The composition and method of treatment of the neoplastic cells
(57) Abstract:The invention relates to medicine and concerns immunotoxin with neoplastic effect, the composition having a neoplastic activity, containing immunotoxin, and method of treatment of neoplastic cells. The objective of the invention is to develop a method for selective direction of the chemotherapeutic agent in the cell. The essence of the invention lies in the fact that the immunotoxin with neoplastic effect, represents a conjugate gelonin and monoclonal antibodies exhibiting a binding specificity against antigenic domain of C - erb b-2, and represents a Tab 250. The composition having a neoplastic activity, is an immunotoxin and a pharmaceutically acceptable carrier. Method of treatment of the neoplastic cells includes the introduction of an effective dose of immunotoxin in this cell. The technical result is to increase the selectivity of delivering a chemotherapeutic agent to the cell. 3 S. and 7 C.p. f-crystals, 7 Il. This invention relates in General to the field of treatment of neoplastic (malignant) diseases. More specifically, this invention relates to new is th problem
Neoplastic disease is one of the main causes of mortality and prevalence of diseases in the Western world. Neoplastic conditions, such as different types of cancer have at least one common characteristic, namely, violations of the regulatory process of cell growth.The process by which normal cells are transformed into malignant cells, has been the subject of intensive research for decades. More recently research has focused on the role of oncogenes in the development of cancer. Oncogenes are genes that have the ability to transform eukaryotic cells so that they grow on the growth of tumor cells.Oncogene formed by mutation, rearrangeable or amplification of the normal gene or proto-oncogene. One of these oncogenes is the proto-oncogene c-erbB-2 (HER -2/neu). In the future, it is called c-erb B-2. This gene encodes a protein similar to receptor epidermal growth factor. Amplification of this proto-oncogene can lead to a cascade of cellular events leading to uncontrolled cell growth.Antibodies are proteins produced by the immune misspecification antigen, to which they are directed. Obtaining specific monoclonal antibodies has provided researchers a possible tool for selective direction of therapeutic agents to cells expressing an excessive amount of certain antigens.Was supposed overexpression of proto-oncogene c-erb B-2 in neoplastic (tumor) cell transformation. Some types of human cancer, including breast cancer and some ovarian carcinoma, find amplificatory gene c-erb B-2. In addition, amplification and subsequent overexpression of the gene c-erb B-2 correlated with poor prognosis of the disease. Therefore, in this research there is a great need and desire to develop a method for selective direction of the chemotherapeutic agent to the cell that detects excessive expression of Onco-gene c-erb B-2 to modulate the growth of cells strongly expressing this protein. This invention provides the means to achieve this goal.Brief description of the invention
This invention provides a novel composition comprising the conjugate of the guide to the cells of the molecule, for example, antigennegative district, the property is engaged in the growth of the reagent. This composition can act as an immunotoxin for specific directions modulator growth of cells to tumor cells, the enhanced expressionwhich protein c-erb b-2.Thus, in one embodiment of the present invention is provided a new composition comprising a conjugate of the guide part of the molecule with binding specificity against protein c-erb b-2, for example, TAb 250 monoklonalnej antibodies and cytotoxic portion of the molecule. Cytotoxic part can be a toxin that causes the destruction of cells drug, cytotoxic agent or modulator of the biological response. In one of the individual variants of cytotoxic part is gelonin.Another variant of the present invention provides a method of treating a neoplastic condition, such as disease, characterized by amplification or over-expression of the oncogene c-erb B-2, introducing effective for cell disruption dose immunotoxin of the present invention to an individual in need of such treatment.Another option provides a method for killing tumor cells in vitro with subsequent reverse the introduction of their master. For example, in the treatment of neoplastic copozitia of the present invention.In another embodiment of the present invention is provided a method of preventing recurrence of neoplastic disease. Relapse prevent the introduction of effective for the destruction of cells number of targeted toxin, for example, such immunotoxin, as TAb 250 antibody-gelonin.In another embodiment of the present invention are provided compositions containing fused design guide parts of the molecules with binding affinity against protein c-erb B-2 and the cytotoxic portion of the molecule. Preferably, the guide part is an antibody that recognizes an extracellular epitope of c-erb b-2, for example, TAb 250, and the cytotoxic portion of the relatively inert when introducing it separately from the guide part, for example, gelonin. In other embodiments of the present invention provided with the means of extending the life time with the tumor of a mammal by introducing targeted toxins of the present invention that mammals, as well as a way to slow the rate of growth of tumors through the introduction of targeted toxins of the present invention. In a typical case, the toxins can be targeted to tumor cells using immunological district linking, for example, binding of the antibody segment. To the STI, conjugated with a monoclonal antibody. Most preferably, the antibody is a TAb 250 and cytotoxic part is gelonin.Fig. 1 shows measured using ELISA actions antibody ZME, antibody TAb 250 or immunoconjugates TAb 250 and gelonin cells SKOV-3.Fig. 2 shows the cytotoxicity design TAb 250-gelonin on cells SKOV-3.Fig. 3 shows the specific competition (as opposed to non-specific) antibodies to the conjugate to cells SKOV-3.Fig. 4 shows the dependence of the response on the dose and the effects of the conjugate TAb 250-gelonin on cells SKOV-3.Fig. 5 shows the cytotoxicity TAb 250 and conjugate TAb 250-gelonin on cells SKOV-3.Fig. 6 demonstrates the ability of the antibody TAb 250 to internalization in different cell lines.Fig. 7 shows the cytotoxicity immunoconjugate TAb 250-gelonin in the MTT-test.A detailed description of the invention
As described here, a guide to the cells of the molecule is capable of selectively contacting the protein c-erb B-2, expressed on the cell, usually on its surface. The guide part includes both the ligand, specifically St. the matter of the fragments. It can be as classic molecule antibodies, chimeric variants, single chain, and modified fragments of antibodies that retain epilepsyusa specificity and affinity.The term "immunoglobulin" or "peptide (peptides), antibodies called a immunoglobulin or whole antibody or any functional binding fragment of immunoglobulin molecules. Examples of such peptides are complete antibody molecules, antibody fragment, such as Fab, F(ab')2, CDR, VLVHand any other part of the antibody, in particular, some of the antibodies that detect antigennegative specificity and affinity. For example, the IgG antibody molecule consists of two light chains, each of which is connected by disulfide bonds and two heavy chains. The heavy chain, in turn, are connected to one another by disulfide bonds in the zone, known as the hinge region of the antibody. Single IgG molecule usually has a mol.the weight of approximately 150-160 KD and contains two antigenspecific site. Fragments of these molecules, e.g. heavy or light chain separately, sometimes can bind antigen. Antibodies, antibody fragments and individual circuits may be functionally equivalent immun what Yu (V) region and C-terminal (-COOH) constant (C) region. The variable region of the heavy chain are referred to as VH(including, for example, V), and the variable region of the light chain is called the VL(including Vor V). Variable region is part of a molecule that binds to a cognate antibody-antigen, whereas the Fc region (the second or third domain (C-region) determines the effector function of antibodies (such as complement fixation, opsonization). "Light chain of the immunoglobulin or antibody is a full length (generally about 25 KD, about 214 amino acids) are encoded gene variable region at the N-end (approximately 110 amino acids) and genome (Kappa or lambda) constant region at the COOH-end. "The heavy chain of the immunoglobulin or antibody is a full length (generally approximately 50 KD, about 446 amino acids) similarly encoded gene variable region (encoding about 116 amino acids) and one gene constant region, e.g., gamma (encoding 330 amino acids). Usually "VL" contains a portion of the light chain encoded by gene segments VLand/or JL(J - or the connecting area), and VH" contains a portion of the heavy chain encoded by gene segments VHand (or) DH(D - and Ilayda.The variable region of the light or heavy chain immunoglobulin consists of a "framework" region interrupted by three hypervariable sites, also called stations, determining complementarity, or CDR. The degree of frame region and CDRs have been defined (see, "Seguences of Profeins of Immunological Interest," E. Kabat, et al., US Department of Health and Human Services (1987), incorporated herein by reference). Sequence frame regions of different light and heavy chains relatively conservative within the species. Frame the field of antibodies, representing a combined frame region light and heavy chains, serve to position and build CDR in three-dimensional space. CDR primarily responsible for binding to the epitope of the antigen. They are commonly called, CDR1, CDR2 and CRD3 sequential numbering from the N-Terminus.Two types of light chains, and referred to as isotypes. Izotopicheskie determinants are usually in a constant region of the light chain, called in General CLand in particular, CKor C. The constant region of the molecule heavy chain, also known as CHdetermines the isotype of the antibody. Antibodies are classified as IgM, IgD, IgG, IgA, and IgE, depending on the isotype of the heavy chain. These isotypes codiroli. In addition, there are several subtypes .
The isotype of the heavy chain determines the different effector functions of antibodies, such as opsonization or fixation of complement. In addition, the isotype of the heavy chain determines the secreted form of the antibody. Secreted isotypes IgG, IgD and IgE are usually found in single-unit or Monomeric form. Secreted IgM isotype detected in pentamers form, while secreted IgA can be detected both in Monomeric and dimeric form.Fragment F(ab')2does not contain C-terminal part of the constant region of the heavy chain and usually has a mol.the weight of approximately 110 KD. He saves two antigenspecific site and disulfide bonds between the chains in the hinge region, but not the effector functions of intact IgG molecules. Fragment F(ab')2can be obtained from the IgG molecule by proteolytic digestion with pepsin at pH 3.0 to 3.5 using standard methods, for example, described in Harlow and Zane, infra.Fragment of "Fab" contains the light chain and the N-terminal part of the heavy chain, which are connected by disulfide bonds. Usually he is saying.the weight of approximately 50 KD and contains one antigennegative website. The Fab fragments can be obtained from frag and reducing agents (See, Harlow and Lane infra). In some cases, the concentration of the reducing agent required to maintain the activity of papain in the presence of atmospheric oxygen, sufficient to fully restore the disulfide bonds between the chains of the antibody. This can lead to loss of recognition of the antigen. To relieve this problem, papain, you can activate and then enter into a buffer with a concentration of the reducing agent that is compatible with maintaining antigennegative activity. Digestion of antibodies is usually carried out in an atmosphere of inert gas to prevent inactivation of papain.The following Protocol is an example of this method:
A) Activation of papain: papain obtained in the form of 10 mg/ml (NH4)2SO4suspension, dissolved in 10 mm EDTA, 20 mm cysteine, pH 8.0, to a final concentration of 2 mg/ml Solution Tegaserod and incubated 2 hours at room temperature under nitrogen.B) Activated papain transferred into 20 mm Na3PO4, pH 7.0, containing 150 mm NaCl, 10 mm EDTA, 30 μm DTT.C) Digestion of antibodies: 1 mg of activated papain is added to each 100 mg of antibody solution cialiswhat against a large excess of 20 mm NaPO4, pH 7.0, containing 150 mm NaCl, 10 mm is ostanavlevaysya agent in the process of digestion.D) After 2-4 hours at room temperature, the digestion is stopped by the addition of iodoacetamide.E) the Fab Fragments are separated from undigested or partially digested antibody standard chromatographic methods.The term "Fab" or any other antibody fragments are classified for use in this invention, similar to the classifications applied to the key terms "antibody" or "immunoglobulin". So, Fab protein "mammals", "chimeric Fab", etc. are used similarly to the corresponding definitions in common use, as set out in the following paragraphs.The terms "chimeric antibody or chimeric peptides" referred to here such antibodies or peptides antibodies, in which one part of the peptide has an amino acid sequence derived from the corresponding sequence or homologous to corresponding sequences in the antibody or the peptide derived from the first source of genes, whereas the remaining segment of this chain (chains) homologous to corresponding sequences other source genes. For example, a chimeric peptide of the heavy chain of the antibody rubs contain a murine variable region and chelovecheskoye representatives of the same species.Chimeric antibodies or peptides are usually obtained using recombinant molecular and / or cell methods. In many cases, chimeric antibodies have variable regions as light and heavy chains that mimic the variable regions of antibodies derived from one species of mammal, whereas constant and (or) the frame homologous to the sequences in antibodies derived from a second, different species of mammals.However, the definition of chimeric antibodies is not limited to this example. Chimera is any antibody in which one or both of the heavy or light chains are composed of combinations of sequences that mimic sequences in antibodies from a variety of sources, regardless of whether these sources to different classes, different response to the antigen, or to different species, and whether a point is the merge in place of communication between the variable and constant regions or outside of this site. For example, the chimeric antibody can be an antibody, in which a frame region, and complementarity determining region (CDR) derived from different sources. For example, nonhuman CDR education "humanized" (humanised" antibodies. See, for example, PCT Application Publication N WO 87/02671; US Patent N 4816567; EP Patent Application 0173494; Jones, et al., Nature, 321:522-525 (1986) and Verhoeyen, et al., Science, 239: 1534-1536 (1988), on all these sources make reference here.Used here, the term "human-like framework region" refers to a frame region of each chain antibodies, which typically contains at least 70 amino acid residues, typically 75-85 or more residues. Amino acid residues of human-like framework region at least 80%, preferably 80-85%, and most preferably more than 85% homologous to the amino acid residues of the human immunoglobulin. This common trait with other endogenous antibodies applicable to create the guide part of the molecule, which gives only a small immune response, for example, a mechanism that minimizes the response to autoantigen markers.Used here, the term "humanized" ("humanised" or "human-like immunoglobulin" called immunoglobulin containing a human-like framework region and constant region, mostly homologous to the constant region of human immunoglobulin, for example, having at least 80% or more, preferred the parts of the immunoglobulin, like a human immunoglobulin, except possibly the CDRs, mostly homologous to the corresponding parts of one or more native sequence of human immunoglobulin.Used here, the term "hybrid antibody" is called antibody, in which each circuit separately homologous chain antibody of a mammal, but their combination represents a new ensemble, so that the antibody can recognize two different antigens. In a hybrid antibodies one pair of heavy and light chains are homologous pair found in the antibody, generated against a single sign of recognition of the antigen, while the other pair of heavy and light chains are homologous pair found in the antibody, generated against a different epitope. This leads to a property of multi-valency, i.e., ability to bind at least two different epitopes simultaneously. Such hybrids can, of course, also be obtained by using chimeric chains.The term "monoclonal antibody" refers to here the song antibody that recognizes a separate antigenic determinant. The term is used without limitations as to the source of the antibody or the manner in which it is received.P the ut can be obtained from hybridoma, producing specific for c-erb b-2 antibodies. The nucleic acid sequences of the present invention capable of ultimately Express the desired chimeric antibodies, can be formed from many different nucleotide sequences (genomic or cDNA, RNA, synthetic oligonucleotides, and so on) and components (e.g., V-, J-, D - and C-areas), as well as a number of different ways. The connection of appropriate genomic sequences is currently one of the usual ways, but can also be used cDNA (see, European Patent Publication N 0239400 and Reichmann, Z., et al., Nature, 332:323-327 (1988), referenced).The DNA sequence of a constant region of a human preferably extracted from immortalized b-cells, see, for example, Heiter, et al., Cell 22: 197-207 (1980), but can be isolated or synthesized from many other sources. The nucleotide sequence of the gene CIhuman immunoglobulin described in Ellison et al., Nucl. Acids Res., 10:4071 (1982); Beidler, et al., J Immunol., 141:4053 (1988); Ziu et al., Proc. Natl. Acad. Sci. USA 84:3439 (1987) (all incorporated herein by reference).CDR to obtain immunoglobulins of the present invention is preferably derived from monoclonal antibodies capable of binding the ISA, rats, rabbits, hamsters or other vertebrate capable of producing antibodies are well known ways. Suitable cells for DNA and host cell for the expression and secretion of immunoglobulin can be obtained from various sources such as the American Type Culture Collection (ATSS) "Catalogue of Cell Lines and Hybridomas", Fifth Edition, (1985) Rpckville, Maryland, USA, included in the references.In addition to the chimeric peptide antibodies described here, it is easy to design and prepare immunoglobulins, mainly homologous, using various methods of recombinant DNA, known to specialists in this field. Modification of these genes can be easily obtained by many well-known methods such as site-directed mutagenesis (see Jillman and Smifh, Jene, 8: 81-97 (1979) and Roberts, S., et al., Nature, 328:731-734 (1987), on which there are links). These modifications can be added amino acid deletions, substitutions, preferably conservative, and other changes in the sequence of the polypeptide, preserving, however, the necessary properties or biological activity. Alternatively, it may be obtained polypeptide fragments containing only part of the primary structure of antibodies and with linking and(or) the effector activities of the ions, each of which has one or more particular activities, these genes can be fused to functional regions from other genes to obtain a fused proteins (for example, immunotoxins) with new properties or new combinations of properties.The cloned variable and constant region can be isolated from the plasmid and ligitamate together in expressing vector mammals, for example, pSV2-neo or pRSV-gpt for the formation of functional transcriptional units. The preferred host is a mouse myeloma cells, such as SP 2/0 or RSH because they do not secrete the protein of endogenous immunoglobulin and contain all components necessary for the expression of immunoglobulin. Myeloma cells can be transliterowany by means of suitable methods, as described above.In this area there are other types of promoters and enhancers (enhancers) that are specific to other host cells. See, Kameyoma, K., et al., Supra.For example, the DNA sequence encoding the amino acid sequence of the chimeric antibodies can be connected with the promoters and enhancers of yeast and transliterowany in yeast are known in this area ways. Cm. Krigler, supra.The W is the ID of the mammal, and frame areas other source, for example, other species of mammal. Then these CDRs can be legirovanyh with frame regions and constant regions with the formation of chimeric antibodies. See, PCT N GB 88/00731 (1989) and the U. S. S. N 07/808 462, filed Decemder 12, 1991, referenced. These CDRs can be cloned in expressing the vector containing, for example, a human skeleton and a constant region.Another example is a recombinant DNA sequence encoding CDR1, CDR2 and CDR3 of the heavy and or light chain of one type, such as a mouse, and a frame region of the heavy chain of the person to produce antibodies specific for c-erb b-2. Other opportunities include the use of CDRs specific for c-erb b-2; application of the variable region encompassing CDR1 and CDR2 from one species of mammals, and then ligation of this sequence with the other, the coding frame region of a mammal of the second species, with CDR3 of the first; or transfection of the cell line host recombinant DNA sequence that encodes a specific for c-erb b-2 CDR heavy chain derived from a first species of mammal, mixed inside the frame area of the second kind with the DNA sequence of light chain, steriade.Expressing the recombinant vectors containing DNA sequences, antibodies, can be transliterowany by electroporation into cells of the host. For selection of clones producing specific for c-erb B-2 chimeric antibodies, use standard selection methods.Antibodies can be expressed with a comfortable degree of folding of the molecule, including in the form of single-chain antibodies, bacteria, such as E. coli. Cm. , Pluckthun, Biotechnology, 9:545 (1991), Huse, et al., Science 246: 1275 (1989) and Ward et al., 341:544 (1989), which reference is made.Sequence DEC peptide antibodies can be amplified by cloning using polymerase chain reaction (PCR), the method for amplification of target DNA using a thermostable DNA polymerase, such as Tag polymerase and oligonucleotide primers, as described in PCR Protocols, ed. Innis, et al., Academic Press, Inc. (1990). Cm. also Orlandi, supra, and Larrick, et al., Biotechnology, 7:934 (1989), included in the references.Protein c-erb B-2 (referred to here simply c-erb b-2) is a membrane glycoprotein mol. weight 185 KD with tyrosinekinase activity and related to the receptor for epidermal growth factor (EGFR), but different from it. Like FGFR protein, protein c-erb VI intracellular kinase domain. In addition, the amino acid sequence of c-erb b-2 protein, as well as the corresponding nucleotide sequence described Coussens, et al., Science, 230:1132 (1985), included in the references.Protein c-erb B-2 is encoded by the c-erb b-2 oncogene, described in 1985 by three different groups of researchers: Semba, et al., Proc. Natl. Acad. Sci. USA, 82: 6497 (gene called c-erb B-2); Coussens, et al., supra (gene called HER-2); and King et al., Science, 229:1132 (a gene called v-erb). Thus, the sequence of the gene c-erb b-2 and the corresponding protein sequence are well known and described. Protein c-erb b-2 has a specific intracellular part, a transmembrane region and an extracellular region. In a typical case, the guides are part of the molecules of the present invention are associated with the extracellular region, which is located on the outer surface of the neoplastic cells. Directing the portion of the molecule, as a rule, recognizes find here a sign or signs, including legendbase.ui district or sites of recognition of antigen, for example, epitopes. The epitopes can be pure polypeptide epitopes, determinants with linear peptide sequence or conformational determinants, but also the epitopes with carbohydrate components. Epitopes may also be obyedinenie or abnormal, can represent also an important epitope determinants.The detection of protein c-erb b-2 can be performed using well-known immunotest using antibodies specific for c-erb B-2 protein, such as described here. Such antibodies are commercially available, for example, from Chemical International, Inc. Temecula, CA, or can be obtained by standard immunological methods. See, for example, Harlow and Lane, Antibodies; A Laboratory Manual, Cold spring Harbor Publication, N. Y. (1988), included in the references.The determination of c-erb b-2 protein will be here also apply to the proteins originating from other systems owners, for example, to proteins that are immunologically similar to a human c-erb b-2. For example, close to the rat gene (designated neu) was reported Schecter, et al., Science, 229:976 (1985).Convenient epitopes, which can be easily obtained antibodies are extracellular epitopes found on the target cells. These epitopes are mainly protein epitopes, for example, linear or conformational epitopes of the protein detected in neoplastic (tumor) cells. Other applicable epitopes are carbohydrate or other modification, usually posttranslational found on the c-erb b-2 protein. Antibodies and other concerns about the Tana against fragments of this protein.Were obtained from mouse monoclonal antibodies against the extracellular part of the c-erb b-2. One example of such antibodies is TAb 250 deposited in ADS Rockville, Maryland under N NW.Alternatively, the guide portion of the molecule can be obtained in any other way, providing the affinity and specificity in relation to expressing c-erb b-2 cells. For example, as the guide part of the molecule could be applied ligand, recognizable and associated c-erb b-2 protein. Cm. for example, Ciccodicola, et al., (1989) Embo J. 3:1987-1991; Ciardiello et al., (1991) Cancer Research 51:1051-1054; Ciardiello et al., (1991) P. N. A. S. USA 88:7792-7796 that describe CRIPTO, a molecule which, apparently, serves as a ligand for the EGF receptor, and binds specifically to the c-erb b-2 protein.With regard to the described sequence, it should be understood that this includes such varieties as mutations in the form of substitutions, additions and / or deletions, or any other sequence having binding activity similar to the activity sequence, from which it is derived or to which it is similar.For this invention, the antibody or other peptide is specific for c-erb B-2 protein, if the antibody or peelo or ligand-receptor for example, competitive tests, tests of saturation or standard immunoassays, such as ELISA or RIA. This definition of specificity applies to single heavy and or light chain, CDR, fused proteins, or fragments of heavy and or light chain, which are also specific protein c-erb b-2, if they bind to the protein c-erb b-2 separately, or they are able to specifically bind the protein c-erb b-2 after inclusion in the conformation of the immunoglobulin with the complementary variable regions and constant regions.In the competitive tests the ability of the antibody or peptide fragment to bind the antigen can be determined by detecting the ability of the peptide to compete with the binding of compounds antigennegative ability known. There are many types of competitive tests, which will be discussed here. Alternatively, you can also apply the tests, which measure the binding of the test compound in the absence of inhibitor. For example, the ability of molecules or other compounds to bind protein c-erb b-2 can be detected by labeling the target molecule or the target molecule may be unlabeled and detected indirectly using the "sandwich" the TEC is, .S. Patent NN 3376110 and 4016043, Harlow and Lane, Antibodies: a Laboratory Manual, Cold Spring Harbor Publications, N. Y. (1988), and Coligan, et al., /eds/, Current Protocol in Immunolory, Wiley and Sons, N. Y., are here referred to). Also suitable tests measuring binding of the test compound with one component without competition. For example, you can use antibodies to identify the presence of the protein c-erb b-2. You can use the standard methods for testing monoclonal antibodies, such as ELISA (see, Harlow and Lane, supra). For a review of different producing signal systems, which can be used, see U. S. Patent N 4391904 included in the references.Further, the specificity of the binding parts of the molecules in relation to c-erb b-2 can be defined by their affinity. Such specificity exists if the dissociation constant (KD=1/K, where K is the affinity constant) of the molecule is less than 1 μm, preferably less than 100 nm and most preferably less than 1 nm. The antibody molecules typically have KDin the lower ranges. K = [R-L] /[R] [L] , where [R], [L] and [R-L] is the concentration at equilibrium of the receptor or c-erb b-2 (R), ligand, antibody, or peptide (L) and receptor-ligand complex (R-L), respectively. In a typical case, the binding interactions between ligand or peptide and receptor or Asil and hydrogen bonds.Other tests may include detection of the presence or absence of various physiological or chemical changes that may be a result of the interaction, such as negative modulation, internalization or increase in phosphorylation, as described in U. S. Patent Application N 07/644361 filed 1/18/91. Cm. also Receptor-Effector Coupling - a Practical approach, ed. Hulme, IRL Press, Oxford (1990).Preferred peptide specific for the protein c-erb b-2, induces an increase in the phosphorylation of this protein when placed in contact with tumor cells expressing the protein c-erb b-2. Molecule, inducing an increase in the phosphorylation of the protein c-erb b-2, causes detective increase in the incorporation of phosphate in the protein compared to switching in the absence of this molecule. Usually see a twofold or greater increase in phosphorylation, preferably more than three-fold increase over the control. Phosphorylation can be measured by methods known in this field, used to detect the phosphorylation of the receptors. See, for example, Cooper et al., Methods in Engymology, 99:387-402 (1983); Antoniades and Pantajis, Methods in Engymology, 147:36-40 (1987); and Lesniak, et al., Methods in Engymology, 150:717-723 (1987), which references are given.In a typical scenario, the phosphorus is investing (Antoniadis, supra). To measure in vivo phosphorylation, for example, tests can be conducted location-carrying protein c-erb b-2 cells in contact with radioactively labeled phosphate. To detect phosphorylation of the receptor protein of the c-erb b-2 in the test ni vivo, it is preferable to incubate the test cells within 12-18 hours with labeled phosphate. Cells are divided into two or more experimental series and some exhibit with molecules, which preferably should increase phosphorylation, while others are the controls. Then aliquots immunoprecipitated, the receptor detects, for example, by means of electrophoresis in SDS page with DDC Na or autoradiography, and a statistically significant increase in phosphorylation is a twofold or greater increase in the sample exposed to the test molecule, compared to the controls.To measure in vitro autophosphorylation, for example, cells or cell extracts can be incubated in the presence or in the absence of peptide, specific for c-erb B-2. After thus antibodies against c-erb B-2 immune complex can be incubated with32P-ATP and analyzed using autoradiography page with DDS Na.Other preferred pietracatella modulation of protein c-erb b-2" is defined detektivami decrease in the presence of the receptor c-erb b-2 in the tumor cells. This negative modulation detects reduction of the ability of an antibody or other specific binding parts of the molecules to bind or to learn receptor protein c-erb b-2 in the tumor cells. For example, negative modulation can be determined by incubation of tumor cells bearing the receptor protein of the c-erb b-2, with a target peptide by washing cells with the subsequent contacting of the cells with labeled (preferably radioactively labeled) antibodies specific for the protein c-erb B-2. The degree of binding soaked antibodies against c-erb b-2 cells exposed to peptide-specific protein c-erb b-2, compared with the degree of binding of these antibodies with the control cells (i.e., not exposed to specific for c-erb b-2 peptide). Preferably in these tests, so that the cells were directly exposed to contact with the labeled antibodies against the c-erb b-2 after washing.The observed negative (downward) modulation usually depends on the dose, i.e. the degree increases with the number of peptide-specific protein c-erb b-2, exposed to protein c-erb B-2. Preferred peptide that causes a reduction of 90% or more link clicks is occhialini peptide, specific for the protein c-erb B-2 is a peptide which binds to tumor cells expressing the protein c-erb B-2, and internalized when placed in contact with tumor cells. "Internalization" occurs when the peptide becomes sequestered (destroyed) in the cytoplasm of cells. When the internalization of the receptor and / or peptide can degradirovali in the lysosomes of cells or can be re-directed to the cell surface. How to determine the internalization of the ligand-receptor complex is also described in Haigler, et al. J. Biol. Chem., 255:1239-1241 (1980). Modulator of cell growth is a molecule that acts on cell growth, to which it is directed. In a typical case, the modulator can be internality in the target cell, but this function is usually provided by the internalization caused by the guide part of the molecule.Modulation is usually a decrease in the rate of metabolism or growth, preferably cytotoxic type, but can be useful and also significant increase in the rate of metabolism or growth. With a significant increase in the rate of metabolism or growth of killing only those cells you can apply poison with a short validity period.For modulators, Ausim high activity. This toxin can be an inorganic or simple organic molecules, but usually, the stronger are the biological molecules. Although there are viral and fungal toxins, the highest specific activities have some bacterial and plant toxins. Stop growth can occur as a result of prevention of any of a number of basic cellular functions, including the synthesis of nucleic acids, protein synthesis and cellular metabolism, primary or specific. For example, Pseudomonas endotoxin and diphtheria toxin irreversibly stop protein synthesis in eukaryotic cells. Both enzyme inactivating elongation factor 2, which is the main component of protein synthesis. Targets other toxins may be other factors elongation. In contrast, ricin, the toxin plants, acts directly on the ribosome, acting on 28 S pPHK.Preferably, the modulators of growth have enzymatic activity with the high rate of turnover, so that the internalization of a very small number of molecules can kill target cells. See, Pastain, et al., Science, 254-1173-1177 given in the link.Gelonin is a glycoprotein (mol. Mac the ohms of plant toxins, other members of this class are chain abrin, ricin and modeccin. Gelonin like abrina and ricin, inhibits protein synthesis by damage to the 60S subunit of ribosomes mammals. Apparently, gelonin is stable to chemical and physical processing. In addition, he gelonin is not associated with cells and usually non-toxic (except for high concentrations) with the introduction of it separately and safe to manipulate in the laboratory. Inactivation of ribosomes is irreversible, does not require cofactors and occurs with efficiency, which suggests that Galanin enzyme acts.Gelonin and ricin inhibits protein synthesis and belong to the most active toxins in the calculation of the weight of protein. Gelonin in 10-1000 times more active in the inhibition of protein synthesis than the A-chain of ricin. Peptides such as ricin and abrina consist of two chains, an A-chain, which is the toxic unit, and the B-chain, which binds to the cells. In contrast to ricin and arino, gelonin consists of one chain and due to the lack of a B-chain to bind to the cell it is in itself relatively inert or non-toxic to intact cells. This property is much lower which is an important property for various variants of the present invention. This differential toxicity is very important for high specificity in relation expressing c-erb B-2 cells.Apparently, mammalian cells do not possess the ability to bind and / or internalizing native molecule gelonin. Conjugates gelonin guide to the tumor with a reagent such as a monoclonal antibody TAb 250 directed to a tumor-specific antigen that is expressed on some tumor cells, provide as a particular way of linking gelonin with the cell, and the path to the internalization of the complex gelonin antibodies.Cytotoxic part of immunotoxin may be a cytotoxic drug or an enzymatically active toxin of bacterial or plant origin, or an enzymatically active fragment ("A-chain") of such a toxin. Preferred enzymatically active toxins and fragments thereof, represented by the example gelonin, the A chain of diphtheria toxin A-chain, exotoxin a (from Pseudomonas aeruginosa), A-chain of ricin. A-chain abrina, A-chain modeccin-sarcina, proteins Aleurites fordii, proteins diantin, proteins Phytoiacca americana (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, Cortina, krotin, an inhibitor of Saponaria officinalis, mitogillin, restrictocin, profesionali and cell growth. Toxins can be metabolic inhibitors or poisons, inhibitors of nucleic acid synthesis, inhibitors of protein synthesis or any other mediators of abnormal or destructive features. The most preferred conjugation with gelonida.Active fragments and derivatives are compounds that have the same structure of the crust, and that the structure gelonin full length, but do not contain the entire primary sequence. These fragments or derivatives have the same or improved biological or cytotoxic activity, as gelonin. Cytotoxicity fragments or derivatives gelonida may be determined by routine methods by experts in this field using test using lysate of rabbit reticulocytes.Modifiers of biological responses that can be connected with antibody TAb 250 and applied in this invention, can be (but are not limited to, lymphokines and cytokines such as IL-1, IL-2, interferons (or ), TNF, LT, TGF - and IL-6. These biological response modifiers have a different effect on tumor cells. Among these effects is the increased killing of tumor cells by direct action, and tecuanhuey antibody TAb 250 with biological response modifiers makes possible selective localization or direction to tumors or cells, excessively expressing c-erb B-2, superior antiproliferative effects. Non-specific effects resulting in toxicity to non-target cells is minimized, because the selected mediator of cell growth ineffective in the absence of the sending component.Cytotoxic drugs (and their derivatives) that are applicable in this invention, can be (but are not limited to, adriamycin, complex CIS-platinum, bleomycin and methotrexate. These cytotoxic drugs applicable for clinical treatment of recurrent tumors, but their use is complicated by serious side effects and injuries, damage to non-target (healthy) cells. Antibody TAb 250 may serve as a convenient medium of such medicines, providing an effective means for delivery to the tumor, and for entry into the tumor cells themselves. In addition, the delivery of cytotoxic drugs using specific antibodies to tumors provides protection from the destructive action of chemotherapeutic agents on sensitive areas, which does not Express excessive c-erb B-2, such as the liver or bone marrow. The use of Lekarstvo ovcu the medicinal product, since all particles medicines conjugated with antibodies, focusing on neoplastic cells and usually internalities in them.Directing the portion of the molecule and a modulator of cell growth can be conjugated using a variety of bifunctional binding protein substances. Examples of such reagents are N-Succinimidyl-3- (2-pyridylthio)(propionate) /SPDP/, 2-IT, 4-succinimidylester- -- methyl )(2-pyridyldithio)toluene /SMPT/, bifunctional derivatives of imidapril, such as dimethylacetamide, HCl, active esters such as disuccinimidyl, aldehydes, such as glutaric aldehyde, bis-etidocaine, such as bis-(p-azidobenzoyl)hexanediamine, derivatives of bis-page, such as bis-(p-diezani-benzoyl)-Ethylenediamine, diisocyanates such as toluene-2,6-diisocyanate, and bis-active fluorine compounds such as 1,5-debtor-2,4-dinitrobenzene.Before applying in these studies, cells Sp2/0-Ag14 growing source in the presence of 0.1 μg/ml native gelonin. After a few months the concentration gelonin gradually increase up until the cells will not be supported at a concentration of gelonin 10 mg/ml the cells are Then clone limi is. the ATEM gelonin removed from the culture medium by two passages and cells again exposed to gelonin to confirm the development of stable clones. After tests to confirm the production and activity of chimeric TAb 250 resistant gelonin cells SP 2/0 producing antibodies, grow and cDNA for antibody TAb 250 is removed from total DNA by incubation with the restriction enzyme. In parallel with cDNA from E. coli J M105 expressing optimized gelonin, delete, clear and coding gelonin DNA release after digestion with Hind 111 and Eco R1. Gene gelonin are ligated in a fragment of the heavy chain and the insertion of introducing resistant gelonin cells SP2/0. The cells are then subcloning limiting dilution and clones are subjected to screening as on the production of chimeric antibodies, as well as the content gelonin. Finally, the positive clones propagated and tested as in tests for cytotoxicity in vitro and in vivo tests distribution in the tissue, pharmacokinetic, therapeutic trials and toxicity tests. To determine the advantages and disadvantages of conducting a comparison of the properties of the fused protein TAb 250 gelonin with the characteristics previously described constructions TAb 250-gelonin. On the basis progressirovaniem breast cancer.The introduction of immunotoxins of the present invention to an individual diagnosed with the presence of neoplastic cells, for example, tumors with an undesirable level of expression of the oncogene c-erb B-2, will allow to concentrate and focus the cytotoxic agent in the place where he needed to kill tumor cells. As a result of such direction cytotoxic agents nonspecific toxicity to other organs, tissues and cells will be eliminated, minimized or at least reduced.When applying for in vivo therapy immunotoxins of the present invention is administered to a patient or an animal in therapeutically effective amounts, i.e. amounts, relieving or reducing the predisposition to tumors. They are usually administered parenterally, preferably intravenously, but can be applied to other routes of administration. Dose or regimen will depend on the nature of the cancer (primary or metastatic) and its population, properties used immunotoxin, e.g., its therapeutic index, the patient, medical history and other factors. Typically, the amount of injected immunotoxin lies in the range 0.1-10 mg/kg weight of the patient. Scheme applications continue to optimize efficiency, although, aston, Penn.; and Joodman and Jilman''s: The Pharmacological Basis of Therapeutics, 8 th Ed /1990/ Pergamon Press; included as links.For parenteral administration immunotoxins is usually prepared in suitable for administration by injection of a standard dose (solution, suspension, emulsion) in combination with a pharmaceutically acceptable parenteral carrier. Such carriers preferably are not toxic and therapeutic. Examples of such carriers are water, saline, ringer's solution, dextrose and 5% human serum albumin. You can also use non-aqueous media, such as fixed oils and etiloleat. As carriers can be used liposomes. The media may contain small amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives. Usually immunotoxin prepared in such media in concentrations of approximately 0.1 mg/ml to 10 mg/mlImmunotoxins of the present invention can also be applied in in vitro method. For example, the method can be applied for killing tumor cells from bone marrow. In this way, the bone marrow is first removed from the individual having a neoplastic diseases is eliminating the remaining tumor cells. The treated bone marrow cells can re-enter the patient for recovery of the immune system after receiving intensive chemotherapy and / or radiotherapy to eliminate all endogenous neoplastically genotoksicheskikh cells.Immunotoxins of the present invention can also be applied to extend the life time of tumor mammals and to slow the rate of growth of tumors containing cancer cells in mammals. For example, mice bearing xenografts of human tumors growing subcutaneously or intraperitoneally, can be treated with doses of immunotoxin, one antibody, a single toxin or saline solution at a dose of 25-100 mg/kg Inhibition of tumor growth can be measured by the change in the physical size of subcutaneous tumors or elongation of the life time of mice bearing intraperitoneal tumors, such as SKOV-3 cells. Such studies can be useful for the development of methodologies and can be applied to other mammals, including primates.The following examples provide a detailed description of the production, characteristics and applications of immunotoxins of the present invention. These examples in no way limit the invention.
Of your synthesis. Test of inhibition of cell-free protein synthesis was performed by sequential addition of 50 μl of the lysate of rabbit reticulocytes with stirring after each addition of the following components: 0.5 ml 0.2 M Tris-HCl (pH 7.8), and 8.9 ml of ethylene glycol and 0.25 ml of 1 M HCl.Then added 20 μl of a mixture of Sol-amino-energy material (SAEM), consisting of 0,375 M KCl, 10 mm Mg(CH3CO2)2, 15 mm glucose, 0.25 to 10 mm of the amino acids except leucine), 5 mm ATP, 1 mm GTP, 50 mm Tris-HCl (pH 7,6), 10 μl of creatinephosphate-creatinephosphate, 12 ál3H-leucine (Amersham, 74 MCI) (mmol) and 1.5 μl of solutions containing varying concentrations janinejay mixture. The mixture is incubated for 60 minutes at 30oC. the Inclusion of H-leucine was determined in an aliquot of the mixture by precipitation of the synthesized protein on the filters of glass fiber, washing in 10% THU and acetone and monitoring of radioactivity in a Beta counter using a scintillation fluid Aquasol theme Park. Gelonin with specific activity of not less than 4 of 109U/mg was used for conjugation with antibodies. Unit activity gelonin is the amount of protein gelonin, which causes 50% inhibition include14C-leucine into protein in a cell-free test.in
Gelonin in saline phosphate buffer was concentrated to approximately 10 mg/ml in a Centriprep concentrator 10. Hydrochloride of triethanolamine (TEA/HCl) pH 8.0, EDTA was added to a final concentration of 60 mm TEA/HCl and 1 mm EDTA, pH 8.0. The original solution of 2-aminothieno (2-IT) (500 mm 60 mm TEA buffer/HCl containing 1 mm EDTA, pH 8.0) was added to a final concentration of 1 mm and the sample is incubated for 30 minutes at 4oC under a current of nitrogen gas with stirring. Excess aminosilane was removed by gel-filtration on a column of Sephadex G-25 (1 of 24 cm) pre-equilibrated with phosphate buffer containing EDTA, pH 7.5, containing 0.01 M Na2HPO4, 0,0018 M KH2PO4, 0,0034 M KCl, 0.001 M EDTA, and 0.17 M NaCl. Fractions were analyzed for protein content in microtiter tablets using test Bio-Rad. Gelonin was suirable in the free volume (approximate fractions 21-23). These fractions were pooled and kept at 4oC.EXAMPLE 4
Obtaining monoclonal antibodies
BalB/c mice were immunized administered intraperitoneally (i.p.) and subcutaneously (s.c.) 2 106- 1 107cells NIH3T3T(cells NIH373, transfetsirovannyh c-erb B-2), emulsified 1: 1 in complete Freund's adjuvant. Animals were reimburseable every 2-4 weeks. After dateinasia for 4 days prior to fusion. Cells of the spleen was merged with myeloma cells RZ-Had 8.653 maintained in RPMI 1640, 10% FBS and 2 mm L-glutamine. Supernatant hybrid was exteremely on positive reactivity in ELISA (see below) and the reactivity of the extracellular domain was determined using an indirect immunofluorescence assay using flow NIH3T3 and NIH3T3tcells at 4oC followed by analysis using flow cytometry. Monoclonal antibody TAb 250 can be charged from any source. Most preferred for use in this invention are human or mouse antibodies.Hybridoma cells producing Tab 250, were grown in 2-liter continuous perfusion bioreactor. The cell supernatant from the bioreactor was filtered and then passed through the system with Protein-G-Trio with subsequent ion exchange chromatography. The material is then concentrated and sterile filtered. Testing of the final product provides tests for total DNA, protein purity, pH (IEF), total protein, endotoxin, power, identity and the antigen protein - G.The invention, Inter alia, uses a chimeric antibody, including hybrid antibodies or "humanized" (humaniged) or human-like the mouse TAb 250 antibody and basically identical. Such chimeric antibody called BACh 250.EXAMPLE 5
Enzyme-linked immunosorbent assay (ELISA)
Sterilnye tablets, 96-cells pre-treated for 2 hours at 37oC bovine collagen in a concentration of 1 mg/ml in sterile PBS. NIH3T3Tcells (NIH353 cells transformed by the vector) were grown to 80% confluently and collected warm versinon of the Bunch (of 0.02% EDTA in PBS), washed and treated with 10% neutral buffer containing formalin followed by stage blocking with 1% bovine serum albumin BSA/PBS. Supernatant sample or dilution of the antibody was then added to the plates and incubated for 2 hours at 37oC, followed by incubation with conjugated with alkaline phosphatase goat secondary antibodies, specific antibodies against Fc mouse IgG for 1 hour at 37oC. the Tablets were washed in PBS, was added p-nitro-phenylphosphate and diethanolamine as substrate and incubated for 15 minutes at room temperature, and then measured A405. Supernatant or antibodies that reacted with transfitsirovannykh cells in the absorption of 0.2 to 1.0 greater than the absorption for the negative control antibody, rassmatrivaemye were labeled using Iodobeads/Piercet in accordance with the manufacturer's specications. Carried out the reaction not containing media Na1251 (400 µci IMS.30. Amersham) with 25 mcg TAb 250 in 100 mm Na-phosphate buffer (200 μl, pH of 7.4) in the presence of 3 Iodobeads (iodophenyl). It was brought to the approximate ratio of 1 atom of iodine per molecule of IgG. The incorporation was carried out at room temperature for 7.5 minutes when interrupted stirring. The reaction mixture was removed from the pellet and after 5 minutes the volume brought up to 0.5 ml Na-phosphate buffer. 2 µl was taken for estimation of the specific activity (see below). The remaining amount was absoluely gel filtration using a column of AR-5 (Pharmacia), equilibrated PBS containing 0.1% BSA and 0.02% azide. Radioactively labeled antibodies were suirable in 1 ml of column buffer and kept at 4oC for up to 6 weeks without apparent loss of binding activity. The desalted material was practically free of neuklyuzhego iodine, since more than 95% was deposited THU.The specific activity of radioactively labeled antibodies was assessed by sedimentation through THU material at the stage of demineralization. So, 2 μl of the reaction mixture was diluted 500 times and a column buffer and aliquots in duplicate were mixed with an equal volume of ice 20% THU. After 25 minutes of standing on ice, the precipitated material was collected center is idemix THU pulses. The inclusion obtained in a separate itinerancy materials, was 27% - 45%, giving an assessment of the specific activity of 3.9 to 7.2 µci/µg. Before each experiment by linking the required number 125I-TAb 250 was absoluely gel-filtration on a NAP-5 column equilibrated with binding buffer. This procedure removed azide and given the material, which is typically more than 98% was deposited THU.EXAMPLE 7
Used cell lines adenocarcinoma human breast SKBR-3, MDA-MB-453, MDA-MB-231 and ovarian adenocarcinoma SKOV-3, SKBR-3, MDA-MB-231 and MDA-MB-453 cells were maintained in minimum basic medium with addition of 10% FBS and 2 mm L-glutamine. Environment for cells MDA-MB-453 also contained 1% essential amino acids and 1% vitamins. Cells SKOV-3 were cultured in Iscove modified medium Dulbecco with 10% FBS and 2 mm L-glutamine. All cultures were incubated at 37oC or 5% or 10% CO2.EXAMPLE 8
The internalization of125I-TAb 250
The internalization of 125I-TAb 250 was assessed by determining the amount of radioactivity in sensitive to acid and insensitive compartments. The cells were collected and resuspendable in ice buffer binding one125I-TAb 250 (6 ng/mlna cell surface radioactively labeled antibodies reached equilibrium, cells were besieged at 200g for 5 minutes at 4oC and washed three times with ice binding buffer to remove unbound antibodies. Cellular precipitation resuspendable in the ice-binding framework and take an aliquot to determine the amount of the original surface binding 125I-TAb 250. To initiate the internalization of radioactively labeled antibodies the cells were heated to 37oC. during periods of from 15 to 150 minutes, aliquots were removed and the cells were collected by centrifugation (1400g, 5 minutes, 4oC). Supernatant, which contained dissociatively or re-circulating antibody was collected. Precipitation resuspendable twice in an acidic rinse (100 µl PBS per tube, 1% glucose, pH 1). Supernatant containing associated with the surfaces of cells antibodies were combined and counted for radioactivity. The tips of the tubes containing the residual associated with the cells radioactivity, cut and counted.Fig. 6 demonstrates that monoclonal antibody TAb 250 is not internalized cells MDA-MB-231 (B). In contrast, cells, SKBR-3 were internalizable antibody TAb 250 is the most effective (A), whereas the internalization of antibodies into cells SKOV-3 and MDA-MB-453 was intermediate (C and D, respectively).The USE of the NAT) (SPDP) in dimethylformamide was prepared in the form of the original solution of 3 mg/ml in dry dimethylformamide. Because crystalline SPDP could be subjected to hydrolysis, the true concentration of chemically reactive cross-linking means were determined spectrometrically by determining the absorption at 260 nm in a double-beam spectrophotometer. The concentration of the original solution of SPDP was calculated from the equation:
< / BR>One milligram of monoclonal antibody TAb 250 in 1.0 ml of saline phosphate buffer (PBS) was added to a glass test tube. Source SPDP solution was slowly added into a test tube with about 5-fold molar excess with constant stirring. The mixture is incubated for 30 minutes at room temperature with stirring every 5 minutes during the incubation period.Excess unreacted SPDP was removed from the sample by gel-filtration chromatography on Sephadex G-25 (1 of 24 cm) pre-equilibrated with 100 mm Na-phosphate buffer, pH 7.0, containing 0.5 mm EDTA (buffer A). Fractions (0.5 ml) were collected and analyzed for protein content by the method of Bradford (Bradford, Anal. Biochem. 72: 248-254 (1976)). Absorbance (600 nm) was traced in the tablet of the 96 wells using an automated reader Bio-TEK Microplate. Antibodies were loirevalley in the free volume (fractions 14-20) and they were United and kept approx buffer with pH 7.0, containing 0.5 mm EDTA. The antibodies were concentrated to a final volume of approximately 0.5 to 0.75 ml.EXAMPLE 10
Conjugation of SPDP modified monoclonal antibody TAb 250 with modified aminosilane gelonida
Conjugation of modified 2-IT gelonin TAb 250
TAb 250-gelonin connected with SMPT, prepared by the connection of the modified 2-IT gelonin modified with SMTP monoclonal antibody TAb 250. Briefly, for the modification TAb 250 SMPT 10 mg of antibody in 1.0 ml of PBS diluted 1: 1 2 borate buffer (0.05 M sodium borate, 1.7% sodium chloride, pH 9,0) and slowly added to a solution of antibodies 52 μl of 4 mm SMPT in dry DMF (dimethylformamide)). The reaction is incubated at room temperature for 2 hours under N2under stirring. Excess SMPT removed by holding the reaction mixture through a Sephadex G-25 column, containing phosphate buffer with EDTA, pH 7.5, containing antibody fractions assessed using the Bio-Rad test. Fractions are combined and stored at 4oC under N2. Stitching with 2-IT is carried out at 27oC under N2with stirring for 96 hours. The final product was then purified as described for SPDP in example 9. One milligram of purified gelonin (2 mg/ml in PBS), obtained as described in example 1, modified them with the Vali with an equal weight of the modified gelonin. This proportion corresponded to 5-fold molar excess gelonin compared to antibodies. the pH of the mixture was brought to 7.0 by addition of 0.05 M TEA/HCl buffer pH 8.0 and the mixture is incubated for 20 hours at 4oC under nitrogen. Iodoacetamide (0.1 M) was added to a final concentration of 2 mm to block the remaining free sulfhydryl groups and the incubation continued for another hour at approximately 25oC. the Reaction mixture was kept at 4oC prior to purification using gel filtration.EXAMPLE 11
Purification of complexes gelonin-monoclonal antibody TAb 250
Unconjugated gelonin and low molecular weight products were removed from the reaction mixtures of example 10 gel filtration on a column of Sephadex S-300 (1,6 31 cm), previously equilibrated PBS.The reaction mixture from example 10 was concentrated to approximately 1 ml using microconcentrators Method 30 before applying on a Sephadex column. The column was washed by PBS. Fractions of 1 ml were collected and aliquots of 50 µl were analyzed for protein using the Bradford method.Unconjugated antibodies were removed from conjugated with gelonida antibodies by affinity chromatography on a column (1 24 cm) Blue Sepharose CL-6B, pre-equilibrated to 10 mm pole complete elution unconjugated antibodies.Conjugated with gelonida antibodies associated with the column, was suirable linear salt gradient of 0.2-2 M NaCl in 10 mm phosphate buffer with a pH of 7.2. The complex of antibody-gelonin was elyuirovaniya at approximately 0.7 M NaCl. The protein content buervenich fractions was determined according to Bradford. Protein fractions were pooled and distribution of proteins in buervenich fractions was confirmed by electrophoresis on a gradient of 5-20% non-polyacrylamide gel. Passing through the column peak (fractions 14-20) contained only available antibodies, whereas the fraction of 50-80, erwerbende high salt concentration, contained the conjugate TAb 250-gelonin free of unconjugated gelonin or antibodies. The final product contained antibody TAb 250 associated with 1, 2, and 3 molecules gelonin. The average content gelonin was 1.5 molecules per molecule of antibody. To evaluate the activity of gelonin almost pure complex gelonin-TAg 250 was used by the translation system in vitro rabbit reticulocytes. One unit of activity in this test was taken by the amount of protein required to provide 50% inhibition of protein synthesis compared with untreated controls. Using this test identified specific active the Eski pure complex gelonin-TAb 250 active in the test with a lysate of reticulocytes. A dilution of 1:1000 initial samples caused approximately 50% inhibition of protein synthesis, i.e. 50% reduction include14C-leucine into protein. Thus, the activity of the original drug was 1000 U/mlThe compositions of this invention also include fused constructs of monoclonal antibody TAb 250 and the cytotoxic portion of the molecule. Fused design immunotoxin of the present invention can be prepared in the following way. The nucleotide sequence of the V regions of H - and L-chains Tab 250 can be identified easily. For example, from producing TAb 250 cells secrete total RNA using thiocyanate quantine. Poly/A/+-RNA can be distinguished by means of chromatography on oligo /dT/-oligoclase. The desired genes can be distinguished using standard methods, including reverse transcription and PCR method (papierosow chain reaction). Chain cDNA can be synthesized from the selected mRNA using oligo-dT primer and reverse transcriptase. Primer near the poly-A tail of mRNA can be based on either sequence poly-A or common related sequences detected in the murine immunoglobulins. Cm. Devereaux, Jenetic Computer Jroup, University of Wisconsin Biofechnology Centre and its associated seguence databases.
For these genes it is possible to apply standard methods of transfection. For example, DNA can be entered in cells of murine hybridomas Sp2/O-AG14 using electroporation. 1-2 103actively growing cells SP2/O-AG14 washed and resuspended in 1.0 ml of sterile S. 30 μg of each of the chimeric, IgK and IgG1, plasmids added to the cell suspension. DNA/cells transferred into pre-cooled electro-cuvette, incubated on ice for at least 5 minutes and then served electrical impulse of 0.5 kV/cm for 10 msec. (Transfector 300, VTH). After electroshock mix DNA/cells return to the ice, incubated for 10 minutes, diluted with 10 ml of DMEM containing 5% NCTC-109 and 10% FCS, and incubated at room temperature for 10 minutes. Nakounou Wednesday, containing 1 μg/ml xanthine. Cells can be applied to the plates with 96 cells at 3/104cell 1 cell. Supernatant cultures analyzed using ELISA for antibodies associated with positive (containing the antigen TAb 250) target cells.EXAMPLE 12
Determination of bromide 3-(4,5-dimethyl/thiazolyl)-2,5-diphenyltetrazolium (MTT ) was performed by removing the cells from the flasks for tissue culture by versinon 1: 5000, centrifugation at 500g for 5 minutes and resuspending cells in medium at a concentration of 1 to 105cells/ml Cells were sown 100 ál of the cell in 96-cellular microtiter plates and incubated in a humidified CO2-incubator at 37oC within 24 hours.The next day was added TAb 250 TAb or 250-gelonin. Immediately after placing a higher concentration of antibodies in the first column of cells was performed dilution 1:2 directly in the microtiter tablets using a multichannel pipette. Then the plates were incubated for 3 days, followed by adding 10 ál of MTT to a cell. MTT was prepared in the form of a solution of 5 mg/ml PBS, sterile filtered and stored at 4oC in the dark. Then the plates were incubated an additional 4 hours in HCl and 3% sodium dodecyl sulfate. The absorbance at 570 nm was determined using a reader enzyme-linked immunosorbent assay (ELISA).EXAMPLE 13
The test cell proliferation (CPA)
The test cell proliferation (CPA) measures the growth of cells by determining the number of cells and their viability. In the 0th day of the cells at 80-90% of confluently released from the flask for tissue culture, besieged at 200g at 20oC for 6 minutes and resuspendable in MEM ISCOVE containing 2 mm glutamine and 10% fetal bovine serum to a concentration of 6000 cells/ml cell Suspension was added to the plates with 24 cells in 1 ml of the cell. The plates were incubated at 37oC, 5% CO2within 24 hours. On the first day of the cells in the three cells were washed in PBS and released from the plate with 1 ml of 0.05% trypsin in PBS and aliquots of 500 ál considered using Caulter Counter. The remaining cells were added 20 μl of PBS, TAb 250 TAb or 250-gelonin at the concentrations indicated on the drawings. The tablets were returned to the incubator. At time points indicated on the drawings, the cells were removed with trypsin and counted as the first day. The remaining 500 μl of cells were stained with iodide of propecia to determine cell viability using flow cytometry. For each point on g is as viable cells. This number was divided by the number of living cells treated with PBS, to Express the percentage of the control on the y axis.EXAMPLE 14
Cytotoxicity gelonin and complex gelonin-antibody TAb 250
As can be seen in Fig. 1, antibody ZME practically no effect on the cells SKOV-Z. In contrast, immunoconjugate TAb 250-gelonin was highly active.Fig. 2 illustrates the cytotoxicity immunoconjugate TAb 250-gelonin on cells SKOV-C compared with one gelonida. When the same concentration immunoconjugate TAb 250-gelonin was approximately 10,000 times more cytotoxic than one geloni.Fig. 3 demonstrates that it is not relevant to the problem under consideration antibodies, monoclonal antibody ZME 18, had no competitive effect on the cytotoxicity immunoconjugate TAb 250-gelonin. In contrast, increasing concentrations of monoclonal antibody TAb 250 reduced cytotoxicity immunoconjugate dependent on dose.Fig. 4 illustrates the dose-dependent effect immunoconjugate TAb 250-gelonin cells SKOV-3. As can be seen in the CPA test, a dose of more than 0.1 μg/ml caused 80% inhibition at 6 days of exposure.Fig. 5 shows the action of one or m is independent of the dose inhibition immunoconjugates TAb 250-gelonin in the CPA test.Fig. 7 depicts the effect of conjugate TAb 250/gelonin on four different cell lines. As expected, the highest toxicity was observed in the cell line SKBR-3. Intermediate toxicity was detected on cells SKOV-C and MDA-MB-453, whereas cells MDA-MB-231 were not observed cytotoxicity. Thus, cytotoxicity immunoconjugate TAb 250-gelonin correlates with the number of receptors on the cell surface in these cells.Thus, it is obvious that this invention is quite suitable to achieve the objectives and obtain the results set forth in the beginning. In the method and device can be made certain changes without departing from the spirit and scope of this invention. It is clear that changes are possible, and it is assumed that each element or stage listed in the claims, should be attributed to all equivalent elements or stages to achieve the same results with the same or equivalent manner. It is planned to expand the scope of the present invention, in whichever form not applied its principles. Thus, this invention is well adapted to achieve goals and get results and the above-mentioned advantages, and t is different, however, he is a conjugate gelonin and monoclonal antibodies exhibiting a binding specificity against antigenic domain of the protein C-erb b-2, and represents a Tab 250.2. Immunotoxin under item 1, characterized in that it has the binding specificity relative to the extracellular epitope of C-erb b-2.3. Immunotoxin under item 1, characterized in that gelonin selected from native or recombinant gelonin.4. The composition having a neoplastic activity, characterized in that it contains immunotoxin under item 1 in a concentration of 0.1 - 10 mg/ml and a pharmaceutically acceptable carrier.5. Method of treatment of the neoplastic cells with excess protein expression of C-erb b-2, providing for the introduction of effective dose immunotoxin under item 1 in this cell.6. The method according to p. 5, characterized in that the above-mentioned immunotoxin slows down the rate of growth of the above-mentioned cells.7. The method according to p. 5, characterized in that the said neoplastic cell is a cell of a human or animal.8. The method according to p. 5, characterized in that the above-mentioned immunotoxin prevents recurrence of a neoplastic condition.9. The way p is I, said neoplastic cell is a cell of the bone marrow.
FIELD: genetic engineering, immunology, medicine.
SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.
EFFECT: improved preparing methods, valuable medicinal properties of antibody.
33 cl, 5 dwg, 1 ex
FIELD: medicine, pharmaceutical industry and technology, pharmacy.
SUBSTANCE: invention relates to a composition eliciting an antiviral effect. The composition comprises hydrophilic conglomerate of immunoglobulins consortium adsorbed with polyethylene glycol 4000-6000, recombinant interferon-α2 and a special additive taken among the following substances: glycine, glucose, maltose, sodium chloride taken in the definite ratio of components. Invention provides elevating solubility of composition eliciting an antiviral effect and enhanced release of biologically active substances to solution.
EFFECT: valuable medicinal properties of composition.
FIELD: medicine, pharmacy.
SUBSTANCE: invention relates to a composition eliciting an antibacterial effect. Composition comprises hydrophilic conglomerate of immunoglobulins consortium adsorbed with polyethylene glycol 4000-6000 and a special additive taken among the following substances: glycine, glucose, maltose, sodium chloride taken in the definite ratio of components. Invention provides sufficient desorption of biologically active substances in resuspending the composition eliciting an antibacterial effect and comprising consortium of immunoglobulins.
EFFECT: valuable medicinal properties of composition.
SUBSTANCE: the innovation deals with new immunogenic conjugates of beta-propionamide-bound polysaccharide and N-propionamide-bound oligosaccharide with protein, and the method to obtain these conjugates has been suggested, as well. Conjugates should be applied to obtain vaccines against infectious diseases and cancer that enables to broaden the number of preparations applied in treating the above-mentioned diseases.
EFFECT: higher efficiency.
1 dwg, 2 ex, 8 tbl
FIELD: microbiology and immunology, in particular immunodiagnosis.
SUBSTANCE: atypical strain of melioidose Burkholderia pseudomallei-111-6-1 with altered phenotype defected with respect to synthesis of 8 antigen and acting as immunosuppressor is used as antigen for animal immunization. Immune serum is obtained after 2 immunization cycles of animal-producer with titer in gel immunodiffusion reaction not less than 1:128.
EFFECT: immune serum with increased specific activity.
2 tbl, 2 ex
FIELD: medicine and immunology, in particular treatment and prevention immunodeficiency conditions and diseases associated with bacterial or viral aggression.
SUBSTANCE: claimed method includes administration to a patient immunoglobulin drug (e.g., pharmaceutical composition containing 6-12 % of specific heterologous secreted immunoglobulin A, isolated from milk or foremilk of immunized ungulates). Administration is performed parenterally wherein single dose is at least 10 IU/kg of patient weight for treatment or at least 5 IU/kg for prophylaxis; or perorally in dose of 0.2-0.5 g and/or topically one-two times per day for 1-5 days. Method of present invention makes it possible to decrease dose of administrating immunoglobulin due to prolonged retention of its high titers in body fluids.
EFFECT: enlarged range of application and assortment of immunoglobulin drugs.
4 cl, 5 ex