EP1056472A2 - Spezifische antikörper gegen brust-tumor assoziiertes mucin, sowie deren herstellung und verwendung - Google Patents

Spezifische antikörper gegen brust-tumor assoziiertes mucin, sowie deren herstellung und verwendung

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Publication number
EP1056472A2
EP1056472A2 EP99917815A EP99917815A EP1056472A2 EP 1056472 A2 EP1056472 A2 EP 1056472A2 EP 99917815 A EP99917815 A EP 99917815A EP 99917815 A EP99917815 A EP 99917815A EP 1056472 A2 EP1056472 A2 EP 1056472A2
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European Patent Office
Prior art keywords
cells
muc1
antibody
antibodies
polypeptide
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EP99917815A
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English (en)
French (fr)
Inventor
Gunther Bastert
Sepp Kaul
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BASTERT Gunter
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BASTERT Gunter
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Priority to EP99917815A priority Critical patent/EP1056472A2/de
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    • 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
    • C07K16/3015—Breast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • the invention relates to novel specific antibodies against the mammary tumor-associated mucin antigen MUC1 , a method for its production and its use in diagnostics and therapeutics.
  • the MUC1 tumor-associated antigens are high molecular weight glycoproteins (> 200 kDa) with multiple oligosaccharide side chains in O-linkage to serine and threonine residues. They are encoded by a hypervariable gene locus on chromosome 1 (region 21 q).
  • MUC1 is a transmembrane glycoprotein of the luminal surface of ductal epithelial cells which extends more than 200-500 nm from the cell surface. It contains a unique extracellular domain consisting mainly of tandem repeats of 20 amino acids.
  • tandem repeats range 20-120, mean 60
  • VNTR variable number of tandem repeats
  • the short sugar side chain starts with N-acetylgalactosamine and galactose (core 1 structure).
  • core 1 structure In normal epithelial cells the core 1 structure is then extended by the addition of N-acetylglucosamine, fucose and siaiic acid.
  • Mucins are secreted in larger amounts by differentiated breast epithelium in milk, and also in other organs with secretorial epithelia.
  • MUC1 expression shows a number of significant changes. These include a) loss of the polarized and restricted luminal expression; b) up-regulation and secretion; c) aberrant glycosylation resulting in the appearance of novel carbohydrate epitopes and unmasking of cryptic peptide epitopes.
  • Mucins therefore have a considerable impact as markers for diagnostic and therapeutic approaches.
  • Underglycosylated MUC1 is highly expressed on breast, ovarian, pancreatic and other cancer cells and can be recognized by HLA-restricted as well as non-MHC-restricted T cells.
  • HLA-restricted HLA-restricted
  • non-MHC-restricted T cells Several groups have demonstrated that the APDTR epitope of the MUC1 VNTR is the target of cytotoxic T lymphocytes isolated from patients with breast and other cancers (overview by Finn et al., 1995).
  • the existence of a B cell immune response was documented in patients with ovarian carcinoma and breast cancer (Rughetti, A., et al., Cancer Res. 53 (1993) 2457-2461).
  • MUC1 is a target for cellular immunotherapy, e.g., with a vaccine of MUC1 -transduced dendritic cells (DCs) generated ex vivo from CD34 enriched peripheral blood cells from breast cancer patients undergoing high-dose chemotherapy and PBSCT (peripheral blood stem cell therapy).
  • DCs dendritic cells
  • Other possibilities may be the active immunization with synthetic MUC1 peptides, underglycosylated MUC1 or MUC1-DNA.
  • Another possibility is the passive immunization using murine, humanized or human mabs with specificity for MUC1, preferably antibodies reactive with glycone or peptide epitopes of underglycosylated mucin produced by carcinoma cells.
  • Carbohydrate residues were involved in many epitopes by regulating epitope accessibility or masking determinants or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Therefore, the characterization of carbohydrate epitopes was more of a problem than the protein core epitopes.
  • the anti-MUC1 -antibody 12H12 (Bastert, G., et al., in Rygaard, Br ⁇ nner eds., Immuno-deficient animals in biomedical research, Basel, Karger, 1987, 224-227) reacts with a tumor-associated antigen known as TAG 12 that is expressted by 96% of all breast cancers.
  • TAG 12 tumor-associated antigen known as TAG 12 that is expressted by 96% of all breast cancers.
  • the antigen can be found in high concentrations in the cytoplasm and cell membrane of breast cancer tissue and metastases and is secreted in tumor cells.
  • This antibody could be used for immunoscintigraphy of human mammary carcinoma xenografts as described by Br ⁇ mmendorf, T.H., et al., Cancer Research 54 (1994) 4162-4168 and Nucl. Med. 34 (1995) 197-202.
  • the extent of specificity of these antibodies too is not sufficiently high for rendering them suitable for routine diagnostics and therapeutic application.
  • Bone metastases are common in breast cancer, large autopsy studies giving their frequency at 47-85% (Weiss, L, and Gilbert, H.A., Bone Metastasis, Boston, GK Hall, 1981).
  • the goal of any adjuvant therapy in breast cancer is the destruction of the smallest subclinical tumor sites, yet there is still no routine diagnostic method that can detect such "micrometastases".
  • breast cancer tends to metastasize to bone in particular, numerous attempts have been made to find malignant cells in bone marrow. Bone marrow as a target organ of metastasis is easily accessible and can be aspirated with relatively little danger and pain from the patient. Detecting "cells at the wrong place" can provide information about early phase of the metastatic cascade and possibly define the metastatic potency of the primary tumor.
  • a subject-matter of the invention is an immunologically active polypeptide which specifically binds to the carbohydrate structure of the 20 amino acid tandem repeat of MUC1 from tumor (carcinoma) cells, wherein a) the quotient between the affinities of said polypeptide for a 200 to 440 kDa underglycosylated glycoprotein fraction (analyzed by SDS-PAGE) from tumor cell- containing ascites from breast cancer patients and for native MUC1 antigen (400 - 440 kDa) from human milk is 100 : 1 or higher;
  • the polypeptide does not bind to nonglycosylated MUC1 antigen
  • the binding of the polypeptide to the said 200 to 440 kDa glycoprotein fraction changes by 10% or less if the glycoprotein fraction was treated with neuraminidase to cleave N-terminal neuraminic acids, or was treated with formalin, preferably with 3.5% formalin, in phosphate buffered saline (PBS) for 15 to 30 minutes.
  • PBS phosphate buffered saline
  • Such antibodies are reactive with carbohydrate epitopes of underglycosylated MUC1 , which are overexpressed and secreted by breast carcinoma and other carcinomas.
  • Such antibodies can be selected by a new immunization protocol. Ascites fluids from patients with advanced breast cancer were used as antigen source. The MUC1 antigen fractions in these fluids were enriched by a two-step-procedure using lectin affinity chromatography, and size exclusion chromatography.
  • An essential aspect of the present invention is such a procedure which favors the selection of antibodies according to the invention with strong reactivity for secreted MUC1 but minimal reactivity for normal milk MUC1 , deglycosylated milk mucin and/or normal urinary mucin.
  • a MUC1- containing glycoprotein fraction which can be isolated via lectin affinity chromatography (preferably, WGA, bound to agarose) from a mixture of ascites from different tumor patients, which contains secreted proteins.
  • WGA lectin affinity chromatography
  • the native glycoprotein peak fraction of 1 ,000 kDa is separated, whereby, amongst others, the immunoglobulins are removed by FPLC gel filtration.
  • Immunization according to the state of the art is carried out with the so purified protein fraction and hybridoma clones are screened, said hybridoma clones having the following properties:
  • Specific binding in the sense of the invention means that the antibody according to the invention does not bind to the peptidic structure of MUC1 and binds slightly to the carbohydrate structure of MUC1 from human milk, while binding strongly to the carbohydrate structure of MUC1 from tumor cells.
  • the WGA fraction from ascites is characterized essentially by the fact that it contains very little of the native fully glycosylated antigen (400 to 440 kDa) but exhibits in the SDS gel strong bands between 200 and 220 kDa (cf. Fig. 1). This difference in molecular weight is due to hypoglycosylation (underglycosylation).
  • "Underglycosylation" on the molecular basis, means that at one or more of the tandem repeats one or more of the glycosylation sites are nonglycosylated or are not occupied with the sugar residues as in the native molecule.
  • each underglycosylated glycosylation site is a potential binding site for the antibodies of the invention. The less the glycosylation of the sugar side chains, the higher the number of antibodies that are capable of binding. Already when at least 10, preferably at least 20, sugar side chains are missing or underglycosylated, an appreciable binding of the antibody according to the invention to MUC1 is to be observed.
  • Another subject-matter of the invention is a method for the production of an immunologically active polypeptide which specifically binds to a hypoglycosylated MUC1 fraction from tumor cells with a molecular weight of 200 to 400 kDa, preferably 200 to 220 kD (as analyzed by SDS-PAGE), wherein the glycoproteins are isolated from tumor cell-containing ascites of breast cancer patients by lectin affinity chromatography, said glycoproteins are optionally separated from the Ig proteins, and the fraction so obtained is used for the immunization of animals, antiserum is obtained, and the polypeptide is isolated therefrom, said polypeptide being characterized in that
  • a) the quotient between the affinities of the said polypeptide for a 200 to 400 kDa underglycosylated glycoprotein fraction (analyzed by SDS-PAGE) from tumor cell- containing ascites of breast cancer patients and of native MUC1 antigen (400 - 440 kDa) from human milk is 100 : 1 or more
  • the polypeptide does not bind to nonglycosylated MUC1 antigen
  • the binding of the polypeptide to the said 200 - 400 kDa glycoprotein fraction changes by 10% or less if the glycoprotein fraction was treated with neuraminidase to cleave N- terminal neuraminic acids, or with formalin.
  • immunologically active polypeptide or antibody refers to a protein consisting of one or more polypeptides substantially encoded by antibody genes.
  • the recognized antibody genes include the different constant region genes as well as the myriad antibody variable region genes.
  • Antibodies may exist of a variety of forms, including, for example, Fv, Fab, and F(ab)2 as well as single chains (e.g., Houston et al., PNAS USA 85
  • Preferred antibodies according to the invention are monoclonal antibodies and fragments thereof.
  • the relative affinity of anti-MUC1 antibodies was determined by the analysis of the K50 value, which is the antibody concentration at which half-maximal binding to MUC1 is achieved
  • the K50 value for 7F11 was 10 M with tumor associated antigen from ascites and T-47D breast cancer cells.
  • the murine monoclonal antibodies 1E4 and 7F11 were selected after immunization with MUC1 antigen fractions purified from ascites of patients with advanced breast cancer.
  • the antibodies are reactive with carbohydrate epitopes of underglycosylated MUC1 which is overexpressed and secreted by breast and other carcinomas.
  • the antibodies are useful tools for tumor diagnosis and therapy.
  • the antibodies according to the invention preferably the antibodies 7F11 and 1 E4 are clearly distinct from all 16 carbohydrate specific mabs submitted to the ISOBM TD-4 international workshop on monoclonal antibodies against MUC1 (1996). Both mabs are reactive with high
  • 7F11 and 1 E4 are unreactive with normal, fully glycosylated milk MUCL They are distinct from clone FH6 by a completely different staining pattern and by the fact that the FH6 immune reactivity is impaired by desialylation of MUC1 (treatment with neuraminidase or formalin).
  • the antibodies can be used as whole monoclonal antibodies, fragments thereof (e.g. Fv, (Fvt ⁇ ,
  • MUC1 in a suitable manner.
  • Short-chain antibody fragments containing only the CDR regions or parts thereof conferring the specific binding to MUC1 are also suitable, especially if the antibody is a labelled one.
  • Antibodies of the lgG1 isotype are preferred.
  • As to production of monoclonal antibodies see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988); Bessler et al., Immunobiol. 170 (1985) 239-244; Jung et al., Angewandte Chemie 97 (1985) 883 or Cianfriglia et al., Hybridoma Vol. 2(1993) 451-457.
  • the use of the antibodies according to the invention for diagnostic use thereby takes place in the known manner by means of an immunological process of determination. Processes of this type are well-known and do not need to be further explained here.
  • the antibodies obtained according to the present invention can be used as unlabelled and/or immobilized receptors.
  • Immobilized receptors can preferably be used for a method for the removal of tumor cells or secreted MUC1 antigen from a patient's body material, wherein the body material is brought into contact with an immobilized antibody as claimed in claim 1 or 2, he tumor cells or said secreted MUC1 antigen are bound to the immobilized antibody, and the tumor cells or said secreted MUC1 antigen so bound to the solid phase are separated along with the solid phase from the body material.
  • the present invention also provides a pharmaceutical composition which comprises one or more antibodies according to the present invention, optionally together with conventional pharmaceutical carrier, adjuvant, filling or additive materials.
  • a medicament according to the present invention is useful for the treatment of micrometastases, especially in the treatment of breast cancer.
  • a suitable dosage of the antibody according to the present invention for the therapeutic treatment is about 2 ⁇ g to 20 mg, preferably 50 ⁇ g to 20 mg /kg body weight, whereby this dosage possibly is to be repeatedly administered.
  • the antibodies according to the invention For the treatment of patients with breast carcinoma with the antibodies according to the invention it is preferred to carry out, after the surgery, an established adjuvant chemotherapy (before menopause) or hormone therapy (after menopause) in lymph-node-positive patients (about 45%).
  • an established adjuvant chemotherapy before menopause
  • hormone therapy after menopause
  • lymph-node-positive patients about 45%
  • the presence of tumor cells in the bone marrow is diagnosed. This is done preferably with the use of the antibodies according to the invention, but can be accomplished also with other tests recognizing usual tumor markers (cytokeratins, etc.).
  • the bone marrow reaction is positive, preferably 6 to 8 immunizations are performed at a 1 to 4- week-interval using the antibody according to the invention.
  • the amount of antibody used is in the range of 0.2 to 50 mg per immunization.
  • This antibody therapy is carried out during adjuvant hormone therapy or after chemotherapy. In the case of hormone therapy, it may be carried out preferably in a parallel manner (no immunosuppression in the latter case).
  • the development of antibodies against the anti-MUC1 antibodies according to the invention is being monitored during the phase of immunization with the antibodies according to the invention or by established tests for human anti-mouse antibodies (HAMA).
  • the desired therapy level is achieved when the antibody titer has been stable for about three to four months.
  • Another determination of the tumor cells in the bone marrow is performed three months after the last immunization. At least 6 x 10 bone marrow cells are analyzed for tumor cell contamination.
  • the antibodies according to the invention exhibit, in addition, an effector function, whereby, after binding to the tumor cells, the tumor cells are recognized as foreign cells and are presumably eliminated by the activation of the complement, anti-idiotypic antibodies, activated NK-cells or T-cells or a combination of the latter.
  • the antibodies of the invention bind also to secreted MUC1 in body fluids such as serum, thereby having a therapeutic effect by elimination of MUC1-induced immunosuppression, by humoral or cellular immune recognition, or as an immunosuppressive agent. This too can be utilized for therapy.
  • MUC1 is overexpressed also in tumor cells of the ovary (>80%), the lung (60%), prostate (50%), pancreas (100%), kidney, and colon.
  • the antibodies of the invention can be utilized here too for diagnosis and therapy.
  • cytotoxic antibodies which impart effector functions (ADCC, CDC) (Br ⁇ ggemann et al., J. Exp. Med. 166 (1987) 1357-1361).
  • the antibody or part of it is conjugated or translationally fused to a toxin molecule (immunotoxin), thus effecting specific killing of tumor cells (Brinkmann et al., Proc. Natl. Acad. Sci. USA 88 (1991) 8616-8620; Pastan et al., Cancer Res. 51 (1991) 3781-3787; FitzGerald and Pastan, J. Natl. Cancer 81 (1989) 1455-1461) or conjugated to a cytokine or interleukin.
  • bispecific antibodies are used for tumor therapy (Bonino et al., BFE 9 (1992) 719-723), which may be constructed by in vitro reassociation of polypeptide chains, by hybrid hybridoma generation or by construction of diabodies (Holliger et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448; Holliger and Winter, Current Opin. Biotechnol. (1993) 446-449).
  • the antibody according to the invention it is preferred to couple the antibody according to the invention to a toxin, such as, for example, Pseudomonas exotoxin, Diphtheria toxin or other toxins (FitzGerald and Pastan, J. Natl. Cancer 81 (1989) 1455-1461). It is also preferred to couple the antibodies to chemotherapeutics, such as, for instance, doxorubicin, or to radioactively labelled substances which have a cytotoxic effect.
  • chemotherapeutics such as, for instance, doxorubicin
  • Conjugates of the antibodies according to the invention in particular of human antibodies, for in vivo imaging, using, for instance, radioactive or fluorescent substances, are also preferred.
  • Immunotoxins can be produced preferably by either of two principally different methods:
  • an antibody or a fragment thereof is chemically coupled in vitro to a toxin or toxin fragment.
  • the antibody part in this type of immunotoxin is either a complete antibody (consisting of two light and two heavy chains) or, more preferably, a Fab-fragment (consisting of one light chain and the VH- and CH1 -regions of the heavy chain).
  • the immunotoxin is generated by recombinant DNA techniques, which leads in any case to a defined, homogeneous molecule.
  • the size of the antibody part should be as small as possible to obtain a small immunotoxin with good tissue penetration.
  • the smallest practically available antibody fragment is not the Fab-fragment, but the functional variable domain of an antibody, consisting of the VH-region of the heavy chain and the VL- region of the light chain only.
  • VH- and VL-region polypeptide chains each of about 100 amino acids
  • VH- and VL-region form very labile complexes only. Therefore, their complex is preferably stabilized by covalent bonds.
  • VH-region a single polypeptide chain is formed, wherein VH- and VL-region, being connected by a peptide linker, fold into a stable variable domain, while the toxin is fused e.g. to VL via a second peptide linker (see Brinkmann et al., Proc. Natl. Acad. Sci. USA 89 (1992) 3075-3079).
  • the length of both peptide linkers is variable and may in some instances even be reduced to a single peptide bond.
  • a molecule of this type has been termed a "single chain immunotoxin", analogous to the term “single chain antibody” or scFV, which is used for a single polypeptide chain containing both VH and VL connected by a peptide linker or bond.
  • VH- and VL-assembly Another possibility to stabilize the VH- and VL-assembly is described in Brinkmann et al., Proc. Natl. Acad. Sci. USA 90 (1993) 7538-7542).
  • amino acids on VH and VL were defined by computer aided modelling, which are closely adjacent in the VH-VL-compiex. The naturally occurring amino acids in these positions were then on the DNA level replaced by a cystein each.
  • two separate polypeptide chains are expressed (in separate cells, e.g. prokaryotic cells, e.g. E.coli), one being the VH-region only, the other the VL-region fused by a peptide linker to the toxin part.
  • Pseudomonas exotoxin preferred fragments of the Pseudomonas exotoxin (P
  • Single chain F v -chain immunotoxin is preferably produced as a single polypeptide chain in
  • the polypeptide is obtained in an inactive form and has to be activated by in vitro renaturation.
  • Polynucleotides of the invention and recombinantly produced antibodies of the invention may be prepared on the basis of the sequence data according to methods known in the art and described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor, New York, and Berger and Kimmel, Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (1987), Academic Press Inc., San Diego, CA, which are incorporated herein by reference.
  • Polynucleotides of the invention are preferably formed from synthetic oligonucleotides.
  • Such recombinant polypeptides can be expressed in eukaryotic or prokaryotic host cells according to standard methods known in the art; preferably mammalian cells, such as lymphocyte cell lines, may be used as host cells.
  • such polynucleotide constructs encode a complete human antibody heavy chain and/or a complete human antibody light chain having at least the amino acid sequences of the antibodies according to the invention, heavy and/or light chain variable regions respectively.
  • Alternative human constant region sequences (heavy and/or light chain) other than those naturally associated with said antibody chains may be substituted, including human constant region isotypes, such alternative human constant region sequences can be selected by those of skill in the art from various reference sources, including, but not limited to, those listed in E.A.
  • a polynucleotide sequence encoding an antibody light chain comprising a human light chain, constant region with an amino terminal peptide linkage (i.e. an inframe fusion) to a variable region of the light chain of the antibody according to the invention and a corresponding heavy chain are expressed and form heavy/light chain dimers and other antibody types.
  • prokaryotes can be used for cloning the DNA sequences encoding an antibody chain according to the invention.
  • E.coli is one prokaryotic host particularly useful for cloning the DNA sequences of the present invention.
  • oligonucleotides may be synthesized chemically by a variety of methods, including phosphoramidite synthesis.
  • the polynucleotide constructs will typically include an expression control sequence operatively linked to the coding sequences, including naturally associated or heterologous promotor regions.
  • the expression control sequences will be eukaryotic promotor systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and the collection and purification of the antibodies according to the invention.
  • eukaryotic host cells mammalian tissue cell cultures may also be used to produce the polypeptides of the present invention. Mammalian cells are actually preferred, because a number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art and include the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, etc.
  • glycosylating cell is a cell capable of glycosylating proteins, particularly eukaryotic cells capable of adding an N-linked "core oligosaccharide” containing at least one mannose residue and/or capable of adding an O-linked sugar to at least one glycosylation site sequence in at least one polypeptide expressed in said cell, particularly a secreted protein.
  • a glycosylating cell contains at least one enzymatic activity that catalyzes the attachment of a sugar residue to a glycosylating site sequence in a protein or polypeptide and the cell actually glycosylates at least one expressed polypeptide.
  • mammalian cells are typically glycosylating cells.
  • Other eukaryotic cells such as insect cells and yeast may be glycosylating cells.
  • antibodies according to the invention can be purified according to standard procedures of the art, including HPLC purification, fraction column chromatography, gel electrophoresis, and the like (see generally, R. Scopes, Protein Purification, Springer Verlag, N.Y. (1982)).
  • the therapeutic compounds of this invention may be administered parenterally, such as intravascularly, intraperitoneally, subcutaneously, intramuscularily, using forms known in the pharmaceutical art.
  • the active drug components of the present invention are used in liquid, powdered or lyophilized form and may be combined with a suitable diluent or carrier, such as water, a saline, aqueous dextrose, aqueous buffer, and the like. Preservatives may also be added.
  • the compounds of the present invention are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.
  • the compounds may also be formulated using pharmacologically acceptable acid or base addition salts.
  • the compounds or their salts may be used in a suitable hydrated form.
  • a non-toxic but therapeutically effective quantity of one or more compounds of this invention is employed in any treatment.
  • the dosage regimen for treating is selected in accordance with a variety of factors including the type, age, weight, sex and medical condition of the patient, type of tumor, the route of administration and the particular compound employed in the treatment.
  • a physician of ordinary skill can readily determine and prescribe the effective amount of the drug required regarding known antibody therapy approaches. In so proceeding, the physician could employ relatively low doses at first, and subsequently, increased dose until a maximum response is obtained.
  • compositions comprising an antibody of the present invention are useful for topical or parenteral administration, i.e. subcutaneously, intramuscularly, intravenously or transdermally.
  • the compositions for parenteral administration will commonly comprise a solution of said antibody dissolved in an acceptable carrier, preferably in an aqueous carrier.
  • aqueous carriers can be used, e.g. water, buffered water, 0.4% saline, 0.3% glycin, and the like.
  • the solutions are sterile and generally free of particulate matter.
  • the compositions may be sterilized by conventional well-known techniques.
  • compositions may contain pharmaceutically acceptable auxiliary substances, such as are required to approximate physiological conditions, such as pH adjusting and buffer agents, toxicity adjusting agents, and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.
  • concentrations of the antibodies according to the invention in these formulations can be varied widely, e.g. from less than about 0.01%, usually at least about 0.1%, to as much as 5% by weight, and will be selected primarily based on fluid volumes, viscosity, etc. or in accordance with the particular mode of administration selected.
  • a typical pharmaceutical composition for intramuscular injection could be made up to contain 1 ml sterile buffered water and about 1 to 50 mg of antibody according to the invention.
  • the antibodies according to the invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use.
  • Conventional lyophilization and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of biological activity loss and that use levels may have to be adjusted to compensate.
  • HAMA HAMA in the range of 1 - 100 ⁇ g/ml and anti-7F11 antibodies (ab ) were reached.
  • the clinical study shows the safety, tolerance, pharmacokinetics of multiple doses of anti-MUC1 antibodies according to the invention in breast cancer patients with minimal residual disease.
  • Micrometastatic tumor cells in bone marrow (mean 3.3 tumor cells per 10 normal bone marrow cells) were eliminated in eighteen patients of the M0 group and in two patients with locoregional disease. No disease progression was seen in these patients during follow up.
  • the study shows, that treatment of breast cancer patients with unconjugated antibodies according to the invention can eliminate tumor cells in the bone marrow either by direct cytotoxicity, passive effector mechanisms or by idiotypic network responses.
  • monitoring micrometastatic cells in bone marrow is presented as an extremely sensitive surrogate marker to monitor the efficacy of adjuvant tumor therapies.
  • TAA tumor associated antigens
  • Breast mucins are highly glycosylated proteins coded by the gene MUC1 on chromosome 1q. The antigen is overexpressed and aberrantly glycosylated in breast and ovarian carcinomas. Tumor-associated mucins are found in cytoplasm, cell membranes and secretory components.
  • MAb 7F11 specific for multiple carbohydrate/peptide binding sites of the tandem repeat region of the human breast mucin MUC1 is reactive with over 96% of breast carcinomas.
  • the 7F11 staining pattern in solid tumors and metastases is homogeneous with usually over 80% of stained tumor cells. Therefore 7F11 can be used for the analysis of tumor cells in bone marrow and for immunoscintigraphy.
  • ADCC antibody-dependent cellular cytotoxicity
  • 7F11 therefore was administered at rather low doses of 2.5, 20 and 50 mg at 4 weeks intervals. Toxic side-effects other than allergic reactions were not encountered in a group of 55 patients. In particular, no anaphylactic reactions were seen. Three patients received a total dose of 450 mg 7F11.
  • the number of immunizations therefore suitably is 1-10, preferably 8-9, and most preferably, 6-8 on a weekly or monthly basis depending on the individual patient and the patient's ability to develop stable and long-lasting antibody titers.
  • Maximal HAMA-titers were in the range of 10 to 100 ⁇ g/ml with a mean serum half live time of 5 months after the last immunization. In four patients (patients 1,2, 4 and 5, Table 1) between 25 - 40 % of the anti-idiotypic response is directed toward the antigen binding site and therefore of ab2 ⁇ -type.
  • the direct mechanisms depend on labeling of tumor cells and are triggered by antibody- dependent cellular cytotoxicity, complement-dependent cytotoxicity and apotosis. Indirect mechanisms may operate via the immune network.
  • Anti-idiotypic antibodies (ab2 ⁇ ) binding to the hypervariable region of the therapeutic mouse antibody induce corresponding T-cells (T2) and trigger the development of anti-anti-idiotypic antibodies (ab3) und T-cell (T3) clones.
  • T2 ⁇ T-cells
  • T3 antibodies recognizing ab2 ⁇ and the nominal antigen epitope of the human breast mucin definded by the therapeutic mouse antibody may be responsible for the favorable outcome of immunized patients.
  • the 7F11 therapy resulted in dramatic reduction or eradication of micrometastatic breast tumor cells in bone marrow of breast cancer patients in the adjuvant situation.
  • the cell lines DSM ACC 2329 (1) and DSM ACC 2328 (2) mentioned in the present invention which secrete the antibodies 7F11 (1) and 1 E4 (2) were deposited by Boehringer Mannheim GmbH with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, on October 31 , 1997.
  • HAMA human anti-mouse antibody
  • PBS phosphate buffered saline
  • APAAP technique alkaline phosphatase anti-alkaline phosphatase complex
  • TCD number of tumor cells
  • TAA tumor-associated antigens
  • PUM polymorphic urinary mucin-like glycoprotein
  • EIA enzyme immunoassay
  • WGA wheat germ agglutinin
  • BSA bovine serum albumin
  • TFMSA trifluoromethane sulfonic acid
  • MAPS monoclonal antibody binding solution (BioRad)
  • PE phycoerythrin
  • Fig. 1 Reactivity of mab 7F11 with different WGA purified MUC1 mucins. Antigen was purified by WGA-Sepharose chromatography as described in Example 1 and subjected to 7.5% SDS-PAGE and Western blotting (500 U in 20 ⁇ l). T47-D supernatant (track 1), T47-D cytosol (track 2), P1.1 ascites (track 3), P1.2 pleural effusion (track 4), P1.3 ascites (track 5), P1.4 ascites (track 6), P1.5 ascites (track 7), P1.6 serum (track 8), P1.6 ascites (track 9), P1.7 ascites (track 10), P1.8 ascites (track 11) (P: Patient). ST, standard molecular weight in kilodalton.
  • Fig. 2 Patient No. 1 : 7F11 pharmacokinetics and humoral immunity, breast cancer T1b, NO, M0.
  • Fig. 3 Patient No. 2: 7F11 pharmacokinetics and humoral immunity, breast cancer T1b, N1b, M0.
  • Fig. 4 7F11 pharmacokinetics. First, second and third immunization.
  • Clone 1 E4 (lgG1,k) was selected from a BALB/c mouse immunized sequentially for 8 times every 4-6 weeks with MUC1 fractions from three patients.
  • Spleenocytes were fused with X63- P3 X 63Ag8.653 (ATCC CRL1580) mouse myeloma cells according to established methods (1) using PEG 6000.
  • Hybridomas were selected in ten 96-well plates using mouse macrophages as feeder cells. Supernatants were screened by ELISA and immunocytology. 19 hybridoma clones with strong MUC1 reactivity were identified and kryopreserved.
  • Main selection criteria for the clone 1 E4 were strong reactivity with various tumor-associated antigen fractions, no reactivity with the immunodominant MUC1-VNTR peptide sequence APDTR (SEQ ID NO:5) und minimal reacticity with normal milk MUC1 , deglycosylated MUC1 and normal urinary mucin (polymorphic urinary mucin-like glycoprotein PUM).
  • Monoclonal antibody 7F11 Monoclonal antibody 7F11 :
  • Clone 7F11 (lgG1 ,k) was derived after 8 immunizations of a BALB/c mouse with MUC1 fractions purified from ascites of two patients and the breast cancer cell line T47-D. After fusion of spleenocytes with P3X63Ag8.653 myeloma cells nine hybridomas with MUC1 specificity were identified. Selection criteria were the same as for 1 E4, namely strong reactivity with secreted MUC1 but minimal reactivity with normal milk MUC1 , deglycosylated MUC1 and PUM.
  • Mycoplasm free myeloma P3X63Ag8.653 was used.
  • the murine Ig nonproducer cell line was grown in Dulbeccos modification of Eagles medium (DMEM) with 4.5 g/l (D+) glucose, 0.7 g/l NaHCO3, 10% fetal calf serum (FCS), 15 mM HEPES, 0.5% penicillin-streptomycin and 2 mM glutamine (DMEM, all Gibco, Eggenstein, DE).
  • DMEM Dulbeccos modification of Eagles medium
  • KS, WA, AR, KM22 and HG15 breast cancer cell lines were established from ascites (KS), primary carcinomas (WA, AR) and bone marrow (KM22 and HG15 of breast cancer patients).
  • Mucin negative control cell lines SW1116 (CCL233) and LS174T (CCL188) are from the ATCC.
  • Human fibroblasts were isolated from connective tissue of breast cancer patients.
  • Ovarian carcinoma cell lines HI and OC were established from ascitic fluids.
  • T47-D cells malignant pleural effusion, ductal breast carcinoma, ATCC HTB133
  • the cell g line KS were grown in Nunc factories (Nunc, Wiesbaden, DE). Batches of 2 x 10 cells were collected by trypsination and washed twice with PBS. Cells were homogenized in 500 ml lysis buffer (10 mM TRIS/HCI pH7.6, 2 mM EDTA, 1% Triton® X-100, 1% aprotinin and 0.1 mM PMSF) for 1 h at 4°C under constant agitation. Nuclei were removed by centrifugation at 10,000 g for 15 min and the supernatant (cytosol) was stored at -70°C or used for antigen purification.
  • 500 ml lysis buffer (10 mM TRIS/HCI pH7.6, 2 mM EDTA, 1% Triton® X-100, 1% aprotinin and 0.1 mM PMSF
  • Cytosol, ascites or pleural effusions were purified by lectin affinity chromatography on Wheat germ agglutinin (WGA) Sepharose (Pharmacia, Freiburg, DE) in 2x30cm columns at a flow rate of 2-5 ml/min. Columns were washed with low and high salt (2 M NaCI) PBS and glycoproteins were eluted with N-acetyl-glucosamin (125 mg/ml) at a flow rate of 1 ml/min, dialysed against PBS and concentrated 10-fold by ultracentrifugation with YM100 membranes (Amicon, Weiden, DE). Protein content was analysed using a micro assay according to Lowry (BioRad, M ⁇ nchen, DE).
  • Lectin affinity purified fractions of ascites and cytosol proteins were purified by Superose 6 fast protein liquid chromatography (FPLC, Pharmacia) in 2.5 x 100 cm columns. Samples of 5 ml were applied and separated at a flow rate of 4 ml/min in PBS, 2 mM EDTA. MUC1 positive fractions in the molecular weight range of 200 to 1 ,000 kDa were analysed by EIA and adjusted to 2-10 ⁇ g/ml and used for immunization of BALB/c-mice and for specificity tests of hybridoma supernatants. Antigen was stored at -20°. 4.2. Purification of MUC1 by immune affinity chromatography
  • the mab 2E11 reactive with native und deglycosylated MUC1 , was coupled to Affi-Gel 10 (BioRad) at a concentration of 10mg antibody per ml gel. Cytosol was applied in batches of 250 ml to the antibody column at a flow rate of 1 ml/min followed by extensive washing with PBS and PBS, 2 M NaCI. Bound antigen was eluted with 7M urea, pH 6, at a flow rate of 0.5 ml/min and dialysed overnight against 5 I PBS. Purified antigen was then passed through a 5 ml goat anti-mouse IgG-Agarose (Sigma, Deisenhofen, DE) column to remove trace amounts of 2E11.
  • BMA breast mucin antigen
  • Proteins were separated under reducing conditions in 7.5 % acrylamide gels (16x16 cm Protean, BioRad), according to Laemmli and transferred to nitrocellulose at 300 mA for 4 h in glycin/methanol buffer. Nitrocellulose was blocked with PBS, 01% Tween 20 and 2% dry milk powder for 1 h. Purified antigen fractions and deglycosylated antigens of different sources were probed with with biotinylated mabs (1 ⁇ g/ml PBS, 1% BSA) for 1 h and detected by strepavidin peroxidase and 4-chloro-1-naphthol as substrate.
  • Affinitiy-purified MUC1 samples were dialysed against 0.1 M PP, pH 5.5 (neuraminidase) or 0.1 M PP pH6.0 (O-glycanase) and incubated at 37°C for 24 h with neuraminidase (2, 5, 10, 20 mU, Vibrio cholerae, Boehringer Mannheim GmbH, Mannheim, DE) or O-glycanase (2, 20 and 200 mU, Diplococcus pneumonie, Boehringer), respectively. Digestion was stopped by heating samples for 5 min to 100°C.
  • T47-D cytosol proteins were partially purified by Superose 6 gel exclusion chromatography.
  • the high molecular weight fraction of 1-2 x 10 Da was used to immmunize BALB/c-mice according to established methods. Final immunizations were performed with neuraminidase and O-glycanase treated antigen. Sleenocytes were fused with X63Ag853 myeloma cells. Hybridoma 2E11 was selected by ELISA, cytology and histology. The mab (lgG3,k) showed strong and broad reactivity with formalin-resistant epitopes of breast and other carcinomas. The antibody could be used alone and in combination with 7F11 and 1E4 for quantitative determination of serum MUC1 levels. A two site sandwich EIA tests with 7F11 as coating ab and peroxidase labeled 2E11 was developed for MUC1 quantification. Mab 2E11 was cloned 11 times by limiting dilution.
  • MUC1 was purified by WGA-chromatography and Superose-6 gelfiltration from ascites of patients FO, OC and ST.
  • Female BALB/c mice were immunized sequentially for 8 times every 4-6 weeks with antigens from diferent patients.
  • Spleenocytes were removed 4 days after a booster immunization and fused with X63-Ag853 mouse myeloma cells according to established methods (Kaul, S., Eck monoklonaler Antik ⁇ rper mit Spezifitat fur Mammakarzinomzellen, Habilitationsarbeit, Zentrum der Biologin Chemie der Universitat Frankfurt, 1983) using PEG 6000.
  • Hybridomas were selected in ten 96-well plates using mouse macrophages (PM) as feeder cells. Supernatants were screened two weeks later by ELISA and cytology. 19 hybridoma clones with strong MUC1 reactivity were identified and kryopreserved. Primary selection criteria for the clone 1E4 were minimal reacticity with normal milk MUC1 , deglycosylated MUC1 and normal urinary mucin (polymorphic urinary mucin-like glycoprotein PUM), strong cytological reactivity with breast cancer cell lines T47D and KS and minimal cross reactivity with human fibroblasts, respectively. 1 E4 was cloned 8 times by limiting dilution.
  • MUC1 was purified by WGA-chromatography and Superose-6 gelfiltration from ascites of patients ST, ZE and from the breast cancer cell line T47-D. BALB/c-mice were immunized sequentially for 8 times with the different antigen fractions. After fusion of spleenocytes with X63-Ag853 myeloma cells nine hybridomas with MUC1 specificity were identified. Clone 7F11 (lgG1 ,k) was selected by ELISA and cytology and was characterized by minimal reactivity with normal milk MUC1 , deglycosylated MUC1 and PUM and strong reactivity with MUC1 from serum, ascites and tumor cells. Mab 7F11 was cloned 12 times by limiting dilution.
  • MUC1 was purified by WGA-chromatography and Superose-6 gelfiltration from serum of patient Zl and from secreted MUC1 (tissue culture supernatant) of T47-D cells. Both antigen fractions were mixed and used for three immunizations of BALB/c-mice. After fusion (Feb. 1990) of spleenocytes with X63-Ag853 myeloma cells 24 hybridomas with MUC1 specificity were identified. Clone 5A6 (lgG1 ,k) was selected by ELISA and cytology using native and deglycoylated antigen fractions from serum, ascites, tumor cells and normal cells. 5A6 was cloned 10 times by limiting dilution.
  • hybridoma cells were seeded at densities of
  • the Ig production of 2E11 , 1 E4, 7F11 and 5A6 in D/H-medium was is in the range of 15-
  • Antibodies were produced in a CellPharmll unit (Unisyn, Heraeus, DE) using hollow fiber g bioreactors type 3570 (70 kDa membrane). 2x10 hybridoma cells were inoculated in complete D/H-medium with 2.5% FCS. Serum was reduced within 3 weks to 1%. During the production phase hybridomas were grown at a medium feed rate of 4 liters/day. Every day 100-200 ml supernatant was harvested, analysed for Ig content, cleared by membrane filtration and stored at -20C. There was no decline in the Ig synthesis rate during two to three months production cycles.
  • Mabs were purified by Protein A-Sepharose 4 Fast Flow (Pharmacia). Bioreactor supernatants were mixed with an equal volume of binding buffer, pH8.9 (MAPS, Bio-Rad) and batches containing of 250 mg mouse Ig were applied on 20 ml protein-A gel at a flow rate of 1 ml/h (FPLC-system Pharmacia). IG was eluted with 0.1 M citrate buffer, ph 3.9, neutralized with 2M HEPES, pH 9.0 and dialysed extensively against either PBS or 0.1 M NaHCO3, pH 8.4.
  • the antibodies were labeled (5 mg, 2 mg/ml in 0.1 M NaHCO3, pH 8.4) with biotin (long arm NHS-Biotin, Vector, Burlingame, USA) and peroxidase (Boehringer Mannheim GmbH, DE) according to the instructions of the manufacturer. Labeling of mabs with FITC, FE and PECy5 for FACS analysis was performed by Cymbus (Dianova, Hamburg, DE).
  • Synthetic peptides of the MUC1 tandem repeat structure were synthesized on a solid peptide synthesizer (Dr. Nastainzyk, Univ. Homburg/Saar) und coated at a concentration of 100ng/well in EIA plates (Maxisorb, Nunc) using 0.1 M carbonate buffer, pH9.4.
  • the peptides M20 (APDTRPAPGSTAPPAHGVTS) SEQ ID NO:1 ,
  • P24 (APDTRPAPGSTAPPAHGVTSAPDT) SEQ ID NO:2, M5-15 (PAPGSTAPPA)
  • SEQ ID NO:3, and M6-17 (APGSTAPPAHG) SEQ ID NO:4 were used for epitope analysis.
  • Binding to KS and T47-D cells (breast carcinoma, MUC1 positive), LS174T (colon carcinoma, MUC1 negative) was analyzed by flow cytometry (Coulter EPICS) after labeling of native or methanol fixed ceils with 7F11 and 1 E4 directly conjugated with FITC, ffp or PE-Cy5.
  • the antibodies of the invention were competed with the commercially available anti-MUC1 antibodies HMFG1 , HMFG2, Ma552, Ma695, b12, DF3, 115D8, BC2, BC3, SM3, VU11 E2, VU12E1 , KC4 and MF06.
  • MUC1 from ascites of breast and ovarian cancer patients, serum from breast cancer patients, human milk, human urine, cytosol fractions from KS, T47-D and LS174T cells were purified according to 4.1.2 and 4.1.3.
  • Maxisorb immunoplates (Nunc) were coated with 0.1 ⁇ g antigen per well in carbonate buffer, pH 9.6 for 18 hours at room temperature. After washing with PBS unspecific binding was blocked by incubation with PBS, 1% BSA.
  • Antibodies of the invention were used as biotinylated reagents at concentrations of 5, 10 and 20 ng/well.
  • the epitope specifictity of 1E4 and 7F11 was analysed on a large panel of MUC1 fractions isolated from different normal and tumor sources (Table 2). In contrast to 2E11 and several other well characterized mabs with specificity for the immundominant MUC1 peptide epitope PDTR (HMFG1 , HMFG2, b12, DF3) the mabs 1E4 and 7F11 are completely negative with several overlapping peptides containing the PDTR sequence. The antibodies did not react with glycoproteins from normal human urin (PUM) and human milk.
  • PUM normal human urin
  • the epitopes of both antibodies are insensitive to mild proteolytic treatment (trypsin) and excessive deglycosylation with neuraminidase, however the antibody reactivity is completely abolished by treatment of purified MUC1 with neuraminidase and O-glycanase.
  • Analysis of MUC1 separated by SDS- polyacrylamide gel electrophoresis and Western immunoblotting shows reactivity of both mabs with high (400-440 kDa) and lower molecular weight fractions (180, 200-220 kDa).
  • 1E4 and 7F11 showed completely different staining profiles with unpurified MUC1 from serum and ascites of breast cancer patients with advanced disease.
  • Both mabs are characterized by a strong reactivity with secreted antigen in sera, ascites and pleural effusions.
  • the MUC1-specific mabs 7F11 , 1 E4 as well as the antibodies 1 A6 and 2E11 were screened for specificity on a large panel of normal tissues and carcinomas of different organs. Staining was performed with biotinylated mabs (0.5 ⁇ g/ml) and streptavidin-alkaline phosphatase with new fuchsin as substrate either manually or using an automated immunostainer (Dako Techmate).
  • the mabs 7F11 and 1E4 showed a much more restricted reactivity with normal epithelial cells.
  • the normal breast 7F11 was positive with acini and ducts in 4 from 12 cases, with heterogenous staining in individual cases.
  • 1 E4 reacted only occasionally with acini, single epithelial cells and secretion products.
  • colon, endometrium, lung, pancreas and placenta 7F11 reacted only weakly with the apical membrane of epithelial cells, while 1 E4 was completely negativ.
  • the only organ with consistently strong reactivity with 2E11 and 7F11 was the kidney, while 1 E4 was weakly positive with ductal membranes in three cases.
  • the tumor reactivity of mabs 2E11 , 5A6, 1 E4 and 7F11 is shown in Table 4. Routine immunhistological staining on formalin fixed, paraffin embedded tissues have shown that 2E11 and 7F11 are reactive with over 96% of primary breast carcinomas, while 1E4 is reactive with more than 90% of primary breast carcinomas.
  • the mabs 7F11 , 1E4 and 5F2 are characterized by a more heterogenous staining pattern in individual tumors. Typically in 50% of the cases there are areas of rather weak and very strong expression of the respective epitops in the same tumor. Approximately 40% of the 1 E4 positive tumors show focal cytoplasmic staining of the golgi complex, while strong reactivity is found only in small tumor areas, single cells and secretion products.
  • Both mucin specific mabs offers new perspectives for the improvement of rare cell detection by immuncytology, ELISA, flow cytometry, immunomagnetic tumor cell enrichment and for the development of effective purging techniques for rare tumor cells in peripheral blood stem cell collections.
  • Epitope specificity of 2E11, 5A6, 1E4 and 7F11 analysis with different MUC1 fractions isolated from normal and tumor tissues, serum, ascites and with the M24 MUC1 peptide
  • E cytoplasmic and membrane staining of erythroid progenitor cells (3-7% of CD34- cells)
  • P plasma cells
  • m membrane staining (expression is limited to the apical border of secretory cells)
  • f focal intracytoplasmic staining (golgi complex)
  • s staining of secretory components
  • w staining intensity weak
  • bm bone marrow
  • m membrane staining, typically not limited to the apical border of tumor cells
  • c homogenous cytoplasmic staining
  • f focal intracytoplasmic staining (golgi complex) s staining of secretory components
  • Pretreatment consisted of infusion of 2 mg clemastinhydrogenfumarat (Tavegil) and oral application of 500 mg Calcium. Patients then received 50 mg 7F11 (1 mg/ml) diluted in isotonic NaCI i.v. over a period of 8 hours. The 20 mg dose was diluted in 500 ml NaCI and infused over 4 hours. These patients could leave the hospital one hour later. Patients of the 2.5 mg had no premedication and received the antibody in 250 ml NaCI by a one hour infusion and could leave the hospital one hour later.
  • tumor markers CA153, CEA, BM27
  • HAMA HAMA
  • anti-7F11 pharmakokinetics was analysed in the serum 4, 12, 48 and 72 h after antibody application. During infusion blood pressure was monitored every hour.
  • Mononuclear cells from peripheral blood were prepared before immunization, a time points when significant ab2 titers were induced and before and 7 days after booster immunizations. Cells were frozen in liquid nitrogen for future phenotyping, B-cell and T-cell culture and FACS analysis.
  • Bone marrow samples were separated by Ficoll densitity centrifugation and mononuclear cells were used for the preparation of cytospin slides with 1x10 bone marrow cells per slide. Cells were air dried and fixed with 3.5% neutral buffered formalin for 15 min., washed with PBS and then fixed for another 5 min. with cold (-20C 0 ) 100% methanol. Slides were washed with PBS, transfered to storage buffer (PBS with 60g/l sucrose, 0,4 g/l MgCl2 and 43% glycerol) and stored at -20 C C.
  • storage buffer PBS with 60g/l sucrose, 0,4 g/l MgCl2 and 43% glycerol
  • Epithelial tumor cells were stained after a blocking step for alkaline phosphatase (20% acetic acid for 10 min., 2.3% NaJO4 for 10 min.) and normal serum (5% normal mouse serum for biotinylated primary antibodies, 5% human serum for the APAAP technique) by immunocytology using biotinylated 7F11 (0.5 ⁇ g/ml), 5D3-biotin (Novocastra,
  • Tumor cells were evaluated and quantitated by the Discovery image analysis system (Becton Dickinson). Tumor cell analysis of patients immunized with 7F11 was controled by additional double staining techniques using alkaline phosphatase kits with Vector Red (biotinylated primary antibody) and Vector Blue (APAAP technique) (Vector, Burlingame, USA) to exclude staining of B-cells and plasma cells with isotype (mouse lgG1 and 7F11) specificity.
  • Vector Red biologicaltinylated primary antibody
  • APAAP technique Vector Blue
  • Results from at least 6 slides were expressed as tumor cells per 10 normal bone marrow cells
  • TCD Analysis of humoral immune response
  • HAMA were assayed with ELISA by coating with mouse IgG.
  • Sera were diluted twofold from 1 :20 to 1 :1000 and binding was assayed by peroxidase labeled mouse IgG according to the instructions of the manufacturer (Medac, Hamburg, DE).
  • Human anti-7F11 Ig was eluted with 0.1 M citrate buffer, pH 3.0, dialysed against PBS and used as standard antigen in the concentration range of 500 to 2.5 ng/ml.
  • the IgG fraction was affinity purified using mouse IgG agarose.
  • Mouse IgG preadsorbed sera were incubated at concentrations of 0.1 to 10 ⁇ g/ml in 7F11- F(ab)2 coated microtiter plates. For competition the plates were incubated 18 hours with purified MUC1 antigen in the concentration range 500 units/ml. Human ab2 ⁇ were determined using goat anti-human IgG labeled with peroxidase.
  • ELISA plates were coated with native MUC1 purified by WGA-Sepharose chromatography from the breast cancer cell lines KS and T47-D. Control antigen were cytosol fractions from MUC1 negative SW1116 colon carcinoma cells. Synthetic MUC1 peptides of the VNTR (p24) were coated at a concentration of 1 ⁇ g/ml. Sera collected before and two weeks after every immunization were screened for anti-MUC1 reactivity. Sera were added at a dilution of 1:25 for 1 h at 37°C. Human IgG and IgM was identified with class specific goat anti-human antibodies labeled with peroxidase (Dianova, Hamburg, DE) and BM-Blue as substrate. Immune phenotyping:
  • TCD tumor cells
  • Table 6 The mean TCD for all patients is 3.2 tumor cells/ 10 mononuclear bone marrow cells before therapy.
  • Clinical data of the tumor stage, type and pretreatment of the study group are presented in Table 2. Staging of the tumor was performed according to the TNM classification. 27 patients were lymph node negative, while 28 were node positive.
  • the 50 mg treatment dose was infused over 120 minutes.
  • Five patients of this 50 mg treatment group had mild immediate allergic reactions (exanthema, hypotension, fever). All symptoms lasted ⁇ 24 hours and resolved spontaneously without specific therapic.
  • Patient Nr. 8 developed symptoms of exanthema, low fever, hyoptension and brochospasm. The side effects were completely reversible by infusion of only 8 mg dexamethason (Fortecortin).
  • the 500mg treatment dose was infused over eight hours. Under these conditions allergic side effects could be avoided, even in patients which had developed allergic symptoms during the first immunizations. No patient developed long lasting toxic side effects. To date, seven patients have received maintenance booster immunizations of 50 mg 7F11 without any side effects. All 12 patients of the 20 mg dosage group (infusion time 4 hours) and all 21 patients of the 2.5 mg treatment group ( infusion time 1 hour) showed no adverse drug effects.
  • the pharmacokinetics of native murine mAb 7F11 in serum is shown in Table 7.
  • Patients receiving 50 mg 7F11 had serum peak concentrations in the range of 6-18 ⁇ g/ml 4 hours post infusion. There was a rather slow decline to 8 ⁇ g/ml after 12 hours and 6 ⁇ g/ml after 48 hours.
  • the peak serum concentrations of 7F11 decreased in parallel with rising HAMA and human anti-7F11 titers.
  • the anti-7F11 titers raised significantly only after the second immunization.
  • all patients of the M0 group developed stable titers in the range of 2-8 ⁇ g/ml during further immunizations.
  • Typical HAMA and anti-7F11 titers of individual patients during 7F11 -therapy are presented in Figs. 2 and 3.
  • HAMA and anti-7F11 titers in the treatment groups with 20 mg and 2.5 mg antibody are not significantly different from that of the 50 mg group.
  • HAMA and 7F11 titers showed the same course during immunization in the majority of the patients.
  • serum titers showed a slow declined with a half life time of 4 to 6 months.
  • Booster immunizations after one year (7 patients) led to dramatic increases and sustained HAMA and anti-7F11 -titers.
  • TCD micrometastatic tumor cells
  • the antibody 7F11 is present at a concentration of 1 mg/ml in physiological saline.
  • Each patient receives on the day of the last immunization an envelope containing a monovette (white) for a further blood withdrawal four weeks after immunization. Arrangement of date for clinical control examination. Fixing a date for the bone marrow control examination three months after the last immunization.
  • the antibody 7F11 is present at a concentration of 1 mg/ml in physiological saline.
  • 20 ml of antibody solution are diluted in 500 ml of physiological saline.
  • Infusion is performed for four hours. Blood pressure is checked every hour. After a follow-up period of one hour, patient can leave the clinic.
  • Each patient receives on the day of the last immunization an envelope containing a monovette (white) for a further blood withdrawal four weeks after immunization. Arrangement of date for clinical control examination. Fixing a date for the bone marrow control examination three months after the last immunization.
  • mice antibody BM-7 when administered in the dose specified above, shows no acute effect on the cardiovascular system in rats.

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Abstract

An immunologically actie polypeptide which specifically binds to the carbohydrate structure of the MUC1 tandem repeat from carcinoma cells, wherein a) the quotient between the affinity of the said polypeptide for a (200 to 400 kDa) glycoprotein fraction from tumor cell-containing ascites of breast cancer patients and or native MUC1 antigen (400 to 440 kDa) from normal cell is 100 : 1 or more, b) the polypeptide does not bind to nonglycosylated MUC1 antigen, and c) the binding of the polypeptide to the said (200 to 440 kDa) glycoprotein fraction changes by 10% or less if the glycoprotein fraction was treated with neuraminidase to cleave N-terminal neuraminic acids, or with formalin, is specific for MUC1 and is useful in the diagnosis and therapy of breast cancer.
EP99917815A 1998-02-13 1999-02-12 Spezifische antikörper gegen brust-tumor assoziiertes mucin, sowie deren herstellung und verwendung Withdrawn EP1056472A2 (de)

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EP1182210A1 (de) * 2000-08-17 2002-02-27 BASTERT, Gunter, Prof.Dr.med.Dr.h.c. Epitope des tumorassoziierten MUC1 Antigens
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