HUT71790A - Humanized antibodies reactive with l-selectin - Google Patents

Description

The present invention is a continuation of USSN Patent Application No. 07 / 983,946, filed January 12, 1992, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the development of novel biological agents related to the combination of recombinant DNA and monoclonal antibody technologies, and more particularly to the production of non-immunogenic (human) immunoglobulins specific for the L-selectin protein and their use in vitro and in vivo.

BACKGROUND OF THE INVENTION

The ability of cells to adhere to each other plays a critical role in development, normal physiology, and disease processes. This ability is mediated by adhesion molecules expressed on cell membranes, generally glycoproteins. Often, an adhesion molecule on one cell type binds to another adhesion molecule expressed on a different cell type, creating a receptor pair against a receptor. Three very important classes of adhesion molecules are: integrins, selectins, and members of the immunoglobulin (Ig) superfamily (see Springer, Naturre 346: 425, 1990; Osborn, Cell 62: 3, 1990; Hynes, Cell 69:11, 1992). , all of which are fully utilized through the link). They are essential for the interaction of leukocytes and platelets with themselves and with extracellular matrix and vascular endothelium.

Integrins are heterodimeric transmembrane glycoproteins consisting of a chain (120-180 kD) and a β chain (90-110 kD), generally short

have cytoplasmic domains. Those subunits all share sequence homology and motifs, as do the β subunits. Known integrins containing the three α-labeled β subunits are important for T cells, neutrophils and monocytes. LFA-1 (α1β2) is widely distributed on lymphocytes, granulocytes and monocytes. Ellen's receptor, ICAM-1 (and perhaps less importantly ICAM-2), is an Ig family molecule that is expressed on a number of cells, including leukocytes, and is up-regulated in the vascular endothelium by cytokines such as TNF and IL-1. Blocking LFA-1 on T cells with antibodies or on α or β subunits strongly inhibits adhesion-dependent functions such as CTL lysis of target cells. Mac-1 (? / 2?) Is distributed ^on neutrophils and monocytes and its counter-receptor is ICAM-1 (and presumably ICAM-2). Among other things, Mac-1 binds the type 3 complementary receptor (CR3) and the C3bi fragment. The third β2 integrin, P150.95 (αχβ2), is also found on neutrophils and monocytes, but appears to be much less important. The subunits of LFA-1, Mac-1, and P150.95 <x also refer to CD11a, CD11b, and CD11c, respectively, while β2 ~ ί also denotes CD18, so LFA-1 CD11a / CD18 and Mac-1 CD11 b / CD18.

There are three known selectins, formerly known as LECCAMs, now known as L-selectin (also called LECAM-1, Mel-1 or LAM-1) and P-selectin (also known as GMP140 or PADGEM). called). These have all been sequenced at the cDNA level, sharing sequence homologies and motifs, including the lectin-like domain. L-selectin has a dual role: a targeting receptor on T cells for highly endothelial venules of peripheral lymph nodes, and an adhesion molecule on neutrophils for endothelium (Hallmann et al., Biochem.

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Biophys. Gap. Commun. 174: 236, 1991; which is incorporated herein by reference in its entirety). E-selectin and P-selectin are induced by cytokines on the endothelium, although with different kinetics. L-selectin is a counter-receptor on neutrophils for L-selectin and P-selectin (Picker et al., Cell 66: 921, 1991, which is hereby incorporated by reference in its entirety), although all three selectins may receptor. More specifically, E-selectin binds the carbohydrate moiety sialyl Lewis x (sLex) (Lowe et al., Cell, 63: 475, 1990, which is incorporated herein by reference in its entirety), and while this carbohydrate is remarkably L- is selective (Picker et al., Cell 66: 921, 1991), it may also be present on other proteins. E-selectin is particularly expressed on inflamed skin surfaces and may serve as an adhesion molecule for T cells in the skin, which may contribute to inflammation (Picker et al., Naturre 349: 796, 1991, which is incorporated herein by reference in its entirety).

In various assays, antibodies to CD11a, CD11b, CD18, L-selectin, and E-selectin inhibit neutrophil binding to activated emdothelial cells to a greater or lesser degree, but the most complete inhibition is generally achieved when CD18 antibody is combined with an antibody to L or E-selectin (see, e.g., Luscinskas, J. Immunol., 142: 2257, 1989, which is incorporated herein by reference in its entirety). A new, but widely accepted, model explains these facts by a three-step process of adhesion (Butcher, Cell, 67.103, 1991, which is incorporated herein by reference in its entirety). In the first step, the neutrophils bind reversibly to the inflamed vascular endothelium through selectins, which bind well under flow conditions, causing the neutrophils to literally roll along the vascular wall. Neutrophils are then activated by a number of stimulants released by the endothelium or in the environment, including IL-8, PAF, and C5a. Activated neutrophils release L-selectin and up-regulate Mac-1. In the final step, binding of Mac-1 to ICAM-1 and possibly other receptors on endothelial cells allows for stable adhesion and extravasation through the endothelium. In principle, antibodies or other antagonists of integrin and selectin adhesion molecules can stop this process by preventing neutrophils from binding to the endothelium and extravasation into tissues. Therefore, such antibodies are useful in the treatment of many diseases for which inflammation is an important ingredient.

For example, in animal models, anti-CD18 antibodies that bind to LFA-1 and Mac-1 have been useful in reducing ischemic reperfusion injury (see, e.g., Vedder et al., J. Clin. Invest. 81: 939, 1988). Vedder et al., Proc. Natl. Acad. Sci. USA 87: 2643, 1990; U.S. Patent 4,797,277. Neutrophil-mediated plaque damage in response to various injuries is also reduced (Doerschuk et al., J. Immunol. 144: 2327, 1990 and Mulligan et al., J. Immunol., 148: 1847, 1992), including Gram-negative sepsis (Walsh). et al., Surgery 110: 205, 1991). In a rabbit model, anti-CD18 antibodies also provide protection against lethality due to meningitis (Tuomanen et al., J. Exp. Med. 170: 959, 1990). They may be useful in preventing or treating organ transplant rejection by inhibiting T cell function.

For example, injection of antibodies against L-selectin or E-selectin into rodents prevented neutrophil accumulation in the peritoneal gland (Jutila et al., J. Immunol. 143: 3318, pp. 143: 3318).

-6 1989 and Mulligan et al., J. Clin. Invest. 88: 1396-1991). Intravital video microscopy revealed that an anti-L-selectin antibody was strongly inhibited by the vascular wall of the mesenteric venules exterior to the leukocyte rabbit endothelium Sci. An anti-E-selectin antibody strongly reduced vascular injury through immune complex deposition in rat skin and lungs and significantly reduced neutrophil accumulation at these sites (Mulligen et al., J. Clin. Invest. 88: 1396, 1991). In a primate model of external asthma, an anti-E-selectin antibody greatly reduced neutrophil influx into the lungs and associated late-phase airway obstruction following antigen inhalation (Gundel et al., J. Clin. Invest, 88: 1407, 1991). Numerous antibodies, including murine DREG-55 antibody, murine DREG-56 and DREG200 antibodies, have been developed that bind human L-selectin (Kishimoto et al., Proc. Natl. Acad. Sci USA 87: 2244, 1990, which is hereby incorporated by reference in its entirety. These antibodies partially or completely inhibit the binding of human lymphocytes to peripheral lymph nodes in high endothelial venules and the binding of human neutrophils to stimulated human umbilical vein endothelial cells (Kishimoto et al., Blood, 78: 805, 1991). of the invention). The capacity to inhibit the binding of these antibodies to neutrophils on endothelial cells indicates that the antigen to which they are bound, L-selectin, may be an appropriate target for potential therapeutic agents.

Unfortunately, non-human monoclonal antibodies, such as murine DREG-200, have certain drawbacks in human treatment, particularly with repeated therapeutic lifestyles, as described below. Mouse monoclonal antibodies, for example, have a relatively short circulation half-life ···

They have -7 t and have no other important functional feature of immunoglobulin when used in humans.

Perhaps more importantly, non-human monoclonal antibodies contain essential amino acid sequence portions that will be immunogenic when injected into a human patient. Numerous studies have shown that after injection of a foreign antibody, the patient's immune response to the antibody can be quite strong, essentially preventing the antibody from being therapeutically useful after an initial treatment. Furthermore, as it is expected that an increasing number of different mouse and other antigenic (human) monoclonal antibodies will be developed to treat various diseases, following the first or multiple treatments with any non-human antibody, subsequent treatments, even with independent therapies, will they can also be dangerous due to cross reactivity. While the production of so-called chimeric antibodies (e.g., mouse variable regions attached to human constant regions) has been somewhat successful, a major immunogenicity problem remains.

Numerous examples of humanized antibodies have been generated for attempts to solve immunogenicity problems. The transition from a rat antibody to a humanized antibody involves a compromise between competing aspects, a solution to which different antibodies belong. To minimize immunogenicity, the immunoglobulin should retain as much of the human acceptor sequence as possible. However, to maintain authentic binding properties, the immunoglobulin framework should contain appropriate substitutions of the human acceptor sequence to ensure that the three-dimensional conformation of the CDR regions is as close as possible to that in the mouse donor immunoglobulin. Aspects of this competition

As a result, many of the humanized antibodies produced today show significant loss in binding affinity compared to the corresponding rat antibodies. See, e.g. Jones, et al., Natura 321: 522-525, 1986; Shearman et al., J. Immunol. 147: 4366-4373, 1991; Kettleborough Protein Engineering 4: -773-783, 1991; Gorman et al., Proc. Natl. Acad. Sci. USA 88: 41814185, 1991; Tempest et al., Biotechnology 9: 266-271, 1991; Riechmann et al., Natura 332: 323, 1988 and EPO Publication 0239400, which are hereby incorporated by reference in their entirety.

Thus, there is a need for improved forms of humanized immunoglobulins specific for the L-selectin antigen, which are essentially non-immunogenic in humans, but can be easily and economically produced for therapeutic and other uses. The invention satisfies these and other needs.

Summary of the Invention

The present invention provides novel formulations useful, for example, in the treatment of human inflammatory disorders, which contain humanized immunoglobulins that are capable of specifically binding L-selectin. Immunoglobulins may have two pairs of light chain / heavy chain complexes, at least one of which comprises one or more mouse complementarity determining regions operably linked to human frame region segments. For example, additional naturally occurring mouse amino acid residues with or without mouse complementarity determining regions can be introduced into the human framework regions, 10? higher affinity levels to generate humanized immunoglobulins capable of binding to Lselectin.

• · · · • · ««

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These humanized immunoglobulins will be able to inhibit the binding of the mouse monoclonal antibody providing CDR to L-selectin.

The immunoglobulins of the invention, including their binding fragments and other derivatives, can be readily produced by various recombinant DNA techniques by final expression in transfected cells, preferably immortalized eukaryotic cells such as myeloma or hybridoma cells. Polynucleotides comprising the first sequence encoding the humanized immunoglobulin framework regions and a second set of sequences encoding the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments.

Humanized immunoglobulins may be used alone, in substantially pure form, or in combination with a chemotherapeutic agent such as a non-steroidal anti-inflammatory drug (e.g. aspirin), a corticosteroid or an immunosuppressive agent. Each of these compounds is particularly useful in the treatment of inflammatory disorders. Humanized immunoglobulins or complexes thereof may be prepared in a pharmaceutically acceptable dosage form which will vary with the route of administration.

Brief Description of the Drawings

Figure 1. CDNA sequences and translated amino acid sequences for the light chain (A) and heavy chain (B) variable regions of mouse DREG-200 antibody. The mature heavy chain begins with the 20 E residue and the mature light chain begins with the 21 D amino acid, preceded by the corresponding signal sequences.

Figure 2. Amino acid sequences of the mature light chain (A) and heavy chain (B) variable regions of mouse DREG-200 antibody (upper rows) and humanized DREG-200 antibody (lower rows). The three CDRs in each chain were underlined. The Frame ····

residues in the humanized antibody that have been replaced by mouse amino acids or typical human amino acids are indicated by double underline.

Figure 3. Nucleotide sequences of the genes encoding the light chain (A) and heavy chain (B) variable regions of the humanized DREG-200 antibody, which begin and end at the Xba1 site and the translated amino acid sequences, including signal sequences.

Figure 4 Competitive binding of mouse and humanized IgG1 and IgG4 DREG-200 antibodies. The target cells are L2-1 cells, a mouse pre-B cell line, which was transfected with the human L-selectin gene and thus expresses human L-selectin (Berg et al., Biochem. Biophys. Res. Comm. 184: 1048, 1992). 5x10 $ cells were incubated with 3 ng 125 | _j _e | OIE _S ü tracer mouse antibody (2 uCi / ug), mouse or humanized competitive antibody together with increasing amounts of, as indicated in 0.2 ml of binding buffer (PBS + 2% FBS + 0 , 1% azide) for 1 hour at 4 ° C. The cells were washed and pelleted and their bound radioactivity was measured. The concentration of bound and free tracer antibody was calculated.

Figure 5 Binding of human neutrophils to IL-1 stimulated human umbilical cord endothelial cells (HUVEC). Neutrophils were first treated with or left untreated control antibody, mouse DREG-200 antibody, or humanized JgG1 DREG-200 antibody, as indicated on the label.

Figure 6 Protection of ischemic reperfused heart tissue with humanized DREG-200. The figure shows a cat treated with humanized DREG-200 or control antibody, from left to right: endangered area / total ventricular area; necrotic tissue area / endangered area; and necrotic tissue area / total left ventricular area. The parentheses represent +/- SEM for six cats; the height of the columns is an average.

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definitions

The term basic identity or basic homologue means that two peptide sequences have at least 65% sequence identity, preferably at least 80 or 90% sequence identity, more preferably at least 95% or greater (e.g., 99% sequence identity) shows sequence identity if optimally sequenced, e.g. with GAP or BESTFIT, using faulty gap weights. Preferably, the non-identical residual positions differ in conservative amino acid substitutions.

For conservative or non-conservative grouping of amino acid substitutions, amino acids are classified into the following group: Group I (hydrophobic side chains): norleucine, met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gin, his, lys, arg; Group V (residues affecting chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions refer to substitutions between amino acids of the same group. Non-conservative substitutions involve the replacement of one member of these groups with another member of the group. Amino acids from the variable regions of the mature heavy and light chains of immunoglobulins are designated as Hx and Lx, respectively, where x is the amino acid sequence of the Kaba (Sequence of Proteins of Immunological Interest, Bethesda, MD, 1987 and 1991). position number. Kába lists the amino acid sequences of several antibodies in each subclass, and lists the most common amino acids for each of the remaining positions in this subclass. Coat of Arms uses a method to denote a residual number for each amino acid in the listed sequence, and the method of denoting residual numbers has become standard in this science ····

-12f area. The Kabak scheme can be extended to other antibodies that are not included in the summary by aligning the antibody in question with one of the consensus sequences in the Kabat scheme. Using the Kabat numbering system, it is easy to identify amino acids at the same positions in different antibodies. For example, an amino acid at the L50 position in a human antibody occupies the same L50 position in the mouse antibody.

From the N-terminus to the C-terminus, both the light chain and heavy chain comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The designation of the amino acids belonging to each domain is in accordance with the definitions of Kabat (1987 and 1991, supra, or Chothia & Lesk, J. Mol. Bioi. 196: 901-917, 1987; Chothia et al., Natur 342: 878-883, 1989). ).

Detailed Description of the Invention

In general, the nomenclature and laboratory techniques used in the invention and described in cell culture, molecular genetics, nucleic acid chemistry, and hybridization methods are well known and commonly used in the art. Standard techniques are used in recombinant nucleic acid methods, polynucleotide synthesis, cell cultures, and transgene insertions (electroporation, microinjection, lipofection). In general, enzymatic reactions, oligonucleotide synthesis, and purification steps are performed according to the manufacturers specifications. The techniques and methods are generally carried out in accordance with conventional methods used in science and various general references provided by the present invention. References to the present invention are believed to be well known in the art and are provided for the convenience of the reader only. THE • ·

All information provided in the present invention is hereby incorporated by reference in its entirety.

Humanized antibodies to L-selectin

In accordance with the invention, humanized immunoglobulins specifically reactive with L-selectin-related epitopes are provided. These immunoglobulins usually at least 10 ^ ^M-1 binding affinity, preferably 10θ M1 - M1 or 10θ bind stronger binding affinity for L-selectin and can, for example. binding to neutrophils. Humanized immunoglobulins have a human framework and have one or more complementarity determining regions (CDRs) derived from an immunoglobulin that is specifically reactive with L-selectin, typically a mouse immunoglobulin. In a preferred embodiment, one or more CDRs are derived from murine DREG-200 antibody and the humanized immunoglobulin will be of the IgG1 or IgG4 isotype. Thus, the immunoglobulins of the invention, which can be produced in large amounts economically, can be used, for example, in human patients for the treatment of inflammatory disorders using a variety of techniques.

The basic antibody structural unit is known to contain a tetramer. Each tetramer consists of two identical polypeptide chain pairs, each pair consisting of a light (about 25 kD) and a heavy (about 50-70 kD) chains. The NH2 terminus of each chain begins with a variable region of about 100-110 amino acids or more, primarily responsible for antigen recognition. The COOH portion of each chain defines a constant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains are gamma, art, alpha, delta or epsilon

-14 is defined and the antibody isotype is defined as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions have an approx. Linked to a J region of 12 or more amino acids, the heavy chain also contains a D region of about 10 or more amino acids. (see, in general, Fundamental Immunology, Paul, W., ed., Chapter 7, 131166, Raven Press, N.Y., 1984, which is incorporated herein by reference in its entirety).

The variable regions of each light / heavy chain pair create antibody binding sites. The chains show the same general structure of relatively conserved framework regions that join three hypervariable regions, also referred to as Complementarity Determining Regions (CDRs) (see Sequences of Proteins of Immunological Interest, E. Kaba, et al., US Department of Health). and Human Services, 1987, and Chothia and Lesk, J. Mol. Biol., 196: 901-917, 1987, which are incorporated herein by reference in their entirety. The CDRs from the two strands of each pair are flanked by the framework regions, allowing binding to a specific epitope.

As used herein, the term immunoglobulin refers to a protein comprising essentially one or more polypeptides encoded by an immunoglobulin gene. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes as well as countless immunoglobulin variable region genes. Immunoglobulins can exist in a variety of forms in addition to antibodies, including, for example, Fv, Fab and (Fab ') 2, and bifunctional antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17: 105) and single chains ( See, e.g., Huston et al., Proc. natl. Acad. Sci. USA 85: 5879-5883, 1988;

-15 et al., Science 242: 423-426, 1988, which are incorporated herein by reference in their entirety, see generally Hood et al., Immunology (Benjamin, N.Y. 2nd ed. 1984), Harlow & Lane, Antibodies: A Laboratory Manual (Cold Sprimg Harbor Laboratory, 1988, and Hunkapiller & Hood, Natura 323: 15-16, 1986, which are incorporated herein by reference in their entirety).

Chimeric antibodies are antibodies whose light and heavy chain genes are typically engineered from immunoglobulin gene segments of various species. For example, variable (V) segments of genes derived from a murine monoclonal antibody can be linked to human constant (C) segments, e.g. for a ·] and «4. A typical therapeutic chimeric antibody is thus a hybrid protein comprising the V or antigen-binding domain of a mouse antibody and a C or effector domain derived from a human antibody, although other mammalian species may be used.

As used herein, the term framework region refers to portions of the immunoglobulin light and heavy chain variable regions that are relatively conserved (i.e., different from CDRs) between different immunoglobulins within a single species, as defined by Kaba et al., Supra. In the use of the present invention, a human framework region is a framework region that is substantially identical (about 85% or greater) to a naturally occurring human antibody framework region, or to the consensus sequence of many such antibodies.

As used herein, the term humanized immunoglobulin comprises a human framework, at least one non-human antibody-derived CDR, and wherein any constant region present is substantially identical to a human immunoglobulin constant region, i.e., at least 85-90%, preferably at least · ··· • · ϊ ”Τ · · · ··» * ·; · *; ·

-16J% identical. Therefore, all parts of a humanized immunoglobulin, with the exception of perhaps CDRs, are substantially identical to corresponding portions of one or more native human immunoglobulins. For example, a humanized immunoglobulin will not enclose a chimeric mouse variable region / human constant region antibody.

Humanized antibodies have at least three advantages over murine and, in some cases, chimeric antibodies for human therapeutic use:

1) since the effector moiety is human, it may be more responsive to other parts of the human immune system (e.g., it is more effective in destroying target cells due to complement dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC).

2) The human immune system will not recognize the framework or C region of the humanized antibody, so the antibody response to such an injected antibody will be less than that of a fully foreign mouse antibody or a partially foreign chimeric antibody.

3) Injected murine antibodies have been reported to have a much shorter half-life in the human circulation than normal antibodies (Shaw, D et al., J. Immunol. 138: 4534-4538, 1987). The half-life of injected humanized antibodies is expected to be much more similar to that of naturally occurring human antibodies, thus allowing for a lower and much less frequent dose administration.

In one aspect, the invention relates to a desired epitope of L-selectin, such as a. monoclonal antibodies, mouse DREG-200, mouse DREG-55, or mouse DREG-56 (Kishimoto et al., 1990, supra, which are incorporated herein by reference in their entirety) capable of binding immunoglobulin derived from ···· · ··· · •: ··· · * ί · ·· ··. ,. ·; ··; ·

-17 targeting recombinant DNA segments encoding heavy and / or light chain CDRs. The DNA segments encoding these regions will typically be linked to the DNA segments encoding the corresponding human framework regions. Sample DNA sequences encoding polypeptides containing CDRs of the murine DREG-200 monoclonal antibody during expression are shown in Figure 1. Due to code degeneracy and non-critical amino acid substitutions, other DNA sequences can be readily used for these sequences, as detailed below. For a detailed description of the design and production of humanized immunoglobulins, see generally Serial Nos. 07 / 290,975 and 07 / 310,252, filed December 28, 1988 and February 13, 1989, which are hereby incorporated by reference in their entirety.

DNA segments typically further include expression control DNA sequences operably linked to humanized immunoglobulin coding sequences having naturally-linked or heterologous promoter regions. Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, however, regulatory sequences for prokaryotic hosts may also be used. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and then, as desired, light chains, heavy chains, light / heavy chain dimers, or intact antibodies, binding fragments, or other collection and purification of immunoglobulin forms may follow.

Nucleic acid sequences capable of finally expressing desired humanized antibodies from a variety of different polynucleotides (genome or cDNA, ···:. ··, .....

• ·. : ··· · '·· ···. * ·; ····

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RNA, synthetic oligonucleotides, etc.) and components (e.g., V, J, D, and C regions) and many different techniques. Linking the appropriate genome and synthetic sequences is currently the most common method of production, but cDNA sequences are also available (see European Patent Publication No. 0239400, and Riechmann, L., et al., Natura 332: 323-327, 1988, which are incorporated herein by reference in their entirety). as part of the invention).

Human constant region DNA sequences can be isolated from a variety of human cells, in particular immortalized B cells, according to well-known procedures (see Kaba, supra, and WP87 / 02671). Similarly, CDRs for the production of immunoglobulins of the invention are derived from monoclonal antibodies which are capable of binding to L-selectin and can be produced from any suitable mammalian source, including mice, rats, rabbits or other vertebrates capable of producing antibodies by well-known methods. Suitable source cells for DNA sequences and host cells for immunoglobulin expression and secretion are available from a variety of sources, such as the American Type Culture Collection (1985), Rockville, MD. is fully part of the invention). In preferred embodiments, the CDRs have sequences corresponding to the CDR sequences of mouse DREG-200, mouse DREG-55, or mouse DREG56, and may include sequences corresponding to mouse DREG-200, mouse DREG-55, or mouse DREG-56. And degenerate nucleotide sequences encoding the CDR amino acid sequences.

In addition to the humanized immunoglobulins specifically described herein, other substantially homologous modified immunoglobulins may be readily designed and prepared using various recombinant DNA techniques known to those of ordinary skill in the art. Other human antibodies other than the Eu antibody discussed in Example 2 may be used as a source of the framework sequence. These frame sequences should exhibit a high degree of sequence identity with the murine DREG-200 variable frame domains from which the CDRs are derived. The heavy and light chain variable framework regions may be derived from the same or different human antibody sequences. In fact, each heavy and light chain framework region may be derived from more than one human antibody. The human antibody sequences may be sequences of naturally occurring human antibodies or may be consensus sequences of many human antibodies. See Carter et al., WO 92/22653, 1992.

The unnatural juxtaposition of the rat CDR regions with the human variable framework region may result in an unnatural conformational constraint which, unless corrected by substitution of certain amino acid residues, may result in loss of binding affinity. Selection of amino acid residues is partially determined by computer modeling. The computer hardware and software required to display the three-dimensional image of immunoglobulin molecules are widely available. Generally, molecular models are generated from soluble structures of immunoglobulin chains or domains thereof. The chains to be modeled are compared for amino acid sequence similarity with the chains and domains of the three-dimensional structures solubilized, and the chains or domains showing the greatest sequence similarity are selected as starting points to construct the molecular model. Dissolved starting constructs are modified to allow for differences in the actual amino acids present in the modeled immunoglobulin chains or domains and in the parent construct. Then the modified

-20 · ······································································································································································································································································································ · Finally, the model is refined by minimizing energy and altered so that all atoms are spaced sufficiently apart and bond lengths and angles are within the chemically acceptable range. Example 2 details the construction of a three-dimensional computer model for the variable regions of the mouse DREG-200 antibody. However, this model can serve as a starting point for predicting the three-dimensional structure of an antibody containing murine DREG-200 complementarity determining regions substituted in human framework constructs. Further structural models can be constructed to introduce additional amino acid substitutions; we will discuss this infra.

Generally, replacement of human amino acid residues with rats should be minimized since the incorporation of rat amino acid residues increases the risk of antibody-induced HAMA response in humans. Amino acids are selected for substitution based on their potential effect on CDR conformation and / or antigen binding. Such potential effects are investigated by modeling, by examining the properties of the amino acids at a given location, or by empirically observing the effect of substitution or mutagenesis on the particular amino acids.

When an amino acid differs between a mouse DREG-200 variable framework region and an equivalent human variable framework region, the human framework amino acid is generally replaced by the corresponding mouse amino acid if it is reasonably expected that the amino acid:

(1) is not covalently attached to the antigen, or (2) interacts with, or otherwise interacts with, a CDR region (e.g., located about 4-6 Å from a CDR region).

• · · · ·

Other candidates for -2 ΙΑ substitution are acceptor human framework amino acids, which are unusual for a human immunoglobulin at this position (eg, the H113 amino acid of the human Eu antibody). These amino acids can be replaced by amino acids derived from the equivalent position of more typical human immunoglobulins. Alternatively, amino acids derived from equivalent positions in the murine DREG-200 may be introduced into the human framework regions, where such amino acids are specific for human immunoglobulin equivalent positions.

Generally, replacement of all or most of the amino acids that meet the above criteria is desirable. Occasionally, however, compliance of a given amino acid with the above criteria is ambiguous and alternative immunoglobulins are produced, one of which has the given substitution but the other does not. The humanized antibodies of the invention generally comprise replacing a mouse light chain framework residue with a corresponding mouse DREG-200 residue at least 1, 2, 3, 4, and more often 5 of positions L87, L54, L66, L76 and L93. Humanized antibodies generally include substitution of a murine heavy chain framework residue at least 1, 3, 5, 7, 9 of positions H93, H95, H98, H111, H112, H115, H30, H98, H111, H27, H48 and H72. , 10, 11, more often 12. In preferred embodiments, when the human heavy chain acceptor is immunoglobulin Eu, the heavy chain also contains a substitution at position H113. This position is generally replaced by an amino acid derived from the equivalent position of a human immunoglobulin with a more typical amino acid residue.

In general, the humanized antibodies have substantially the same CDR regions and more often the corresponding CDR regions in the murine DREG-200 antibody. Occasionally, however, it is desirable to alter one of the residues in a CDR region, for example, an L-selectin ligand binding site. · · · · · · · · · · · · · · · · · · · · · · ·

Δ »). Although generally undesirable, it is sometimes possible to effect substitution of one or more conservative amino acids on CDR residues without appreciably affecting the binding affinity of the resulting humanized immunoglobulin.

In addition to the specific amino acid substitutions discussed above, the framework regions of humanized immunoglobulins are generally substantially the same, more often the framework regions of the human antibodies from which they are derived. However, in some embodiments, framework regions differ in amino acid substitutions, terminal and intermediate additions and deletions (etc.) from native sequences at the primary structural level. Stereoisomers of twenty conventional amino acids (e.g., D-amino acids), non-natural amino acids such as α, α-double-substituted amino acids, N-alkyl amino acids, lactic acids and other non-conventional amino acids may also be suitable components of the polypeptides of the invention. Of course, many of the amino acids present in the framework region contribute little or no direct contribution to the specificity or affinity of an antibody. Thus, some conservative substitutions of framework residues can be tolerated without appreciably altering the specificity or affinity of the resulting humanized immunoglobulin. However, such substitutions are generally undesirable. Modification of genes can easily be accomplished by a number of well-known techniques, such as site-directed mutagenesis (see, Gillman & Smith, Gene, 8: 81-97, 1979, and Roberts et al., Natura 328: 731-734, 1987). are hereby incorporated by reference in their entirety.

Alternatively, a polypeptide fragment comprising only a portion of the primary antibody construct having one or more immunoglobulin activities (e.g., binding activity) may be generated. These are polypeptide fragments

-23 can be produced by proteolytic cleavage of intact antibodies by methods well known in the art, or by inserting stop codons at the desired location in the pVk and pVg1-dhfr vectors using site directed mutagenesis such as after CH1 to generate Fab fragments or after the comprehensive region (Fab ') 2 fragments. Single chain antibodies can be produced by linking VL and VH to a DNA binder (see Huston et al., Supra, and Bírd et al., Supra).

For example, Fv or Fab fragments can be produced in E. coli according to the methods described (Buchner and Rudolph, Bio / Technology 9: 157-162, 1991, and Skerra et al., Bio / Technology 9: 273-277, 1991). are hereby incorporated by reference in their entirety. Fv and Fab can be produced by expression of the encoding polynucleotides in eukaryotic, preferably mammalian cells. Similarly, as with many genes, immunoglobulin-related genes containing distinct functional regions, each with one or more distinct biological activities, may be fused to functional regions derived from other genes (e.g., enzymes, see U.S. Patent No. 132,387, filed December 15, 1987). which is incorporated herein by reference in its entirety) for the production of fusion proteins with novel properties (e.g., immunotoxins).

Expression of humanized immunoglobulin sequences in bacterial hosts can be used to facilitate the selection of humanized immunoglobulin sequences for higher affinity by mutagenizing CDR regions and creating a bacteriophage display library that has humanized immunoglobulin with high affinity for L-selectin and / or highly specific binding to CDR. A potential advantage of such an affinity realization is the humanized immunoglobulin CDR variant

-24 · ··· · ···················································································································································································································· have binding affinity and / or reduced cross-reactivity. Science provides a method for producing phage display libraries with immunoglobulin variable region sequences, e.g., Cesareni FEBS Lett 307: 66-70, 1992; Swimmer et al., Proc. Natl. Acad. Sci. USA 89: 3756-60, 1992; Gram et al., Proc. Natl. Acad. Sci. USA 89: 3576-80, 1992; Clackson et al., Natura 352: 624-8, 1991; Scott & Smith, Science 249: 386-90, 1990; Garrard et al., Bio / Techniques 9: 1373-1377, 1991, which are incorporated by reference in their entirety. The resulting affinity-excited CDR variant humanized immunoglobulin sequences are then expressed in a suitable host for efficient expression.

As stated previously, DNA sequences in hosts will be expressed after operably linked to an expression control sequence (i.e., positioned to provide its functionality). These expression vectors are typically replicable in the host organism, either as episomes or as an integral part of the host chromosomal DNA. In general, expression vectors contain selectable markers, such as tetracycline resistance (tet ^R ), G418 resistance (neo ^R ), mycophenolic acid resistance (gpt), or HSV-tk, to allow detection of cells transformed with the desired DNA sequences. (See, e.g., U.S. Patent No. 4,704,362, which is incorporated herein by reference in its entirety).

E. coli is a prokaryotic host that is particularly useful for cloning the DNA sequences of the present invention. Other useful microbial hosts include bacilli, such as. Bacillus subtilis; and other enterobacteriaceae such as Salmonella, Serratia, and various species of Pseudomonas. In these prokaryotic hosts, expression vectors can also be produced, which are typically

• ·

-25 contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, a number of well known promoters may be present, e.g. a lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a broad lambda promoter system. Promoters typically regulate expression, optionally with an operator sequence, and have a ribosome binding site sequence and the like to initiate and complete transcription and translation.

Other microbes like yeasts, can also be used for expression. Saccharomyces is a preferred host that has expression control sequences such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences, and the like, as desired.

Plants and plant cell cultures can be used to express humanized immunoglobulins of the invention. (Larrick & Fry, Hom. Antibodies Hybridomas 2 (4): 172-89, 1991; Benvenuto et al., Plant Mol. Bioi. 17 (4): 865-874, 1991; Durin et al., Plant Mol. Bioi. 15 (1991)). 2): 281-293, 1990; Hiatt et al., Natur 342: 76-8, 1989, which are incorporated herein by reference in their entirety. include Arabidopsis, Nicotiana tabacum, Nicotiana rustica and Solanum tuberosum. The preferred expression cassette for expressing polynucleotide sequences encoding humanized anti-L-selectin antibodies of the invention is plasmid pMOG18, wherein the inserted polynucleotide sequence encoding the humanized immunoglobulin chain is operably linked to a CaMV 35S promoter by a double amplifier sequence; plasmid pMOG18 was used according to the method of Simons et al., Bio / Technology 8: 217-221, 1990, which is hereby incorporated by reference in its entirety.

-26 • · · «· · · · · · · · · · · · · ·

Λ · · · • * * * * ♦ ♦ ♦ ♦

Alternatively, the preferred embodiment for expressing humanized immunoglobulins in plants follows the method of Hiatt et al., Supra, in which the polynucleotide sequences encoding the humanized anti-L-selectin antibodies are replaced by the immunoglobulin sequences used by Hiatt et al. Aqrobacterium tumefaciens TDNS-based vectors may also be used to express humanized immunoglobulin sequences, preferably such vectors include a marker gene encoding spectinomycin resistance or other selectable markers.

Insect cell culture can also be used to produce the humanized immunoglobulins of the invention, typically using a baculovirus-based expression system. Humanized immunoglobulins may be produced by expression of polynucleotide sequences expressing humanized immunoglobulins according to the method of Putlitz et al., Bio / Technology 8: 651-654, 1990, which is hereby incorporated by reference in its entirety. The method of Putlitz et al. Can be accomplished by modifying the insertion of polynucleotide sequences encoding the humanized anti-L-selectin antibodies of the invention into the site of Putlitz et al., Mouse monoclonal Ab 6A4 heavy chain and light chain cDNA sequences.

In addition to microorganisms and plants, mammalian tissue cells may also be used to express and produce the polypeptides of the invention (see Winnacker, From Genes to Clones (VCHPublishers, N.Y., 1987), which is hereby incorporated by reference in its entirety). In fact, mammalian cells are preferred because a number of appropriate host cells capable of secreting intact immunoglobulins have been developed in the art, including CHO cell lines, various COS cell lines, HeLa cells, preferably myeloma ···· · ······················································ ··· • · · · · · · · ···

-27 cell lines, etc., or transformed B cells or hybridomas. Expression vectors for such cells may include expression control sequences such as origin of replication, a promoter, an enhancer sequence (Queen et al., Immunol. Rev. 89: 49-68, 1986, which is incorporated herein by reference in its entirety). as well as required processing information sites such as ribosome binding sites, RNA cleavage sites, polyadenylation sites, and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, cytomegalovirus, and the like. Generally, a selectable marker, such as a neo ^R expression cassette, is incorporated into the expression vector. Transgenes encoding the humanized immunoglobulin of the present invention can be used to generate transgenic non-human animals that express the desired humanized immunoglobulin, typically in a digestible body fluid such as milk or serum. Such transgenes contain a polynucleotide sequence encoding the humanized immunoglobulin (s) operably linked to a promoter, generally through an enhancer sequence such as the murine immunoglobulin enhancer sequence or the casein gene promoter / enhancer sequence (Buhler et al., Bio. Technology 8: 140-143, 1990; Meade et al., Bio / Technology 8: 443-446, 1990, which are incorporated by reference in their entirety. Transgenes can be transferred into cells and embryos according to methods known in the art (infra) to generate homologous recombination structures. Preferred non-human animals include: mice, rats, sheep, cattle, and goats, particularly expressing cow's milk. See WO 91/08216, 1991 (which is incorporated herein by reference in its entirety).

-28 • * ♦ · ♦ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · being · being ·

Purification of humanized antibodies is carried out according to immunoglobulin purification procedures well known in the art.

Vectors containing the DNA segment of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be introduced into the host cell by well known methods, which will vary depending on the type of cellular host. For example, calcium chloride transfection is often used to transfect prokaryotic cells, while calcium phosphate treatment, lipofection, biolytics, viral-based transduction or evaporation can be used to transfect other cellular hosts. Tungsten particle ballistic transgenesis is preferred for plant cells and tissues. (See, generally, Maniatis et al., Molecular Cloning: The Laqboratory Manual (Cold Spring Harbor Press, 1982), which is hereby incorporated by reference in its entirety.)

Once expressed, the complete antibodies of the invention, their dimers, individual light and heavy chains, or other forms of immunoglobulin may be purified according to standard procedures known in the art, including ammonium sulfate precipitation, affinity columns, gel electrophoresis and similar methods (see generally, Scopes, R., Protein Purification (Spinger-Verlag, NY, 1982), which are incorporated herein by reference in their entirety). Substantially pure immunoglobulins having a homogeneity of at least 90-95%, more preferably 98-99% or more, are preferred for pharmaceutical use. Once purified, partially or to the desired homogeneity, the polypeptides can be used therapeutically (including extracorporeal use) or in the development or execution of assays, immunofluorescence staining, and the like. ·· · · · · · · · · · · · · · · · · · · · · · · · · · · 29-29. (See, generally, Immunological Methods, Vols. I and II, Lefkovits and Pernis, eds., Academic Press, N.Y., 1979 and 1981.)

In a preferred embodiment, an L-selectin using standard coupling conditions (phosphate supplemented buffered eg. 2% fetal calf serum in saline solution, at temperatures of 25 °), we produce bind at least 1 x 10 ⁷ M ^_1 binding affinity humanizáltimmunoglobulinokat, such humanized immunoglobulins is a An example is the humanized DREG-200 antibody having the amino acid sequences shown in Figure 2. (Hereinafter, the humanized DREG-200 antibody will sometimes be referred to as hu DREG-200.) Humanized immunoglobulins containing CDRs derived from mouse DREG-55 or mouse DREG-56 are at least 1 x 10? M'1 can bind to L-selectin with binding affinity. Standard coupling conditions, preferably humanized antibodies of the invention bind to human L-selectin of at least 1 x 10θ ^_1 M and preferably at least 1 x 10 ¹⁰ IVH or better binding affinity. Generally, the binding affinity of the humanized immunoglobulin is within the three factors of the mouse immunoglobulin from which it is derived. For example, the mouse DREG-200 antibody has an affinity of about 10θ Μ V

computers

In another embodiment of the invention, computers are provided which are programmed to display a three-dimensional representation of the antibodies. For example, a Silicon Graphics IRIS 4D workstation running under the UNIX operating system and using the QUANTA molecular modeling package (Poilgen Corp. USA). Computers can be used to generate variants of humanized antibodies. In general, the antibodies of the invention have sufficient binding affinity. However, it is likely that even stronger binding affinities can be identified by further variation of certain amino acid residues. The three-dimensional image will also identify a number of non-critical amino acids, which can then be subjected to conservative substitutions without apparently affecting the binding affinity of the antibody. Collectively, even conservative substitutions can have a significant effect on the properties of an immunoglobulin. However, many single conservative substitutions are unlikely to significantly affect the properties of immunoglobulins.

Human antibodies to L-selectin

In another embodiment of the invention, human antibodies to L-selectin are provided. These antibodies are produced by a number of techniques described below. Some human antibodies are selected by competitive binding assay, or alternatively, with the same murine antibody, e.g. mouse epitope specificity for DREG-200 or a humanized version thereof.

Such antibodies are particularly likely to share useful therapeutic properties such as those already described for the humanized DREG-200. Antibodies with the desired epitope specificity can also be identified by screening for the ability to inhibit neutrophil-endothelial cell interaction. An assay for the simple visual detection of such an interaction is described in Kishimoto et al., Supra, 1991. Briefly, monolayers of human umbilical vein cells are stimulated with IL-1. Neutrophils, pretreated or untreated with the antibody to be tested, are added to the monolayer under defined conditions and the number of neutrophils attached is determined microscopically. In one method, the neutrophils are deficient in human leukocyte adhesion · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

We get it from 31 patients. See Anderson et al., Ann. Port. Med. 38: 175, 1987. Neutrophils from such patients lack integrin receptors, the binding of which to neutrophils may mask the effect of inhibiting L-selectin binding.

the. Trio methodology

The basic approach and the exemplary cell fusion partner used in this approach, SPAZ-4, have been described by Oestberg et al., Hybridoma 2: 361-367, 1983; U.S. Patent No. 4,634,664 to Oestberg. Patent; and Engleman et al., U.S. Patent No. 4,634,666, which are incorporated herein by reference in their entirety. The antibody-producing cell line obtained by this method is called a triome because they are derived from three cells: two human cells and one mouse. Initially, a murine myeloma line was fused to a human B lymphocyte to obtain a non-antibody producing xenogeneic hybrid cell, such as the supra, SPAZ-4 cell line described by Oestberg. The xenogeneic cell is then fused with an immunized human Blimphocyte to generate an antibody-producing trioma cell line. The triomas were found to produce the antibody much more stable than ordinary hybridomas made from human cells.

Immunized B-lymphocytes are obtained from human donors' blood, spleen, lymph nodes or bone marrow. In vivo immunization of a living person with L-selectin is generally undesirable due to the risk of inducing a dangerous reaction. Thus, B-lymphocytes are generally immunized in vitro with the Lselectin polypeptide, an antigenic fragment thereof, or a carrier cell: If antibodies to a specific antigen or epitope are desired, the antigen or epitope thereof should be used in vitro.

-32vitro for immunization. B lymphocytes are typically exposed to antigen for 7-14 days in media such as RPMI 1640 (see Engleman, supra) supplemented with 10% human plasma.

Immunized B-lymphocytes are used in a xenogeneic hybrid cell, e.g. fused to SPAZ-4 by known methods. For example, cells are treated with 40-50% polyethylene glycol 1000-4000 molecular weight at approximately 37 ° C for 5-10 minutes. Cells are removed from the fusion mixture and grown in the desired hybrids in a selective medium (e.g., HAT or AH). Clones secreting antibodies with the desired binding specificity are identified by assaying the ability of the trioma culture medium to bind to L-selectin or a fragment thereof. Triomas producing human antibodies with the desired specificity are subcloned using the limiting dilution technique and grown in vitro in culture medium. The resulting trioma cell line is then tested for its ability to bind to Lselectin or a fragment thereof.

Although triomas are genetically stable, antibodies are not produced at very high levels. Expression levels can be increased by cloning the antibody genes from the triomas into one or more expression vectors, and the vector is transformed into a cell line discussed supra for expression of recombinant or humanized immunoglobulins.

b. Transgenic non-human mammals

Human antibodies to L-selectin can be generated from non-human transgenic mammals having transgenes encoding at least one segment of the human immunoglobulin locus. Generally, the endogenous immunoglobulin locus of such transgenic mammals is functionally inactivated. Preferably, the human immunoglobulin locus segment is a heavy and light chain. V · 4 4 4 4 4 4 4 4 4 4 · · · ·· · ·

-33 contains disordered sequences of components. Inactivation of endogenous immunoglobulin genes and delivery of exogenous immunoglobulin genes can be accomplished by targeted homologous recombination or by introduction of YAC chromosomes. The transgenic mammals obtained as a result of this method are capable of functional rearrangement of immunoglobulin component sequences and the repertoire of various isotype antibodies encoded by human immunoglobulin genes without expression of endogenous immunoglobulin genes. The properties and production of mammals having such properties are described in detail e.g. Lonberg et al., WO93 / 12227 (1993); Kutcherlapati, WO 91/10741 (1991) (which are incorporated herein by reference in their entirety). Transgenic mice are particularly suitable. Anti-Ls selectin antibodies are obtained, e.g. immunization with transgenic non-human mammalian L-selectin or a fragment thereof as described by Lonberg or Kutcherlapati (supra). Monoclonal antibodies are prepared, e.g. by fusing B cells from such mammals to appropriate myeloma cell lines using conventional Kohler-Milstein technology.

c. Faz detection methods

A further approach to recovering human anti-L-selectin antibodies is to screen a human B cell derived DNA library according to the general protocol described by Huse et al., Science 246: 1275-1281 (1989). Antibodies that bind to L selectin or a fragment thereof are selected. The sequences (or binding fragments) encoding such antibodies are then cloned and amplified. The protocol described by Huse is much more effective when used in conjunction with phage detection technology. See, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, which are incorporated herein by reference in their entirety.

-34 ············································································ed · · In these methods, phage libraries are created in which the members have different antibodies. Antibodies are generally designated as Fv or Fab fragments. A phage containing antibodies with the desired specificity is selected by affinity enrichment of an L-selectin polypeptide or fragment thereof.

In a variant of the phage detection method, human antibodies having binding specificity for a selected rat antibody can be prepared. See Winter, WO 92/20791. In this method, the variable region of the heavy or light chain of a selected rat antibody (e.g., mouse DREG-200) is used as a security material. If, for example, a light chain variable region is selected as the starting substance spy, and a phage library is constructed in which the members have the same light chain variable region (i.e., the rat starting material) and a different heavy chain variable region. The heavy chain variable regions are obtained from a library of rearranged human heavy chain variable regions. A phage displaying a strong specific binding to L-selectin (e.g., at least 10θ and preferably at least 1Οθ M'1) is selected. The human heavy chain variable region derived from this phage is then used as a starting material to construct an additional phage library. In this library, each phage has the same heavy chain variable region (e.g., the region identified from the first library shown) and a different light chain variable region. The light chain variable regions are obtained from a library of rearranged human variable light chain regions. Phages showing strong specific binding to L-selectin are also selected in this case. These phages have variable regions of fully human anti-L-selectin antibodies. These antibodies have the same or similar epitope specificity as the rat starting material (e.g., mouse DREG-200).

· «·· * ·· * ··· * · · · · · · · · · · · · · · · · ···

-3 5 Application Methods

Antibodies of the invention are typically useful in the treatment of disease states characterized by an inflammatory component, particularly those mediated by neutrophils or T cells. Preferred applications include myocardial infarction, cerebral ischemic event (e.g., cerebral haemorrhage), kidney, liver or spleen infarction, cerebral surgery, shock cardiac surgery (eg, coronary artery bypass grafting), elective angioplasty and similar ischemic injury due to interventional ischemic injury prophylactic treatment. Other preferred applications include sepsis, adult respiratory distress syndrome, and the treatment of multiple organ dysfunctions. The antibodies can be used to treat injuries caused by trauma, burns, frost damage or spinal cord injury. They may be used in the treatment of autoimmune disease including, but not limited to, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, Ies type diabetes and uveitis, inflammatory diseases of the skin, e.g. in the treatment of psoriasis and in the treatment of meningitis and encephalitis. Other typical applications are the prevention and treatment of organ transplant rejection and transplantation versus host disease.

Any immunoglobulin of the invention may be used in combination with other antibodies, in particular with humanized or human antibodies reactive against various adhesion molecules. For example, suitable immunoglobulins include immunoglobulins specific for CD11a, CD11b, CD18, E-selectin, P-selectin and ICAM-1. Other suitable antibodies with lymphokinases such as specific antibodies to IL-1, IL-2 and IFN-α and their receptors.

·· «·

-36s

The antibodies of the invention can also be used as separate formulations when used in combination with other therapeutic agents. Typically, the substances may include non-steroidal anti-inflammatory drugs and corticosteroids, but many other substances well known to those skilled in the art (e.g., cyclosporine) may be used. In fact, the immunoglobulins of the invention are typically used in conjunction with drugs currently used by those skilled in the art to treat particular diseases.

In some therapeutic procedures, for example, anti-L-selectin antibodies are used together with thrombolytic agents. In previous methods, patients with acute myocardial infarction are treated by opening a closed coronary artery. The reopening of a blocked coronary artery is accomplished by the use of thrombolytic agents that dissolve the blockage-causing knot, thereby restoring coronary blood flow. Vascular reperfusion can also be accomplished by acute percutaneous transluminal coronary artery angioplasty (PTCA) by balloon dilation of the occluded and narrower segment of the coronary artery. However, restoration of coronary blood flow leads to ischemic reperfusion injury in previous methods.

In the present methods, ischemic reperfusion injury is reduced or prevented by the use of a thrombolytic agent or a PTCA in combination with humanized or human anti-L-selectin antibodies. Antibodies are generally administered prophylactically or at the same time prior to initiation of thrombolytic agents or PTCA. Subsequent doses of antibody are often administered after thrombolytic or angioplasty treatment. Between prophylactic administration of antibodies and initiation of thrombolytic or angioplasty treatment ···· · • ·

Φ ··· ··

• · • · · ······

The duration of -37 is generally 5 to 30 minutes, preferably 5 to 20 minutes, and most preferably minutes. The antibodies are administered parenterally, preferably by intravenous injection, at doses of 0.01 to 10 mg / kg body weight, preferably 0.14 to 5 mg / kg body weight, most preferably 0.3 to 3 mg / kg body weight. The antibodies may be administered in high dose injections, e.g. 1-5 minutes as repeated injections of smaller doses or as an intravenous infusion. High dose injection is particularly useful as a prophylactic dose or in an emergency. Additional doses of antibodies may be repeated (e.g., every 4 to 6 hours) during or after acute myocardial infarction by thrombolytic or angioplasty, in the same proportions as described above, to obtain optimal antibody plasma levels.

Thrombolytic agents are drugs that are capable of stimulating the in vivo dissolution of thromboses, either directly or indirectly. Thrombolytic agents include tissue plasminogen activator (see EP-B 0 093 619), activase, alteplase, duteplase, cileplase, streptokinase, anistreplase, urokinase, heparin, warfarin and coumarin. Other thrombolytic agents include saruplasm and vampire bat plasminogen activator. (See Harris, Protein Engineering 6: 449-458, 1987; PCT / EP 90/00194; U.S. Pat. No. 4,970,159.) The thrombolytic agent is administered to the patient in an amount sufficient to prevent the formation of thromboses and their complications and to partially disperse the thromboses. The appropriate amount required to achieve this effect is called the pharmaceutically effective amount. The use of effective amounts for this will depend on the severity of the disease condition, the general condition of the patient, the route of administration and the combination with other drugs. Often, the pharmacologically effective doses of thrombolytic agents and the routes of administration for such agents are those that the FDA ··· ::

-38 », for independent use of thrombolytic agents e.g. 100 mg alteplase or 1.5 million IU streptokinase.

A preferred pharmaceutical composition of the invention includes the use of said immunoglobulins in immunotoxins for killing L-selectin-expressing cells. Immunotoxins are characterized by two components and are particularly useful for killing selected cells in vitro or in vivo. One component is a cytotoxic agent that is usually fatal to the cell to which it is attached or to which it is absorbed. The second ingredient, known as a vehicle for transporting toxic material to a particular cell type, such as provides a means for delivery of an L-selectin epitope expressing cells. Often, the two components may be chemically bonded to each other by any of a number of well-known chemical processes. For example, when the cytotoxic agent is a protein and the second component is an intact immunoglobulin, the linkage may be accomplished by heterobifunctional crosslinks, e.g. SPDP, carbodiimide, glutaraldehyde or the like. The production of various immunotoxins is well known in the art and e.g. See Monoclonal Antibody-Toxin Conjugates, Aiming the Magic Bullet, Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, p. 168-190, 1982, which is hereby incorporated by reference in its entirety. The components may also be linked genetically (see Chaudhary et al., Natura 339: 394, 1989, which is hereby incorporated by reference in its entirety).

Many cytotoxic agents are suitable for use in immunotoxins. Cytotoxic agents may include radionuclides, such as the like. iodine-131 or other iodine isotopes, yttrium-90, rhenium-188, and bismuth-212 or other alpha emitters; many chemotherapeutic drugs such as vindesine, methotrexate, adriamycin and cisplatin; and cytotoxic proteins like • · • «

-39- ........

ribosomal inhibitory proteins such as sub-claw antiviral protein, Pseudomonas exotoxin A, ricin a diphtheria toxin, ricin A chain, etc. or a cell surface active agent such as phospholipase enzymes (e.g. phospholipase C). (See generally U.S. Patent No. 07 / 290,968 to Chimeric Toxins Olsnes and Phill, Pharmac. There. 25: 355-381, 1982, and Monoclonal Antibodies for Cancer Detection and Therapy, Eds. Baldwin and Byers, Academic Press 1985). .159179, 224-266, which are hereby incorporated by reference in their entirety.

The immunotoxin delivery component comprises the immunoglobulins of the invention. Preferably intact immunoglobulins or binding fragments thereof, such as Fab or Fv, are used. Typically, the antibodies in the immunotoxins are human IgM or IgG isotype, but other mammalian constant regions may be used as desired.

The antibodies of the invention and pharmaceutical compositions thereof are particularly useful for parenteral administration, i.e., subcutaneously or intramuscularly or intravenously. The antibodies of the invention may also be administered, typically for topical administration, as artificial nutrition or washing, intraperitoneal injection, ophthalmic ointment, topical ointment, intracranial injection (typically in a ventricular chamber), intrapericardial injection, or intrabural injection. Formulations for parenteral administration generally contain a solution or mixture of immunoglobulin dissolved in an acceptable carrier, preferably in an aqueous carrier. Many aqueous carriers can be used, e.g. water, buffered water, phosphate buffered saline (PBS), 0.4% saline, 0.3% glycine, human allobumin solution and the like. These solutions are sterile and generally free of particles. These formulations may be sterilized in conventional manner using well known sterilization techniques. The formulations may contain pharmaceutically acceptable excipients as required to approach physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents and the like, e.g. sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of antibody in these formulations may vary within wide limits, i.e., from less than 0.005% by weight, and generally from at least about 1% by weight, to a lower value of 15 to 20% by weight, and primarily to liquid volumes, viscosities, etc. is selected according to the selected application mode.

Thus, a typical pharmaceutical composition for injection may be formulated containing 1 ml of sterile buffered water and 1-70 mg of immunoglobulin. For intravenous infusion, a typical formulation may be prepared containing 250 ml of sterile Ringer's solution and 150 mg of antibody. Current methods for the preparation of parenteral formulations are known or obvious to those skilled in the art, e.g. described in Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pennsylvania, 1980), which is hereby incorporated by reference in its entirety. Formulations for laundry or other applications are selected for their intended use. Some formulations also contain anti-L-selectin antibodies and thrombolytic agents.

The antibodies of the invention may be frozen or lyophilized for storage and reconstituted in a suitable vehicle prior to use. This technique has been shown to be effective with conventional immune globulins and to use the lyophilization and regeneration techniques known in the invention. Those skilled in the art will recognize that lyophilization and regeneration can result in varying degrees of loss of antibody activity (e.g., conventional immune globulins, IgM antibodies lose more of their activity,

-41 like IgG antibodies) and application levels need to be added to compensate.

Compositions containing the present antibodies or mixtures thereof may also be used for prophylactic and / or therapeutic treatments. In therapeutic applications, the compositions are administered to a patient suffering from an inflammatory disease in an amount sufficient to cure or at least partially prevent the disease and its complications. A dose sufficient to achieve this is defined as a therapeutically effective dose. The amounts effective for this application will depend on the severity of the disease and the general condition of the patient's own immune system, but will generally be from about 1 to about 1 to about 10 minutes. It ranges from 200 mg antibody / dose, more frequently at doses ranging from 5 to 70 mg / patient. The dosage regimen will vary depending on the condition of the disease and the patient's condition and will typically range from a single high dose or continuous infusion to multiple daily dosing (e.g. every 4-6 hours) or as advised by the attending physician or according to the patient's condition. dependent. It will be appreciated that the materials of the invention are generally used in the treatment of serious illnesses, i.e., life-threatening or potentially life-threatening cases. In such cases, in view of the reduced likelihood of extraneous matter and the risk of rejection of foreign matter, which is achieved with the immunoglobulins of the invention, it is possible and desirable for the attending physician to employ substantially excess amounts of these antibodies.

For prophylactic use, compositions comprising the present antibodies or mixtures thereof are administered to a patient not yet afflicted with a particular disease to enhance patient resistance. Such an amount is called a prophylactically effective amount. In this application,

Amounts of -42 points again depend on the patient's state of health and general level of immunity, but generally range from 1-70 mg / dose. Preferred prophylactic use is for the prevention of adult respiratory distress syndrome in patients with sepsis or trauma; to prevent organ transplant rejection and to prevent reperfusion injury in patients with ischemia. In patients with severe disease, doses of about 50-150 mg of humanized or human immunoglobulin / administration are frequently used and high doses are indicated.

Single or multiple administrations of the compositions may be administered at dosage levels and in such manner as the physician will determine. In any event, the pharmaceutical compositions should provide an amount of the antibody (s) of the invention that are effective in treating the patient.

The antibodies of the invention may be used in a variety of other in vitro applications. For example, antibodies can be used to detect L-selectin antigens, to isolate specific leukocytes, or for similar purposes. For example, without limitation, a humanized DREG-200 immunoglobulin can be immobilized and contacted with blood from a patient to remove blood cells carrying the L-selectin antigens, and the resulting blood released from the L-selectin-bearing cells can be returned to the patient. Any residual humanized antibody present in purified blood returned to the patient (e.g., as a result of detachment from the immobilization support) has reduced or negligible antigenicity to a rat antibody.

For diagnostic purposes, antibodies may be labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (a second antibody) that are reactive with humanized or human antibodies such as

43 Antibodies specific for the human immunoglobulin constant region. Alternatively, the antibodies may be directly labeled. A number of markers can be used, e.g. radionuclides, fluorines, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially haptens), etc. Many different types of immunoassays can be used and are well known to those skilled in the art.

The following examples are provided to illustrate and not limit the invention. It will be appreciated that although the examples relate to the mouse DREG-200 antibody, the production of humanized antibodies with high binding affinity for L-selectin can also be achieved using CDRs derived from mouse DREG-55, DREG-56 or other monoclonal antibodies. They bind to an epitope of L-selectin.

Examples

Example 1: Cloning of heavy and light chain cDNA

The cDNAs of the murine DREG-200 heavy and light chain variable domain genes were cloned using fixed polymerase chain reactions as described (see Co et al., J. Immunol. 148: 1149, 1992, and U.S. Pat. No. 07 / 634,278). 3 'primers hybridized to constant regions and containing HindIII sites and 5' primers hybridized to the dG tail and containing EcoRI sites. The amplified PRC fragments were digested with EcoRI and HindIII and cloned into pUC18 vector for sequencing. At least two gamma-1 specific and two kappa specific clones were sequenced for murine DREG-200. Gamma-1 clones and kappa clones are identical in sequence. The cDNA variable domain sequences and deduced amino acid sequences are shown in Figure 1.

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · pit ·

Example 2: Computer modeling of humanized antibodies

In order to maintain the high binding affinity of humanized antibodies, the general procedure of Queen et al., See Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029, 1989, and WO 90/07861, are incorporated herein by reference in their entirety. of the invention). The more homologous an acceptor human antibody to the original rat donor antibody is, the less likely it is that combining rat CDRs with a human framework would cause distortions in the CDRs, which could reduce affinity. At least 65% homology (i.e., percent sequence identity) between the humanized immunoglobulin heavy chain variable region framework and the donor immunoglobulin heavy chain variable region framework is preferred. Normally, the heavy chain and light chain from the same human antibody are chosen to provide the framework sequences so as to reduce possible incompatibilities between the two strands. Based on sequence homology studies with the NBRF protein sequence database (performed by MicroGenie Sequence Analysis Software (Beckman)), the Eu antibody is selected to provide framework sequences for humanizing the DREG-200 mouse.

In order to construct the mouse DREG-200 variable region model, the computer program ENCAD (Levitt, J. Mol. Bioi. 168: 595, 1983, which is incorporated herein by reference in its entirety) is selected. The model is used to determine the amino acids in the murine DREG-200 frame that are close enough to the CDRs to interact with (Category 4, below). To design the humanized light and heavy chain DREG-200 variable regions, the amino acids at each position are chosen to be the same as in the Eu antibody, unless this position falls into one or more of the following five categories:

(1) the position is within the CDR, (2) the Eu residue is unusual for the human antibody at this position, while the mouse DREG-200 residue is typical for the human antibody at this position, (3) the position is direct. is adjacent to the CDR, (4) the model described above suggests that the amino acid may be physically close to the antigen binding region (CDRs).

For positions in this category, the amino acid derived from the mouse DREG-200 antibody is used.

In addition, a position falls into the fifth category if:

(5) the Eu amino acid is very unusual for human antibodies at this position and the mouse DREG-200 amino acid is different but also unusual. Then, an amino acid representative of the human antibody at this position is used.

The amino acids in each category are shown in Table 1. Some amino acids may fall into more than one category. The final sequences of the humanized DREG-200 light and heavy chain variable domains are shown in Figure 2 compared to rat DREG-200 sequences.

Table 1

Category Light chain Heavy chain 1 24-40, 56-62, 95-103 31-35, 50-66, 99-110 2 87 93, 95, 98, 111, 112, 115 3 - 30, 98, 111 4 54, 66, 76, 93 27, 30, 48, 72 5 __ 113

• ·

-46- .....

To construct genes for humanized antibodies, nucleotide sequences that encode the protein sequences of humanized heavy and light chains, including signal peptides, generally using codons found in the mouse sequence, are selected. Many degenerate codons are altered to create restriction sites or to remove unwanted sites. The nucleotide sequences of the genes also contain linker donor signals and an Xba I site at each end. The nucleotide sequences and the encoded humanized light and heavy chain variable domains are shown in Figure 3. Each gene is constructed from four overlapping synthetic oligonucleotides as described (see Co et al., J. Immunol. 148: 1149, 1992, and U.S. Patent No. 07 / 634,278, which are incorporated herein by reference in their entirety). The heavy and light chain variable region genes are then sequentially ligated to the Xba I sites of the pVg1-dhfr or pVk expression vectors (see U.S. Patent No. 07 / 634,278, U.S. Pat. No. 7), in the proper orientation to produce the complete heavy and light chain genes. The reactions are carried out under conditions well known in the art (Maniatis et al., Supra).

The heavy and light chain plasmids were transfected by electroporation into Sp2 / 0 mouse myeloma cells and the cells were selected for expression of gpt. Clones are screened by ELISA for the production of human antibody in culture supernatant and purified from the best producing clone. The humanized DREG-200 IgG1 antibody is then purified by passing the tissue culture supernatant through a staphilococcal protein A-Sepharose CL-4B (Pharmacia) column. Bound antibody was eluted with 0.2 M Glycine-HCl at pH3.0 and neutralized with 1 M Tris (pH8.0). The buffer was changed to PBS by passage through a PD10 column (Pharmacia) or by dialysis. Cells that produce antibodies at higher levels

Ü7 • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · can be achieved by increasing concentrations of methotrexane.

To produce a humanized DREG-200 antibody of the IgG4 isotype, another pVg4-dhfr vector was first constructed. To do this, the XbaI-BamHI fragment of pVg1-dhfr containing the a1 constant region is replaced by an approximately 2000 base pair fragment of the human a4 constant region gene (Ellison and Hood, Proc. NAtl. Acad. Sci. USA 79: 1982). | - | from HindIII site upstream of exon 1 to 270 base pairs downstream of Nsil site following exon Cf- | This substitution is accomplished by methods well known to those skilled in the art, including polymerase chain reaction. The humanized DREG-200 heavy chain variable region gene is then cloned into the XbaI site of pVg4-dhfr. The heavy chain plasmid, together with the above light chain plasmid, is then transfected into Sp2 / 0 cells, the clones are selected and the humanized DREG-200 IgG4 antibody is purified as described for IgG1.

Example 3

Properties of humanized antibodies

The affinity of humanized DREG-200 antibodies for L-selectin was determined by comparison with radioactive iodine-labeled mouse DREG-200 antibody (Figure 4). Binding affinities were calculated according to the methods of Berzofsky (J.A. Berzofsky and I.J. Berkowe, in W.E. Paul, Ed., In Essential Immunology, ed., New York, 595, 1984, which is incorporated herein by reference in its entirety). Humanized DREG-200 antibodies have about twice the affinity of murine DREG-200 antibody. Similar results are found in the measurement of the affinity of human neutrophils for L-selectin.

···· 4 • ··

-18t

The ability of murine and humanized DREG-200 antibodies to inhibit the adhesion of human neutrophils to endothelial cells was determined by modifying the method described by Hallmann et al (Biochem. Biophys. Res. Comm. 174: 236, 1991). Specifically, human umbilical cord endothelial cells (HUVEC, from Clonetics, San Diego) were grown to confluency in EGM medium (Clonetics) on Lab-Tek 8-well plates (Nunc, Naperville, IL). HUVEC cells were stimulated with 20 ng / ml ILΙβ (R&D System, Minneapolis, MN) 4 hours prior to use. Neutrophils were isolated by density gradient centrifugation from a membrane coating, which was extracted from erythrocytes by dextran sedimentation and adjusted to 10 µm. Neutrophils (100 µl) were preincubated with various concentrations of antibody (100 µl in RPMI) for 20 min on ice. The HUVEC plates were washed free of IL-1B and placed on a rotary shaker (100 rpm) at 4 ° C. Untreated and antibody-treated neutrophils were added to the wells and the plates were incubated at 4 ° C for 30 minutes on a shaker. The plates are then washed with 10x immersion in a beaker RPMI, fixed with 1% glutaraldehyde in RPMI and allowed to air dry. Neutrophil adhesion is determined by microscopic counting of the number of neutrophils attached to a specific area of the endothelial cell monolayer. As shown in Figure 5, both humanized IgG1 and murine DREG-200 antibodies effectively inhibited the binding of neutrophils to HUVEC, whereas a different control antibody did not. The humanized DREG-200 IgG4 antibody likewise blocks the binding of neutrophils to endothelial cells.

• the*

Example 4

Effect of hu DREG-200 on myocardial injury following reperfusion

The effect of IgG4 isotype humanized DREG-200 (hu DREG-200) on the extent of true salvage following reperfusion is investigated. Adult male cats (2.8-4.2 kg) are anesthetized with sodium pentobarbital (30 mg / kg). An intratracheal cannula was passed through a midline incision and the cats were periodically placed on a positive pressure ventilator (Harvard Small Animal Ventilator, Dover, MA). A polyethylene catheter is inserted into the right external jugular vein for further infusion of pentobarbital to maintain the level of surgical anesthesia and for the administration of antibodies. Another polyethylene catheter was inserted through the left femoral artery and placed in the abdominal aorta to measure mean arterial blood pressure via a pressure transducer (Cobe Instruments, Lakewood, CO) (MABP). After a midsternal thoracotomy, the anterior pericardium is incised and a 3-0 silk suture is placed around the left anterior descending (LAD) coronary artery 8 to 10 mm from its origin. A high-credibility catheter-end pressure transducer (Model MPC 500, Model TCB 500, Millar Instruments Inc., Houston, TX) is delivered to the left ventricle via the apical foveola. Through observation, the catheter is adjusted to LV pressure and dP / dt waveform and secured in place with silk suture. A standard II-lead Scaling Electrocardiogram (ECG) is used to determine the heart rate (HR) and to elevate the ST segment. ST segment elevation was determined by analyzing the ECG record (50 mm / sec, every 20 minutes). ECG, MABP, LVP, and dP / dt values were continuously monitored on a Hewlett-Packard 7830 A unit (Hewlett-Packard, Palo Alto, CA) and on a Gould oscillographic recorder (Gould Inc., Cleveland, OH) every 20 ♦ ♦♦ · ·

-50percbe. The Pressure Ratio Index (PRI) is defined as the approximate value of myocardial oxygen demand as a fraction of one thousandth of MABP & HR.

After all surgical procedures, cats were stabilized for 30 minutes, during which time ECG, MABP, LVP and dP / dt baseline records were recorded. Myocardial ischaemia (M1) is first induced by tightly reversing the vascular ligation around the LAD by completely occluding the vessel. This is denoted as zero time. Two mg / kg body weight hu DREG200 (IgG4 isotype) or a control MAb hu ABL-364 (i.e. isotype paired humanized control IgG4 MAb) were administered intravenously at high dose 80 minutes after coronary occlusion (i.e. 10 minutes before reperfusion, R). Ten minutes later (i.e., after a total of 90 minutes of ischemia I), the LAD vascular ligation is released and the ischemic myocardium is reperfused for 4.5 hours.

Cats are randomly divided into 3 groups. Six false M1 + R cats hu DREG-200 (2 mg / kg), six M1 + R cats control MAb hu ABL-364 (2 mg / kg) and six M1 + R cats hu DREG-200 (2 mg) / kg). False M1 + R cats are subjected to the same surgical procedure as M1 + R cats except that the LAD coronary artery is not blocked.

At the end of the 4.5 hour reperfusion, the vascular ligation around LAD is tightened again. Twenty ml of 0.5% Evans blue was rapidly injected into the left ventricle to stain the area of the myocardium that had been perfused through the coronary artery of the sample. Following this injection, the heart is immediately excised and placed in warm oxygenated K-H solution. Left circumflex (LCX) and LAD coronary artery were isolated and subtracted from subsequent coronary ring vasoactivity and PMN binding assay. The right ventricle, large vessels and adipose tissue are carefully removed and the left ventricle is sliced in parallel with the atrioventricular furrow into 3 mm thick slices. The non-stained sections of the myocardium (i.e., the entire risk area or the ischemic area) are separated from the Evans blue stained myocardium section (i.e. the non-risk area or non-ischemic area). The risk area is cut into small cubes and placed in 0.1% nitro blue tetrazolium in phosphate solution (pH 7.4, 37 ° C for 15 minutes). Tetrazolium dye forms blue formazan complex in the presence of myocardial cells containing active dehydrogenases and their cofactors. The irreversibly damaged or necrotic part of the risk myocardium that is not stained is separated from the stained part of the myocardium (i.e., the ischemic but not necrotic part). The three parts of the myocardium (i.e., non-lysemic, ischemic, necrotic, and ischemic necrotic tissue) are then weighed. Results are expressed as necrotic heart tissue area, either as a percentage of risk area or as a percentage of total left ventricular mass.

According to these criteria, cardiac tissue damage was significantly attenuated (p <0.001) in cats treated with hu DREG-200. While 30% of compromised myocardium developed to necrotic tissue in the control antibody-treated group, the amount of necrotic tissue was less than 15% (p <0.01) in the hu DREG200-treated group, a 50-60% decrease. See Figure 6. There is no significant difference between the wet weights of the risk areas, expressed as a percentage of the total left ventricle, between the two ischemic groups, indicating that comparable myocardial ischemia occurs in both groups. Therefore, the hu DREG-200 provides significant protection against reperfusion injury.

Significant hu DREG-200 retention of ischemic tissue is also illustrated by measuring the biochemical marker of myocardial injury, plasma creatine kinase activity. Arterial blood samples (2 ml) were taken just before and one hour after ligation. Blood is collected in polyethylene test tubes containing 200 IU of heparin sodium. Samples at 2000 x g rpm • ··

Centrifuged at -52 * for 4 minutes at 4 ° C and the plasma was drained for biochemical analysis. Plasma protein concentration was assayed using the Buret method of Gornall et al., J. Biol. Chem. 177: 751-766, 1949. Plasma creatine kinase (CK) activity was measured using the method of Rosalki (J.L. Clin. Med. 69: 696-705, 1967) and expressed as IU / pg protein.

Plasma CK activity in mammalian cats receiving hu DREG-200 increased slightly during the 6 hour observation period and reached 3.8 ± 0.9 IU / pg protein. In the two ischemic groups, plasma CK activity increased slightly during the period of myocardial ischemia. In cats receiving hu ABL-364, CK activity in the circulating blood increased significantly during the first 30 minutes after reperfusion and continued to increase during the remaining 4 hours of reperfusion. Conversely, ischemic cats treated with hu DREG200 exhibit significantly lower plasma CK activities compared with ischemic cats receiving hu ABL-364 (p <0.05). The effect is maintained throughout the perfusion period, further demonstrating the essential protection of hu DREG-200 against myocardial reperfusion injury.

Example 5

Effect of hu DREG-200 on heart function

The effect of hu DREG-200 (IgG4 isotype) on cardiac function was determined by measuring left ventricular pressure (LVP) and determining LVP, dP / dt max, the first derivative of myocardial contractility. Data is obtained with a catheter tip manometer inserted into the left ventricle cavity. The three groups of cats discussed in the previous example all show comparable individual values for these heart variables. There was no significant change in dP / dt max in the sham M1 group over the entire six-hour experimental period. However, in both M1 / R groups, ca. The dP / dt max decreases to 65% after closure of the LAD. In cats receiving hu ABL364, contractility is not significantly reversed. However, in hu DREG-200 treated M1-R cats, dP / dt max returns to control values three hours after reperfusion. Thus, 4.5 hours after reperfusion, dP / dt max in hu ABL-364 treated cats is significantly lower than in hu DREG-200 treated cats (p <0.01). Not only do these results indicate that hu DREG-200 reduces myocardial necrosis following ischemic myocardial reperfusion, but this myocardial rescue also results in an improvement in the mechanical filling of the heart.

It will be appreciated from the foregoing that the immunoglobulins of the invention offer several advantages over L-selectin specific antibodies. Compared to murine monoclonal antibodies, the present immunoglobulins are more economically produced and contain significantly less foreign amino acid sequences. Reduced antigenicity after injection into a human patient represents a significant therapeutic advance. All cited publications and patent applications are hereby incorporated by reference in their entirety, as if each individual notice or patent application were specifically and individually indicated as being part of the invention by reference. While the invention has been described in detail with the help of illustrations and examples, for the sake of clarity and clarity, it will be understood that certain changes and modifications within the scope of the appended claims may be made.

Claims (29)

PATENT CLAIMS Claims 1. A humanized immunoglobulin having complementarity determining regions (CDRs) corresponding to heavy and light chain variable region frames derived from a donor immunoglobulin corresponding to a human acceptor immunoglobulin heavy and light chain framework. humanized immunoglobulin specifically binds to human L-selectin with an affinity constant of at least 107 M1, wherein the humanized immunoglobulin heavy chain variable region framework is 65% or more identical to that of the donor immunoglobulin heavy chain variable region framework. 2. The humanized immunoglobulin of claim 1, wherein the antibody comprises two light chain / heavy chain dimers. The humanized immunoglobulin of claim 2, wherein said antibody is derived from IgG1 or IgG4 isotype. 4. The humanized immunoglobulin of claim 1, wherein it has binding affinity for human L-selectin of at least 10θ M'1. The humanized immunoglobulin of claim 1, wherein said donor immunoglobulin is represented by the murine DREG-200 antibody. The humanized immunoglobulin of claim 1, wherein said acceptor immunoglobulin heavy and light chain frames are derived from said human antibody. The humanized immunoglobulin according to claim 6, wherein said human antibody is represented by the human antibody Eu. 8. A humanized immunoglobulin having complementarity determining regions (CDRs) corresponding to CDRs derived from a donor immunoglobulin and heavy and light chain variable region conforming to the acceptor. immunoglobulin heavy and light chain frameworks, which humanized immunoglobulin specifically, at least 10 ^ M ^- 'affinity binds to human L-selectin with a constant, wherein the sequence of the acceptor immunoglobulin heavy chain variable region is among the 5 sequences in a collection of representative human immunoglobulin heavy chain variable region most homologous to that of the donor immunoglobulin heavy chain variable region. 9. A humanized immunoglobulin having complementarity determining regions (CDRs) corresponding to CDRs derived from a donor immunoglobulin and having heavy and light chain variable regions corresponding to the heavy and light region of the acceptor immunoglobulin. chain, which specifically humanized immunoglobulin, has at least 10 ⁷ affinity! is bound to human L-selectin, wherein said humanized immunoglobulin comprises amino acid residues derived from a donor immunoglobulin framework replacing the corresponding amino acids in the light or heavy chain of the acceptor immunoglobulin, said amino acids are not located at positions 26-30 of the heavy chain, and each: (i) is adjacent to the CDR in the donor immunoglobulin sequence, or (ii) contains atoms within 5 angstroms of the CDR in said humanized immunoglobulin. 10. A humanized immunoglobulin having complementarity determining regions (CDRs) corresponding to CDRs derived from a donor immunoglobulin and heavy and light chain variable regions corresponding to the heavy and light chain of the acceptor immunoglobulin. which humanized immunoglobulin specifically, at least 10? It binds to human L-selectin with an IVH affinity constant, wherein said humanized immunoglobulin comprises amino acid residues derived from a donor immunoglobulin framework replacing the corresponding amino acids in the light or heavy chain of the acceptor immunoglobulin, said amino acids not being located at positions 26-30 of the heavy chain. each of the following amino acids: (i) is adjacent to the CDR in the donor immunoglobulin sequence, or (ii) contains atoms 4 NM from the CDR in said humanized immunoglobulin. 11. A humanized immunoglobulin according to claim 9 or 10, characterized in that the distance of said atom from the CDR is determined on the basis of a computer generated immunoglobulin model. A humanized immunoglobulin according to claim 9 or 10, wherein said donor immunoglobulin is represented by the murine DREG-200 antibody. 13. A humanized immunoglobulin according to claim 9 or 10, wherein the antibody comprises two light / heavy chain dimers. 14. A humanized immunoglobulin according to claim 13, wherein said antibody is represented by the IgG1 or IgG4 isotype. 15. A humanized immunoglobulin according to claim 9, characterized in that said acceptor immunoglobulin heavy and light chain frames are derived from the human antibody Eu. A humanized immunoglobulin according to claim 1 or 9, characterized in that it is substantially purified. . . · ··· · · .57 ·· '* · ^; ”♦ A humanized immunoglobulin according to claim 1 or 9, characterized in that it inhibits the binding of human neutrophils to human endothelial cells. 18. A composition comprising the humanized immunoglobulin of claims 1 or 9. 19. A recombinant immunoglobulin that specifically binds human L-selectin, characterized in that the amino acid sequence of the mature light chain variable region is as shown in the bottom lines of Figure 2A. 20. A recombinant immunoglobulin that specifically binds to human L-selectin, characterized in that the amino acid sequence of the mature heavy chain variable region is as shown in the bottom lines of Figure 2B. 21. A method of treating an inflammatory disease or condition comprising administering to a human subject a therapeutically effective dose of a humanized immunoglobulin that specifically binds to human L-selectin. 22. The method of claim 21, wherein the inflammatory disease or condition is selected from the group consisting of ischemic reperfusion injury, adult respiratory distress syndrome, sepsis, and autoimmune disease. The method of claim 21, wherein the humanized immunoglobulin comprises the amino acid sequence of the mature light chain variable region shown in the lower rows of Figure 2A and the amino acid sequence of the mature heavy chain variable region shown in the lower rows of Figure 2B. 24. The method of claim 21, wherein the humanized immunoglobulin binds human L-selectin with an affinity of at least 1 x 10 4 Hg. · ♦ · • é! ··· · · · _ _sg _ ·· ··· ·· * · * ··: * 25. The method of claim 24, wherein at least 10 mg of the humanized immunoglobulin is administered parenterally to the patient. 26. The method of claim 22, wherein the inflammatory disease is represented by ischemic reperfusion injury. 27. The method of claim 26, further comprising the step of administering a therapeutically effective amount of a thrombolytic agent. 28. The method of claim 27, wherein the ischemic reperfusion injury is due to myocardial infarction or balloon angioplasty. 29. The humanized immunoglobulin of claim 1, comprising a humanized heavy chain and a humanized light chain: (1) the humanized light chain comprises three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions from a murine DREG-200 immunoglobulin light chain and a variable region frame from a human light chain variable region framework. with the exception that at least one position is selected from a first group comprising L87, L54, L66, L76 and L93, wherein said amino acid position is a murine DREG-200 immunoglobulin light chain variable region occupied by the same amino acid at the same position in the frame; and (2) the humanized heavy chain having three complementary amino acid sequences from regions corresponding to complementarity determining regions from a murine DREG-200 immunoglobulin heavy chain and a variable region frame derived from a human heavy chain variable region framework sequence. ···· · • .... • J. : · ♦ ·· -59 * Contains the complementarity determining region (CDR1, CDR2 and CDR3) with the exception that at least one position is H93, H95, H98, H111, H112, H115, H30 H98, H111, H27, H30, H48, and H72, wherein said amino acid position is the same as the same amino acid residue at the same position in the murine DREG200 immunoglobulin heavy chain variable region frame. wherein the immunoglobulin binds to the L-selectin ligand with an affinity within three times the binding affinity of the mouse DREG-200 immunoglobulin.