Antibody raised against to human gastroenteric tract epithelial tumor antigen related with alpha-6,beta-4-integrin

FIELD: medicine, oncology, gastroenterology, immunobiotechnology.

SUBSTANCE: invention describes an antibody or its derivative, or its fragment showing the structure able to bind the target structure. Antibody is located inside and on surface of human gastroenteric tract epithelial tumor cells and in subpopulation of normal gastroenteric tract epithelial cells. Indicated binding structures comprise sequences determining the complementarity of the region (CDR) in light chain comprising in main amino acids at number 23-33 (CDR 1), 49-55 (CDR 2), 88-98 (CDR 3) of amino acid sequence represented in SEQ ID NO:2, and CDR sequence in heavy chains comprising in main amino acids at number 158-162 (CDR 1), 177-193 (CDR 2), 226-238 (CDR 3) of amino acid sequence represented in SEQ ID NO:2, or other binding structures with similar unique binding properties. Also, invention describes the target-structure located inside or on surface of tumor cells: vaccine composition designated for treatment of malignant disease in human and comprising abovementioned antibody. Also, invention describes methods for treatment and diagnosis of malignant disease. Using this invention provides preparing antibodies that relieve identification of new phenotype-specific tumor-associated antigens, to predict and treat metastatic human diseases. Invention can be used in medicinal practice.

EFFECT: valuable medicinal properties of antibodies.

37 cl, 21 dwg, 4 tbl, 8 ex

 

The present invention relates to an antibody or its derivative, or its fragment having the structure of the linking structure of the target located on the cell surface and in cells of epithelial tumors of the gastrointestinal tract of man and in a subpopulation of normal epithelial cells of the gastrointestinal tract; and to the structures of the targets in tumor cells or on their surface; to vaccine compositions; pharmaceutical compositions; and to methods related to malignant diseases.

The BASIS of the INVENTION

Surgery is the main treatment for colorectal cancer, leading to survival for five years in 90-40% of cases depending on the stage of tumor development from a to C [classification] Dukes. Traditional therapy, which includes radiation and chemotherapy can reduce the rate of death of approximately 30% (1). Despite these achievements, cancer of the colon is the main cause of death among cancer in humans. Widely attempted immunological treatment. However, cancer of the colon is usually resistant to immunotherapy and, as I believe, has low immunogenicity. Patients with colon cancer not amenable to either IL-2 treatment, no treatment related the adoptive transfer of in vitro lymphocyte, infiltrating the tumor, which in other cases may have another activity in patients with immunogenic malignancies, such as melanoma. However, the most encouraging messages are Riethmuller, etc. about 32 percent decrease in the rate of death within seven years for colorectal cancer Dukes on stage when treatment after resection of the primary tumor pure murine mAb directed against the tumor and cons associated with normal epithelial antigen (EP-CAM)(2), showing that other immunotherapy techniques may be effective.

Significant improvements additional immunotherapy and treatment of the later stages of cancer requires more powerful effector mechanisms than provide a clean mAb. In principle, increased efficiency should be associated with increased selectivity of antibodies against tumor target.

A limited number of antigens associated with cancer of the colon detected using hybridoma producing murine mAb in the xenogeneic immunization of human cancers (3).

It can be expected that the use of the extensive library of the distribution of phages to identify new tumour-related antigens, will greatly accelerate the process of detection of target molecules suitable for swollen the left immunotherapy and diagnostics. Such identification of target molecules can be effected by the selection and screening of a library of phage antibodies on cultured tumor cells and tissue sections to obtain specific reagents, determining in vitro and in vivo expressed antigens (4). A technology placement of phages as an effective tool for the production of monoclonal or antibody-based test reagents for various purified antigens, and a few studies have described the design and successful breeding based on immune, intact and synthetic libraries of phage antibodies (5).

Immune libraries are convenient from the point of view of their habitual use, which eliminates unique libraries for each individual target. On the other hand, sufficiently large and of high quality non-immune libraries create difficulties for design, and the process of detecting the target using these libraries should require effective ways of eliminating selection on the basis of a complex antigens.

Currently has a library of phages of more moderate size from non-human primates immunized with the complex human antigens. This approach creates the advantages of pre-selection repertoire in vivo. Such libraries should be enriched with whom Azizi specificity against tumor-specific epitopes in reduced background reactivity against xenogenic antigens (6). In addition, in contrast to murine antibodies primates with a sequence highly homologous to human antibodies, should not be immunogenic to humans (7).

Currently defined new antibodies primates from a library of phage that define selectively expressed antigens associated with cancer of the colon. Therapeutic potential, demonstrated by mediated T-cell destruction of cultured cancer cells of the colon, covered two of these condensed antibodies, merged with designed superantigens, comparable to those of superantigens, fused with mouse Fab fragments specific antigens associated with cancer of the colon, such as the EP ITSELF, which was previously established therapeutic properties in experimental systems (8).

Also the proposed method is effective positive and excluding cell selection of phage antibodies, which should facilitate future identification of new genotypespecific antigens, including antigens associated with the tumor using antibodies from a large phage libraries.

A BRIEF DESCRIPTION of the INVENTION

The present invention relates in a first aspect the antibody or its derivative, or its fragment having the structure is round, connecting structure of targets located on the surface or inside the cells, gastrointestinal epithelial tumors of the gastrointestinal tract of man and in a subpopulation of normal epithelial cells of the gastrointestinal tract of humans, and specified connecting structure includes a sequence of complementarity determining region (CDR) in the light chain containing principally amino acids 23-33 (CDR1), 49-55 (CDR2), 88-98 (CDR3) amino acid sequence shown in SEQ ID NO:2, and sequences of CDRs in the heavy chain, containing principally amino acids 158-162 (CDR1), 177-193 (CDR2), 226-238 (CDR3) amino acid sequence shown in SEQ ID NO:2, or other connecting structure with such a unique binding properties.

In one embodiment the antibody is a selected phage. In another embodiment the sequence will Masasa fascicularis. Another aspect of the invention relates to a derivative of the indicated antibodies of human origin. The sequence is preferably at least 84% identical to the corresponding sequences of human origin. Preferably the antibody has a low immunogenicity or absence of immunogenetic for a person.

In another embodiment the antibody derivative as a result of genetic binding on the natives of the polypeptides and/or by chemical conjugation with organic or inorganic chemical molecules, and/or by di-, oligo - or multimerization.

In another embodiment of the specified antibody is genetically linked or chemically conjugated with a cytotoxic polypeptide or cytotoxic organic or inorganic polypeptides or cytotoxic organic or inorganic chemical molecules.

In another embodiment of the specified antibody is genetically linked or chemically conjugated with biologically active molecules.

In another embodiment of the specified antibody is genetically linked or chemically conjugated with immunoactive molecules.

In another embodiment of the specified antibody modified to increase or decrease the avidity and/or affinity.

In another embodiment of the specified antibody modified to increase output at its output.

In another embodiment of the specified antibody modified to impact on its pharmacokinetic properties.

In another embodiment of the specified antibody is modified to obtain a new pharmacokinetic properties.

In another embodiment of the specified antibody is labeled, and its binding is inhibited by its form of this antibody, but not the other connecting structures, while not inhibiting the binding of the other binding structures with a friend who Yu specificity.

Another embodiment is an antibody, binding structure which recognizes unrestored form α6β4-integrin.

In another aspect the invention relates to the structure of the target located in the tumor cells or on their surface, and these patterns of the target

a) have the ability to specifically block and specifically block the binding structure of the antibody as defined in any of paragraphs 1-14, and other connecting structures with similar binding specificity,

b) are located in the epithelial cells of the gastrointestinal tract of man or on their surface,

c) have a substantial degree of homology with α6 - and/or β4-integrirovanie chains or their variants, representing a widespread or unique epitope

d) are highly expressed in a high degree on the surface of tumor cells, and

e) are a target for cytotoxic effector mechanisms.

In this context, the terms "substantial homology" means the homology in those parts of the structure of the target, which are suitable for antibody binding sites.

In one embodiment of this structure-target binding structure is labeled, and binding is inhibited by unlabeled form specified connecting patterns, and the e other connecting structures, and does not inhibit the binding of other connecting structures having a different binding specificity.

In another embodiment of this structure is the target specified linking structure contains one or more of the sequences of the region complementarity determining (CDR)comprising mainly of amino acids number 23-33, 49-55, 88-98, 158-162, 177-193, 226-238 amino acid sequence SEQ ID NO: 2, or other connecting structures with similar unique binding properties.

In another embodiment of this structure-specified target binding structure is an antibody, which in one embodiment contains a variable region light chain comprising mainly of amino acids, room 1-109 amino acid sequence SEQ ID NO: 2, and the variable region of the heavy chain, comprising mainly of amino acids number 128-249 amino acid sequence of SEQ ID NO: 2.

This structure is the target of yet another embodiment of homogeneity is expressed in the epithelial cells of the colon of humans and to a lesser extent in cells of the pancreatic duct and bile duct.

In another embodiment, the expression of the specified structure of the target correlates with the differentiation of the gastrointestinal epithelium.

In another embodiment of the specified structure of the target includes serial is inost amino acids α 6β4-integrin, α6-part of which is shown in SEQ ID NO: 3, and β4-part shown in SEQ ID NO: 4. Another embodiment of the structure of the target includes a Homo - or heterogeneity or Homo - or heteropolymer specified α6β4-integrin and/or the specified one or more slices and/or subunits. Preferably these patterns target have apparent molecular weight in its unrestored form from 90 to 140 kDa, more preferably from 80 to 160 kDa.

In another embodiment of the structure of the target includes a peptide or polypeptide(s), containing principally amino acid sequence shown in SEQ ID NO: 5-51, or contains a molecule forming a complex with the specified(and) the polypeptide(s).

In the case when the structure of the target comprises the amino acid sequence of α6β4-integrin, in another embodiment of the specified structure of the target can also be recognized, exclusively or not, its not recovered form with binding patterns, included antibody as defined above.

In another aspect, the invention relates to a substance which is associated with the structure of the target, as defined above, which is an organic chemical molecule or peptide. In one embodiment of the specified substance is an anti-idiotype of the specified structure of the s-target. Specified anti-idiotype can be specifically blocked and can specifically block the binding structure having similar binding specificity of the specified structure of the target.

In another aspect the invention relates to a substance that blocks the function of the structure of the target, as defined above, which is an organic molecule or peptide.

In another aspect the invention relates to a connecting structure that recognizes the structure of the target, as defined above, and which has an organic chemical nature.

In another aspect the invention relates to pharmaceutical compositions containing as active principle an antibody, as defined above, or the structure of the target, as defined above, or substance, as defined above.

In another aspect the invention relates to vaccine compositions containing as active principle abn antibody, as defined above, or the structure of the target, as defined above, or substance, as defined above.

In another aspect, the invention relates to a method for treatment of conditions which are based on anti-angiogenic mechanism, where the person is injected antibody, as defined above, or the structure of the target, as defined above, or substance that is defined above.

In another aspect the invention relates to spoorwegen metastatic human diseases, where the person is put defined above antibody.

In another aspect, the invention relates to a method of conducting in vitro histopathological diagnosis and determine the prognosis of malignant diseases of man, where the sample is in contact with the antibody, as defined above, and with the indicator.

Embodiments of this method include determining the type of tumor, cancer screening, diagnosis and prognosis, and monitoring of precancerous lesions.

In another aspect, the invention relates to a method of conducting in vitro diagnostics and determine the prognosis of malignant diseases, where investigated, the concentration of antigen in body fluids, including the structure of the target, as defined above, or anti-idiotype specified structure of the target, defined above.

In another embodiment the invention relates to a method of conducting in vitro diagnostics and determine the prognosis of malignant diseases, and investigates the concentrations defined above antibodies in body fluids.

In another aspect, the invention relates to a method of conducting in vitro diagnostics and determine the prognosis of malignant diseases, where the investigated concentration in the body fluids of the complex (a) antigen, which includes the structure of the target, as defined above, or anti-idiotype specified with the touch target defined above, and b) antibodies, as defined above.

In another aspect, the invention relates to a method of conducting in vitro diagnostics and determine the prognosis of malignant diseases of man, where is determined by the localization of the antibody, as defined above, relative to the location of the tumor in humans. The specified antibody preferably is administered to the subject prior to determination. In one embodiment, the specified antibody accumulates in the tumor tissue. In another embodiment of this method is quantitative.

Another aspect of the invention relates to a method for treatment of malignant diseases of the person, where the person is injected antibody, as defined above. In one embodiment of this method called the antibody is modified by genetic linkage to a molecule that gives the modified pharmacokinetic properties of the combined molecule. In another embodiment of the specified antibody is replaced by its derivative.

Detailed description of the invention

Identification of new tumour-related antigens (TAA) is a major factor for development in the field of immunotherapy and diagnosis of tumors. In connection with the present invention were first developed based on the flow cytometrical assessment and the use of mini-libraries, consisting of clones of specific antibodies associated the various markers of antibiotic resistance, how positive and exclusive selection of phage antibodies using intact cells as a source of antigens. the scFv phage library (2,7×107constructed in primates (Masasa fascicularis), immunized with a pool of carcinoma of the colon of a person. This library was chosen by three of conduct binding l205 cell adenocarcinoma of the colon, and proteolytic elution, followed by amplification of the phage.

Several antibodies that interact with carcinoma of the colon and limited reactivity against several types of normal epithelial tissues, identified using immunohistochem. One clone, A3 scFv that recognizes the epitope, which was homogeneous expressed in 11 of the 11 investigated carcinomas of the colon and in 4 of 4 investigated in pancreatic carcinoma, and its expression in normal tissues was limited to subtypes of the epithelium of the gastrointestinal tract. A3 scFv had a seeming total affinity, about 100 times higher than A3 Fab, indicating that the binding of scFv homodimers. Density A3 epitopes on the cell surface, calculated on the basis of Fab binding was exceptionally high, approximately 3 million for the cell.

Also described effective mediated T-cell destruction is ukovich cells of the colon, covered As scFv fused with superantigens mutant SEA (D227A) with low affinity for MHC class II. Identified A3 molecule, thus, represents a TAA-molecule with properties that allow us to offer the use in immunotherapy of cancer of the colon and pancreas.

DISCUSSION

In connection with the present invention developed an efficient sampling scheme phages from the library of phages for use in identifying fragments of antibodies specific for defined phenotype cells. Misheneva specificity are presented in the examples was presented to antigens associated with tumors of the colon.

First analyzed the frequency of surface fused protein distribution pIII-scFv in a population of phages, using present here hamidou design for distribution of phage. Achieved a higher level of location C215 scFv compared with the previous messages. This should have a favourable effect on the efficiency of the exclusive selection, as well as to increase the likelihood Abednego selection of libraries of antibodies with low affinity.

Clearly demonstrated binding specificity C215 scFv phage cells l205 adenocarcinoma of the colon. Bound phage can be effectively blueraven using Protea the s Genenase, which specifically cleaves the sequence of the target between rahovym protein III and scFv antibody, leaving after elution of intact cells. This method of non-chemical elution should with equal efficiency eluted as phage antibodies regardless of their binding affinity, and only the phage bound scFv relationships, adding to the specificity of the process.

The enrichment achieved after three laps of selection on ln205-cells (500 g) using this scheme selection, like enrichment described by other authors for the selection of complex antigens.

After confirmation of the execution of the various methodological steps for the selection of the library used a mixed methodology using l205 cells.

The library was designed from a species of humanoid immunized with human cancers. Pool of antibodies obtained in this way may include affine Mature antibodies to ofwholesale antigens with a limited background Xeno reactivity to common normal human tissue antigens (6). Identified antibodies recognize tumor antigens and tissue differentiation with restricted distribution in normal tissues. All selected antibodies, identified as reactive in primary screening for tissue cancer of the colon, so the e interact with viable cells l205 in flow cytometry. This restriction specificity of cell surface should reflect the selection process and not part of the library, if used for immunization suspension of the mixture components of the tumor tissue.

In a similar previous study of intra - and intercellular specificity identified in antimelanoma the library obtained in the same way and selected using tissue slices as a source of antigen (4). Tissue sections of resected colorectal tumors and normal colon man (placed in the same hole) used for primary screening, using immunohistochemistry, in order to ensure clinical compliance of the selected specificdate to improve efficiency and to obtain better information compared with screening by flow cytometry.

Selected antibodies can be classified into four enciclopedicheskii groups, differing in the nature of reactivity to the epithelium of various organs (see example 1, table 1). Among these groups the specificity A3scFv determined the majority of the selected tumor antigens. This A3 TAA was highly homogeneous and often expressed in samples of primary and metastatic colon cancer and pancreatic cancer. Moreover, the level of its expression on the gunning surface, defined using the fused protein A3 Fab (3 million epitopes/cell)was exceptionally high and is available for mediated cell surface cytotoxic effects.

Few, if any, from a defined often expressed tumor antigens are ofwholesale, they typically refer to tissue differentiation, such as A3 and the EP ITSELF. However, increased expression of these antigens in tumors should provide the basis for therapeutically active doses. The availability of revenues from the circulation of normal tissue compartments expressing the antigen may also be more limited in the limited capillary permeability and localization of their expression in the body (for example, exposure of the apical side of the epithelial cells of the intestine for circulating antibodies should be very limited).

Clinical experience with pan-epithelial Er-HIMSELF-reactive 17-1A mAb confirms the ability to identify effective non-toxic dose of antibody. Limited expression in the epithelium of all selected in this work scFv clones indicates that these clones can principally be regarded as candidates for immunotherapy applications, similar to 17-1A, for example, as fully mAb. However, a special advantage A3 TAA than the s with the EP ITSELF is no expression in most normal epithelium, such as the epithelium of the lungs and kidneys, although expression in the colon the same.

Tissue distribution of subtypes of normal epithelium supported by the selective expression in subtypes of carcinomas originating from the gastrointestinal tract (see example 2, table 2).

Several of the previously well-known antigens associated with cancer of the colon (CEA, CA, CA19-9, CA, Tag-72) (3), compared with A3 epitope, equally or more restrictedly expressed in normal tissues. However, unlike the A3 and S EP ITSELF they are more heterogeneously expressed in tumors.

Using antibodies to Er-HE gave good clinical results, including the advantage in survival in patients with colon cancer, when conducting adjuvant therapy. To induce tumor response even in patients with more advanced stage, you can enter a strong effector molecules in combination with this antibody, which will cause the resistance of normal tissue"observed in the treatment of pure 17-1A mAb. In preclinical studies it can be studied on model systems using toxin-conjugated antibodies specific against murine version of this antigen, or animal transgenic for antigens associated with cancer of the colon of a person.

Previously immunotoxins antibodies successfully ispolzovalis is to treat mice in models with metastatically growing tumors, expressing Xeno (human) tumor antigens not expressed in the tissues of mice (10). However, used TAA are really ofwholesale, and the model does not reflect the potential toxicity to normal tissue target.

In previous studies the authors have reported on the possible superantigenic as immunostimulatory toxins to tumor immunotherapy (8). Mediated by antibody targeting of superantigens attracted a large number of cytotoxic and cytokine-producing T cells to the site of the location of the tumor. Superantigen SEA (D227A), mutated to acquire low affinity of binding MHC class II was genetically linked to targeted tumor antibodies. This "owholeheartedly agent was applied to the young T-cells, regardless of the expression of MHC in the tumor, thereby reducing the problems associated with tax regulation and polymorphism, which are a significant obstacle for other active immunotherapy approaches.

A mini library of the obtained clones of antibodies "pwholesalebingo8", 1F scFv-phage, "xerocreative" C215 phage, and nonspecific D1.3-phage was an essential basis for the development of effective exclusive selection. The requirement of this exclusionary rule is that for negative selection of the m should "rescue" phage and amplification, due to the high frequency distributed phage particles. Alternative, not distributed phage can be made non-infectious through selective proteolysis (G. Winter, pers. Comm.). This technique allows the creation of "inert libraries, i.e. libraries that are pre-selected by the extensive negative selection (for example, against the cage in a state of rest or capable of transfection parent cell).

In conclusion, the "junk" model ragovoy specificity can be selectively excluded from the population of phages with approximately a factor of 100 in each cycle of selection. Further exclusive selection with the help of the developed scheme in combination with the use of a large non-immune library of phages to identify antigens differentially expressed on the cell surface, will show, will be whether this approach is better than the strategy used by the inventors in this study, i.e. positive selection using in vivo pre-selective immune repertoire, including limitations and bias, such as immunodominance (4). Low affinity and a high density of epitopes shown in the case of A3 binding Fab with tumor cells, compared with the merged protein A3 scFv, involves the formation of scFv-multimers that interact with EPI is apami, which form clusters on the cell surface. Should develop a monovalent options A3 Fab with a higher affinity or alternative stable bivalent constructs, such as full-grafted mAb A3 Fv comparable to A3 with suspected low immunogenicity. Such designs are suitable for targeting the appropriate effector molecules to those expressed in a large number of antigens associated with tumors of the gastrointestinal tract.

Further, the invention is illustrated by the following non-limiting experimental part of the description.

EXPERIMENTAL PART

Materials and methods

Animals

Monkeys cynomolgus Macaque (Macaca fascicularis) were kept and were immunized at the Swedish Institute for Communicable Diseases (SIIDC), Stockholm. Water and drink animals always received ad libitum. Four monkeys were immunized subcutaneously with 2 ml of the crude suspension tissues of colon cancer in 10% normal serum in PBS. A reinforcing dose was administered at 21, 35 and 49 days. Or antibody-based test responses were obtained from two monkeys, where the antigen was mixed with adjuvant containing alum. All animals were kept in accordance with Swedish legislation, and the experiments were approved by local ethical Committee.

Tissues and cells

Samples of tumors and normal tissues receive the Lee of the National University hospital General hospital Malmö , Sweden. Colorectal human cell lines l205 In cell lymphoma person line Raji and the mouse cell line B16 melanoma cells were obtained from American tissue culture collections (ATS, Rockville, MD). Cells of mouse melanoma B16-C215+subjected to transfection with expression vector pKGE839, containing the gene for EP-CAM-1 (S215), described previously (9).

Human cells were maintained in RPMI1640 medium (Gibco, Middlesex, UK)with 10% V / V heat inactivated fetal bovine serum (Gibco) and 0.1% mg/ml gentamicin.almost (Biological Industries, Kibbutz Beit Haemek, Israel). Mouse cells were cultured in the medium supplemented with 1 mm glutamine (HyClone, Cramlington, UK), 5×10-5M β-mercaptoethanol (ICN, Costa Mesa, CA), and 0.2% NaHCO3(Seromed Biochrome, Berlin, Germany), 1 × 10-2M HEPES (HyClone, UT) and 1 × 10-3M sodium pyruvate (HyClone). Cells are re-tested for Mycoplasma contamination using the test genetic sample Mycoplasma T.S. (San Diego, CA).

Formigny vector and constructing libraries of phage

Total spleen RNA was extracted from one of the meet the monkeys, using the kit for RNA extraction from Promega (Mannheim, Germany), and cDNA amplified using a set of PCR RNA from PE Biosystems (Stockholm, Sweden). Primers for cDNA synthesis gene light chain lambda, and heavy chain and for the Association of these genes in the scFv genes were previously published (4). scFv-ligated cDNA in formigny vector (4) where SL is anii with the remnants 249-406 M13 gene III. scFv-gIII gene expressed from the phoA promoter, and the resulting protein was directed depletability signal peptide toxin II E. Coli.

Re electroporation 7 µg library of vectors with inserts of a gene of scFv leads to an initial increase in the amount of 2.7×107transformirovannykh E. Coli TG-1 in the form colonies on minimal agar dies. Colonies were removed from the plates and were grown in 2×YT at 150 rpm and 37°C for 1 hour.

The culture was superificial helper phage MK (Promega) in 50-fold excess. Added ampicillin concentrations up to 100 mg/l and the culture was grown for another hour. After adding kanamycin to a concentration of 70 mg/l culture was grown for 15 hours at 30°C and 250 rpm Phage particles were collected from the culture supernatant by two re-PEG/NaCl precipitation. Precipitated phage was dissolved in PBS with 1% BSA.

Western blotting

Series two-fold dilution of scFv-C215 phage particles (from original undiluted PEG-precipitated/concentrated phage) was applied for the separation of regenerating a 12% polyacrylamide gel with 1% and 2% β-mercaptoethanol. Then the protein by electrophoresis were transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% low-fat milk (Semper AB, Stockholm, Sweden) and then incubated with rabbit anticorodal FR the peptide sequence AEGDDPAKAAFNSLQASATEC, derived from protein III, conjugated with hemocyanin Molucca the saucer. Secondary conjugated with horseradish peroxidase (HRP) goat anti-rabbit antibodies (Bio-Rad) were incubated for 30 minutes. Between any steps, the membrane was washed 3 times for 5 minutes in PBS/0.5% tween-20. The membrane was incubated with the substrate (Amersham Pharmacia Biotech, Little Chalfon Buckinghamshire, UK) for 1 minute. The light-sensitive film (hyperfilm ECL, Amersham) were exposed on the membrane and kept for 0.5-5 minutes

Similarly, to analyze the integrity of the purified Fab (A3, including cynomolgus CH1 and C-lambda domains), scFv and Fab (including mouse CH1 m s-Kappa) -SEA (D227A) fused proteins (obtained as described previously (9)), were taken by 12% SDS-PAGE. Membranes with transferred proteins were incubated with purified polyclonal rabbit anti-SEA antibodies with subsequent stages of processing described above.

The choice of model and library of phage to cells

Suspension of phage library of light chain lambda (or model phage), 1012in 100 μl PBS/1% BSA were incubated with 3 million l205 cells for 1 hour on ice. Cells were washed 3 times, including a 10-minute incubation with 2 ml PBS/1% BSA for each washing. The phage was suirable by adding 50 μl of 33 µg/ml generasi (Genenase) cell draught, and Incurabili within 15 minutes. Genedata, which is a mutant BPN- subtilisin, S24C/H64A/E156S/G169A/Y217L, was kindly provided by Dr. Paul Carter (San Francisco, CA). After centrifugation the supernatant was transferred into a new tube and add 250 ál of 1% BSA in PBS. For "salvation" and amplification of selected libraries and models of phage particles in reusable experiment) elyuirovaniya rahovym particles were allowed to infect 1 ml of E. coli DH5αF' (OD600Nm=1,0). Infected bacterial culture was diluted 100-fold environment 2×YT, enriched with the appropriate antibiotic and grown until OD>1,0 (up to 2 days).

Finally, to obtain the soluble scFv amber-suppressor shtam NV E. coli infected library, selected from the second and third cycle. After growth on agar tablets containing ampicillin, single colonies were cultured in 96-well-microplate 2×YT medium supplemented with ampicillin at 30°C for 17 hours. After centrifugation, removal of supernatant, to which was added an equal volume of PBS/1% BSA, individual scFv were analyzed for immunoreactivity against sections of tumor and normal human tissues. In short, C-terminal tag ATPAKSE was determined using rabbit anticigarette, and then biotinylated goat anti-rabbit antibodies (DAKO A/S, Copenhagen, Denmark) and Streptogramins HRP (DAKO A/S) (see "Immunohistochemistry").

Immunohistochemistry

Frozen is crisisi (8 μm) were air dried on slides, were fixed with acetone at -20°C for 10 minutes and was registrirovali 20% fetal bovine serum in PBS (FBS). Endogenous Biotin was blocked by Avidya (diluted 1/6) for 15 minutes, and then Biotin (diluted 1/6) within 15 minutes (Vector Laboratories, Burlingame, CA). Affinity purified and biotinylated rabbit anti-SEA antibody, 5 μg/ml, were incubated for 30 minutes followed by the addition of Streptokinase HRP (DAKO A/S, Copenhagen, Denmark), diluted 1/110 50 mm Tris pH 7.6 for 30 minutes. Between all stages of the sections were washed 3 times in TBS. Staining was carried out for 8 minutes in 0.5 mg/ml 3,3'-diaminobenzidine (Sigma)dissolved in Tris, pH to 7.6 with 0.01% of N2O2. After 10 minutes of staining in 0.5% methyl green glass slides were washed for 10 minutes with tap water and were slowly degidrirovanie in 70-99% ethanol and xylene before being placed in the environment DPX (Sigma).

Flow cytometry

Cells l205 colon cancer person disconnected with 0.02% weight/volume of EDTA and washed with PBS. To develop or antibody-based test response in monkeys, cells were incubated sequentially with diluted serum for 1 hour at 4°, biotinylating rabbit anti-human IgG-antibodies (Southern Biotechnology Ass. Inc., A1, USA) for 30 minutes and, finally, with Avidya-PE (Becton Dickinson, Mountain View, CA) for 30 minutes is.

The binding model of the phage to the cells was analyzed using rabbit anti-M13 antibody obtained by immunization of rabbits particles M13) and FITC-conjugated donkey anti-rabbit antibodies (Amersham Pharmacia Biotech). The binding of the antibody, merged with the SEA (D227A), was determined using biotinylated rabbit anti-SEA antibodies and avidin RE. All reagents were diluted in PBS/1% BSA. Cells were washed twice in PBS/1% BSA after incubations with reagents and three times, including a 10-minute incubation after binding of phage particles.

Running cytometrics analysis was performed using a FACSort flow cytometer (Becton Dickinson).

Determination of affinity for cultured cells

Slit proteins A3 scFv-SEA(D227A), A3 Fab-SEA(D227A) and 1F scFv-SEA(D227A), 80 µl of each protein was labelled with iodine, as described by Bolton and Hunter to the specific activity 10-15 µci/µg. Cells l205 and Raji cells, 30000/sample were incubated with iodinated fused protein, 100 ál/tube, in a series of twofold dilutions of 1% BSA for 1 hour, and then washed three times in PBS before measuring binding activity. The concentration of added and linked fused protein were used for Scatchard analysis. To calculate specific binding to l205 cells subtracted background binding to Raji cells.

Analysis of cytotoxicity

Dependent T-cell cytotoxicity superantigen the aqueous fused protein (cell-mediated cytotoxicity, dependent super antigenic antibody) was measured in a standard 4-hour analysis with the release of chromium using51Cr-labeled l205 cells as target cells, and human cells as effector cells (9). The percentage of specific lysis was calculated as follows:

[cpm - number of pulses per minute].

Example 1

Output communicating with tumor monoclonal antibodies cynomolgus

Cynomolgus monkeys, Macaca fascicularis (four individuals) were immunized again with a slurry of carcinoma of the colon of a man four times each week. After a gradual development or antibody-based test response in monkeys spent running cytometrical staining of cultured colorectal cells, l205 using a series of dilutions preimmune and immune serum. Or antibody-based test IgG response was detected only when using the suspension of precipitated alum tumor tissues (two individuals).

The monkey with the highest level of binding immune from praemunire serum antibodies used for constructing a large combinatorial library of phage scFv, approximately 2.7×107(estimated by the number of primary transformants). Library phage primates selected using cells l205. The total yield of phage (number lirovannomu/dobavlennogo, counted as kolonialismus units, CFU) of three successive cycles of selection was increased gradually from 1.9×10-7, 1,4×10-5to 1.2×10-3. Flow cytometry showed that five percent (12/246) monoclonal soluble scFv:s, obtained from a library of phage after three cycles of selection associated with the tissue section of colorectal tumors with intact cells l205. All the selected antibodies detected individual unique nucleic acid sequences corresponding to the picture Hinf I restriction upon analysis by gel electrophoresis on 1% agarose.

Genes of antibodies amplified using the polymerase chain reaction using 5 µl of the bacterial culture and primers complementary to regions 5'- and 3'of the gene of scFv, fahmida vector (the area of the phoA promoter in M13 gene III).

Selected scFv has an individual unique reactivity against epithelium of normal tissues.

Reactive scFv colorectal cancer classified into specific groups based on the nature of their immunochemical reactivity with normal tissues (table 1). Antibodies were investigated in detail, are A3 scFv (and A3 scFv-SEA(D227A), A10 scFv, 3D scFv and ID scFv. Typical antibodies could differ from each other in their extreme specificity in relation to the epithelium of various organs and p is their binding to leukocytes. 1D scFv strongly interacts with the intestinal epithelium and is the only antibody that interacts with cells with the morphology of polymorphonuclear granulocytes. 1D scFv is also different from other antibody staining luminale surface of the renal glomeruli and collecting ducts, where A10 scFv reacts homogeneous (non-polar) with these epithelial cells, and 3D and A3 scFv scFv are negative. 1D, A10 and 3D, but not A3 scFv, also react with macrophagecolony cells in the lungs.

The fifth group of antibodies is not extensively evaluated and is therefore not included in Table 1, interacted with the epithelium of the colon, leukocytes and kupferschmid cells in the liver. A3 scFv differs in that it shows the most limited reactivity with the used set of normal tissues. Among normal tissues, the most significant reactivity A3 with respect to the normal epithelium of the colon. Weak staining is also defined in the small ducts of the pancreas and bile ducts of the liver and the substructures of the epithelium of the small intestine. The surface epithelium of one of the two samples of the stomach heavily stained A3 antibodies.

Picture reactivity A3 scFv was confirmed using a fused protein A3 scFv-SEA (D227A). This format allows the use of polyclonal rabbit anti-SEA antibodies for immunogen chimicheskogo detection, which is a more sensitive detection system which is characterized by a lower background and tissue cross-reactivity compared to the use of secondary antibodies to the peptide tag ATPAKSE-the end of the scFvs.

EXAMPLE 2

Associated with tumor antigen A3 is homogeneous and is often expressed in colorectal tumors and tumors of the pancreas

Fused protein A3 scFv-SEA (D227A) used for immunohistochemical staining of various tumors of epithelial origin (table 2 and figure 1). Protein is homogeneous and strongly stains 11/11 tissue samples of primary colon cancer and 4/4 samples with metastatic colon cancer, resected from the ovary, lymph node and liver. Tumor pancreatic cancer, samples 4/4, equally strongly positive. Conversely, tissue samples of carcinoma of the stomach, prostate, breast and non-small cell lung cancer were negative.

EXAMPLE 3

A3 TAA highly expressed on the surface of cancer cells of the colon

The results of several Scatchard curves to determine the affinity-based binding fused protein A3 scFv-SEA(D227A), A3 Fab and 1F scFv-SEA(D227A) (1F was rate the new group A3 specificity) with cells l205, summarized in table 3. Specific binding was calculated by subtracting nonspecific binding to Raji cells In cell lymphoma person, not expressing A3 1F TAA, from binding to cells l205. Used linear regression to calculate the slope and stutter extrapolated curve Scatchard. Fused protein A3 scFv-SEA(D227A) were fed approximately 10-fold less binding sites per cell compared with the merged protein A3 Fab (approximately 3 million sites per cell), showing that the bivalent (multivalent) binding occurred in the case of scFv. This is confirmed by more than 100-fold increase in the overall affinity (3,6-5,5 nm) in respect of the fused protein A3 scFv compared with A3 Fab (580-780 nm).

The only experiment conducted using a fused protein 1F scFv-SEA(D227A), showed the same binding affinity and saturation of binding sites, as fused protein A3 scFv-SEA (D227A).

Table 3
Scatchard analysis of the binding of iodized fused protein with l205 cells
Fused proteinn*Kd (nm)a million sites/cell
A3 Fab-SEA(D227A)2580-7803,0-3,9
A3 scFv-SEA(D227A) 33,6-5,50,11-0,39
1FscFv-SEA(D227A)14,20,18
* Conducted experiments

EXAMPLE 4

Indirect A3 1F scFv-SEA(D227A) T-cell lysis of the cells l205

The ability of mediating the two fused proteins A3 1F scFv-SEA(D227A) antibody-superantigen-dependent cellular toxicity (SADCC) in the cells l205 investigated and compared with positive control fused protein S215 Fab-SEA(D227) and negative control fused protein D1.3 scFv-SEA(D227A). Titration of the fused protein A3 scFv-SEA (D227A) reached a plateau at the maximum lysis, which was similar to approximately 50 percent of those in the 4-hour analysis for the fused protein S215 Fab-SEA(D227A), although the concentration was 10 times higher (figure 2). 1F scFv-SEA(D227A) operatonal a similar level of cytotoxicity at higher concentrations compared to A3 scFv-SEA(D227A). Negative control fused protein D1,3 scFv-SEA(D227A) did not cause any cytotoxicity.

EXAMPLE 5

Cleansing associated with tumor antigen that is recognized reactive antibody A3 colon cancer

Tumor extracts were obtained from cells xenoprofile tumor lines l205. The extract was applied on pratolongo combined with C215Fab-SEAm9, and the column, the volume is United with A3scFv-SEAm9. The columns were connected in series during application of the sample, but separated before elution in alkaline conditions.

A single peak was determined during elution by means of UV-spectroscopy (figure 3). This elyuirovaniya faction of the latest A3-column were collected, neutralized, concentrated and then analyzed by SDS-PAGE in non conditions (figure 4). Two bands, prominent staining of silver marks I and II in figure 4) with an apparent molecular weight of approximately 90-140 kDa was cut out and examined by standard methods of peptide mapping. These two bands correspond to the bands, which is defined by A3 Western-blotting, see example 8. Of the bands I got 47 different masses trypticase peptides (see SEQ ID NO:3, table 4, and figure 5 for sequences and the corresponding weights of the masses), which is fully consistent with different masses trypticase peptides identified by MALDI-TOF human α6-integrin and β4-integrin (see SEQ ID No: 5-51 and 3-4, respectively, and figures 3A and b respectively, where figure 3A underline corresponds to peptides that appear on figure 3B/SEQ ID No:5-51). Of the strip II received 22 separate mass trypticase peptides, which fully corresponded to the different masses trypticase peptides β4-integrin (data not shown). The data show that heterodimer α 6β4-integrin specifically allocated using A3-affinity column.

1026.608
Table 4
Peptide/polypeptide derived from a human α6β4-integrin and their weight
The village-there are noSequenceMeasured weightEstimated weight
5LLLVGAPR838.568838.551
6ANRTGGLYSCDITARGPCTR2226.1312226.050
7VVTCAHRYEK1262.6371262.631
8RQHVNTK882.524882.490
9CYVLSQNLR1152.6181152.583
10FGSCQQGVAATFTK1501.7061501.710
11DFHYIVFGAPGTYNWK1914.8811914.917
12DEITFVSGAPR1191.6251191.600
13ANHSGAVVLLK1108.6001108.647
14DGWQDIVIGAPQYFDR1879.8651879.897
15DGEVGGAVYVYMNQQGR1842.8111842.844
16WNNVKPIR1026.584
17NIGDINQDGYPDIAVGAPYDDLGK2520.2132520.189
18GISPYFGYSIAGNMDLDR1975.9131975.922
19NSYPDVAVGSLSDSVTIFR2026.9922027.008
20SRPVINIQK1054.6441054.637
21LRPIPITASVEIQEPSSR1993.0661993.108
22VNSLPEVLPIINSDEPK1863.9201864.006
23TAHIDVHFLK1180.6651180.647
24FSYLPIQK995.601995.556
25DIALEITVTNSPSNPR1726.8661726.897
26SEDEVGSLIEYEFR1672.7641672.770
27VESKGLEKVTCEPQK1731.8661731.895

28REITEKQIDDNRK1644.7921644.866
29FSLFAER869.476869.452
30YQTLNCSVNVNCVNIR1954.0031953.927
31LNYLDILMR1150.6441150.629
32 AFIDVTAAAENIR1390.7391390.733
33LPNAGTQVR955.523955.532
34VSVPQTDMRPEK1386.7271386.705
35EPWPNSDPPFSFK1547.7301547.717
36NVISLTEDVDEFR1536.7441536.754
37TQDYPSVPTLVR1375.7181375.722
38RGEVGIYQVQLR1417.8011417.791
39ALEHVDGTHVCQLPEDQK2075.9652075.981
40GNIHLKPSFSDGLK1512.7491512.817
41MDAGIICDVCTCELQK1928.9011928.822
42YEGQFCEYDNFQCPR2012.7952012.790
43SCVQCQAWGTGEKKGR1879.8651879.890
44DEDDDCTYSYTMEGDGAPGPNSTVL VHK3103.2293103.278
45QEVEENLNEVYR1521.7791521.718
46VAPGYYTLTADQDAR1640.7791640.791
47VPLFIRPEDDDEK1572.7781572.790
48DVVSFEQPEFSVSR165.758 1625.781
49LLELQEVDSLLR1427.7601427.810
50VCAYGAQGEGPYSSLVSCR2060.8832060.916
51VLVDNPKNR1054.6441054.600

Materials and methods

Solubilization tumor tissue

Fabric colon cancer person expressing A3 antigen, was provided by hospitals in Sweden and was stored frozen at -70°in the Bank of the tissue at ABR. Frozen tissues colon cancer scalpel did the cut and transferred into a test tube containing cold isotonic sucrose buffer (0.25 M sucrose, 10 mm KCl, 1.5 m MgCl2, 50 mm Tris-HCl pH 7.4 at 25° (C)containing 1% (V/V) Nonidet P-40 (NP-40) and protease inhibitors (Completet™ Protease Inhibitor Coctail Tablet, Boehringer Mannheim). The tissue is homogenized in a homogenizer Ultra-Turrax and left to solubilize at 0°C. the solubilized preparation was centrifuged at 11000 rpm (universal centrifuge Hettich rotor 30 RF) to remove cellular debris. The supernatant continued centrifuged at 108000 g at 4°C. (Ultracentrifuge Backman rotor Ti-60), and in the end was filtered through a filter of 0.2 μm Minisart plus (Sartoriuis AG Gottingen Germany).

Affinity purification of tissue antigens

A3scFv-SEAm9 combined with NHS-activated HiTrap column (Pharmacia Biotech, Uppsala, Sweden is), in accordance with the recommendations of the manual. Control and pratolongo combined with C215Fab-SEAm9, and control and pratolongo connected in series. All columns were washed with buffer for pre-rinse (20 mm Tris HCl pH 7.5 at 4°containing 0.2% NP 40). The extract was applied on the column at 0.1 ml/min, and the flowing stream is recycled. Then the column was washed with the starting buffer. Bound antigen was suirable gradient pH diethylamine, starting from pH 7.5 to 11.0. 2.5 ml of eluent was collected and concentrated to 75 ml. Purification was performed at 4°using system ACT FPLC (Amersham Pharmacia Biotrch Uppsala, Sweden). Suirvey protein was analyzed using SDS PAGE and silver staining. Individual strips were dissected and subjected to trypsin digestion and peptide mass was determined using Maldi-TOF production Protana A/S (Odense, Denmark). Then the peptide mass was compared in the computer study with all the masses trypticase peptides for each protein in the SWISSPROT database service, presents Protana A/S (Odense, Denmark).

EXAMPLE 6

A3scFv-SEAm9 defines a new epitope α6β4-integrin

Commercial antibodies human α6-integrin and β4-integrin compared with A3 on sections of normal and malignant colon. Reactivity is shown in figure 6, shows that A3 is limited to what epithelium colon (Fig.6 [i]) and malignant tumor cells (Fig.6 [ii]). Commercial antibody NKI-GoH3 against α6-integrin also reacted with normal colon rectum (6 [iii]) and colon cancer (6 [iv]). The reaction is seen in the epithelial cells and malignant cells (arrows)and blood vessels (BV), some components of the stroma (s) and in the mucosa of the mouse (mm). The reaction is observed using a commercial antibody ASC-3 against β4-integrin, was similar to that obtained with the use of anti-α6 antibodies, but its weaker, as in normal colon (v)and cancerous tissue of the colon (vi).

Materials and methods

Antibodies

A3 scFv were selected from the library of M. fascicularis. The genes of VH and VL were isolated by digestion of restrictase and poured with chimeric mutant staphylococcal enterotoxin AE (D227A) obtaining A3scFv-SEAm9. It had very low levels of nonspecific binding was sensitive to detektirovanie secondary antibodies. Antibody ASC-3 against human β4-integrin and antibody NKI-GoH3 against human α6-integrin were acquired by Becton Dickinson (Copenhagen, Denmark).

Immunohistochemistry

Samples of tumor and normal tissue received in the surgical Department of the National hospital. They were quick frozen in isopentane pre-cooling in liquid nitrogen. The samples were stored at - 70°to essakane. After cryo is Secunia sections were air dried overnight, were fixed in cold acetone and blocked with Avidya/Biotin (Vector Burlingam, CA). Then slice within one hour was added to the primary antibody.

Secondary antibodies were incubated for 30 minutes with subsequent processing of the streptavidin-Biotin/HRP (Dakopatts Copenhagen, Denmark) for 30 minutes. Between all of these stages have conducted extensive washing with 50 mm Tris pH of 7.6 to 0.15 NaCl. Diaminobenzidin (DAB) was used as Chromogen and the sections were stained in 0.5% methyl green. Controls included netchanay reactive Fab and SEA D227A or there is no primary antibody. All antibodies were used at a final concentration of 5 μg/ml Results were classified as negative, medium and strong.

EXAMPLE 7

Associated with tumor antigen A3 interacts with α6 β4-integrirovanie antibodies in the ELISA analysis of binding.

Untreated tumor extract or A3-antigen, purified by A3-affinity chromatography (see example 5), were analyzed using binding ELISA. Commercial antibody ASC-3, specific beta-4-integrin, was used as catching antibody, which caused various dilutions of untreated tumor extract. Then it was covered A3scFv-SEAm9. Linking A3scFv-SEAm9 then detected with anti-SEA-HRP (figure 7A). In figure 7B commercial antibody NKI-GoH3 α 6-integrin were used to capture various dilutions of concentrated affinity purified eluate A3. In the same way as in figure 7A, the bound proteins were monitored using A3scFv-SEAm9 and was detected by anti-SEA-HRP. In both experiments was determined depending on the concentration signal. These results confirmed the specificity of the A3 towards heterodimer α6β4-integrin, indicating that they specifically selected from A3-affinity column in example 5.

Materials and methods

Commercial antibodies NKI-GoH3 or ASC-3 (Becton Dickinson Copenhagen, Denmark), 100 μl of a 0.05 M NaHCO3pH of 9.6 was used to cover the wells E.I.A./R.I.A (Costar). The reaction proceeded overnight at 4°after which all the tablets were washed 4 times in DPBS +0.05% tween-20. Then the wells were blocked with 200 μl of 3% skim milk powder in DPBS+0.05% Tween-20 for 1-2 hours at room temperature (RT) with shaking. The wells are again washed as described above and 100 μl of the extract antigen, diluted in 3% fat-free milk powder in DPBS+0.05% Tween-20, was applied for 2 hours at room temperature with shaking. The wells are again washed (4×DPBS+0.05% tween-20), after which 100 ál of primary antibody diluted in 3% fat-free milk powder in DPBS+0.05% tween-20, incubated for 2 hours at room temperature with shaking. L the NCI again washed, as described above, and 100 μl of secondary antibody diluted in 3% fat-free milk powder in DPBS+0.05% tween-20 was added to each well for 1 hour at room temperature with shaking. The wells are again washed as described above and stained by adding 100 μl of peroxidase substrate (Sigma Fast OPD Substrate Buffer Tablet Set P-9187). The reaction proceeded for 30 minutes at room temperature in the dark and under stirring, and then the reaction was stopped by adding 50 μl of 3M H2SO4. The absorption was measured at 490 nm.

EXAMPLE 8

Western blotting of tumor antigen A3

Extracts affinity purified tumor antigen A3 were separated by SDS-PAGE and transferred to membranes for Western blotting. The extracts were applied directly or heated to 100°C for 5 minutes, or heated to 100°C for 5 minutes, but in the presence of mercaptoethanol (TOGETHER) (figure 8). Then the membrane was investigated using A3scFv-SEAm9 and anti-SEA-HRP or antibodies against α6-integrin or β4-integrin. Antibodies against β4-integrin person not interacted with any of the proteins on the membrane (figure 8 [ii]). Antibodies against α6-integrin person interacted with the majority of samples corresponding to molecular masses of from 90 to 140 kDa, A3-affinity purified extract of tumor antigens (figure 8 [iii]). Same ol whom were also detected using A3scFv-SEAm9, moreover, the detection is also carried out after heating, but in this case, the result was much weaker in reducing conditions (in the presence TOGETHER) (figure 8 [i]). The main band in the range 90-140 kDa corresponded to the bands in example 5, which were analyzed by peptide mapping and found the content they α6-integrin and β4-integrin.

Materials and methods

Antibody ASC-3 against β4-integrin person and antibody NKI-GoH3 against α6-integrin person received from Becton Dickinson (Copenhagen, Denmark). The samples were dissolved in SDS-PAGE in 0.25 M Tris-glycine pH of 8.9 and 0.1% SDS at 100 V through the upper gel, then 170V after allowing the gel. Standards of molecular weights (Biorad broad Range, Biorad) were applied in all gels. Separated samples were transferred to nitrocellulose (Biorad) in a figurative buffer (10 mm Tris main, 2M glycine, 40% (V/V) methanol) at 100V for 1 hour. Then the membrane was blocked with 5% (wt/V) BSA/TBS for at least 2 hours at 4°C, then incubated with the appropriate antibody diluted in 5% BSA/TBS/0.2% of the azide. The reaction proceeded for at least 2 hours at room temperature, after which extensive membrane was washed in TBST-t, the bound antibodies were determined, incubare membrane for 1 hour with HRP conjugated antibody, diluted in TSB-T containing 5% milk powder. Sitemember incubated with reagents, enhance the chemiluminescence (ECL) detection (Renaissance® NEN™ Life Science Products, Boston, MA)for 5 minutes and exposed to film within 1 hour.

DESCRIPTION of FIGURES

Figure 1

Associated with tumor antigen A3 homogeneity expressed in primary and metastatic tumors

Immunohistochemical staining of frozen and fixed with acetone sections of tumor tissues using A3 scFv-SEA(D227A) and S215 Fab-SEA(D227A) at 70 nm. Fused protein A3 scFv interacts strongly and homogeneous as with primary carcinoma of the colon and pancreas obtained during resection of the tumor patients. Characteristic staining of primary cancer of the colon is shown in the case of S215 Fab-SEA (D227A) (A) in the case of A3 scFv-SEA(D227A) in (In). Staining with A3 scFv-SEA(D227A) of liver metastases of colon cancer is shown in (C), and primary cancer of the pancreas in (D).

Figure 2

Covered A3 scFv-SEA(D227A) tumor cells l205 effectively killing T-cells.

Superantigen-antibody-dependent cellular cytotoxicity against cells l205 mediated A3 scFv-SEA (D227A), reached the same maximum level of cytotoxicity, as in the case of a fused protein anti-EP-HIMSELF S215 Fab-SEA (D227A), although 10-fold higher concentration. The absence of cytotoxicity, mediated by D1,3 cFv-SEA(D227A), indicates whether the merged protein fragment antibodies aimed at the tumor.

Figure 3

Immunoaffinity chromatography tumor extract on 3scFv-SEAm9-hand column. Protein associated with A3-containing columns, extensively washed, then suirable, as described in "Materials and methods of example 5. Erwerbende fraction was investigated by means of UV-spectroscopy (arrow) and was determined by a single peak. Samples were suirable pH gradient, as indicated in X.

Figure 4

Preparation of antigen A3 were separated in non SDS PAGE and stained with silver. Using the previous Western analysis was determined interval of molecular weights, which you can find A3. Distinct bands within this region (marked I and II) was investigated by the method of peptide mapping.

Figures 5A and 5B

The epithelial integrin α6β4: complete primary structure α6 and various forms of β4 (predecessor) (Tamura et al., J. Cell Biol 111:1593-1604 (1990)). The corresponding peptides shown in SEQ ID No: 5-1, are underlined in the sequence α6-integrin person (figa) and β4 (predecessor)-integrin person (pigv), as published.

Figure 6

Immunohistochemistry of normal and malignant colon, using A3scFv and commercial monoclonal antibodies against α6 β4-integrin person.

Figures 7A and 7B

Binding analysis by ELISA. On figa monoclonal antibody ASC-3, specific β4-integrin, was used as the binding of the antibody, which was applied in various dilutions of untreated tumor extract. On FIGU used monoclonal antibody NKI-GoH3 against α6-integrin for binding A3-affinity purified eluate in various dilutions. As Figo and figv associated integrity antigen then successfully determined using A3scFv-SEAm9.

Figures 8A and 8B

Western blotting of the eluate from A3-affinity column. Used primary antibodies are (i) and (ii) A3scFv-SEAm9, (iii) antibody ASC-3 against β4-integrin person, and (iv) the antibody NKI-GoH3 against α6-integrin person. Line And the eluate was applied directly line In the eluate was heated to 100°C for 5 minutes, and the line With the eluate was heated to 100°C for 5 minutes, but in the presence of mercaptoethanol. Shows the position for the standard molecular weights.

Sources of information

1. DeCosse JJ, Tsioulias GJ, Jacobson JS. Colorectal cancer: detection, treatment, and rehabilitation. CA Cancer J Clin 1994; 44: 27-42.

2. Riethmuller G, et al. Monoclonal antibody therapy for resected Dukes'With colorectal cancer: seven-year outcome of a multicenter randomized trial. J Clin Oncol 1998; 16: 1788-1794.

3. Kuhn JA, Thomas G. Monoclonal antibodies and colorectal carcinoma: a clinical review of diagnostic applications. Cancer Invest 1994; 12: 314-323.

4. Tordsson J, et a2. Efficient selection of scFv antibody phage by asorption to in situ expressed antigens in tissue sections. J Immunol Methods 1997; 210: 11-23.

5. Aujame L, Geoffroy F, R. Sodoyer High affinity human antibodies by phage display. Hum Antibodies 1997; 8: 155-168.

6. Clark RK, Trainer DL, Bailey DS, Greig RG Immunohistochemical analysis of antiserum from rhesus monkeys immunized with human colon carcinoma. Cancer Res 1989; 49: 3656-3661.

7. Lewis AR, et al. Cloning and sequence analysis of kappa and gamma cynomolgus monkey immunoglobulin cDNAs. Dev Comp Imniunol 1993; 17: 549-560.

8. Brodin TN, et al. Man-made superantigens: Tumor-selective agents for T-cell-based therapy. Adv Drug Deliv Rev 1998; 31: 131-142.

9. Dohlsten M,et a2. Monoclonal antibody - superantigen fusion proteins: tumor-specific agents for T-cell-based tumor therapy. Proc Natl Acad Sci USA 1994; 91: 8945-8949.

10. Liu S, et al. Eradication of large colon tumor xenografts by targeted delivery of maytansinoids. Proc Natl Acad Sci USA 1996; 93: 8618-8623.

1. The antibody or its derivative, or a fragment having the structure of the linking structure of the target, inside and on the surface of epithelial tumor cells of the gastrointestinal tract of man and in a subpopulation of normal epithelial cells of the gastrointestinal tract, and specified connecting structure contains a sequence of complementarity determining region (CDR) in the light chain containing principally amino acids under non-23-33 (CDR1), 49-55 (CDR2), 88-98 (CDR3) amino acid sequences shown in SEQ ID NO:2, and sequences of CDRs in the heavy chain, containing principally amino acids under rooms 158-162 (CDR1), 177-193 (CDR2), 226-238 (CDR3) amino acid sequences shown in SEQ ID NO:2, or other connecting structure with such a unique binding properties.

2. The antibody according to claim 1, which is selected rahovym method.

3. The antibody according to claim 1, where the sequence will Masasa fascicularis.

4. The antibody or its derivative according to claim 1, which is derived from human.

5. The antibody according to claim 1, where the sequence of at least 84% identical to the corresponding sequences of human origin.

6. The antibody according to claim 1, which is characterized by low immunogenicity or absence of immunogenicity in humans.

7. The antibody according to claim 1, of which the gain derived by genetic linkage with other polypeptides and/or by chemical conjugation with organic or inorganic chemical molecules and/or by di-, oligo - or multimerization.

8. The antibody according to claim 1, which is genetically linked or chemically conjugated with a cytotoxic polypeptide or cytotoxic organic or inorganic chemical molecules.

9. The antibody according to claim 1, which is genetically linked or chemically conjugated with biologically active molecules.

10. The antibody according to claim 1, which is genetically linked or chemically conjugated with immunoactive molecules.

11. The antibody according to claim 1, which is modified by genetic binding molecules or by derivatization, leading to changes in the pharmacokinetic properties of the combined molecule.

12. The antibody according to claim 1, which is labeled, and the binding of labeled antibody to the structure of the target is specifically inhibited by unlabeled form of this antibody.

13. The antibody according to claim 1, where the specified linking structure recognizes neuregulirovannaya form α6β4-integrin.

14. The structure of the target inside or on the surface of tumor cells, and this structure is the target

a) has the ability to specifically block and specifically block the binding structure of the antibody according to any one of claims 1 to 11, and other connecting structures with similar binding properties

b) located inside or on the surface e is italianni cells of the gastrointestinal tract of man,

c) has significant homology with α6 - and/or β4-integrirovanie chains or their variants, representing a widespread or unique epitope

d) is highly expressed on the surface of tumor cells and

e) is a target for cytotoxic effector mechanisms.

15. The structure of the target by 14 where the binding structure of the antibody is labeled and its linking with the structure of the target inhibited by unlabeled form binding antibody structures.

16. The structure of the target by 14 where the specified linking structure contains one or more sequences of the complementarity determining regions (CDR), containing principally amino acids under non-23-33, 49-55, 88-98, 158-162, 177-193, 226-238 amino acid sequence represented in SEQ ID NO:2, or other connecting structure with such a unique binding properties.

17. The structure of the target by 14, where the specified antibody contains a variable region light chain containing principally amino acids under room 1-109 amino acid sequence represented in SEQ ID NO:2, and the variable region of the heavy chain, containing principally amino acids numbered 128-249 amino acid sequence represented in SEQ ID NO:2.

18. The structure of a target according to any one of p-17, cat heaven homogeneity is expressed in epithelial cells of the colon of a person and to a lesser extent - in cells of the pancreatic duct and bile duct.

19. The structure of the target on p, the expression of which correlates with the differentiation of the epithelium of the gastrointestinal tract.

20. The structure of the target according to claim 19, which mainly contains the amino acid sequence of α6-integrin, is presented in SEQ ID NO:3, and/or β4-integrin, is presented in SEQ ID NO:4, and/or one or more fragments and/or variants, or the United variants, and/or their subunits.

21. The structure of the target in claim 20, which includes Homo - or heterogeneity, or Homo - or heteropolymer specified α6β4-integrin, and/or the specified one or more of its fragments and/or variants and/or subunits.

22. The structure of the target in claim 20, which has an apparent molecular weight in his neuregulirovannaya form from 90 to 140 kDa, most preferably from 80 to 160 kDa.

23. The structure of the target in claim 20, which contains a peptide or polypeptide(s), containing mainly any of the amino acid sequence presented in SEQ ID NO: 5-51, or contains a molecule forming a complex with the specified polypeptide (s).

24. The structure of a target according to any one of p-23 recognized, exclusively or not, in his neuregulirovannaya form a connecting structure that is included in the antibody according to any one of claims 1 to 13.

25. A peptide capable of blocking the function of the structure of the URS target according to any one of p-24.

26. The pharmaceutical composition intended for the treatment of malignant disease in humans, comprising as active principle an antibody according to any one of claims 1 to 13.

27. The pharmaceutical composition intended for the treatment of malignant disease in humans, containing as active principle the structure of a target according to any one of p-24.

28. Vaccine composition intended for the treatment of malignant disease in humans, comprising as active principle an antibody according to any one of claims 1 to 13, or structure of a target according to any one of p-24, or the peptide according A.25.

29. Method of treating conditions based on antiangiogenic mechanism, characterized in that person injected antibody according to any one of claims 1 to 13 or structure of a target according to any one of p-24, or the peptide according A.25.

30. A method for the treatment of metastatic human diseases, according to which man is injected antibody according to any one of claims 1 to 13.

31. The way histopathology in vitro diagnostics and forecasting of development of malignant disease in humans, characterized in that the sample is brought into contact with an antibody according to any one of claims 1 to 14 and indicator, signal indicator provides an indication of the malignant disease in humans.

32. The method according to p, which involves the typing of the tumor.

33. SPO is about on p, which means cancer screening.

34. The method according to p, which involves the monitoring of previous malignancy conditions.

35. The method of in vitro diagnostics and forecasting of development of malignant disease in humans, characterized in that the body fluids to determine the concentration of antigen structure containing a target according to any one of p-24, with higher concentration of antigen provides an indication of the malignant disease in humans.

36. The method of in vitro diagnosis and prognosis of malignant diseases of man, characterized in that the body fluids to determine the concentration of the antibody according to any one of claims 1 to 13, with higher concentration of antibodies is an indication of malignant disease in humans.

37. The method of in vivo diagnosis and prognosis of malignant diseases of man, characterized in that determine the localization of the antibody according to any one of claims 1 to 13 relative to tumor deposits in humans, the identification of antibodies in tumor deposits is an indicator of malignant diseases in humans.



 

Same patents:

FIELD: pharmacology, medicine.

SUBSTANCE: as initial raw material one should apply tick culture of domestic dust. Extracting should be conducted for 45 h with 0.125 M ammonium hydrocarbonate solution, supernatant should be poured and centrifuged for 45 min, extract should be filtered followed by dialysis for 52 h at 6±2 C, twice per 24 h against 0.002 M ammonium hydrocarbonate solution and once for 4 h against distilled water. Dialyzed extract should be centrifuged for 1 h, filtered through paper filter to fulfill freeze drying up to residual moisture being 4%, not more. Dry residue should be dissolved in 0.1 M phosphate buffer at pH being 7.5. Then one should add 1%-formaldehyde solution and perform sterilizing filtration. Formalinized allergoid should be kept for 32 d at 32 C. Then comes dialysis at 6±2 C against 0.1 M phosphate buffer solution at pH being 7.5. The innovation enables to develop the allergen of decreased allergenic activity.

EFFECT: higher efficiency.

1 ex

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: one should isolate mononuclear cells in peripheral blood to register there the parts of Helicobacter pylori genome due polymerase chain reaction (PCR). Thus, the innovation provides low-invasive and specific way for predicting chronic persisting helicobacter pylori-infection that enables to evaluate systematic nature of the disease and prescribe adequate therapy in due time.

EFFECT: higher efficiency of diagnostics.

1 cl, 3 dwg, 3 ex, 1 tbl

FIELD: veterinary science, virology.

SUBSTANCE: method for evaluation of semen from bull-sires for contamination with cattle infectious rhinotracheitis virus involves taking semen samples, combining semen samples obtained from bull-sire for one month and their analysis as a single sample. The analysis is carried out by method of molecular hybridization. For evaluation of semen from rejected bull-sires the semen samples are taken obtained for all period of their exploitation. Method provides reducing labor intensity and time for analysis and enhanced sensitivity of analysis in assay of semen contamination with indicated virus.

EFFECT: improved method for evaluation of semen.

2 cl, 1 tbl, 4 ex

FIELD: veterinary science.

SUBSTANCE: one should detect positively reacting animals in RIA to conduct additional serological RIA testing, moreover, repeated serological testing in sero-negative animals should be carried out after provocation of immune response due to three-fold injection of inactivated vaccine against cattle leucosis at the dosage of 2 ml at 14-d-long interval and at obtaining positive result in RIA one should diagnose an animal to be infected one. The innovation accelerates detection of animals being virus-carriers, shortens leucosis-recovery period in farms and enables to decrease financial expenditures for recovery.

EFFECT: higher accuracy of diagnostics.

1 ex

FIELD: agriculture, poultry.

SUBSTANCE: the present innovation deals with carrying out transplantation of small flaps of donor's skin and registration for the duration of transplants' detachment period, and, also, their safety up to certain terms after transplantation, moreover, the mentioned transplantation of donor's skin should be carried out reciprocally among partner groups of the same age, moreover, such transplantation should be performed in youngsters of neonatal age.

EFFECT: higher accuracy of comparative evaluation.

1 ex, 4 tbl

FIELD: veterinary science.

SUBSTANCE: the suggested method deals with isolating lymphocytes out of peripheral blood and lymphoid organs to achieve their concentration up to working concentration followed by reaction of blast-transformation with mitogen as phytohemagglutinin, incubation of lymphocytes and registration of reaction results by radiometric method: concentration of lymphocytes should achieve working concentration value of 10-15x106 cells/ml, incubation of lymphocytes should be performed for 48-54 h at 37.5-40.0 C, index of lymphocytic stimulation should be calculated by the following formula:index of stimulation = (average amount of impulses/min in experiment) / average amount of impulses/min in control) The method provides increased efficiency in evaluating immune state in minks due to increased sensitivity in reaction of blast-transformation.

EFFECT: higher accuracy and efficiency of detection.

1 ex, 5 tbl

FIELD: veterinary science.

SUBSTANCE: the suggested method deals with isolating lymphocytes out of peripheral blood and lymphoid organs to achieve their concentration up to working concentration followed by reaction of blast-transformation with mitogen as phytohemagglutinin, incubation of lymphocytes and registration of reaction results by radiometric method: concentration of lymphocytes should achieve working concentration value of 10-15x106 cells/ml, incubation of lymphocytes should be performed for 48-54 h at 37.5-40.0 C, index of lymphocytic stimulation should be calculated by the following formula:index of stimulation = (average amount of impulses/min in experiment) / average amount of impulses/min in control) The method provides increased efficiency in evaluating immune state in minks due to increased sensitivity in reaction of blast-transformation.

EFFECT: higher accuracy and efficiency of detection.

1 ex, 5 tbl

FIELD: medicine, immunology, allergology.

SUBSTANCE: one should detect the parameters of immune state and objective anamnesis, before carrying out specific immunotherapy (SIT) with allergens one should detect IgM level (g/l), the quantity of monocytes (Mon), lymphocytes (Lymph) in total blood analysis in %, the quantity of B lymphocytes in % (CD19), immunoregulatory index (IRI) (CD4/CD8), phagocytic value in % (PhV), IgE serumal level in IU/ml, the quantity of points in evaluating accompanying nonallergic anamnesis (Point) {one point in case of one chronic disease, 2 points in case of two, etc.}, then one should calculate the changes in immunity parameters on the 7th d of SIT-therapy, obtained values should be applied in the following equation of regression (Y= -195.4 + 117.3·IgM + 10.8·Mon - 0.35·IgE - 17.1·Point + 7.8·Dlymph + 20.4·Dmon + 3·Dbl + 22.8·Diri) and calculating the possibility of complications it is necessary to perform by the following formula: P = 100/(1+Exp(-Y)).

EFFECT: higher accuracy of prediction and improved quality of therapy.

3 ex

FIELD: medicine, cardiology, clinical biochemistry and medicinal immunology.

SUBSTANCE: invention relates, in particular, to immunologic composition and set used in diagnosis of heart diseases by using human mitochondrial adenylate kinase isozymes. As a marker, methods involve using human mitochondrial adenylate kinase isozymes presenting in myocardium muscle cells. These markers provide carrying out the precise diagnosis of heart disease being more easily.

EFFECT: expanded assortment of agents used in diagnosis of heart diseases.

14 cl, 17 dwg, 5 tbl, 7 ex

FIELD: biotechnology, medicine, immunology.

SUBSTANCE: method involves preparing control samples by preparing solution of heterologous chimeric antibodies consisting of whole molecules or fragments of immunoglobulin isolated from immunized animal serum and associated with whole molecules or fragments of human immunoglobulin in phosphate-saline buffer, pH 6.0 followed by preparing different dilutions in indicated buffer, their control in IFA and selection of dilutions at optical density values 1.0, not less. Invention provides preparing control samples comprising specific antibodies that elicit high specificity and capacity for detection in combination with their infectious safety, standard indices and availability with respect to economy aspects.

EFFECT: improved preparing method.

3 cl

FIELD: immunology; treatment of mediated diseases IL-1 and failures.

SUBSTANCE: bonding molecule IL-1β which is antibody to human IL-1β and especially human antibody to human IL-1β where hypervariable sections CDRs of heavy and light chains have definite amino acid sequences. Antibody may be used for treatment of mediated disease IL-1, for example osteoarthritis, osteoporosis and other inflammatory processes of bones of rheumatism or podagra nature. Constructions of deoxyribonucleic acid are described which code heavy and light chains or their fragments and expressive vectors which may be replicated in cells including deoxyribonucleic acid constructions. Method of obtaining bonding molecule IL-1β by means of cell transformed by vector is described. Proposed antibody may be used both in prophylactic and treatment of diseases.

EFFECT: enhanced efficiency.

15 cl, 3 dwg, 5 ex

FIELD: biotechnology, medicine, proteins.

SUBSTANCE: invention describes new polypeptide in isolated form relating to subfamily of superfamily human immunoglobulins (Ig-Sf). This polypeptide shows at least 70% of homology level with amino acid sequence of murine molecules CRAM-1 or CRAM-2 regulated by the confluence of adhesive (figures 3, 6 are represented in the claim). Also, invention relates to antibodies showing specificity with respect to the polypeptide. Antibodies and soluble polypeptide can be used for treatment of inflammation and tumors. Invention describes polynucleotide or oligonucleotide encoding the full-size polypeptide or its moiety and represents primer, probe, anti-sense RNA and shows the nucleotide sequence that is identical conceptually with human CRAM-1. Invention provides preparing new adhesive proteins from superfamily Ig-Sf that are regulated at the transcription level in endothelium by effect of tumors. Invention can be used for treatment of different diseases, in particular, inflammatory responses.

EFFECT: valuable medicinal properties of polypeptide.

19 cl, 33 dwg, 1 ex

The invention relates to the field of biotechnology and medicine, namely, to new sequences of DNA nucleotides and amino acids sequences of monoclonal antibodies (MABS) generated against lymphoblastoid cells, and peptides that bind MAT

The invention relates to the field of immunology, in particular to new monoclonal antibodies against the calcium-binding protein S100, application of antibodies for the development of immunoassay-specific for the determination of total S100 or S100 isoforms in serum, plasma, cerebrospinal fluid and other biological fluids, with the claimed method it is possible to determine S100, S100and S100isoforms

The invention relates to medicine, in particular to the treatment and pulmonology, and for the treatment of acute lung injury and fibrosis

The invention relates to genetic engineering

The invention relates to the field of biotechnology, in particular to bioengineered product for ant Richter and method of manufacturing such a product

The invention relates to medicine and relates to methods and compositions for immunomodulation

The invention relates to medicine and related methods of use antifibrin antibodies for inhibition of in vivo blood clots, as well as pharmaceutical compositions and a kit containing a pharmaceutical composition for use in such methods

Chalcone coumarins // 2266291

FIELD: organic chemistry, medicine, oncology, pharmacy.

SUBSTANCE: invention relates to compounds of the formula (I): or their pharmaceutically acceptable salts or solvates wherein Ar represents a substituted or unsubstituted (preferably aromatic one) carbocyclic or heterocyclic group wherein abovementioned carbocyclic or heterocyclic group comprises 5 or 6 atoms in cyclic structure wherein a heteroatom is taken among the group consisting of nitrogen (N) and sulfur (S) atom and any substitutes at Ar group are taken independently of one another of the group consisting of Cl, Br, F atoms and OR10 wherein R10 represents saturated or unsaturated lower hydrocarbon (C1-C6)-radical of normal or branched structure; R represents OR10 wherein R10 corresponds to above given value; R1 represents lower hydrocarbon (C1-C6)-radical of normal or branched structure under condition that if R1 represents -CH3 and R means -OCH3 or -OH then Ar group can't represent 4-methoxyphenyl or 3,4-dimethoxyphenyl. Also, invention proposes a component of medicinal agent used in treatment or prophylaxis of neoplasms. Also, invention proposes a pharmaceutical composition possessing with an anti-proliferative activity and comprising the effective amount of one or some compounds of the formula (I) in combination with one or some pharmaceutically acceptable additives. Invention provides the development of chalcone coumarins possessing with the enhanced anti-proliferative effect with respect to sensitive tumor cells, cells with resistance to conventional chemotherapeutic agents, among them, to anti-tumor medicinal agents of the last generation represented by paclitaxel and docetaxel.

EFFECT: valuable medicinal properties of compounds and compositions.

1 tbl, 21 ex

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