JP2002502621A - Antibodies specific for mucins with breast tumors, methods for producing and using the same - Google Patents

Abstract

  (57) [Summary] An immunologically active polypeptide that specifically binds to the carbohydrate structure of MUC1 tandem repeats from carcinoma cells, a) to a 200 to 440 kDa glycoprotein fraction from ascites containing tumor cells of breast cancer patients Quotient of the polypeptide's affinity for the untreated MUC1 antigen (400 to 440 kDa) from normal cells is 100: 1 or more (molecular weight region analyzed by SDS gel electrophoresis) B) the polypeptide does not bind to the unglycosylated MUC1 antigen; c) the glycoprotein fraction is treated with neuraminidase to cleave the N-terminal neuraminic acid or treated with formalin Sometimes the binding of the polypeptide to the 200 to 440 kDa glycoprotein fraction changes by less than 10% and is specific for MUC1 , Useful in the diagnosis and therapy of breast cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel and specific antibody against the mucin antigen MUC1 associated with breast tumors, a method for producing the same, and a method for diagnostic or therapeutic use.

[0002] Antigens associated with MUC1 tumors (polymorphic epithelial mucins, PEMs) are high molecular weight glycoproteins (> 200 k) with poly-oligosaccharide side chains connecting O-linked serine and threonine residues.
Da). These are very widely variable loci on chromosome 1 (region 21q
). MUC1 is a membrane-permeable glycoprotein on the luminescent surface of tubular epithelial cells extending 200-500 nm or more from the cell surface. It contains a single extracellular domain consisting primarily of tandem repeats of 20 amino acids. The number of tandem repeats (regions 20-120, average 60) in MUC1 (VNTR, variable number of tandem repeats) varies greatly as a result of gene polymorphism. In each tandem repeat,
Up to four serine / threonine residues are used for glycosylation. Short sugar side chains begin with N-acetylgalactosamine and galactose (core 1 structure).
In normal epithelial cells, the nuclear 1 structure extends with the addition of N-acetylgalactosamine, fucose and sialic acid.

[0003] The physiological functions of mucins have not been fully elucidated. In many organs, mucins are likely to protect epithelial cells against pH changes and from mechanical and microbial damage. Mucins are secreted in large quantities by differentiated breast epithelium in milk and by other secretory epithelia in other organs. MUC1 expression in tumor cells shows many important changes. These include: a) loss of polarized and restricted luminescence expression, b) up-regulation and secretion, c) the appearance of novel carbohydrate epitopes resulting from altered glycosylation and unmasking of cryptic peptide epitopes.

[0004] Therefore, mucins have great impact as markers for diagnostic and therapeutic approaches. Underglycosylated MUC1 is highly expressed in breast, ovary, pancreas and other cancer cells and can be recognized as non-MHC restricted T cells as well as HLA restricted T cells. Many groups have described that the APDTR epitope of MUC1 VNTR is a target for cellular T-lymphocytes isolated from patients with breast and other cancers (Reviewed by Finn et al., 1995). Furthermore, the presence of a B cell immune response has been demonstrated in patients with ovarian and breast cancers (A. Ruggetti et al., Cancer Res.
53 (1993) 2457─2461). Thus, MUC1 is produced ex vivo from CD34-enriched peripheral blood cells of breast cancer patients undergoing immunotherapy of cells, such as high dose chemotherapy or PBSCT (limbic blood stem cell therapy). Targeted by dendritic cell (DCs) vaccines. Another possibility is that the synthetic M
Active immunization with UC1 peptide, underglycosylated MUC1 or MUC1-DNA. Other possibilities are mice with specificity for MUC1;
Passive immunization using antibodies that react with the glycones or peptide epitopes of underglycosylated mucins, which are humanized or produced by human mabs, preferably carcinoma cells.

At the ISOBM TD-4 workshop in San Diego, November 17-23, 1996, 56 monoclonal antibodies against MUC1 mucin were analyzed (MR Price, et al., ISOBM TD-4 study). Summary report of the rally, "Analysis of 56 Monoclonal Antibodies to MUC1 Mucin", Tumor Biol. 19, suppl. 1 (1998) 1-20, S. Carger AG, Basel, Switzerland). The study group was attended by 16 research groups. Many antibodies (34/56) have defined epitopes located within the non-glycosylated MUC1 mucin protein core 20 amino acid tandem repeat sequence. Many of these mabs reacted with hydrophilic determinants of the MUC1 protein core.

[0006] Among the remaining 22 antibodies, there was evidence that carbohydrate residues were included in the epitope of 16 antibodies (Table 1). There was no clear relationship between the type of immunogen and each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody, either by direct binding assay or by determining the ability of the synthetic ligand to suppress antibody binding interactions. Characterizing carbohydrate epitopes was more difficult than with peptide epitopes (OE Galania et al., Tumor Bi.
ol. 19, suppl. 1 (1998) 79-87). By controlling epitope accessibility,
Or, by masking the determinants, or by stabilizing the preferred conformation of the peptide epitope within the MUC1 protein nucleus, carbohydrate residues become included in many epitopes. Therefore, characterization of carbohydrate epitopes was more problematic than for protein core epitopes.

[0007] The number of possible substitutions for the linear and branched isomers of small oligosaccharides is
Can reach 10 out of 1000 (RA line, Glycobiology, 4 (1994)
1-9). The findings from this workshop are highlighted in the complexity of the assignment and definition of carbohydrate epitopes for antibodies to MUC1 (KO Lloyd, Tumor Biol. 19, suppl. 1 (1998) 118-121. ). Eight of these mabs did not react with MUC1 in soluble, tumor-bearing, ZR75 breast carcinoma cells. Only four mabs were non-reactive with the repetitive MUC1 tandem repeat peptide, but had strong binding to MUC1 with lactation secreted into milk. Although very limited reactivity is carried out by cloning FH6, the Mab with sialosyl -Le ^x glycolipid specificity has only weak reactivity with membranes spot and Golgi complex in T-47D cells (J. Fukushi et al., J. Biol. Chem. 259 (1984) 10
511-10517). Binding of FH6 to MUC1 is strongly affected by neuraminidase treatment.

[0008] Anti-MUC1-antibody 12H12 (G. Bastert et al., Immunodeficient animals in pharmaco-pharmaceutical research in the Regard Bruner Edition, Basel-Carger, 1987,
224-227) reacts with a tumor-associated antigen known as TAG12 that is expressed in 96% of all breast cancers. The antigen can be found in high concentrations in the cytoplasm and cell membrane of breast cancer tissue and in metastase and is secreted into tumor cells. This antibody is T.A. H. As described by Brumendorf et al. (Canc
er Research 54 (1994) 4162-4168), which could be used for immunoscintigraphy of human breast cancer xenografts. However, the degree of properties of these antibodies is not high enough to make them suitable for routine diagnostic and therapeutic applications.

Furthermore, 12H12 is a MUC1 peptide-specific Mab2E11 (JJ Deal, Natl. Cancer Inst. 88 (1996) 1652-) for analysis of micrometastatic tumor cells in bone marrow.
1658). Bone metastases are common in breast cancer and extensive anatomical studies have resulted in a frequency of 47-85% (L. Weiss and HA Gilbert, GK Hall, Boston, 1981). "Bone metastasis"). Although the goal of immunotherapy of breast cancer is the minimal destruction of semi-clinical tumor sites, there is still no routine diagnostic method that can detect such "micrometastasis". Numerous attempts have been made to find malignant cells in the bone marrow, especially as breast cancer tends to metastasize to bone. Bone marrow as a target organ for metastasis is expected to be easily accessible and can be performed with relatively little risk and patient distress. Detecting "bad cells" can provide information about the early stages of the metastatic cascade, and can also determine the metastatic potential of major tumors.

[0010] It is known that analysis of tumor cell contamination in the bone marrow of breast cancer patients is a strong predictor of patient outcome (JJ Deal, Natl. Cancer Inst. 88 (1996) 165).
2-1658). Enriching rare tumor cells with anti-MUC1 antibodies immobilized on magnetic beads dramatically improves the sensitivity of cytochemical tumor cell detection (S. Carl et al., Abstract 51, Kongress der Deutschen Gesellscahft. fur Gynakologie u
nd Geburtshilfe, Dresden, 1-5, 10.96).

However, the value of such a method is limited by the specificity of the antibody used. Numerous antibodies to different epitopes of human neonatal breast tissue have been previously studied. Many of these are cancer therapeutic antigens (FH Deland et al., J. Nucl. Med. 20).
(1979) 1243-1250), TAG72 (M. Ramki et al., J. Nucl. Med. 32 (1991) 1326
-1332), MAbs B6.2 (Korcher et al., Proc. Natl. Acad. Sci. USA 78 (1
981) 3199-3203), cell surface antigens such as anti-MME (T. Willbanks, Ca.
ncer 48 (1981) 1768-1775), or various others. Many of the MAbs used for breast cancer have been shown to recognize epitopes on mucin molecules (J. Taylor-Papadimitruu, Int. J. Cancer 49 (1991) 1-5). However, highly specific and sensitive monoclonal antibodies for diagnostic and therapeutic purposes have not yet been found (MJ Merino et al., Nucl. Med. Biol. 18 (1991) 437-443.
). However, the known anti-MUC1 antibodies are not highly specific enough and are not suitable for widespread use in diagnosis and therapy.

It is an object of the present invention to provide new and highly specific antibodies, methods for their production, and methods for their diagnostic and therapeutic use. Accordingly, the present invention is directed to immunologically active polypeptides that specifically bind to the tandem repeat carbohydrate structure of the 20 amino acids of MUC1 from tumor (cancer) cells, comprising: a) a breast cancer patient 200 to 400 K from ascites containing tumor cells
The quotient between the affinity of the underglycosylated glycoprotein fraction of Da (analyzed by SDS-PAGE) and the native MUC1 antigen (400 to 440 kDa) from human milk is greater than 100: 1; b) The polypeptide does not bind to the unglycosylated MUC1 antigen; c) the glycoprotein fraction is treated with neuraminidase to cleave the N-terminal neuraminic acid, or in formalin, preferably in phosphate buffered saline (PBS). Is 3.5%
From the 200 of the polypeptide when treated with formalin for 15 to 30 minutes.
10% binding of the 400 kDa glycoprotein fraction (analyzed by SDS-PAGE)
It is a polypeptide characterized by the following changes.

[0013] Polypeptides having such antibody activity and properties have surprisingly been found to have a high degree of specificity for MUC1 presented by tumor cells. Thus, such antibodies are useful tools for the diagnosis and treatment of tumors. It has been surprisingly found that such antibodies have underglycosylated MUC1 reactive with carbohydrate epitopes and are overexpressed and secreted by breast and other cancer types. It is. Such antibodies can be selected by a novel immunization protocol. Ascites fluid from patients with advanced breast cancer was used as a source of antigen. The MUC1 antigen fraction in these ascites fluids was enriched by a two-step procedure using lectin affinity chromatography and size exclusion chromatography. An essential aspect of the invention is that it has strong reactivity against secreted MUC1, but minimal reactivity against normal milk MUC1, deglycosylated milk mucin and / or normal urinary mucin This is an operation according to the present invention as described above, which is advantageous for selecting an antibody having the following.

For the characterization of the antibodies according to the present invention, they are isolated by a lectin affinity chromatography (preferably WGA bound to agarose) from a mixture of ascites fluid from different tumor patients containing secreted proteins. In addition, it is essential to use a glycoprotein fraction containing MUC1. In the second step, the native glycoprotein peak fraction of 1000 kDa (determined by FPLC, 200-440 kD in SDS gel)
(Corresponding to Da), thereby removing immunoglobulins from among others by FPLC gel filtration. Prior art immunizations have been performed using purified protein fractions to screen for hybridoma clones, which have the following properties: -Minimal reaction with antigens isolated from human milk and / or urine. -Perform maximal reaction with WGA fraction. It does not bind to non-glycosylated (synthetic) peptides, for example as described in International Application No. WO 90/05142. -Binds to WGA fraction even after N-terminal neuraminic acid is separated from WGA fraction by neuraminidase or formalin. -Binds the carbohydrate structure of the 20 amino acid tandem repeat of MUC1 from tumor cells.

“Specific binding” in the sense of the present invention means that the antibodies according to the invention do not bind to the peptide structure of MUC1, but only slightly to the carbohydrate structure of MUC1 from human milk, while the tumor cells Binding strongly to the carbohydrate structure of MUC1 from The WGA fraction from ascites (MUC1 underglycosylated)
It is a very low level, natural, fully glycosylated antigen (400 to 440 kD
a) but is essentially characterized by showing a strong band between 200 and 220 kDa in the SDS gel. This difference in molecular weight is due to hypoglycosylation (underglycosylation). The molecular-based “underglycosylation” is 1
Or means that one or more glycosylation sites in two or more tandem repeats are not glycosylated or are occupied by sugar residues as in the case of natural molecules.

Since the average number of repeats contained in MUC1 is about 60 and a maximum of three sugar chains are located in each repeat, fully glycosylated MUC1 is about 150
From 200 to 200 sugar side chains. The average number of sugar residues per side chain is about 10. In underglycosylated MUC1, each underglycosylated glycosylation site is a potential binding site for an antibody of the invention. Less glycosylation of the sugar side chains increases the number of antibodies that can bind. If at least 10 and preferably at least 20 sugar side chains have already been lost or underglycosylated, considerable binding of the antibodies according to the invention to MUC1 should be observed.

Another object of the present invention is that the object is 200 to 400 kDa, preferably 200 to 220 kDa (
A method for producing an immunologically active polypeptide that specifically binds to a hyperglycosylated MUC1 fraction from a tumor cell having a molecular weight of (analyzed by SDS-PAGE), wherein the glycoprotein comprises lectin affinity chromatography. The glycoprotein is isolated from the tumor cell-containing ascites of a breast cancer patient by chromatography, the glycoprotein is separated from the lg protein as needed, and the fraction thus obtained is used for immunization of animals, and In a manner whereby the polypeptide is isolated therefrom, comprising: a) 200 to 400 k from ascites containing tumor cells of a breast cancer patient;
The quotient between the affinity of the underglycosylated glycoprotein fraction of Da (analyzed by SDS-PAGE) and the native MUC1 antigen (400 to 440 kDa) from human milk is greater than 100: 1; b) The polypeptide does not bind to the unglycosylated MUC1 antigen; c) said polypeptide when said glycoprotein fraction is treated with neuraminidase to cleave the N-terminal neuraminic acid or treated with formalin The above
It is characterized in that the binding of a glycoprotein fraction of 200 to 400 kDa (analyzed by SDS-PAGE) changes by less than 10%.

As used herein, “immunologically active polypeptide or antibody” refers to a protein consisting of one or more polypeptides that is substantially decoded by an antibody gene. The recognized antibody genes contain different constant region genes in addition to the myriad antibody variable region genes. Antibodies can be in various forms, eg, in addition to a single chain, Fv,
Fab and F (ab) ₂ (eg, Houston et al., PNAS U
SA 85 (1988) 5879-5883 and, generally, Food et al., Immunology, Benjamin,
New York, 2nd edition (1984) and Funkapillar and Hood, Nature 323 (1986
) 15-16). Preferred antibodies according to the invention are monoclonal antibodies and fragments thereof. The relative affinity of the anti-MUC1 antibody was determined by analysis of the K ₅₀ value, which is the antibody concentration at which binding to MUC1 is achieved at half the maximum (V. Calanicus et al.
Tumor Biol. 19, suppl. 1 (1998) 71-78). For the affinity of the ELISA binding assay, purified MUC1 antigen was coated in microtiter wells. With a molecular weight of 160,000 for the IgG, K ₅₀ value 7F11 is 10 ^-10 M, ascites and T
Had antigens with tumors from -47D breast cancer cells.

After immunization with MUC1 antigen fraction purified from ascites of patients with advanced breast cancer, murine monoclonal antibodies 1E4 and 7F11 were selected.
The antibody has reactivity with an underglycosylated MUC1 carbohydrate epitope that is overexpressed and secreted by breast and other cancer types. The antibody is a useful tool for diagnosing and treating tumors. Antibodies according to the invention, preferably antibodies 7F11 and 1E4, are clearly distinguished from a total of 16 carbohydrate-specific mabs presented at the ISOBM TD-4 International Workshop on Monoclonal Antibodies to MUC1 (1996). Both mabs react with high affinity (K ₅₀ = 10 ⁻¹⁰ M) with MUC1 with tumors from different sources (early tumors, metastatic cells, secreted antigens). In contrast to TD-4 mab (Table 1), 7F11 and 1E4 do not react with normal, fully glycosylated milk MUC1. They are distinguished from clone FH6 by a completely different coloring pattern and by the fact that FH6 immunoreactivity is impaired by desialylation of MUC1 (treatment with neuraminidase or formalin).

As long as it binds to MUC1 in an appropriate manner, the antibody is a whole monoclonal antibody,
Fragments (eg Fv, (Fv)_Two, Fab, Fab 'and F (ab) _Two ), Chimeric, humanized or human antibodies. To MUC1
Short chain antibody fragments containing only CDR regions or portions thereof that confer specific binding
This is particularly preferred when the antibody is labeled. lgG1 isoto
Group antibodies are also preferred. For production of monoclonal antibodies, see, for example, E. Harlow
And D. Rain's Antibody: Laboratory Manual, Cold Spring Herba
-Press (1988), Besler et al., Immunobiol. 170 (1985) 239-244, and Yun
K. et al., Angewandte Chemie 97 (1985) 883 or Cyan Friglia et al., Hybridoma Vo
See l.2 (1993) 451-457. The diagnostic use of the antibodies of the invention has been implemented as a known method by immunological determination processes.
Give. This type of process is well known and requires no further explanation. The present invention
Can be used as unlabeled and / or immobilized receptors
Wear.

The immobilized receptor can be used for a method for removing tumor cells or secreted MUC1 antigen from tissue of the patient's body, wherein as claimed in claims 1 or 2, The body tissue is contacted with the immobilized antibody, the tumor cells or the secreted MUC1 antigen are bound to the immobilized antibody, and the tumor cells or the secreted MUC1 thus bound to the solid phase are It is separated from the body tissues together with the solid phase. In each case of such a diagnostic immunization method, a change in signal is assessed after binding of at least one antibody according to the invention.

The monoclonal antibodies obtained by the method according to the invention bind to the surface of tumor cells and can be used in human organisms. Thus, the present invention can also provide a pharmaceutical composition comprising one or more antibodies according to the present invention, optionally used with conventional pharmaceutical carriers, adjuvants, filling or additional substances. You can also. The administration of the drug according to the present invention is useful for treating micrometastasis, especially for treating breast cancer. The dosage of the antibody according to the invention for a therapeutic treatment is about 2 μg to 20 mg, preferably 2 μg to 20 mg / kg-body weight, and this administration should be repeated.

In order to treat a patient with breast cancer type with an antibody according to the present invention, after a surgical procedure,
It is preferred that lymph node-positive patients receive chemotherapy with established adjuvants (before menopause) or hormonal therapy (after menopause) (about 45%). The next step is to diagnose the presence of tumor cells in the bone marrow. This is preferably achieved by using the antibodies of the present invention, but can also be achieved by other tests that identify common tumor markers (such as cytokeseratin). If the bone marrow response is positive, preferably 6 to 8 immunizations are performed using the antibodies of the invention at 1 to 4 week intervals. The amount of antibody used is in the range of 0.2 to 50 mg per immunization. This antibody treatment is given during adjuvant hormone treatment or after chemotherapy. In the case of hormonal treatment, it is preferably carried out in a parallel manner (in the latter case there is no immunodrug suppression). The development of antibodies against the anti-MUC1 antibodies according to the invention has been followed during the immunization phase with the antibodies according to the invention or by established tests for human anti-mouse antibodies (HAMA). A desirable therapeutic level is that the antibody titer is stable for about 3 to 4 months. Another measurement of tumor cells in the bone marrow was three times after the last immunization.
Perform after months. At least 6 × 10 ⁶ bone marrow cells are analyzed for contamination of tumor cells. Using the antibodies of the invention, 17 out of 18 patients were found to show a negative response in the assay of tumor cells in the bone marrow, as a result of the therapeutic application of the antibodies of the invention. This suggests that tumor cells in the bone marrow were clearly destroyed. Surprisingly, the antibodies of the invention also show effector effects, which, after binding to tumor cells, allow the tumor cells to be recognized as foreign cells, complement, anti-dementia antibodies,
Probably eliminated by activation with activated NK-cells or T-cells, or a combination thereof.

It has been shown that the antibodies of the present invention also bind to secreted MUC1 in body fluids such as lymph, which eliminates the immunosuppression caused by MUC1 due to immune recognition by body fluids or cells. To have a therapeutic effect or an effect as an immunosuppressive drug. This can also be used for treatment. In addition, MUC1 contains ovary (> 80%), lung (60%), prostate (50%), pancreas (100%).
%), Was also found to be overexpressed in kidney and colon tumor cells. The antibody of the present invention can be used for diagnosis and treatment of such substances. Cytotoxic antibodies that provide effector function for therapeutic purposes (ADCC, CDC)
It is particularly preferred to use (Bruggemann et al., J. Exp. Med. 166 (1987) 1357-1361).

In another approach, an antibody or a portion thereof is conjugated or migrated and bound to a toxin molecule (immunotoxin) to produce the effect of specifically killing tumor cells (Brinkman 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 the antibody or a portion thereof may be conjugated to a cytokine or interleukin. In another preferred embodiment of the invention, bispecific antibodies are used for the treatment of tumors (Bonino et al., BFE 9 (1992) 719-723), which are used to generate peptide chains in vitro by hybrid hybridoma development or diabody construction. Reconstitution (Holiger et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448, Holliger and Winter, Current Opin. Biotechnol. (1993) 446-449).

With respect to immunotoxins, the antibodies of the invention are preferably conjugated 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 that the antibody be conjugated to a chemotherapeutic agent such as, for example, doxorubicin, which has a cytotoxic effect, or a radiolabeled substance. Also preferred are antibodies of the present invention, especially conjugates of human antibodies for imaging in vivo, for example using radioactive or fluorescent materials. Immunotoxins can preferably be produced in one of two principally different ways.

The first method is to use an antibody or a fragment thereof (usually produced by proteolysis,
For example, Fab fragments) are chemically coupled to a toxin or a fragment of a toxin in vitro. For practical reasons, the antibody portion of this type of immunotoxin can be a complete antibody (consisting of two light and two heavy chains), or a more preferred Fab fragment (one light and one heavy chain). VH- and CH1 regions). In another method, the immunotoxin is produced by recombinant DNA technology, which results in a defined, homogeneous molecule in any case. The size of the antibody portion is made as small as possible to obtain a small immunotoxin with good tissue permeability. In this method, the smallest antibody fragment actually available is not a Fab fragment, but a functionally variable domain of an antibody consisting solely of the heavy chain VH-region and the light chain VL-region. VH and VL regions (polypeptide chains of about 100 amino acids each)
Must form a variable domain that confers functional assembly or antigen binding. In the absence of the rest of the antibody, the VH and VL regions form only very unstable complexes. Therefore, these complexes are preferably stabilized by covalent bonds.

One possibility is to bring the VH and VL regions to the DNA level (or vice versa).
The condensation does the same for toxins. After expression, a single polypeptide chain is formed, in which the VH- and VL-regions connected by a peptide linker are incorporated into a stable and variable domain, while the toxin is linked via a second peptide linker. For example, to VL (Brinkman et al., Proc. Natl. Acad. Sci.
USA 89 (1992) 3075-3079). The length of both peptide linkers is variable and may even be reduced to a single peptide bond. Molecules of this type are designated by the term "single-chain antibody" or the term "single-chain immunotoxin", analogous to scFV, where the terms VH and V are connected by a peptide linker or bond.
Used for a single polypeptide chain containing both L.

Another possibility for stabilizing the VH and VL assemblies is that of Brinkman et al.
Natl. Acad. Sci. USA 90 (1993) 7538-7542. In this technique, the VH and VL amino acids were defined by computer modeling and were adjacent to each other in the VH-VL complex. The amino acids naturally occurring at these positions were then each replaced by cysteine at the DNA level. In order to obtain a functional immunotoxin in this case, two separate polypeptide chains appear (in separated cells, for example, prokaryotic cells, such as E. Coli), one of which is only the VH-region, The other is a VL-region linked to the toxin moiety by a peptide linker. The two polypeptide chains are mixed under appropriate conditions, thereby incorporating a functional immunotoxin, where the VH and VL in the variable antibody domain are between the two engineered cysteines. Are connected by a disulfide bond. The antibody portion of this type of immunotoxin is denoted as dsFV,
Therefore, the entire molecule is designated as "dsFV-immunotoxin".

Of course, immunotoxins can be obtained by recombinant DNA technology, for example by means of large Fab-fragments (VH-CH1 non-covalently incorporated into VL-CL, one of which is linked to the toxin by a peptide linker). There are other possibilities to produce using. However, Brinkman et al., Proc. Natl. Acad. Sci. USA 89 (199
2) Proc. Natl. Acad. Sci. USA 90 (1993) by 3075-3079 and Brinkman et al.
The possibilities described in 7538-7542 are preferred. Regarding the toxin portion of the immunotoxin, Pseudomonas exotoxin (PE)
Preferred fragments are PE38 and PE40 and derivatives of Yura (
I. Pastan et al., WO 92/07271 and WO 90/12592). Single-chain Fv-chain immunotoxins can preferably be produced as a single polypeptide chain in E. coli using the T7 RNA Holimerase expression system. The polypeptide is obtained in an inactive form and must be activated by in vitro regeneration.

Another method for the production of peptide-linked single-chain immunotoxins is described in WO88.
/ 09344. Single chain antibodies with a peptide linker between the light and heavy chains are described in WO 88/01649. The production of chimeric antibodies comprising at least the variable region of the heavy or light chains, whereby one of these chains is linked to the non-lg molecule by a peptide bond is described in EP-B0193276. The T7 RNA polymerase expression system is disclosed in US Pat. No. 7,648,9.
Nos. 71, 4,952,496 and 6,595,016. The polynucleotides of the invention and the recombinantly produced antibodies of the invention can be prepared by known methods and by the methods of Sunblock et al., Molecular Cloning, Laboratory Manual, Second Edition, Cold Spring Harbor Press, New York, 1989), and Berger and Kimmel, Methods of Enzymology, Volume 152, Molecular Cloning Techniques (1987
) ", Prepared based on the sequence data described in Academic Press, Inc. of San Diego, California, which is incorporated herein by reference. Preferably, the polynucleotide of the present invention is formed from a synthetic oligonucleotide.

[0032] Such recombinant polypeptides can be expressed in eukaryotic or prokaryotic host cells according to known standard methods, preferably mammalian cells such as lymphocyte cell lines can be used as host cells. Typically, such a polynucleotide structure is the amino acid sequence of an antibody according to the invention, the heavy chain of a fully human antibody and / or the light chain of a fully human antibody, each having at least the variable region of a heavy and / or light chain. Break the chain. Alternative human constant region sequences containing human constant region isotopes other than the antibody chains and naturally occurring sequences can be substituted, and such alternative human constant region sequences can be obtained by those skilled in the art from various sources. And the literature is referred to by E. A. These include, but are not limited to, those listed in Kabat et al., "Sequences of Immunologically Interesting Proteins (1987)" (National Institute of Health, Bethesta, MD). In one aspect of the invention, an amino-terminal peptide bond (to the variable region of the light chain of an antibody according to the invention)
(I.e., in-frame binding) and the corresponding heavy chains appear, forming light / heavy chain dimers and other types of antibodies.

In general, prokaryotic cells can be used for cloning DNA sequences that pirate the antibody chains according to the present invention. E. coli is a prokaryotic host cell particularly useful for cloning the DNA sequences of the present invention. Alternatively, oligonucleotides may be chemically synthesized by various methods, including phosphoramidite synthesis. Polynucleotide structures typically include an expression control sequence operably linked to a coding sequence that includes a naturally occurring or heterologous tissue promoter region. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of converting or transfecting eukaryotic host cells. Once the vector has been incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of antibodies according to the present invention. As eukaryotic host cells, cultures of mammalian tissue cells can also be used to produce the polypeptides of the invention. Mammalian cells are indeed preferred, since a number of suitable host cell lines have been previously developed that can secrete native heterologous tissue proteins, including CHO cell lines, various COS cell lines, HeLa cells, Includes myeloma cell lines.

Typically, a polynucleotide sequence that decodes the heavy and / or light chains of an antibody according to the present invention is introduced and expressed in glycosylated cells that glycosylate the antibody. As used herein, a "glycosylated cell" is a cell capable of glycosylating a protein, especially at least one polypeptide expressed in the cell, especially at least one glycosylation site sequence in a secreted protein. An N-linked "nuclear oligosaccharide" containing at least one mannose residue and / or
Or eukaryotic cells to which O-linked sugars can be added. Thus, a glycosylated cell contains at least one enzymatic activator that catalyzes the addition of a sugar residue to a glycosylation site sequence in a protein or polypeptide, wherein the cell actually contains at least one expressed polypeptide. To glycosylation. For example, but not by way of limitation, mammalian cells are typically glycosylated cells. Insect cells and yeast are also glycosylated cells.

Once expressed, antibodies according to the present invention can be purified by standard techniques of the prior art, including HPLC purification, fractional column chromatography, gel electrophoresis, etc. [R.
See Scopes, "Protein Purification (1982)" (Springer Verlag, New York). Therapeutic compounds of the invention can be administered intravascularly using forms known in the pharmaceutical arts.
It can be administered parenterally, such as intraperitoneally, subcutaneously, intramuscularly. The active pharmaceutical ingredient of the present invention can be used in liquid, powder or lyophilic form, and may be combined with a suitable diluent or carrier such as water, saline, dextrin, buffer and the like. A preservative may be added.

Regardless of the route of administration selected, the compounds of the present invention are formulated into pharmaceutically acceptable dosage forms according to conventional methods known to those skilled in the art. The compounds may be formulated using pharmaceutically acceptable acid or base addition salts. Further, the compound or a salt thereof may be used in a suitable hydrolyzed form. Regardless of the route of administration chosen, a non-toxic but therapeutically effective amount of one or more of the compounds of the present invention is used for any treatment. The method of administration for treatment will be selected according to a variety of factors including the type of patient, age, weight, sex and medical condition, type of tumor, route of administration and special compounds used in the treatment. A physician of ordinary skill can readily determine and prescribe the effective amount of the drug required for the known antibody therapy approaches. In such a process, the physician was able to first use a relatively low dose and subsequently increase the dose until a maximal response was obtained.

Pharmaceutical compositions comprising an antibody according to the present invention are useful for topical or parenteral administration, ie, subcutaneously, intramuscularly, intravenously or through the skin. Compositions for parenteral administration comprise a solution of the antibody dissolved in a generally accepted carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, eg, water, buffered water, 0.4% saline, 0.3% glycine, and the like. The solution is sterile and generally free of particulate matter. The compositions are sterilized by conventional, well-known techniques. The composition is required to bring it to a nearly appropriate physiological state such as a pH adjusting and buffering agent such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. It may contain acceptable auxiliary substances. The concentration of the antibodies according to the invention in these formulations can vary widely, for example in the range of less than about 0.01%, usually at least about 0.1% to as high as 5% by weight, mainly on the basis of liquid volume, viscosity and the like. Or according to the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intramuscular injection would contain up to 1 ml of sterile buffered water and about 1 to 50
mg of the antibody according to the invention. Antibodies according to the present invention may be rendered lyophilic for storage and reconstituted in a suitable carrier prior to use. Conventional lyophilic and reconstitution techniques can be used. It can be appreciated by those skilled in the art that lyophilicity and reconstitution can vary the degree of loss of biological activity and that use levels must be adjusted for compensation.

The safety, dosage and therapeutic potential of the anti-MUC1 antibody according to the present invention was evaluated in a clinical case study of 55 breast cancer patients. Minimal residual disease (n = 47)
And patients with metastatic disease (n = 8) participated in the study. Prior to standard adjuvant treatment and treatment with antibody 7F11, all patients demonstrated contamination of bone marrow with tumor cells during the initial procedure. Eighteen patients received multiple infusions of 50 mg of natural 7F11 every four weeks. One patient with advanced metastatic breast cancer received four 50 mg doses at weekly intervals. The second group of 14 patients was 7F11
A third group of 22 patients received the 2.5 mg treatment, while being treated with the 20 mg dose.
In general, antibody infusions were well tolerated. Only 6 patients are quick (2 hours) 7F11
Received a mild allergic reaction immediately after receiving a 50 mg infusion. These side effects are 50mg
This could be avoided by extending the infusion time to 8 hours. No systemic side effects were encountered. No adverse side effects were observed in the 20 mg and 2.5 mg treatment groups, with infusion times of 4 and 1 hour, respectively. Human anti-mouse antibody (HAMA)
The immunization was repeated until a stable titer was obtained in the range of 1 to 100 μg / ml and the anti-7F11 antibody (ab ² ) ゅ stable titer. Three months later, bone marrow was analyzed for micrometastase detection and assay. Up to this point, 24 patients had bone marrow 7F
11 Re-examination during and after treatment, other patients still on treatment. Eighteen patients in the M0 group were found to be free of contaminated tumor cells in the bone marrow.
In 10 patients, an increase in tumor cells from 3 to 7 out of 10 ⁶ bone marrow cells was recorded. During the follow-up, all immunized patients remained tumor-free. By the way, 7 of these patients received enhanced prophylactic immunization as maintenance treatment, resulting in a dramatic increase in HAMA and 7F11 titers. These boost immunizations are tolerated without causing adverse events, indicating that maintenance treatment with murine 7F11 is feasible despite increased HAMA and 7F11 titers.

In the group of patients with clinically proven local disease or metastasis before 7F11 treatment, in two cases there was depletion of tumor cells in the bone marrow, a decrease was recorded in two patients, Cell proliferation was seen in one other patient. Three patients are still being treated. One patient with advanced breast cancer received a 50 mg infusion of 7F11 at weekly intervals. Twenty-four hours after the first treatment lymphocytes, bone marrow and ascites fluid were analyzed for 7F11 biodistribution. Lymphocyte 7F11 levels of 10 μg / ml resulted in 8 μg / ml in bone marrow and 6 μg / ml in ascites, respectively. Under these conditions, the tumor cells in the bone marrow (> 100/10 ⁶ normal bone marrow cells) and tumor cells in ascites (10 cells / ml)
Demonstrated uniform and strong 7F11 labeling of the cell membrane and cytoplasm.

Overall, clinical studies show the safety, tolerance, and pharmacokinetics of the difference between multiple doses of anti-MUC1 bone marrow according to the present invention in breast cancer patients with minimal residual disease.
Micrometastasis tumor cells in the bone marrow were removed in 18 patients in the MO group and 2 patients with locoregional disease. The study shows that treatment of breast cancer with unjoined bone marrow according to the present invention can eliminate tumor cells in the bone marrow either by a passive cytotoxic passive effector mechanism or by a mental illness network response. Is shown. In addition, the tracking of micrometastases in the bone marrow offers an extremely sensitive surrogate marker for the efficacy of adjuvant tumor therapy.

Induction of immunity to an antigen (TAA) such as GA733-2 (CO17-1A) with a tumor removes tumor cells or delays the reappearance of the disease. Studies on colon cancer patients using natural mAb 17-1A repeatedly soaked at high doses (500 mg) have reduced mortality to 30% (G. Rietmuller, Lancet 343).
(1994) 1177-1183). J. in a group of patients with advanced colon cancer treated with 17-1A. Recent results from Fagerberg et al. Cancer Immunol. Immunother. 38 (1994) 149-159 show that a) patients with significant ab2 responses had a long survival time, b) 2 g Higher total doses of the range of native mAbs led to lower concentrations of human ab2, and c) that recombinant humanized 17-1A greatly reduced titers of antipsychotic antibodies. .

Breast mucin is a highly glycosylated protein encoded by the gene MUC1 on chromosome 1q. The antigen is overexpressed in breast and ovarian carcinomas,
Modified and glycosylated. Mucins with tumors are found in the cytoplasm, cell membrane and secretory components. These cell membranes and secreted TAA are humoral (A. Ruggetti et al., Cancer Res. 53 (1993) 2457-2461) and cell-mediated immunity (HR Jerome, J. Immunol. 151) in breast cancer patients. 1993) 1654-1662). Thus, mabs with MUC1 specificity are candidates for selection of interventions in immunotherapy (for review, see V. Apostolobouros and FC Mackenzie Critical Rev. Immunology 14 (1994) 293-309).
).

Our research group has developed a number of monoclonal antibodies (mAbs) to different epitopes of human breast mucin (BM). MAb 7F11 specific for the carbohydrate / peptide binding site of the tandem repeat region of human breast mucin MUC1
Is reactive with over 96% of breast cancer types. The 7F11 color pattern during solid tumors and metastases is generally homogeneous with more than 80% of the colored tumor cells. Therefore, 7F11 can be used for analysis of tumor cells in bone marrow and for immunoscintigraphy.

Flow cytology and histological analyzes demonstrated high tumor selectivity and minimal cross-reactivity with normal cells of different tissues for Mab7F11. For this reason, mAbs were selected for immunotherapy treatment of breast cancer patients with minimal residual disease. This immunization protocol was designed for a) psychiatric disease network activation, b) B-cell activation, c) T-cell activation, or d) antibody-dependent cellular cytotoxicity (ADCC). Thus, 7F11 has relatively low doses of 2.5, 20 and 4 at 4-week intervals.
It was administered at 50 mg. No toxic side effects other than allergic reactions were encountered in a group of 55 patients. In particular, no anaflexic reaction was observed. Three patients received a total of 450 mg of 7F11.

All patients in the study group received HAMA and anti-7 after 3 to 10 immunizations.
With a stable and long-lasting titer of F11 antibody, one patient obtained a stable titer after one immunization (average 5.6). Thus, the number of immunizations is suitably 1-10, preferably 8-9, most preferably 6-8, depending on the individual patient and the patient's ability to obtain a stable and long-lasting titration of the antibody. It is. Maximum HAMA titers are in the range of 10 to 100 μm / ml with an average lymphocyte half-life of 5 months after the last immunization. In 4 patients (patients 1, 2, 4 and 5 in Table 1) a 25-40% antipsychotic response is related to ab2β-type according to the antigen binding site.

Three months after the last immunization, the patient underwent a retest for tumor cell contamination in the bone marrow. Bone marrow cells from each patient were analyzed immunologically and by automated fraction analysis with at least 6 cell spins. Eighteen patients in the MO group and two patients with local disease were negative for micrometastatic tumor cells. Only one patient in the M0 group had increased tumor cells in the bone marrow. These results clearly show that passive immunotherapy with 7F11 can eliminate individual micrometastatic tumor cells in the bone marrow of most breast cancer patients in situations using adjuvants. Unconjugated murine mAbs7F11 affects the human immune system in different ways. The direct mechanism relies on tumor cell labeling and is triggered by antibody-dependent cytotoxicity, complementation-dependent cytotoxicity and apoptosis. Indirect mechanisms occur through the immune network. Antipsychotic antibody (ab) that binds to the hypervariable region of a therapeutic mouse antibody
2β) leads to T-cells (T2) and induces the development of anti-antipsychotic antibodies (ab3) and T-cell (T3) clones. The normal antigenic epitope of human breast mucin, defined by a human ab3 antibody recognizing ab2β and a therapeutic mouse antibody, responds to the favorable outcome of the immunized patient.

In conclusion, 7F11 treatment resulted in a dramatic reduction or eradication of micrometastatic breast tumor cells in the bone marrow of breast cancer patients in the adjuvant setting. The cell line DSM described in the present invention which secretes antibodies 7F11 (1) and 1E4 (2)
ACC2329 (1) and DSM ACC2328 (2) were issued on October 31, 1997 by Deutsche D. 38124, Bradschweig, Germany, Macheroder Bek 1b.
It was marketed by Samlung von Microorganismen und Zerkulturen Gämbehr (DSM) and Boehringer Mannheim Gämbehr. The following tables, examples, references, sequence listings and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is to be understood that modifications can be made in the operations described without departing from the spirit of the invention.

Abbreviated list HAMA = human anti-mouse antibody PBS = phosphate buffered saline APAAP technology = alkaline phosphatase anti-alkaline phosphatase complex TCD = number of tumor cells TAA = antigen with tumor PUM = polymorphic urinary mucin glycoprotein EIA = Enzyme immunoassay PMSF = phenylmethylsulfonyl fluoride WGA = wheat malt agglutinin BSA = bovine serum albumin PP = phosphate buffer TFMSA = trifluoromethanesulfonic acid MAPS = monoclonal antibody binding solution (BioRad) PE = phycoerythrin PECy5 = tandem conjugate phycoerythrin And cyanine 5

[0050]

[Table 1]

Example 1 Identification and Purification of Antibodies Monoclonal antibody 1E4 Clone 1E4 (lgG1, k) was selected from BALB / c mice immunized eight times with MUC1 fractions from three patients every 4 to 6 weeks. did. Splenocytes were converted to X63-P3 by an established method (1) using PEG6000.
X63Ag8.653 (ATCC CRL1580) conjugated to mouse myeloma cells. Hybridomas were selected in ten 96-well plates using mouse macrophages as feeder cells. Supernatants were screened by ELISA and immunocytological techniques. Nineteen hybridoma clones with strong MUC1 reactivity were identified and cryopreserved. The main selection criterion for clone 1E4 is that it has strong reactivity with antigen fractions with various tumors,
With no reactivity with the VNTR peptide sequence APDTR (SEQ ID NO: 5) and with normal milk MUC1, deglycosylated MUC1 and normal urinary mucin (polymorphic urinary mucinous glycoprotein PUM) It was determined to have minimal reactivity.

Monoclonal antibody 7F11 Clone 7F11 (lgG1, k) was induced by immunizing BALB / c mice eight times with MUC1 fraction purified from ascites fluid and breast cancer cell line T47-D from two patients. did. After conjugation of splenocytes with P3X63Ag8.653 myeloma cells, nine hybridomas with MUC1 specificity were identified. The selection criteria were the same as for 1E4, that is, it had strong reactivity with secreted MUC1, but had minimal reactivity with normal milk MUC1, deglycosylated MUC1 and PUM.

Method 1. Myeloma P3X63Ag8.653 without cellular mycoplasm was used. Rat
g non-producing cell lines were prepared with 4.5 g / l (D +) glucose, 0.7 g / l NaHCO ₃ , 10% rescue bovine serum (FCS), 15 mM HEPES, 0.5%
Pericillin-streptomycin and 2 mM glutamine (DMEM, all Gibco, Eggenstein, Germany).

Breast cancer lines T47D (HTB133), SKBR3 (HTB30), MCF7 (
HTB22) and ZR75 (CRL1500) were received from the ATCC. KS
, WA, AR, KM22 and HG15 breast cancer lines were established using ascites (KS), early cancer types (WA, AR) and bone marrow (KM22 and HG15 in breast cancer patients). The 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 using ascites fluid.

[0055] 2. Purification of MUC1 Glycoprotein Fraction from Ascites Fluid and Pleural Exudates Ascites and pleural exudates were collected from 26 patients with advanced breast cancer, clarified by membrane filtration, and analyzed for MUC1 content by EIA. 2-3 liters of highly positive samples from individual patients were frozen at -20 ° C and used for antigen purification.

[0056] 3. Production of Standard Antigen (BMA) from Breast Cancer Lines KS and T47-D T47-D cells (malignant pleural effusion, breast carcinoma, ATCC HTB133) and cell line KS at Nunc factory (Nunc, Wiesbaden, Germany). Grew. Batches of 2 × 10 ⁹ cells were collected by trypsination and washed twice with PBS. The cells are incubated for 1 hour at 4 ° C. under constant agitation in 500 ml lysis buffer (10 mM TR).
IS / HCl, pH 7.6, 2 mM EDTA, 1% Triton® X-
100, 1% aprotinin, and 1 mM PMSF). 10000 g
The supernatant (cytosol) was stored at -70 ° C or used for antigen purification.

[0057] 4. Purification of MUC1 4.1 Antigen purification for hybridoma production 4.1.2 Lectin affinity chromatography Cytosol, ascites mab pleural effusion was transferred to wheat malt agglutinin (WGA) Sepharose (WGA) in a 2 × 30 cm column at a flow rate of 2-5 ml / min. Purification by lectin affinity chromatography on Pharmacia, Freiburg, Germany). The column was washed with low and high concentrations of salt (2M sodium chloride) in PBS, and the glycoprotein was eluted with N-acetylglucosamine (125 mg / ml) at a flow rate of 1 ml / min, dialyzed against PBS, and diluted with YM100. It was concentrated 10 times by ultracentrifugation on a membrane (Amicon, Biden, Germany). Protein content was analyzed using a microassay by Raleigh (Biorad, Munich, Germany).

4.1.3 Gel Exclusion Chromatography Lectin affinity fractions of ascites and cytosolic proteins were purified by Sperose 6 rapid protein liquid chromatography (FPLC, Pharmacia) in a 2.5 × 100 cm entanglement. The MUC1 positive fraction in the molecular weight region from 200 to 1000 kDa was
Analyzed in A, adjusted to 2-10 μg / ml and used for immunization of BALB / c mice and for specificity testing of hybridoma supernatants. Antigen was stored at -20 ° C.

4.2 Immunoaffinity Chromatography Mab 2E11, which has reactivity with native and glycosylated MUC1, was loaded on 1 ml gel.
The antibody was bound to Affi-Gel 10 (Bio-Rad) at a concentration of 10 mg of antibody per antibody. The cytosol was applied as a 250 ml batch to the antibody column at a flow rate of 1 ml / min and then washed extensively with PBS and PBS-2M sodium chloride. Bound antigen was eluted with 7M urea at pH 6 at a flow rate of 0.5 ml / min and dialyzed against 5IPBS overnight. The purified antigen was then passed through a 5 ml goat anti-mouse IgG-agarose (Zicuma, Daisenhofen, Germany) column to remove traces of 2E11.
The protein was analyzed by Raleigh (Biorad) and adjusted to 200 μg protein / ml. This breast mucin antigen (BMA) is 1:35 to 1: 100 for the 500 U standard.
Can be used as a standard in MUC1 assays at dilutions of.

[0060] 5. SDS-PAGE and Western immunoblotting proteins were separated according to Laemmli under reducing conditions in 7.5% acrylamide gel (16 × 16 cm, Biorad Protean) and purified in 30% chrysin / methanol buffer.
Converted to nitrocellulose at 0 mA for 4 hours. Nitrocellulose is PBS, 0.1
Blocking was performed for 1 hour with 20% Tween 20 and 2% dry milk powder. Purified antigen fractions and deglycosylated antigens of different sources were tested for 1 hour with biotinylated mab (1 μg / ml PBS, 1% BSA), and strepavidin peroxidase and 4-chloro-1-naphthol. Was used as a substrate for detection.

[0061] 6. A sample of MUC1 purified with enzymatic and chemical deglycosylation affinity of MUC1 is dialyzed against 0.1 M PP, pH 5.5 (neuraminidase) or 0.1 M PP, pH 6.0 (O-glycanase), Neuraminidase (2, 5, 10, 20 mU, Boehringer from Mannheim, Germany-Vibrio cholere from Mannheim Gämbecher) or O-glycanase (2,
20 and 200 mU, using the Beringer's Dipiococcus pneumoniae)
The cells were cultured at 37 ° C for 24 hours. Digestion was stopped by heating the sample to 100 ° C. for 5 minutes. Chemical deglycosylation with TFMSA is described in S. B. Anal by Edge et al.
Biochem. 118 (1981) 131-137. Purified antigen (0.1 mg
) Was made lyophilic, cultured at 200C for 1 to 3 hours at 4 ° C, cooled to -80 ° C, neutralized with 50% aqueous pyridine, and dialyzed against 0.1% ammonium bicarbonate.

[0062] 7. The T47-D cytosolic protein was partially purified by developed Sucrose 6 gel exclusion chromatography of the MUC1 peptide-specific mab 2E11 . BALB / c mice were immunized using a high molecular weight fraction of 1-2 × 10 ⁶ Da according to established methods. The last immunization was performed with antigen treated with neuraminidase and O-glycanase. Splenocytes were conjugated to X63Ag853 antigenoma cells. Hybridoma 2E11 for EL
ISA, cytological and histological selections. Mabu (lgG3, k) showed strong and broad reactivity with formalin resistant epitopes in breast and other cancer types. Antibodies are
It could be used alone or in combination with 7F11 and 1E4 for quantitative determination of lymph MUC1 levels. A two-site sandwich EIA test with 7F11 as coating and 2E11 labeled with peroxidase was developed for MUC1 quantification. Mab 2E11 was cloned with eleven limiting dilutions.

[0063] 8. Immunization and selection of clone 1E4 MUC1 was purified from ascites of patients FO, OC and ST by WGA-chromatography and Suprose 6 gel filtration. Female BALB / c mice were immunized sequentially with antigens from different patients eight times every 4-6 weeks.
Splenocytes are removed 4 days after enhanced immunization and established methods using PEG6000 (S. Cowl, "Development of monoclonal antibodies with specificity for mammalian cells", Center for Biochemistry, University of Frankfurt, Germany) (Visual Acquisition Paper, 1983) using X63-Ag853 mouse myeloma cells. Hybridomas were selected in 10 96-well plates using mouse macrophages (PM) as feeder cells. Two weeks later, supernatants were screened using ELISA and cytological techniques. Nineteen hybridoma clones with strong MUC1 reactivity were identified and cryopreserved. The primary selection criteria for clone 1E4 is that it has minimal reactivity with normal milk MUC1, deglycosylated MUC1 and normal urine mucin (polymorphic urinary mucinous glycoprotein PUM), Cancer cell line Y
It had strong cytological reactivity with 47D and KS and minimal cross-reactivity with human fibroblasts. 1E4 was cloned at eight limiting dilutions.

9. Immunization and selection of clone 7F11 MUC1 was obtained from the ascites of patients ST and ZE and ST and from the breast cancer cell line T47.
-D was purified by WGA-chromatography and Superose 6 gel filtration. BALB / c mice were immunized eight times sequentially with different antibody fractions. After conjugation of splenocytes to X63-Ag853 myeloma cells, MUC
Nine of these hybridomas with one specificity were identified. Clone 7F11 (lgG1
, K) were selected using ELISA and cytological techniques and were converted to normal milk MUC1
With minimal reactivity with deglycosylated MUC1 and PUM and with MU from lymph
It was characterized by having strong reactivity with C1, ascites and tumor cells.
Mab 7F11 was cloned at 12 limiting dilutions.

10. Immunization of clone 5A6 and selection of MUC1 from lymph of patient ZI and secreted MUC1 of T47-D cells
(Tissue culture supernatant) was purified by WGA-chromatography and Superose 6 gel filtration. Both antigen fractions were mixed and used for three immunotherapy treatments of BALB / c mice. After splinocytes were conjugated to X63-Ag853 myeloma cells (February 1990), 24 hybridomas with MUC1 specificity were identified. Clone 5A6 (lgG1, k) was selected by ELISA and cytological techniques using native and deglycosylated antigen fractions from lymph, ascites, tumor cells and normal cells. 5A6 was cloned at 10 limiting dilutions.

[0066]11. Cloning of hybridoma In order to select stable clones with high productivity, 1 × 10 ^Four 10% FCS, 50 U penicillin / ml, 50 U in 96 well plates containing PM
0.5, 1, 2 and 5 cells in DMEM with μg streptomycin / ml
Seeds formed at the density of the wells. Six days later, the plate is examined under a microscope and a single
An hybridoma clone was identified. In antigen-coated plates, specific anti-
Tested for body production. 6-8 high productivity clones from each cloning step
The lawn grew as a bulky culture and was frozen in liquid nitrogen. All clones
, Transferrin, insulin, selen (Boehringer Mannheim Geh
Mbeher product) and 0.5% bovine serum albumin (ALBMAC from GIPCO)
15 mM HEPES, 0.8 g NaHC, supplemented with (complete D / H medium)
O_Three/ Liter, 4.5 g / liter no glucose, 2.5% FCS (Gipco,
DMEM / Nutrient Mix F without antibiotics
Finally grown in 12 (Gipco). 2E11, 1E4: 7F in D / H medium
Ig production of 11 and 5A6 ranged from 15-25 μg / ml in static cultures.

12. Production of antibodies Using a type 3570 bioreactor with hollow fibers (70 kDa membrane),
Antibodies were produced in self-firm units (Unidin, Heraus, Germany). 2 × 10 ⁹ hybridoma cells were inoculated in complete D / H medium with 2.5% FCS. Lymph decreased to 1% in 3 weeks. During production, hybridomas grew at a media feed rate of 4 liters / day. Every day 100-200 ml of supernatant was taken, analyzed for lg content, clarified by membrane filtration and stored at -20 ° C. There was no decrease in lg synthesis rate during the 2-3 month production cycle.

13. The antibody purified mab was purified by "Protein A-Sepharose 4-Fast-Flow (Pharmacia). The supernatant of the bioreactor was mixed with the same amount of binding buffer (Biorad MAPS) at pH 8.9, 1 ml batch containing 250 mg mouse lg
Per hour at a flow rate of / h to 20 ml of Protein A gel (Pharmacia FPLC system). 1G was eluted with 0.1 M citrate buffer at pH 3.9 and 2 M HE at pH 9.0.
Neutralized with PES and dialyzed against either PBS or 0.1 M NaHCO ₃ at pH 8.4. Gel filtration using Superdex 200 (Pharmacia) and 15
% Acrylamide gel (Violad ready gel) and silver coloring (Biorad)
Purity was analyzed by SDS-PAGE using. Monoclonal antibodies were sterilized by membrane filtration and stored at concentrations of 1, 2 and 5 mg / ml at -20 ° C.

14. For mab labeling cytology, histology and EIA, the antibody was conjugated to biotin (long arm NH).
S- biotin, vector, Inc. of the United States Burlingame) and bar oxidase (labeled in accordance with the instruction of using the Boehringer Mannheim GmbH) of Germany producers (5mg, in NaHCO ₃ of 0.1 M, pH8.4). FA
Mab labeling with FITC, PE (shaded) and PECy5 for CS analysis was performed with Simbus (Dianova, Hamburg, Germany).

15. Quantification of MUC1 by Two-Site Enzyme Immunoassay (EIA) Maximum adsorbed immunoplates (Nunc) were prepared using phosphate buffered saline (PBS, pH 7.6).
Using mucin-glycone specific mab 7F11 (500 ng / well) at 37 ° C
For 1 hour. Plates were washed with PBS, blocked with PBS and 1% bovine serum albumin (BSA) for 1 hour, buffered water (WB, 150 mM sodium chloride, p7.6 50 mM Tris, and 0.05% Tween 20). ) Three times. Test samples and standard antibodies (100 yl) were added at appropriate dilutions in PBS, 0.5% BSA, 0.5% Triton X100 (AD, antibody diluent) for 1 hour at 37 ° C. The plate was washed four times with WB and peroxidase (2E11-PO, in AD,
MUC1 was determined using a peptide (APDTR) specific mab 2E11 labeled with 2 mg / ml, 1: 40,000, 30 minutes, 37 ° C.). 100 μg of substrate BM-blue (
Boehringer) was added at 37 ° C. for 30 minutes. Standard antigens (BMA, breast mucin antigen) were immunoaffinity substances purified from breast cancer lines KS and T47-D.

16. Synthetic MUC1-VNTR Peptide MUC1 A synthetic peptide with a tandem repeat structure was synthesized on a solid peptide composite (Dr. Nastindig, Hamburg / Saar University), using 0.1 M carbonate buffer at pH 9.4, 100 ng / well. EIA plate (Nunc maxisorb) at a concentration of Peptide M20 (APDTRPAPGSTAPPAHGVTS) SEQ ID NO: 1 P24 (APDTRPAPGSTAPPAHGVTSPDRT) SEQ ID NO: 2, M5-15 (PAPGSTAPPA) SEQ ID NO: 3, and M6-17 (APGSTAPPAHG) SEQ ID NO: 4 for epitope analysis. used.

17. Characterization of Epitopes by Flow Cytometry and Competitive ELISA Binding to KS and T47-D cells (breast carcinoma, MUC1 positive), LS174T (colon carcinoma, MUC1 negative) was performed by natural or direct FITC, PE (shadow). ) Or 7F11 and 1E4 conjugated to PE-Cy5 and labeled with methanol-fixed cells, and analyzed by flow cytometry. For epitope characterization,
The antibody of the present invention was prepared using commercially available anti-MUC1 antibodies HMFG1, HMFG2, Ma5
52, Ma695, b12, DF3, 115D8, BC2, BC3, SM3, V
Competed with U11E2, VU12E1, KC4 and MF06.

General information on the epitopes identified by the anti-MUC1 antibodies of the invention was obtained by ELISA using different sources of antigen. Ascites of breast and ovarian cancer patients, lymph from breast cancer patients, human milk, human urine, KS, T47-D
And the cytosol from LS174T were purified according to 4.1.2 and 4.1.3. Maximally adsorbed immunoplates (Nunc) were coated with 0.1 μg antigen per well for 18 hours at room temperature in carbonate buffer pH 9.6. After washing with PBS, unspecified binding was blocked by culturing with PBS and 1% BSA. Antibodies of the invention were used as biotinylation reagents at concentrations of 5, 10 and 20 ng / well. For the competitive reaction, unlabeled anti-MUC1 antibody was added at a concentration range of 1-100 ng / well. Plates were incubated at 37 ° C for 2 hours. After washing, strepavidin peroxidase (1.20000, Dianova, Hamburg, Germany) was added for 20 minutes at room temperature. After washing, the OD with BM Blue POD (Boehringer-Mannheim, Germany) substrate was determined at 450 nm after 20 minutes of culture.

Results 1.1 Epitope Specificity of 1E4 and 7F11 The epitope specificity of 1E4 and 7F11 was analyzed on a large panel of MUC1 fractions isolated from different normal and tumor sources (Table 2). The immunodominant MUC1 peptide epitope FDTR (HMFG1, HMFG2, b12,
In contrast to 2E11 and other well-characterized mabs with specificity for DF3), mabs 1E4 and 7F11 are completely negative for some overlapping peptides, including PDTR sequences. The antibody does not react with glycoproteins from normal human urine (PUM) and human milk. The epitopes of both antibodies are less sensitive to mild proteolytic treatment (trypsin) and excessive deglycosylation by neuraminidase, but by treating purified MUC1 with neuraminidase and O-glycanase, the reactivity of the antibody is complete. Disappears. Analysis of MUC1 separated by SDS-polyacrylamide gel electrophoresis and western immunoblotting was high (400-440 kDa) and low (180, 200-220 kDa).
The reactivity of both mabs with a molecular weight fraction is shown. Furthermore, 1E4 and 7
F11 showed a completely different coloring profile from crude MUC1 from lymph and ascites from patients with advanced disease breast cancer. Generally, there is a wide range of reactivity with the antigen in the molecular weight region of 180 to 440 kDa. These data show that 1E4 and 7F11
It is overexpressed and secreted in carcinomas, suggesting that it reacts with an epitope present in the underglycosylated MUC1 fraction that is not present in MUC1 of normal (epithelial) cells or is only expressed by chance. Both mabs are characterized by strong reactivity with antigens secreted in lymph, ascites and pleural effusions.

2. Comparative histological analysis of mabs 2E11, 1E4, 7F11 and 5A6 2.1 In addition to the normal tissue antibodies 1A6 and 1E11, the MUC1 specific mabs 7F11 and 1E4 also show normal tissue and a large panel of different organ cancer types. Screening was performed for specificity. Coloring was performed with either biotinylated mab (0.5 μg / ml) and strepavidin-alkaline peroxidase with the novel fuchsin as temperament, either by humans or by an automated immunocoloring device (Dako Techmate).

As can be seen from Table 3, normal cell and skin tissue, limbic blood, connective and adipose tissue, smooth and striated muscle, blood vessels, cartilage, bone, heart, liver, tongue, spleen, kidney and brain Is negative for all antibodies in all cases. Chest (Asini, tube), colon (
Enterocit), endometrium, spleen (asini, duct), prostate, placenta, stomach (wall cells), kidney (distal duct, collecting duct) and lung (ciliated epithelium) And MUC1 is detected in the secreted product along with the peptide-specific mab 2E11. Almost all mabs (HMFG1, HMFG
2, 115D8, DF3, F36 / 22, M26, b12; R. Price et al., Summary Report of ISOBM TD-4 Workshop, "Analysis of 56 Monoclonal Antibodies to MUC1 Mucin", Tumor Biol. 19, suppl. 1 (1998) 1-20).
Are characterized by this coloring pattern. MUC1 expression was also demonstrated for mab 2E11 in a small fraction of erythroblasts and bone marrow plasma cells. In contrast, 7F11 and 1E4 showed more limited reactivity with normal epithelial cells. In a normal chest, 7F11 is a non-uniformly colored 4
12 cases were positive with Asini and tube. 1E4 reacted only by chance with asini, single epithelial cells and secreted products. In the colon, endometrium, lung, spleen and placenta, 7F11 reacted only weakly with the ridge membrane of epithelial cells, while 1E4 was completely negative. The only organ with consistently strong reactivity with 2E11 and 7F11 was the kidney, and 1E4 was weakly positive for tubular membranes in three cases.

2.2 Tumor Tissue The tumor reactivity of Mabu 2E11, 5A6, 1E4 and 7F11 is shown in Table 4. Routine immunohistological staining of formalin-fixed, paraffin-embedded tissues shows that 2E11 and 7F11 are more than 96% reactive with early breast carcinomas, while 1E11
4 shows that it has a reactivity of more than 90% with the early breast carcinoma. MUC1-peptide specific mab with breast and various other cancer types (2E11, HMFG1)
In contrast to the uniform coloring pattern of Mab 7F11, 1E4 and 5F2, they are characterized by a more uneven coloring pattern in the individual tissues. Typically, in 50% of cases, there is a relatively weak and very strong expression area of each epitope in the same tumor. Approximately 40% of 1E4 positive tumors show cytoplasmic coloration at the center of the Golgi complex, while strong reactivity is seen only in small tumor areas, single cells and secreted products.

2.3 Analysis of micrometastasis tumor cells in bone marrow Comparative analysis of micrometastasis tumor cells in bone marrow was performed using 2E11, 7F11 and 1E4 (0.5 μg / ml) with APAAP technology (Dako Techmate).
Performed with methanol-fixed cell spins (1 × 10 ⁶ cells / slide). All slides were evaluated by image discovery analysis (Becton Dickson). Control antibodies for tumor cell analysis were CAM5.2 (cytokeratin 8/18) and 5D
3 (cytokeratin 8/18/19). 2E11, 1E4 and 7F11 showed strong labeling of tumor cells. Application of 2E11 is hampered by large cross-reactivity with erythroblasts and plasma cells, with 7F11 having only modest reactivity with erythroblasts, and 1E4 with normal bone marrow cells (n = 12) and Normal peripheral blood lymphocytes (n =
48) There were 42 non-neoplastic patients with no cross-reactivity with other blood disorders. The sensitivity of standard cells by Mab 7F11 and 1E4 was the region of 1 tumor cells in 10 ⁶ bone marrow cells or PBL. A comparative analysis of micrometastasis tumor cells in the bone marrow of 416 patients with early breast cancer is shown in Table 5. Two cell spins (2
× 10 ⁶ BM cells) were stained with each mab. Evaluation of slides by image analysis showed sensitivity and specificity to compare mucin-specific 7F11, 1E4, and cytokeratin-specific 5D3 for the detection and quantification of micrometastasis cells. Both mucin-specific mabs are used for improved immunocytology, ELISA, flow cytometry, detection of rare cells by immunomagnetically enriching tumor cells, and stem cells in peripheral blood. It provides a new perspective for development to effectively clarify rare tumor cells in cells.

[0079]

[Table 2]

[0080]

[Table 3]

[0081]

[Table 4]

[0082]

[Table 5]

[0083] including the breast cancer patients with minimal residual disease after clinical studies patients standard treatment immunotherapy of breast cancer patients with Example 2 the minimum residual disease (Table 6). 49
There was no evidence of clinical metastasis (MO) in one patient. The criteria included in the patient was that the bone marrow demonstrated evidence of tumor cell contamination after initial diagnosis or / and standard adjuvant treatment.

After clinical protocol informed consent, the patients were treated in gynecology and placed under medical supervision (50 mg and 20 mg treatment groups). Pretreatment was infusion of 2 mg clemastine hydrogen fumarate (Tavegil) and oral administration of 500 mg calcium. The patient was then treated with 50 mg of 7F11 (1 mg / ml) diluted in isotonic NaCl.
) Was administered over 8 hours. The 20 mg dose was diluted in 500 ml of NaCl and infused over 4 hours. These patients were able to leave the hospital one hour later.
The 2.5 mg patient did not receive any preliminary drug treatment, was given the antibody in 250 ml of NaCl by infusion over 1 hour, and was able to leave the hospital 1 hour later.

Prior to treatment, a sample of venous blood was collected and a) immune status, tumor markers (CA
153 CEA BM27), HAMA and anti-7F11. The pharmacokinetics of 7F11 in selected patients was analyzed in lymph at 4, 12, 48 and 72 hours after drug administration. During the infusion, blood pressure was measured hourly. Venous blood samples were analyzed 2 and 4 weeks after each immunization for HAMA, ab2 ab2β and ab3. Mononuclear cells from peripheral blood were prepared prior to immunization, at which time the critical ab2
Titration decreased, before and 7 days after boost immunization. Cells were frozen in liquid nitrogen for future phenotypic realization, B-cell and T-cell culture and FACS analysis.

Detection of Micrometastasis Tumor Cells in Bone Marrow A sample of bone marrow was separated by Ficoll density centrifugation and mononuclear cells were prepared to prepare a cell spin slide having 1 × 10 ⁶ bone marrow cells per slide. used. Cells are air-dried, fixed in 3.5% neutral buffered formalin for 15 minutes, PB
Washed with S, then fixed with cooled (-20 ° C) 100% methanol for another 5 minutes. The slides were washed with PBS, transferred to storage buffer (PBS with 60 g / l sucrose, 0.4 g / l magnesium chloride, 43% glycerin) and stored at -20 ° C. Alkaline immunity was achieved by immunocytological techniques using biotinylated 7F11 (0.5 μg / ml), 5D3-biotin (Novocastra, 1: 100) and MOC31 (biogenex anti-epithelial specific antigen, 1: 300). phosphatase (10 min with 20% acetic acid, 10 minutes at 2.3% NaJO ₄₎ and normal lymph (
After the step of blocking 5% of normal mouse lymph for biotinylated primary antibody (5% human lymph for APAAP technology), epithelial tumor cells were stained. Stereptavidin-alkaline phosphatase, either goat anti-mouse IgG alkaline phosphatase F (ab) ₂ (Kurta, 1: 250) or the APAAP system (Dako), and Dako technomate 500 immunization with the novel fuctin as substrate The slides were processed using colorants. Tumor cells were evaluated and quantified by an image discovery analysis system (Becton Dickinson). Tumor cells from patients immunized with 7F11 consisted of B-cells and isotopes (mouse IgG1 and 7).
F11) Addition using an alkaline phosphatase kit with Vector Red (biotinylated primary antibody) and Vector Blue (APAAP technology) (Vector from Burlingame, USA) to eliminate plasma cells with specificity Was controlled by a typical double coloring technique.

Analysis of Humoral Immune Response, Expressing Results from at least 6 Slides as Tumor Cells per 10 ⁶ Bone Marrow Cells ( TCD) HAMA was assayed by mouse IgG coated ELISA. Lymphocytes were diluted in duplicate from 1:20 to 1: 1000 and binding was assayed with peroxidase-labeled mouse IgG according to the manufacturer's instructions (Medac, Hamburg, Germany).

Anti--7F11 antibody patient's lymph was adsorbed to mouse IgG-agarase over 18 hours, and the pre-adsorbed lymph was added to an ELISA plate coated with 7F11-F (ab) ₂ fragment. The human ab2 antibody was determined with peroxidase-labeled 7F11. A standard antigen was prepared from a pool of lymph from a patient that had a low HAMA but a high 7F11 titer. 7F11 was coupled to Affigel 10 (Biorad) at a concentration of 5 mg / ml according to the manufacturer's instructions. High anti-7F11 titer of human lymph
Diluted 1: 2 with H8.9 binding buffer and applied to a 7F11 gel at a flow rate of 0.2 ml / min. 11 lg of human anti-7F was eluted with 0.1 M citrate buffer at pH 3.0, dialyzed against PBS, and used as a standard antigen in a concentration range of 500 to 2.5 ng / ml.
For ab2β analysis, the IgG fraction was affinity purified using mouse IgG agarose.

Competitive ELISA for ab2β determination Mouse IgG IgG preadsorbed lymph was used at a concentration of 0.1 to 10 μg / ml 7F11-F
(Ab) Cultured in ₂ coated microtiter plates. For competition, the cells were cultured for 18 hours with the purified MUC1 antigen in a concentration range of 500 units / ml. Human ab2β
Was determined using peroxidase-labeled goat anti-human IgG.

ELISA plates were coated with native MUC1 purified by WGA-Sepharose chromatography from human anti-MUC1 reactive breast cancer cell lines KS and T47 ロ ー D. The control antigen was cytosol from MUC1 native SW1116 colon carcinoma cells. V
The synthetic MUC1 peptide of NTR (p24) was coated at a concentration of 1 μg / ml. Lymph collected before and 2 weeks after each immunization was screened for anti-MUC1 reactivity. Lymph was added at a dilution of 1:25 for 1 hour at 37 ° C. Human lg
G and IgM were identified with a sphere-specific goat anti-human antigen labeled with peroxidase (Dianova, Hamburg, Germany) and BM-blue as a substrate.

Lymphocyte activation during immunophenotypic treatment was analyzed using a panel of CD markers by four-color flow cytometry (Curter Epix).

[0092] The clinical aspect patient 55 people of the breast cancer patients were repeatedly immunized with rat mAb7F11. Table 6 shows the number of bone marrow tumor cells (TCD) before and after the treatment and the number of times of immunization and administration of 7F11. Average TCD of all patients is the treatment before 10 ⁶ mononuclear marrow cells per 3.2 tumor cells. The clinical data for tumor stage, type and study group pretreatment are shown in Table 2. Tumor staging was performed according to TNM classification. 27 patients had a negative lymph node and 28 patients had a positive lymph node. Chemotherapy with adjuvant in 32 cases was prioritized over 7F11 treatment, and 18 patients continued on standard hormonal treatment during 7F11 treatment.

7F11 Immunization A 50 mg treatment dose was initially infused over 120 minutes. 5 of this 50 mg treatment group
Chen's patient immediately developed a mild allergic reaction (rash, hypotension, fever).
All signs lasted <24 hours and resolved spontaneously without special treatment. Patient No. 8 developed signs of rash, hypothermia, hypotension, and brocospam. Side effects could be completely reversed by infusion of only 8 mg of dexamethasone (fortecortin). A 500 mg treatment dose was then infused over 8 hours. Under these conditions, allergic side effects could be avoided even in patients who showed signs of allergy during the first immunization. None of the patients showed long-lasting toxic side effects.
To date, 7 patients have received 50 mg of 7F11 without side effects.
Has undergone strong maintenance immunization. All 12 patients in the 20 mg dose (4 hours drip time) group and all 21 patients in the 2.5 mg treatment group (1 hour drip time) did not show adverse efficacy.

Pharmacokinetics of 7F11 Pharmacokinetics of the native murine mAb 7F11 in lymph is shown in Table 7. 50mg of 7
Patients receiving F11 had regional peak concentrations in the region of 6-18 μg / ml 4 hours after infusion. There was a rather slow decrease of 8 μg / ml after 12 hours and 6 μg / ml after 48 hours. 7F11 peak lymph concentration is increased with HAMA and human anti-7F
Decreased in parallel with 11 titers.

HAMA and ab2 antibodies At present, 18 patients in the 50 mg group and 6 patients in the 50/20 mg group
Immunization has been completed. HAMA in these treatment groups showed significant changes in time and titer. In three patients, slightly elevated levels were recorded before treatment. During the procedure, one group of patients (patients 3, 6, 11, 13, 14, and 20)
It was characterized by a very slow increase not exceeding 1 μg / ml and a very stable titration of HAMA. However, after four to six immunizations, many patients have continuous HA
An increase in MA was indicated, resulting in a titer greater than 10 μg / ml. In contrast, the titer of anti-7F11 increased significantly with the second immunization. During treatment, all patients in the MO group received 2-8 μg / ml between subsequent immunizations.
The stable titer was shown in the region of. Typical HA of an individual patient during 7F11 treatment
MA and anti-7F11 titration isolates are shown in FIGS. HAMA and anti-7F11 titers in the 20 mg and 2.5 mg antibody treatment groups did not differ significantly from those in the 50 mg group.

In general, for most of the patients, HAMA and 7F11 showed the same pathway during immunotherapy. After treatment, lymph titration showed a slow decrease with a half-life of 4 to 6 months. One year later, boost immunization (7 patients) showed a dramatic increase and retained HAMA and 7F11 titers. Purified mucin 7F for competition in selected patients (numbers 1, 2, 4 and 5)
Suppression assays using 11 indicate that a significant fraction of the ab2 antibody
b7F11 (ab1), thus clearly showing that it was specific for the variable region of the ab2β antibody.

Analysis of Surrogate Markers of Micrometastatic Tumor Cells (TCD) in Bone Marrow and Follow- Up 19 patients in the M0 group completed the immunotherapy attempt. Before treatment (mean 3.
Table 6 shows a comparison between TCD after treatment and 3 tumor cells). Two patients (numbers 1 and 3)
) Were analyzed twice after treatment. Other patients were analyzed at the times indicated in the table. In each case,
At least six cytosine preparations with 6 × 10 ⁶ bone marrow cells were prepared using cytokeratin (5D3) and MUC1 (7F11, 1E4) and human epithelial-specific antigen (M
It was stained with an antibody against OC31). Cases that showed isotope reactivity were analyzed by additional double coloration. Bone marrow aspiration of 18 patients in the M0 group was scored negative after 7F11 treatment, while one patient with significant anti-mouse reactivity was scored positive (71 × 10 ⁶ ) and re-analyzed in the future. You.

Patients with proven metastases were included in the 7F11 immunization protocol.
Prior to treatment, tumor markers CA153 (patient no. 34), CEA (patient no. 27) and C
Three patients with increased lymph levels of a125 (patient no. 16) showed significant slow and delayed development of HAMA and anti-7F11 titers (Table 7). To date, six patients in this group have TCDs during and after immunotherapy.
Analysis was completed. Removal of tumor cells in the bone marrow in two cases (number 12, local disease,
And No. 17), and no changes were recorded in other patients (No. 10, local disease). A steady increase in TCD was recorded in a third patient (number 16) with proven bone metastases and increased tumor marker levels of CA125 before treatment.

Example 3 3.1 Treatment Scheme for 2.5 mg Dose Patients with positive bone marrow findings were immunized 6-8 times at 4-week intervals. The purpose is stable titration of HAMA.

Blood collection for laboratory tests Prior to each 7F11 treatment 1) Blood count 2) One white monobet for tumor marker with patient 3) HAMA, one to determine anti-7F11 White monobet 4) One blue monobet to determine immune status

No preliminary treatment

Use of 7F11 Antibody 7F11 is present in saline at a concentration of 1 mg / ml. For intravenous use, dilute 2.5 ml of the antibody solution in 250 ml of saline. The infusion is performed over 1 hour. Check blood pressure 1 hour later. After one hour of tracking time, the patient can leave the hospital.

Last Immunization Each patient receives an envelope containing a monobet (white) for further blood collection on the day of the last immunization four weeks after the immunization. Adjustment of date and time for clinical control studies. Three months after the last immunization, the day of the bone marrow control test was fixed.

3.2 Treatment Scheme for 20 mg Administration Patients with positive bone marrow receive 6-8 immunizations at 4-week intervals. The goal is stable HAMA titration. Blood collection for laboratory tests 1) Blood count 2) One white monobet for tumor marker with patient 3) One white monobet to determine HAMA, anti-7F11 before each 7F11 treatment 4) One blue monobet to determine immune status

[0105] pre-treatment 1) calcium of 2 tablet 2) Tabegiru by short infusion. One ampoule of 2 ml of 5 ml in 100 ml of NaCl.

Use of 7F11 Antibody 7F11 is present in saline at a concentration of 1 mg / ml. For intravenous use, dilute 20 ml of the antibody solution in 500 ml of saline. The infusion is performed over 1 hour. Check blood pressure 1 hour later. After one hour of tracking time, the patient can leave the hospital.

Last Immunization Each patient receives an envelope containing a monobet (white) for further blood collection on the day of the last immunization 4 weeks after the immunization. Adjustment of date and time for clinical control studies. Three months after the last immunization, the day of the bone marrow control test was fixed.

Example 4 Evaluation of Sharp Effects To test the sharp toxic effects of the MUC1 antibody, 2.28 mg of the antibody in 1.2 ml of PBS corresponding to 9.5 mg / kg was administered to 12 female Sprague-Dawley rats. (Obtained from Charles River Wiga, age: 8-9 weeks, weight: 231 ± 9 g). This dose corresponds to 25 doses of the drug intended for female patients (0.
38 mg / kg = 25 mg per 65 kg body weight).

Despite this external use, no changes in behavior, clinical signs of resistance and especially signs of allergic reactions (no shock, no change in respiratory rate) during the intravenous injection of the mouse antibody Animal behavior after waking from anesthesia (3-4 minutes after injection,
Anesthetics: N ₂ O, O ₂ , halothane) were not without complete attention. From this, mouse antibody B when administered at the specified dose as described above
M-7 has been shown to have no sharp effect on the cardiovascular system in rats.

[0110]

[Table 6]

[Continuation of Table 6]

[Table 7]

List of References Apostolobouros and F. C. Mackenzie, Critical Rev. Immunolo
gy 14 (1994) 293-309). G. FIG. Bastert et al., Immunodeficient animals in pharmaco-pharmaceutical studies in the Regard Bruner Edition, Basel-Carger, 1987, 224-227) Berger and Kimmel, "Enzymology Methods, Volume 152, Molecular Cloning Techniques (198
Academic Press, Inc., San Diego, CA; Besler et al., Immunobiol. 170 (1985) 239-244 Brinkman et al., Proc. Natl. Acad. Sci. USA 88 (1991) 8616 ─8620 Brinkman et al. Proc. Natl. Acad. Sci. USA 89 (1992) 3075 ─3079 Brinkman et al., Proc. Natl. Acad. Sci. USA 90 (1993) 7538 ─7542 Brueggemann et al., J. Exp. Med. 166 (1987) 1357 $ 1361 T.R. H. Brumendorf et al., Cancer Research 54 (1994) 4162-4168 H. Brumendorf et al., Nucl. Med. 34 (1995) 197-202.

Cyanfriglia et al., Hybridoma Vol. 2 (1993) 451-457 Korcher et al., Proc. Natl. Acad. Sci. USA 78 (1981) 3199-3203 F. H. Deland et al., J. NUcl. Med. 20 (1979) 1243-1250 J. Deal et al., Natl. Cancer Inst. 88 (1996) 1652-2658. Harlow and D.M. Rein's "Antibodies: Laboratory Manual" (Cold Spring Harbor Press (1988) ASB Edge et al., Anal. Biochem. 118 (1981) 131-137 EP-B 0193276 J. Fagerberg et al., Cancer. Immunol. Immunother. 38 (1994) 149-159 Finn et al., 1997 Fitzgerald and Pastan, J. Natl. Cancer 81 (1989) 1455-1461 J. Fuchsi et al., J. Biol. Chem. 259 (1984) 10511- 10517

O. E. FIG. Garania et al., Tumor Biol. 19, suppl. 1 (1998) 79-87 Holliger and Winter, Current Opin. Biotechnol. (1993) 446-449 Holliger et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444 ─ 6448 Hood et al., Immunology, Benjamin, New York, 2nd edition (1984 Houston et al., PNAS USA 85 (1988) 5879-5883 Funkapillar and Hood, Nature 323 (1986) 15-16). R. Summarized report of Price et al. ISOBM TD-4 workshop, "MU
Analysis of 56 Monoclonal Antibodies to C1 Mucin ", Tumor Biol. 19, suppl. 1 (1
998) 1-20, S.M. Carger AG H. R. Jerome, J. Immunol. 151 (1993) 1654-1662 Junk et al., Angewandte Chemie 97 (1985) 883 A. Kabat et al., "Sequences of Proteins of Immunological Interest (1987)" (National Institute of Health, Bethesta, MD)

V. Kalanikas et al., Tumor Biol. 19, suppl. 1 (1998) 71-78 S.M. Cowl, "Development of Monoclonal Antibodies with Specificity for Mammalian Cells",
Vision Acquisition Paper at the Biochemical Center of the University of Frankfurt, 1983 Kaul et al., Abstract 51, German Society for Gynecology and Obstetrics, Dresden, October 1-5, 1196. A. Line, Glycobiology 4 (1994) 1-9. Ramiki et al., J. Nucl. Med. 32 (1991) 1326-1332 K. O. Lloyd, Tumor Biol. 19, suppl. 1 (1998) 118-121. J. Merino et al., Nucl. Med. Biol. 18 (1991) 437-443 Pastan et al., Cancer Res. 51 (1991) 3781-3787. R. Summarized report of Price et al. ISOBM TD-4 workshop, "MU
Analysis of 56 Monoclonal Antibodies to C1 Mucin ", Tumor Biol. 19, suppl. 1 (1
998) 1-20

G. Lietmüller, Lancet 343 (1994) 1177-1183 A. Ruggetti et al., Cancer Res. 53 (1993) 2457-2461 "Molecular Cloning, Laboratory Manual, Second Edition" by Sunbrook et al., Cold Spring Harbor, New York. Press (1989) R. Scopes, Protein Purification, 1982, Springer Verlag, J. Taylor-Papadimitruu, New York, Int. J, Cancer 49 (1991) 1-5 US Patent Specification No. 4,952,496 US Patent L. Weiss and HA Gilbert, "Bone metastases" at GK Hall, Boston, 1981, T. Willbanks, Cancer 48 (1981) 1768-1775 WO 90 / 12592 WO 88/01649 WO 88/09344 WO 90/05142 WO 92/07271

List of Sequences (1) General Information (i) Applicant (A) Name: Bastert Günther, Professor, School of Medicine, Doctor (B) Street: Fosstraße 9 (C) City: Hakidelberg (E) Country: Germany (F) Postal code (zip): D-69115

(Ii) Title of the Invention: Antibody Specific to Mucin with Breast Tumor, Method for Producing and Using the Same (iii) Number of Sequences: 5 (iv) Computer-readable Form (A) Medium Type: Floppy Disc (B) Computer: IBM PC compatible (C) Operating system: WINDOWS NT 40 (D) Software: MICROSOFT WORD7

(2) Information of sequence ID number 1 (i) Sequence characteristics (A) Length: 20 amino acids (B) Type: amino acid (C) Strand: single (D) Topology: linear (ii) Molecular type: Peptide (iii) Description of sequence: Sequence ID number 1

Embedded image

(3) Information of sequence ID number 2 (i) Sequence characteristics (A) Length: 24 amino acids (B) Type: amino acid (C) Strand: single (D) Topology: linear (ii) Molecular type: Peptide (iii) Sequence description: sequence ID number 2

Embedded image

(4) Information of sequence ID number 3 (i) Sequence characteristics (A) Length: 10 amino acids (B) Type: amino acid (C) Strand: single (D) Topology: linear (ii) Molecular type: Peptide (iii) Sequence description: sequence ID number 3

Embedded image

(5) Information of sequence ID number 4 (i) Sequence characteristics (A) Length: 11 amino acids (B) Type: amino acid (C) Strand: single (D) Topology: linear (ii) Molecular type: Peptide (iii) Sequence description: sequence ID number 4

Embedded image

(6) Information of sequence ID number 5 (i) Sequence characteristics (A) Length: 5 amino acids (B) Type: amino acid (C) Strand: single (D) Topology: linear (ii) Molecular type: Peptide (iii) Sequence description: sequence ID number 4

Embedded image

[Sequence list]

[Brief description of the drawings]

FIG. 1. Reactivity of mab7F11 with different WGA purified MUC1 mucins. Antigen was purified by WGA-Sepharose chromatography as described in Example 1 and 7.5% S
DS-PAGE and Western blotting (50 U in 20 μl)
. T47-D supernatant (Track 1), T47-D cytosol (Track 2), P1.1
Ascites (Track 3), P1.2 Pleural outflow (Track 4), P1.3 Ascites (Track 5), P1.4 Ascites (Track 6), P1.5 Ascites (Track 7), P1.6 Ascites (Track) 8), P1.6 ascites (track 9), P1.7 ascites (track 10), P1.8 ascites (track 11), (P is patient). ST, standard molecular weight in kilodaltons. Detection of highly glycosylated or underglycosylated MUC1 fragments. 400 in lanes 1, 2, 5 and 7
Double band at ~ 440 kD. Lanes 2, 5, 6, 8, 9, 10 and 11 at 200-220k
Underglycosylated MUC1 fragment of the Da region.

FIG. 2. Patient No. 1: 7F11 Pharmacokinetics and Humoral Immunity, Breast Cancer T1b, N0, M0.

FIG. 3. Patient No. 2: 7F11 pharmacological kinetics and humoral immunity, breast cancer T1b, N1b, M0.

FIG. 4. 7F11 pharmaceutical kinetics. First, second and third immunizations.

FIG. 5. HAMA grown in patients immunized with 50, 20 and 2.5 g of 7F11.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 21/08 G01N 33/574 A G01N 33/574 33/577 A 33/577 C12N 15/00 C (81 ) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA ( AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, Z, DE, DK, EE, ES, FI, GB, GE, GH, GM, 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 F term (reference) 4B024 AA01 AA12 BA36 BA45 GA03 GA18 GA23 HA15 4B064 AG27 CA10 CA20 CC24 CE06 CE07 CE12 DA05 DA14 4C085 AA13 BB01 BB12 CC03 DD21 DD32 4 DDEEEEA EE01A03EE03A AA11 AA20 AA30 BA53 BA72 CA41 DA76 DA86 EA28 EA50 FA71 FA74 GA22 GA26

Claims (17)

[Claims] 1. An immunologically active polypeptide that specifically binds to the carbohydrate structure of MUC1 tandem repeats from carcinoma cells, a) 200 to 440 kDa from ascites containing tumor cells of a breast cancer patient. Affinity of the polypeptide for the glycoprotein fraction of natural MUC1 from normal cells
The quotient of the polypeptide's affinity for the antigen (400 to 440 kDa) is greater than 100: 1 (molecular weight region analyzed by SDS gel electrophoresis); b) the polypeptide is non-glycosylated Does not bind; c) when the glycoprotein fraction is treated with neuraminidase to cleave the N-terminal neuraminic acid or when treated with formalin,
An immunologically active polypeptide, wherein the binding of said polypeptide to the glycoprotein fraction of Da changes by no more than 10%. 2. The cell line DSM ATCC2328 or DSM ATCC2.
329. The immunologically active polypeptide of claim 1 obtainable from 329. 3. An antibody specifically bound to hypoglycosylated MUC1, especially
A method for producing a carcinoma cell fraction from a tumor cell having a molecular weight of 200 to 440 kDa, comprising the steps of: converting a glycoprotein into a body fluid containing a tumor cell, particularly a body fluid containing a carcinoma cell, particularly a carcinoma of a breast cancer patient; The glycoprotein may be isolated from the ascites fluid containing the cells by lectin affinity chromatography, the glycoprotein may be isolated from the lg protein, and the fraction thus obtained is used for immunization of an animal, and the antiserum is A method for producing an antibody, wherein the antibody is obtained and isolated therefrom. 4. The antibody as claimed in claim 1, wherein the antibody comprises: a) 200 to 440 cells from the ascites of carcinoma cells, especially of carcinoma patients.
The quotient of the polypeptide's affinity for the kDa glycoprotein fraction and the affinity of the polypeptide for the native MUC1 antigen (420 to 440 kDa) from normal cells is
B) the polypeptide does not bind to the non-glycosylated MUC1 antigen, c) the glycoprotein fraction is treated with neuraminidase to cleave the N-terminal neuraminic acid, or 200 to 440 K when treated with formalin
4. The method of claim 3, wherein the binding of the polypeptide to the glycoprotein fraction of Da changes by no more than 10%. 5. The method according to claim 3, which is used for breast cancer patients. 6. The polypeptide produced by expression of a recombinant nucleic acid encoding for the polypeptide in a prokaryotic or eukaryotic host cell and subsequent isolation of the desired polypeptide from the host cell or supernatant. A method for producing an immunologically active polypeptide according to any one of claims 1 to 5. 7. An antibody that inhibits the growth of tumor cells, especially cancerous cells of a patient, wherein a pharmaceutically effective amount of the antibody according to any one of claims 1 to 6 is used in the patient. Antibodies. 8. A method of producing a pharmaceutical composition for inhibiting the growth of tumor cells, particularly cancerous cells of a patient, wherein the pharmaceutical composition is in a pharmaceutically effective amount.
A method for producing a pharmaceutical composition, comprising the antibody according to any one of 1 to 7 as an essential component. 9. The antibody according to claim 7, wherein the antibody is used 1 to 10 times at weekly intervals or monthly intervals. 10. An antibody in an amount of 2 μg to 20 mg / kg body weight.
10. The antibody according to any one of claims to 9. 11. The antibody is used so that the titer of said antibody in a patient's body fluid is maintained at a stable level over a period of more than three months.
The antibody according to any one of the above. 12. The antibody according to any one of the preceding claims for the removal of carcinoma cells or secreted MUC1 antigen from tumor cells, especially body material of a patient, wherein the body material is Claims 1. The tumor cell or the secreted MUC1 antigen is contacted with the immobilized antibody of claim 1, and the tumor cell or the secreted MUC1 antigen is bound to the immobilized antibody, and the tumor cell or the secreted MUC1 antigen bound to a solid phase is a body substance. An antibody separated from the solid phase together with the solid phase. 13. A method comprising using the antibody of any of the preceding claims in a patient to affect the activity of T-cells in a tumor patient. 14. A conjugate comprising the polypeptide of claim 1 or 2 covalently linked to a toxin or cytotoxin substance. 15. A method for determining a tumor cell, particularly a carcinoma cell, by culturing a sample from a patient having the polypeptide according to claim 1 or 2,
A method comprising measuring the binding of an antibody to MUC1. 16. M in a body fluid, comprising the polypeptide according to claim 1 or 2.
A composition for determining UC1. 17. A method for systematically distinguishing a normal cell from a tumor cell, particularly a cancerous cell, comprising examining the binding of the polypeptide according to claim 1 or 2 to a cell. Method.