AU678474B2 - Kit and method for pretargeting - Google Patents

Description

Kit and method for pretargeting.

The invention relates to the fields of immunotherapy and immunodiagnostics, more particular to the field of pretargeting. The invention describes a kit and method for the delivery of pharmaceutical compounds or diagnostics to target cells in the body. These kits comprise an antibody or a fragment thereof, or a derivative of said antibody or fragment, which antibody or fragment recognize a target associated antigen, and a ligand-effector moiety conjugate to comprising a ligand specific for said antibody, and an effector moiety linked to the ligand.

S

The in vivo use of antibodies for the selective delivery of diagnostic or therapeutic agents to target tissues is known in the art.

L Since the advent of methodology for the production of monoclonal antibodies, antibodies of predefined specificities (K6hler and Milstein, Nature 256, 495-497, 1975), numerous studies aiming at specific targeting of radionuclides, plant or bacterial toxins, biological Qo agents and cytostatic agents (drugs) to target cells or tissues with the help of said antibodies have been performed, as reviewed by Bator and Reading (in: Therapeutic Monoclonal Antibodies, eds: C.A.K. Borrebaeck and J.W. Larrick, Stockton Press, pp. 35-56, 1990 In in I vitro studies the great potential of these immunoconjugates has been amply demonstrated. Application of such immunoconjugates in vivo in tumor bearing animals as well as in cancer patients has met with much less success. A major problem has been the inability to attain 3o a sufficient difference in uptake of the immunoconjugates between the target tissues as compared to the non-target tissues. As a result it has been difficult for instance to effect an increase in therapeutic index of clinically used anti-cancer drugs by administration in the form of a drug- 3J immunoconjugate. Similarly, due to the lack of operational selectivity of therapeutic radio-immunoconjugates when administered in vivo, normal tissues are unduly exposed to destructive radiation. The therapeutic and diagnostic limitations of immunoconjugates, apart from a number of factors adversely affecting antibody delivery, are partly due to changes in antibody affinity, specificity and immunogenicity upon chemically linking a diagnostic or therapeutic agent to an antibody.

In order to overcome part of the problems mentioned, lt newer approaches to delivery of diagnostic or therapeutic agents have recently been suggested, that are characterised in that the localisation phase of the S antibody component is separated in time from the phase in which the diagnostic or therapeutic agent is administered.

I In these so-called pretargeting schemes sufficient time is permitted for accumulation of non-labelled antibody at the target site and for elimination of the antibody from other non-target tissues. The latter process, the clearing phase, may be aided by, for example, plasmaphoresis or use of a second antibody reactive with the first. At the time S of optimal antibody accumulation at the target site as compared to the non-target tissue, the diagnostic or therapeutic agent to be recognised by the antibody already localised, is administered.

S. f The pretargeting approaches which have been so far described in literature require that the antibody must have a dual specificity: a specific site that allows binding to a target tissue associated antigen and a specificity that enables specifically binding of the 3c diagnostic or therapeutic agent.

Early on, bifunctional antibodies were constructed by chemically linking univalent fragments of antibodies with different specificities. Raso and Griffin (Cancer Res. 41, 207, 1981) for instance constructed a bifunctional J~ antibody that was able to bind human IgG and to ricin, a toxic lectin. Later on, technology has been developed to produce hybrid hybridomas that produce monoclonal antibodies of dual specificity (Reading, US Patent 4,474,893). A biologically produced bifunctional antibody recognising both carcinoembryonic antigen (CEA) and vinca alkaloids was described by Corvalen et al. (Cancer Immunol. Immunother. 24, 133, 1987). Studies on xenografts in nude mice revealed the combined use of the bifunctional antibody and vinblastin, given 7-11 days followingadministration of the antibody, to be more effective than drug alone in suppressing tumor growth.

p Recently pretargeting techniques have been proposed in which the specific antibody recognises a haptenic group, i.e. an EDTA derivative chelating indium (Stickney et al., Cancer Res. 51, 6650, 1991).

In a closely related domain of the prior art 5 monovalent, target site selective antibodies have been endowed with a second specificity by linkage, through either chemical or biological methods, to a member of a specifically interacting binding pair. The high affinity system avidin-biotin has been adopted in such an approach.

o (European Patent No.:0 251 494, to the Board of Trustees of the Leland Stanford University). The pretargeting system used in this patent application employs an antibody that has been linked to the protein avidin or streptavidin and a biotinylated metal chelate.

S A major problem with the bifunctional antibodies mentioned above, and with most other pretargeting systems S described in the literature, is that the antibodies are of murine origin. These, like all other foreign proteins, are highly immunogenic in man. The phenomenon of HAMA, human Jo anti mouse antibodies, is well known in the field and severely limits the use of mouse derived antibodies in diagnostic and especially in therapeutic applications in human beings. A single application of a murine antibody is usually sufficient to mount an immune response that will 3S prevent subsequent applications to be effective. This immunogenicity problem will even be aggrevated when antibody-conjugates are made with non-human proteins, such as avidin.

Another problem which is inherent to the pretargeting schemes described above is that the immunoconjugates in most cases have to be made chemically, which introduces not only possible loss of specificity, but also loss in yield of the conjugate due to incomplete reactions and losses during purification of the conjugate.

According to a first embodiment of this invention, there is provided a method for pretargeting comprising administration of an antibody or a fragment thereof or a derivative of said antibody or fragment, which antibody or fragment recognizes a target associated antigen, followed by administration of a ligand-effector moiety conjugate comprising a ligand specific for said antibody, and an effector moiety linked to the ligand, characterized in that: said antibody is a mono specific antibody with multiple binding sites; and said ligand is derived from the target associated antigen or a crossreactive analogue thereof recognisable by said antibody.

According to a second embodiment of this invention, there is provided a ligandeffector moiety conjugate for therapeutical use comprising a ligand derived from the tumor associated antigen reactive with human monoclonal antibody 16-88, or a crossreactive analogue thereof, and an effector moiety linked to said ligand.

According to a third embodiment of this invention, there is provided a 2 pharmaceutical preparation comprising the ligand-effector moiety conjugate of the second embodiment and a pharmaceutically acceptable carrier.

According to a fourth embodiment of this invention, there is provided use of the ligand effector moiety conjugate of the second embodiment for the manufacture of a pharmaceutical preparation for anti-cancer treatment.

25 Using a monospecific antibody gives the advantage that there is no need for .o.

chemical conjugation methods.

The present invention proposes to administer to a patient a mono specific antibody S in a dose, or in successive doses, sufficient to maximise target site accumulation of the antibody. At some optimal time later, when most of the non-bound antibody has been 30 cleared, a diagnostic or therapeutic effector molecule carried by the target associated •antigen or a cross-reactive analogue thereof, is administered. As target associated antigen every antigen that is specific for a population of target cells can be used. Examples of antigens which are specifically useful are: antigens related to cancer cells, like CEA (Carcino-embryonic antigen), AFP (alphafoetoprotein), FHAP (fast homoarginine-sensitive alkaline phosphatase), p97 (melanome specific), and EL-1 (elongation factor antigens related to viruses or bacteria, like HIV gp 120, HIV gp 160, Hepatitis B core 6 antigen and Outer Membrane Protein of Borrelia burgdorferi, autoimmune related antigens, like small nuclear RNA's and Mycobacterium 65 kD HS-protein.

Preferably the ligand derived from the target associated antigen is the cognate epitope, which is (o recognised by the antibody. This epitope is a small, lowmolecular weight part of the antigen to which the antibody or fragment of antibody specifically binds. If the antigen is a glycoprotein the epitope can be formed by a small sequence of amino acids, by the carbohydrate moieties of S: the protein or by a combination of those two. In the case of a sequence of amino acids this can be formed by a sequence which is linearly present in the antigen (linear epitope), but it is also possible that due to the threedimensional conformation of the protein the epitope has l been formed by a sequence of amino acids which is not linearly present in the antigen, but which, according to the folding of the protein is presented as an adjacent sequence (conformational epitope).

To be able to bind more or more efficiently to the antibody another embodiment of the invention is to present the epitope as a multimer. Multimerization can be achieved by common chemical techniques, in case of peptide epitopes it is also possible to produce a multimer by recombinant DNA techniques. In this last way the nucleotides coding Jc for the epitope are introduced as repeating sequences in an expression system.

Next to the epitope also epitope-analogues which are cross-reactive with the epitope can be used. Crossreactive means here that the analogue is able to compete .S with the epitope for binding with the antibody.

Monospecific antibodies to an epitope can be obtained by affinity purification from polyspecific antisera by a modification of the method of Hall et al. (Nature 311, 379-387, 1984). Polyspecific antisera can be obtained by 6 immunizing rabbits according to standard immunisation schemes. Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogeneous binding characteristics for the relevant antigen. Homogeneous binding as used herein refers to the Io ability of the antibody species to bind to a specific antigen or epitope.

The antibody is preferably a monoclonal antibody, more preferably a humanised monoclonal antibody.

Monoclonal antibodies can be prepared by immunizing inbred mice, preferably Balb/c with the appropriate protein by techniques known in the art (Kohler and Milstein, Nature 256, 495-497, 1975). Hybridoma cells are subsequently selected by growth in hypoxanthine, thymidine and aminopterin in an appropriate cell culture medium such as Dulbecco's modified Eagle's medium (DMEM). Antibody producing hybridomas are cloned, preferably using the soft agar technique of MacPherson, (Soft Agar Techniques, Tissue Culture Methods and Applications, Kruse and Paterson, eds., Academic Press, 276, 1973). Discrete E colonies are transferred into individual wells of culture S plates for cultivation in an appropriate culture medium.

Antibody producing cells are identified by screening with the appropriate immunogen. Immunogen positive hybridoma cells are maintained by techniques known in the art.

Jo specific anti-monoclonal antibodies are produced by cultivating the hybridomas in vitro or preparing ascites fluid in mice following hybridoma injection by procedures known in the art.

It is preferred to use humanized antibodies. Methods for humanizing antibodies, such as CDR-grafting, are known (Jones et al., Nature 321, 522-525, 1986). Another possibility to avoid antigenic response to antibodies reactive with polypeptides according to the invention is the use of human antibodies or fragments or derivatives thereof Human antibodies can be produced by in vitro stimulation of isolated B-lymphocytes, or they can be isolated from (immortalized) B-lymphocytes which have been harvested from a human being immunized with at least one polypeptide according to the invention. This last method S has been described in the European Patent Application no: 0 488 470 of Akzo N.V.

Antibodies are formed from basic Y-shaped immunoglobulin molecules, of which the variable parts are able to bind with the antigen. Five distinct classes of immunoglobulin molecules are recognized in most higher mammals, three of which (IgG, IgD and IgE) are monomeric "o proteins, while IgA i-f mostly dimeric and IgM is pentameric. Each monomer has two identical regions which can bind to the antigen, rendering the antibody monospecific.

Preferably the antibody is of the IgM type. This type of antibodies is characterized by 5 times 2 identical binding places. Multiple identical binding places enable both the attachment to more than one antigen at the target site, thus creating a more stable binding, and/or the attachment of more than one ligand-effector moieties, thus Jo creating the opportunity to introduce more diagnostic label or therapeutic drug at the target site.

These IgM type antibodies can be obtained through cells normally producing IgM-type antibodies or they can be obtained by causing a class switch of the immunoglobulin antitype from IgG or IgA producing cells, said IgG or IgA having the cesired binding characteristics.

I

A preferred human monoclonal IgM-type antibody is Mr o 16-88 produced by a hybridoma cell line which is depot ,-ed under number HB 8495 with the American Type Culture Collection on January 30, 1984. Reactivity of this monoclonal antibody has been specified in US Patent No, 5,106,738.

The cognate epitope for monoclonal antibody 16-88 is an epitope which is related to cytokeratines 8, 18 and 19, to which 16-88 has been demonstrated to show activity. More (0 particularly, the epitope is a polypeptide formed by the amino acid sequence -Thr-Leu-Gln-Gly-Leu-Glu-Ile-Glu-Leu- Gln-Ser-Gln-Leu-Ser-Met-Lys- or a fragment or functional equivalent thereof.

The term "polypeptide" refers to a molecular chain of amino acids, does not refer to a specific length of the S product and if required can be modified in vivo or in vitro, for example by glycosylation, amidation, carboxylation or phosphorylation; thus inter alia peptides, oligopeptides and proteins are included within the definition of polypeptide.

SThe term "fragment" refers to any sequence of amino acids that is part of the polypeptide defined above, 0 having common elements of origin, structure and mechanism of action that are within the scope of the present invention because they can be prepared by persons skilled in the art, once given the teachings of the present invention.

As used herein, "functional equivalent" means variations of the described sequence still maintaining 3~ functional characteristics of the above given amino acid sequence. These functional characteristics are the ability to react with the antibody 16-88.

The variations that can occur in a sequence may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino J acid(s) in said sequence. Amino acid substitutions that are expected not to essentially alter biological and immunological activities, have been described. Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution o are, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. Based on this information Lipman and Pearson developed a method for rapid and F sensitive protein comparison (Science 227, 1435-1441, 1985) and determining the functional similarity between homologous polypeptides.

A polypeptide which shows slight variations to the polypeptide with a sequence as mentioned above is the a 180-188 amino acid fragment of the 65 kD HS-protein of Mycobacterium.

The effector moiety can be either a therapeutic drug or a diagnostic compound. As a diagnostic compound preferably a 7-emitter is used, such as 99 mTc, 111 In or 123I (Goldenberg et al., Antib. Immunoconj. and Radiopharm., 151-167, 1990).

When using a therapeutic drug as effector moiety preferably a compound selected from the group consisting of a- and p-emitting radioisotopes, drugs, toxins, boron 3t addends, photosensitizers and radiosensitizers is used (Bos et al., J. Contr. Release 16, 101-112, 1991). As radioisotopes preferably 90 y, 1 31 186Re, 188 Re, 211At, 212 Bi, 213 Bi or 225 Ac are used.

Also part of the invention are ligand-effector moieties for therapeutic use in which the ligand has been derived from a target associated antigen or a cross-reactive analogue thereof and in which the effector has been conjugated to the ligand.

Preferably the ligand which has been derived from a target associated antigen is the epitope of that antigen or a cross-reactive analogue thereof.

A preferred embodiment is the epitope that is o1 recognised by monoclonal antibody 16-88, especially the epitope which has the amino acid sequence -Thr-Leu-Gln- Gly-Leu-Glu-Ile-Glu-Leu-Gln-Ser-Gln-Leu-Ser-Met-Lys- or a S 00 fragment or functional equivalent thereof or a multimere of this epitope. Such a multimere provides a multitude of binding places, or a multitude of sites to which the effector moiety can be conjugated. This last is especially "0 advantageous because in such a way an effective dose of effector can be multiplied.

Numerous methods for the preparation of the ligandeffector moiety conjugates of the present invention are known in the art. Use can be made of the chemical a* methodology that has been developed in the field of S protein-protein conjugation and antibody-drug conjugation.

Overviews of these methods are given by Means and Feeney 2 (Bioconj. Chem.1, 2-12, 1990) and Reisfeld et.al.

(Antibody, Immunoconjugates and Radiopharmaceuticals 2, 217-224, 1989), which are incorporated herein by reference. For instance, the use of well known homo- or heterobifunctional protein cross-linking reagents allow 3 the coupling of an effector moiety to a ligand by means of either a readily cleavable disulfide bond, or a more stable thioether or amide bond, or the like. A preferred general conjugation methodology makes use of a heterobifunctional reagent, such as N-succinimidyl 4-(N- JS maleimidomethyl)cyclohexane-l-carboxylate (SMCC), or derivatives thereof, which have a maleimido group at one end and an activated ester group at the other end. Either the ligand, or the effector moiety is derivatized with SMCC through reaction with a nucleophilic amino group, or the like. The other conjugate component, which either contains a free thiol group, or which is derivatized for instance with the well known N-succinimidyl 3-(2pyridyldithio)propionate (SPDP) or with 2-iminothiolane (Traut's reagent) to contain a free thiol group, is reacted with the maleimido-derivatized component to give a oconjugate in which the components are joined by a stable thioether bond.

When the effector moiety is to be a diagnostic or therapeutic radioisotope, the conjugation with the ligands of the invention can be performed, for instance, jby the methods described by Fritzberg et.al.

(Pharmaceutical Research 5, 325-334, 1988), which is incorporated herein by reference. Many of the suitable radioisotopes require conjugation through the use of bifunctional chelates, while radioiodination is preferably o done employing oxidants like chloroamine-T and iodogen (Fraker and Speck, Biochem. Biophys. Res. Comm. 80, 849- 857, 1978).

o Another embodiment of the invention is formed by a kit in which a diagnostic moiety is coupled to the antibody.

Diagnostic immunoconjugates are commonly known in the art.

In this setting a diagnostic immunoconjugate is advantageous because it provides a way to investigate the biodistribution of the antibody used in the pretargeting step. Preferably the diagnostic moiety is a '-emitting J3 radioistope, preferably from the group of 99mTc, 111In or 123I. Coupling of the radioisotope to the antibody can be performed through known chemical methods, either by direct coupling or by coupling to bridging or linker molecules.

I

For pharmaceutical use the antibody and the ligandeffector conjugate should be in a pharmaceutically acceptable form.

The useful dosage to be administered will vary Sdepending on the age, weight and mode of administration.

Preferably the pharmaceutical compound is mixed with one or more pl.armaceutically acceptable carriers, e.g. as described in the standard reference Chase et ai., Remington's Pharmaceutical Sciences. Examples of \o pharmaceutically acceptable carriers or diluents useful in the present invention include stabilizers such as SPGA, carbohydrates sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin, tetanus toxoid, keyhole limpet haemocyanin or casein, protein 1 containing agents such as bovine serum or skimmed milk and buffers phosphate buffer).

By means of pharmaceutically suitable liquids the compound can also be applied as an injection preparation in the form of a solution, suspension, emulsion, or as a L spray, e.g. a nasal spray.

Kits and conjugates according to the present invention can be used for the treatment and detection of tissue specific cell disorders such as cancer, auto-immune diseases and inflammatory diseases.

The invention further comprises a method for pretargeting, this method being characterized in that an antibody or a fragment thereof or a derivative of said fragment or antibody is administered followed by a subsequent administration of a ligand-effector moiety, 3o further characterized in that said fragment or antibody is monospecific and that said ligand is derived from the target-associated antigen or a cross-reactive analogue thereof recognisable by said antibody. Furthermore all specific embodiments mentioned above apply for use in such 3Y a method for pretargeting.

The present invention is further described by way of example with reference to the accompanying figures, in which: fig. 1 shows the pretargeting of a conjugate with biotin and the 180-188 peptide of the 65 kD HS-protein of Mycobacterium, conjugated involving an intermediate with NHS. Specific reaction with increasing concentrations of antibody 16-88 is shown; fig. 2 shows the same pretargeting as fig. 1 but now the is made involving an intermediate with LC-SPDP.

Specificity for antibody 16-88 is preserved; fig. 3 shows an autoradiogram of 125 I-PhCG bound to IgM 593B and not to control antibody IgM fig. 4 shows the radioactivity count of fractions eluted I from a Sepharose column. The amount of radioactivity from 125 I-hCG is related to a peak in the concentration of IgM 593B.

5* *e e o e e eeee e 14 EXAMPLE 1 1. Pretarqeting with IqM 16.88, with biotin as effector molecule.

1.1 Synthesis of peptide-biotin conjugate.

6 The peptide TF. ,QLELT (amino acid 180-188 of the kD HS-protein of Mycobacterium) was solved in 50 pl DMSO (dimethylsulphoxide) in a concentration of 30 mMol/l.

Next, 50 ;l of a 300 mMol/l solution of biotin-NHS conjugate (Pierce) was mixed with the peptide solution 14 and incubated for 1 hour at room temperature. Then ml 100% ethanol was added and the solution was vortexed.

The mixture was centrifuged fro 2 minutes 1400 rpm and the pellet was washed twice with 1.0 ml 100% ethanol.

PBS (7.65 g/l NaCI, 0.21 g/l KH 2

PO

4 0.91 g/l IS Na 2

HPO

4 .2H 2 0, pH 7.2) was used to dissolve the pellet to a final concentration of ca. 1 mg/ml of the peptidebiotin conjugate.

1.2 Coating of microtiter plates.

The peptide Thr-Leu-Gln-Gly-Le-u-luIle-Glu-Leu-Gln- )o Ser-Gln-Leu-Ser-Met-Lys was dissolved in PBS to a concentration of 1 gg/ml. Microtiter wells were filled with 110 pl of this solution per well and incubated for 1 hr at 37"C. After incubation the plate was washed three times with PBS/Tween and three times with PBS 250 l Cl/well. The wells were blocked with 250 /l/well of skimmed milk (Difco) in PBS/Tween and incubated for 1 hr at 37*C. After incubation the plate was washed three times with PBS/Tween and three times with PBS 250 Al/well.

I, 1.3 Pretarcetinq To the coated wells of a plate prepared as described above various concentrations of antibody 16-88 or control antibody were added and incubated for 1 hr at 6 room temperature. After a wash step as described above the wells were incubated for 1 hr at room temperature with a 20 ig/ml solution of the peptide-biotin conjugate dissolved in PBS (from Ex. 1.1) in the presence of 1% BSA (bovine serum albumin). After a wash step the wells o1 were incubated for 1 hr at room temperature with a 1:1000 solution of Streptavidin-HRP (Streptavidin-Horse Radish Peroxidase, Sanbio) in PBS and 1% BSA. After a wash step the wells were incubated with 100 Al/well TMBsubstrate (10.2 ml MQ, 0.2 ml TMB, 1.08 ml buffer and 0.12 ml UPO; Organon Teknika). Next the colouring reaction was stopped with 4M H 2

SO

4 50 il/well. Colour intensities were measured by absorption spectrometry at 450 nm. Results are depicted in fig. 1.

S Example 2 o 2. Pretargeting of Example 1, conjugates made through

SPDP.

o 2.1 Synthesis biotin-HPDP and 180-188-LC-SPDP conjugates.

180-188 peptide and LC-SPDP (Pierce) were dissolved in small volumes of DMSO and mixed in a proportion of 1 part peptide to 1.5 part LC-SPDP. This mixture was incubated for 30 minutes at room temperature, then 1 M DDT was added to a final concentration of 10 mM. After again 30 minutes the peptide was precipitated with 1 ml do 100% ethanol. The pellet was washed twice with ethanol and dissolved in a small volume of DMSO.

Biotin-HPDP (Pierce) was dissolved in DMSO and added to I I the solution of peptide-LC-SPDP in a 10:1 proportion.

After 60 minutes the peptide-biotin conjugate was precipitated with 1 ml 100% ethanol, washed twice with ethanol and dissolved in PBS.

S Pretargeting and enzyme-immunoassay were performed as in Example 1. Results are depicted in fig. 2.

EXAMPLE 3 3. Pretargeting with GhCG and antibody to 3hCG.

3.1 Binding of 125I-BhCG to IqM 593B.

1 A range of concentrations of IgM 593B and a control eo antibody (SPM5B) were spotted on a spotblot (Immobilon PVDF membrane). After addition with Blotto the spots were incubated with 20 g/ml 125 I-phCG in PBS for 1 hour at room temperature. After a washstep the blot was I( autoradiographed, Results are depicted in fig. 3.

3.2 Binding of IM 593B to hCG-Sepharose.

hCG 555 (which contains 40% hCG) was conjugated to tresyl activated Sepharose. To an amount of hCG-Sepharose equivalent with 1 mg hCG excess IgM 593B (20mg in PBS) was o added. After incubation for 15 hr at 4°C and a wash step the extinction at 280 nm was measured. It appeared that 1 mg IgM 593B had been bound. In a control experiment with IgM SPM5B only 0.03 mg was bound.

3.3 Pretargeting.

as 5 Ag 125 I-hCG in 0.5 ml PBS was added to hCH-Sepharose complexes loaded with IgM 593B as described above. Free 12 5I-.hCG was removed by washing with PBS. hCG-Sepharose was eluted with 10 mM NaOH (pH 12.0) and the fractions 17 were analysed for radioactivity. Results are depicted in fig. 4.

18 Sequence Listing INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO o1 (iii) ANTI-SENSE: NO FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Thr Leu Gin Gly Leu Glu lie Glu Leu Gin Ser Gin Leu Ser Met Lys 1 5 10 so a ease s [N:\LIBF]05203:GSA

Claims (28)

1. A method for pretargeting comprising administration of an antibody or a fragment thereof or a derivative of said antibody or fragment, which antibody or fragment recognizes a target associated antigen, followed by administration of a ligand-effector moiety conjugate comprising a ligand specific for said antibody, and an effector moiety linked to the ligand, characterized in that: said antibody is a mono specific antibody with multiple binding sites; and said ligand is derived from the target associated antigen or a cross-reactive analogue thereof recognisable by said antibody.

2. The method of claim 1, characterised in that said ligand comprises the cognate epitope.

3. The method of claim 1 or 2, characterised in that said ligand comprises a multimere of the cognate epitope or a cross-reactive analogue thereof.

4. The method of any one of claims 1 to 3, characterised in that said antibody is a monoclonal antibody. The method of claim 4, characterised in that said antibody is a human monoclonal antibody.

6. The method of claim 5, characterised in that said antibody is an antibody of the IgM isotype.

7. The method of claim 6, characterised in that said antibody is the human monoclonal antibody 16.88, deposited with the American Type Culture Collection under number HB 8495.

8. The method of claim 4, characterised in that said ligand comprises the cognate epitope of human monoclonal antibody 16.88.

9. The method of claim 4 or 8, characterised in that the ligand comprises a multimere of the cognate epitope of the MoAb 16.88, or a peptide analogue of the cognate epitope of the MoAb 16.88. The method of claim 4 or 8, characterised in that the ligand comprises the epitope of claim 8, having the amino acid sequence -Thr-Leu-Gln-Gly-Leu-Glu-Ile-Glu- 30 Leu-Gln-Ser-Gln-Leu-Ser-Met-Lys-, a fragment thereof or a functional equivalent thereof.

11. The method of any one of claims 1-10, characterised in that the effector moiety is a diagnostic agent.

12. The method of claim 11, characterised in that the effector moiety is a y- emitting radioisotope.

13. The method of claim 12, characterised in that the effector moiety is a radioisotope selected from the group consisting of 99 mTc, 111 In and 1231

14. The method of any one of claims 1-13, characterised in that the effector moiety is a therapeutic agent. L The method of claim 14, characterised in that the effector moiety is selected from the group consisting of p- and a-emitting radioisotopes, drugs, toxins, boron addends, photosensitizers and radio-sensitizers.

16. The method of claim 14, characterised in that the therapeutic radioisotope is selected from the group consisting of 90y, 1311, 186 Re, 18 8Re, 211 At, 2 12 Bi, 2 13 Bi and 225Ac

17. The method of any one of claims 14 to 16, characterized in that a diagnostic agent is attached to said antibody.

18. The method of claim 17, characterised in that said diagnostic agent is a y- emitting radioisotope.

19. The method of claim 18, characterised in that said diagnostic agent is selected from the group consisting of 99 mTc, 111 In and 1231. A ligand-effector moiety conjugate for therapeutical use comprising a ligand derived from the tumor associated antigen reactive with human monoclonal antibody 16-88, or a cross-reactive analogue thereof, and an effector moiety linked to said ligand.

21. The ligand-effector moiety conjugate of claim 20, characterized in that the ligand comprises the cognate epitope of human monoclonal antibody 16.88.

22. The ligand-effector conjugate of claim 20 or 21, characterized in that the 20 ligand comprises a multimere of the cognate epitope of human monoclonal antibody 16- 88, or a peptide analogue of the cognate epitope of monoclonal antibody 16-88.

23. The ligand-effector conjugate of claim 21 or 22, characterized in that the ligand comprises the amino acid sequence -Thr-Leu-Gln-Gly-Leu-Glu-Ile-Glu-Leu-Gln- Ser-Gln-Leu-Ser-Met-Lys-, a fragment thereof or a functional derivative thereof.

24. The ligand-effector moiety conjugate of any one of claims 20-23, characterised in that said effector moiety is a therapeutic agent.

25. The ligand-effector moiety conjugate according to claim 24, characterised in that the therapeutic agent is selected from the group consisting of P- or c-emitting radioisotopes, drugs, toxins, boron addends, photosensitizers and radiosensitizers. 30 26. The ligand-effector moiety conjugate according to claim 25, characterised in that the therapeutic radio-isotope is selected from the group consisting of 90y, 1311, 18 6 Re. 138 Re, 2 11 2 12 Bi, 213 Bi and 225 Ac.

27. The ligand-effector moiety conjugate to any one of claims 20-23, characterised in that the effector moiety is a diagnostic agent.

28. The ligand-effector moiety conjugate according to claim 27, characterised in that the diagnostic agent is a y-emitting radioisotope.

29. The ligand-effector moiety conjugate of claim 28, characterised in that said y-emitting radioisotope is selected from the group consisting of 99 mTc, 11lIn and 1231. Pharmaceutical preparation comprising the ligand-effector moiety conjugate f according to any one of claims 20-29 and a pharmaceutically acceptable carrier.

31. A method for pretargeting comprising administration of an antibody or a fragment thereof or a derivative of said antibody or fragment, which antibody or fragment recognizes a target associated antigen, followed by administration of a ligand-effector moirty conjugate comprising a ligand specific for said antibody, and an effector moiety linked to the ligand, which method is substantially as herein described with reference to any one of Examples 1 to 3.

32. A ligand-effector moiety conjugate for therapeutical use, substantially as herein described with reference to any one of Examples 1 to 3.

33. Pharmaceutical preparation comprising the ligand-effector moiety conjugate of claim 33 and a pharmaceutically acceptable carrier.

34. A method of treating cancer in a patient requiring such treatment comprising administering to said patient an effective amount of a ligand effector moiety conjugate according to any one of claims 20 to 29. Dated 25 March, 1997 15 Akzo Nobel N.V. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON e o e e IN:\LIBVV100663:SAK:TCW Kit and Method for Pretargeting ABSTRACT The invention describes a novel kit or method for the delivery of pharmaceutical or diagnostic compounds to target cells in the body. This kit comprises a monospecific antibody with multiple binding sites, which antibody recognizes a target associated antigen, and a ligand-effector moiety, of which the ligand has been derived from the target associated antigen or analogue thereof. Specifically the invention relates to targeting cytotoxic drugs or radioisotopes to tumor cells. The preferred antibody is a human IgM and the ligand is preferably the epitope cognate to said antibody. 0 *ee 0 00 «r No-:Figure