CA2126819A1 - Kit and method for pretargeting - Google Patents
Kit and method for pretargetingInfo
- Publication number
- CA2126819A1 CA2126819A1 CA002126819A CA2126819A CA2126819A1 CA 2126819 A1 CA2126819 A1 CA 2126819A1 CA 002126819 A CA002126819 A CA 002126819A CA 2126819 A CA2126819 A CA 2126819A CA 2126819 A1 CA2126819 A1 CA 2126819A1
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- Prior art keywords
- antibody
- ligand
- effector moiety
- epitope
- fragment
- Prior art date
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- 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
- A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
- A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
-
- 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
- A61K51/1045—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 animal or human tumor cells or tumor cell determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2121/00—Preparations for use in therapy
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- Medicinal Chemistry (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
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.
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.
Description
.`J~ ~ .
2 1 2 6 8 1 ~
Kit and method for pretargeting.
The invention relates to the filelds 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 comprising a ligand specific for said antibody, and an effector moiety linked to the ligand. `
.: -The in vivo use of antibodies for the selective delivery of diagnostic or therapeutic agents to target tissues is known in the art.
Since the advent of methodology for the production of monoclonal antibodies, antibodies of predefined specificities (Kohler and Milstein, Nature 256, 495-497, 1975), numerous studies aiming at specific targeting of radionuclides, plant)or bacterial toxins, biological 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 vitro studies the great potential of these immunoconjugates has be~n 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 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-immunoconjugate. Similarly, due to the lack of operational 2 2~6~9 `
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, `
newer approaches to delivery of diagnostic or therapeutic agents have recently been suggested, that are characterised in that the localisation phase of the antibody component is separated in time from the phase in which the diagnostic or therapeutic agent is administered.
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 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.
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 ! ~ ,'". ''.,'~
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 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 .
Kit and method for pretargeting.
The invention relates to the filelds 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 comprising a ligand specific for said antibody, and an effector moiety linked to the ligand. `
.: -The in vivo use of antibodies for the selective delivery of diagnostic or therapeutic agents to target tissues is known in the art.
Since the advent of methodology for the production of monoclonal antibodies, antibodies of predefined specificities (Kohler and Milstein, Nature 256, 495-497, 1975), numerous studies aiming at specific targeting of radionuclides, plant)or bacterial toxins, biological 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 vitro studies the great potential of these immunoconjugates has be~n 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 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-immunoconjugate. Similarly, due to the lack of operational 2 2~6~9 `
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, `
newer approaches to delivery of diagnostic or therapeutic agents have recently been suggested, that are characterised in that the localisation phase of the antibody component is separated in time from the phase in which the diagnostic or therapeutic agent is administered.
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 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.
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 ! ~ ,'". ''.,'~
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 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 .
3 ~12 6'~
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 following administration of the antibody, to be more effective than drug alone in suppressing tumor growth.
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 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.
(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. `
A major problem with the bifunctional antibodies mentioned above, and with most other pretargeting systems `~
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 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 prevent subsequent applications to be effective. This immunogenicity problem will even be aggrevated when 4 2 ~ ~ 6 ~ ~ 9 ! ~;
`~ '`'``,`''`
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 con~ugate. `~
The present invention provides a kit, comprising an -~
antibody or a fragment thereof or a derivative of said antibody or fragment, which fragment or antibody recognizes a target associated antigen, and a ligand-effector moiety conjugate comprising a ligand specific for ~ `~
said antibody, and an effector moiety linked to the ;~
ligand, characterised in that~
- said antibody is a mono specific antibody with `~
multiple binding sites, - said ligand is derived from the target associated antigen or a cross-reactive analogue thereof recognisable by said antibody.
Using a monospecific antibody gives the advantage that there is no need for chemical conjugation methods. -The present invention proposes to administer to a i -patient a mono specific antibody in a dose, or in ; -successiva doses, sufficient to maximise target site `
accumulationlof the antibody. At some optimal time later, when most of the non-bound antibody has been 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 useEul are: antigens related to cancer cells, like CEA (Carcino-embryonic antigen), AFP (alpha-:~`
--' -~
: :
1 5 ~ 2 ~ ~ 1 9 foetoprotein), FHAP (fast homoarginine-sensitive alkaline phosphatase), ps7 (melanome specific), and EL-l (elongation factor 1), antigens related to viruses or bacteria, like HIV gp 120, HIV gp 160, Hepatitis B core 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 recognised by the antibody. This epitope is a small, low-molecular 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 ;~
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 three-dimensional conformation of the protein the epitope has 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. ~ultimerization can be achieved by common chemical techniques, in case of peptide epitopes -it is also posslble to produce a multimer by recombinant DNA~techniques. In this last way the nucleotides coding 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. Cross-reactive means here that the analogue is able to compete -with the epitope for binding with the antibody.
6 ~ 1 9 ~ --Monospecific antibodies to an epitope can be obtained by affinity purification from polyspecific antisera by a modification of the method of Hal:L et al. (Nature 311, 379-387, 1984). Polyspecific antisera can be obtained by 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 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 colonies are transferred into individual wells of culture plates for cultivation in an appropriate culture medium.
Antibody producing cells are identified by screenin~ with the appropriate immunogen. Immunogen positive hybridoma ~
cells are maintained by techniques known in the art. ! ;'`, 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.
7 ~ 8 1 9 ' ::
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 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 proteins, while IgA is mostly dimeric and IgM is pentameric. Each monomer has two identical regions which `
can bind to the ant.igen, 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, andjor the I `~
attachment of more than one ligand-effector moieties, thus 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 desired binding characteristics.
' ~'-,.'',,' `.'',"', :'."' 8 ~ 9 .
A preferred human monoclonal IgM-type antibody is MoAb 16-88 produced by a hybridoma cell line which is deposited und~r 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 ~`
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 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.
The tarm "fragment" refers to any sequence of am.ino acids that is part of the polypeptide defined above, 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 ! I variations of the described sequence still maintaining I ~`
functional characteristics of the above given amino acid sequence. These functional characteristics are the ability to react with the antibody 16-88.
~ 1 2 6 g ~ 9 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 acid(s) in said sequence. Amino ac:id 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 are, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M.D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3). Based on this information Lipman and Pearson developed a method for rapid and 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 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 ~-emitter is used, such as 99mTc, lllIn or 123I ~`
(Goldenberg et al., Antib. Immunoconj. and Radiopharm., 3(3), I51-167, 1990). :~
When using a therapeutic drug as effector moiety preferably a compound selected from the group consisting of a- and ~-emitting radioisotopes, drugs, toxins, boron addends, photosensitizers and radiosensitizers is used (Bos et al., J. Contr. Release 16, 101-112, 1991). As radiOiSotope5 preferably 90y, 131I, 186Re 188Re 211At ~;
212Bi 213Bi or 225Ac are used. ' `-'`
. ...- :;:
":,, "-,.
: :
lo ~ 9 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 lepitope of that antigen or a cross-reactive analogue thereof.
A preferred embodiment is the epitope that is 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 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 advantageous because in such a way an effective dose of effector can be multiplied.
Numerous methods for the preparation of the ligand- ~;
effector moiety conjugates of the present invention are known in the art. Use can be made of the chemical ;
methodology that has been developed in the fiald of protein-protein conjugation and antibody-drug conjugation.
Overviews of these methods are given by Means and Feeney (Bioconj. Chem.l, 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 -~
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 1~ ;
general conjugation methodology makes use of a heterobifunctional reagent, such as N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), or derivatives thereof, which have a maleimido group at one 2 ~
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-(2-pyridyldithio)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 conjugate 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, by 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 ;` ;~
done employing oxidants like chloroamine-T and iodogen (Fraker and Speck~ Biochem. Biophys. Res. Comm. 80, 849-857, 1978). `-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 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.
~.: :-;.. ..
.. :.:~
.''''.,' ~""~ ''''`,"', . . ~ -,, 12 ~ ~9 ~ ;
For pharmaceutical use the antibody and the ligand-effector conjugate should be in a pharmaceutically acceptable form.
The useful dosage to be administered will vary depending on the age, weight and mode of administration.
Preferably the pharmaceutical compound is mixed with one or more pharmaceutically acceptable carriers, e.g. as described in the standard reference Chase et al., Remington's Pharmaceutical Sciences. Examples of pharmaceutically acceptable carriers or diluents useful in the present invention include stabilizers such as SPGA, -carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin, tetanus toxoid, keyhole limpet haemocyanin or casein, protein containing agents such as bovine serum or skimmed milk and buffers (e.g. 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 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 antiibody or a fragment thereof or a derivative of said fragment or antibody is administered followed by a subsequent administration of a ligand-effector moiety, 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 a method for pretargeting. ~;
13 21~ 9 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 conjugate is made involving an intermediate with LC-SPDP.
Specificity for antibody 16-88 is preserved;
fig. 3 shows an autoradiogram of 125I-~,'hCG bound to IgM
593B and not to control antibody IgM SPM5B; ~ ;
fig. 4 shows the radioactivity count of fractions eluted ~`;`
from a Sepharose column. The amount of radioactivity from `~
125I-~-,'hCG is related to a peak in the concentration of IgM
593B. ~ ~ ~
-.~; ,.
;''.',',"''' .. ~ . ., ,`` ~`''~i - ;'..-:, .
`,`,;~
! ' :, ~ ' ' -EXAMPLE 1 ~-1. Pretargetin~ with IqM 16.88. with biotin as effector molecule.
1.1 Synthesis of pe~tide-biotin coniuqate.
The peptide TFGLQLELT (amino acid 180-188 of the 65 kD HS-protein of Mycobacterium) was solved in 50 ~1 DMSO
(dimethylsulphoxide) in a concentration of 30 mMol/l.
Next, 50 ~1 of a 300 mMol/l solution of biotin-NHS
conjugate (Pierce) was mixed with the peptide solution and incubated for 1 hour at room temperature. Then 1.0 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 NaCl, 0.21 g/l KH2P04, 0.91 g/l Na2HP04.2H2O, pH 7.2) was used to dissolve the pellet to a final concentration of ca. 1 mg/ml of the peptide-biotin conjugate.
1.2 Coatinq of microtiter e~ates.
The peptide Thr-Leu-Gln-Gly-Leu-Glu-Ile-Glu-Leu-Gln-Ser-Gln-Leu-Ser-Met-Lys was dissolved in PBS to a concentration of 1 ~g/ml. Microtiter wells were filled with 110 ~1 of this solution per well and incubated for 1 hr at 37C. After incubation the plate was washed three times with PBS/Tween and three times with PBS 250 well. Theiwells were blocked with 250 ~l/well of 5%
skimmed milk (Difco) in PBS/Tween and incubated for 1 hr at 37C. After incubation the plate was washed three times with PBS/Tween and three times with PBS 250 ~l/well.
. .
~ 9 1.3 Pretargeting ~-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 ;;
room temperature. After a wash stlep as described above the wells were incubated for 1 hr at room temperature ~ ~
with a 20 ~g/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 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 ~l/well TMB-substrate (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 H2S04 50 ~l/well. Colour intensities were measured by absorption spectrometry at 450 nm. Results are depicted in fig. 1.
Example 2 2. Pretarqetinq of Example lr conlugates made throuqh ~ `~
SPDP. ;~`
2.1 Synthesis biotin-HPDP and 180-188-LC-SPDP .`~
con~uqates.
180-188 peptid~ 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 100% ethanol. The pellet was washed twice with ethanol and dissolved n a small volume of DMSO. -;
Biotin-HPDP (Pierce) was dissolved in DMSO and added to . -,,'- .
16 ~1 2'~
the solution of peptide-LC-SPDP in a 10:1 proportion.
After 60 minutes the peptide-biot:in conjugate was precipitated with 1 ml 100% ethanol, washed twice with ethanol and dissolved in PBS.
Pretargeting and enzyme-immunoassay were performed as in Example 1. Results are depicted in fig. 2.
:
3. Pretarqetinq with ~hCG and antibody to ~hCG.
3.1 Bindinq of 125I-~hCG to IgM 593B.
A range of concentrations of IgM 593B and a control antibody (SPM5B) were spotted on a spotblot (Immobilon PVDF membrane). After addition with Blotto the spots were incubated with 20 ~g/ml 125I-~hCG in PBS for 1 hour at room temperature. After a washstep the blot was autoradiographed. Results are depicted in fig. 3.
3.2 Bindinq of IqM 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 added. After incubation for 15 hr at 4C 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 Pretarqeting.
5 ~g 125I-~hCG in 0.5 ml PBS was added to hCH-Sepharose complexes loaded with IgM 593B as described above. Free l25I-~hCG was removed by washing with PBS. hCG-Sepharose was eluted with 10 mM NaOH (pH 12.0) and the fractions -: ' .
17 ~2~9 :-~
`:
- , . .;.
were analysed for radioactivity. Results are depicted in fig. 4- :~
'"~ ';:
, ~ ~
.~ ~ ~ .....
.. ~.~,...
,: , . . . ~
" ','~",,"'`' . '` ' `~
.
~,. ... . . .
~,::: , .
`''~.'.'`~ `''' `' ..:'.'.". `
; ~ ,. ..
, . . ., ~
`,',~
,,.,,"~ ", .'~".~"~
' 18 ~6~19 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) A~PLICANT:
(A) NAME: Akzo Nobel N.V.
(B) STREET: Velperweg 76 (C) CITY: Arnhem (E) COUNTRY: The Netherlands (F) POSTAL CODE (ZIP): 6824 BM
(G) TELEPHONE: 04120-66911 (H) TELEFAX: 04120-50592 (I) TELEX: 37503 akpha nl (ii) TITLE OF INVENTIQN: Kit and method for pretargeting (iii) NUMBER OF SEQUENCES: 1 (iv) COMPUTER READABLE FORM: -(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: EP 93.202.016.7 (B) FILING DATE: 09-JUL-1993 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: peptide :-(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Thr Leu Gln Gly Leu Glu Ile Glu Leu Gln Ser Gln Leu Ser Met Lys ~-. ~: . . ~ , . . . , :
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 following administration of the antibody, to be more effective than drug alone in suppressing tumor growth.
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 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.
(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. `
A major problem with the bifunctional antibodies mentioned above, and with most other pretargeting systems `~
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 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 prevent subsequent applications to be effective. This immunogenicity problem will even be aggrevated when 4 2 ~ ~ 6 ~ ~ 9 ! ~;
`~ '`'``,`''`
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 con~ugate. `~
The present invention provides a kit, comprising an -~
antibody or a fragment thereof or a derivative of said antibody or fragment, which fragment or antibody recognizes a target associated antigen, and a ligand-effector moiety conjugate comprising a ligand specific for ~ `~
said antibody, and an effector moiety linked to the ;~
ligand, characterised in that~
- said antibody is a mono specific antibody with `~
multiple binding sites, - said ligand is derived from the target associated antigen or a cross-reactive analogue thereof recognisable by said antibody.
Using a monospecific antibody gives the advantage that there is no need for chemical conjugation methods. -The present invention proposes to administer to a i -patient a mono specific antibody in a dose, or in ; -successiva doses, sufficient to maximise target site `
accumulationlof the antibody. At some optimal time later, when most of the non-bound antibody has been 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 useEul are: antigens related to cancer cells, like CEA (Carcino-embryonic antigen), AFP (alpha-:~`
--' -~
: :
1 5 ~ 2 ~ ~ 1 9 foetoprotein), FHAP (fast homoarginine-sensitive alkaline phosphatase), ps7 (melanome specific), and EL-l (elongation factor 1), antigens related to viruses or bacteria, like HIV gp 120, HIV gp 160, Hepatitis B core 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 recognised by the antibody. This epitope is a small, low-molecular 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 ;~
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 three-dimensional conformation of the protein the epitope has 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. ~ultimerization can be achieved by common chemical techniques, in case of peptide epitopes -it is also posslble to produce a multimer by recombinant DNA~techniques. In this last way the nucleotides coding 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. Cross-reactive means here that the analogue is able to compete -with the epitope for binding with the antibody.
6 ~ 1 9 ~ --Monospecific antibodies to an epitope can be obtained by affinity purification from polyspecific antisera by a modification of the method of Hal:L et al. (Nature 311, 379-387, 1984). Polyspecific antisera can be obtained by 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 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 colonies are transferred into individual wells of culture plates for cultivation in an appropriate culture medium.
Antibody producing cells are identified by screenin~ with the appropriate immunogen. Immunogen positive hybridoma ~
cells are maintained by techniques known in the art. ! ;'`, 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.
7 ~ 8 1 9 ' ::
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 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 proteins, while IgA is mostly dimeric and IgM is pentameric. Each monomer has two identical regions which `
can bind to the ant.igen, 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, andjor the I `~
attachment of more than one ligand-effector moieties, thus 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 desired binding characteristics.
' ~'-,.'',,' `.'',"', :'."' 8 ~ 9 .
A preferred human monoclonal IgM-type antibody is MoAb 16-88 produced by a hybridoma cell line which is deposited und~r 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 ~`
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 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.
The tarm "fragment" refers to any sequence of am.ino acids that is part of the polypeptide defined above, 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 ! I variations of the described sequence still maintaining I ~`
functional characteristics of the above given amino acid sequence. These functional characteristics are the ability to react with the antibody 16-88.
~ 1 2 6 g ~ 9 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 acid(s) in said sequence. Amino ac:id 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 are, inter alia Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M.D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3). Based on this information Lipman and Pearson developed a method for rapid and 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 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 ~-emitter is used, such as 99mTc, lllIn or 123I ~`
(Goldenberg et al., Antib. Immunoconj. and Radiopharm., 3(3), I51-167, 1990). :~
When using a therapeutic drug as effector moiety preferably a compound selected from the group consisting of a- and ~-emitting radioisotopes, drugs, toxins, boron addends, photosensitizers and radiosensitizers is used (Bos et al., J. Contr. Release 16, 101-112, 1991). As radiOiSotope5 preferably 90y, 131I, 186Re 188Re 211At ~;
212Bi 213Bi or 225Ac are used. ' `-'`
. ...- :;:
":,, "-,.
: :
lo ~ 9 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 lepitope of that antigen or a cross-reactive analogue thereof.
A preferred embodiment is the epitope that is 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 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 advantageous because in such a way an effective dose of effector can be multiplied.
Numerous methods for the preparation of the ligand- ~;
effector moiety conjugates of the present invention are known in the art. Use can be made of the chemical ;
methodology that has been developed in the fiald of protein-protein conjugation and antibody-drug conjugation.
Overviews of these methods are given by Means and Feeney (Bioconj. Chem.l, 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 -~
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 1~ ;
general conjugation methodology makes use of a heterobifunctional reagent, such as N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), or derivatives thereof, which have a maleimido group at one 2 ~
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-(2-pyridyldithio)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 conjugate 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, by 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 ;` ;~
done employing oxidants like chloroamine-T and iodogen (Fraker and Speck~ Biochem. Biophys. Res. Comm. 80, 849-857, 1978). `-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 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.
~.: :-;.. ..
.. :.:~
.''''.,' ~""~ ''''`,"', . . ~ -,, 12 ~ ~9 ~ ;
For pharmaceutical use the antibody and the ligand-effector conjugate should be in a pharmaceutically acceptable form.
The useful dosage to be administered will vary depending on the age, weight and mode of administration.
Preferably the pharmaceutical compound is mixed with one or more pharmaceutically acceptable carriers, e.g. as described in the standard reference Chase et al., Remington's Pharmaceutical Sciences. Examples of pharmaceutically acceptable carriers or diluents useful in the present invention include stabilizers such as SPGA, -carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin, tetanus toxoid, keyhole limpet haemocyanin or casein, protein containing agents such as bovine serum or skimmed milk and buffers (e.g. 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 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 antiibody or a fragment thereof or a derivative of said fragment or antibody is administered followed by a subsequent administration of a ligand-effector moiety, 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 a method for pretargeting. ~;
13 21~ 9 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 conjugate is made involving an intermediate with LC-SPDP.
Specificity for antibody 16-88 is preserved;
fig. 3 shows an autoradiogram of 125I-~,'hCG bound to IgM
593B and not to control antibody IgM SPM5B; ~ ;
fig. 4 shows the radioactivity count of fractions eluted ~`;`
from a Sepharose column. The amount of radioactivity from `~
125I-~-,'hCG is related to a peak in the concentration of IgM
593B. ~ ~ ~
-.~; ,.
;''.',',"''' .. ~ . ., ,`` ~`''~i - ;'..-:, .
`,`,;~
! ' :, ~ ' ' -EXAMPLE 1 ~-1. Pretargetin~ with IqM 16.88. with biotin as effector molecule.
1.1 Synthesis of pe~tide-biotin coniuqate.
The peptide TFGLQLELT (amino acid 180-188 of the 65 kD HS-protein of Mycobacterium) was solved in 50 ~1 DMSO
(dimethylsulphoxide) in a concentration of 30 mMol/l.
Next, 50 ~1 of a 300 mMol/l solution of biotin-NHS
conjugate (Pierce) was mixed with the peptide solution and incubated for 1 hour at room temperature. Then 1.0 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 NaCl, 0.21 g/l KH2P04, 0.91 g/l Na2HP04.2H2O, pH 7.2) was used to dissolve the pellet to a final concentration of ca. 1 mg/ml of the peptide-biotin conjugate.
1.2 Coatinq of microtiter e~ates.
The peptide Thr-Leu-Gln-Gly-Leu-Glu-Ile-Glu-Leu-Gln-Ser-Gln-Leu-Ser-Met-Lys was dissolved in PBS to a concentration of 1 ~g/ml. Microtiter wells were filled with 110 ~1 of this solution per well and incubated for 1 hr at 37C. After incubation the plate was washed three times with PBS/Tween and three times with PBS 250 well. Theiwells were blocked with 250 ~l/well of 5%
skimmed milk (Difco) in PBS/Tween and incubated for 1 hr at 37C. After incubation the plate was washed three times with PBS/Tween and three times with PBS 250 ~l/well.
. .
~ 9 1.3 Pretargeting ~-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 ;;
room temperature. After a wash stlep as described above the wells were incubated for 1 hr at room temperature ~ ~
with a 20 ~g/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 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 ~l/well TMB-substrate (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 H2S04 50 ~l/well. Colour intensities were measured by absorption spectrometry at 450 nm. Results are depicted in fig. 1.
Example 2 2. Pretarqetinq of Example lr conlugates made throuqh ~ `~
SPDP. ;~`
2.1 Synthesis biotin-HPDP and 180-188-LC-SPDP .`~
con~uqates.
180-188 peptid~ 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 100% ethanol. The pellet was washed twice with ethanol and dissolved n a small volume of DMSO. -;
Biotin-HPDP (Pierce) was dissolved in DMSO and added to . -,,'- .
16 ~1 2'~
the solution of peptide-LC-SPDP in a 10:1 proportion.
After 60 minutes the peptide-biot:in conjugate was precipitated with 1 ml 100% ethanol, washed twice with ethanol and dissolved in PBS.
Pretargeting and enzyme-immunoassay were performed as in Example 1. Results are depicted in fig. 2.
:
3. Pretarqetinq with ~hCG and antibody to ~hCG.
3.1 Bindinq of 125I-~hCG to IgM 593B.
A range of concentrations of IgM 593B and a control antibody (SPM5B) were spotted on a spotblot (Immobilon PVDF membrane). After addition with Blotto the spots were incubated with 20 ~g/ml 125I-~hCG in PBS for 1 hour at room temperature. After a washstep the blot was autoradiographed. Results are depicted in fig. 3.
3.2 Bindinq of IqM 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 added. After incubation for 15 hr at 4C 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 Pretarqeting.
5 ~g 125I-~hCG in 0.5 ml PBS was added to hCH-Sepharose complexes loaded with IgM 593B as described above. Free l25I-~hCG was removed by washing with PBS. hCG-Sepharose was eluted with 10 mM NaOH (pH 12.0) and the fractions -: ' .
17 ~2~9 :-~
`:
- , . .;.
were analysed for radioactivity. Results are depicted in fig. 4- :~
'"~ ';:
, ~ ~
.~ ~ ~ .....
.. ~.~,...
,: , . . . ~
" ','~",,"'`' . '` ' `~
.
~,. ... . . .
~,::: , .
`''~.'.'`~ `''' `' ..:'.'.". `
; ~ ,. ..
, . . ., ~
`,',~
,,.,,"~ ", .'~".~"~
' 18 ~6~19 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) A~PLICANT:
(A) NAME: Akzo Nobel N.V.
(B) STREET: Velperweg 76 (C) CITY: Arnhem (E) COUNTRY: The Netherlands (F) POSTAL CODE (ZIP): 6824 BM
(G) TELEPHONE: 04120-66911 (H) TELEFAX: 04120-50592 (I) TELEX: 37503 akpha nl (ii) TITLE OF INVENTIQN: Kit and method for pretargeting (iii) NUMBER OF SEQUENCES: 1 (iv) COMPUTER READABLE FORM: -(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: EP 93.202.016.7 (B) FILING DATE: 09-JUL-1993 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: peptide :-(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Thr Leu Gln Gly Leu Glu Ile Glu Leu Gln Ser Gln Leu Ser Met Lys ~-. ~: . . ~ , . . . , :
Claims (20)
1. A kit, comprising a) an antibody or a fragment thereof or a derivative of said antibody or fragment, which antibody or fragment recognizes a target associated antigen, and b) a ligand-effector moiety conjugate comprising a ligand specific for said antibody, and an effector moiety linked to the ligand, characterised in that:
- said antibody is a mono specific antibody with multiple binding sites, - said ligand is derived from the target associated antigen or a cross-reactive analogue thereof recognisable by said antibody.
- said antibody is a mono specific antibody with multiple binding sites, - said ligand is derived from the target associated antigen or a cross-reactive analogue thereof recognisable by said antibody.
2. A kit according to claim 1, characterised in that said ligand comprises the cognate epitope.
3. A kit according to claim 1 or 2, characterised in that said ligand comprises a multimere of the cognate epitope or a cross-reactive analogue thereof.
4. A kit according to any of the claims 1 to 3, characterised in that said antibody is a monoclonal antibody, preferably a human monoclonal antibody, more preferably an antibody of the IgM isotype, most preferably the human monoclonal antibody 16.88, deposited with the American Type Culture Collection under number HB 8495.
5. A kit according to claim 4, characterised in that said ligand comprises the cognate epitope of human monoclonal antibody 16.88.
6. A kit according to claim 4 or 5, 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.
7. A kit according to claim 4 or 5, characterised in that ligand comprises the epitope of claim 5, having 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 equivalent thereof.
8. A kit according to any of the claims 1-7, characterised in that the effector moiety is a diagnostic agent, preferably a .gamma.-emitting radioisotope, most preferably a radioisotope selected from the group consisting of 99mTc, 111In and 123I.
9. A kit according to any of the claims 1-7, characterised in that the effector moiety is a therapeutic agent, preferably selected from the group consisting of .beta.- and .alpha.-emitting radioisotopes, drugs, toxins, boron addends, photosensitizers and radio-sensitizers.
10.A kit according to claim 9, characterised in that the therapeutic radioisotope is selected from the group consisting of 90Y, 131I, 136Re, 188Re, 211At, 212Bi, 213Bi and 225Ac.
11. A kit according to any of the claims 9-10 characterized in that a diagnostic agent is attached to said antibody, said diagnostic agent preferably being a .gamma.-emitting radioisotope, more preferably selected from the group consisting of 99mTc, 111In and 123I.
12.A ligand-effector moiety conjugate for therapeutical use comprising a ligand derived from a target associated antigen, preferably an epitope, or a cross-reactive analogue thereof, and an effector moiety linked to said ligand.
13.A ligand-effector moiety conjugate according to claim 12, characterised in that said ligand is a multimere of an epitope or a cross reactive analogue thereof.
14.A ligand-effector moiety conjugate according to claim 12 or 13, characterised in that said ligand is the epitope recognised by human monoclonal antibody 16.88.
15.A ligand-effector moiety conjugate according to any of the claims 12-14, characterised in that said effector moiety is a therapeutic agent, preferably selected from the group consisting of .beta.- or .alpha.-emitting radioisotopes, drugs, toxins, boron addends, photosensitizers and radiosensitizers.
16.A ligand-effector moiety conjugate according to claim 15, characterised in that the therapeutic radio-isotope is selected from the group consisting of 90Y, 131I, 186Re, 188Re, 211At, 212Bi, 213Bi and 225Ac.
17.A ligand-effector moiety conjugate according to any of the claims 12-14, characterised in that the effector moiety is a diagnostic agent, preferably a .gamma.-emitting radioisotope, more preferably selected from the group consisting of 99mTc, 111In and 123I.
18.Pharmaceutical preparation comprising a ligand-effector moiety conjugate according to any of the claims 12-16 and a pharmaceutical acceptable carrier.
19.Use of a ligand effector moiety conjugate according to any of the claims 12-16 for the manufacture of a pharmaceutical preparation for treatment of tissue specific cell disorders, preferably anti-cancer treatment or treatment of inflammatory diseases.
20.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, - said ligand is derived from the target associated antigen or a cross-reactive analogue thereof recognizable by said antibody.
- said antibody is a mono specific antibody with multiple binding sites, - said ligand is derived from the target associated antigen or a cross-reactive analogue thereof recognizable by said antibody.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP93202016 | 1993-07-09 | ||
| EP93202016.7 | 1993-07-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2126819A1 true CA2126819A1 (en) | 1995-01-10 |
Family
ID=8213969
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002126819A Abandoned CA2126819A1 (en) | 1993-07-09 | 1994-06-27 | Kit and method for pretargeting |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPH07181186A (en) |
| KR (1) | KR960014510A (en) |
| AU (1) | AU678474B2 (en) |
| CA (1) | CA2126819A1 (en) |
| FI (1) | FI943269A (en) |
| ZA (1) | ZA944893B (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994013805A1 (en) * | 1992-12-10 | 1994-06-23 | Celltech Limited | Humanised antibodies directed against a33 antigen |
-
1994
- 1994-06-27 CA CA002126819A patent/CA2126819A1/en not_active Abandoned
- 1994-07-06 ZA ZA944893A patent/ZA944893B/en unknown
- 1994-07-08 FI FI943269A patent/FI943269A/en unknown
- 1994-07-08 AU AU67360/94A patent/AU678474B2/en not_active Ceased
- 1994-07-08 KR KR1019940016440A patent/KR960014510A/en not_active Application Discontinuation
- 1994-07-08 JP JP6157499A patent/JPH07181186A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| KR960014510A (en) | 1996-05-22 |
| AU6736094A (en) | 1995-01-19 |
| ZA944893B (en) | 1995-02-20 |
| JPH07181186A (en) | 1995-07-21 |
| AU678474B2 (en) | 1997-05-29 |
| FI943269A0 (en) | 1994-07-08 |
| FI943269A (en) | 1995-01-10 |
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