomedics, Inc., Morris Plains, NJ) was radio- labeled at room temperature with 31 mCi Tc-99m in 1 mL added to the lyophilized kit. A 50- ⁇ L aliquot (1.55 mCi) was removed and diluted with saline to 3 mL (500 ⁇ Ci/mL).
- a group of 5 Ramos tumor bearing nude mice with similar sized tumors were used per time point. Each animal was injected with 50 ⁇ L of the labeled antibody fragment (25 ⁇ Ci Tc-99m). The animals were anesthetized and sacrificed by cervical dislocation at 4 hr and 24 hr. The following tissues and organs were collected and counted: Tumor, Blood, Muscle, Liver, Kidneys, Spleen, Stomach. The ratio of tumor-to-normal organ is given for each organ.
- the stability of the Tc-99m labeled conjugates was screened by labeling the conjugates by exchange with Tc-99m-glucoheptonate followed by purification of the labeled protein through two successive Sephadex G 50-80 gel columns which removed all of the unbound small molecular weight Tc-99m species.
- the purified material was then analyzed by ITLC (0.1 M citrate, pH 5), size exclusion HPLC and reverse phase HPLC.
- ITLC 0.1 M citrate, pH 5
- the stability of the different conjugates was assessed by incubation in the gel filtration buffer or labeling buffer at room temperature over 24 hr, by challenge with 1 mM cysteine at 37°C over 24 hr and by incubation in serum at 37°C over 24 hr.
- ITLC strips were spotted with an aliquot (0.2 ⁇ Ci) of the labeled solution and eluted with pH 5, 0.1 M citrate buffer.
- the labeled protein remained at the origin while 99m-TcO4 " , and Tc-99m-glucoheptonate were eluted up the strip.
- Reverse Phase HPLC was performed on a Waters Radial-Pak, C-18, Nova-Pak (4 ⁇ , 100 x 8 ram) column.
- the column was eluted with a gradient using two buffers (Buffer A, 0.1 % aqueous TFA, Buffer B 90% acetonitrile, 10 % water, 0.1 % TFA).
- the gradient was as follows: Flow Rate 3 mL/min 100 % Buffer A to 100 % Buffer B over 10 min, flow rate 5 mL/min for 5 min.
- the reverse-phase HPLC method was useful for identifying the nature of the Tc-99m labeled small molecular weight species. The stable, Tc-99m labeled proteins did not elute well on reverse-phase HPLC.
- the crude labeled protein prepared analogously to labeled IMP-155-LL2-F(ab')2, was purified on a Sephadex G 50-80 gel column in pH 7.3, 0.1 M phosphate buffer. An aliquot was removed and diluted five-fold in saline. The saline solution was incubated for 1 hr at 37°C. Size-exclusion HPLC analysis showed that approximately 5 % of the activity was dissociated from the protein after 1 hr.
- Tc-99m-IMP 126-LL2-F(ab')2 Incubation of Tc-99m-IMP 126-LL2-F(ab')2 in pH 7.3, 0.1 M phospate buffer containing 1 mM cysteine at 37°C for 1.5 hr caused 31 % of the labeled protein to be reduced to the Tc-99m IMP 126-Fab' fragment. There was 56 % of the Tc-99m-IMP 126-LL2-F(ab') 2 left intact and 12 % of the activity was converted to low molecular weight species such as Tc- 99m cysteine.
- Tc-99m-IMP 126-LL2-F(ab)2 Incubation of Tc-99m-IMP 126-LL2-F(ab)2 diluted ten-fold in fresh human serum at 3 7°C for 21 hr caused 34 % of the activity to be lost as low molecular weight species. There were three Tc-99m labeled protein species all about the same percentage (20 %) of the activity. The peaks appeared to be due to aggregate, Tc-99m-IMP 126-LL2-F(ab')2, and Tc- 99m-IMP 126-LL2 Fab' Tc-99m-IMP 140-LL2-F(ab') 2
- the labeled peptide prepared analogously to labeled IMP-155-LL2-F(ab')2, was purified and stored in pH 7.3, 0.1 M phosphate buffer over night. Size-exclusion HPLC analysis showed that approximately 30 % of the activity was dissociated from the protein after 24 hr. Reverse phase HPLC analysis indicated that the bulk of the Tc-99m labeled small molecular weight species was present as the Tc-99m labeled peptide that had hydrolyzed off the protein. The conclusion was that the acylhydrazone linkage to the antibody was unstable.
- the labeled peptide was purified and stored in pH 7.3, 0.1 M phosphate buffer over night. Size exclusion HPLC analysis showed less than 5 % decomposition to small molecular weight species after incubation for 24 hr at room temperature.
- Tc-99m-IMP 155-LL2-F(ab') 2 Incubation of Tc-99m-IMP 155-LL2-F(ab') 2 in pH 7 3, 0.1 M phospate buffer containing 1 mM cysteine at 37°C caused the bulk of the labeled protein (61.5 %) to be reduced to the Tc-99m IMP 155-Fab' fragment. There was 8 % of the Tc-99m-IMP 155-LL2 F(ab')2left intact and 30 % of the activity was converted to low molecular weight species such as Tc-99m cysteine.
- Tc-99m-IMP 155-LL2-F(ab')2 Incubation of Tc-99m-IMP 155-LL2-F(ab')2 diluted ten-fold in fresh human serum at 37°C for 21 hr caused 10 % of the activity to be lost as low molecular weight species. There were three Tc-99m labeled protein species. The peaks appeared to be due to aggregate (38 %), Tc-99m-IMP 155-LL2-F(ab') 2 (42 ), and Tc-99m-IMP 155-LL2-Fab' (9 %).
- the aggregate formation may be an artifact due to disulfide formation caused by exposure to oxygen.
- the conjugations of IMP 155 were carried out as described above and the antibody concentration was measured by the UV absorbance of an aliquot at 280 nm.
- the thiol content was measured using an Ellman's assay and the thiol concentration in the aliquot was correlated to the antibody content as measured by UV to obtain the thiol loading on the antibody. It was later determined that the UV measurement method was inaccurate because the peptide has a significant absorbance at the wave length used to determine the protein content thus giving an artificially high number for the protein content.
- the analysis was then switched to matrix assisted laser desorption ionization (MALDI) mass spectrometry to unambiguously determine the number of peptides attached to the protein.
- MALDI matrix assisted laser desorption ionization
- a conjugation control experiment was carried to determine if the free thiol containing peptide was reacting with disulfides on the antibody to generate protein thiols or if the peptide was attached to the antibody by some means other than to the oxidized carbohydrate.
- unoxidized LL2-F(ab')2 was treated with 50 fold excess IMP 155 at the same time as a lot of periodate oxidized LL2 F(ab')2.
- the periodate oxidized LL2-F(ab')2 formed a conjugate which contained 2.3 thiols/ab as determined by UV (4.3 peptides/ab by MALDI) and the unoxidized LL2-F(ab')2 contained 0.2 thiols/ab as determined by UV.
- This experiment demonstrated that periodate oxidation was necessary in order to add the peptide to the antibody and that the thiols present were on the peptide and not a product of disulfide interchange.
- a control experiment was carried out in which a batch of LL2-F(ab')2 was treated with periodate and conjugated as described above with 100 fold excess of the peptide IMP 140 except that 10 mg/mL sodium cyanoborohydride was added after two hours of conjugation and the conjugation was continued for two hours more before purification.
- the control batch of the LL2 -F(ab')2 was treated the same except there was no periodate present in the oxidation control.
- the two antibody preparations were labeled by exchange with Tc-99m- glucoheptonate.
- IMP 171 a peptide which contains two metal binding ligands, was conjugated to LL2-F(ab')2 using the same process as described for IMP-155. It was necessary to use a peptide to antibody ratio of 50:1 or less with this peptide because the increased number of thiols could cause reduction of the LL2-F(ab')2 to the LL2-Fab' .
- This reaction did produce a conjugate, IMP 171- LL2-F(ab')2 with twice the number of thiols (5.3 thiols/ab) as compared to the IMP 155-LL2-F(ab')2 conjugation (2.3 thiols/ab) carried out on the same batch of oxidized antibody.
- Rhenium labeling using a pre-reduction procedure for the Re-188 was performed using a pre-reduction procedure for the Re-188.
- conjugates may be labeled with rhenium isotopes (primarily Re- 186 and Re- 188), which are then useful for radioimmunotherapy. Because reduction of perrhenate requires more stannous ion (typically above 200 ug/mL final concentration) than is needed for the reduction of technetium, extra care needs to be taken to ensure that the higher levels of stannous ion do not reduce sensitive disulfide bonds such as those present in the hinge region of F(ab')2 fragments.
- rhenium isotopes primarily Re- 186 and Re- 188
- the peptide, IMP 162 (0.0012 g) was dissolved in 30 mL of an aqueous solution containing 10 % HPCD, 200 mM glucoheptonate, 14 mM sodium acetate, and 12 mM ascorbic acid at pH 5.29.
- a stannous solution was prepared by mixing 0.2 mL of 200 mg/mL SnCk in 6 M HC1 with 3.8 mL of the HPCD glucoheptonate solution.
- a 0.2 mL aliquot of the stannous /HPCD solution was added to the peptide solution and the solution was then filtered through a 0.22 ⁇ m Millex GV filter in 1.5 mL aliquots into lyophilization vials. The vials were then frozen, lyophilized and sealed under vacuum.
- the lyophilized kit was reconstituted with 20 mCi Wm TcO4 " in 1.5 mL saline and incubated at room temperature for 10 min and then heated in a boiling water bath for 15 min. Labeling is complete and quantitative.
- An analog of octreotide having an appended N-terminal serine residue attached to the original octapeptide is oxidized using sodium m-periodate to generate an N-terminal aldehyde.
- This intermediate is reacted with an amount of IMP-155 peptide sufficient to convert all aldehyde groups present on the serine-octreotide to N-terminal hydrazones.
- the hydrazones are reduced to alkyl hydrazines using sodium cynanoborohydride, and the intermediate thiol- appended IMP-155-hydrazinyl-octreotide is purified and coupled with the anti-cancer drug calicheamicin via thiol exchange with the trisulfide group of the latter.
- the product calicheamicin-octreotide is comprised of a disulfide-IMP 155-peptidyl-hydrazine linker.
- a solution of a human (scFv)2 fragment bearing an N-terminal serine amino-acid, at 3 mg/mL, is treated with 10 mM sodium periodate for 2 h in the dark at 4°C.
- Glycerol is added to a 20 mM final concentration, to destroy excess periodate, and the reaction stirred for a further 20 minutes.
- the N-terminal oxidized (scFv is purified from low molecular weight contaminants on a column of G-10-Sephadex equilibrated and run in argon-degassed 0.1 M sodium acetate, pH 5.5, containing ImM EDTA. The product is concentrated to 5 mg/mL prior to further reaction.
- the N-terminal aldehyde-(scFv)2 intermediate, obtained as in a), is treated with a freshly-prepared DMSO solution of p-(2-thioethyl)-phenylhydrazine (TEPH) at a molar excess of 20:1, and the reaction is stirred for 2 h at 4°C.
- TEPH-(scFv)2 containing a hydrazone bond is obtained by purification on a column of G-10-Sephadex equilibrated and run in argon-degassed 0.1 M sodium acetate, pH 6.5, containing ImM EDTA.
- the phenylhydrazone is reduced to a phenylhydrazine linkage by a 2 h reaction in the presence of 10 mM sodium borohydride, after pH adjustment of the initial reaction mixture from 5.5 to 7.0 with a small amount of sodium carbonate.
- the product TEPH-(scFv)2 is analyzed by SE-HPLC on a Bio-Sil GF-125 column equilibrated in 0.5 M sodium phosphate, pH 6.5, to confirm the lack of breakdown into scFv monomers.
- MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester
- the mixture is extracted with 3 x 20 mL of a substantially water-immiscible organic solvent such as ethyl acetate, and the combined organic extracts washed with 3 x 20 mL 0.1 M sodium bicarbonate, pH 8.0.
- the organic solution is dried over anhydrous sodium sulfate, filtered, and evaporated to obtain the maleimido-doxorubicin .
- TEPH-(scFv)2 in argon-degassed 0.1 M sodium acetate, pH 6.5, containing ImM EDTA, at 4°C, is made 15% in DMSO, and treated with a 2 x molar excess (to thiol content) of maleimido-doxorubicin in DMSO, added in one portion with rapid stirring. Stirring is continued for 1 h at 4°C, and the doxorubicin-(scFv)2 is purified by column chromatography on a Sephadex G-10 gel column equilibrated in 0.2 M sodium phosphate buffered 0.9% sodium chloride, pH 7.5.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Optics & Photonics (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Semiconductor Memories (AREA)
- Crystals, And After-Treatments Of Crystals (AREA)
- Dicing (AREA)
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU64244/99A AU755783B2 (en) | 1998-10-13 | 1999-10-13 | Site-specific labeling of disulfide-containing targeting vectors |
AT99951901T ATE293996T1 (en) | 1998-10-13 | 1999-10-13 | LOCATION-SPECIFIC LABELING OF DISULFIDE-CONTAINING TARGETED VECTORS |
JP2000575545A JP4680387B2 (en) | 1998-10-13 | 1999-10-13 | Site-specific labeling of disulfide-containing target vectors |
EP99951901A EP1121153B1 (en) | 1998-10-13 | 1999-10-13 | Site-specific labeling of disulfide-containing targeting vectors |
CA002346170A CA2346170C (en) | 1998-10-13 | 1999-10-13 | Site-specific labeling of disulfide-containing targeting vectors |
DE69925009T DE69925009T2 (en) | 1998-10-13 | 1999-10-13 | LOCATION-SPECIFIC MARKING OF DISULFIDE-CONTAINING TARGET-ORIENTED VECTORS |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10390498P | 1998-10-13 | 1998-10-13 | |
US60/103,904 | 1998-10-13 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2000021573A1 true WO2000021573A1 (en) | 2000-04-20 |
Family
ID=22297637
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1999/023614 WO2000021573A1 (en) | 1998-10-13 | 1999-10-13 | Site-specific labeling of disulfide-containing targeting vectors |
Country Status (8)
Country | Link |
---|---|
EP (1) | EP1121153B1 (en) |
JP (1) | JP4680387B2 (en) |
AT (1) | ATE293996T1 (en) |
AU (1) | AU755783B2 (en) |
CA (1) | CA2346170C (en) |
DE (1) | DE69925009T2 (en) |
ES (1) | ES2242426T3 (en) |
WO (1) | WO2000021573A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2287149A1 (en) * | 2009-08-20 | 2011-02-23 | Max-Delbrück-Centrum Für Molekulare Medizin | Enhancers of protein degradation |
EP3015116A4 (en) * | 2013-06-24 | 2017-03-08 | ABLBio | Antibody-drug conjugate having improved stability and use thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996040289A1 (en) * | 1995-06-07 | 1996-12-19 | Immunomedics, Inc. | Thiolation of peptides for radionuclide-based radiodetection and radiotherapy |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IL113610A0 (en) * | 1994-06-03 | 1995-08-31 | Immunomedics Inc | A method of radiolabeling a protein, radiolabeled proteins prepared thereby and diagnostic kits containing the same |
-
1999
- 1999-10-13 AT AT99951901T patent/ATE293996T1/en not_active IP Right Cessation
- 1999-10-13 JP JP2000575545A patent/JP4680387B2/en not_active Expired - Fee Related
- 1999-10-13 DE DE69925009T patent/DE69925009T2/en not_active Expired - Lifetime
- 1999-10-13 EP EP99951901A patent/EP1121153B1/en not_active Expired - Lifetime
- 1999-10-13 ES ES99951901T patent/ES2242426T3/en not_active Expired - Lifetime
- 1999-10-13 CA CA002346170A patent/CA2346170C/en not_active Expired - Fee Related
- 1999-10-13 AU AU64244/99A patent/AU755783B2/en not_active Ceased
- 1999-10-13 WO PCT/US1999/023614 patent/WO2000021573A1/en active IP Right Grant
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996040289A1 (en) * | 1995-06-07 | 1996-12-19 | Immunomedics, Inc. | Thiolation of peptides for radionuclide-based radiodetection and radiotherapy |
Non-Patent Citations (2)
Title |
---|
BRIDGER ET AL: "comparison of cleavable and noncleavable hydrazinopyridine linkers for the 99mTc labeling of Fab' monoclonal antibody fragments", BIOCONJUGATE CHEMISTRY, vol. 7, no. 2, 1996, pages 255 - 264, XP000558423 * |
ZARA ET AL: "a carbohydrate-directed heterobifunctional crosss-linking reagent for the synthesis of immunoconjugates", ANAL. BIOCHEM., vol. 194, 1991, pages 156 - 162, XP000872283 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2287149A1 (en) * | 2009-08-20 | 2011-02-23 | Max-Delbrück-Centrum Für Molekulare Medizin | Enhancers of protein degradation |
WO2011020883A1 (en) * | 2009-08-20 | 2011-02-24 | Max-Delbrück-Centrum für Molekulare Medizin | Enhancers of protein degradation |
US9512066B2 (en) | 2009-08-20 | 2016-12-06 | Max-Delbruck-Centrum Fur Molekulare Medizin | Enhancers of protein degradation |
EP3015116A4 (en) * | 2013-06-24 | 2017-03-08 | ABLBio | Antibody-drug conjugate having improved stability and use thereof |
US10071170B2 (en) | 2013-06-24 | 2018-09-11 | Ablbio | Antibody-drug conjugate having improved stability and use thereof |
Also Published As
Publication number | Publication date |
---|---|
JP2002527409A (en) | 2002-08-27 |
CA2346170C (en) | 2010-01-12 |
DE69925009T2 (en) | 2006-03-02 |
CA2346170A1 (en) | 2000-04-20 |
EP1121153B1 (en) | 2005-04-27 |
ATE293996T1 (en) | 2005-05-15 |
ES2242426T3 (en) | 2005-11-01 |
DE69925009D1 (en) | 2005-06-02 |
JP4680387B2 (en) | 2011-05-11 |
EP1121153A1 (en) | 2001-08-08 |
AU6424499A (en) | 2000-05-01 |
AU755783B2 (en) | 2002-12-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6576746B2 (en) | Site-specific labeling of disulfide-containing targeting vectors | |
US5057301A (en) | Modified cellular substrates used as linkers for increased cell retention of diagnostic and therapeutic agents | |
AU597903B2 (en) | Antibody complexes of hapten-modified diagnostic or therapeutic agents | |
AU619738B2 (en) | Metal-radionuclide-labeled proteins and glycoproteins for diagnosis and therapy | |
US6010680A (en) | Thiolation of proteins for radionuclide-based radioimmunodetection and radioimmunotherapy | |
DE69434086T2 (en) | Preparation and Use of Immunoconjugates Containing a VL Chain Glycosylated at the Asn in Position 18 | |
EP0294294B1 (en) | Amine derivatives of anthracycline antibiotics | |
US5807879A (en) | Biotinidase-resistant biotinylated compound and methods of use thereof | |
EP0385601B1 (en) | Cross-linked antibodies and processes for their preparation | |
JPH0470320B2 (en) | ||
JP2001514236A (en) | Stable radioactive iodine conjugates and their synthesis | |
US5354554A (en) | Crosslinked antibodies and processes for their preparation | |
NO881077L (en) | Chemo-RADIO IMMUNO conjugates. | |
CN109963620A (en) | Cell-targeting conjugate | |
CA2503647C (en) | Method for the selective and quantitative functionalization of immunoglobulin fab fragments, conjugate compounds obtained with the same and compositions thereof | |
JPH06507918A (en) | In vivo binding versus pre-targeting | |
US5795560A (en) | Targeting protein-diagnostic/therapeutic agent conjugates having Schiff base linkages | |
EP1121153B1 (en) | Site-specific labeling of disulfide-containing targeting vectors | |
JPH05500953A (en) | Methods for reducing non-target retention of immune conjugates and their metabolites | |
JPH06505795A (en) | chelating agent | |
KR100238558B1 (en) | Technetium-99m labeling of proteins | |
JPH06505480A (en) | stabilized antibody fragment | |
JPH09507488A (en) | Novel polyaminocarboxylate chelating agent | |
JPH0692869A (en) | Polymerizing carrier for releasing agent covalent |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
ENP | Entry into the national phase |
Ref country code: AU Ref document number: 1999 64244 Kind code of ref document: A Format of ref document f/p: F |
|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
ENP | Entry into the national phase |
Ref document number: 2346170 Country of ref document: CA Ref country code: CA Ref document number: 2346170 Kind code of ref document: A Format of ref document f/p: F |
|
ENP | Entry into the national phase |
Ref country code: JP Ref document number: 2000 575545 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 64244/99 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1999951901 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1999951901 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWG | Wipo information: grant in national office |
Ref document number: 64244/99 Country of ref document: AU |
|
WWG | Wipo information: grant in national office |
Ref document number: 1999951901 Country of ref document: EP |