Monoclonal antibody, nucleic acid and the vector encoding the antibody, and the use of antibodies for the diagnosis and treatment of malignant diseases

 

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 the MAT. The invention also relates to diagnostic assays, in which the foregoing antibodies or peptides are used to identify individuals with a high probability of malignant diseases and, sometimes, to identify individuals with specific malignant disease. In addition, the invention relates to pharmaceutical compositions comprising a MAT or peptides according to the present invention and intended for use in the treatment of various malignant diseases, and to methods of treatment of malignant diseases using MAT or peptides according to the present invention. The use of the invention will allow for the differential diagnosis of cancer. 7 N. and 23 C.p. f-crystals, 18 ill., 5 table.

The technical FIELD,

The present invention relates to new sequences of monoclonal antibodies, peptide sequences by Albanien monoclonal antibodies and peptides.

PRIOR art

Joint international Application filed under the PCT (patent cooperation Treaty), no publication WO 95/20605, reveals the essence of immunostimulatory monoclonal antibodies. Antibodies which are the subject of the International Application was developed against lymphoblastoid b-cells and have been shown to have immune-stimulating effect. With the introduction of their animals native tumors was also found that these antibodies exert antitumor effect.

Diagnosis of cancer in accordance with modern medical procedures typically is a multistage process that includes medical examination, the use of different techniques of visualization, the use of different markers of cancer and so on, In the art there has long been a need for methods of cancer diagnosis, which would ensure detection of cancer, as well as determining the type of cancer that occurs in the surveyed individual.

The INVENTION

The present invention is based on the opening sequences of monoclonal antibodies to lymphoblastoid cells. The present invention also based on the discovery that the binding of these ant the antibodies with T-cells of healthy people.

In accordance with one aspect of the invention provides a monoclonal antibody having a variable region selected from the group consisting of:

(a) a monoclonal antibody having a variable region heavy chain comprising the amino acid sequence depicted in Fig.1;

(b) a monoclonal antibody having a variable region of the Kappa light-chain including amino acid sequence depicted in Fig.2;

(C) a monoclonal antibody having a variable region heavy chain comprising the amino acid sequence depicted in Fig.1, and the variable region of the Kappa light-chain including amino acid sequence depicted in Fig.2;

(g) a monoclonal antibody having a variable region heavy chain having at least 70% identity to amino acid sequence depicted in Fig.1;

(d) a monoclonal antibody having a variable region light chain having at least 70% identity to the sequence depicted in Fig.2.

According to the present invention, the term "antibody" refers to monoclonal antibodies of any of the classes IgG, IgM, IgD, IgA and IgE. example, for antibodies, which do not have the Fc-fragment, single-chain antibodies, fragments of antibodies, consisting essentially only of the variable antigennegative domain antibodies, etc.,

In addition, the invention also relates to antibodies that bind with the antigen which specifically binds to any of the above MAT, that is, antibodies that have cross-reactivity with the above antibodies.

In accordance with one example embodiment of the invention a monoclonal antibody (MABS) is a chimeric antibody human and mouse, namely MAT with a constant region derived from human, and a variable region derived from a mouse. With this purpose, the variable region of Kappa light-chain and heavy chain MAT according to the present invention has cloned by PCR, and their DNA sequenced. In accordance with another example embodiment of the invention the antibody is a fully humanized antibody, that is, variable and constant region it received from the man.

The term "having at least X% identity" refers to the percentage of amino acid residues that are identical in the two compared sequences, the EU is ti means, 70% of the amino acids in two or more optimally mapped polypeptide sequences are identical.

Preferably, the identity is equal to at least 80%, more preferably at least 90%.

In accordance with an additional aspect of the invention is provided a hybridoma line of mouse cells that produce any MAT according to the present invention. Hybridoma can be obtained by any method known in the art (e.g., Kohler, G. and Milstein, C., Nature, 256:495-497 (1975)). The supernatant hybridoma cell lines were subjected to typical screening antigennegative activity in any of the ways known in the art, for example, using enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay analysis (RIA RIA). Supernatant were subjected to screening for MAT products that are associated with any of the peptides according to the present invention (explained below) or which are associated with cells that bind these peptides, for example, cells of Daudi or T-lymphocytes.

The DNA sequence encoding any of the amino acid sequences of the heavy chain or light chain of the above MAT, also included the particular genetic code MAT according to the present invention can encode a multitude of nucleotide sequences, in addition to the sequences depicted in Fig.1 or 2.

The invention also provides expressing vectors, such as plasmids comprising the above DNA sequences, and cells of the host that contains one or more of these expressing vectors.

In accordance with another aspect of the invention is provided a peptide sequence of antigens In cells, which can join MAT according to the present invention. Was conducted the search in the database of the Bank non-redundant genes and EST Department has determined that these peptide sequences are new ones.

In accordance with this additional aspect of the invention provides a peptide selected from the group consisting of:

(a) a peptide having the amino acid sequence depicted in Fig.10;

(b) a peptide having the amino acid sequence depicted in Fig.11;

(b) a peptide having the amino acid sequence depicted in Fig.12;

(d) a peptide having at least 85% identity to any of the amino acid sequences of the peptides according to paragraphs (a), (b) and (C) above, and

(d) a protein or peptide that includes one or more peptides according to (agnosticism analyses, for example, such as a competitive immunological assays, which determine the level of binding of the MAT according to the present invention with their natural antigen, which is present on T-cells. In addition, peptides can be used for antibody production in immunized animals, and these antibodies can then be used for any of the purposes described above and below.

Analogues of all the above peptides also form an additional aspect of the present invention. As will be clear to a person skilled in the technical field of amino acid sequences of the peptides according to the present invention can be modified, for example by addition, deletion or conservative or non-conservative substitution of one or several amino acids without significant disturbance of the ability of peptides to bind antibodies.

The term "conservative substitution" refers to substitution of an amino acid of one class of amino acid of the same class, where the class is defined the same physico-chemical properties of the side chains of amino acids and a high frequency of substitutions in homologous proteins occurring in nature, which can be determined, for example, using standard matrix frequency of Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys); Class V (Ile, Ley, Val, Met) and Class VI (Phe, Tight, Thr)]. For example, substitution of Asp for the remainder of Class III, such as Asn, Gln or Glu, is a conservative substitution. The term "non-conservative substitution "refers to substitution of an amino acid of one class of amino acid from another class; for example, substitution of Ala, the residue Class II, the rest of the Class III, such as Asp, Asn, Glu or Gln.

Individual letters used above (and below) to identify specific amino acids (AA), correspond to the 1-letter symbols of amino acids recommended by the Commission on biochemical nomenclature of the IUPAC-IUB.

Analogues of the above peptides that fall within the scope of the present invention are those peptides, which have approximately the same level of binding MAT according to the present invention, as the peptides depicted in Fig.10-12. The level of binding can be determined by any method known in the art.

Peptides and analogues according to the present invention can also be modified chemically, such chemically modified peptides and their analogues also form part of the invention. The term "chemically modified" refers to a protein, in which such as processing or other post-translational modifications either by chemical modification techniques which are well known to specialists in this field of technology. Among the many known modifications of a typical, but not exclusive, examples include acetylation, acylation, amination, ADP-ribosylating, glycosylation, formation of GPI-anchors, covalent joining of liquids or derived lipids, methylation, miesterioasa, tahilramani, prenisolone, phosphorylation, originalarbeit and any similar process.

The second discovery on which the invention is that the MAT according to the present invention can, in various degrees of contact with T-cells derived from people with malignant disease, compared with the degree of binding of the same MAT with T-cells of healthy people.

Therefore, in accordance with another aspect of the present invention, there is analysis to identify the surveyed people with a high likelihood of the presence of malignant disease, including:

(a) obtaining a sample of body fluid from the above-mentioned person;

(b) contact the above sample with at least one MAT according to the present invention; and

(C) determining the extent to which ivania, determined in step (C), with the degree of binding of the MAT according to the present invention with T-cells contained in a sample obtained from a healthy person;

no significant difference between the two degrees of binding indicates that there is a high probability that the examined person's malignant disease.

In accordance with the invention, a break received from the person who needs to analyze, can be any fluid of the body, which contains a detectable amount of T-cells. In a typical case, the sample of body fluid, a test of blood or lymphatic fluid. Preferably, before the contact MAT according to the present invention obtained with the breakdown of mononuclear cells from peripheral blood (MCPC) contained in the sample is separated using any of the methods known in the art, for example, by centrifugation in a density gradient Ficoll Hypaque, and then the separated cells are brought into contact with the test antibodies.

The term "malignant disease" in accordance with the invention should be understood as any malignant disease, known in the art, at any stage.

This term the definition of the symptoms. Preferably, the term refers to a dense tumors.

The term "healthy person" refers to someone who is not a malignant disease, and may also refer to the average value for several people, or to the value obtained in the formation of a pool of bodily fluids obtained from several people. It should be noted that, if the standard degree of binding for healthy people is defined, there is no need to re-define the standard for each assay, and the resulting number can be used constantly. According to the invention, it was found that in healthy people, approximately 25% of CD3+T cells bind antibodies according to the present invention.

The term "high probability" means that the analysis according to the present invention is an analysis performed at the initial screening and are able to identify people who presumably have malignant disease. The true presence of malignant disease in humans, identified using the method according to the present invention, must be later confirmed using additional methods known in the art.

The term "degree svyazyvaniem this level can be determined by any known in the art methods of determining levels of antibody binding, for example ELISA or Western blot testing. The degree of binding can be determined using any detection system, for example, use of an antibody to antigens of the mouse or its fragments connected with a detectable marker. Examples of such detectable markers are radioactive group, fluorescent group, an enzyme capable of catalyzing a reaction product which is detectable product (e.g., a color reaction), bitenova group, which can be detected using avidin, etc., In the preferred embodiment of the invention, the degree of binding of the MAT according to the present invention with T-cells was determined by double labeling, in which the antibody to T-cell (e.g., antibody to BD3+cell) attached to one fluorescent marker, a MAT according to the present invention to another type of fluorescent marker. The degree of binding is then determined using an apparatus for sorting cells activated by fluorescein (FACS). Quantitative assessment of the degree of binding is produced by determining the percentage of CD3+T cells (defined by their binding with antibodies to CD3+cells), which also bind the MAT according to the present isotropy people having malignant disease, similar to the number of CD3+cells in blood samples obtained from healthy people, so the normalization degree of binding MAT and CD3+T cells using the total number of CD3+binding of T cells in cancer patients and healthy people legitimate. However, the percentage of CD3+binding of T cells that bind and MAT according to the invention (hereinafter: "CD3+MAT cells") in people with malignant disease, significantly different from the percentage of CD3+MAT cells in the blood of healthy people. The percentage of CD3+MAT cells in humans with malignant disease may be significantly higher or significantly lower than the percentage D3+MAT cells in the blood of healthy people, depending on the type of malignant disease.

The degree of binding MAT according to the present invention T-cells isolated from the examined person is found to be "significantly different" from the degree of binding to T-cells obtained from a healthy person, if the difference in binding MAT statistically significant when evaluated using any of the statistical methods known in the art (for example, using t-test, Studentds.

The invention not only allows you to identify people with a high probability of having any type of malignant disease (because sick people have a different degree of binding of T cells with a MAT according to the present invention, in comparison with healthy people), but can also help to identify people with a specific type of cancer by determining whether the above-mentioned degree of binding is higher or lower than the corresponding degree of binding in healthy people.

In a typical case, the percentage of binding of the MAT according to the present invention T-cells isolated from healthy individuals is approximately 25%, that is 25% of cells expressing the marker CD3+T cells (as measured by binding of antibody to CD3+cells), also bind and MAT according to the present invention.

According to the present invention, it has been shown that in samples obtained from patients with prostate cancer, the percentage of CD3+T cells that bind MAT according to the present invention, is approximately 50%.

In addition, it was shown that, if CD3+T cells originate from samples taken from patients with cancer of the colon or breast cancer, is respectively.

Therefore, according to the present invention, there is an opportunity to identify high probability of the presence of specific types of cancer on the sample of body fluid obtained from the examined person, with a simple and single analysis based on the degree of binding of the MAT according to the present invention with CD3+cells present in the sample of body fluid. Easy diagnostic analysis according to the present invention, for which it is sufficient to use only one type of MAT to identify a person with a certain type of cancer, is very well suited for a wide population screening.

Therefore, the present invention in one of its aspects provides analysis to identify the surveyed person with a high probability of the existence of specific malignant diseases, including:

(a) obtaining a sample of body fluid from the above-mentioned person;

(b) contact the above sample with a MAT according to the present invention; and

(C) determining the degree of binding of the above MAT with T-cells contained in the above sample, and

(g) comparing the extent of binding determined in step (C), the obtained cells with Stephen the project with a high probability indicates what in the examined person has a malignant disease, and whether the degree of binding MAT with T-cells obtained from the above person, higher or lower than the bonding MAT with T-cells of healthy people indicates a specific type of malignant disease, which with high probability has examined the person.

In particular, if the degree of binding with a MAT according to the present invention, significantly higher than in healthy people, there is a high probability that the examined person has prostate cancer.

If the degree of binding was significantly lower than in healthy people, there is a high probability that the examined person has colon cancer or breast cancer.

According to the diagnostic aspect of the invention, the compositions (compositions), including MAT according to the present invention can be used for diagnosis in order to identify people who have high probability there is a malignant disease (in General), or to identify specific malignant disease, which probably has a person. Therefore, the invention in one of its escasany antibodies together with a suitable carrier. The carrier can be either soluble carrier, for example, any physiologically acceptable buffer solutions known in the art (for example, phosphate buffer solution (FBI)), or a solid carrier, such as, for example, latex beads.

The present invention also provides kits, for example, kits for diagnostic tests intended for use MAT according to the present invention and perform diagnostic tests, the essence of which is disclosed above. In one example of the invention, the diagnostic kit may typically include at least one of the above MAT in one or more containers, the conjugate substances partner that specifically bind to the MAT (e.g., antigen or analogue according to the present invention), the label is able to give a detectable signal, and instructions for their use. The label can, a priori, be contacted with a monoclonal antibody, or alternatively, the label may be contacted with a molecule carrier, which is then specifically binds to the MAT. Incubation of the sample with the composition, which is a diagnostic reagent for a specific time is provided pharmaceutical compositions comprising, as active ingredient, one or more MAT according to the present invention. The use of the above MAT for the preparation of pharmaceutical preparations for the treatment of various malignant diseases in humans is also in the scope of the invention.

In another aspect the present invention provides a method of treatment of malignant diseases by introducing the needy in this human a therapeutically effective amount of the above-mentioned MAT. Therapeutically effective amount is the amount that can alleviate the symptoms of malignant disease, reducing the symptoms or completely eliminating them.

Pharmaceutical compositions comprising the peptides according to the present invention, also form an aspect of the invention. Such compositions can be used, for example, for the active immunization of a person for the purpose of obtaining antibodies, which can then be contacted with T cells and induce an immune response in humans.

Further, the basic aspects of the invention will be described with occasional reference to the accompanying graphics. In the following description and graphic materials, the term "Wat antibody" is used as Trichet DNA and peptide sequences of variable regions of the heavy chain MAT according to the present invention.

Fig.2 shows the DNA and peptide sequences of variable region of Kappa light-chain MAT according to the present invention.

Fig.3 shows the analysis of amino acid sequences of variable regions of heavy chain antibodies according to the present invention (labeled "BAT"). "Wat" means amino acid sequencen-the area of Wat antibodies, while "VMS2" refers to the amino acid sequence of a gene VMS2/VGK4 germ line cells. If Wat sequence and the sequence of the germline of identical, sequence germ lines denote by a dot (.); if there are inconsistencies, specify a different balance of the germ line. The table, below, and sequence shown on the following pages describe the frequency with which certain amino acids can be detected at a certain position of rest, as in the mixed subgroup heavy chain of the mouse (Mouse vnMixed.) (Kabat et al., Sequences of proteins of immunological interest, (1991)), and a larger database of all known sequences of the VHmouse (all sequences VHmouse).

Fig.4 shows the results of amino acid analysis the settlement of the tion in Fig.4 as "Wat"). "Mouse" refers to amino acid sequence To aTothe area of Wat antibodies, whereas the "Embryo" refers to the amino acid sequence of a gene H4 germ line cells. If Wat sequence and the sequence of the germline of identical, sequence germ line denoted by a dot (.); if there are inconsistencies, specify a different balance to the germ line. The table below and on the following pages describe the frequency with which it is possible to detect specific amino acid at a certain position of rest, as in the subgroup VI heavy chain of the mouse according to Kabat, and a larger database of all known sequences of the VTomouse (all sequences VTomouse).

Fig.5 shows the DNA and peptide sequences of the variable regions of Kappa light-chain chimeric antibody according to the present invention.

Fig.6 shows the DNA and peptide sequences of variable regions of the heavy chain of the chimeric antibody according to the present invention.

Fig.7 shows a schematic representation expressing vector pKN 110 mammals, used for the expression of Kappa light-chain himaruya vector pG1D 110 mammals, used for the expression of the heavy chain of the chimeric antibody according to the present invention.

Fig.9 shows the graphical representation of the results of ELISA analysis, measuring the characteristics of the binding of the chimeric antibody and mouse1/Kappa according to the present invention with cells of Gaudi.

Fig.10 shows the amino acid sequence of peptide 1 according to the present invention.

Fig.11 shows the amino acid sequence of peptide 2 in accordance with the present invention.

Fig.12 shows the amino acid sequence of peptide 3 in accordance with the present invention.

Fig.13 is a schematic depiction showing the percentage of CD3+cells that connect MAT according to the present invention (labeled "BAT"), compared with the total number of CD3+cells in blood samples of healthy people, defined by analyzing the FAX.

Fig.14 shows the percentage of CD3+cells that connect MAT according to the present invention (labeled "BAT"), compared with the total number of CD3+cells in blood samples obtained from patients with carcinoma of the large intestine, in particular the present invention (labeled "BAT"), compared with the total number of CD3+cells in blood samples obtained from patients with carcinoma of the breast.

Fig.16 shows the percentage of CD3+cells that connect MAT according to the present invention (labeled "BAT"), compared with the total number of CD3+cells in blood samples obtained from patients with carcinoma of the prostate.

Fig.17 is a schematic depiction showing the average percentage of CD3+cells that bind MAT according to the present invention (designated as BAT), in healthy subjects compared to patients with carcinoma of the breast, carcinoma of the colon or carcinoma of the prostate.

Fig.18 is a photograph of the results of Western blotting of peptides derived from T cells of people with prostate cancer, otorhinolaryngological carcinoma (ENT), carcinoma of the breast, or peptides derived from cell membranes, Daudi. The blot was incubated with a MAT according to the present invention and identified the increased concentration of the antigen in T cells obtained from patients with carcinoma of the prostate, compared with undetectable levels of antigen to T-cells, polycentricity

I. SEQUENCING MAT

(A) Abbreviations

Fetal serum of calves (FTS); ribonucleic acid (RNA); the messenger RNA (mRNA); deoxyribonucleic acid (DNA); copy DNA (cDNA); polymerase chain reaction (PCR); minute (min); second (); Tris-borate buffer (TBB).

(B) Materials

Components of environments and all other materials for tissue culture were obtained from Life Technologies (UK). Kit for RNA extraction was obtained from the company Stratagene (USA), while the set for the synthesis of 1st cDNA strands were purchased in the company of Pharmacia (UK). All components and equipment for PCR reactions, including AmpliTaqDNA polymerase were purchased from the company Perkin Elmer (USA). Set TA Cloningwas obtained from the company Invitrogen (USA). Agarose (UltraPure) was obtained from Life Technologies (UK). Pre-set to cycle sequencing Thermo Sequencesand apparatus for DNA sequencing Vistra 725 were purchased in the company Amersham (UK). All other products for molecular biology were obtained from New England Biolabs (USA).

(C) Methods of experiment: PCR klonirovannyi and line cells Daudi were successfully transferred to MRC-CC, both cell lines were cultured in suspension using RPMI (without glutamine) supplemented with 10% (volume) of the FTS, 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mm L-glutamine, 1 mm sodium pyruvate and 12.5 units/ml nystatin.

Had accumulated approximately 108viable cells hybridoma cell line BAT, and from 108cells was isolated total RNA using a kit for RNA extraction according to the manufacturer's instructions. The set was used for one-step extraction procedure guanidine the thiocyanate-phenol-chloroform as described Chromczynski and Sacchi, Anal. Biochem., 162:156, 1987. Also in accordance with the manufacturer's instructions set for the synthesis of 1st cDNA strands was used to obtain single-stranded DNA copies of mRNA Wat hybridoma with Notl-(dT)18primer supplied in the kit. Each volume of the final reaction mixture, equal to 33 μl, was used at approximately 5 µg of total RNA. After completion of the reaction, the reaction mixture was heated to 90C for 5 min for denaturation of duplex RNA-cDNA and inactivation of reverse transcriptase before cooling on ice.

For PCR amplification of the gene variable regions of the heavy chain of the mouse (VNgene) and the gene variabel the Jones and Bendig, Bio/Technology, 9:8, 1987. Essentially, there were used two series of degenerate primers is one that is designed to hybridization leader sequences of the heavy chain of the mouse (i.e. MHV1-12; table 1), and the second designed for hybridization leader sequences of genes Kappa light-chain mouse (i.e. MKV1-11; table 2), with primers designed for the hybridization of the 5'end of the corresponding gene constant region, for PCR gene cloning of variable region of mice.

Separate PCR reactions were prepared for each of the degenerate primers together with the corresponding primer constant region in a special room for PCR using specific protocols designed to minimize the possibility of cross-contamination. In all cases, amplification matrix cDNA used DNA polymerase Amplitaq®. Tubes for PCR reaction was then loaded into the device for the cyclic heating and implemented cycles (after the initial melting at 94C for 1.5 min) at 94C for 1 min and at 72C for 10 min in the amount of 25 cycles. After completion of the last tion was cooled to 4C. except For a break between stages of annealing (50C) and elongation (72(C) when used enhanced the ramp time, equal to 2.5 min, between all stages of the cycle, the ramp time was 30 s

The aliquots of 10 µl of each PCR reaction was passed through a gel containing 1% agarose/FBI (pH 8.8), in order to determine which reactions was formed PCR product of the correct size. The CRL-reaction, in which, apparently, were fully amplified variable regions genes, were repeated to obtain independent PCR clones and thus minimized the effect of PCR errors. The aliquots of 1-6 ál of these PCR products of the correct size were cloned directly into the pCRIIthe vector supplied in the kit TA Cloningand transformed in competent cells INAF’, as described in the manufacturers instructions. Colonies containing the plasmid with the insert of the correct size were identified by PCR screening of colonies using the direct (pCRII) and reverse (pCRII) oligonucleotide primers described in table 3, according to the wayin accordance with the manufacturer's instructions.

Example 1: Cloning and sequencing of variable regions of the heavy chain Wat antibodies

As with any plans humanization, strictly followed the Protocol of PCR-cloning and sequencing. This was done to minimize the possibility of introducing errors in the sequence of the wild type gene variable regions VHmouse from hybridoma cell line BAT. Only if all the data on DNA sequences from at least two different clones of VHgenes from the hybridoma cell line expressing Wat antibody mouse, perfect match, the sequences of genes were recognized correctly.

Three separate PCR product, each of the separate preparation of total RNA and subsequent reaction of the synthesis of the first chain cDNA were PCR-cloned, and were completely sequenced DNA of both threads. Although analyzed all twelve of the heavy chain primers (table 1), only primer MHV9 (together with MHCG3 - designed for hybridization CH1domain gene(data in table 1 are not represented).

Analysis of the DNA sequences of several individual clones from each of the three PCR products (each of the separate reactions of synthesis of the 1st thread and subsequent PCR reaction) resulted in the sequencing of the variable region of the heavy chain Wat antibodies described in Fig.1. This sequence was confirmed on both strands of DNA for all three investigated PCR clones.

Example 2: Kodirovanie and sequencing of the variable region of Kappa light-chain Wat antibodies

Single-strand matrix cDNA obtained in synthesis process 1 thread, PCR-amplified using a series of degenerate primers Kappa light-chain (table 2). However, this resulted in the amplification of multiple PCR products with more than one degenerate primer, which meant that at least more than one gene variable regions were transcribed Wat hybridoma cell line.

First, was detected PCR product when sharing V2-primer (which, like all series MKV primers, it hybridises 5'-end of the DNA sequence of the signal peptide easy kappe conducted by the authors of the present invention, the experiment showed, what MKV2 primer will amplify in the PCR reactions aberrant mRNA transcript. This aberrant of pseudogene was present in all standard partners merge, derived from the original cell line plasmacytoma MORSE-21, and was known as the MORSE-21 (Deyev, S. M., et al., Genetica, 85:45, 1991). NO-0 was a line of cells derived from line MORSE-21, and this line was used as a merge partner to get Wat hybridoma. Because this was not unexpected that the use MKV2 primer was detected PCR product. This product was analyzed and it was shown that it is non-functional pseudo (data not shown).

The strange thing was that the other pseudogene, previously identified as secretory related cell line NS-1 (Hamlyn, P. H., et al., Nucl. Acid Res., 9:4485, 1981) and in normal PCR cloned using MKV7 primer together with the ISS primer, was not detected in any of the PCR products analyzed to date. Because cell line NS-1 and NS-0 were closely related, it was not very surprising. However, it also demonstrated the complex nature of transcription Kappa light-chain, which was present in the hybridoma cell line BAT.

Another PCR-clone, which is Domna cell lines using primers MKV5 and ISS. After transformation product, containing approximately 450 base pairs, INVF’ competent cells, likely positive transformants were identified by PCR-screening, and then was sequenced DNA.

When analyzing sequences of two separate clones MKV5 product (each of which was obtained from a separate synthesis reaction of the 1st chain and subsequent PCR reactions was determined the DNA sequence of a gene variable region of Kappa light-chain Wat antibodies (Fig.2). This sequence was also confirmed in both strands of DNA for each clone.

Example 3: Analysis of sequences of the variable regions Wat antibodies mouse

The amino acid sequence of VKand VHregions Wat was compared with the consensus sequences of the subgroup variable regions of rodents that are defined in the Kabat database (see above).

In this analysis, it was found that VHthe area is Wat most closely corresponds to the subgroup VI Kappa-chain of the mouse. When a similar comparison of the VHthe area of Wat with a Kabat database, it was found that it is closest matches the consensus sequence "mixed" subgroup heavy chain we shall radisheva lines of cells of rodents has revealed, which is closest to VHgene Wat the genome of the germ line was VMS/VGK4 (Fig.3), whereas the closest to VKgene Wat the genome of the germ line was H4 (Fig.4). As can be seen in Fig.3, the discrepancies that exist between the VHgenome Wat and its nearest gene germ lines were (which were not unexpected) are primarily based in the CDR2 and CDR3. There were only three changes to the frame, and all of them were located in FR3. As for the VKgene Wat (Fig.4), it was not unexpected that the majority of discrepancies were located in the CDR. All four differences, which were located in FR, were highly conservative changes, except cysteine at position 72 (numbering according to Kabat) in FR3. Its location directly next to the important canonical residue (position 71) suggest that cysteine may play a key role in binding to the antigen. However, only by modeling the Fv domain could confirm this assumption.

However, these analyses confirmed that VHregion and VKthe field variable regions Wat mouse, apparently, are typical for the variable regions of a mouse.

(A) Abbreviations

We used the following units outside the SI system, and other abbreviations are:

Polymerase chain reaction (PCR); deoxyribonucleic acid (DNA); copy DNA (cDNA); the variable region of the Kappa light-chain (VK); the variable region of the heavy chain (VH); minute (min); Tris-borate buffer (TBB); phosphate buffer solution (FBI); room temperature (CT); a serum albumin of a bull (as); hydrochloric acid (HCl); horseradish peroxidase (HRP); milk, low fat (MNSI); HR (hour); percentage (%); O-phenylenediamine the dihydrochloride (OPD); a multiple cloning site (QMS).

(B) Materials

Components of environments and all other materials for tissue culture were obtained from Life Technologies (UK). Components for PCR reactions, including AmpliTaqDNA polymerase were purchased from the company Perkin Elmer (USA). However, the set TA Cloningand INVF’ competent cells were obtained from the company Invitrogen (USA). DH5competent cells and agarose (UltraPure) were obtained from Life Technologies (UK).

A pre-mixed set to cycle is the business though Amersham (UK). Ready-to-use set of reagents to complete the cyclic sequencing (Big DyeTerminator Cycle Sequencing Ready Reaction Kit) used in conjunction with the analyzer genes ABI Prism 310 Genetic Analyzer was purchased in the company's PE Applied Biosystems (UK). All other described products for molecular biology were purchased either New England Biolabs (USA) or Promega (USA). Immunological tablets Nunc-Measurement Plate MaxiSorpwere purchased from Life Technologies (United Kingdom), while easily leached tablets Corning for ELISA were purchased from Corning Laboratory Sciences Company (UK). Antibody IgG goat to antigens person (specific for Fc-fragment, conjugate Kappa light-chain antibodies goat to antigens of human and horseradish peroxidase and affinity purified IgG conjugate goat to antigens person (specific for Fc-fragment) and horseradish peroxidase were obtained from Jackson ImmunoResearch Laboratories Inc. (USA). The substrate (K-Blue TMB solution Red Stop were purchased in the company Neogen Inc. (USA). All other products for ELISA were obtained from Sigma (UK). The software package Microplate Managerdata analysis was brandy filter RM were obtained from Amicon company PLC (UK), while the cleaning kit Immunopure IgG(G) was purchased from the Pierce company PLC (UK).

(C) Methods of experiment

B1. The creation of chimeric1/K Wat antibodies

Pre-selected gene variable region of Kappa light chain mouse (VK) (Fig.1) and gene variable regions of the heavy chain (VH) (Fig.2) were modified at the 5'- and 3'-ends using a specially designed PCR primers (table 1) for the purpose of gene expression variable regions Wat in mammalian cells as part of a chimeric antibodies from mouse and man. To achieve this, conducted separate PCR reactions for each gene variable regions in a special room for PCR using specific protocols designed to minimize the possibility of cross-contamination. As matrices were used plasmids VH-R2.1 and BATVK-pCR2.1, a AmpliTaqDNA polymerase used for amplification of these matrices. Primers V and V (table 4) were used for PCR modification VHgene Wat, whereas primers S and S (table 4) were used for PCR mutation VKgene Wat.

Tubes -�p://img.russianpatents.com/chr/176.gif">C for 50 s, 72C for 1 min 30 s 30 cycles. After completion of the last cycle produced the final stage of elongation at 72C for 10 min, then cooled reaction mixture on ice. Then aliquots of 5 µl of each PCR reaction was passed through a gel containing a 1.2% agarose/TBB (pH 8.8), to determine which response was obtained PCR product of the correct size.

The aliquots of 1-2 ál of these PCR products of the correct size directly cloned into the vector pCR2.1available in a set TA Cloningand transformed in competent cells INVF’, as described in the manufacturer's instructions. Colonies that contained a plasmid with an insert of the correct size were identified by PCR screening of colonies using oligonucleotide primers 1212 and 1233 (table 5) according to the method Gussow and Clackson (see above). We identified the probable positive clones was sequenced Dunaeva plasmid DNA using the apparatus for DNA sequencing Vistra and analyzer genes ABI Prism 310. A pre-mixed set to cycle sequencing Thermo Seque the Oia Big Dyeused according to the manufacturer's instructions with primers 1212 and 1233 (table 5).

Clones containing the correctly adapted VKand VHgenes Wat (Fig.5 and 6, respectively) were subclinically, in the form of fragments of HindIII-BamHi, expressing vectors N110 (Fig.7) and pG1D110 (Fig.8), respectively, for expression of a chimeric light and heavy chains in mammalian cells. Legirovannye expressing vectors (i.e. N110-VKand pG1D110-VHthen transformed into competent DH5 cells. In conclusion, the identified positive clones containing the correctly constructed expressing vectors, using analysis restricteduse enzymes.

B2. Joint transfection of vector DNA encoding chimeric1/K Wat antibody in COS cells

For transliterowany expressing vectors mammalian COS cells followed the way Kettleborough et al. Briefly, DNA (10 μg of vector expressing Kappa light-chain G1D110-VKand vector that expresses a heavy chain PG1D110-VH) was added to the aliquot volume to 0.70 ml, containing 1107cells/ml FBI, and gave pulsating voltage equal In 1900, TCA was added to 8 ml of DMEM, containing 5% FTS, and incubated for 72 h with 5% content of CO2at 37C. After incubation for 72 h, the medium was collected, centrifuged to remove residual cells and analyzed using ELISA for products Wat antibodies.

B3. Quantitative evaluation of chimeric1/to antibodies using ELISA

In each cell 96-cell immunological tablet Nunc-Measurement Plate MaxiSorpin the beginning, was applied aliquots of 100 μl containing 0.4 ng/µl IgG (with specific Fc-fragment) goats to antigens man, bred FBI, incubated overnight at 4With and removed before use. Then aliquots of the experimental samples (i.e. collected supernatants COS cells was centrifuged to remove residual cells) with a volume of 100 μl of each cell and the sample diluted in the ratio 1:2 buffer solution for dilution and conjugated enzymes (0.1 M Tris-Hcl (pH 7.0), 0.1 M NaCl, 0.02 percent (volume/volume) TWEEN-20 and 0.2% (weight/volume) SAB), distributed by immunological tablet. In addition, refined1/to antibody man (1000 ng/µl), which was used as standout incubated at 37C for 1 h, and then three times washed with wash buffer (FBI/0,1% (volume/volume) TWEEN-20) in a volume of 200 μl/cell. 100 ál conjugate Kappa light-chain antibodies goat to antigens of human and horseradish peroxidase, diluted 5000 times in the buffer for cultivation of algae samples and enzymes were added to each cell, then immunological tablet incubated at 37C for 1 h, after which it was again washed as described above. Thereafter, each cell was added aliquots of 150 μl of substrate (K-Blue, then immunological tablet incubated for 10 min in the dark at room temperature. The reaction eventually stopped the distribution of 50 ál Red Stop in each cell. Then was determined by optical density at a wavelength of 655 nm using devices microplate reader Bio-Rad 3550 in conjunction with the software package Microplate Manager.

B4. Purification of chimeric Wat antibodies

Chimeric1/K Wat antibody was isolated from supernatants COS cells in two stages. First used microcase ultrafiltration with a stirrer and a membrane filter RM according to the manufacturer's instructions to reduce the mg> for affinity separation chimeric Wat antibodies from the concentrated supernatant, also according to the manufacturer's instructions.

B5. Enzyme-linked immunosorbent assay (ELISA) cells Daudi

Enzyme-linked immunosorbent assay (ELISA) cells was performed using cells Daudi, cultivated from the source material Dr. Hardy (Felsenstein Medical Research Center, Rabin Medical Center, Beilinson Campus, Petach Tikva, 49100, Israel). Have made a small modification analysis, depending on the analyzed whether Wat antibodies mouse or chimeric antibodies mouse-man. In the analysis of affinity Wat antibodies mouse as the second antibody used conjugate IgG goat to antigens of mouse (Fab-specific) horseradish peroxidase (diluted in a ratio of 1:15000). In contrast, when the measurement of the affinity of chimeric Wat antibodies used conjugate affinity purified goat IgG antigens to the person (specific for Fcslice) with horseradish peroxidase (diluted 1000 times).

Cells Daudi (two days after passage), was first applied in a quantity of 105cells/cell 96-cell tablet Corning easy wash ELISA plate (easily leached tablet for ELISA produced by Corning), and then incubated overnight at 37C), twice washed with 200 μl/cell FBI, containing 5% milk low-fat (MNSI), and allowed to dry. Then add 50 μl/cell conjugated with horseradish peroxidase secondary antibodies, and then produced a series of six different leaching (namely the one washing the FBI, containing 5% MNSZ, three washes with the same buffer with the addition of 0.05% TWEEN-20 and two subsequent leaching FBI/MNSI buffer). Then add 200 ál of cell substrate solution ID at a concentration of 0.4 mg/ml in 0.05 M citrate buffer (pH 5.0) and 60 mg/ml of hydrogen peroxide, after which the tablet for ELISA were incubated in the dark at room temperature until color development (usually within about 30 min). In conclusion, the reaction was stopped by adding 50 μl/cell 2.5 M sulfuric acid, and then measured the optical density at a wavelength of 490 nm using devices microplate reader Bio-Rad measures 4. Construction of chimeric1/K Wat antibodies

As with all programs, followed a strict Protocol of PCR-cloning and sequencing. This was done to minimize the possibility of introducing errors in the sequence of the wild type gene variable regions of the mouse during stage PCR modification. Using primers C and S (table 1) gene VKmouse (Fig.2) was modified by PCR to obtain the sequence of 418 base pairs (data not shown). This PCR product was Legerova to plasmid pCR2.1 and transformed into INVF’ competent cells. Similarly gene VHBAT mouse (Fig.1) was mutated by PCR using primers V and V (table 1) to obtain the sequence of 436 base pairs (data not shown). This PCR product was also Legerova in plasmid pCR2.1 and transformed into INVaF' competent cells.

Then the probable positive transformants were identified by PCR-screening (data not shown) with subsequent final DNA sequencing on the ABI Prism 310 Genetic Analyzer. Fig.3 and 4 show the results of this analysis DNA sequences of chimeric genes VKand VHto demonstrirovat the presence of any PCR errors, which could be embedded in the genes. Only one PCR reaction is actually carried out for each gene variable regions, and only two clones from each of these PCR reactions, ultimately, were completed by the DNA sequences.

However, it was enough to highlight at least one clone for each of the modified gene variable regions that contained the correctly modified DNA sequence.

Mutated VHand VKgenes were then subcloned into the corresponding expressing vectors, as hindIII/BamHI fragments, with the formation of pKN110-BATVK(7,88 thousand bases) and G1D110-VK(7,55 thousands of bases), respectively. The correctness of the constructed expressing vector was then confirmed using restriction enzyme analysis (data not shown). Being transfitsirovannykh in COS cells, these vectors were provided temporary expression1/chimeric variant Wat antibodies.

In addition, as an additional component of the program of humanization of Wat antibodies, was also subcloned VHgene, in the form of a HindIII/BamH1 fragment in both expression vector of the heavy chain pG3D110 and pG4D1100. the 947.gif">1 person, or a cDNA copy of the gene constant region 3(in case pG3D110) or cDNA gene constant region3 (in the case of pG3D110) or cDNA gene constant region4 (in the case of pG3D110). The construction of these vectors (i.e., PG3D110-BATVKboth3/K and4/to variants of chimeric BAT antibody in COS cells.

Example 5. Transient expression of chimeric1/K Wat antibodies

Two vectors N110-VKand pG1D110-BATVHshare transfusional in COS cells in a series of repeated experiments with a time expression. After expression within 72 h of chimeric Wat1/to antibody mouse-man was discovered in supernatant together transfected COS cells by ELISA on1/K. Based on the results of these analyses, it was estimated that the average concentration of chimeric Wat1/to antibodies detected in the environment, was equal to 509272 ng/ml

Interestingly,3/K and4/go to the options chimeric Wat antibodies, apparently, was produced authentically borovany 3D110-Vnand pKN110-BATVTowhen the initial analysis of the supernatant (using the ELISA method described in Section B3, and antibodies lg3/Kappa person as a standard) were measured levels of expression of a chimeric3/to Wat antibodies 6.7 µg/ml. moreover, if in COS cells expressibility G4D110-VHand N110-VKin the same ELISA assays (using antibodies lg4/Kappa person as a standard) were measured levels of expression of a chimeric4/to Wat antibodies, equal to 8.2 µg/ml.

Example 6. Purification of chimeric1/K Wat antibodies

Accumulating approximately 8 ml from each co-transfection, performed a series of transfection until then, until it was collected 200 ml supernatant of COS cells. The volume of the supernatant was then reduced to 15 ml by passing the supernatant through microcase ultrafiltration with a stirrer and a membrane filter RM having a molecular weight cutoff of 30 kDa.

Set to highlight Immunopure IgG(G) actually included a column in 2 ml of immobilized Protein g Antibody was suirable from the column 6 ml of eluting buffer, the eluate was collected fractions 1 ml Concentration of chimeric1/K Wat antibodies. It was stored at -20To transfer to the Curetech for further analysis.

Example 7. Analysis of the binding of cells Daudi with chimeric1/K Wat antibody

Using ELISA analysis of cells Daudi was clearly shown that the purified chimeric1/K Wat antibody binds to cells Daudi. Fig.9 shows a typical example of the experiment. However, less convincing was the linking of similar concentrations Wat antibodies mouse, in the same ELISA assays, which, apparently, was lower than the binding of the chimeric antibody. However, since conjugated secondary antibody used for detection of antibody binding to cells Daudi differed from all of recombinant antibodies, it was not feasible to make a direct comparison of the binding of the two options.

IV. AMINO ACID SEQUENCE OF 3 PEPTIDES.

OBTAINED FROM p>Three peptide contained in the antigenic epitope In lymphoblastoid cells of Gaudi with whom contact MAT according to the present invention, were sequenced. Their sequence is shown in Fig.10, 11 and 12.

The search, conducted in the Bank's database of non-redundant genes and EST Department found no matches in the transmission of these three peptides in the form of a query using the TBLASTN algorithm (Version 2) with the level of EXPECTATION is equal to 10, and matrix BLOSUM 62.

However, because these peptides were small peptides, they were presented again at a higher level of EXPECTATION to make the search less strict. In addition, the filter has not been masked from low complexity that would eliminate a potential mixed combination (for example, the matched regions rich in Proline, or cast new And all) of the report, leaving only the area of statistics which reflect the specificity of their pairwise alignment. For the three peptides according to the present invention was not detected coincidences when working with databases gene Bank and the Department EST even at very low severity.

Therefore, in accordance with the above results, three of the above peptide presented new peptides.

V. DIAGNOSIS PLACECAST and, received from the surveyed people were double-labeled with antibodies to CD3 and one of the MAT according to the present invention. Determined the percentage D3+cells that bound MAT according to the present invention. It was shown that the number of CD3+MAT+cells in people who have had malignant disease, differs from the percentage of these cells in blood samples obtained from healthy individuals. The fact that there is a significant difference between the percentage of CD3+cells in people with malignant disease, and that this percentage is higher or lower percentage of CD3+MAT+cells in samples obtained from healthy individuals suggests high probability to determine whether a person's malignant disease, as well as the specific type of malignant disease, which may be in the examined person.

Usually lymphocytes in human peripheral blood was obtained from 20 ml of blood taken either in a healthy person or a patient with cancer by centrifugation in density gradient Ficoll Hypaque. Cells were washed and suspended in the FBI, containing 0.5% of SAB and 0.05% acid. Samples containing 0,5106cells, modulating the structure for 45 min at 0S, followed by incubation with their MAT to antigens of mouse conjugated with FITZ, for 30 min on ice. After two washes and centrifugation at 1200 rpm, the cells were incubated with antibodies to human CD3, conjugated with PE, for 30 min on ice. After this incubation, the cells twice washed, and the sample was analyzed using FACS scan (Bectan Dickinson). The results are shown in Fig.13-17.

As can be seen in Fig.13, and in Fig.17, the percentage of CD3+Wat+cells (compared with the total number of CD3+cells) in blood samples obtained from healthy people, lies in the range of about 25%. As shown in Fig.14, the percentage of CD3+Wat+cells in blood samples obtained from patients with cancer of the large intestine, significantly lower compared with healthy people, and lies in the range of about 7%. Similarly, the percentage of CD3+Wat+cells in blood samples obtained from patients with breast cancer, was in the range of about 10% (Fig.15). These results clearly show that carcinoma of the colon and breast cancer can be identified by the fact that the percentage of CD3+Wat+cells in comparison with healthy people.

The percentage of CD3+Wat+cells in the samples Kirovabad blood from healthy people, as shown in Fig.16, and lies in the range of about 50%. These results clearly show that carcinoma of the prostate can be identified by the fact that the percentage of CD3+Wat+cells higher in comparison with healthy people. As shown in Fig.18, the amount of antigen to which are attached MAT according to the present invention, found on T-cells isolated from patients with carcinoma of the prostate is very large, whereas in T-cells obtained from patients with carcinoma of the breast, the antigen cannot be detected.

The above results show that the MAT according to the present invention can be used to determine that an individual suffers a defined type of malignant disease. Therefore, if a sample of blood is obtained from the subject person, and the degree of binding MAT according to the present invention is significantly high (in the range of about 50%), there is a very high probability that a subject suffering from prostate cancer. On the contrary, if the percentage of CD3+cells in the sample was significantly lower compared with healthy people (in the range between about 7 and 10%), there is a very high probability togradually man is a man, there is a very high probability that he is suffering from a carcinoma of the colon.

The above examples are not limiting, and additional correlation between the percent of CD3+cells that bind MAT according to the present invention, and other malignant diseases are also within the scope of the invention.

Claims

1. Monoclonal antibody for the diagnosis and treatment of malignant diseases with variable region selected from the group consisting of (a) a monoclonal antibody having a variable region heavy chain comprising the amino acid sequence depicted in Fig.1; (b) a monoclonal antibody having a variable region of the Kappa light-chain including amino acid sequence depicted in Fig.2; (C) a monoclonal antibody having a variable region heavy chain comprising the amino acid sequence depicted in Fig.1, and the variable region of the Kappa light-chain including amino is here the heavy chain, holding at least 70% identity to amino acid sequence depicted in Fig.1, and having the same binding specificity as a monoclonal antibody as defined in subparagraph (a); (d) a monoclonal antibody having a variable region light chain having at least 70% identity to amino acid sequence depicted in Fig.2, and having the same binding specificity as a monoclonal antibody as defined in subparagraph (b).

2. Monoclonal antibody under item 1, characterized in that it has a variable region heavy chain comprising the amino acid sequence depicted in Fig.1.

3. Monoclonal antibody under item 1, characterized in that it has a variable region of the Kappa light-chain including amino acid sequence depicted in Fig.2.

4. Monoclonal antibody under item 1, characterized in that it has a variable region heavy chain comprising the amino acid sequence depicted in Fig.1, and the variable region of the Kappa light-chain including amino acid sequence depicted in Fig.2.

5. Monoclonal antibody under item 1, characterized in that the sledovatelnot, is depicted in Fig.1.

6. Monoclonal antibody under item 1, characterized in that it has a variable region light chain having at least 70% identity to amino acid sequence depicted in Fig.2.

7. Monoclonal antibody according to any one of paragraphs.1-6, characterized in that it is a chimeric antibody mouse-man.

8. The nucleotide sequence of the nucleic acid encoding the amino acid sequence characterizing monoclonal antibody on PP.1-7.

9. Expressing plasmid vector containing the nucleotide sequence of the nucleic acid under item 8.

10. Expressing plasmid vector under item 9, characterized in that it is a plasmid pKN110.

11. Expressing plasmid vector under item 9, characterized in that it is a plasmid pG1D110.

12. The way to identify the man, thought to have malignant disease, characterized in that it comprises (a) obtaining a sample of body fluid from the subject person; and (b) the specified contact of the sample with at least one of the monoclonal antibodies on the PP.1-7; (C) determining the degree of binding of these monoclonal antibodies to T-cells, stereoscopically antibodies to T-cells, contained in a sample obtained from a healthy person, and the presence of the examined person's malignant disease determine if the existence of significant differences between the degrees of binding compared to step (g).

13. The method according to p. 12, wherein the fluid body is blood.

14. The method according to p. 12, characterized in that the specific type of malignant disease, which presumably has surveyed people, specifies whether the degree of binding of monoclonal antibodies to the T cell sample person higher or lower compared to the degree of binding of monoclonal antibodies to T-cells of healthy people.

15. The method according to p. 13 or 14, characterized in that before step (b) from the sample emit mononuclear cells of peripheral blood (MNCPC), then MNCPC lead contact at the step (C) with monoclonal antibodies.

16. The method according to p. 14 or 15, characterized in that the degree of binding of monoclonal antibodies to T-cells obtained from the subject person, higher than the degree of binding of the same monoclonal antibodies to T-cells of healthy people.

17. The method according to p. 16, characterized in that the specific zlokacestvennoe binding of monoclonal antibodies to T-cells, obtained from the sample person, lower than the degree of binding of the same monoclonal antibodies to T-cells of healthy people.

19. The method according to p. 18, characterized in that the specific malignant disease is a carcinoma of the breast.

20. The method according to p. 18, characterized in that the specific malignant disease is a carcinoma of the colon.

21. Pharmaceutical composition for the treatment of malignant diseases, containing one or more monoclonal antibodies according to any one of paragraphs.1-7, and a pharmaceutically acceptable carrier.

22. The pharmaceutical composition according to p. 21, characterized in that it contains a monoclonal antibody as an active ingredient.

23. The pharmaceutical composition according to p. 22, wherein the malignant disease is cancer.

24. Pharmaceutical composition for p. 23, wherein the cancer is a solid tumor.

25. The pharmaceutical composition according to p. 24, wherein the solid tumor is a carcinoma of the prostate.

26. The pharmaceutical composition according to p. 24, wherein the solid tumor is a carcinoma of the breast.

27. The pharmaceutical composition according to p. 24, azizia on p. 22, characterized in that it is intended for the treatment of malignant diseases in humans.

29. Kit for detection of malignant disease, comprising one or more monoclonal antibodies on PP.1-7, a conjugate which specifically binds with the above monoclonal antibody, and at least one label, able to give a detectable signal.

30. The method of treatment of malignant diseases, including the introduction of the required treatment to the person a therapeutically effective amount of one or more monoclonal antibodies according to any one of paragraphs.1-7.

 

Same patents:
The invention relates to medicine, namely to a method of assessing the compensatory capacity of the organism in patients with aseptic necrosis of the femoral head and chistovodnoe restructuring of the articular ends of II-III degree

The invention relates to the medical industry and refers to polypeptides that are specific against CD19 and CD3, and their application

The invention relates to medicine, namely to Pediatrics, and can be used to predict local infectious-inflammatory disease in full-term newborns in the first month of life
The invention relates to medicine, namely to Allergology, and can be used to identify sensitization to allergens in adults and children

The invention relates to medicine, in particular to immunology

The invention relates to the field of veterinary Virology, in particular to a method for the diagnosis of sheep pox and goat

The invention relates to the field of biotechnology and medicine, specifically to the diagnosis of opisthorchiasis

The invention relates to veterinary Virology, in particular to a method for the diagnosis of sheep pox and goat pox

The invention relates to medicine, namely to identify the expression of cytokines in cells

The invention relates to immunology and biotechnology and can be used to test rapamycin

The invention relates to biotechnology, can be used in medical practice for a polypeptide, which is excreted through the kidneys and does not contain in its original form Fc-region of IgG

The invention relates to the production potential of drugs aimed at the destruction of protein antigens, in particular glycoprotein gp 120 of the main surface protein of human immunodeficiency virus

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

The invention relates to the field of immunobiotechnology and medicine and relates to monoclonal antibodies (MCAT) to SD person

v-integrins (options), receive (options) and use hybridoma, polypeptide, dna" target="_blank">

The invention relates to immunobiotechnology and applies to new monoclonal antibodies, methods for their production using hybridoma technology and our-integrin or vitronectin as antigens, polypeptides, representing light and heavy chains of the antibody and DNA encoding these polypeptides

The invention relates to veterinary medicine, namely to genetic engineering, in particular to a method for the original nucleotide sequence of the EgF, which, when cloned into a vector expressing the system pQE-SG 13009, you can get unlimited amount of antigen of hydatid cyst of E. granulosus used as a main component enzyme diagnosticum for in vivo detection of sick people and animals by larval hydatidosis
Up!