Humanitarianly immunoglobulin specific for the protein l - selectin person

 

(57) Abstract:

The invention relates to biotechnology and concerns gumanitarnogo immunoglobulin specific for the protein L-selectin person. The essence of the invention lies in the fact that humanitarianly immunoglobulin specific for the protein L-selectin person, is a chimeric immunoglobulin having a region complementarity determining (CDR) corresponding CDR regions of the donor mouse immunoglobulin, and a frame region of the variable region heavy and light chain acceptor human immunoglobulin, and specifically bind to L-selectin person with a constant affinity of 107M-1. The invention allows to obtain non-immunogenic to a human immunoglobulin specific for the protein L-selectin. 2 C. and 17 C.p. f-crystals, 6 ill. , 1 table.

This application is a partial continuation application of U.S. reg. N 07/983946 filed 12/1/92, which in all cases and is entered in its entirety in the present description by reference.

The invention relates to a combined technology of recombinant DNA and monoclonal antibodies used for the development of new biological materials, and in particular, for polzovaniyu of these immunoglobulins in vitro and in vivo.

The ability of cells to mutual adhesion plays an important role in the maturation, normal physiology and in pathological processes. This ability is mediated by adhesion factors, mainly glycoproteins expressed on the cell membranes. In many cases, the factor of adhesion of one cell type is associated with another factor of adhesion, expressed in another cell type, forming a pair contraceptor-receptor". There are three main classes of adhesion factors, namely integrins, selectins, and members of the superfamily of immunoglobulins (Ig) (see Springer, Nature 346:425 (1990); Osborn, Cell 62:3 (1880); Hynes, Cell 69:11 (1992), which in its entirety are introduced in the present description by reference). These molecules are particularly important for the interaction of leukocytes and platelets to each other, with the extracellular matrix and vascular endothelium.

Integrins are heterodimeric transmembrane glycoproteins consisting of a-chain (120-180 kDa) and a-chain (90-110 kDa), with mainly short cytoplasmic domains. All subunits are homologous sequence and contain common elements, as well as-subunit. Three known integrin containing-subhed is B>) is widely distributed on lymphocytes, granulocytes and monocytes. His contraceptor is a molecule ICAM-1 (and probably molecule smaller values of ICAM-2) belonging to the family of Ig, which is expressed on many cells, including white blood cells, and is activated vascular endothelium by cytokines, such as TNF and IL-1. Blocking LFA-1 on T-cells by antibody - or-subunit significantly inhibits adhesion-dependent functions, such as CTL-mediated lysis of target cells. Mac-1 (M2) is located on neutrophils and monocytes, and his contraception is ICAM-1 (and possibly ICAM-2). In addition, Mac-1 is a receptor of activated complement 3 (CR3) and is associated with C3bi-fragment. Third2-integrin, P15095 (x2), is also found on neutrophils and monocytes, but, in all probability, he plays a less significant role. -Subunit, LFA-1, Mac-1 and P150.95 also have the appropriate CD - labeling CD11a, CD11b and CD11c, and2also denoted CD18, so that LFA-1 is a CD11a/CD18, Mac-1 is a CD11b/CD18.

There are three known selectin, which were previously identified LECCAM, and currently referred to as L-selectin (referred to as the selectins were sequenced at the cDNA level and have homologous sequences and identical fragments, including lectin-like domain. L-selectin has a dual function: on the one hand, he is "homing-receptor on T-cells for outdoor endothelial venules of peripheral lymph nodes; and on the other hand, it is a factor of adhesion of the neutrophils to the endothelium (Hallmann et al., Biochem. Biophys. Res. Commun. 174:236 (1991); this work in its entirety is introduced into the present description by reference). E-selectin and P-selectin induced on the endothelium by cytokines, but with different kinetics. L-selectin on neutrophils is contraception E-selectin and P-selectin (Picker, etc., Cell 66:921 (1991); this work in its entirety is introduced into the present description by reference), although it is possible that all three of these receptors are also other contraceptor. In particular, E-selectin binds to the carbohydrate group sialyl-Lewis x (SLex)(Lowe et al., Cell 63:475, (1990); this work in its entirety is introduced into the present description by reference), whereas the carbohydrate, as it is known, is present on L-selectin (Picker et al., Cell 66:921 (1991)) and may, obviously, be present in other proteins. E-selectin is expressed mainly in inflammatory skin cells, and also serves as an adhesion factor for homing" T cells of the skin, the entirety by reference).

As shown by various tests, all antibodies to CD11a, CD11b, CD18, L-selectin and E-selectin in more or less block the binding of neutrophils to activated endothelial cells, but the most complete inhibition is achieved mainly by a combination of antibodies to CD18 and antibodies to L - or E-selectin (see, for example, Luscinskas, J. Immunol., 142:2257 (1989); this work in its entirety is introduced into the present description by reference). Only recently gained wide recognition model, which explains the above facts three-stage process of adhesion (Butcher, Cell 67:1033(1991); this work is entirely introduced into the present description by reference). In the first stage neutrophils bind reversibly to the inflamed vascular endothelium via selectins, which are very well connected in terms of flow, causing neutrophils to literally "roll" along the walls of blood vessels. Then the neutrophils are activated by a number of stimulants that are around or released by the endothelium, including IL-8, PAF and C5a. Activated neutrophils secrete L-selectin and activated Mac-1. In the final stage, the binding of Mac-1 with ICAM-1 and probably with other contraception on endothelial cells promotes stable adhesion and tra is to integrin and selectin, can stop this process by preventing the binding of neutrophils to the endothelium and their transudate in the tissue. Therefore, these antibodies can be used to treat a variety of pathological conditions, which are based on the inflammatory process.

For example, in live models antibodies against CD 18 that are associated with LFA-1 and Mac-1 were used to reduce damage during ischemic reperfusion (see , for example, Vedder et sl., J. Clin. Invest., 81:939 (1988); Vedder et al. , Proc. Natl. Acad. Sci. USA 87:2643 (1990); U.S. patent N 4797277). These antibodies also contribute to the reduction of neutrophil-mediated damage in the lungs, caused by various insults (Doerschuk et al., J. Immunol 144:2327 (1990) and Mulligan et al., J. Immunol. 148:1847 (1992)), including sepsis caused by gram-negative bacteria (Walsh et al., Surgery 110:205 (1991)). In experiments with rabbit antibodies against CD 18 also protected from death due to meningitis (Tuomanen et al. , J. Exp. Med., 170:959 (1990)). These antibodies can also be used for the prevention or treatment of transplant rejection, because they block the function of T-cells.

For example, injection of antibodies against L-selectin or E-selectin rodents suppresses the accumulation of neutrophils within vospitannyh studies have also identified the antibody against L-selectin highly inhibited "rolling" of leukocytes along the vascular endothelium of mesenteric venules rabbit, temporarily placed on the surface of the body (Von Andrian et al., Proc. Natl. Acad. Sci. USA. 88:7538 (1991)). The antibody against E-selectin contributes to a significant reduction in vascular disorders induced by deposition of immune complexes in the skin or the lungs of rats, and a significant decrease in the accumulation of neutrophils in these areas (Mulligen et al. , J. Clin. Invest. 88:1396 (1991)). In addition, the model acquired bronchial asthma in primates antibody against E-selectin contributes to a significant reduction in the influx of neutrophils into the lungs, and associated with him late obstruction of the respiratory tract after inhalation of antigen (Gundel et al. , J. Clin. Invest. 88:1407 (1991)).

There were several antibodies, including mouse DREG-55, mouse DREG-56 and the mouse DREG-200 that are associated with L-selectin person (Kishimoto et al. , Proc. Natl.Acad. Sci. USA 87:2244 (1990); this work is entirely introduced into the present description by reference). These antibodies partially or completely block the binding of human lymphocytes with outer endothelial venules of peripheral lymph nodes, as well as the binding of neutrophils h is camping in the present description by reference). The ability of these antibodies to block the binding of neutrophils to endothelial cells suggests that the antigen with which they are associated, namely L-selectin may be a suitable target for potential therapeutic agents.

Unfortunately, the use of monoclonal antibodies, non-human, such as mouse antibody DREG-200, to ensure the person has some drawbacks, especially in the modes of re-treatment, as explained below. For example, murine monoclonal antibodies have a relatively short half-life in the bloodstream, and do not possess other important immunoglobulin functional properties required for use in treatment of humans.

And probably more importantly, non-human monoclonal antibodies contain significant portions of the amino acid sequences that can be immunogenic when administered to humans. Numerous studies have shown that after injection the patient alien antibody immune response induced in the body of the patient against this antibody can be so strong that it will negate therapeutic effect of the antibody so the other antigen (human) monoclonal antibody for the treatment of various diseases, it may be that after the first or multiple introductions man alien antibody followed by treatment even using a totally different therapy will be ineffective or dangerous due to cross-reactivity. Some success was achieved with the production of so-called "chimeric antibodies" (in which, for example, murine variable regions linked to human constant regions), but despite this, the problem associated with significant immunogenicity, still has not been resolved.

To solve immunogenicity alien antibodies several attempts were made to obtain a "humanized" (humanized) antibodies. The transition from mouse to "humanized" antibody provides a certain compromise in relation to some of the competing factors, and the choice of solution depends on the specific antibodies used. To minimize the immunogenicity of the immunoglobulin should remain a large part of the human sequence of the acceptor. However, to preserve the authentic ability to bind immunoglobulin frame should contain a sufficient number of substitutions in the sequence of the acceptor celonova donor immunoglobulin. As a result, many "humanized" antibodies produced up to the present time, to find a significant loss of affinity of binding compared with the corresponding murine antibody. See, for example, Jones et al., Nature 321: 522-525 (1986); Shearman et al., J. Immunol. 147:4366-4373 (1991); Kettleborough Protein Engineering 4: 773-783 (1991); Gorman et al., Proc. Natl. Acad. Sci. USA 88:4181-4185 (1991); Tempest et al., Boitechnology 9:266-271 (1991); Riechmann et al., Nature 332:323 (1988) and EPO publication N 0239400 (each of these works is entered in its entirety by reference).

In accordance with the above it is obvious that it is necessary to develop improved forms of humanized antibodies specific to the antigen, L-selectin, which are not only mostly non-immunogenic to humans, but could be easily and without large economic costs produced by a method acceptable to the manufacture of medicines, and for other purposes. These and other problems were solved by the present invention.

The invention relates to new compositions that can be used, for example, for the treatment of inflammatory diseases in humans and which contain the "humanized" antibodies capable of specific binding of L-se is at least one chain contains one or more hypervariable regions, functionally connected to the segments of the frame regions of human immunoglobulin. For example, murine hypervariable region with additional natural amino acid residues of the mouse, or they can be put into a frame region of a human immunoglobulin for the production of "humanized" immunoglobulins capable of binding to L-selectin at levels of affinity, constituting more than 107M-1. These "humanized" immunoglobulins also have the ability to block the binding of CD R-donor mouse monoclonal antibody to L-selectin.

The immunoglobulins of the present invention, including binding fragments and other derivatives thereof, can be easily produced using techniques of recombinant DNA, where the final expression in transfected cells is carried out preferably in the immortalized eukaryotic cells, such as myeloma cells or hybridoma. Polynucleotide containing the first sequence encoding a frame region "humanized" immunoglobulin, and a second series of sequences encoding the desired hypervariable region of immunoglobulin can be produced si the immunoglobulins can be used separately i.e., basically, in pure form or in combination with chemotherapeutic agent, such as non-steroidal anti-inflammatory agent (e.g., aspirin); corticosteroid or immunosuppressant. All these compounds are particularly effective in the treatment of inflammatory diseases. "Humanized" immunoglobulins or their complexes can be obtained in the form of a pharmaceutically acceptable drug, the shape of which may vary depending on the method of administration.

In Fig.1 shows the cDNA sequence and translated amino acid sequences of the variable regions of the light chain (A) and heavy chain (B) mouse antibody DREG-200. Mature heavy chain begins with amino acids 20 E, and a Mature light chain begins with amino acids 21 D, which are preceded by a signal sequence.

In Fig. 2 - amino acid sequence of the variable regions of the Mature light chain (A) and heavy chain (B) mouse antibody DREG-200 (top row) and "humanized" antibody DREG-200 (bottom row). The three CDRs in each chain are underlined. Amino acid residues in the framework regions, which were replaced by the murine amino acids, or typical amino acids person "ones, encoding the variable region of the light chain (A) and heavy chain (B) "humanized" antibody DREG-200 and beginning and ending XbaI sites; and translated amino acid sequence, including the signal sequence.

In Fig. 4 - competitive binding of murine and humanized IgG1 and IqG4 DREG-200. Target cells are cells 2-1, line murine pre-B-cells that have been transfected with the gene for human L-selectin, and therefore expressed human L-selectin (Berg et al., Biochem. Biophys. Res. Comm. 184: 1048 (1992)). 5105cells were incubated with 3 ng1251-labeled mouse antibodies (2 µci/µg) together with increasing amounts of mouse or humanized" antibodies-a competitor in 0.2 ml buffer for binding (PBS + 2%FBs +0,1% azide) for 1 hour at 4oC. Cells were washed and precipitated, and then measured their associated radioactivity. Then calculate the concentration of bound and free labeled antibody.

In Fig. 5 binding of human neutrophils with IL-I-stimulated endothelial cells of the umbilical vein in human (HU V EC). Neutrophils are first treated with irrelevant control antibody, mouse antibody DREG-200 or a humanized IgG1 antibody DREG-Ni heart with "humanized" antibody DREG-200. Figure illustrated by an experiment conducted on cats treated "humanized" antibody DREG-200 or the control antibody; from left to right: area under threat/complete ventricular region; a region of necrotic tissue/region under threat, and the area of necrotic tissue/full region of the left ventricle. Values in parentheses represent +/- standard deviation for six cats; height segments means the average value.

Definition

The term "essentially identical" or "mostly homologous" means that two peptide sequences, when optimally comparison of their primary structure, for example, using the programs GAP or BESTFIT using weights estimated gaps have an identity of at least 65%, preferably at least 80-90%, and more preferably at least 95% or more (e.g., 99%). In this case, preferably, if the position of the residues that are not identical, differ from each other conservative amino acid substitutions.

For classification conservative or non-conservative amino acid substitutions of the amino acids can be divided into the following groups: Group I (hydrophobic side chains): norepi): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues influencing chain orientation): gly, pro, and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions are substitutions between amino acids of the same class. Non-conservative substitutions are substitution of the amino acid of one class to an amino acid other class.

Amino acids from the variable regions of the Mature heavy and light chains of immunoglobulins are designated Hx and Lx, respectively, where x is a number that indicates the position of amino acids in accordance with the scheme Kobat in "Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991). In this work Kabat transferred a lot of amino acid sequences of antibodies for each subclass, as well as a listing of the most frequently occurring amino acids for each position of the residue in this subclass. Kabat used method, in which each amino acid in the listed sequences were ascribed to the room, and this method of numbering amino acid residues has been accepted by experts. The Kabat scheme can be used for other antibodies, is not included in the list Kabat, by comparing the primary structure of ASCS and Kabat, allows for easy identification of amino acids in equivalent positions of different antibodies. For example, the amino acid at position L 50 human antibodies occupies the equivalent position in relation to the position of the amino acid L 50 mouse antibodies.

Starting from N-Terminus to the C-end, light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat (1987) and (1991), (see above), or Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).

Detailed description of the invention

Basically, the classification used below and in the description of laboratory procedures; methods of molecular genetics, chemistry of nucleic acids and hybridization described below are well known and widely used by specialists in this field. In the methods of recombinant DNA, synthesis of polynucleotides, the cultivation of cells and transgenic introduction of genetic material (e.g., electroporation, microinjection, lipofection) uses the traditional technique. Basically, enzyme reactions, synthesis of oligonucleotides and the stage of purification is carried out in accordance with the manufacturer's instructions. Sposabella, and in accordance with the descriptions available in the literature, links to which are given in this application. For the reader's convenience in the present application also describes a well-known procedures. All necessary information is entered into the present description by reference.

"Humanized" antibodies against L-selectin

The present invention relates to the production of "humanized" immunoglobulins that specifically bind to epitopes of L-selectin. These antibodies, in General, have high affinity binding to L-selectin, comprising at least about 107M-1and preferably from 108M-1up to 109M-1or more, and have the ability to communicate, for example, with neutrophils. "Humanized" immunoglobulins have the skeleton of a human immunoglobulin, and one or more areas, complementarity determining (CDR Complementarity determining regions), derived from the immunoglobulin, typically a mouse immunoglobulin capable of specific reaction with L-selectin. In a preferred embodiment of the present invention, one or more CDR regions derive from a mouse antibody DREG-200, a "humanized" immunoglobulin, in whole is in large quantities in a cost effective way, designed for use in the treatment of inflammatory diseases in humans using various techniques.

It is known that the basic structural unit of an antibody consists of a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, this pair has one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). NG2the end of each chain begins with the variable region consisting of approximately 100-110 or more amino acids, which are responsible mainly for the recognition of antigen. COOH-portion of each chain defines a constant region, which is responsible mainly for effector function.

Light chains are classified Kappa - and lambda-chain. Heavy chains are classified as gamma, mu, alpha, Delta, or Epsilon-chains, which determine the isotype of the antibody, namely IgG, IgM, IgA, IgD and IgE, respectively. In the light and heavy chain variable and constant region connected "J"region of about 12 or more amino acids; and in the heavy chain, in addition, there is an area "D", consisting of about 10 or more amino acids (See. for example, Fundamental Immunology, Paul, W., Ed., Chapter 7 pp. 131-166, Raven Press, N. Y. (1984); this work is introduced at present, on enswathe center. Conservative frame region all circuits have basically the same structure and are connected by three hypervariable regions, also called areas, complementarity determining (CDR) (see, e.g., "Sequences of Proteins of Immunological Interest", Kabat E., and others, U. S. Department of Health and Human Services (1987) and Chothia &Lesk, J. Mol. Biol., 196:901-917 (1987); these works are wholly entered into the present description by reference). CDR two chains of each pair are moved with the frame sections having the ability to bind to a specific epitope.

Used in the present description, the term "immunoglobulin" refers to a protein consisting of one or more polypeptides, mainly encoded by immunoglobulin genes. Such genes are Kappa, lambda, alpha, gamma, Delta, Epsilon and mu-genes constant regions, as well as many gene variable regions of immunoglobulin. Immunoglobulins may exist in addition to antibodies, in the form of other shapes, such as FV, Fab, and (Fab')2and also in the form of heterovalent antibodies (for example, Lanzavechia and others , Eur. J. Immunol. 17:105 (1987)) and single chains (e.g., Huston et al. , Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988) and Bird et al., Science 242: 423-426 (1988); these works in its entirety entered into this the nual (Cold Spring Harbor Laboratory, 1988), and Hunkapiller & Hood, Nature, 323: 15-16 (1986); all of these works are introduced in the present description in its entirety by reference).

Chimeric antibodies are antibodies in which the genes for the light and heavy chains were usually constructed by means of genetic engineering of segments of immunoglobulin genes belonging to different species. For example, the variable (V) segments of the genes from a mouse monoclonal antibody can be connected to a constant (C) segments of the human immunoglobulin, such as V1and V4. Thus, a typical therapeutic chimeric antibody is a hybrid protein consisting of the V or antigennegative domain, derived from a murine antibody, and C or ejector domain derived from human antibodies, although can be used antibodies and other mammalian species.

Used in the present description, the term "framework region" refers to those parts of the variable regions of light and heavy chains of immunoglobulin, which are relatively conservative (i.e. less variable than CDR) of immunoglobulins of the same species (as defined by Kabat and others, see above). Used in this Opir, 85% or more) frame the field of natural human antibodies or consensus sequences of several of these antibodies.

Used in the present description, the term "humanized immunoglobulin" refers to an immunoglobulin, which includes the skeleton of a human immunoglobulin; and at least one hypervariable region (CDR) derived from antibodies, non-human and in which any present constant region, mostly identical to the constant region of human immunoglobulin at least about 85-90%, preferably at least 95 %. Therefore, all of the "humanized" immunoglobulin, except possibly the CDRs, are basically identical to the corresponding parts of one or more sequences of native human immunoglobulin. So, for example, a "humanized" immunoglobulin does not apply chimeric antibody with mouse variable region and human constant region.

Compared to antibodies, and in some cases, and chimeric antibodies, "humanized" antibodies have at least three potential advantages when used for the treatment of cloughvalley, this antibody interacts better with the other parts of the human immune system (for example, more efficiently destroys target cells due complementability cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC)).

(2) the Human immune system should not recognize the frame or C-region "humanized" antibody as foreign, and therefore, or antibody-based test the body's response against such an injected antibody should be less than the response against wholly foreign mouse antibody or a partially foreign chimeric antibody.

(3) Reported (Shaw D., et al., J. Immunol., 138:4534-4538 (1987)), that injected mouse antibodies have a shorter half-life in human blood flow than normal antibodies. Injected "humanized" antibodies, in all probability, have the same half-life as natural human antibodies, when you consider that these antibodies into the blood stream in smaller and not so frequent doses.

In one of its variants the present invention relates to segmental recombinant DNA encoding the CDR region of the heavy and/or light chain immunoglobulin able to contact the right Apache; this work in its entirety is introduced into the present description by reference). DNA segments encoding these areas, sew with DNA segments encoding the respective frame region, originating from human immunoglobulin. Characteristic DNA sequence, which, after expression code for the polypeptide chain, containing the CDR region of the heavy and light chains of the monoclonal antibody DREG-200, shown in Fig. 1. Due to the degeneracy of the codon and non-critical amino-acid substitutions, these sequences can be easily replaced by other DNA sequences, such as described below. Detailed description of the design and production of "humanized" immunoglobulins is described in General terms in the assigned applications per. N 07/290975 and 07/310252, filed on 28 December 1988 and February 13, 1989, respectively, which in their entirety are introduced in the present description by reference.

Furthermore, DNA segments can contain a DNA sequence that regulates the expression and appropriately attached to sequences coding for the "humanized" immunoglobulin, for example, natural or heterologous to the promoter region. Predosa in the vectors, capable of transformation or transfection of eukaryotic host cells, although it can also be used and the regulatory sequences for prokaryotic cells. After the introduction of the vector into an appropriate cell host this cell is incubated under conditions suitable for high level expression of the nucleotide sequences, and then, if necessary, collect and clean light chains, heavy chains, dimers of light/heavy chain or intact antibodies, binding fragments or other immunoglobulin.

Thus, nukleinovokisly sequence of the present invention, with the ability to producing the desired "humanized" antibodies can be constructed from various polynucleotides (such as genomic or cDNA, RNA, synthetic oligonucleotides, etc) and components (such as V-, J-, D - and C-region) using different techniques. The most common method of such a design is the connection of the respective genomic and synthetic sequences, although can also be used and cDNA sequences (see publication of Europatent N 0239400 and Riechmann, L. et al., Nature 332: 323-327 (1988); both of these works constant regions can be selected using well-known procedures from a variety of human cells, and preferably immortalizing B-cells (Kabat see above, and WP87/02671). CDR used to obtain the antibodies of the present invention, can be obtained from monoclonal antibodies capable of contact with L-selectin, and produced in any suitable source, taken from a mammal, such as mouse, rat, rabbit or other vertebrate animal capable of producing antibodies using methods well known in the art. Suitable cell sources for DNA sequences, as well as cell hosts for the expression and secretion of immunoglobulins can be obtained from a number of sources, such as the American type culture collection ("Catalogue of Cell Lines and Hybridomas, 5-th ed. (1985) Rockville, MD; this work in its entirety is introduced into the present description by reference). In preferred embodiments of the present invention CDR sequences correspond to the CDR sequences of the antibody DREG-200, antibodies mouse DREG-55 or antibody DREG-56, and may contain degenerate nucleotide sequences encoding the corresponding amino acid sequences of CDRs of murine antibody DREG-200, DREG-55 or DREG-56.

In addition to the "humanized" immunoglobulin is m, homologous" modified immunoglobulins using well-known techniques of recombinant DNA. As the source frame sequences can be used, and other human antibodies that differ from antibodies Eu, discussed in Example 2. These frame sequence should be highly identical variable frame domains of murine antibody DREG-200, from which originate the field of the CDR. Variable framework region of the heavy and light chains can occur from sequences of the same antibodies, or sequences from different human antibodies. For example, each of the frame regions of the heavy and light chains may occur from several human antibodies. Such sequences can be sequences of natural human antibodies, or consensus sequences of several human antibodies (see Carter and others WO 92/22653 (1992)).

Direct and unnatural neighborhood murine CDR regions with variable frame area of a person may lead to conformational difficulties, which, if not corrected by the substitution of some amino acid residues, can lead to loss of Aterno modeling. For these purposes, a widely used computer equipment and software for three-dimensional projection of the invention, molecules of the immunoglobulin. In General, molecular models produce based on the known structures of immunoglobulin chains or domains. The simulated circuit is compared with chains or domains of known three-dimensional structures for the analysis of similarity of their amino acid sequence and, if the chains or domains reveal a high degree of similarity, then use them as a starting point for constructing molecular models. Selected similar patterns change with regard to the differences between tactical existing amino acids in simulated chains or immunoglobulin domains and amino acids present in the original structure. Then modified patterns collected with obtaining a model compound of immunoglobulin. Finally, the resulting model improve by energy minimization and control that all atoms were located at an appropriate distance from each other and to the length of the links and the angles were within a chemically acceptable range. Stage produced a three-dimensional computer model for varit starting point for predicting the three-dimensional structure of antibodies contains a hypervariable region of the mouse DREG-200 is replaced in the frame people. structures. Additionally, there may be constructed a model representing the structure in the case, if we introduce additional amino acid substitution, as discussed below.

Generally speaking, the substitution of human amino acid residues of murine residues should be minimized, since the introduction of murine residues increases the risk of inducing a person HAMA response. Amino acids for substitution can be chosen according to their potential impact on CDR conformation and/or binding to the antigen. The study of such potential impact is carried out by simulation, performance evaluation of amino acids in specific positions or empirical observation of the influence of substitution or mutagenesis of specific amino acids.

If the amino acid in the variable frame region of the mouse antibody DREG-200 differs from the amino acid at the equivalent variable frame region of the person, the human amino acid must be replaced by an equivalent mouse amino acid if there is reason to expect that this amino acid:

/1/ ecovalence contact directly with the antigen or
located at a distance of from about CDR-region/.

Other candidates for substitution is of amino acid acceptor framework of human immunoglobulin, which is unusual for human immunoglobulin at that position (for example, amino acid H113 human antibody Eu/. These amino acids can be substituted amino acids from equivalent Poloniny more typical of human immunoglobulins. Alternatively, amino acids from equivalent provisions mouse antibody DREG-200 can be entered in a frame region of a person, if such amino acids are typical of human immunoglobulin in the equivalent position.

Generally speaking, replacing all or most of the amino acids that meets the above criteria is desirable. However, sometimes, there is some uncertainty about exactly which amino acid meets the above criteria, and in this case produce an alternative of antibodies, one of which contains the given substitution, and the other is not. "Humanized" antibodies of the present invention typically have a substitution in the frame region of the light chain corresponding residue murine antibody DREG-200 at least 1, 2, 3, 4, and usually 5 positions selected from CL is the second region of the mouse heavy chain is at least 1, 3, 5, 7, 9, 10, 11, and usually 12 positions, selected from the following provisions: H93, H95, H98, H111, H112, H115, H30, H98, H111, H27, H48 and H72. In a preferred embodiment of the present invention, if the acceptor immunoglobulin heavy chain of human antibody is Eu, the heavy chain also has a substitution in H113. This position is usually replaced by amino acids from equivalent provisions of the human immunoglobulin with a more typical amino acid residues.

Usually, CDR field in the "humanized" antibodies are mainly and preferably identical to the corresponding CDR regions of a murine antibody DREG-200. However, in some cases, it may be preferable to replace one residue in a CDR region, for example, to create a similarity to the binding site of the ligand of L-selectin. Sometimes it is possible, though not desirable, to make one or more conservative amino acid substitutions CDR residues, unless, of course, does not appreciably affect the binding affinity of the resulting "humanized" immunoglobulin.

With the exception of specific amino acid substitutions discussed above, the frame region "humanized" immunoglobulins, mainly and in most cases, identical frame areas entity may differ from the native sequence by its primary structure, for example, several amino acid substitutions, terminal or intermediate insertions and deletions, etc. Suitable components for polypeptides of the present invention can also serve as stereoisomers (for example, D-amino acids/ twenty major amino acids, unnatural amino acids, such as ,-disubstituted amino acids, N-alkyl amino acids, lactic acid and other minor amino acids. It is obvious that many of the amino acids of the frame region can contribute small or indirect contribution to the specificity or affinity of the antibody. So many individual conservative substitutions of residues of the frame region are perfectly acceptable if they do not adversely affect the specificity or affinity of the resulting "humanized" immunoglobulin. In General, however, such substitution is undesirable. Gene modifications can be easily implemented using well-known techniques, such as site-specific mutagenesis (see Gillman & Smith, Gene 8:81-97 /1989/ and Robers et al. , Nature 328:731-734 /1987/; both of these works in their entirety are introduced in the present description by reference/.

An alternative can be producirovanie activities of immunoglobulin /for example, activity link/. These polypeptide fragments may be produced by proteolytic cleavage of intact antibodies with known methods, or by inserting stop codons at the desired positions in the vector pVk and pVg1 - dhfr using site-specific mutagenesis, for example, so that after CH1 was producirovanie Fab fragments or after the hinge region producirovanie /Fab'/2-fragments. Single-chain antibodies can be produced by connecting the VL and VH with a DNA linker (see Huston et al., see above, and Bird et al. see above/. For example, an FV or Fab fragments can be produced in E. coli in accordance with the methods described Buchner & Rudolph, Bio/Technology 9: 157-162 /1991/ and Skerra et al., Bid Technology 9:273-277 /1991/ /these works in their entirety are introduced in the present description by reference/. FV and Fab fragments can be produced by expression of the coding polynucleotide in eukaryotic cells, preferably mammalian cells. In addition, since, like many genes, genes associated with immunoglobulins contain separate functional regions, each of which has one or more characteristic biological activities, these genes can be legirovanyh with fu the 15 th Dec.1987, which is entered in its entirety in the present description by reference, the result can be obtained hybrid proteins /for example, immunotoxins/ possessing new properties.

The expression of sequences "humanized" immunoglobulin in bacterial hosts can be used for a favorable choice sequences "humanized" immunoglobulin with higher affinity by mutagenesis of CDR-regions and production of phage display libraries, which can be skanirovaniya on the availability of CDR variants "humanized" immunoglobulins with high affinity and/or high specificity of binding to L-selectin. One of the potential advantages of such an improved selection of affinity is the generation of a CDR-options "humanized" immunoglobulin having a higher affinity of binding and/or lower cross-reactivity with respect to the molecules, which is not L-selectins. Methods of production of phage display libraries, comprising sequences of the variable regions of immunoglobulin, known in the art (see, for example, Cesareni, FEBS Lett 307: 66-70 /1992/; Swimmer et al., Proc. Natl. Acad. Sci. USA 89:3756-s 9:1373-1377 /1991/; all these works in their entirety are introduced in the present description by reference/. The resulting sequence of CDR variants "humanized" immunoglobulin then Express in a suitable host that provides an effective expression.

As mentioned previously, DNA sequences can be expressed in the appropriate cell hosts after the sequences are properly attached /i.e., so as to ensure their operation/ sequence regulating expression. For these purposes can be used for expressing the vectors are capable of replication in the host organisms either as episome, or as an integral part of the chromosomal DNA of the host. Typically, the expression vectors contain a selective marker, for example, resistance to tetracycline /tetR/, G418-resistance /neoR/, resistance to mycophenolic acid /gpt/, HSV-tk, which allow to detect cells transformed with the desired DNA sequences (see for example, U.S. patent 4704362, which is fully introduced into the present description by reference/.

One of prokaryotic hosts, they are suitable microbial hosts are bacilli, such as Bacillus Subtilis; and other enterobacteria such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts can also be expressed by vectors, which typically contain regulatory sequences that are compatible with this cell host /for example, the site of replication initiation/. In addition, there may also be any other well-known promoters such as the promoter system of the lactose promoter system, a tryptophan /trp/, promoter system, a beta-lactamase promoter or system of phage lambda. These promoters typically control expression, optionally, through an operator sequence, and are sequence binding site with the ribosome, etc. for the initiation and termination of transcription and translation.

For expression can also be used and other microbes, such as yeast. The preferred host is Saccharomyces, and suitable vectors are vectors that have a sequence controlling the expression, such as promoters, such as promoters for 3-phosphoglycerate or other glycolytic enzymes, and is also the site of initiation of replication, termination sequences, etc. if necessary.

< and culture of plant cells /Larrick & Fry, Hum. Antibodies Hybridomas 2/4/:172-89 /1991/; Benvenuto et al., Plant Mol. Biol. 17/4/:865-74 /1991/; Durin et al., Plant Mol. Biol. 15/2/:281-93 /1990/; Brian Harvey et al., Nature 342:76-8 /1989/; all these works are introduced in the present description by reference/. Preferred plant hosts are, for example, Arabidopsis, Nicotiana tabacum, Nicotiana rustica and Solanum tuberosum. Preferred polygenic expressing cluster suitable for expression of polynucleotide sequences encoding humanized" antibodies of the present invention, directed against L-selectin, is a plasmid pMOG18 in which insertiona polynucleotide sequence encoding a chain of "humanized" immunoglobulin is respectively sewn with CAMV35S-promoter associated with duplicated enhancers; and the indicated plasmid used in accordance with the method described Sijmons et al., Bio/Technology, 8:217-221 /1990/; this work is introduced in the present description by reference/. Alternatively, the expression "humanized immunoglobulins in plants carried out preferably according to the method of Brian Harvey and others above, except that the immunoglobulin sequences used Brian Harvey and others above, replace the polynucleotide sequences encoding humanized the immunoglobulin can be also used vectors on the basis of the T-DNA of Agrobacterium tumifaciens, and preferably such vectors, which contain a marker gene that encodes resistance to spectinomycin, or other marker gene.

For the production of "humanized" immunoglobulins of the present invention can also be used for the culture of insect cells, but is mainly used expressing the system on the basis of baculovirus. "Humanized" antibodies can be produced by expression of a polynucleotide sequence encoding humanized" antibodies, using techniques Putlitz, etc. /Bio/Technology, 8:651-654 /1990/ this work is fully introduced into the present description by reference/. Method Putlitz, etc. can be modified, for example, instead of the cDNA sequences of the heavy and light chains of murine monoclonal antibodies Ab6A4 used Putlitz, etc. can be introduced polynucleotide sequence encoding humanized" antibodies against L-selectin.

For the expression and production of polypeptides of the present invention, in addition to microorganisms and plants can be used in cell culture mammals (see Winnacker, From Genes to Clones (VCH publishers, NY, 1987; this work is fully introduced in the present description Posada cell line host, able to secrete intact immunoglobulins, for example cell line CHO, different cell line COS, HEIa cells, preferably cell lines, myeloma, etc., or transformed B-cells or hybridoma. Expressing the vectors used for these cells can include regulatory sequences, such as the site of initiation of replication, a promoter, enhancer /Queen and others, Immunol. Rev. 89:49-68 /1986/; this work is fully introduced into the present description by reference, as well as the necessary sites, regulatory processing, such as sites of ribosome binding sites, RNA splicing, polyadenylation sites, and the sequence termination of transcription. Preferred regulatory sequences are promoters derived from immunoglobulin genes, SV 40, adenovirus, bovine papilloma virus, cytomegalovirus, etc., Usually expressing a vector containing the marker gene such as neoR.

Transgenes encoding "humanized" immunoglobulin of the present invention, can be used to generate transgenic animals, non-human, which Express a "humanized" immunoglobulin, usually in secreted fluids orderyou "humanized" immunoglobulins and properly connected to the promoter, usually with attached enhancer, such as immunoglobulin rodents or promoter/enhancer casein gene /Buhler et al., Bio/Technology 8: 140-143, /1990/; Meade et al., Bio/Technology 8:443-446 /1990/; these works are entered entirely into the present description by reference/. Transgenes can be introduced into cells and embryos in accordance with known methods, resulting in /see below/ can be obtained homologous recombinant constructs. Preferred animals, non-human, are mice, rats, sheep, cows and goats; however, preferably, if the expression is used cow's milk. Cm. WO 91/08216 /1991/ /this work is introduced in the present description by reference/. Cleaning "humanized" antibodies of the present invention is carried out according to known purification methods commonly used for the purification of immunoglobulins.

The vectors containing the DNA segments of /for example, sequences encoding heavy and light chain, and regulatory sequences/, can be transferred into cells-the owners of the known methods, the choice of which depends on the type of host cell. For example, prokaryotic cells are usually applied transfection using calcium chloride, whereas for listica, transduction with virus or electroporation. For plant cells and tissues, it is preferable to use the ballistic transgenesis using tungsten particles In General, see Maniatis et al. , Molecular Cloning: A Laboratory Manual /Cold Spring Harbor Press, 1982/; this work is introduced in the present description by reference/.

After expression of intact antibodies, their dimers, individual light and heavy chains, or other immunoglobulin of the present invention can be purified according to standard procedures such as, for example, precipitation with ammonium sulfate, affinity chromatography, column chromatography, gel electrophoresis/in General, see Scopes, R., Protein Purificatin (Springer-Verlag, NY, 1982; this work is entirely introduced into the present description by reference/. Basically, purified immunoglobulin, having, preferably, a homogeneity of at least about 90-95%, and most preferably 98-99% can be used for pharmaceutical purposes. Then cleared /partially or to homogeneity/ polypeptides can be used for therapeutic purposes/for example, in vitro and/ or in research and analytical procedures, immunofluorescent staining, etc. /in General terms, the invention produced a "humanized" immunoglobulins, associated with L-selectin with affinity binding component of at least 1107M-1under standard binding conditions /for example, phosphate-buffered saline containing 2% fetal calf serum; if 25oC/. One example of such "humanized" immunoglobulins is "humanized" antibody DREG-200, having the amino acid sequence shown in Fig. 2. /Further, in some cases, the "humanized" antibody DREG-200 to read "people DREG-200"/. Humanized immunoglobulins that contain a CDR derived from a murine antibody DREG-55 or from the mouse antibody DREG-56, can also contact L-selectin with affinity, which constitutes at least 1107M-1.

Humanized antibodies of the present invention connected in a standard binding conditions) with L-selectin preferably with an affinity, which constitutes at least 1 of 108M-1more preferably with affinity, which constitutes at least 1108M-1; and most preferably with an affinity, which constitutes at least 11010M-1or higher. Usually, the binding affinity of humanized" immunoglobulin approximately t is finest mouse antibody DREG-200 is about 108M-1.

Computers

In another embodiment, its implementation of the present invention relates to computers for generating a three dimensional image of antibodies on the monitor. For example, for the purposes of the present invention can be used in automated working installation of Silicon Graphics IRIS 4D, working in the UNIX operating system, and a software package for molecular simulation QUANTA (Polygen Corp. USA). Computers can be used to generate variants of "humanized" antibodies. Generally speaking, antibodies of the present invention already have sufficient binding affinity of. However, it is clear that antibodies even with stronger affinity binding can be identified by subsequent variations of some amino acid residues. Using three-dimensional images can also identify many non-critical amino acids, which can be objects of conservative substitutions that do not have a significant effect on the binding affinity of this antibody. However, even a conservative substitution, taken together, can have a significant impact on the properties of the immunoglobulin. But many individual conservative substitution, in all probability, against L-selectin

In another embodiment, the present invention relates to human antibodies against L-selectin. These antibodies produced using various techniques, described below. Some human antibodies chosen by experiments on competitive binding or otherwise, by determining whether these antibodies with the same specificity of the antigenic determinants that specific mouse antibody, such as a murine antibody DREG-200 or "humanized" version. These antibodies have, in all probability, shared valuable therapeutic properties demonstrated for "humanized" antibody DREG-200.

Antibodies having the desired specificity of the antigenic determinants can be identified by screening for the ability to block the interaction between neutrophils and endothelial cells. A simple visual analysis to detect such interaction was described by Kishimoto and others (1991) (see above). Briefly, this method consists in the fact that the monolayers of cells derived from the umbilical vein of a person, stimulate interleukin IL -1. Then these layers under certain conditions, add neutrophils, raw or pre-processed test anticelulitic from patients with deficiency of adhesion of leukocytes. Cm. Anderson and others Ann. Rev. Med. 38:175 (1987). Neutrophils from these patients do not have receptors integrin, which is very convenient, since binding of these receptors with neutrophils could obscure the effects of blocking binding to L-selectin.

A. Methodology using creamy

The essence of this method and suitable partner to obtain cell hybrids (SPAZ-4) used in this method, described Oestberg and others (Hybridoma 2:361-367 (1983); Oestberg (U.S. patent 4634664) and Engleman and others (U.S. patent N 4634666) (each of these works in their entirety is introduced into the present description by reference). Cell line producing the antibody and obtained in this way were called triomune, because they come from three cells, two human and one mouse. First, the cell line of mouse myeloma was hybridisable with B-lymphocyte person, resulting in a received xenogenic hybrid cell that is not producing the antibody, such as cell line SPAZ-4, described Oestberg (see above). Then the xenogenic cell was hybridisable immunized with B-lymphocyte person, resulting in a received cell line trioma producing the antibody. It was found that trisomy produce antibody more than you got from the blood, spleen, lymph nodes or bone marrow donor. In vivo immunization of people living with L-selectin undesirable because of the risk of producing an adverse response. Therefore, B-lymphocytes, usually subjected to immunization in vitro with the use of the polypeptide of L-selectin, its antigenic fragment, or cells carrying the indicated polypeptide or its fragment. If you want to obtain antibodies against a specific antigen or epitope, for in vitro immunization is preferable to use this antigen or epitope. B-lymphocytes, usually exhibit antigen within 7-14 days in this environment, as RPMI-1640 (see Engleman, see above) containing 10% human plasma.

Immunized B lymphocytes hybridizing with xenogenic hybrid cells, such as SPAZ-4, a well-known ways. For example, cells treated with 40-50% polyethylene glycol (MW 1000-4000) approximately at 37oC for about 5-10 minutes. The cells are then isolated from the hybridization mixture and cultured in selective medium to obtain a desired hybrids (e.g., HAT or AH). Clones secreting antibodies with the desired binding specificity, identified by analysis of the environment trimnell culture on the ability to bind to L-selectin or podologie serial dilution and cultured in vitro in culture medium. After that, the obtained cells creamy examined for the ability to bind to L-selectin or its fragment.

Although trisomy are genetically stable, they do not produce very high levels of antibodies. The levels of expression can be increased by cloning of antibody genes derived from trisomy, in one or more expressing vectors with subsequent transformation of the vector into the cell line, such as discussed cell line used for expression of recombinant or humanized" immunoglobulins.

b. Transgenic mammals, non-human

Human antibodies against L-selectin can also be produced from a transgenic mammal that is not a person, and containing transgenes that encode at least a segment of the locus of human immunoglobulin. Usually endogenous immunoglobulin locus such transgenic mammals are functionally inactivate. Preferably, if the segment is the locus of human immunoglobulin includes prearrangement sequence components of heavy and light chain. Inactivation of endogenous genes of the immunoglobulin and the introduction of exogenous genes immunocom. The resulting transgenic mammals have the ability to function rearrangeable sequence components of the immunoglobulin, as well as the expression of serum spectrum of antibodies of different isotypes encoded by genes of the immunoglobulin, but these mammals do not have the ability to Express genes of endogenous immunoglobulin. The production of these mammals and their properties are described Lonberg and others, 093/12227 (1993); Kucherlapati, WO 91/10741 (1991) (each of these works is completely introduced into the present description by reference). Especially suitable are transgenic mice. Antibodies against L-selectin can be obtained by immunization of transgenic mammals, non-human (e.g., described by Lonberg or Kucherlapati (see above)), using L-selectin or its fragment. Monoclonal antibodies can be obtained, for example, by hybridization of B-cells obtained from the above-mentioned mammals, with appropriate lines of myeloma cells using known techniques Kohler-Milstein.

c. Methods ragovoy display

Another way to get people of antibodies against L-selectin is screening a DNA library vyzyvayushaya with L-selectin, or its fragments. Then clone and amplified sequences encoding these antibodies (or binding fragments). The Protocol developed Huse, can be used more effectively in combination with the methodology ragovoy display. See, for example, Dawer and other WO 91/17271 and Mc Cafferty and other WO 92/01047 (each of these works is entered in its entirety in the present description by reference). This technique produced library of phage, the members of which display on their surface a variety of antibodies. These antibodies are usually FV - or Fao-fragments. Selection of phage, indicating that the antibody with the desired specificity, carry out the highest affinity in respect of the polypeptide of L-selectin or its fragment.

In one of its variants method ragovoy indication is that can be produced human antibodies having the binding specificity of the selected mouse antibodies. Cm. Winter, WO 92/20791. In this method, as the source material used variable region or a heavy or light chain selected mouse antibodies (for example, mouse antibody DREG-200/. If, for example, as the source material is chosen variable region l of the region of light chain (so E. murine source material) and different variable regions of the heavy chain. Variable regions of the heavy chain is obtained from the library rearanging variable regions of the heavy chain of human immunoglobulin. Then select phage that detect strong specific binding to L-selectin (e.g., at least 108M-1and preferably at least 109M-1). Variable region of the heavy chain of the person obtained from this phage is used later as a starting material for constructing additional ragovoy library. In this library, each phage points to the same variable region of the heavy chain (i.e., the region identified from the first ragovoy library) and on different variable region of the light chain. Variable region of the light chain is obtained from the library of rearranged variable regions of the light chain of a human. And similarly select phage that detect strong specific binding to L-selectin. This phage indicates variable regions that belong to the entire human antibody against L-selectin. These antibodies are usually of identical or similar to the specificity of the antigenic determinants, Stela of the present invention can be used for the treatment of diseases, associated with inflammatory processes, in particular, mediated by neutrophils or T cells. The preferred application of the antibodies of the present invention is the treatment or prevention of conditions such as ischemic-reperfusion damage caused by myocardial infarction, stroke (for example, "blow"), renal, hepatic or splenic infarction, brain surgery, heart surgery (for example, arteriogenesis bypass surgery), plastic surgery on vessels, etc., Other preferred uses of the antibodies of the present invention is the treatment of sepsis, respiratory distress syndrome in adults and failure of various organs. Antibodies of the present invention can be used to treat a variety of injuries caused by trauma, burns, frostbite, or spinal cord injury. In addition, antibodies of the present invention can be also used for the treatment of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, diabetes I and uveitis; for the treatment of inflammatory skin diseases such as psoriasis, and for the treatment of meningitis and encephalitis. Other typical p is ewusi "graft versus host".

Any immunoglobulin of the present invention can be used in combination with other antibodies, particularly human or humanized" antibodies that bind with other adhesion molecules. For example, suitable for this purpose are immunoglobulins with specificity to CD11A, CD11b, CD18 and E-selectin, P-selectin, and ICAM-1. Other suitable antibodies are antibodies specific to lymphokines, such as IL-1, IL-2 and IFN- , and other receptors.

Antibodies of the present invention can also be used as a separate input compositions used in combination with chemotherapeutic agents. Such agents can be non-steroidal anti-inflammatory drugs and corticosteroids or other well-known drugs (eg, cyclosporine). Basically, the immunoglobulins of the present invention are intended for use in combination with other drugs commonly used in modern medicine for the treatment of specific diseases.

In some therapeutic methods antibodies against L-selectin can be used in combination with thrombolytic agents. In reneerenee coronary artery. Reopen blocked coronary artery can be achieved by the introduction of thrombolytic agents, dissolving clots caused by obstruction of the artery, thereby reducing coronary blood flow. Reperfusion of blood vessels can be also achieved by an emergency transcutaneous catheter coronary angioplasty (PTCA), carried out with the help of a balloon dilatation of the arteries are clogged and narrowed segment of the coronary artery. However, when using these methods restore coronary blood flow leads to ischemic - reperfusion damage of the blood vessels.

In the methods of the present invention ischemic-reperfusion injury of blood vessels can be reduced or prevented by the use of combination thrombolytic funds or PTCA, and human or humanized" antibodies against L-selectin. Antibodies used for preventive purposes, usually do before the introduction of or simultaneously with the introduction of thrombolytic agent or the initiation of the PTCA. Often after or during the introduction of thrombolytic agent or holding angioplasticheskih treatment administered additional doses of the antibody. The interval between the introduction of preventive antic is but 5-20 min, and more preferably 5-10 minutes Antibodies of the present invention is administered parenterally, preferably by intravenous injection, in doses, comprising 0.1 to 10 mg/kg body weight, preferably 0.14 to 5 mg/kg, and most preferably 0.3 to 3 mg/kg of the Indicated antibodies can be introduced in the form of an intravenous loading dose, for example, within 1 to 5 minutes in the form of repeated injections with lower doses or intravenous infusion. Injection loading dose is typically used for prophylactic doses or in critical cases. Additional doses of antibodies can be re-entered (e.g., every 4-6 hours), during or after thrombolytic or angioplasticheskih the treatment of acute myocardial infarction in the same amounts as described above, in order to achieve optimal levels of antibodies in the plasma.

Thrombolytic agents are drugs with the ability to directly or indirectly stimulate the dissolution of blood clots in vivo. Such thrombolytic agents are tissue plasminogen activator (see EP-B0 093619), activase, alteplase, dataplate, citiplaza, streptokinase, anistreplase, urokinase, heparin, warfarin and coumarin. Dopolnitelnie, 6:449-458 (1987); PCT-EP 90/00194; U.S. patent 4 970 159). Thrombolytic tools administered to the patient in a quantity sufficient to partial resorption, or for the prevention of blood clots and their complications. The amount adequate to accomplish this purpose, is defined as "therapeutically effective dose" or "effective dose". Amount, effective for use for the above purposes, depend on the severity of the condition, the General state of the patient, the route of administration and used in combination with other drugs. Often therapeutically effective dose of thrombolytic agents and schemes of their introduction are approved by Management under the control over quality of the food and drug administration (FDA) for independent use, for example, for alterati this dose is 100 mg, and for streptokinase 1.5 million ME.

The preferred composition of the present invention provides for the use of immunoglobulin of the present invention, included in immunotoxins, which neutralize the cells expressing L-selectin. Immunotoxins are characterized by the fact that they consist of two components and can be used for lysis of selected cells in vitro or in vivo. One component of immunotoxin prebble. Another component, known as "hard media", is a means of delivering the toxic agent to the cells of a particular type, such as cells expressing the antigenic determinants of L-selectin. These two components are usually connected with each other by chemical means using well-known procedures. For example, if the cytotoxic agent is a protein, and the second component is an intact immunoglobulin, these components can be linked by cross-linking agents (cross-linkers), such as SPDP, carbodiimide, glutaraldehyde, or so on, Producing different immunotoxins well known in the art, see, e.g., "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982), which is introduced in the present description by reference. These components can also be linked genetically (see Director et al., Nature 339: 394 (1989); this work is introduced in the present description by reference).

To obtain immunotoxins can be used in a variety of cytotoxic agents. Such cytotoxic agents can be radionuclides, such as iodine-131 or other isotopes of iodine, yttrium-90, rhenium-188, and bismuth-212 or d is n and cisplatin; and cytotoxic proteins, such as proteins, inactivating the ribosome, for example antiviral protein Fallaci American, exotoxin A of Pseudomonas, ricin, diphtheria toxin a chain of ricin A, etc., or an agent active at the cell surface, such as a phospholipase (e.g., phospholipase C). (See in General, the assigned application for U.S. patent reg. N 07/290968; "Chimeric Toxins', Olsnas & Phil, Pharmac. There., 25:355-381 (1982) and Monoclonal Antibodies for Cancer Detection and Therapy (ed. Baldwin & Byers, Academic Press, 1985), pp. 159-179, 22-266; all these works are introduced in the present description by reference).

As a component used to deliver cytotoxic agent, can be used immunoglobulins of the present invention. Preferred are intact immunoglobulins or their binding fragments, such as Fab or Fv. Typically, the antibodies used in immunotoxin are human immunoglobulin isotype IgM or IqG, but, if necessary, can also be used constant region of immunoglobulins of other mammals.

Antibodies and pharmaceutical compositions of the present invention are used preferably for parenteral administration, such as subcutaneous, intramuscular or vnutri gavage feeding or lavage, for intraperitoneal injection, for the manufacture of ophthalmic ointments and ointments for external use, for intracranial injection (usually in a ventricle of the brain), for intrapericardially injection or for intrasynovial injection. Compositions for parenteral administration generally contain the solution of the immunoglobulin or its mixture, dissolved in an acceptable carrier, preferably an aqueous carrier. For these purposes, can be used in a variety of aqueous media such as water, sauverny water, a phosphate buffer solution (PBS); and 0.4% saline, 0.3% glycine, albumin human, etc., These solutions are sterile and generally do not contain any particles. Compositions of the present invention can be sterilized by standard methods, well known to specialists. These compositions can contain pharmaceutically acceptable additives needed to create the physiological conditions such as pH adjustment and tabularasa agents, agents regulating toxicity, and so on, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The concentration of antibody in these compositions can vary widely, for example, from about less cheat, primarily based on the volume of fluid, viscosity, etc., and depending on the selected method of administration.

So, for example, a typical pharmaceutical composition for injection may contain 1 ml sterile buffered water and 1-70 mg of immunoglobulin. A typical composition for intravenous infusion may contain 250 ml of sterile ringer's solution, and 150 mg of antibody. Methods of receiving parenteral compositions is well known in the art and are described in detail in the literature (see , for example Remington''s Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pennsylvania, 1980; this work is introduced in the present description by reference). Compositions suitable for lavage or for other routes of administration may be made in appropriate forms depending on the purpose of their use. Some pharmaceutical compositions may contain antibody against L-selectin, and thrombolytic agent.

To store the antibodies of the present invention can be frozen or liofilizovane and then restore them in the appropriate media immediately before use. It was found that when using conventional immunoglobulins can be applied with success , recovery can lead to varying degrees of loss of antibody activity (for example, in the case of normal immunoglobulins IgM antibodies detect a greater tendency to activity loss than IgG antibodies), and therefore it may be necessary to adjust the levels used for compensation of possible losses.

Compositions of the present invention containing antibodies or their mixture can be introduced in the preventive and/or therapeutic purposes. For therapeutic purposes the composition is administered to a patient suffering from inflammatory diseases, in a quantity sufficient to cure or at least partial suppression of this disease and its complications. The amount adequate to accomplish this purpose, is defined as "therapeutically effective dose". The effective dose depends on the severity of the disease, the General condition of the patient's immune system and is largely comprised of from about 1 to about 200 mg of antibody per dose, and usually from 5 to 70 mg per patient. The scheme is the introduction of a medicinal product may vary depending on the specific disease and the patient's condition and usually ranges from one loading dose or continuous infusion daily to multiple injections within 1 day (for example, every 4-5 hours) in accordance with the purpose lechago to be used in case of serious diseases in life-threatening or potentially life-threatening situations. In such cases, to minimize the presence of foreign substances and reduce the likelihood of rejection of "foreign material", which is achieved through the use of immunoglobulins of the present invention, it may be desirable introduction of excessive amounts of these antibodies.

In the prophylactic use of the composition, the content of the antibodies of the present invention or a mixture thereof, is administered to the patient, yet not suffering from a specific disease, in order to strengthen its resilience. The number of antibodies that are entered for these purposes, is defined as "prophylactically effective dose". In such cases, the exact number of antibodies also depend on the General health of the patient and General level of immunity and generally comprise from 1 to 70 mg per dose. The preferred prophylactic purposes by use of the compositions of the present invention is the prevention of respiratory distress syndrome in adult patients suffering from trauma or sepsis; prevention of rejection of transplanted organ and the prevention of reperfusion injury in patients suffering from ischemia. For seriously ill patients can be used dose comprising from about 50 to 150 m schemes (i.e., single or multiple introduction) compositions of the present invention is usually performed by the attending physician. But in any case, the pharmaceutical composition should include a certain amount of antibody sufficient for effective treatment of the patient.

In addition, antibodies of the present invention can find wide application in in vitro-issledovaniyakh. For example, these antibodies can be used for detection of antigens of L-selectin in order to highlight specific cells or etc.

Other examples of such applications include, but are not limited to, the "humanized" immunoglobulin DREG-200, which can be immobilized and subjected to interaction with blood taken from the patient, to remove blood cells bearing the antigen, L-selectin, then the remaining blood with remote L-selectin-bearing cells can be reintroduced to the patient. Any residual "humanized" antibodies are present in L-selectin-depleted and reintroduced to the patient's blood (e.g., the removal from immobilizing media) will have very small or very small antigenicity compared with the antibody.

When used to diagnose the other labeled antibodies ("second" antibody), which are reactive toward "humanized or human antibodies, such as antibodies, specific for the constant regions of a human immunoglobulin. Alternatively, antibodies can be subjected to tagging. This can be used various labels a wide range, such as radionuclides, fluorescent substances, enzymes, enzyme substrates, conferment, enzyme inhibitors, ligands (particularly haptens), etc. for this purpose can be used various types of immunoassays, well-known specialists.

The following examples illustrate the present invention and should not be construed as a limitation of the scope of the invention. It should be noted that although the following examples relate to the use of mouse antibody DREG-200 for the production of "humanized" antibodies against L-selectin with high affinity binding, however, for the production of "humanized" antibodies can also be used hypervariable region (CDR) derived from a murine antibody DREG-55, mouse antibody DREG-56 or other monoclonal antibodies which are able to bind epitope L-selectin.

Premeeting antibody DREG-200 cloned using polymerase chain reactions (described Co and other C. J. Immu. Vol. 148:1149 (1992), and assigned application U.S. reg. N 07/634278) and 3'-primers, which hybridize with a constant regions and contain HindIII sites, and 5'-primers, which hybridize with IgG-ends and contain EcoRI sites. PCR amplificatoare fragments were digested with the enzymes EcoRI and HindIII and cloned into the vector pUC18 for sequencing. For mouse antibody DREG-200 were sequenced at least two gamma-1-specific clone and two Kappa-specific clone. Gamma 1-clones and Kappa-clones had identical sequences. cDNA sequence of the variable domain and introduced amino acid sequence shown in Fig. 1.

Example 2: Computer simulation "humanized" antibodies

To maintain the high affinity binding in the "humanized" antibodies were carried out following the procedure described Queen and others (see the Queen and other Proc. Natl. Acad. Sci. USA 86:10029 (1989) and WO 90/07861; these works are fully entered into the present description by reference). For the original donor mouse antibodies it is necessary to choose more homologous to human acceptor antibody, as in this case, when combining murine CDR regions with the person of frame areas less likely is if homology (i.e., the percentage of sequence identity) between the frame variable region of the heavy chain humanized" immunoglobulin and frame variable region of the heavy chain donor immunoglobulin is at least 65%. Usually, to obtain frame sequences of the heavy and light chain chosen from the same human antibodies in order to reduce the incompatibility in the Assembly of these two circuits. In the search for homologous sequences from a database of protein sequences (performed using the software Micro Genie Sequence Analysis Soft Ware (Beckman)) was selected antibody Eu, which was then used to obtain frame sequences for the "humanization" of the mouse antibody DREG-200.

To construct a model of the variable regions of the mouse DREG-200 used a computer program ENCAD(Levitt, J. Mol. Biol. 168:595 (1983); this work is introduced in its entirety by reference). The resulting model was used to determine amino acid sequence in the frame region DREG-200 mouse, which was close enough to the CDR regions, in order to be able to interact with these obla" DREG-200 at each position the amino acids were chosen so that so that they coincided with the amino acids in the antibody Eu, unless this provision does not fall under one or more of the following 5 categories:

(1) This regulation is within the scope CDR;

(2) amino acid at Eu is not typical for human antibodies at that position, and the amino acid mouse DREG-200 is typical for human antibodies at that position;

(3) This provision is in the immediate vicinity of the CDR;

(4) In accordance with the above described model has the assumption that this amino acid may be in close proximity to antigennegative region (CDR).

For provisions that fall under these categories were used amino acids from the murine antibody DREG-200.

In addition, a specific provision could fall under the fifth category, namely:

(5) the Amino acid Eu is highly unusual for human antibodies at that position, the amino acid DREG-200, though differs from the amino acid Eu, but is also uncharacteristic. In these cases, for the given position used amino acid, typical for human antibodies.

Amino acids each category are shown in Table 1. Some aminoxy and heavy chain humanized" antibody DREG-200 shown in Fig. 2 in comparison with the sequences of the mouse DREG-200.

For constructing genes encoding the "humanized" antibodies, were selected (mainly using codons found in the mouse sequence) a nucleotide sequence encoding the protein sequence of the heavy and light chains "humanized" antibodies, including signal peptides. To create restriction sites or to remove unwanted sites some degenerate codons were replaced. Nucleotide sequence of the genes also include donor splicing signals and XbaI site at each end. The nucleotide sequence and encoded the variable domains of the light and heavy chain humanized" immunoglobulin shown in Fig. 3. Each gene was constructed from four overlapping synthetic oligonucleotides described in the literature (see Co and others, J. Immunol. 148:1149 (1992), and assigned application U.S. reg. N 07/634278, these works are wholly entered into the present description by reference). Then the genes encoding the variable regions of heavy and light chains, ligated into XbaI sites expressing vectors pVg1-dhfr or pVk (see assigned application U.S. reg. N 07/634278) sootvetstvuyuschih, well known in the art (Maniatis and others, see above).

Plasmids for the heavy and light chains were introduced into cells of the mouse myeloma Sp2/O by electroporation, and cells were selected for expression of gpt. Clones were skanirovali by assessing the production of human antibodies in the culture supernatant using ELISA and clones of the best production was isolated antibody. Then "humanized" IgGI-antibody DREG-200 was purified by passing the supernatant tissue culture through the column with Separate staphylococcal protein CL-4B (Phafmacia). Bound antibody was suirable 0.2 M Glycine-HCl (pH 3.0) and neutralized 1 M Tricom (pH 8.0). Then the buffer was replaced with PBS by passing through a PD10 column (Pharmacia) or by dialysis. To obtain cells that produce high levels of antibodies, transfected clones can be cultured in increasing concentrations of methotrexate.

For the production of "humanized" antibody DREG-200 isotype IgG4 was first constructed another vector pVg4-dnfr. For this XbaI-BamH1 fragment of the vector pVg1-dnfr containing a constant 1-region, was replaced by a fragment (about 2000 p. O.) gene constant 4-region of human immunoglobulin (Ellison and Hood, Proc. Natl. Acad. Sci. USA 79:1984 (1982)) to the th for CH4-exon of this gene, using known techniques, including polymerase chain reaction. Then, the gene encoding the variable region of the heavy chain humanized" antibody DREG-200, cloned into the Xba1 site of the vector pVg4-dnfr. The resulting plasmids carrying the gene for the heavy chain, together with the above-described plasmid carrying the gene for the light chain, was introduced into cells Sp2/O, clones were selected,

a "humanized" antibody DREG-200 isotype IgG4 was purified as described above for antibody IgG1.

Example 3. Properties "humanized" antibodies

The affinity of the humanized antibody DREG-200 against L-selectin was determined by competitive binding with mouse antibody DREG-200, labeled with radioactive iodine (Fig. 4). The affinity of binding was calculated according to the method Berzofsky J. A. Berzofsky, and L. J. Berkower, in"Fundamental Immunology (ed. W. E. Paul, Raven Press (New York), 595 (1984), this work is entirely introduced into the present description by reference). The affinity of "humanized" antibody DREG-200 approximately 2 times higher than the affinity of a murine antibody DREG-200. Similar results can be obtained if the affinity of antibodies against L-selectin to measure in human neutrophils.

The ability of murine and humanized" antibody DREG-200 block adhesion of neutrophils to indotel is particularly endothelial cells of the umbilical cord (HUVEC, Clonetics, San Diego) were cultured to the condition of continuity in EGM medium (Clonetics) on slides with wells Lab-Tek 8 (Nunc, Naperville, IL). Before using HUVEC cells stimulated for 4 hours with 20 ng/ml IL - 1 (R&D Systems, Minneapolis, MN). By centrifugation in density gradient neutrophils were isolated from bright layers of a blood clot, which was purified from erythrocytes by means of precipitation with dextran, after which their number was brought up to 107/ml Neutrophils (100 ál) pre-incubated for 20 minutes on ice with different concentrations of antibody (100 μl RPMI). Slides with HUVEC cells were washed in the absence of 1L - 1 and were placed in a rotary shaker (100 rpm./min) at 4oC. Then the wells were added to untreated or treated with antibody neutrophils and slides were incubated on a shaker for 30 minutes at 4oC. Then slides were washed by dipping 10 times in a vessel with RPMI-medium, fixed in 1% glutaraldehyde in RPMI and left in air for drying. Quantitative evaluation of adhesion of neutrophils was performed by counting neutrophils related to specific areas of the monolayer of endothelial cells, when SIP is of neutrophils to HUVEC, while irrelevant control antibody for such blocking unable. "Humanized" IgG4-antibody DREG-200 similarly blocks the binding of neutrophils to endothelial cells.

Example 4.

The effect of antibodies people. DREG-200 damage myo - after reperfusion.

Investigated the extent of real protective effect "humanized" antibody DREG-200 isotype IgG4 (Ochel. DREG-200) to the ischemic tissue of the myocardium after reperfusion. Male adult cats (2.8 to 4.2 kg) were anestesiologi sodium pentobarbital (30 mg/kg, i. v.). In the middle section was introduced tracheotomies cannula, after which the cats were placed in conditions of artificial lung ventilation under variable positive pressure (small respirator for animals, Harvard, Dover, MA). In the right external jugular vein was injected polyethylene catheter for the introduction of additional pentobarbital to maintain the desired level of anesthesia during surgery and for the introduction of antibodies. Another polyethylene catheter was inserted through the left femoral artery and placed in the abdominal aorta for measurement of mean arterial pressure (MOOR) using a pressure sensor (Kobe Instruments, Lakewood, CO). After sternal thoracotomy dissected anterior pericardium is from its beginning. Then through the apical hole to the left of the perch were introduced high-precision pressure transducer-tipped catheter (Model MPC 500 with control unit - Model TCB 500, Milla Instruments Inc., Houston, TX). The catheter was determined by monitoring LV-pressure and form dP/dt-wave, and then fixed in place with a silk suture. To determine heart rate (HR) and elevation ST-segment used a standard lead II scalar electrocardiogram (ECG). Elevation of the ST segments were determined by analyzing the ECG recording at 50 mm/s every 2O minutes. ECG, MABP, LVP and dP/dt controlled by the monitor unit, Hewlett - Packard 78304 A(Hewlett - Packard, Palo Alto, CA) and recorded on oscillographically recorder (Gould Inc., Cleveland, OH) every 20 minutes. The PRI index, the approximate requirement of a myocardium in oxygen, was calculated as the ratio of MABP and HP/1000.

After completion of all surgical procedures cats were left for 30 minutes to stabilize and during this time wrote a basic ECG readings, MABP, LVP and dP/dt. Ischemia, infarction (MI) was induced by tightening reversible ligatures, initially placed around the LAD, in order to achieve a complete vessel occlusion. This situation was defined as zero time. 80 minutes after coronary occlusion was intravenously injected the Chennai" control Mab, appropriate isotype IgG4) (10 minutes before reperfusion, R). Then after 10 minutes (i.e., generally within 90 minutes after coronary occlusion, (I) the ligature around the LAD started and ischemic myocardium subjected to reperfusion for 4.5 hours.

Animals were randomly divided into three basic groups. The group of six cats with false MI + R was introduced Ochel. DREG-200 (2 mg/kg), group of 6 cats MI + R was administered to the control antibody Mab hu ABL - 364 (2 mg/kg) and the group of 6 cats MI + R was introduced Ochel. DREG-200 (2 mg/kg). A group of cats with false MI + R were subjected to the same surgical procedures except that the LAD coronary artery was not subjected to occlusion.

After a 4.5-hour period of reperfusion the ligature around the LAD was again delayed. Then in the left ventricle quickly were injected with 20 ml of 0.5% Evans blue for staining zone of the myocardium, which were subjected to perfusion through the open coronary arteries. This zone is "under threat" was determined by negative staining. Immediately after this injection, the heart was quickly excised and placed in a heated, oxygenated K-H solution. Left circumflex (LCX) and LAD coronary artery was isolated and removed for subsequent research on vasoactivity coronary rings and the section thickness of 3 mm parallel to the atrioventricular sulcus. Unpainted part of the myocardium (i.e., the full risk or ischemic area) was separated from the part of the myocardium, painted blue Evans (i.e., safe zone or neishemicescoy area). The area under threat (or risk) cut into small cubes and incubated in 0.1% nitrocine of tetrazole in phosphate solution (pH of 7.4) at 37oC for 15 minutes. Tetrazolium dye forms a blue formosanus complex in the presence of myocardial cells containing active dehydrogenase and their cofactors. Irreversibly damaged or necrotic part of infarction risk zones, which were not stained, was separated from the painted parts of the myocardium (i.e., ischemic, but not necrotic zone). After that, all three parts of the myocardium (i.e., neishemicescoy, ischemic, but not necrotic and ischemic necrotic tissue) were weighed. The results were expressed as the area of necrotic cardiac tissue, calculated as % or from areas of risk, either to the total weight of the left ventricle.

In accordance with these criteria the damaged cardiac tissue was significantly reduced (p < 0,001) in cats treated Ochel. DREG-200. Then, as the group treated with the control antibody, about 30% of infarction risk zones developed in necroth,01), i.e. decreased 50-60% (see Fig. 6). Significant differences between wet mass risk areas, expressed as a percentage of the total mass of the left ventricle, for two ischemic groups were observed, which indicates that myocardial ischemia is exposed to relatively the same area in these two groups. Thus, the humanized antibody DREG-200 provides significant protection from damage after reperfusion.

Notable ability Ochel. DREG-200 to protect ischemic tissue was also illustrated by measurements of the activity of creatine kinase in plasma biochemical marker of myocardial damage. Samples of arterial blood (2 ml) were taken immediately after the application of ligatures, and then every hour. This blood was collected in plastic tubes containing 200 ME of sodium salt of heparin. The obtained sample was centrifuged at 2000 x g at 4oC for 20 minutes, after which the plasma decantation for biochemical analysis. The protein concentration in plasma was determined using biureta by the method of Gornall, etc., J. Biol. Chem. 177:751-766 (1949). The activity of creatine kinase (ck) in plasma was measured according to the method of Rosalki, J. Lab. Clin. Med. 69:696-705 (1967) and expressed as ME/µg protein.

Cats with false MI/R receiving Ochel. DREG-200, p is two ischemic groups CK activity in plasma was slightly increased during myocardial ischemia. In cats treated with hu ABL-364, the CK activity in the blood increased significantly during the first 30 minutes after reperfusion and remained elevated for at least 4 hours of reperfusion period. In contrast, ischemic group treated Ochel. DREG-200, had a significantly lower CK activity in plasma than in ischemic group treated with hu ABL-364 (p < 0,05). This effect was observed during the entire reperfusion period, testified that Ochel. DREG-200 has a significant protective effect against myocardial injury after reperfusion.

Example 5: Effect of Ochel. DREG-200 on the function of the heart

The influence of Ochel. DREG-200 (IgG4 isotype) on heart function was determined by measuring the pressure in the left ventricle (LVP), the first derivative of LVP, dP/dtmax, index of contractility. All data were obtained using a pressure gauge, introduced by the catheter tip into the cavity of the left ventricle. All three groups of cats, discussed in the previous example showed relatively the same initial values of the cardiac parameters. For the group with false MI during the entire experimental period (6 hours) was not observed significant changes in the values of dP/dt<5%. In cats treated with hu ABL-364, myocardial contractility did not show significant recovery. However, Ochel. DREG-200-treated MI/R-cats, dP/dtmaxrecovered to control values after three hours after reperfusion. Thus, after a 4.5-hour reperfusion dP/dtmaxwas significantly lower in hu ABL-364-treated cats than Ochel. DREG-200-treated cats (p < 0,01). The results showed that Ochel. DREG-200-antibody not only reduces postreperfusion necrosis of the myocardium during ischemia, but also contributes to the improvement of the mechanical functions of the heart.

In accordance with the foregoing it should be noted that the immunoglobulins of the present invention have several advantages compared with other antibodies specific for L-selectin. Compared with mouse monoclonal antibodies, the immunoglobulins of the present invention can be produced with lower economic costs and contain significantly less alien amino acid sequence. This reduces the likelihood of production of antigenicity after their introduction to the man, which significantly increases therapeutic effect of these antibodies.

All publications and patentem links. Although the present invention is described in detail with specific examples of its implementation, however, it should be stated that these examples are only illustrative purposes, and therefore it is understood that in the present invention can be made various changes and modifications without leaving the scope of the following claims.

1. Humanitarianly immunoglobulin specific for the protein L-selectin person having a region defining complementarity (DR) corresponding DR-areas of donor mouse immunoglobulin, and a frame region of the variable region heavy and light chain acceptor human immunoglobulin, and the specified humanitarianly immunoglobulin specifically binds to L-selectin person with a constant affinity, which constitutes at least 107M-1and the sequence of the frame region of the variable region of the heavy chain of the specified gumanitarnogo immunoglobulin 65% or more identical to the sequence of frame region of the variable region of the heavy chain donor mouse immunoglobulin.

2. Humanitarianly immunoglobulin under item 1, characterized in that it is an antibody, soda, is the antibody has an isotype Ig or Ig G1 G4.

4. Humanitarianly immunoglobulin under item 1, characterized in that specifically binds to L-selectin person with affinity, which constitutes at least 108M-1.

5. Humanitarianly immunoglobulin under item 1, characterized in that the donor immunoglobulin is a murine antibody DREG-200.

6. Humanitarianly immunoglobulin under item 1, characterized in that the donor immunoglobulin is a murine antibody DREG-55.

7. Humanitarianly immunoglobulin under item 1, characterized in that the said frame region heavy and light chain acceptor immunoglobulin come from the same human antibodies.

8. Humanitarianly immunoglobulin under item 7, characterized in that said antibody is the antibody Eu person.

9. Humanitarianly immunoglobulin specific for the protein L-selectin person having a region defining complementarity (DR) corresponding DR-areas of donor mouse immunoglobulin, and a frame region of the variable region heavy and light chain acceptor human immunoglobulin, nteu affinity, at least 107M-1and contains amino acids of the frame region of the donor immunoglobulin, which replace the corresponding amino acids of the frame regions of the heavy and light chain acceptor immunoglobulin, and are not in position 26 - 30 heavy chain, in this case, each of these amino acids is located near DR in the sequence of the donor immunoglobulin, or contains an atom at a distance from DR in the specified humanitariannet immunoglobulin.

10. Humanitarianly immunoglobulin under item 9, wherein the specified distance of the atom from DR determined on the basis of the model of immunoglobulin, built with the help of computer.

11. Humanitarianly immunoglobulin under item 9, characterized in that said donor immunoglobulin is mouse antibody DRG-200.

12. Humanitarianly immunoglobulin under item 9, characterized in that it is an antibody that contains two dimers light/heavy chain.

13. Humanitarianly immunoglobulin under item 12, characterized in that the antibody has an isotype Ig or Ig G1 G4.

14. Humanitarianly immunoglobulin under item 9, characterized in that the said frame region heavy and lay noglobulin under item 1 or 9, characterized in that it is essentially pure .

16. Humanitarianly immunoglobulin under item 1 or 9, characterized in that it inhibits the binding of human neutrophils with endothelial cells.

17. Humanitarianly immunoglobulin under item 1, characterized in that specifically binds to L-selectin and amino acid sequence of the variable region of the Mature light chain is shown in the lower rows in Fig.2A.

18. Humanitarianly immunoglobulin under item 1, characterized in that specifically binds to L-selectin and amino acid sequence of the variable region of the Mature heavy chain is shown in the lower rows in Fig.2B.

19. Humanitarianly immunoglobulin under item 1, characterized in that it includes humanitarian heavy chain humanitarianlaw light chain, namely: (1) humanitarian light chain that contains three areas that define complementarity (DR1, DR2 and DR3), having the amino acid sequence of a corresponding region defining a complementary light chain of mouse immunoglobulin DREG-200 and DREG-55, and frame the scope of the variable region derived from a sequence s, selected from the first group, which includes L-87, L-54, L-66, L-76, L 93, where the specified amino acid position is occupied by the same amino acid present in the equivalent position of a frame region of the variable region of light chain of mouse immunoglobulin DREG-200 and DREG-55, and (2) humanitarian heavy chain that contains three areas that define complementarity (DR1, DR2 and DR3), having the amino acid sequence derived from the respective regions determining the complementarity of the heavy chain of mouse immunoglobulin DREG-200 and DREG-55 and frame the scope of the variable region, derived from the sequence of frame region of the variable region of the heavy chain of a human, except for at least one position selected from the group comprising H93, H95, H98, H111, H112, H115, H30, H98, H111, H27, H30, H78, H72, where the specified amino acid position is the same amino acid, which is present in the equivalent position of the frame region of the variable region of the heavy chain of mouse immunoglobulin DREG-200 or DREG-55, where the specified immunoglobulin binds to L-selectionism ligand and the affinity of binding, which is almost three times higher than the affinity of murine immuno the

 

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