JPH08503617A - Humanized antibody reactive with L-selectin - Google Patents

Abstract

  (57) [Summary] Humanized immunoglobulins that react specifically with L-selectin are prepared using recombinant DNA technology, for example, for use in the treatment of inflammatory diseases.

Description

Detailed Description of the Invention             Humanized antibody reactive with L-selectin   Reference to related inventions   This application is related to US patent application serial number 07 / 983,946 (1992). Filed on Dec. 1), which is cited for all purposes. As a result, it becomes a part of this specification as a whole.   Field of the invention   The present invention is generally directed to recombinant DNA and mono-nucleotides for developing new biologics. The invention relates to a combination of clonal antibody technologies, and in particular to eg L-selectin protein. Production of non-immunogenic immunoglobulins specific for white matter (in humans),Inn Vitro as well asIn vivoRegarding their use in.   Background of the Invention   The ability of cells to adhere to each other influences developmental, normal physiology, and disease processes. Play a crucial role. This ability is due to adhesion molecules (typically glycoproteins, Is expressed on the cell membrane). Often on one cell type Adhesion molecules bind to other adhesion molecules expressed on different cell types and It forms a putter counter-receptor pair. Three very important adhesion molecules Kura Is an integrin, selectin and immunoglobulin (Ig) superfamily -Member (Springer, Nature 346: 425 (1990), Oh Sbourne, Cell 62: 3 (1990); Hines, Cell 69:11 (1992) , All of which are hereby incorporated by reference for all purposes. Will be part of))). These molecules interact with white blood cells and platelets with themselves. Particularly important for interaction and interaction with extracellular matrix and vascular endothelium Is.   Integrins are α-chain (120-180 kD) and β-chain (90-110 kD). A heterodimeric transmembrane glycoprotein consisting of, and generally having, a short cytoplasmic region. Is. All α subunits are aligned with each other, as are β subunits. Shares column homology and motif. β₂A β subunit called Three known integrins, including, are important for T cell, neutrophil and monocyte function Is. LFA-1 (α_Lβ₂) Is widely distributed on lymphocytes, granulocytes and monocytes There is. The counter-receptor is ICAM-1 (and probably less ICAM-2) of high importance, which is expressed on many cells including white blood cells. And is amplified on the vascular endothelium by cytokines such as TNF and IL-1. It is an up-regulated Ig family molecule. α or Block LFA-1 on T cells with antibodies to either of the β subunits. It is important to know that adhesion-dependent functions such as targeting Strongly inhibits CTL-mediated lysis of vesicles. Mac-1 (α_Mβ₂) Is a neutrophil , And its counter-receptor is also ICAM-1. (And possibly ICAM-2). In particular, Mac-1 is type 3 It is a complement receptor (CR3) and binds the C3bi fragment. Third β₂ Integrin, P150, 95 (α_Xβ₂) Was also found on neutrophils and monocytes But it seems less important. LFA-1, Mac-1 and P15 The 0,95 α-subunits are also labeled CD name, CD11a, and CD11, respectively. b and CD11c, while β₂Is also referred to as CD18, so LF A-1 is CD11a / CD18 and Mac-1 is CD11b / CD18. It   There are three known selectins, which were previously known as LECCAM. L-selectin (also LECAM-1, Mel-14 or LAM-1), E-selectin (also called ELAM-1) and And P-selectin (also called GMP140 or PADGEM) ing. They have all been sequenced at the cDNA level and It shares homology and modification and contains a lectin-like domain. L-selectin has a dual role Homin on T cells for high endothelial venules in peripheral lymph nodes Gluceptor and adhesion molecule on neutrophils for endothelium (Harman Et al., Biochem. Biophys. Res. Commun. 174: 236 (1991), which is cited for all purposes. Is made a part of this specification as a whole). E-selectin and P- Although selectins are both induced on the endothelium by cytokines, they differ Have kinetics. All three selectins are probably similar to other counsels. Although it has a ter-receptor, L-selectin contains E-selectin and P-selectin. -A counter-receptor on neutrophils for both selectins (Picker -Er., Cell 66: 921 (1991), which is cited for all purposes. It becomes a part of this specification as a whole). In particular, E-selectin is charcoal water Compound group sialyl Lewis x (sLex) was attached (Lou et al., Cell 63: 475 (1 990), which is hereby incorporated by reference in its entirety for all purposes. , And this carbohydrate is predominantly present on L-selectin. (Picker et al., Cell 66: 921 (1991)), but that It can occur on the white matter as well. E-selectin is specifically expressed in inflammatory skin sites And also with adhesion molecules for skin homing T cells that may contribute to inflammation (Picker et al., Nature 349: 796 (1991), this is all Incorporated herein by reference in its entirety).   In various assays, CD11a, CD11b, CD18, L-selectin and And antibodies to E-selectin are all Block the binding of neutrophils to activated endothelial cells to a greater or lesser extent. However, the most complete inhibition is generally with antibodies against CD18 and L- or E-se. Achieved by a combination of antibodies against lectins (eg lucinuscus J. Immunol. 142: 2257 (1989), for all purposes Incorporated herein by reference in its entirety))). Recent , But now widely accepted models take these facts It is explained using the process of the floor (Butcher, Cell 67: 1033 (199 1), which is incorporated herein by reference in its entirety for all purposes. Be part of). In the first stage, neutrophils are infected with blood that is inflamed via selectins. Reversibly binds to the vascular endothelium, which binds well under flow conditions, literally neutrophil Roll the ball along the vessel wall. The neutrophils are then released by the surrounding or endothelium. Is activated by various stimulants including IL-8, PAF and C5a. Activated neutrophils release L-selectin and the amplification-regulated Mac-1. Most In the final step, ICAM-1 and possibly other counter-relays on endothelial cells Binding of Mac-1 to sceptors allows for stable adhesion and extravasation through the endothelium. Noh.   In principle, antibodies to integrin and selectin adhesion molecules or other antagonis Strokes prevent neutrophils from binding to the endothelium and extravasating into tissues. By this, this process can be suppressed. Therefore, such antibodies are Can be used in the treatment of a large number of different diseases where is an important factor.   For example, in an animal model, an antibody that binds to both LFA-1 and Mac-1 CD18 antibodies are useful in reducing ischemia-reperfusion injury (eg, Vue). Dar et al. Clin. Invest, 81: 939 (1988); Vedder et al. , Proc. Natl. Acad. SCi. USA. 87: 2643 (1990 ); U.S. Pat. No. 4,797,277). They are also Gram Yin Including sexual sepsis (Walsh et al., Surgery 110: 205 (1991)) Reduces neutrophil-mediated injury in the lung in response to various lesions (dwell Shook et al. Immunol. 144: 2327 (1990) and Milligan. Et al., J. Immunol. 148: 1847 (1992)). To the rabbit model Thus, anti-CD18 antibodies also protect against death from meningitis (Tumannen et al., J. ． Exp. Med. 170: 959 (1990)). They also have T cell function It is useful in preventing or treating organ transplant rejection. obtain.   For example, injection of antibodies against L-selectin or E-selectin into rodents Suppressed the accumulation of neutrophils in the inflamed peritoneum (Jutira et al., J. I. mmunol. 143: 3318 (1989) and Milligan et al. Clin. Invest. 88: 1396 (1991)). In-vivo video microscopy is L-selectin antibody Inhibits leukocyte rolling along the vascular wall endothelium of mesenteric venules explanted from Gui (Von Andrian et al., Proc. Natl. Aca d. Sci. USA 88: 7538 (1991)). Anti-E-selectin antibody Vascular lesions induced by immune complex deposits in rat skin or lung Are reduced and substantially reduce the accumulation of neutrophils at those sites (Murigi En, et al. Clin. Invest. 88: 1396 (1991)). Also, In a primate model of exogenous asthma, anti-E-selectin antibody binds neutrophils into the lung Significantly reduces late airway obstruction after influx of E. coli and associated inhalation of antigen (Gundel et al. J. Clin. Invest. 88: 1407 (1991)).   Mouse DREG-55 and mouse DREG-56 that bind to human L-selectin And several antibodies have been developed, including mouse DREG-200 (Kishimoto et al., Proc. Natl. Acad. Sci. USA 87: 2244 (1990). It is hereby incorporated by reference in its entirety for all purposes as part of this specification. Become). These antibodies are partially or completely isolated from the peripheral lymph nodes of human lymphocytes. Binding to high endothelial venules and human neutrophils to stimulated human umbilical vein endothelial cells Block binding (Kishimoto et al., Blood 78: 805 (1991) Are incorporated herein by reference in their entirety for all purposes. ). These antibodies block the binding of neutrophils to endothelial cells. The ability to lock depends on the antigen they bind to, L-selectin, as a potential therapeutic agent. It may be a suitable target for.   Unfortunately, the use of non-human monoclonal antibodies such as mouse DREG-200 Is used in the treatment of humans, especially in repeated treatment regimens as described below. It has certain drawbacks. Mouse monoclonal antibodies are, for example, relatively short Other important immunoglobulins that have a circulating half-life and when used in humans Lacks the functional properties of.   Perhaps more importantly, the non-human monoclonal antibody was infused into a human patient. Includes substantial lengths of amino acid sequences that are sometimes immunogenic. A lot of research After injection of foreign antibody, the immune response to the antibody evoked by the patient is Can be very strong, essentially eliminating the therapeutic utility of the antibody after the first treatment. Is shown. Moreover, an increasing number of different macromolecules are being used to treat various diseases. Us or other (human) antigenic monoclonal antibodies are expected to be developed So after the first or several treatments with any of the different non-human antibodies it Even treatment for unrelated therapies following is ineffective due to cross-reactivity Or it can itself be dangerous. So-called "chimeric antibodies" (eg The murine variable region linked to the human constant region) was somewhat successful. While proven, significant immunogenicity issues remain.   To try to solve the immunogenicity problem, Humanized antibodies have been produced. The change from murine to humanized antibody is a competitive consideration The solution, including the compromise of what to do, depends on the different antibodies. Exemption To minimize epidemiology, immunoglobulins contain as many human receptor sequences as possible. Should be retained. However, because it retains its authentic binding properties, it does not The briin framework is a three-dimensional representation of CDR regions in mouse donor immunoglobulins. Guarantees a 3D conformation in the CDR region as close as possible to the conformation It should include sufficient substitutions of the human receptor sequence. Of these competing considerations As a result, many humanized antibodies produced to date are comparable to the corresponding murine antibodies. All show a marked loss of binding affinity. For example, Jones et al. Nature 32 1: 522-525 (1986); Scharman et al., J. Am. Immunol. 14 7: 4366-4373 (1991); Ketterborov, Protein Engineering. Alling 4: 773-783 (1991); Goalman et al., Proc. Natl ． Acad. Sci. USA 88: 4181-4185 (1991); Tempe. Str et al., Biotechnology 9: 266-271 (1991); Leachman et al., Nature 332: 323 (1988) and European Patent Publication No. 023940. Publication No. 0 (each of which is incorporated by reference for all purposes And become part of this specification).   Thus, it is substantially non-immunogenic in humans but suitable for therapeutic formulations or other uses. Easily and economically manufactured in a fashion An improved form of humanized immunoglobulin specific for L-selectin antigen is required Is. The present invention meets these and other needs.   Summary of the invention   The present invention provides novel compositions useful, for example, in the treatment of inflammatory human conditions. Provided is a humanized immunoglobulin capable of specifically binding L-selectin. Including Immunoglobulins can have two pairs of light / heavy chain complexes. At least one chain is operably linked to a human framework region segment. One or more mouse complementarity determining regions. For example, the mouse complementarity determining region Is a human frame with or without additional naturally associated mouse amino acid residues. Introduced into the work area, about 10⁷M^-1L-select at stronger affinity levels Humanized immunoglobulins capable of binding to tin can be produced. These humanized immunoglobs Briin also binds to CDR-donating mouse monoclonal antibody L-selectin Can block the binding of.   Immunoglobulins Containing Binding Fragments and Other Derivatives Thereof of the Invention Can be easily produced by various recombinant DNA techniques and Cells, preferably immortalized eukaryotic cells such as myeloma or It can be finally expressed in hybridoma cells. Humanized immunoglobulin frame No. code work area One sequence and a second set of sequences encoding the desired immunoglobulin complementarity determining regions A polynucleotide containing a synthetic or suitable cDNA and genomic DNA segment Can be manufactured by combining the components.   Humanized immunoglobulins alone or in chemotherapeutic agents in substantially pure form For example, non-steroidal anti-inflammatory drugs (eg aspirin), corticosteroids or It can be used with immunosuppressants. All these compounds, especially for inflammatory diseases Useful in therapy. Humanized immunoglobulins or their conjugates are Can be prepared in a pharmaceutically acceptable dosage form, depending on the mode of administration. Exist and change.   Brief description of the drawings   Figure 1. C of light chain (A) and heavy chain (B) variable regions of mouse DREG-200 antibody DNA sequences and translated amino acid sequences. The mature heavy chain begins at the 20th amino acid (E). And the mature light chain begins at the 21st amino acid (D) and is preceded by a signal There is an array.   Figure 2. Mouse DREG-200 antibody (top) and humanized DREG-200 antibody (Lower row) Amino acid sequences of mature light chain (A) and heavy chain (B) variable regions. 3 for each chain One CDR is underlined. Mouse amino acids or typical in humanized antibodies Residues in the framework that were replaced with human amino acids are double underlined.   Figure 3. Light chain of humanized DREG-200 antibody starting and ending at the Xbal site (A) and heavy chain (B) the nucleotide sequence of the gene encoding the variable region, and And the translated amino acid sequence including the signal sequence.   Figure 4. Competitive binding of mouse and humanized IgG1 and IgG4 DREG-200 antibodies Go. The target cells are L2-1 cells (mouse pre-B cell line), which Human L-selectin gene and therefore human L-select Express Chin (Berg et al., Biochem. Biophys. Res. Com. m. 184: 1048 (1992)). 5 × 10^FiveCells of¹²⁵I labeled Using 3 ng of tracer mouse antibody (2 μCi / μg), sequence as shown. Subsequent to the addition of 0.2 ml binding with increasing amounts of mouse or humanized competitor antibody. Incubate in buffer (PBS + 2% FBS + 0.1% azide) for 1 hour at 4 ° C. I started. The cells were washed, pelleted, and their bound radioactivity measured. Decided The concentration of bound and free tracer antibody was calculated.   Figure 5. Human preference for IL-1 stimulated human umbilical cord endothelial cells (HUVEC) Coupling of spheres. Neutrophils were first treated with an unrelated control antibody, mouse D, as shown. Treated or untreated with REG-200 antibody or humanized IgG1 DREG-200 antibody I left it.   Figure 6. Protection of ischemia-reperfused heart tissue by humanized DREG-200. Figure Is a humanized DREG-200 or pair Left-to-right area at risk / total ventricular area for cats treated with larval antibodies , Necrotic tissue area / risk area, and necrotic tissue area / total left ventricular area. Bracket Tos represents +/− SEM in 6 cats, bar height is average.   Definition   The term “substantially identical” or “substantially homologous” means that two peptide sequences are For example, in the program GAP or BESTFIT using the default gap weight At least 65 percent sequence identity, or better, when more optimally aligned. Preferably at least 80 or 90% sequence identity, more preferably less 95% sequence identity or higher (eg, 99% sequence identity). I) means to share. Preferably, non-identical residue positions are defined by Depends on mino acid substitution.   For purposes of classifying amino acid substitutions as conservative or non-conservative, the amino acids are Grouped into: Group I (hydrophobic side chain): norleucine, met, al a, val, leu, ile, group II (neutral hydrophilic side chain): cys, ser , Thr, group III (acidic side chain): asp, glu, group IV (basic side) (Chain): asn, gln, his, lys, arg, group V (affects chain orientation Residues): gly, pro, and group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions include substitutions between amino acids within the same class. Non-conservative substitution is , One of these classes To exchange members for members of other classes.   Amino acids from the variable regions of the mature heavy and light chains of immunoglobulin are x and Lx, where x is the Kabat diagram (design of the protein of immunological interest). Row, National Institutes of Health, Bethesda, MD, 198 7 and 1991). Kabat is , Lists a number of amino acid sequences for antibodies for each subclass, and List the most commonly occurring amino acids for each residue position in the subclass There is. Kabat assigns a residue number to each amino acid in the listed sequences This method for assigning residue numbers is standard in the art. It is quasi. The Kabat diagram shows the target antibody as one of the common sequences in Kabat. By lining it up to other antibodies not included in his general introduction It The use of the Kabat numbering system allows for the equivalent position in various antibodies. Easily identify amino acids. For example, the amino acid at the L50 position of human antibody is Occupies a position equivalent to amino acid position L50 of the antibody.   From the N-terminus to the C-terminus, both the light chain and the heavy chain are FR1, CDR1, FR2, C It contains the DR2, FR3, CDR3 and FR4 regions. Amino acids for each domain Is assigned to Kabat (1987) and (1991), supra, or Chochia. Rescu, J. Mol. Biol. 196: 901-917 (1987); Chochi Oh, Nature 342: 878-883 (1989).   Detailed Description of the Invention   In general, the scientific terms used below and the cell culture, molecular genetics, and And laboratory methods in nucleic acid chemistry and hybridization are well known in the art. It is well known and commonly used. Standard technology is recombined Nucleic acid methods, polynucleotide synthesis, cell culture, and transgene transfer (eg, , Electroporation, microinjection, lipofection) Used for. Generally, enzymatic reactions, oligonucleotide synthesis, and purification steps Is performed according to the product specifications. Techniques and methods are generally conventional in the art. Method and various general references provided throughout this specification. It The methods in it appear to be well known in the art and are convenient for the reader. Provided for All information contained therein is hereby incorporated by reference. Become part of.   Humanized antibody against L-selectin   In accordance with the present invention, a humanized immune response specifically reacting with an L-selectin related epitope Epiglobulin is provided. These immunoglobulins are usually at least about 1 0⁷M^-1) Preferably 10⁸M^-1-10⁹M^-110,^TenM^-1Or more L-ku It has a binding affinity for tin and can bind, for example, neutrophils. Humanization exemption Epiglobulin is a human Immunoglobulin having a framework and specifically reacting with L-selectin , Typically one or more complementarity determining regions (CDRs) from mouse immunoglobulin Have. In a preferred embodiment, the one or more CDRs are mouse DREG-200. Derived from an antibody and humanized immunoglobulin is IgG1 or IgG4 isotype It is Therefore, the immunoglobulin of the present invention should be produced economically in large quantities. Various techniques, for example, for the treatment of inflammatory diseases in human patients. It turned out to be used.   The basic antibody structural unit is known to constitute a tetramer. Each tetra A mer consists of two identical pairs of polypeptide chains, each pair consisting of one "light" It has a chain (about 25 kD) and one "heavy" chain (about 50-70 kD). N of each chain H₂The ends are approximately 100-110 or more amino acids that contribute primarily to antigen recognition. It begins with the variable region of the mino acid. The COOH part of each chain is mainly an effector machine It defines a steady region that contributes to Noh.   Light chains are classified as either kappa or lambda. Heavy chains are gamma, mi , Alpha, delta, or epsilon. Ip has been identified as IgG, IgM, IgA, IgD and IgE. Light chain and Within the heavy chain, the variable and constant regions are comprised of about 12 or more amino acids "J Linked by "regions, the heavy chain is also a" D "region of more than about 10 amino acids. Range (generally, fundamental immunology, Paul W. Ed., Chapter 7, pp. 131-166, Raven Press, N. et al. Y. (1984) (This It is hereby incorporated by reference in its entirety for all purposes. See)).   The variable regions of each light / heavy chain pair form the antibody binding site. All chains are three Hypervariable region (this region is also called the complementarity determining region or CDR ("immunological Protein Sequence of Interest, "Kabat E. et al., US Department of Hell Suand Human Service (1987); and Cho Chi And Rescu, J. Mol. Biol. 196: 901-917 (1987) (this Are incorporated by reference in their entirety for all purposes. Same) of relatively conserved framework regions linked by It shows a ubiquitous structure. The CDRs from the two chains of each pair depend on the framework regions. Are aligned in a row, allowing binding to specific epitopes.   As used herein, the term "immunoglobulin" refers to an immunoglobulin gene. Is a protein consisting of one or more polypeptides that are substantially encoded. recognition These immunoglobulin genes are kappa, lambda, alpha, gamma, delta, Epsilon and mu constant region genes, and myriad immunity groups Contains the lobulin variable region gene. Immunoglobulins include, for example, Fv, Fab and ( Fab ')₂Aligned bivalent antibodies (eg, Lanzabetia et al., Eur. J. Immun ol. 17: 105 (1987)) In addition to antibodies, various forms and single chains (eg, Houston et al., Proc. N. atl. Acad. Sci. USA) 85: 5879-5883 (1988) and Byrd et al., Science, 242: 423-426 (1988) (these are all Incorporated by reference in its entirety for all purposes). Can exist (Generally, Hood et al., Immunology (Benjamin, N .; Y. , 2nd edition, 1984), Harlow and Lane, Antibodies. : A Laboratory Manual (Cold Spring Harbor   Laboratories, 1988) and handkerchiefs and foods, nature, 323: 15-16 (1986), each of which is cited for all purposes. It becomes a part of this specification as a whole)).   Chimeric antibodies have light chain genes and heavy chain genes, typically by genetic engineering, An antibody composed of immunoglobulin gene segments belonging to different species is there. For example, the variable (V) segment of a gene from a mouse monoclonal antibody Is a human constant (C) segment, such as γ₁And γ_FourCan be connected with. Therefore , Although other mammalian species may be used, typical therapeutic chimeric antibodies are V or antigen-binding domain from an antibody and C or an effector from a human antibody It is a hybrid protein consisting of main.   In this specification, the term "framework area" is used by Kabat et al. (Supra). As defined by A relatively conserved (ie, non-CDR) immunoglobulin of the epidemic glorin Light chain and heavy chain variable region. As used herein, "human framework "Region" refers to the framework regions of naturally-occurring human antibodies or some of these Is substantially identical (about 85% or more) to the consensus sequence of such antibodies. This is a framework area.   As used herein, the term "humanized immunoglobulin" refers to the human framework, An immunoglobulin containing at least one CDR from a non-human antibody, in which Any constant region present in human is substantially identical to the human immunoglobulin constant region. Is at least about 85-90%, preferably at least 95% identical . Therefore, presumably all parts of the humanized immunoglobulin, except the CDRs, have more than one Is substantially identical to the corresponding portion of the native human immunoglobulin sequence of. For example , Humanized immunoglobulins do not include chimeric mouse variable region / human constant region antibodies. Yes.   Humanized Antibodies Compared to Mice and Go for Use in Human Therapy In some cases it has at least three potential advantages over chimeric antibodies.   1) The effector part is human, which makes it better than other parts of the human immune system. Can interact (eg, complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity) More efficient destruction of target cells by harm (ADCC)).   2) The human immune system recognizes the framework or C region of humanized antibody as a foreign substance Without and is thus injected like this The antibody response to the antibodies is Less chimeric antibody.   3) Injected mouse antibody is much shorter than the half-life of normal antibody in humans. It has been reported to have a half-life in the circulation in (Show, D. et al., J. Im. munol. 138: 4534-4538 (1987)). Injected humanized anti The body probably has a half-life closer to that of naturally occurring human antibodies, It is possible to give a dosage of less or less frequency.   In one aspect, the present invention is capable of binding to a desired epitope of L-selectin. Immunoglobulin, eg, monoclonal antibody mouse DREG-200, Mau DREG-55 or mouse DREG-56 (Kishimoto et al., (1990), supra. , Which is hereby incorporated by reference in its entirety for all purposes. And a recombinant DNA segment encoding heavy and / or light chain CDRs from It is directed to the ment. The DNA segment encoding these regions is Typically a DNA segment encoding a suitable human framework region Connected to. Exemplary DNA sequences (which, when expressed, are monoclonal Polypeptide chain containing heavy and light chain CDRs of mouse antibody DREG-200 Are included in FIG. 1. Due to codon degeneracy and minor amino acid substitutions , Other DNA sequences are easily substituted for those sequences, as detailed below. Can be Design of humanized immunoglobulin and For a detailed description of manufacture, a commonly assigned serial number, 07/29 0,975 and 07 / 310,252 (December 1988, respectively) Filed 28th and 13th February 1989), both of which are for all purposes. Used as part of the present specification as a whole).   The DNA segment is typically additionally humanized immunoglobulin coding Contains an expression control DNA sequence operably linked to a sequence and is naturally associated Alternatively, it contains a heterologous promoter region. Preferably, the expression control sequences are eukaryotic host cells. Cells in a vector capable of transforming or transfecting eukaryotes. Control system, but control sequences for prokaryotic hosts may also be used. vector Once incorporated into a suitable host, the host will have a high level of nucleotide sequence. Of the light chain, heavy chain, light chain / Of heavy chain dimers or whole antibodies, binding fragments or other immunoglobulin forms Collection and purification can follow.   Nucleic acid sequences of the present invention that are capable of ultimately expressing the desired humanized antibody will vary widely. Polynucleotide (genomic DNA or cDNA, RNA, synthetic oligonucleotide Etc.) and constituents (eg, V, J, D and C regions), and various different Can be formed by various techniques. Linking the appropriate genomic sequence to the synthetic sequence At present, although the most common method of manufacture, cDNA sequences are also available (yeo). Lock PA Patent No. 0239400 and Reachman, L.S. Et al., Nature 332: 3 23-327 (1988) (both of which are cited for all purposes) Becomes a part of the present specification as a whole)).   Human constant region DNA sequences can be isolated from various human cells, preferably from human cells, according to well known methods. More preferably it can be isolated from immortalized B cells (Kabat, supra, and WP87. // 02671). CDRs for producing the immunoglobulins of the present invention are similar Derived from a monoclonal antibody capable of binding L-selectin, and A convenient mammalian source (mouse, rat, rabbit or by well-known methods (Including other vertebrates capable of producing antibodies). Suitable for DNA sequences Suitable source cells, and suitable host cells for immunoglobulin expression and secretion are Many sources, such as the American Type Culture Collection (Cellular In and Hybridoma Catalog, 5th Edition (1985), Rockville, MD, It is hereby incorporated by reference in its entirety for all purposes as part of this specification. Can be obtained from. In a preferred embodiment, the CDRs are each , Mouse DREG-200, mouse DREG-55 or mouse DREG-56 Having a sequence corresponding to the CDR sequences, and mouse DREG-200, mouse DR Encoding the corresponding CDR amino acid sequences of EG-55 or mouse DREG-56 Can include synonymous nucleotide sequences.   In addition to the humanized immunoglobulins detailed herein, other "substantially homologous" Modified immunoglobulins are available in various combinations well known to those skilled in the art. It can be easily designed and manufactured using recombinant DNA technology. Example 2 Human antibodies other than the Eu antibody discussed above were used as the source of the framework sequences. Can be done. These framework sequences are the sequences from which the CDRs were derived. A high degree of sequence identity with the murine DREG-200 variable framework domain Show. The heavy and light chain variable framework regions may contain the same or different human antibody sequences. It can come from a row. In fact, the heavy and light chain framework regions are each 1 It may be derived from more human antibodies. Human antibody sequences are sequences of naturally occurring human antibodies. It can be a string or it can be the consensus sequence of some human antibodies. Carter Et al., WO 92/22653 (1992).   Unnatural arrangement of human variable framework regions and murine CDR regions is Natural steric inhibition can result, which is the substitution of certain amino acid residues. If not calibrated by, it results in loss of binding affinity. Amino for substitution The choice of acid residue is determined in part by computer modeling. Immunity Computer hardware for creating 3D images of globulin molecules And software is widely available. Generally, the molecular model is the immunoglobulin. It is made with the known structure of the chain or its domain as the root. Model made The chain that should be Chain or domain of the three-dimensional structure in which the amino acid sequence is similar. And the chains or domains showing the greatest sequence similarity are Is selected as the starting point for Known starting structure is modeled Between the actual amino acids in the immunoglobulin chain or domain and the amino acids in the starting structure Qualified to account for differences. The modified structure is then combined with the composite immune group. Assembled into Roblin. Finally, the model is First, all the atoms are within a suitable distance from each other, and the bond length and angle are It is improved by making sure that it is within the acceptable limit. Example 2 , And more particularly, a three-dimensional computer for the variable region of the mouse DREG-200 antibody. Discussing the steps taken to create a motor model. This model is now Degrees of mouse DREG-200 complementation with human framework structures determined It can serve as a starting point for predicting the three-dimensional structure of an antibody containing regions. less than If additional amino acid substitutions to be discussed were introduced, additional amino acids showing their structure A model can be built.   In general, murine substitution of human amino acid residues should be minimized. Because , The introduction of murine residues increases the risk of antibodies eliciting a HAMA response in humans. This is because it can be done. Amino acids for substitution may have CDR conformations and / or Are selected based on their potential impact on binding to the antigen. This Studying the possible effects of eels For model building, testing the properties of amino acids at specific positions, or Based on empirical observation of effects of substitution or mutagenesis.   Amino acids are murine DREG-200 variable framework regions and human equivalents If there are differences between the variable framework regions, if the amino acid is (1) directly in non-covalent contact with the antigen, or (2) adjacent to the CDR region or otherwise interacting with the CDR region Yes (eg, within approximately 4-6 Angstroms of CDR region) Human framework amino acids are usually equivalent to Should be replaced by a sub-amino acid.   Other candidates for substitution are unusual for human immunoglobulins at that position Receptor human framework amino acids (eg, amino acid H of human Eu antibody) 113). These amino acids are the equivalent positions of the more typical human immunoglobulin. Can be replaced with an amino acid from Alternatively, the equivalent in mouse DREG-200 Amino acids from positions are located in the human immunoglobin at positions where such amino acids are equivalent. Can be introduced into the human framework regions when it is representative of brin .   Generally, it is desirable that all or most of the amino acid substitutions meet the above criteria. New However, sometimes it is necessary to check whether individual amino acids meet the above criteria. Ambiguous , And various alternative immunoglobulins have been produced, one of which is With certain substitutions of, but not others. The humanized antibody of the present invention is usually At least in the lower position, namely L87, L54, L66, L76 and L93 Also in 1, 2, 3, 4 and more usually 5 corresponding mouse DREG-20 Included substitutions with mouse light chain framework residues with 0 residues. Humanized antibody Also, usually, the following positions, that is, H93, H95, H98, H111, H112 , H115, H30, H98, H111, H27, H48 and H72 At least 1, 3, 5, 7, 9, 10, 11 and more usually 12 mouse weights Includes substitutions at chain framework residues. In a preferred embodiment, the human heavy chain receptor If the body immunoglobulin is Eu, the heavy chain also contains a H113 substitution. This position is usually that of a human immunoglobulin, etc., which has more typical amino acid residues. Substituted with an amino acid from the valency position.   The CDR regions in humanized antibodies are usually substantially identical, and more usually It is identical to the corresponding CDR regions in the mouse DREG-200 antibody. But Naga Occasionally, one of the residues in the CDR region is linked, for example, to the ligand of L-selectin. Alterations are also desirable to create similarities to the junction. Don't usually want Nonetheless, the binding affinity of the resulting humanized immunoglobulin was appreciably affected. Alternatively, it is sometimes possible to make one or more conservative amino acid substitutions in a CDR residue.   In addition to the specific amino acid substitutions discussed above, humanized immunoglobulin frames The work areas are usually substantially the same, and more usually, where they originated. It is the same as the framework region of human antibody. However some implementations In an aspect, the framework region comprises several amino acid substitutions, termini and Changes from the natural sequence at the primary structure level due to additions and deletions in the middle Can be Stereoisomers of 20 conventional amino acids (eg D-amino acids) , Unnatural amino acids, eg α, α-disubstituted amino acids, N-alkylamino Acids, lactic acids, and other non-conventional amino acids also may be present in the polypeptides of the invention. It may be a suitable constituent. Of course, many of the amino acids in the framework are Little or no direct contribution to antibody specificity or affinity. Obedience Thus, many individual conservative substitutions of framework residues can be generated in the resulting humanized immunoglobin. It can be tolerated without appreciable changes in the specificity or affinity of brin. Shi However, such substitutions are generally undesirable. Gene modifications can be Well-known techniques such as site-directed mutagenesis (Gillman and Smith Su, Gene 8: 81-97 (1979) and Roberts et al., Nature 328. : 731-734 (1987), both of which are cited for all purposes. , Which is hereby incorporated by reference in its entirety)). sell.   Alternatively, a polypeptide containing only part of the primary antibody structure Fragments can be produced which contain one or more immunogroups. Has lobulin activity (eg, binding activity). These polypeptide fragments Are capable of proteolyzing intact antibodies by methods well known in the art. Or by using site-directed mutagenesis to add a stop codon to the vector pVk. And the desired position of pVg1-dhfr, for example the Fab fragment. After CH1 or (Fab ')₂Hinge area for manufacturing fragments It can be manufactured by inserting after the zone. Single-chain antibody uses DNA linker Can be prepared by linking VL and VH (Houston et al., Supra, And Bird, et al., Supra). As an example, the Fv or Fab fragment is , Buchner and Rudolf (Bio / Technology 9: 157- 162 (1991)) and Skera et al. (Bio / Technology 9:27). 3-277 (1991) Method (These should be cited for all purposes. As a whole)E. FIG. coliManufactured in sell. Fv and Fab may also be expressed in eukaryotic cells, preferably mammalian cells, in Can be produced by expressing a polynucleotide encoding the same. Also, Like many genes, immunoglobulin-related genes contain segregated functional regions. However, the genes are novel because each has one or more distinct biological activities. In order to produce a fusion protein having various properties (for example, immunotoxin), other Functional regions from genes (eg enzymes, commonly assigned US patent serial numbers No. 132,387 (filed Dec. 15, 1987), which describes all Incorporated herein by reference in its entirety). Can be   Expression of humanized immunoglobulin sequences in a bacterial host causes ablation of the CDR regions. Mutagenizing and high affinity and / or high specific binding for L-selectin To be screened for humanized immunoglobulin CDR variants with sex Produce a bacteriophage display library capable of This is advantageous for selecting higher affinity humanized immunoglobulin sequences. Can be used for. The potential advantage of such sensitive affinity is that L-selectin Humanized immunity with improved binding affinity to external molecules and / or reduced cross-reactivity Manufacture of a globulin CDR variant. Has immunoglobulin variable region sequence Methods for producing phage display libraries are known in the art. , For example, Cesarini, FEBS Lett 307: 66-. 70 (1992); Swimmer et al., Proc. Natl. Acad. Sci. US A 89: 3756-60 (1992); Gram et al., Proc. Natl. Ac ad. Sci. USA 89: 3576-80 (1992); Crackson et al., Nature 352: 624-8 (1991); Scott and Smith, Sai. Enth 249: 386-90 (1990), Garrard et al., Bio / Techniques 9: 1373-1. 377 (1991) (these are incorporated by reference for all purposes) As part of the present specification). The resulting affinity-sensitized CD The R-mutated humanized immunoglobulin sequence is then prepared in a host suitable for efficient expression. Expressed.   As mentioned previously, the DNA sequence is operably linked to the expression control sequence (immediately Then, after arranging so as to ensure that the expression control sequence functions, To be revealed. These expression vectors are typically episomal or as a host. It is capable of replicating in the host as an integral part of chromosomal DNA. Usually an expression vector Is a selectable marker, for example tetracycline resistance (tet^R), G418 resistance ( neo^R), Mycophenolic acid resistance (gpt) or HSV-tk, Allows detection of cells transformed with a DNA sequence (see, eg, US Pat. No. 4, 704,362, which is incorporated by reference for all purposes. As part of the specification as a body))).   E. FIG. coliAre particularly useful for cloning the DNA sequences of the invention. One prokaryotic host. Other microbial hosts suitable for use are Bacillus, such as Herb fungi, and other Enterobacteriaceae, such as Salmonella, Serratia, and various pseudo. Including Monas species. To make expression vectors in these prokaryotic hosts And the vector typically contains expression control sequences compatible with the host cell (eg, , Duplicate Including the starting point). In addition, any number of various well-known promoters, such as Kutose promoter system, tryptophan (trp) promoter system, β-lac There may be a tamase promoter system or a promoter system from lambda phage Yes. Promoters typically express (optionally with operator sequences) Ribosome binding sites to regulate and initiate and complete transcription and translation It has an array, etc.   Other microorganisms, such as yeast, can also be used for expression. Saccharomyces Contains expression control sequences such as 3-phosphoglycerate kinase or other glycolytic enzymes. Suitable promoters and origins of replication, terminal sequences, and the like, as desired Is a preferred host with various vectors.   Plants or plant cell cultures are used for expression of the humanized immunoglobulins of the invention. Can be (Lalic and Fry, Hum. Antibodies Hy bridomas 2 (4): 172-89 (1991); Benvenuto et al., P. ant Mol. Biol. 17 (4): 865-74 (1991); Dew. Lin et al., Plant Mol. Biol. 15 (2): 281-93 (1990) ); Hyatt et al., Nature 342: 76-8 (1989), all of which Incorporated herein by reference in its entirety). Preferred New plant hosts include, for example, Arabidopsis, Nicotiana Anatabacum (Nicotiana tabacu) m), Nicotiana rustica and Sora Includes Solanum tuberosum. The present invention To express a polynucleotide sequence encoding a modified anti-L-selectin antibody. A preferred expression cassette for encoding a humanized immunoglobulin chain therein CaMV with an enhancer in which the inserted polynucleotide sequence overlaps Plasmid pMOG1 operably linked to the 35S promoter 8 pMOG18 is a product of Simmons et al., Bio / Technology 8 : 217-221 (1990) (which is quoted for all purposes) More generally, it becomes part of this specification). Or plant A preferred embodiment for the expression of humanized immunoglobulins in a subject is the Hyatt Follow these methods (supra). However, the humanized anti-L-selectin antibody of the present invention is The polynucleotide sequence to be used is the same as that used by Hyatt et al. (Supra). Used in place of the globulin sequence. Agrobacterium tumefaciens T -DNA-based vectors also for expressing humanized immunoglobulin sequences And preferably such a vector is spectinomycin. It contains a marker gene encoding a syn resistance or other selectable marker.   Insect cell cultures have also been used to produce the humanized immunoglobulins of the invention. Can be, and typically A culovirus-based expression system is used. Humanized immunoglobulin Ritz et al., Bio / Technology 8: 651-654 (1990). Method, which is incorporated herein by reference in its entirety for all purposes. Polynucleotides encoding humanized immunoglobulins according to It can be produced by expressing a sequence. The method of the present invention is applied to the method of Putritz et al. The polynucleotide sequence encoding the anti-L-selectin antibody is In place of the mouse monoclonal Ab 6A4 heavy and light chain cDNA sequences. You can make the change to enter.   In addition to microorganisms and plants, mammalian cell cultures also include polypeptides of the invention. Can be used to express and produce (Winacker, gene to clone (VCH Publishers, NY, 1987 (This is for all purposes. Incorporated herein by reference in its entirety))). Mammal Cells are actually preferred. Because of the many suitable secretory immunoglobulins that can be secreted. Host cell lines have been developed in the art and are available in CHO cell lines. , Various COS cell lines, HeLa cells, preferably myeloma cell lines Etc. or transformed B cells or hybridomas. Expression of these cells Vectors include expression control sequences, such as origins of replication, promoters, enhancers ( Queen et al., Immunol. Rev. 89: 49-68 (1986), , All eyes Incorporated herein by reference in its entirety), and necessary Processing information site such as ribosome binding site, RNA splice site , A polyadenylation site, and a transcription terminator sequence. Preferred expression control Sequences are immunoglobulin gene, SV40, adenovirus, bovine papillomau It is a promoter derived from Ils, cytomegalovirus and the like. In general, the choice Sex markers, eg neo^RThe expression cassette is included in the expression vector.   The transgene encoding the humanized immunoglobulin of the present invention can be used to Conjugated immunoglobulin is typically collected in a recoverable body fluid such as milk or serum. Can be used to produce transgenic non-human animals that express. this Such transgenes are usually linked to enhancers such as rodent immunity. Globulin enhancer or casein gene promoter / enhancer (boo Ra et al., Bio / Technology 8: 140-143 (1990); Ade et al., Bio / Technology 8: 443-446 (1990), They are hereby incorporated by reference in their entirety for all purposes and are hereby incorporated by reference. With a humanized immunoglobulin operably linked to a promoter. Encoding the polynucleotide sequence. Transgenes are shown in the art. Cells for homologous recombinant constructs and according to the methods described below. And can be transferred into the embryo. Preferred non-human animals are Expression in bovine milk is particularly preferred, including mouse, rat, sheep, cow and goat. Good. WO 91/08216 (1991) (This is cited for all purposes Which is hereby incorporated by reference in its entirety). Purification of humanized antibody , Purification methods known in the art for immunoglobulin purification.   DNA segments of interest (eg, sequences encoding heavy and light chains and Vectors containing current control sequences are well known with modifications depending on the host cell type. It can be introduced into a host cell by the method described. For example, calcium chloride trans Ffection is commonly utilized for prokaryotic cells, while calcium phosphate Treatment, lipofection, biolistics, Virus-based transduction, or electroporation is another Cell host. Tungsten particle ballistic transgene Cis is preferred for plant cells and tissues. (In general, Maniatis et al., Mo regular Cloning: A Laboratory Manual ( Cold Spring Harbor Press, 1982) (This is for all purposes Incorporated herein by reference in its entirety))).   Once expressed, the complete antibodies of the invention, their dimers, individual light chains. Chains and heavy chains, or other forms of immunoglobulins, follow standard methods in the art. Refined The method includes ammonium sulfate precipitation, an affinity column, and a column chromatograph. , Gel electrophoresis, etc. (generally scopes, R., protein purification (spread) Nger-Verlag, N.N. Y. , 1982) (this is for all purposes Used as a part of the present specification as a whole)). For pharmaceutical use Substantially pure immunoglobulins of at least about 90-95% homogeneity are preferred for this purpose. Most preferably, a homogeneity of 98-99% or more is preferred. Once partial Once purified to the desired homogeneity, the polypeptide can then be used therapeutically (in the body). (Including externally), or in the development and implementation of assay methods, such as immunofluorescent staining Can be put. In general, immunological methods, Volumes I and II (Lufkowitz and P. Lunis, Academic Press, NY, 1979 and 1981).   In a preferred embodiment, standard binding conditions (eg, 2%, at 25 ° C.) are used. At least 1 x 10 with phosphate buffered saline containing fetal bovine serum)⁷M^-1 A humanized immunoglobulin that binds L-selectin with a binding affinity of Be done. One example of such a humanized immunoglobulin is the amino acid shown in FIG. A humanized DREG-200 antibody comprising a sequence. (Hereinafter, humanized DREG-20 0 antibody is sometimes referred to as "hu DREG-200"). Mouse DREG-5 5 or a humanized immunoglobulin containing CDRs from mouse DREG-56 is also At least 1 x 10⁷M^-1It can bind to L-selectin with affinity. The humanized antibody of the present invention is preferably at least 1 x 10 under standard binding conditions.⁸M^{- 1} With an affinity of, more preferably at least 1 × 10⁹M^-1With the affinity of , Preferably at least 1 × 10^TenM^-1Or with a stronger affinity, human L -Binds selectin. Usually, the binding affinity of humanized immunoglobulins is It is within the factor of 3 of the mouse immunoglobulin from which it is derived. For example, mouse DR The affinity of the EG-200 antibody is about 10⁸M^-1Is.   computer   In another aspect of the invention, a three-dimensional image of the antibody is shown on a monitor. A computer program is provided. For example, UNIX operating system It moves under the stem, and the molecular modeling package QUANTA (Polygen C orp. USA) Silicon Graphics IRIS 4D Workstation Is suitable. Computers are useful for making variants of humanized antibodies. is there. In general, the antibodies of the invention already provide a satisfactory binding affinity. Shi However, an antibody with a still stronger binding affinity can be used to further change certain amino acid residues. Further identification is expected. Three-dimensional images also have many non-significant No acid and the amino acid has no appreciable effect on the binding affinity of the antibody. It can be the target of class substitution. Overall, even conservative substitutions are notable for immunoglobulin properties. May affect you. However, many individual like Substitution does not appear to significantly impair the properties of immunoglobulins.   Human antibody against L-selectin   In another aspect of the invention, a human antibody against L-selectin is provided. Be done. These antibodies are produced by the various techniques described below. Some humans Antibodies are specific mouse antibodies, such as mouse, determined by competitive binding experiments or other methods. To have the same epitope specificity as DREG-200 or its humanized version To be selected. These antibodies are specifically shown for humanized DREG-200. It seems to share useful therapeutic properties.   Antibodies with the required epitopic specificity also interact with neutrophil-endothelial cell interactions. Can be identified by screening for the ability to block. Such interaction One simple visual assay for the detection of is described by Kishimoto et al. (1991) (supra). Therefore, it is described. In summary, human umbilical vein cell monolayers were stimulated with IL-1. To do. Neutrophils, either with or without pretreatment with antibodies under test, under certain conditions The number of neutrophils added to the monolayer at and adhered is measured under a microscope. Some way In, the neutrophils are obtained from a human leukocyte adhesion deficient patient. Anderson et al. , Ann. Rev. Med. 38: 175 (1987). Is this a patient Their neutrophils lack the integrin receptor. To the neutrophils of the receptor The binding to L-selection is May obscure the effect of blocking binding.   a. Trioma methodology   Basic approach and representative cell fusion pars for use in this approach Toner, SPAZ-4, is described by Ostberg et al., Hybridoma 2: 361-. 367 (1983); Ostberg, U.S. Pat. No. 4,634,664; And Englemann et al., U.S. Pat. No. 4,634,666, each of which Incorporated herein by reference in its entirety). Has been done. The antibody-producing cell line obtained by this method is called a trioma. Be done. Because they have 3 cells, 2 human cells and 1 mouse This is because it is derived from cells. First, the mouse myeloma line was replaced with human B lymphocytes. Fused to a non-antibody producing xenogeneic hybrid cell, such as Ostberg (previously Obtain the SPAZ-4 cell line described in (ibid.). The xenogeneic cells are then immunized Fused human B lymphocytes are fused to obtain an antibody-producing trioma cell line. To Liomas produce antibodies more stably than normal hybridomas made from human cells. I knew it would live.   Immunized B lymphocytes were obtained from human donor blood, spleen, lymph nodes or bone marrow. Can be Living human using L-selectinIn vivoImmunization with is harmful It is usually undesirable because of the risk of causing a false response. Therefore, B lymphocytes are usually L-selectin polynucleotide, its antigenicity Using cells that produce Lagment or either of these,In vitroImmunized with Be done. If antibodies against a particular antigen or epitope are desired,INBI Toro It is preferred to use the antigen or its epitope for immunization. B Impa spheres are typically RPMI supplemented with medium, such as 10% human plasma. -1640 (Englemann, supra), exposed to antigen for a period of 7-14 days. Be done.   Immunized B lymphocytes can be transformed in a well-known manner into xenogeneic hybrid cells, eg For example, it is fused with SPAZ-4. For example, cells may be stored at about 37 ° C for about 5-10 minutes. For 40 to 50% polyethylene glycol (MW, 1000 to 4000) Is managed. The cells are separated from the fusion mixture and made into the desired hybrid. Are grown in a selective medium (eg HAT or AH) for Required binding A clone that secretes an antibody having specificity is L-selectin or its fragment. Identified by assaying the trioma culture medium for its ability to bind to . Triomas that produce human antibodies with the desired specificity are subdivided by the limiting dilution method. Subcloned and in culture mediumIn vitroAre propagated in. Then get The resulting triomacell line binds to L-selectin or its fragments. Are tested for their ability to survive.   Although triomas are genetically stable, they have very high levels Does not produce antibodies. The expression level was 1 or more for the antibody gene from trioma. Expression vector And clone the vector into a cell line, such as recombinant or human. To transform into the cell line described above for expression of a modified immunoglobulin. Can be increased more.   b. Transgenic non-human mammal   Human antibodies to L-selectin also have low numbers of human immunoglobulin loci. Non-human Tran with a transgene encoding at least one segment It can be produced from a transgenic mammal. Usually such transgenics The mammalian endogenous immunoglobulin loci are functionally inactivated. Preferably Is a segment of the human immunoglobulin locus that is a rearrangement of the heavy and light chain components. Contains unplaced arrays. Inactivation of endogenous immunoglobulin genes and exogenous immunity Both the introduction of the globulin gene is carried out by targeted homologous recombination or by YAC staining. Achieved by body introduction. Transgenic mammal obtained from this method Can functionally rearrange immunoglobulin component sequences, and Encoding by human immunoglobulin genes without expressing the lobulin gene It is possible to express a repertoire of antibodies of different isotypes. These properties Manufacture and properties of mammals having the same are described in Romberg et al., WO 93/12227 (1 993); Kuchel Lapaty, WO 91/10741 (1991) (these Is quoted for all purposes as a whole Are incorporated herein by reference). Transgenic ma Us is particularly suitable. Anti-L-selectin antibody is a transgenic non-human Described by animals, such as Romberg and Kucherrapati (supra) Transgenic non-human mammals with L-selectin or fragments thereof Obtained by immunization. Monoclonal antibodies are, for example, B cells from the animal were placed in a suitable myeloma cell line in a conventional Kohler-Mills It is prepared by fusing using the Tain method.   c. Phage display method   A further approach for obtaining human anti-L-selectin antibodies is described by Hughes et al. Jens, 246: 1275-1281 (1989). Screening a DNA library from human B cells according to standard protocols It is to be. Antibodies that bind to L-selectin or fragments thereof are selected. Be done. The antibody encoding such antibody (or binding fragment) is then encoded. Rows are cloned and amplified. The protocol described by Hughes is Combined with phage expression technology, it changes more efficiently. For example, Dwar et al., W O91 / 17271 and McCaferty et al., WO92 / 01047 (these are Each is hereby incorporated by reference in its entirety for all purposes. See). In these methods, the members are Antibody table The manifest phage library is produced. Antibodies are usually Fv or F Expressed as an ab fragment. A library expressing antibodies with the desired specificity. To increase the affinity for L-selectin polypeptide or fragments thereof. Larger choice.   Has the binding specificity of a murine antibody of choice in a variant of phage expression Human antibodies can be produced. See Winter, WO 92/20791. This way In a heavy chain variable of a selected murine antibody (eg, mouse DREG-200) Either the region or the light chain variable region is used as the starting material. If for example , If the light chain variable region is selected as a starting material, That is, the murine initiator) and a phage vector expressing different heavy chain variable regions. The Ibrary is built. The heavy chain variable region is a sequence of rearranged human heavy chain variable regions. Obtained from Ibrary. Strong specific binding to L-selectin (eg less With 10⁸M^-1, Preferably at least 10⁹M^-1) Is selected. The human heavy chain variable region from this phage is then added to the additional phage library. It works as a starting material for building. In this library, each phage is , The same heavy chain variable region (ie, the region identified from the original expression library) and Represents different light chain variable regions. The light chain variable region is a rearranged human variable light chain. Obtained from a library of regions. Again, the strong specific binding to L-selectin The phages shown are selected. these Phage express the variable region of fully human L-selectin antibody. these Antibody is usually the same as the murine starting material (eg, mouse DREG-200) or Or have similar epitope specificity.   how to use   The antibodies of the invention are typically mediated by inflammatory components, especially neutrophils or T cells. It finds use in the treatment of disease states involving inflammatory components. preferable Uses include myocardial infarction, cerebral ischemic conditions (eg stroke), renal, hepatic or spleen Infarction, brain surgery, shock, heart surgery (eg coronary artery bypass), elective Therapeutic or prophylactic treatment of ischemia-reperfusion injury caused by selective angiogenesis It is a place. Other preferred uses are sepsis, adult respiratory distress syndrome, and multiple organ failure. Is. Antibodies are used to treat injury due to trauma, burns, frostbite or injury to the spinal cord. The use to kick out is found. Antibodies can also be used in autoimmune diseases, which are, for example, chronic Rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, type I diabetes and throat (Including but not limited to uveitis), inflammatory diseases of the skin, such as dryness. It finds use in the treatment of tinea and in the treatment of meningitis and encephalitis. Other typical Specific applications are the prevention and treatment of organ transplant rejection and graft-versus-host disease.   Any of the antibodies of the invention may be other antibodies, particularly humans that are reactive with various adhesion molecules. Antibody or can be used in combination with human antibodies. For example, a suitable immunoglobulin is , CD11a, Specific for CD11b, CD18, E-selectin, P-selectin and ICAM-1 Contains foreign immunoglobulins. Other suitable antibodies are lymphokines such as IL- 1, antibodies specific for IL-2 and IFN-γ, and their receptors.   The antibodies of the present invention may also be given separately in a composition given with a chemotherapeutic agent. Can also be used as Typically, the drug is a non-steroidal anti-inflammatory drug and a Many additives, which may include thicosteroids, are well known to those skilled in the pharmaceutical arts. (Eg cyclosporine) may also be used. In fact, the immunoglob of the present invention Phosphorus is typically used by those skilled in the art for the treatment of certain diseases. Used in combination with currently used drugs.   In some therapeutic methods, for example, anti-L-selectin antibody is used for thrombolysis. Used in combination with agents. In the previous method, patients with acute myocardial infarction Individuals are often treated by opening an occluded coronary artery. Blocked Reopening the coronary arteries lyses the blood clot that causes the obstruction, and thereby the coronary artery. It can be achieved by administering a thrombolytic agent that restores blood flow. Reperfusion of blood vessels Also, an acute percutaneous transluminal tube by balloon dilation of the occluded or stenotic coronary artery It can be achieved by coronary artery dilation (PTCA). However, coronary blood flow Restoration leads to ischemia-reperfusion injury in the conventional manner.   In the method of the invention, ischemia-reperfusion injury is thrombolysis. Agent or PTCA with humanized or human anti-L-selectin antibody Is reduced or prevented by. Antibodies are usually administered by administration of thrombolytic agents or PTCA It is administered prophylactically before or at the same time. Then a further dose of antibody is often given. For example, during or after thrombolytic or angioplasty procedures. Prophylactic administration of antibodies, The interval between initiation of thrombolysis or thrombosis treatment is usually 5 to 30 minutes, preferably 5 to 20 minutes, most preferably 5-10 minutes. The antibody is parenterally, preferably iv Intravenous infusion, 0.01-10 mg / kg body weight, preferably 0.14-5 mg / Kg and most preferably 0.3-3 mg / kg. Anti The body may administer less as an intravenous bolus infusion, eg over 1-5 minutes. It can be given as a repeated infusion of the dose or as a single intravenous infusion. Bolus Infusion is particularly useful for prophylactic doses and in emergency situations. Antibody changes At the same dose as above, thrombolytic or angioplasty treatment for acute myocardial infarction During or after, repeat (eg, to achieve optimal plasma levels of antibody). (Eg, every 4-6 hours).   Thrombolytic agents can be either directly or indirectlyIn vivoHas the ability to stimulate the dissolution of blood clots It is a drug that does. The thrombolytic agent is a tissue plasminogen activator (Europe U.S. Pat. No. 0,936,19), an activase, Alteplase, duteplase ), Silteplase (si lteplase), streptokinase, anistreplase (anistr) eplase), urokinase, heparin, warfarin and coumarin. Additional thrombolytic agents include saruplase and blood-sucking bats. Includes rasminogen activator. Harris, Protein Engineering   6: 449-458 (1987); PCT / EP 90/00194; See U.S. Pat. No. 4,970,159. Thrombolytic agents include thrombus or its co-products. It is administered to the patient in an amount sufficient to disperse in part or prevent their formation. This An amount sufficient to accomplish this is defined as the "therapeutically effective dose" or "effective dose". Is defined. An effective amount for this purpose will be the severity of the illness, the normal condition of the patient, the route of administration. And depending on the combination with other drugs. Often, thrombolytic agents are used therapeutically and effectively. Dosages and methods of administration of such agents include FAD for the use of thrombolytic agents alone. Approved by, for example, 100 mg alteplase or 1.5 million units Is a streptokinase.   A preferred pharmaceutical composition of the present invention is for killing L-selectin expressing cells. Includes the use of immunoglobulins, the subject of the present invention, in munotoxins. Immunoto Toxins are characterized by two components, in particular:In vitroOrINBI Bo Useful for killing cells selected in. One component is a cytotoxic agent It is usually lethal to cells when attached or absorbed . First The second component, known as the "delivery vehicle", is a specific cell type. Cells, for example cells expressing the L-selectin epitope, are delivered a toxic drug. Provide a means to barry. The two components are usually of various well-known chemistries. Chemically bound to each other by any of the following methods: For example, cytotoxic agents Is a protein and the second component is a complete immunoglobulin, the binding Is a heterodivalent cross-linking substance such as SPDP, carbodiimide, glutarua. Such as Rudehid. The production of various immunotoxins is well known in the art. For example, "Monoclonal antibody-toxin conjugate: Aiming at a mysterious bullet "Torpe et al., Monoclonal Antibodies in Cli academic, Academic Press, pp. 168-190 ( 1982), which is incorporated by reference in its entirety for all purposes. Be part of the booklet). The components are also genetically linked. (Chowdaly et al., Nature 339: 394 (1989)) Incorporated by reference in its entirety for all purposes). ).   Various cytotoxic agents are suitable for use in immunotoxins. Cytotoxic agents Radionuclides, eg iodine-131 or other iodine isotopes, it Lithium-90, Rhenium-188 and Bismuth-212 or other alpha emitters , Many chemotherapeutic agents, eg vindesine, metho Lexate, adriamycin and cisplatin, and cytotoxic proteins such as , Ribosome inhibitory protein-like pokeweed antiviral protein, Pseudomonas spp. Active on cell surface such as xotoxin A, ricin, diphtheria toxin, ricin A chain Agents, such as phospholipase enzymes (eg, phospholipase C). Commonly assigned US patent application serial number 07 / 290,968 "Chimeric Toxin" Orsnes and Phil, Pharmac. There ． 25: 355-381 (1982), and monok for detecting and treating cancer. Ronal Antibody (Baldwin and Bayerz, Academic Press, 1985 ) 159-179, 224-226 (all of these for all purposes) Incorporated by reference in its entirety))).   The delivery component in the immunotoxin comprises an immunoglobulin of the invention. Perfect Immunoglobulins or binding fragments thereof, such as Fab or Fv It is preferably used. Typically, the antibody in the immunotoxin will be human IgM or IgG isotype, but other mammalian constant regions may be used if desired .   The antibodies and their pharmaceutical compositions according to the invention are especially suitable for parenteral administration, i.e. subcutaneously, It is useful in intramuscular or intravenous administration. The antibodies of the invention will also typically be local. Gastrostomy or (gastric) lavage, intraperitoneal injection, eye ointment, topical ointment for local application Agent, intracranial injection (typically in the ventricles), pericardium It can be administered by internal injection, or intrabursal injection. Non-currency Compositions for buccal administration will usually be dissolved in an acceptable carrier, preferably an aqueous carrier. Solution of the prepared immunoglobulin or a mixture thereof. Various aqueous carriers, such as Water, buffered water, phosphate buffered saline (PBS), 0.4% saline, 0.3 % Glycine, human albumin solution and the like can be used. These solutions should be sterile. And generally no particulate matter is present. These compositions are conventional, good It can be sterilized by well-known sterilization methods. The composition should be close to physiological conditions. In order to meet the requirements, pharmaceutically acceptable auxiliary substances such as pH adjustment and buffering Agents, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, salts It may include calcium chloride and sodium lactate. The concentration of antibody in these formulations is Widely, ie less than about 0.005% by weight (usually at least about 1% by weight) Can vary up to as much as 15 or 20% by weight and is mainly selected. Depending on the particular mode of administration, the choice is based on fluid volume, viscosity, etc.   Thus, a typical pharmaceutical composition for injection would be 1 ml sterile buffered water, and It can be made to contain ˜70 mg of immunoglobulin. References for intravenous infusion A typical composition should contain 250 ml of sterile Ringer's solution and 150 mg of antibody. Can be made as follows. Practical methods for preparing parenteral compositions are well known in the art. Known or obvious to those skilled in the field, for example Remington's Pharmacy. Te Sical Science (15th Edition, Mac Publishing Company, Ys) T., Pennsylvania, 1980), which is quoted for all purposes. And become a part of this specification in its entirety). Washing Or, a composition suitable for other routes may be selected according to the particular intended use. Selected. Some pharmaceutical compositions include an anti-L-selectin antibody and a thrombolytic agent. Including.   The antibody of the present invention may be frozen or lyophilized for storage, and may be stored in a suitable capacity prior to use. Can be reconstituted in the body. This technique is effective in conventional immunoglobulins Freeze drying and reconstitution techniques known in the art can be used. Freeze-drying And reconstitution can lead to varying degrees of antibody activity loss (eg, conventional immunity). Globulin, IgM antibodies tend to cause greater loss of activity than IgG antibodies. And the level of use must be adjusted to compensate. It may be recognized by those skilled in the art. It   The composition containing the antibody of the present invention or a mixture thereof is prophylactic and / or therapeutic. It can be administered for treatment. In therapeutic application, the composition already has an inflammatory disease. To prevent or at least partially prevent the disease and its complications. Administered in an amount sufficient to stop. A sufficient amount to achieve this is Effective dose ". An effective amount for this use is the severity of the disease And the patient's own immune system depends on normal conditions, but in general Ranges from about 1 to about 200 mg of antibody per dose, more usually per patient A dose of 5 to 70 mg is used. The dosing regimen will depend on the condition of the disease and the condition of the patient. More variable, typically from a single bolus dose or continuous infusion per day Multiple doses (eg, every 4-6 hours) or of the treating physician and patient. It changes as indicated by the state. The substances of the present invention are commonly used in serious disease states. I.e. can be used in life threatening or potentially threatening situations That must be taken into consideration. In such cases, Of the "heterologous" rejection reactions produced by the immunoglobulins of the present invention. It is possible to overdose such antibodies due to their low probability And may be felt by the treating physician as desirable.   In prophylactic applications, compositions containing the antibodies of the invention or mixtures thereof are still It is administered to patients who do not have a particular disease to increase their resistance. This Such an amount is defined as a "prophylactically effective dose." Accurate in this use The amount again depends on the patient's health status and normal immunity level, but in general, The range is 1 to 70 mg per administration. Preferred prophylactic use is sepsis or external Of adult respiratory distress syndrome in patients already suffering from injury, of organ transplant rejection Use for prophylaxis and prevention of reperfusion injury in patients suffering from ischemia. Heavy About 50 humanized or human immunoglobulins per dose in patients with major illnesses Dosages of ~ 150 mg are often used, and higher doses are indicated. sell.   Single or multiple doses of the composition may be administered at a dose level selected by the treating physician. And patterns. In all cases, pharmaceutical formulations are effective for patients Should provide a sufficient amount of the antibody of the invention to treat   The antibody of the present invention is furtherIn vitroIt finds widespread utility in. As an example , Antibodies for the detection of L-selectin antigen, isolation of specific white blood cells, or It can be used for others. For example, but not limited to, humanized DRE Blood immobilizing G-200 immunoglobulin and having L-selectin antigen Can be contacted with blood drawn extravascularly from the patient to remove cells, And the remaining blood, which has been depleted of L-selectin-bearing cells, It can be reintroduced into the patient. Humanized antibody remaining in depleted blood reintroduced into the patient (Eg, as a result of desorption from an immobilized support), compared to murine antibodies, Antigenicity is diminished or negligible.   For diagnostic purposes, antibodies are either labeled or unlabeled. Can be The unlabeled antibody may be humanized or otherwise labeled with a human antibody. Antibody (second antibody), eg, an antibody specific for human immunoglobulin constant regions. Can be used with the body. Alternatively, the antibody can be directly labeled. Wide range of la Bell, eg radionuclide, fluorescent dye, enzyme, enzyme substrate, enzyme cofactor, fermentation Elementary inhibition Agents, ligands (particularly haptens) and the like can be used. Many types of immuno Says are possible and are well known to those skilled in the art.   The following examples are provided by way of illustration, not limitation. Although the example relates to the mouse DREG-200 antibody, it is directed against L-selectin. Producing humanized antibodies with high binding affinity for 55, mouse DREG-56, or other binding to L-selectin epitopes It will be appreciated that this can be accomplished using CDRs from monoclonal antibodies.   Example   Example 1 Cloning of heavy and light chain cDNAs   The cDNAs of the heavy and light chain variable domain genes of mouse DREG-200 are Ko et al. Immunol. 148: 1149 (1992) and commonly assigned U.S. Patent Application Serial No. 07 / 634,278). Hybridized to the constant region using an anchored polymerase chain reaction. And a 3'primer containing a HindIII site, and hybridized to the dG tail Cloned with a 5'primer that was cloned and contains an EcoRI site. PCR The amplified fragment was digested with EcoRI and HindIII and sequenced. Cloned into the pUC18 vector for For mouse DREG-200 At least two gamma-1 specific clones and a few At least two kappa-specific clones were sequenced. Gamma-1 clone and Each of the kappa clones has the same sequence. cDNA variable domain sequence and The derived amino acid sequence is shown in FIG.   Example 2 Computer modeling of humanized antibodies   To retain high binding affinity in humanized antibodies, the general method of Queen et al. (Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029 (1989) and WO 90/07861, (these are for all purposes Incorporated herein by reference in its entirety))). Receptor The more homologous the human antibody is to the original murine donor antibody, the more human the murine CDR Introducing strains in CDRs that can reduce affinity when combined with frameworks Less likely to be done. Humanized immunoglobulin heavy Chain variable region framework and donor immunoglobulin heavy chain variable region framework At least 65% homology (ie, percent sequence identity) between sequences. Good. Normally, the heavy and light chains from the same human antibody will contain framework sequences. To reduce the likelihood of incompatibility in the assembly of the two chains. Let Based on a sequence homology search against the NBRF protein sequence database (M microGenie sequence analysis software (Beckman)), Antibody Eu provides framework sequences for humanization of mouse DREG-200. Selected to serve.   Computer program ENCAD (Levitt, J. Mol. Biol. 1 68: 595 (1983), which is cited for all purposes. Which is incorporated herein by reference in its entirety) is a model of the mouse DREG-200 variable region. Used to construct the The model is sufficient to potentially interact with the CDRs In the mouse DREG-200 framework, which approximates the CDRs in It was used to determine the no acids (category 4 below). Humanized light chain and heavy chain DR To design the EG-200 variable region, at each position, the position is Amino acids are the same as in the Eu antibody unless they meet one or more of the five categories of Was chosen to be. (1) The position was within the CDR range, (2) The Eu amino acid is abnormal for the human antibody at that position, while Us DREG-200 amino acid is typical for human antibodies in that position. there were, (3) The position was right near the CDR, (4) In the above model, the amino acid is physically close to the antigen binding region (CDR). It was indicating that it may be.   Because of the position in these categories, from the mouse DREG-200 antibody Was used.   Furthermore, if (5) below, the position was in the fifth category. Ie if,   (5) The Eu amino acid is unique to the human antibody at that position. Very abnormal, and the mouse DREG-200 amino acid is different If it is abnormal, then the position is typical for human antibodies. Specific amino acids were used.   The amino acids in each category are shown in Table 1. Some amino acids are more than one Can fall into a category. Maximum of humanized DREG-200 light and heavy chain variable domains The final sequence is shown in Figure 2 as compared to murine DREG-200.   To construct genes for humanized antibodies, humanized heavy and light chain protein sequences were constructed. It encodes a sequence and contains a signal peptide, commonly found in mouse sequences. A nucleotide sequence utilizing the codons was selected. Some synonymous codons Were modified to create restriction sites or to remove unwanted restriction sites . The nucleotide sequence of the gene also contains a splice donor signal and X at each end. ba Includes I site. Nucleotide sequences and encoded humanized light and heavy chain variables The domains are shown in FIG. Each gene is described in (Ko et al., J. Immunol. 148: 1 149 (1992) and commonly assigned U.S. patent application serial number 07/6 34,278 (these are incorporated by reference for all purposes). As described in (1)))), which is a part of this specification as a body, Constructed from synthetic oligonucleotides. Then the heavy and light chain variable region genes Are pvg1-dhfr or pvk expression vectors (commonly assigned) Xbal part of U.S. patent application serial number 07 / 634,278) Position, ligated in the proper orientation to produce the complete heavy or light chain gene. . The reaction was performed under conditions well known in the art (Maniatis, (See above).   Heavy and light chain plasmids were electroporated into Sp2 / 0 mice Transfecting myeloma cells and selecting cells for gpt expression did. Clones are assayed for human antibody production in culture supernatants by ELISA Screened and the antibody purified from the best producing clones . The humanized DREG-200 IgG1 antibody was then added to the tissue culture supernatant to remove the supernatant. Color of Tahirococcus Protein A-Sepharose CL-4B (Pharmacia) It was purified by passing through a column. The bound antibody was treated with 0.2 M glycine-HCl, Elution at pH 3.0 and neutralization with 1M Tris, pH 8.0. PD10 column The buffer was exchanged for PBS by passage through (Pharmacia) or by dialysis. To obtain cells that produce higher levels of antibody, the transfected clones Lawns can be cultured in increasing concentrations of methotrexate.   To produce a humanized DREG-200 antibody of IgG4 isotype, another vector was prepared. The vector pVg4-dhfr was constructed first. In order to do so, the γ1 constant region is The XbaI-BamHI fragment of pvg1-dhfr containing the Using methods well known to those skilled in the art, including chain reactions, the γ4 gene C_HFrom the HindIII site before the 1 exon to the C of the gene_HN following 4 exons Approximately 2000b of human γ4 constant region gene extending 270bp behind siI p fragment (Ellison and Food, Proc. Natl. Acad ． Sci. USA 79: 1984 (1982)). Then human Modified DREG-200 heavy chain variable region gene at the Xbal site of pvg4-dhfr. Cloned in. This heavy chain plasmid was then combined with the light chain plasmid described above. Cotransfected into Sp2 / 0 cells together, selecting clones and humanized The DREG-200 IgG4 antibody was purified as described above for the IgG1 antibody. It was   Example 3 Properties of humanized antibody   Humanized DREG-200 antibody against L-selectin Affinity was determined by competition with radioiodine-labeled mouse DREG-200 antibody. It was measured (Fig. 4). The binding affinity is determined by the method of Berzovsky (JA Versofus). Key and I. J. Belcower, Basic Immunology (WE Paul), Raven   Press (New York), 595 (1984), for all purposes By reference, which is hereby incorporated by reference in its entirety). Humanized DREG-200 antibody is less than about 2-fold the parent of mouse DREG-200 antibody. It had compatibility. Similar results show affinity for L-selectin on human neutrophils. Seen when sex is measured.   Adhesion of human neutrophils to endothelial cells by mouse and humanized DREG-200 antibody The ability to block is described in Harman et al., Biochem. Biophys. Res. Comm. 174: 236 (1991) using a modification of the assay method. Specifically, human umbilical cord endothelial cells (from HUVEC, Clonetics, San Diego ) Is Lab-Tek 8-chamber slide (Nunc, Naperville, IL). Growing to confluent growth in EGM medium (Clonetics). HUVE C cells were loaded with 20 ng / ml IL-1β (R & D system, (Apolis, MN) for 4 hours. Neutrophils were depleted by density gradient centrifugation. Isolate from soft layer depleted of erythrocytes by quislorane sedimentation, then 1 per ml 0⁷Was prepared. Neutrophils (100 μl) were treated with various concentrations of antibody (100 μl R During PMI) Both were preincubated on ice for 20 minutes. Wash HUVEC slides I Remove L-1β and at 4 ° C on a rotary shaker (100 rpm) placed. Add untreated or antibody-treated neutrophils to chamber and slide Were incubated for 30 minutes on a shaker at 4 ° C. Then slide 1 Wash by immersing in a beaker containing RPMI 0 times, It was fixed with 1% glutaraldehyde and air dried. Neutrophil adhesion in a microscope To count the number of neutrophils attached to a given area of a monolayer of endothelial cells using More quantified. As shown in FIG. 5, humanized IgG1 and mouse DREG-20 0 antibody both effectively blocked the binding of neutrophils to HUVEC, while The control antibody with no was not blocked. Humanized DREG-200 IgG4 anti The body also blocks the binding of neutrophils to endothelial cells.   Example 4 huDREG-200 in myocardial injury following reperfusion Effect of   IgG4 in the actual degree of rescue of myocardial ischemic tissue following reperfusion To investigate the effect of isotyped humanized DREG-200 (hu DREG-200) It was Adult male cats (2.8-4.2 kg) have sodium pentobarbital ( 30 mg / kg, i. v. ) Was used for anesthesia. Midline cut of endotracheal cannula Insert through open, and place the cat in intermittent positive pressure ventilation (Harvard Small Animal). Maru   Respirator, Dover, MA). To maintain a surgical plan for anesthesia Polyethylene for additional pentobarbital injection and for antibody administration A catheter was inserted into the right external jugular vein. Pressure transducer (Kobe Instrument , Lakewood, CO) for measurement of mean arterial blood pressure (MABP) , Another polyethylene catheter was inserted through the left femoral artery, and the abdominal aorta I placed it inside. After midsternal thoracotomy, cut the anterior pericardium , And 8-10m from its origin around the left anterior descending (LAD) coronary artery m, 3-0 silk thread suture was applied. High performance catheter tip pressure transducer (model   Equipped with MPC500 and converter control unit model TCB500. Instruments, Inc., Houston, TX) through the hollow on the tip to the left Introduced into the room. Place the catheter through observation of LV pressure and dP / dt waveform And then fixed in place by silk suture. Scalar electrocardiogram (ECG) Standard lead II was used to measure heart rate (HR) and ST-segment elevation It was ST-segment elevation analyzes ECG recording at 50 mm / s every 20 minutes It was decided by. ECG, MABP, LVP and dP / dt are T-Packard 78304 A Unit (Hewlett-Packard, Palo   (Alto, CA) continuously monitored, and Guld Oscillograph Record Dur (Gould Inc., Cleveland, OH) was recorded every 20 minutes. Pressure -Speed Degree Index (PRI), the approximate value of myocardial oxygen demand, is MABP and HR Calculated as the value obtained by dividing the product of by 1000.   After completing all surgical procedures, cats were left to stabilize for 30 minutes. That At this time, record baseline readings of ECG, MABP, LVP and dP / dt. I recorded it. Myocardial ischemia (MI) involves the first placement of a reversible ligature around the LAD. Triggered by tightening and completely occluding the vessel. This time is represented as time point 0 . 2 mg / kg body weight huDREG-200 (IgG4 isotype) or control MAb hu ABL-364 (ie, humanized control Ig of the same isotype) G4MAb) was bolated 80 min after coronary occlusion (ie 10 min before reperfusion, R). Was given parenterally. 10 minutes later (ie after 90 minutes total ischemia, I) , LAD ligature was loosened and the ischemic myocardium was reperfused for 4.5 hours.   Cats were randomly divided into three major groups. 6 fake MI + R cats Hu DREG-200 (2 mg / kg) to 6 MI + R cats MAb hu ABL-364 (2 mg / kg) and 6 MI + R Cats received hu DREG-200 (2 mg / kg). Fake MI + R cat , Same procedure as MI + R cat, but LAD coronary artery is not occluded? It was.   At the end of the 4.5 hour reperfusion period, the ligature around the LAD was retightened. 20 ml of 0.5% Evans Blue Is rapidly infused into the left ventricle and the region of the myocardium perfused by the open coronary artery is The area was stained. Areas at risk were determined by not staining. This injection Immediately following, the heart was rapidly excised and placed in warm, oxygenated KH solution. placed. Left coronary (LCX) and LAD coronary arteries, followed by coronary ring vascular activity Isolated and removed for study of sex and PMN adhesion. Right ventricle, giant blood vessels and adipose tissue Carefully, and the left ventricle in a 3 mm thick section parallel to the coronary groove. Sliced. The unstained part of the myocardium (ie the total risk or ischemic area) ) Is the area of myocardium stained with Evans blue (ie non-risk areas or non-ischemia). Area). Divide the hazardous area into small cubes and store in a phosphate solution. 15 minutes at pH 7.4 and 37 ° C. in 0.1% nitroblue tetrazolium Incubated for a period of time. Tetrazolium dyes are active dehydrogenases and Forms blue formazan complex in the presence of cardiomyocytes containing these cofactors It The portion of the myocardium at risk of irreversibly damaged or necrotic (which is stained No) from the stained portion of the myocardium (ie ischemic but non-necrotic area) did. Next, three parts of the myocardium (ie, non-ischemic tissue, ischemic non-necrotic tissue, and imaginary tissue). Blood necrosis tissue). Results are in the necrotic heart tissue area, at risk or all left Expressed as a percentage of either ventricular mass.   According to both of these criteria, heart tissue damage In cats treated with hu DREG-200, there was a significant reduction (p <0. 001). In a group where about 30% of the dangerous myocardium was treated with control antibody A group treated with hu DREG-200, whereas it developed into necrotic tissue The amount of necrotic tissue was less than 15% (p <0.01), It is a decrease. See FIG. Percentage of total left ventricle between the two ischemic groups There is no significant difference in the wet weight of the hazardous area, expressed as It shows that similar areas of blood occurred in the two groups. Hence h u DREG-200 shows significant protection against reperfusion injury.   Notable protection of ischemic tissue by hu DREG-200 is plasma clair. From the measurement of tin kinase activity, which is a biochemical marker of myocardial damage Further description. Arterial blood sample (2 ml), 1 hour before and after ligation It was quickly withdrawn. Blood is a porosity containing 200 IU of heparin sodium. Collected in a polyethylene tube. Sample at 2000 xg, 4 ° C for 20 minutes While centrifuging and plasma decanted for biochemical assay. plasma The protein concentration was determined according to Gornal et al. Biol. Chem. 177: 751-766 It was assayed using the Burett method of (1949). Plasma creatine kinase (CK) activity is described by Rosarki, J. et al. Lab. Clin. Med. 69: 696-7 05 (1967) and was expressed as IU / μg protein.   In fake MI / R cats fed hu DREG-200, plasma CK activity was , Slightly increased over the 6-hour observation period, with 3.8 ± 0.9 IU / μg protein The final value has been reached. In the two ischemic groups, plasma CK activity is the phase of myocardial ischemia. It increased slightly during the whole period. In cats given hu ABL-364, C in circulating blood K activity increased significantly within the first 30 minutes after reperfusion, and at 4 o'clock following reperfusion It increased further during the period. In contrast, treated with hu DREG-200 Ischemic cats have significantly lower plaques than ischemic cats fed hu ABL-364. Zuma CK activity was shown (p <0.05). The effect is maintained over the entire reperfusion period. Which further provided substantial protection against myocardial reperfusion injury, hu DREG-. Prove that given by 200.   Example 5 Effect of hu DREG-200 on cardiac function   The effect of hu DREG-200 (IgG4 isotype) on cardiac function, The maximum dP / dt (myocardial contraction), which is the temporary derivative of left ventricular pressure (LVP) and LVP. It was determined by measuring the sex index). Data are inserted into the left ventricle cavity. Obtained from an inserted catheter tip sphygmomanometer. Cats discussed in previous examples All three groups showed comparable initial values for these cardiac variables . In the sham MI group, maximal over the entire 6-hour experimental period There was no significant change in dP / dt. However, both MI / R groups In max, the maximum dP / dt was reduced by approximately 65% shortly after LAD occlusion. . Contractility was not significantly restored in cats fed hu ABL-364. . However, in MI-R cats treated with hu DREG-200, maximum dP / dt returned to control values 3 hours after reperfusion. Therefore, 4.5 of reperfusion After hours, the maximum dP / dt of cats treated with hu ABL-364 was hu   Significantly lower compared to cats treated with DREG-200 (p <0.01) . These results indicate that hu DREG-200 follows reperfusion of the ischemic myocardium. Not only does myocardial necrosis decrease, but this myocardial rescue also helps the heart's mechanical performance. It shows that it has also been improved.   From the above, the immunoglobulin of the present invention is non-toxic compared to other L-selectin specific antibodies. It will be appreciated that it will always offer many advantages. Mouse monoclonal antibody The immunoglobulin of the present invention can be produced more economically, and It contains substantially fewer heterologous amino acid sequences. Antigen after injection into human patients This reduced likelihood of sex represents a significant therapeutic improvement.   All documents and patent applications cited above are individual documents or patent applications. Is clearly and individually shown to be part by being quoted. To the same extent as a whole by being quoted for all purposes Become a part of this specification. The present invention has been described and described for purposes of clarity and understanding. Although described in more detail in the examples, certain modifications and improvements are It is obvious that it can be done within the scope of the following claims.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification code Internal reference number FI A61K 39/395 ACD ADZ C07K 16/26 8318-4H C12P 21/08 9358-4B (81) Designated country EP (AT , BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN) , ML, MR, NE, SN, TD, TG), AT, AU, BB, BG, BR, BY, CA, CH, CZ, DE, DK, ES, FI, GB, HU, JP, KP, KR. , KZ, LK, LU, LV, MG, MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SK, UA, US, VN

Claims (1)

[Claims] 1. A CDR corresponding to a complementarity determining region (CDR) from a donor immunoglobulin and a heavy and light chain variable region framework corresponding to a human acceptor immunoglobulin heavy and light chain framework, and at least 10 A humanized immunoglobulin that specifically binds to human L-selectin with an affinity constant of ⁷ M ^-1 , wherein the sequence of the humanized immunoglobulin heavy chain variable region framework is a donor immunoglobulin heavy chain variable region frame. A humanized immunoglobulin having 65% or more homology to the sequence of the work. 2. The humanized immunoglobulin of claim 1, wherein the antibody comprises two light / heavy chain dimers. 3. The humanized immunoglobulin according to claim 2, wherein the antibody is IgG1 or IgG4 isotype. 4. The humanized immunoglobulin of claim 1, which specifically binds human L-selectin with an affinity of at least 10 ⁸ M ^-1 . 5. The humanized immunoglobulin according to claim 1, wherein the donor immunoglobulin is a mouse DREG-200 antibody. 6. The humanized immunoglobulin according to claim 1, wherein said receptor immunoglobulin heavy and light chain frameworks are from the same human antibody. 7. The humanized immunoglobulin according to claim 6, wherein the human antibody is a Eu human antibody. 8. A CDR corresponding to the complementarity determining regions (CDRs) from the donor immunoglobulin and a heavy and light chain variable region framework corresponding to the acceptor immunoglobulin heavy and light chain frameworks, and at least 10 ⁷ A humanized immunoglobulin that specifically binds to human L-selectin with an affinity constant of M ^-1 , wherein the sequence of the acceptor immunoglobulin heavy chain variable region is most similar to that of the donor immunoglobulin heavy chain variable region. A humanized immunoglobulin that is one of five sequences in a representative collection of sequences of human immunoglobulin heavy chain variable regions that are homologous. 9. A CDR corresponding to the complementarity determining regions (CDRs) from the donor immunoglobulin and a heavy and light chain variable region framework corresponding to the acceptor immunoglobulin heavy and light chain frameworks, and at least 10 ⁷ A humanized immunoglobulin that specifically binds human L-selectin with an affinity constant of M ^-1 , wherein the humanized immunoglobulin binds a corresponding amino acid in a receptor immunoglobulin heavy or light chain framework. Including an amino acid from the donor immunoglobulin framework to replace, wherein the amino acid is not at positions 26-30 of the heavy chain, and each of the amino acids is (1) adjacent to a CDR in the donor immunoglobulin sequence. Or (2) contains an atom within a distance of 5 angstroms from the CDR in the humanized immunoglobulin, Humanized immunoglobulin according to claim. 10. CDRs corresponding to complementarity determining regions (CDRs) from the donor immunoglobulin and heavy and light chain variable region frameworks corresponding to the acceptor immunoglobulin heavy and light chain frameworks, and at least 10 ⁷ A humanized immunoglobulin that specifically binds human L-selectin with an affinity constant of M ^-1 , wherein the humanized immunoglobulin binds a corresponding amino acid in a receptor immunoglobulin heavy or light chain framework. Including an amino acid from the donor immunoglobulin framework to replace, wherein the amino acid is not at positions 26-30 of the heavy chain, and each of the amino acids is (1) adjacent to a CDR in the donor immunoglobulin sequence. Or (2) contains an atom within 4 angstroms of the CDR in the humanized immunoglobulin, Humanized immunoglobulin according to claim. 11. 11. The humanized immunoglobulin according to claim 9 or 10, wherein the distance from the atom to the CDR is determined from a computer generated model of the immunoglobulin. 12. The humanized immunoglobulin according to claim 9 or 10, wherein the donor immunoglobulin is a mouse DREG-200 antibody. 13. The humanized immunoglobulin according to claim 9, which is an antibody containing two light chain / heavy chain dimers. 14. The humanized immunoglobulin according to claim 13, wherein the antibody is IgG1 or IgG4 isotype. 15. 10. The humanized immunoglobulin according to claim 9, wherein the receptor immunoglobulin heavy and light chain frameworks are both from Eu human antibodies. 16. 10. A humanized immunoglobulin according to claim 1 or 9 which is substantially pure. 17． The humanized immunoglobulin according to claim 1, which inhibits binding of human neutrophils to human endothelial cells. 18. A composition comprising the humanized immunoglobulin according to claim 1 or 9. 19. A recombinant immunoglobulin that specifically binds to human L-selectin, wherein the amino acid sequence of the mature light chain variable region is shown in the lower part of FIG. 2A. 20. A recombinant immunoglobulin which specifically binds to human L-selectin, wherein the amino acid sequence of the mature heavy chain variable region is shown in the lower part of FIG. 2B. 21. A method of treating an inflammatory disease or condition comprising administering to a human patient a therapeutically effective dose of a humanized immunoglobulin that specifically binds human L-selectin. 22. 22. The method of claim 21, wherein the inflammatory disease or condition is selected from the group consisting of ischemia-reperfusion injury, adult respiratory distress syndrome, sepsis and autoimmune disease. 23. 22. The humanized immunoglobulin according to claim 21, wherein the humanized immunoglobulin comprises the amino acid sequence of the mature light chain variable region shown in the lower part of FIG. 2A and the amino acid sequence of the mature heavy chain variable region shown in the lower part of FIG. 2B. Method. 24. 22. The method of claim 21, wherein the humanized immunoglobulin binds human L-selectin with an affinity of at least 1 x 10 ⁷ M ^-1 . 25. 25. The method of claim 24, wherein at least 10 mg of humanized immunoglobulin is administered by the parenteral route. 26. 23. The method of claim 22, wherein the inflammatory disease or condition is ischemia-reperfusion injury. 27. 27. The method of claim 26, further comprising the step of administering a therapeutically effective dose of a thrombolytic agent. 28. 28. The method of claim 27, wherein the ischemia-reperfusion injury is due to myocardial infarction or balloon angioplasty. 29. A humanized immunoglobulin (1) having the following humanized heavy chain and humanized light chain, having an amino acid sequence from the corresponding complementarity determining region of the mouse DREG-200 immunoglobulin light chain. And having a variable region framework from a human light chain variable region framework sequence except at least one position selected from the first group consisting of L87, L54, L66, L76 and L93 (wherein the amino acid The position is a humanized light chain comprising three complementarity determining regions (CDR1, CDR2 and CDR3) occupied by its amino acids present at equivalent positions in the mouse DREG-200 immunoglobulin light chain variable region framework. And (2) the amino acid sequence from the corresponding complementarity determining region of mouse DREG-200 immunoglobulin heavy chain And a human heavy chain variable region framework sequence except at least one position selected from the group consisting of H93, H95, H98, H111, H112, H115, H30, H98, H111, H27, H30, H48 and H72. Complementarity Determining Regions with the variable region frameworks from which the amino acid positions are occupied by the amino acids located at equivalent positions in the mouse DREG-200 immunoglobulin heavy chain variable region frameworks. A humanized heavy chain comprising (CDR1, CDR2 and CDR3), wherein the immunoglobulin binds to L-selectin ligand with a binding affinity within 3 times that of mouse DREG-200 immunoglobulin.