[go: up one dir, main page]

WO1999043817A1 - Anticorps monoclonaux anti-ace humanises et faisant preuve d'une grande affinite - Google Patents

Anticorps monoclonaux anti-ace humanises et faisant preuve d'une grande affinite Download PDF

Info

Publication number
WO1999043817A1
WO1999043817A1 PCT/US1998/003680 US9803680W WO9943817A1 WO 1999043817 A1 WO1999043817 A1 WO 1999043817A1 US 9803680 W US9803680 W US 9803680W WO 9943817 A1 WO9943817 A1 WO 9943817A1
Authority
WO
WIPO (PCT)
Prior art keywords
humanized antibody
humanized
ser
thr
gly
Prior art date
Application number
PCT/US1998/003680
Other languages
English (en)
Inventor
William Henry Kerr Anderson
Philip R. Tempest
Frank J. Carr
William J. Harris
Kathryn Armour
Original Assignee
The Dow Chemical Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Dow Chemical Company filed Critical The Dow Chemical Company
Priority to BR9815775-2A priority Critical patent/BR9815775A/pt
Priority to PCT/US1998/003680 priority patent/WO1999043817A1/fr
Priority to AU63388/98A priority patent/AU752494B2/en
Priority to CA002321947A priority patent/CA2321947A1/fr
Priority to IL13802298A priority patent/IL138022A0/xx
Priority to EP98907630A priority patent/EP1056859A1/fr
Priority to JP2000533557A priority patent/JP2002504372A/ja
Publication of WO1999043817A1 publication Critical patent/WO1999043817A1/fr
Priority to NO20004251A priority patent/NO20004251L/no

Links

Classifications

    • 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
    • C07K16/3007—Carcino-embryonic Antigens
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6853—Carcino-embryonic antigens
    • 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
    • A61K51/1048—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 the tumor cell determinant being a carcino embryonic antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to humanized monoclonal antibodies and fragments or derivatives thereof which specifically bind carcinoembryonic antigen (CEA), which is an antigen expressed by various human carcinomas including breast, lung, and gastrointestinal carcinomas such as stomach and colon cancers. More specifically, the present invention relates to humanized monoclonal antibodies and humanized antibody fragments and derivatives thereof which are derived from murine monoclonal antibody COL-1 , a high affinity anti-CEA antibody. The present invention further relates to methods for producing such humanized monoclonal antibodies specific to CEA, pharmaceutical and diagnostic compositions containing such humanized monoclonal antibodies, and methods of use thereof for the treatment or diagnosis of cancer.
  • CEA carcinoembryonic antigen
  • Anti- tumor monoclonal antibodies exhibit potential application as both therapeutic and diagnostic agents.
  • Such monoclonal antibodies have potential application as diagnostic agents because they specificaliy bind tumor antigens and thereby can detect the presence of tumor cells or tumor antigen in an analyte.
  • use of monoclonal antibodies which bind tumor antigens for in vitro and in vivo imaging of tumor cells or tumors using a labeled form of such a monoclonal antibody is conventional in the art.
  • monoclonal antibodies which bind tumor antigens have well known application as therapeutic agents.
  • the monoclonal antibody functions as a targeting moiety, i.e. it directs the effector moiety (which typically possesses therapeutic activity) to the antibody's target, e.g., a tumor which expresses the antigen bound by the monoclonal antibody.
  • the monoclonal antibody itself operates as a therapeutic agent, the antibody functions both as a targeting moiety - i.e. it will specifically bind a cell which expresses the antigen - and as an effector which mediates therapeutic activity, typically tumor cell lysis.
  • a monoclonal antibody may
  • CEA carcinoembryonic antigen
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • CEA is an antigen complex having a molecular weight of about 180,000 D, which is expressed by numerous carcinomas including gastrointestinal carcinomas, colorectal carcinomas, breast carcinomas, ovarian carcinomas, and lung carcinomas. See, e.g., Robbins et al, Int'I J.
  • Muraro et al. report generation of monoclonal antibodies designated COL-1 through COL-15, which exhibit a strong, selective reactivity for human colon carcinomas versus normal adult tissues. These antibodies react with distinct, restricted epitopes on CEA.
  • the COL-1 antibody has been the focus of considerable attention because of its high affinity for CEA (1.4x10 9 M " ') and also because it comprises no detectable reactivity for CEA-related antigens such as the nonspecific cross- treating antigen (NCA) and the normal fecal antigen (NFA).
  • NCA nonspecific cross- treating antigen
  • NFA normal fecal antigen
  • COL-1 Because of its binding properties, COL-1 is currently being evaluated for use as a therapeutic agent.
  • Siler et al. Biotech. Ther., 4(3-4):163-181 (1993) report the administration of 3 l-labeled COL-1 to LS-M4T human colon carcinoma xenograft- containing athymic mice. They report that this treatment resulted in reduction of the rate of tumor growth, within little or no toxicity, and that their results demonstrate the potential therapeutic efficacy of radiolabeled COL-1 in clinical trials.
  • Yu et al. J. Clin. Oncol, 14(6): 1798-1809 (1996)
  • 31 I-labeled COL-1 is now in phase 1 clinical trials in patients having gastrointestinal malignancies.
  • conjugates of COL-1 and ⁇ -galactosidase has been shown to specifically kill in vitro tumor cells from a variety of tumor cell lines. Abraham et al, Cell Biophys., 24-25:127-133 (1994).
  • murine antibodies such as COL-1 and other anti-CEA murine antibodies
  • COL-1 and other anti-CEA murine antibodies have applicability as therapeutic agents in humans
  • they are disadvantageous in some respects.
  • murine antibodies are of foreign species origin, they may be immunogenic in humans. This may result in a neutralizing antibody response - a human anti-murine antibody (HAMA) response - which is particularly problematic if the antibodies are desired to be administered repeatedly, e.g., for treatment of a chronic or recurrent disease condition.
  • HAMA human anti-murine antibody
  • This is a significant drawback, as some cancer treatments are effected over a prolonged time period, e.g., over several years or longer.
  • these antibody molecules contain murine constant domains they may not exhibit human effector functions.
  • Chimeric antibodies contain portions of two different antibodies, typically of two different species. Generally, such antibodies contain human constant regions attached to variable regions from another species, typically murine variable regions. For example, some mouse/human chimeric antibodies have been reported which exhibit binding characteristics « of the parental mouse antibody and effector functions associated with the human constant region. See, e.g., U.S. Patent 4,816,567 to Cabiily et al; U.S. Patent 4,978,745 to Shoemaker etal; U.S. Patent 4,975,369 to Beavers etal; and U.S. Patent 4,816,397 to Boss et al.
  • these chimeric antibodies are constructed by preparing a genomic gene library from DNA extracted from pre-existing murine hybridomas. Nishimura etal, Cancer Res., 47:999 (1987). The library is then screened for variable region genes from both heavy and light chains exhibiting the correct antibody fragment rearrangement patterns. Alternatively, cDNA libraries are prepared from RNA extracted from the hybridomas and then screened, or the variable regions are obtained by polymerase chain reaction. The cloned variable region genes are then ligated into an expression vector containing cloned cassettes of the appropriate heavy or light chain human constant region gene. The chimeric genes are then expressed in a cell line of choice, usually a murine myeloma line. Such chimeric antibodies have been used in human therapy.
  • chimerized monoclonal antibodies typically exhibit lesser immunogenicity, they are still potentially immunogenic in humans because they contain murine variable sequences which may elicit antibody responses. Thus, there is the possibility that these chimeric antibodies may elicit an anti-idiotypic response if administered to patients. Saleh et al, Cancer Immunol. Immunother., 32:185-190 (1990).
  • variable light chain (V L ) and variable heavy (V H ) domains have been noted (Colman et al, Nature, 326:358-363 (1987)) and those differences are ostensibly due to variations in the residues involved in the inter-domain contact (Padlan et al, Molec. Immunol, 31:169-217 (1994)).
  • the residues which potentially affect antigen binding fall into several groups.
  • the first group comprises residues that are contiguous with the combining site surface and which could therefore make direct contact with antigens. These residues include the amino- terminal residues and those adjacent to the CDRs.
  • the second group includes residues that could alter the structure or relative alignment of the CDRs by contacting either the CDRs or the opposite chains.
  • the third group comprises amino acids with buried side chains that could influence the structural integrity of the variable domains.
  • the residues in these groups are usually found in the same positions (id.) according to the adopted numbering system. See Kabat etal, Sequences of Proteins of Immunological Interest, NIH Pub. No.
  • humanized antibodies are desirable because of their potential low immunogenicity in humans, their production is unpredictable. For example, sequence modification of antibodies may result in substantial or even total loss of antigen binding affinity, or loss of binding specificity. Alternatively, “humanized antibodies” may still exhibit immunogenicity in humans, irrespective of sequence modification.
  • ⁇ CEA human carcinoembryonic antigen
  • ⁇ CEA anti-CEA
  • pharmaceutical compositions containing humanized ⁇ CEA antibodies It is a more specific object of the invention to provide pharmaceutical compositions containing humanized antibodies derived from the high affinity murine ⁇ CEA antibody, COL-1.
  • compositions for detecting cancer cells the compositions containing a humanized ⁇ CEA antibody, preferably derived from COL-1 , which antibody is in labeled or unlabeled form. It is another object of the invention to provide immunodiagnostic compositions for detecting cancer cells, the compositions containing a humanized ⁇ CEA antibody, preferably derived from COL-1 , which antibody is in labeled or unlabeled form. It is another object of
  • compositions which contain a humanized ⁇ CEA antibody, preferably derived from COL-1 , which is in labeled or unlabeled form.
  • nucleic acid sequences which encode humanized ⁇ CEA antibodies or fragments thereof It is a more specific object of the invention to provide nucleic acid sequences which encode humanized antibodies derived from the high affinity murine ⁇ CEA antibody, COL-1. It is another object of the invention to provide vectors which provide for the expression of humanized ⁇ CEA antibodies, in particular humanized antibodies derived from the high affinity murine ⁇ CEA antibody, COL-1.
  • Figure 1 contains an alignment of the amino acid sequences of murine COL-1 V H (COLI MuVH), a NEWM FR template, and a humanized NEWM-based V H (COL1 NMVH or "HuVH").
  • the CDRs are boxed.
  • Murine FR residues retained in the various humanized VHs exemplified herein are indicated by the symbols ( T) , (A) , (T) , (s) , ( T) , ( A ) , and ( Y ) , according to the table below .
  • HuVHSTAY is the version of COL1NMVH expressed from the deposited cell line, ATCC CRL-12208.
  • Figure 2 contains an alignment of the amino acid sequences of murine COL-1 V ⁇ (COLI MuVK or "HuVK”), an REI FR template, and a humanized REI-based V ⁇ (COL1 REVK). The CDRs are boxed. The murine FR residues retained in the humanized
  • HuVKVL is the version of COL1 REVK expressed from the deposited cell line, ATCC CRL- 12208.
  • Figure 3 shows the lgG1 expression vectors used to express the subject humanized antibodies in NSO myeloma cells.
  • Figure 4 shows binding of different COL-1 antibodies to CEA, as measured by an ELISA assay.
  • Figure 5 contains results of ELISA assays with COL-1 antibodies including those produced according to the invention.
  • Figure 6 contains results of ELISA assays with COL-1 antibodies including those produced according to the invention.
  • Figure 7 contains results of ELISA assays with COL-1 antibodies including those produced according to the invention.
  • Figure 8 contains results of ELISA assays with COL-1 antibodies including those produced according to the invention.
  • Figure 9 contains results of ELISA assays with COL-1 antibodies including those produced according to the invention.
  • Figure 10 contains results of ELISA assays with COL-1 antibodies including those produced according to the invention.
  • Figure 11 contains results of ELISA assays with COL-1 antibodies including those produced according to the invention.
  • Figure 12 contains results of ELISA assays with COL-1 antibodies including those produced according to the invention.
  • Figure 13 contains the amino acid sequences of the humanized V H expressed by the deposited cell line ATCC CRL-12208.
  • Figure 14 contains the amino acid sequences of the humanized V ⁇ expressed by the deposited cell line ATCC CRL-12208.
  • Figure 15 presents the nucleotide sequence of the DNA template use to produce the initial humanized COL-1 heavy chain variable region, HuVH.
  • Figure 16 presents the nucleotide sequence of the DNA template used to produce a variety of HuVH derivatives.
  • Figure 17 presents the nucleotide sequence of the DNA template used to produce the initial humanized COL-1 light chain variable region, HuVK.
  • Figure 18 presents the nucleotide sequence of the DNA template used to produce a the HuVKVL derivative of HuVK.
  • Antibody - This refers to single chain, two-chain, and multi-chain proteins and glycoproteins belonging to the classes of polyclonal, monoclonal, chimeric, and hetero immunoglobulins (monoclonal antibodies being preferred); it also includes synthetic and genetically engineered variants of these immunoglobulins.
  • Antibody fragment includes Fab, Fab', F(ab') 2 , and Fv fragments, as well as any portion of an antibody having specificity toward a desired target epitope or epitopes.
  • Humanized antibody - This will refer to an antibody derived from a non-human antibody, typically murine, that retains or substantially retains the antigen-binding properties of the parent antibody but which is less immunogenic in humans. This may be achieved by various methods including (a) grafting only the non-human CDRs onto human framework and constant regions with or without retention of critical framework residues, or (b) transplanting the entire non-human variable domains, but "cloaking" them with a human-like
  • CDR Complementarity Determining Region
  • CDR refers to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site as delineated by Kabat et al (1991).
  • Framework Region - refers to amino acid sequences interposed between CDRs. These portions of the antibody serve to hold the CDRs in an appropriate orientation for antigen binding.
  • Constant Region The portion of the antibody molecule which confers effector functions.
  • murine constant regions are substituted with human constant regions.
  • the constant regions of the subject chimeric or humanized antibodies are derived from human immunoglobulins.
  • the heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu. Further, heavy chains of various subclasses (such as the IgG subclasses of heavy chains) are responsible for different effector functions and thus, by choosing the desired heavy chain constant region chimeric antibodies with desired effector function can be produced.
  • Preferred constant regions are gamma 1 (lgG1 ), gamma 3 (lgG3) and gamma 4 (lgG4). More preferred is a constant region of the gamma 1 (lgG1 ) isotype.
  • the light chain constant region can be of the kappa or lambda type, preferably of the kappa type.
  • Chimeric antibody - This is an antibody containing sequences derived from two different antibodies, which typically are of different species. Most typically chimeric antibodies comprise human and murine antibody fragments, generally human constant and murine variable regions.
  • Mammals Animals that nourish their young with milk secreted by mammary glands, preferably warm blooded mammals, more preferably humans.
  • Immunogenicity A measure of the ability of a targeting protein or therapeutic moiety to elicit an immune response (humoral or cellular) when administered to a recipient.
  • the present invention is concerned with the immunogenicity of the subject humanized antibodies or fragments thereof.
  • -10- Humanized reduced immunogenicity This refers to a humanized antibody exhibiting reduced immunogenicity relative to the parent antibody, typically a murine antibody such as COL-1.
  • Humanized antibody substantially retaining the binding properties of the parent antibody -
  • the humanized antibody will exhibit the same or substantially the same antigen- binding affinity and avidity as the parent antibody, e.g., COL-1.
  • the affinity of the antibody will not be less than 5% of the parent antibody affinity, more preferably not less than about 30%, and most preferably the affinity will not be less than 50% of the parent antibody.
  • Methods for assaying antigen-binding affinity are well known in the art and include half-maximal binding assays, competition assays, and Scatchard analysis. Suitable antigen binding assays are described in this application.
  • the present invention is directed to humanized antibodies which specifically bind CEA, an antigen expressed by various human cancers, in particular gastrointestinal, colorectal, breast, lung, and ovarian cancers.
  • humanized antibodies will be derived from antibodies having good binding affinity to CEA, such as COL-1 through COL-15 disclosed by Muraro et al, Cancer Res., 45(1 1 Pt. 2):5769- 5780 (1985).
  • such humanized antibodies will be derived from COL-1 , a murine antibody of the lgG2a isotype, which has been reported to bind to CEA with high affinity (1.4x10 9 M '1 ) with no detectable cross-reactivity for CEA-related antigens, such as the nonspecific cross-reacting antigen (NCA) and the normal fecal antigen (NFA).
  • CEA-related antigens such as the nonspecific cross-reacting antigen (NCA) and the normal fecal antigen (NFA).
  • humanized antibodies afford potential advantages over murine and also chimeric antibodies, e.g., reduced immunogenicity in humans. This is advantageous because it should reduce and potentially eliminate the eliciting of a HAMA response when such humanized antibodies are administered in vivo, e.g., either for treatment of cancer or for diagnosis of cancer as by tumor imaging. Also, such antibodies may exhibit improved plasma clearance, pharmacokinetic, and tumor targeting properties. However, as noted above, humanization may in some instances adversely affect antigen binding.
  • the humanized ⁇ CEA antibodies of the present invention will possess a binding affinity for CEA of not less than about 5% and more preferably not less than about 30%, and most preferably not less than 50% of the CEA binding antigen affinity of the parent murine antibody, preferably COL-1.
  • the humanized antibodies will possess a binding affinity for CEA of not less than about 5% and more preferably not less than about 30%, and most preferably not less than 50% of the CEA binding antigen affinity of the parent murine antibody, preferably COL-1.
  • the humanized antibodies will possess a binding affinity for CEA of not less than about 5% and more preferably not less than about 30%, and most preferably not less than 50% of the CEA binding antigen affinity of the parent murine antibody, preferably COL-1.
  • the humanized antibodies will possess a binding affinity for CEA of not less than about 5% and more preferably not less than about 30%, and most preferably not less than 50% of the CEA binding antigen affinity of the parent murine antibody, preferably COL-1.
  • -11- of the present invention will possess a binding affinity for CEA of not less than about 5% and more preferably not less than about 30% and most preferably not less than about 50% of the CEA binding affinity of COL-1 , or a chimeric antibody derived therefrom.
  • the humanized antibodies of the present invention will bind the same epitope as COL-1.
  • Such antibodies can be identified based on their ability to compete with COL-1 for binding to CEA or to CEA-expressing cells.
  • the subject humanized antibodies are produced by obtaining nucleic acid sequences encoding the variable heavy (V H ) and variable light chains (V L , e.g., V ⁇ ) of an antibody which binds CEA (preferably COL-1), identifying the CDRs in said V H and V L sequences, and grafting such CDR-encoding nucleic acid sequences onto selected human framework-encoding nucleic acid sequences.
  • V H variable heavy
  • V L variable light chains
  • the human framework amino acid sequences are selected such that the resulting antibody is likely to be suitable for in vivo administration in humans. This can be determined, e.g., based on previous usage of antibodies containing such human FRs. Preferably, the human FRs will not themselves be significantly immunogenic. Examples of such human frameworks include NEWM and REI.
  • the amino acid sequences of the FRs of the antibody to be humanized (e.g., COL-1 ) will be compared to those of known human FRs, and the human FRs to be used for CDR-grafting will be selected based on their comprising sequences highly similar to those of the parent antibody, e.g., a murine antibody which binds CEA.
  • a murine antibody which binds CEA.
  • the FRs of REI and NEWM antibodies have been identified as having amino acid sequences which are likely to allow the CDRs of COL-1 To retain a significant degree of antigen binding affinity.
  • the selected human framework regions will preferably be those that are expected to be suitable for in vivo administration, i.e., not immunogenic. Based on their amino acid sequences, REI and NEWM human framework regions are expected to be substantially non-immunogenic.
  • the DNA sequences encoding the V H and V L regions of the preferably murine ⁇ CEA antibody must be obtained.
  • Methods for cloning nucleic acid sequences encoding immunoglobulins are well known in the art. Such methods will generally involve the amplification of the immunoglobulin-encoding sequences to be cloned
  • the amino acid sequences encoding the CDRs are then identified (deduced based on the nucleic acid sequences and the genetic code and by comparison to previous antibody sequences) and the CDR-encoding nucleic acid sequences are grafted onto selected human FR-encoding sequences. This may be accomplished by use of appropriate primers and linkers. Methods for selecting suitable primers and linkers to provide for ligation of desired nucleic acid sequences is well within the purview of the ordinary artisan.
  • the selected human FRs used for humanization will preferably be those that are likely to be suitable for in vivo administration, i.e. they are not in themselves immunogenic in humans (e.g., because of aliotypic differences); examples thereof are REI and NEWM human FRs.
  • the human FRs will be selected such that they comprise amino acid sequences which are highly similar to those of the parent antibody's FR sequences. This may be effected by comparing the amino acid sequences of the murine FRs to those of previously reported human FRs (see, e.g., Kabat et al, id.).
  • the resultant DNA sequences encoding the "humanized" variable heavy and variable light sequences will then be expressed to produce a humanized Fv or humanized antibody which binds CEA.
  • the humanized V H and V L sequences will be expressed as part of a whole ⁇ CEA antibody molecule, i.e. as a fusion protein with human constant domain sequences whose encoding DNA sequences have been obtained from a commercially available library or which have been obtained using, e.g., one of the above-described methods for obtaining DNA sequences.
  • the V H and V L sequences can also be expressed in the absence of constant sequences to produce a
  • DNA sequences which encode the subject humanized V L and V H sequences are synthesized, and then expressed in a vector system suitable for expression of recombinant antibodies. This may be effected in any vector system which provides for the subject humanized V L and V H sequences to be expressed as a fusion protein with human constant domain sequences and to associate to produce functional (antigen binding) antibodies or antibody fragments.
  • Useful methods are set forth, e.g., in U.S. Patent 4,816,397 to Boss et al. and U.S. Patent 5,225,539 to Winter et al.
  • vectors suitable for expression of recombinant antibodies are commercially available.
  • the vector may, e.g., be a bare nucleic acid segment, a carrier-associated nucleic acid segment, a nucleoprotein, a plasmid, a virus, a viroid, or a transposable element.
  • Host cells known to be capable of expressing functional immunoglobulins include, e.g.: mammalian cells such as Chinese Hamster Ovary (CHO) cells; COS cells; myeloma cells, such as NSO and SP2/O cells; bacteria such as Esche ⁇ chia coli; yeast cells such as Saccharomyces cerevisiae; and other host cells.
  • mammalian cells such as Chinese Hamster Ovary (CHO) cells
  • COS cells such as myeloma cells, such as NSO and SP2/O cells
  • bacteria such as Esche ⁇ chia coli
  • yeast cells such as Saccharomyces cerevisiae
  • other host cells e.g., CHO cells are used by many
  • NSO cells are one of the preferred types of host cells useful in the present invention.
  • recombinant expression of humanized antibodies is obtained by one of two general methods.
  • the host cells are transfected with a single vector which provides for the expression of both V H and V L variable sequences optionally fused to selected constant regions.
  • the second method host cells are transfected with two vectors, each of which provides for expression of either the V H or V L sequence, each optionally fused to a selected constant region.
  • Human constant domain sequences are well known in the art, and have been reported in the literature.
  • Preferred human constant light chain sequences include the kappa and lambda constant light sequences.
  • Preferred human constant heavy chain sequences include human gamma 1 , human gamma 2, human gamma 3, human gamma 4, and mutated versions thereof which provide for altered effect or function, e.g., enhanced in vivo half-life, reduced Fc receptor binding, and the like.
  • the antigen binding affinity of the resultant humanized antibody will be assayed by known methods, e.g., Scatchard analysis.
  • the antigen-binding affinity of the humanized antibody will approximate that of the parent antibody, e.g., COL-1.
  • the affinity of the humanized antibody will not be less than 5% of the parent antibody, more preferably not less than 30%, and most preferably not less than 50% of that of the parent antibody, e.g., COL-1.
  • humanized antibodies produced by grafting CDRs (from an antibody which binds CEA) onto selected human FRs may provide humanized antibodies having the desired affinity to CEA.
  • those framework residues of the parent (e.g., murine) antibody which maintain or affect combining-site structures will be retained. These residues may be identified by X-ray crystallography of the parent antibody or Fab fragment, thereby identifying the three-dimensional structure of the antigen-binding site. These residues may potentially be identified by X-ray crystallography of the parent
  • framework residues which may be involved in antigen binding may be putatively selected based on previously reported humanized murine antibody sequences. Thus, it may be beneficial to retain these and other murine framework residues from the parent murine
  • the present invention further embraces variants and equivalents which are substantially homologous to the humanized antibodies and antibody fragments set forth herein. These may contain, e.g., conservative substitution mutations, i.e. the substitution of one or more amino acids by similar amino acids.
  • conservative substitution refers to the substitution of an amino acid with another within the same general class, e.g., one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art.
  • the phrase "substantially homologous" is used in regard to the similarity of a subject amino acid sequence (of an oligo- or poly-peptide or protein) to a related, reference amino acid sequence.
  • This phrase is defined as at least about 75% "correspondence" ⁇ i.e. the state of identical amino acid residues being situated in parallel - between the subject and reference sequences when those sequences are in "alignment,” i.e. when a minimal number of "null” bases have been inserted in the subject and/or reference sequences so as to maximize the number of existing bases in correspondence between the sequences.
  • "Null" bases are not part of the subject and reference sequences; also, the minimal number of "null” bases inserted in the subject sequence may differ from the minimal number inserted in the reference sequence.
  • a reference sequence is considered "related" to a subject sequence where both amino acid sequences make up proteins or portions of proteins which are either ⁇ CEA antibodies or antibody fragments with ⁇ CEA binding affinity.
  • Each of the proteins comprising these ⁇ CEA antibodies or antibody fragments may independently be antibodies or antibody fragments or bi- or multi-functional proteins, e.g., such as fusion proteins, bi- and multi-specific antibodies, single chain antibodies, and the like.
  • the present invention is further directed to nucleic acid sequences from which such humanized antibodies and antibody fragments may be expressed, as well as expression vectors from which these humanized antibodies and antibody fragments may be expressed in transfected host cells.
  • such humanized antibodies and corresponding nucleic acid sequences will be derived from COL-1.
  • the humanized V H sequence and the humanized V L sequence will have the sequences substantially as depicted in Figure 1 or 13 or in Figure 2 or 14, respectively, and as discussed in the Examples set forth below.
  • the invention further contemplates other modifications of these humanized V H and V L sequences, e.g., sequences which further comprise one or more conservative amino acid substitutions or which retain one or more additional murine framework residues which affect or do not significantly reduce antigen binding.
  • the subject humanized antibodies because they specifically bind CEA (an antigen expressed on many different cancer cell types, e.g., lung carcinomas, breast carcinomas, gastrointestinal carcinomas such as stomach cancers, colorectal carcinomas such as colon cancers, ovarian carcinomas, etc.) and further because they will not be significantly immunogenic in humans -- should be suitable for use as: therapeutics for the treatment or prevention of cancers characterized by CEA expression; diagnostic agents, e.g., for diagnosis and evaluating the prognosis of cancers characterized by CEA expression (based on levels of CEA expression); tumor imaging agents; or radiolabeled antibodies in the Radioimmunoguided Surgery® System (RIGS®). See Hinkle etal, Antibody, Immunoconjugates and Radiopharmaceuticals, 4(3):339-358 (1991).
  • CEA an antigen expressed on many different cancer cell types, e.g., lung carcinomas, breast carcinomas, gastrointestinal carcinomas such as stomach cancers, colorectal carcinomas such as colon cancers,
  • an effective dosage will be in the range of about 0.05 to 100 milligrams per kilogram body weight per day.
  • the humanized antibodies or humanized antibody fragments of the invention may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect.
  • the antibodies of the subject invention can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody of the invention with a conventional, pharmaceutically acceptable carrier, diluent, and/or excipient according to known techniques. It will be recognized by one of ordinary skill in the art that the form and character of the pharmaceutically acceptable carrier, diluent, and/or excipient is dictated by the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables.
  • compositions may include, e.g., a suitable solvent, preservatives such as benzyl alcohol if desired, and a buffer.
  • a suitable solvent e.g., benzyl alcohol if desired
  • a buffer e.g., benzyl alcohol
  • Suspension- type formulations may include a liquid suspending medium as a carrier, e.g., aqueous polyvinylpyrrolidone, inert oils such as vegetable oils or highly refined mineral oils, or aqueous cellulose ethers such as aqueous carboxymethylcellulose.
  • a thickener such as gelatin or an alginate may also be present, one or more natural or synthetic surfactants or antifoam agents may be used, and one or more suspending agents such as sorbitol or another sugar may be employed therein.
  • Such formations may contain one or more adjuvants.
  • the route of administration of the antibodies (or fragment thereof) of the present invention may be oral, parenteral, by inhalation, or topical.
  • parenteral as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, or intraperitoneal administration.
  • the subcutaneous, intravenous, and intramuscular forms of parenteral administration are generally preferred.
  • the daily parenteral and oral dosage regimens for prophylactically or therapeutically employing humanized antibodies of the present invention will generally be in the range of about 0.005 to 100, but preferably about 0.5 to 10, milligrams per kilogram body weight per day.
  • the antibodies of the present invention may also be administered by inhalation.
  • inhalation is meant intranasal and oral inhalation administration.
  • Appropriate dosage forms for such administration such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.
  • the preferred dosage amount of a compound of the invention to be employed is generally within the range of about 0.1 to about 100, more preferably about 10 to 100, milligrams per kg body weight.
  • the antibody of the invention may also be administered topically.
  • topical administration is meant non-systemic administration.
  • a humanized antibody (or humanized antibody fragment) formulation of the invention externally to the epidermis or to the buccal cavity, and instillation of such an antibody into the ear, eye, or nose, and wherever it does not significantly enter the bloodstream.
  • systemic administration is meant oral, intravenous, intraperitoneal, subcutaneous, and intramuscular administration.
  • the amount of an antibody required for therapeutic, prophylactic, or diagnostic effect will, of course, vary with the antibody chosen, the nature and severity of the condition being treated and the animal undergoing treatment, and is ultimately at the
  • a suitable topical dose of an antibody of the invention will generally be within the range of about 1 to 100 milligrams per kilogram body weight daily.
  • the active ingredient may comprise, for topical administration, from 0.001 % to 10% w/w, e.g., from 1 % to 2% by weight of the formulation, although it may comprise as much as 10% w/w but preferably not in excess of 5% w/w and more preferably from 0.1% to 1% w/w of the formulation.
  • the topical formulations of the present invention comprise an active ingredient together with one or more acceptable carrier(s) therefor and optionally any other therapeutic ingredients(s).
  • the carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear, or nose.
  • Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent.
  • the resulting solution may then be clarified and sterilized by filtration and transferred to the container by an aseptic technique.
  • bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01 %).
  • Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
  • Lotions according to the present invention include those suitable for application to the skin or eye.
  • An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops.
  • Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
  • Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing
  • the basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogels.
  • hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogels.
  • the formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surface active such as sorbitan esters or polyoxyethylene derivatives thereof.
  • suitable surface active agent such as an anionic, cationic or non-ionic surface active such as sorbitan esters or polyoxyethylene derivatives thereof.
  • Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
  • Kits according to the present invention include frozen or lyophilized humanized antibodies or humanized antibody fragments to be reconstituted, respectively, by thawing (optionally followed by further dilution) or by suspension in a (preferably buffered) liquid vehicle.
  • the kits may also include buffer and/or excipient solutions (in liquid or frozen form) - - or buffer and/or excipient powder preparations to be reconstituted with water - for the purpose of mixing with the humanized antibodies or humanized antibody fragments to produce a formulation suitable for administration.
  • kits containing the humanized antibodies or humanized antibody fragments are frozen, lyophilized, pre-diluted, or pre-mixed at such a concentration that the addition of a predetermined amount of heat, of water, or of a solution provided in the kit will result in a formulation of sufficient concentration and pH as to be effective for in vivo or in vitro use in the treatment or diagnosis of cancer.
  • a kit will also comprise instructions for reconstituting and using the humanized antibody or humanized antibody fragment composition to treat or detect cancer.
  • the kit may also comprise two or more component parts for the reconstituted active composition.
  • a second component part - in addition to the humanized antibodies or humanized antibody fragments - may be bifunctional chelant, bifunctional chelate, or a therapeutic agent such as a radionuclide, which when mixed with the humanized antibodies or humanized antibody fragments forms a conjugated system therewith.
  • a therapeutic agent such as a radionuclide
  • the optimal course of treatment i.e., the number of doses of an antibody or fragment thereof of the invention given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
  • the subject humanized antibodies may also be administered in combination with other anti-cancer agents, e.g., other antibodies or drugs. Also, the subject humanized antibodies or fragments may be directly or indirectly attached to effector moieties having therapeutic activity.
  • Suitable effector moieties include by way of example cytokines (IL-2, TNF, interferons, colony stimulating factors, IL-1 , etc.), cytotoxins (Pseudomonas exotoxin, ricin, abrin, etc.), radionuclides, such as 90 Y, 131 l, 99m Tc, l11 ln, 125 l, among others, drugs
  • the subject humanized ⁇ CEA antibodies or fragments may be used as immunodiagnostic agents both in vivo and in vitro.
  • a particularly preferred usage is for in vivo imaging of cancer cell lesions which express CEA.
  • the subject antibodies are preferred because they should elicit no significant HAMA or allergic response. Thus, they may be used repeatedly to monitor the disease status of a patient.
  • Radioimmunoguided System® RIGS®
  • This technique involves the intravenous administration of a radiolabeled monoclonal antibody or its fragment prior to surgery. After allowing for tumor uptake and blood clearance of radioactivity, the patient is taken to the operating room where surgical exploration is effected with the aid of a hand-held gamma activity probe, e.g., the Neoprobe® 1000. This helps the surgeon identify the tumor metastases and lessen the complications of excision.
  • the RIGS® system is advantageous because it allows for the detection of tumors not otherwise detectable by visual inspection and/or palpation. See, O'Dwyer et al, Arch. Surg.,
  • the subject humanized antibodies or fragments thereof may be radiolabeled with radionuclides which are suitable for in vivo administration, e.g., iodine radionuclides such as 131 l and 125 l. Moreover, 111 ln and 99m Tc are also suitable.
  • the subject humanized antibodies may be used alone or in combination with other antibodies. Also, the subject humanized antibodies may be prepared in the form of a diagnostically effective composition. Generally, this will entail the incorporation of diagnostically acceptable carriers and excipients, and labels which provide for detection.
  • Suitable labels include diagnostic radionuclides, enzymes, etc. Methods for using antibodies for tumor imaging are well known in the art.
  • the source of the NEWM framework regions for producing the initial humanized COL-1 VH was an M13 construct bearing -- between the M13 SamH I and Hind III sites -- a DNA segment having the nucleotide sequence shown in figure 13.
  • the source of REI framework regions for producing the initial humanized COL-1 VL was an M13 construct bearing -- between the M13 BamH I and Hind III sites -- a DNA segment encoding the REI amino acid sequence of Figure 2.
  • M13 DNA sequence double stranded M13 DNA was utilized.
  • extension-ligation procedures were used instead, the oligonucleotides were designed to anneal to only one of the two DNA strands.
  • the M13 DNA was first treated to substitute uridine for every thymidine base in the DNA, to produce uridinylated DNA. This was
  • Hercules, CA are also suitable) by combining the following ingredients.
  • the culture was shaken for 5 hours at 37°C and the resulting single-stranded plasmid
  • DNA was isolated and dissolved in 50 ⁇ L Tris-EDTA buffer. The DNA was then treated with uracil glycosylase by mixing together:
  • oligonucleotide primers were used throughout the process of preparing the humanized COL-1 VHs and VLs exemplified below.
  • antibodies having a chimeric heavy chain i.e. a heavy chain having a murine COL-1 VH region and a human lgG1 constant region
  • a chimeric light chain i.e. a light chain having a murine COL-1 VL and a human K constant region
  • T4 polynucleotide kinase (Gibco BRL).
  • the phosphorylation reaction was started with the addition of the enzyme and allowed to proceed for one hour at 37°C.
  • the annealing step for non-overlap extension-ligations involved performing one annealing in which all mutation-carrying oligonucleotides and one primer oligonucleotide were annealed to a single stranded DNA template in which all thymidine bases had been replaced with uridine bases.
  • the mutating oligonucleotides were first phosphorylated according to the above oligonucleotide phosphorylation protocol. In a final volume of 20 ⁇ L, the following ingredients were combined:
  • the mixture was then heated to 90°C for 30 sec, then quickly cooled to 70°C, and finally allowed to slowly cool to 37°C.
  • extension-ligation was performed as follows. In a final volume of 30 ⁇ L, the following ingredients were combined:
  • Reactions were initiated with the addition of the DNA polymerase and then treated with about 15 cycles of: (1 ) 94°C for 30 sec, (2) 50°C for 30 se , and (3) 30-60 seconds at either 75°C (for Vent DNA polymerase) or 72°C (for Thermalase). Reactions were brought
  • Humanized antibodies were expressed in pSV vectors grown in NSO cells as follows.
  • the humanized variable region constructs which were produced in the plasmid, M13 were obtained by ethanoi precipitating the M13 plasmids (as cytosolic DNA), redissolving them in aqueous solution, and digesting them with 10U each of Hind III and SamH I (both from BRL, i.e. Gibco BRL) for 1 hour at 37°C in a final volume of 100 ⁇ L with Tris-EDTA buffer.
  • the resulting DNA fragments were then run on a low melting point agarose gel, the band containing the humanized construct DNA was cut out, and the DNA was purified using an ELUTIP 'd' column with 20 ⁇ L Tris-EDTA buffer. 10 ⁇ L of the purified DNA preparation was then combined with 1 ⁇ L of a Hind III and BamH l-digested pSV preparation, 3 ⁇ L of 5x ligase buffer, and 1 U of T4 DNA ligase (BRL), in order to insert the construct into a pSV plasmid.
  • a Hind III and BamH l-digested pSV preparation 3 ⁇ L of 5x ligase buffer
  • 1 U of T4 DNA ligase (BRL) T4 DNA ligase
  • Humanized COL-1 VH constructs were inserted into pSVgpt vectors bearing a human lgG1 heavy chain constant region; the pSVgpt vector used is the "aLYS-30" shown in Figure 5.
  • Humanized COL-1 VL constructs were inserted into pSVhyg vectors bearing a human K light chain constant domain; the pSVhyg vector used in the "aLys-17" shown in
  • Each humanized variable region construct was inserted adjacent to the respective constant region, i.e. so as to replace either the HuVHLYS or the HuVLLys segment illustrated in Figure 5.
  • the resulting vectors were transfected into NSO cells as follows. About 3 ⁇ g of the VH vector, or about 6 ⁇ g of the VL vector, produced by the pSV-insertion procedures, was then linearized by digestion with 10U Pvu ⁇ (Gibco BRL). The digested DNA was then precipitated with ethanoi and redissolved in 50 ⁇ L of water. NSO cells were collected by centrifugation and resuspended in 0.5mL Dulbecco's Modified Eagle's Medium (DMEM) and then transferred to a Gene Pulser cuvette (Bio-Rad). The DNA from both one VH and one VL construct was gently mixed with the cells by pipetting and the cuvette was left on ice for 5 minutes.
  • DMEM Dulbecco's Modified Eagle's Medium
  • the cuvette was inserted between the electrodes of the Bio-Rad Gene Pulser and a single pulse of 170V at 960 ⁇ F was applied.
  • the contents of the cuvette were then transferred to a flask containing 20mL DMEM and the cells were allowed to rest for 1-2 days at 37°C. Cells were again harvested by centrifugation and resuspended in 36mL selective DMEM. 1.5mL aliquots of this resuspension were placed in each well of a 24-well plate and
  • the concentration of IgG secreted from transfected cells was measured using an enzyme-linked immunosorbent assay (ELISA) procedure which is set forth as follows.
  • ELISA enzyme-linked immunosorbent assay
  • Polyvinyl chloride (PVC) microtiter plates (Dynatech Laboratories, Chantilly, VA, catalog # 001-010-2101 ) were coated with goat anti-human IgG (10mg/mL, GAHIG, Southern Biotechnology Associates, Inc., Birmingham, AL, catalog # 2010-01 ) diluted with Milli-Q® water and placed on the plates using 50mL/well. Plates were air-dried overnight at ambient temperature or at 37°C for 3 hours. Prior to use, non-specific binding was blocked the addition of 0.2 mlJweil of 1% (w/v) bovine serum albumin (Sigma, St.
  • PBS/BSA phosphate buffered saline
  • All incubations were carried out in a humidified container. Plates were incubated for 1 -2 hours at 37°C and the blocking solution removed prior to sample addition. Two-fold serial dilutions of samples or a standard IgG solution set at 500 ng/mL (50 ⁇ l/well) were made in triplicate in the PBS/BSA solution. The plate was incubated at 37°C for 3 hours or overnight at 4°C.
  • the plate was washed 3-times with 0.025% Tween-20 (v/v, Sigma) using an automatic plate washer. 50 ⁇ l/well of 1 :1000 dilution of a goat anti-human IgG conjugated to Horseradish Peroxidase (Southern Biotechnology Associates Inc.) was added and incubated at 37°C for 1.5 hours. The wells were washed 3 times with 0.025% Tween-20 (v/v, Sigma) using an automatic plate washer and 50 ⁇ l/well OPD substrate buffer added. The color was developed for 4 minutes, stopped with 12.5 ⁇ l 12.5% H 2 SO 4 and the absorbance at 492 nm read. The concentration of IgG in the test sample was estimated by comparison of the mean of the optical densities to a standard curve constructed from the standard IgG.
  • Two-fold serial dilutions (starting concentration range of 1.0 ⁇ g/ml - 10 ⁇ g/ml) of the samples to be tested in the PBS/BSA solution were added to triplicate wells of the TAG-coated plate (50 ⁇ l/well). The plate was incubated overnight at 4°C or 1 -2 hours at 37°C. The plate was washed 3-times with 0.025% Tween-20 (v/v, Sigma) using an automatic plate washer. 50 ⁇ l/well of 1 :1000 dilution of a goat anti-human IgG conjugated to Horseradish Peroxidase (Southern Biotechnology Associates Inc.) was added and incubated at 37°C for 1.5 hours.
  • An 125 l-labeled goat anti-human IgG probe (ICN Biomedicals, Inc., catalog # 68088) was diluted to 75,000 cpm/25 ⁇ l in PBS/BSA and added (25 ⁇ l/well) to all wells. This TAG plate was incubated for 1 hour at 37°C.
  • the trap plate was washed as described above and 125 l-labeled GAHIG probe was added. This plate was incubated for 1 hour at 37°C.
  • This NEWM-grafted humanized COL-1 V H was accomplished using two rounds of a dual PCR procedure: the standard PCR protocol (for overlap-extension) followed by PCR amplification with oligonucleotide primers 10 and 11. This procedure was carried out as described above using a single-stranded M13 DNA template bearing, between the Hind III and BamH I sites thereof, a DNA segment having the nucleotide sequence shown in Figure 13.
  • the mutating oligonucleotides used in the first round (with Vent DNA polymerase)were designed and synthesized with the following sequences:
  • the production of the REI-grafted humanized COL-1 V L was accomplished by using two rounds of a procedure involving performing the annealing and extension-ligation protocols described above - followed by amplification by the standard Thermalase PCR protocol -- using a ssM13 template bearing, between the Hind III and BamH I sites thereof, a ssU-DNA segment produced by the above procedure using the REIVK sequence shown in Figure 17 (or in the second round, the mutated M13 template resulting from the first round).
  • the mutating oligonucleotides used in round number 1 were designed and synthesized with the following sequences:
  • a second humanized COL-1 VL was made by the same procedure using the following mutating oligonucleotides.
  • the resulting VL was termed "HuVKF.”
  • HuVHT (using the HuVH DNA shown in Figure 16 as a template) 954. 5'-GACAATGCTGACAGACACCAGCAA-3' and 955. 5'-TGCTGGTGTCTGTCAGCATTGTCA-3';
  • HuVHS (using the HuVH DNA as a template) 684. 5'-CACCAGCAGCAACCAGTTCAG-3' and 683. 5'-ACTGGTTGCTCGTGGTGTCTA-3',
  • HuVHSTAY (using the HuVHS DNA as a template) 1026. 5'-ACCAGCAGCAACACAGCCTACCTGAGACTCAGCAG-3' and 1028. 5'-TGCTGAGTCTCAGGTAGGCTGTGTTGCTGCTGGTGT-3';
  • HuVHA using the HuVH DNA as a template
  • HuVHAA using the HuVHA DNA as a template
  • HuVHAT (using the HuVHA DNA as a template) 1071. 5'-CCTGAGACTCAGCAGCGTGACAG-3' and
  • HuVHAY (using the HuVHA DNA as a template) 1071. 5'-CCTGAGACTCAGCAGCGTGACAG-3' and 1073. 5'-CGCTGCTGAGTCTCAGGTAGAACTGGTTCTTGC-3';
  • HuVHATAY (using the HuVHA DNA as a template) 1071. 5'-CCTGAGACTCAGCAGCGTGACAG-3' and
  • HuVHASTAY (using the HuVHS DNA as a template)
  • HuVKVL (using the HuVK DNA shown in Figure 18 as a template)
  • the above-produced M13 DNA constructs were transferred to pSV vectors which were then transfected into NSO host cells, according to the above-described procedures.
  • the VH construct- and VL construct-containing pSV vectors were transfected into NSO cells in the following combinations (to produce antibodies having the indicated combinations of VH and
  • HuVH/HuVKVL HuVHS/HuVKVL;
  • HuVHSTAY/HuVKVL Combinations of the other VHs with the various VKs are expected to behave in an analogous manner, though having an unpredictable variation in degree of binding. Final selection of the optimum combination will be a function of obtaining an antibody having the best, selective antigen binding properties with the fewest murine amino acid substitutions.
  • the humanized antibodies disclosed herein exhibit antigen-binding characteristics, i.e., CEA affinities comparable to the parent monoclonal antibody, nCOL-1 (murine antibody), and to chimeric antibodies derived from nCOL-1 , e.g., ChCOL-1 ⁇ 1 (ATCC No. CRL 11217).
  • these antibodies possess properties which will render them well suited for usage as in vivo diagnostics or therapeutics, e.g., improved serum clearance, metabolic properties, and little or no immunogenicity in humans.
  • the subject humanized antibodies will enable the subject humanized antibodies to be administered repeatedly, in large dosages, and over a prolonged period of time without significant adverse effects, e.g., a HAMA response or non-specific cytotoxicity. This is important for cancer treatment as well as for cancer diagnosis as it enables these antibodies to be used over prolonged time periods.
  • the clearance properties of the subject human antibodies will enable these antibodies to effectively target desired target sites, e.g., CEA expressing carcinomas (because of the effects of serum clearance on targeting efficiency). Therefore, the humanized antibodies of the present invention comprise a substantial improvement in relation to previously disclosed antibodies specific to CEA.
  • Other embodiments of the invention will be apparent to those skilled in the art from a consideration of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.
  • NAME The Dow Chemical Company STREET: 1790 Bldg. Washington Street CITY : Midland STATE : MI COUNTRY: U.S.A. POSTAL CODE: 48674 TELEPHONE: 517-636-1687 TELEFAX: 517-638-9786
  • Gly Trp lie Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60
  • Gly Trp lie Asp Pro Glu Asn Xaa Xaa Xaa Xaa Tyr Ala Pro Lys Phe 50 55 60
  • NAME/KEY Humanized COL-1 VH, HuVH LOCATION: 1..124
  • NAME/KEY Humanized COL-1 VH, HuVHA LOCATION: 1..124
  • Gly Trp lie Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60
  • NAME/KEY Humanized COL-1 VH, HuVHAT LOCATION: 1..124
  • Gly Trp lie Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60
  • NAME/KEY Humanized COL-1 VH, HuVHAA LOCATION: 1..124
  • Gly Trp lie Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60
  • NAME/KEY Humanized COL-1 VH, HuVHAY LOCATION: 1..124
  • Gly Trp lie Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60
  • NAME/KEY Humanized COL-1 VH, HuVHATAY LOCATION: 1..124
  • Gly Trp lie Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60
  • NAME/KEY Humanized COL-1 VH, HuVHASTAY LOCATION: 1..124
  • Gly Trp lie Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60
  • NAME/KEY Humanized COL-1 VH, HuVHT LOCATION: 1..124
  • Gly Trp lie Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60
  • NAME/KEY Humanized COL-1 VH, HuVHS LOCATION: 1..124
  • NAME/KEY Humanized COL-1 VH, HuVHSTAY LOCATION: 1..124
  • Gly Trp lie Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe 50 55 60
  • Lys Leu Leu lie Tyr Leu Ala Ser Asn Leu Gin Ser Gly Val Pro Ala 50 55 60
  • NAME/KEY Humanized COL-1 VK, HuVK LOCATION: 1..110
  • Lys Leu Leu lie Tyr Leu Ala Ser Asn Leu Gin Ser Gly Val Pro Ser 50 55 60
  • NAME/KEY Humanized COL-1 VK, HuVKVL LOCATION: 1..110
  • Lys Leu Leu lie Tyr Leu Ala Ser Asn Leu Gin Ser Gly Val Pro Ser 50 55 60
  • NAME/KEY Humanized COL-1 VK, HuVKF LOCATION: 1..110
  • Lys Leu Leu lie Tyr Leu Ala Ser Asn Leu Gin Ser Gly Val Pro Ser 50 55 60
  • NAME/KEY Humanized COL-1 VH, ATCC CRL-12208 VH LOCATION: 1..125
  • Gly Trp lie Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Gly Phe 50 55 60
  • NAME/KEY Humanized COL-1 VK, ATCC CRL-12208 VK LOCATION: 1..110
  • Lys Leu Leu lie Tyr Leu Ala Ser Asn Leu Gin Ser Gly Val Pro Ser 50 55 60
  • NAME/KEY Template used to produce HuVH variants LOCATION: 1..372
  • NAME/KEY Template used to produce HuVK variants LOCATION: 1..330
  • SEQ ID NO: 34 TTCTACTCAC GTGTGATTTG CAGCTTGGTC CCTTGGCCGA ACGTAGGAAG CTCCCTACTG 60 TGCTGGCAGT AG 72

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Public Health (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Epidemiology (AREA)
  • Urology & Nephrology (AREA)
  • General Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Optics & Photonics (AREA)
  • Hospice & Palliative Care (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention concerne de nouveaux anticorps monoclonaux humanisés ainsi que des fragments ou des dérivés de ces anticorps, lesquels se lient de manière spécifique à l'antigène carcino-embryonnaire (ACE). Cette invention concerne également des procédés de production des ces anticorps et de leurs fragments ou dérivés. Ces anticorps humanisés sont utiles dans le traitement des cancers qui expriment l'ACE, ainsi qu'à des fins diagnostiques comme, par exemple, l'imagerie in vivo de tumeurs ou de cellules cancéreuses qui expriment l'ACE.
PCT/US1998/003680 1998-02-25 1998-02-25 Anticorps monoclonaux anti-ace humanises et faisant preuve d'une grande affinite WO1999043817A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
BR9815775-2A BR9815775A (pt) 1998-02-25 1998-02-25 Anticorpos monoclonais anti-antìgeno carcinoembriÈnico (cea) humanizados de elevada afinidade
PCT/US1998/003680 WO1999043817A1 (fr) 1998-02-25 1998-02-25 Anticorps monoclonaux anti-ace humanises et faisant preuve d'une grande affinite
AU63388/98A AU752494B2 (en) 1998-02-25 1998-02-25 High affinity humanized ANTI-CEA monoclonal antibodies
CA002321947A CA2321947A1 (fr) 1998-02-25 1998-02-25 Anticorps monoclonaux anti-ace humanises et faisant preuve d'une grande affinite
IL13802298A IL138022A0 (en) 1998-02-25 1998-02-25 High affinity humanized anti-cea monoclonal antibodies
EP98907630A EP1056859A1 (fr) 1998-02-25 1998-02-25 Anticorps monoclonaux anti-ace humanises et faisant preuve d'une grande affinite
JP2000533557A JP2002504372A (ja) 1998-02-25 1998-02-25 高親和性ヒト型化抗−ｃｅａモノクロ−ナル抗体
NO20004251A NO20004251L (no) 1998-02-25 2000-08-24 Humaniserte anti-CEA monoklonale antistoffer med høy affinitet

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1998/003680 WO1999043817A1 (fr) 1998-02-25 1998-02-25 Anticorps monoclonaux anti-ace humanises et faisant preuve d'une grande affinite

Publications (1)

Publication Number Publication Date
WO1999043817A1 true WO1999043817A1 (fr) 1999-09-02

Family

ID=22266464

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/003680 WO1999043817A1 (fr) 1998-02-25 1998-02-25 Anticorps monoclonaux anti-ace humanises et faisant preuve d'une grande affinite

Country Status (7)

Country Link
EP (1) EP1056859A1 (fr)
JP (1) JP2002504372A (fr)
AU (1) AU752494B2 (fr)
CA (1) CA2321947A1 (fr)
IL (1) IL138022A0 (fr)
NO (1) NO20004251L (fr)
WO (1) WO1999043817A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003025565A3 (fr) * 2001-09-21 2003-05-30 Caprion Pharmaceuticals Inc Preparation de membranes plasmiques fortement purifiees
US9556438B2 (en) 2005-05-27 2017-01-31 Fondazione Centro San Raffaele Del Monte Tabor Gene vector
US10316104B2 (en) 2011-04-29 2019-06-11 Roche Glycart Ag Immunoconjugates
CN111533805A (zh) * 2016-12-04 2020-08-14 深圳市国创纳米抗体技术有限公司 一种抗癌胚抗原的高亲和力纳米抗体及其应用
WO2020259550A1 (fr) 2019-06-26 2020-12-30 江苏恒瑞医药股份有限公司 Anticorps anti-cea et son utilisation
US11433100B2 (en) 2020-08-20 2022-09-06 A2 Biotherapeutics, Inc. Compositions and methods for treating ceacam positive cancers
US11602543B2 (en) 2020-08-20 2023-03-14 A2 Biotherapeutics, Inc. Compositions and methods for treating mesothelin positive cancers
US12435124B2 (en) 2022-07-22 2025-10-07 A2 Biotherapeutics, Inc. Compositions and methods for treating CEACAM positive cancers

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012117002A1 (fr) * 2011-03-02 2012-09-07 Roche Glycart Ag Anticorps anti-cea

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001059A1 (fr) * 1990-07-05 1992-01-23 Celltech Limited Anticorps diriges contre l'antigene carcino-embryonnaire (cea) et a greffe de zones determinant la complementarite (cdr) et production de ces anticorps
WO1994019466A2 (fr) * 1993-02-16 1994-09-01 The Dow Chemical Company Nouvelle famille d'anticorps chimeres diriges contre l'antigene carcino-embryonnaire
WO1996011013A1 (fr) * 1994-10-05 1996-04-18 Immunomedics, Inc. Anticorps monoclonaux de souris humanises humain anti-cea, de type iii greffes a la region determinante de complementarite (cdr)
WO1997042329A1 (fr) * 1996-05-04 1997-11-13 Zeneca Limited Anticorps monoclonal anti-cea, conjugues contenant cet anticorps et leur utilisation therapeutique dans un systeme adept

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001059A1 (fr) * 1990-07-05 1992-01-23 Celltech Limited Anticorps diriges contre l'antigene carcino-embryonnaire (cea) et a greffe de zones determinant la complementarite (cdr) et production de ces anticorps
WO1994019466A2 (fr) * 1993-02-16 1994-09-01 The Dow Chemical Company Nouvelle famille d'anticorps chimeres diriges contre l'antigene carcino-embryonnaire
WO1996011013A1 (fr) * 1994-10-05 1996-04-18 Immunomedics, Inc. Anticorps monoclonaux de souris humanises humain anti-cea, de type iii greffes a la region determinante de complementarite (cdr)
WO1997042329A1 (fr) * 1996-05-04 1997-11-13 Zeneca Limited Anticorps monoclonal anti-cea, conjugues contenant cet anticorps et leur utilisation therapeutique dans un systeme adept

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
R. SHARKEY ET AL.: "Evaluation of a complementarity-determining region-grafted (humanized) anti-carcinoembryonic antigen monoclonal antibody in preclinical and clinical studies.", CANCER RESEARCH SUPPLEMENT, vol. 55, no. 23, 1 December 1995 (1995-12-01), Baltimore, MD, USA, pages 5935s - 5945s, XP002079262 *
S. LEUNG ET AL.: "Bacterial expression of a kemptide fusion protein facilitates 32P labeling of a humanized, anti-carcinoembryonic antigen (hMN-14) antibody fragment.", CANCER RESEARCH SUPPLEMENT, vol. 55, no. 23, 1 December 1995 (1995-12-01), Baltimore, MD, USA, pages 5968s - 5972s, XP002079261 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003025565A3 (fr) * 2001-09-21 2003-05-30 Caprion Pharmaceuticals Inc Preparation de membranes plasmiques fortement purifiees
US9556438B2 (en) 2005-05-27 2017-01-31 Fondazione Centro San Raffaele Del Monte Tabor Gene vector
US10000757B2 (en) 2005-05-27 2018-06-19 Ospedale San Raffaele S.R.L. Gene vector
US11753643B2 (en) 2005-05-27 2023-09-12 Ospedale San Raffaele S.R.L. Gene vector
US10316104B2 (en) 2011-04-29 2019-06-11 Roche Glycart Ag Immunoconjugates
US11130822B2 (en) 2011-04-29 2021-09-28 Roche Glycart Ag Immunoconjugates
CN111533805A (zh) * 2016-12-04 2020-08-14 深圳市国创纳米抗体技术有限公司 一种抗癌胚抗原的高亲和力纳米抗体及其应用
CN111533805B (zh) * 2016-12-04 2022-02-08 深圳市国创纳米抗体技术有限公司 一种抗癌胚抗原的高亲和力纳米抗体及其应用
WO2020259550A1 (fr) 2019-06-26 2020-12-30 江苏恒瑞医药股份有限公司 Anticorps anti-cea et son utilisation
US11433100B2 (en) 2020-08-20 2022-09-06 A2 Biotherapeutics, Inc. Compositions and methods for treating ceacam positive cancers
US11602543B2 (en) 2020-08-20 2023-03-14 A2 Biotherapeutics, Inc. Compositions and methods for treating mesothelin positive cancers
US12435124B2 (en) 2022-07-22 2025-10-07 A2 Biotherapeutics, Inc. Compositions and methods for treating CEACAM positive cancers

Also Published As

Publication number Publication date
CA2321947A1 (fr) 1999-09-02
AU752494B2 (en) 2002-09-19
NO20004251D0 (no) 2000-08-24
AU6338898A (en) 1999-09-15
EP1056859A1 (fr) 2000-12-06
JP2002504372A (ja) 2002-02-12
IL138022A0 (en) 2001-10-31
NO20004251L (no) 2000-10-24

Similar Documents

Publication Publication Date Title
US6417337B1 (en) High affinity humanized anti-CEA monoclonal antibodies
EP1736163B1 (fr) Anticorps monoclonaux de souris humanisés anti-cea, de type III greffés à la région déterminante de complementarité (CDR)
US6753420B2 (en) High affinity humanized anti-Tag-72 monoclonal antibodies
AU752494B2 (en) High affinity humanized ANTI-CEA monoclonal antibodies
AU758358B2 (en) High affinity humanized anti-TAG-72 monoclonal antibodies
MXPA00008391A (en) High affinity humanized anti-cea monoclonal antibodies
TW523521B (en) High affinity humanized anti-TAG-72 monoclonal antibodies
TW553949B (en) High affinity humanized anti-CEA monoclonal antibodies
MXPA00008390A (en) High affinity humanized anti-tag-72 monoclonal antibodies

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 1998907630

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 138022

Country of ref document: IL

ENP Entry into the national phase

Ref document number: 2321947

Country of ref document: CA

Ref country code: CA

Ref document number: 2321947

Kind code of ref document: A

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: KR

WWE Wipo information: entry into national phase

Ref document number: PA/a/2000/008391

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 63388/98

Country of ref document: AU

WWP Wipo information: published in national office

Ref document number: 1998907630

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWG Wipo information: grant in national office

Ref document number: 63388/98

Country of ref document: AU

WWW Wipo information: withdrawn in national office

Ref document number: 1998907630

Country of ref document: EP