WO1996040942A1

WO1996040942A1 - Humanized antibodies to e-selectin

Info

Publication number: WO1996040942A1
Application number: PCT/US1996/009204
Authority: WO
Inventors: Mark A. Williams; Carl Perez; Mary M. Bendig; S. Tarran Jones; Jose W. Saldanha
Original assignee: Cytel Corporation
Priority date: 1995-06-07
Filing date: 1996-06-06
Publication date: 1996-12-19
Also published as: AU6155896A

Abstract

The present invention provides humanized antibodies that specifically bind E-selectin, wherein the antibodies comprise substantially the complementarity determining regions of a non-human antibody and substantially the framework regions (FRs) of a human antibody, where the FRs can be substituted in one or more amino acid positions. The invention also provides nucleic acid molecules encoding the humanized anti-E-selectin antibodies, vectors comprising the nucleic acid molecules, and cells containing the vectors. The invention further provides pharmaceutical compositions comprising a humanized anti-E-selectin antibody and provides methods of using a humanized anti-E-selectin antibody to detect the presence of E-selectin in a human subject and methods to diagnose a pathology characterized by the involvement of E-selectin in a human subject. In addition, the invention provides methods of reducing or inhibiting E-selectin mediated cell adhesion to an endothelial cell in a subject and methods of treating a human subject having a pathology characterized by the involvement of E-selectin by administering a humanized anti-E-selectin antibody to the subject.

Description

HUMANIZED ANTIBODIES TO E-SELECTIN

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

This invention relates generally to the fields of antibody engineering and immunology and more specifically to humanized antibodies that specifically bind E-selectin.

BACKGROUND INFORMATION

E-selectin is a cell surface receptor that is expressed by endothelial cells and mediates the binding of various white blood cells (leukocytes), including neutrophils, monocytes and some types of memory T cells, to the endothelial cells. Leukocyte adhesion to endothelial cells is a critical step in the migration of a leukocyte out of a blood vessel and into a tissue.

Leukocytes are an important component of inflammatory and immunological responses. For example, neutrophil migration to the site of an injury in a tissue is involved in the earliest stages of an inflammatory response. Following such an injury, neutrophils are mobilized from the circulation to the site of the injury. E-selectin is involved in mediating neutrophil adhesion to the vascular endothelium as a first step in this mobilization. Thus, E-selectin mediated adhesion of leukocytes to endothelial cells is necessary for maintaining the health of an individual.

In some cases, however, E-selectin mediated cell adhesion can produce or contribute to a pathological condition. For example, E-selectin can mediate the binding of tumor cells to endothelial cells and, therefore, may have a role in the process of tumor cell metastasis. In addition, higher than normal levels of E-selectin, including a soluble form of E-selectin, are associated with various pathological conditions. For example, increased levels of soluble E-selectin are present in the fluid associated with gross cystic breast disease, which may be a precancerous condition, and in the serum of workers exposed to high levels of hydrocarbons and other solvents. These observations suggest that E-selectin expression is associated with various pathological conditions and may have a role in the pathogenesis of these conditions.

A means for identifying the presence of

E-selectin can provide useful information as to the course or progress of a condition involving E-selectin. Anti-E-selectin antibodies have been raised in various animals and can be useful, for example, to detect the presence of E-selectin in a sample in vitro. However, such antibodies cannot be administered to a human subject over an extended period of time because the antibodies are recognized as foreign by the person and an immune response is induced against the antibodies. As a result, the utility of non-human antibodies in humans is limited.

Thus, a need exists to obtain antibodies that specifically bind E-selectin and can be administered to a human subject without inducing an immune response against the antibodies. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides humanized antibodies that specifically bind E-selectin. The humanized antibodies of the invention contain substantially the complementarity determining regions (CDRs) of a non-human antibody and substantially the framework regions (FRs) of a human antibody, which can be substituted in one or more amino acid positions. The invention is exemplified by various humanized anti-E-selectin antibodies comprising CDRs that correspond to the CDRs of murine monoclonal antibody CY1787, which is produced by the cell line designated Accession # HB 10591, and FRs that correspond to the FRs in human monoclonal antibody FK-001 variable light chain (V_L)and to the FRs of human monoclonal antibody NE M variable heavy chain (V_H) . Furthermore, the human V_L can be substituted at amino acid position 45 and the human V_H can be substituted at one or more of amino acid positions 20, 27, 29, 30, 48, 67, 68, 69, 70, 71, 73, 76, 78, 82, 82c and 94.

The invention also provides reshaped antibodies, which contain the CDRs of a first species and the FRs of a second species. In addition, the invention provides an antibody of the invention that is detectably labeled and a pharmaceutical composition containing an antibody of the invention and a pharmaceutically acceptable carrier.

The invention also provides nucleic acid molecules encoding humanized anti-E-selectin antibodies of the invention. These nucleic acid molecules encode humanized antibodies containing various humanized V_H and V_L chains as disclosed herein. In addition, the invention provides vectors comprising the nucleic acid molecules encoding humanized anti-E-selectin antibodies and cells that contain and express the vectors.

The invention also provides methods of using a humanized anti-E-selectin antibody to detect the presence of E-selectin in a human subject. Such methods are useful, for example, to diagnose the presence of an inflammatory response or of a pathology characterized by the involvement of E-selectin in a human subject. In addition, the invention provides methods of reducing or inhibiting E-selectin mediated cell adhesion in a human subject. The invention further provides methods of reducing or inhibiting the severity of an inflammatory response or of a pathology involving E-selectin in a human subject by administering a humanized anti-E-selectin antibody to the subject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides humanized antibodies that specifically bind E-selectin. As disclosed herein, a humanized antibody of the invention comprises substantially the complementarity determining regions (CDRs) of a non-human antibody and substantially the framework regions (FRs) of a human antibody. If desired, the human FRs in a humanized antibody of the invention can be substituted in at least one amino acid position with an amino acid present in the corresponding position of the non-human antibody.

The present invention is exemplified by various humanized anti-E-selectin antibodies containing CDRs that correspond to those of murine monoclonal antibody CY1787 (mu MAb CY1787; see SEQ ID NOS: 2 and 4), which is produced by the cell line designated ATCC Accession # HB 10591, and FRs that correspond to human monoclonal antibody variable light chain (V_L) and variable heavy chain (V_H) FRs. In the examples provided herein, the human V_H FRs correspond to those of human monoclonal NEWM and the human V_L FRs correspond to those of human monoclonal FK-001. A humanized antibody can have various amino acid substitutions in the FRs, including one or more substitutions of amino acid positions 20, 27, 29,

30, 48, 67, 68, 69, 70, 71, 73, 76, 78, 82, 82c and 94 in the V_H FRs and a substitution at amino acid position 45 in V_L FR2. Numbering of amino acid positions in an antibody follows that of Kabat et al. , Sequences of Proteins of Immunological Interest (National Institutes of Health; Bethesda MD; 1987 and 1991), each of which is incorporated herein by reference) . Six versions of humanized V_H chains are shown in SEQ ID NOS: 6, 8, 10, 12, 14 and 16 and two versions of humanized V_L chains are shown in SEQ ID NOS: 18 and 20. The humanized V_H and V_L chains can be combined in various combinations and can be combined with a constant heavy chain (C_H) and constant light chain (C_L) to produce humanized anti-E-selectin antibodies.

E-selectin, which also has been designated ELAM-1, LECAM-2 and CD62E, is a cell surface receptor that mediates the binding of neutrophils, monocytes and some types of memory T cells to the endothelial cells that line blood vessels (see, for example, Bevilacqua et al.. Cell 67:233 (1991); see, also. The Adhesion Molecule FactsBook (ed. Pigott and Power, Academic Press; 1993), pages 1-20 and 60-63, and references cited therein, each of which is incorporated herein by reference) . E-selectin is a member of the selectin family of proteins that also includes L-selectin and P-seleσtin. The selectins are structurally related in that each member of the family contains an N-terminal Ca^-dependent lectin domain, an EGF-like domain, two to nine consensus repeat units designated CRP domains, a transmembrane domain and a C-terminal cytoplasmic domain (see Collins, J. Biol. Chem. 2466-2473 (1991), which is incorporated herein by reference; see, also, Bevilacqua et al., supra , 1991)). Mammalian E-selectins, for example, generally contain six CRP repeats, although the rabbit E-selectin contains five CRP repeats.

The lectin domain present in the selectins was identified by its homology to the carbohydrate binding domains present in various lectins. The lectin binding domain is involved in selectin mediated binding, which can be inhibited by antibodies directed against the lectin domain. The role of the EGF-like domain in the selectins is less clear. However, deletion of the EGF domain in E-selectin abolishes cell adhesion (Piggot et al., J. Immunol. 147:130-135 (1991)). The role of CRP domains, which resemble similar units present in complement binding protein, is not yet known.

Each selectin can recognize several different ligands and different selectins can recognize the same ligand. For example, E-selectin can bind the sialyl

Lewis³⁵ determinant(sLe^x) , which is present on neutrophils, sialyl Lewis³, and related fucosylated N-acetyl- lactosamines present in leukocyte glycolipids and glycoproteins (Piggot and Power, supra , 1993). E-selectin binds sLe^x with a dissociation constant of about 0.1 to 1 mM. L-selectin and P-selectin also can bind sLe^x, although with lower affinity than E-selectin (see McEver et al., supra , 1995).

E-selectin occurs in membrane-bound form and in soluble form in a human subject. For example, E-selectin is transiently expressed on cytokine-activated endothelial cells and, therefore, is expressed on endothelial cells near regions of inflammation. In addition, circulating soluble forms of E-selectin are produced due to proteolytic cleavage of E-selectin or to expression of alternatively spliced forms of E-selectin mRNA (McEver et al., J. Biol. Chem. 270:11025-1102 (1995), which is incorporated herein by reference). The humanized antibodies of the invention can specifically bind the membrane-bound and soluble forms of E-selectin.

As used herein, the term "antibody" is synonymous with the term "immunoglobulin" and is used broadly to mean a protein that can specifically bind a particular antigen by virtue of an interaction of the antibody variable region with one or more epitopes on the antigen. The term "antibody" refers to an intact antibody, which is a tetrameric protein having two identical heavy chains and two identical light chains, wherein the heavy chains and light chains each contain a variable region and a constant region, and to functional fragments of the intact antibody, including, for example, fragments such as Fab, Fab", F(ab')₂ and Fv fragments, which can specifically bind a particular antigen (Kuby, J. , Immunology 2nd ed. (W.H. Freeman and Co. 1994); see Chapter 5, which is incorporated herein by reference). Such fragments can be produced by treating an intact antibody with an enzyme such as pepsin, which produces an F(ab')₂ fragment, or by chemical synthesis using well known methods of peptide synthesis (see, for example, Bodanszky, M. , Principles of Peptide Synthesis 2nd revised ed. (Springer-Verlag 1988 and 1993); see, also, Houghten, Proc. Natl. Acad. Sci.. USA 82:5131 (1985), each of which is incorporated herein by reference) or by methods of recombinant DNA technology as disclosed herein (see Example II) or otherwise known in the art.

The amino acid sequences of various humanized V_H and V_L chains are disclosed herein. Numbering of amino acid positions in an antibody follows that of Kabat et al. ( supra , 1987 and 1991). The skilled artisan will recognize that such humanized V_H and V_L chains can be combined with one or more domains of a C_H chain such as ^cvir ^cy2, ^cγ3' ^cv4 ^cαir ^Cα2 r ^c _μ, ^cδ and C_e and with a C_L chain such as C_κ and C_λ, respectively, to produce an intact antibody of a particular isotype or any of various functional fragments of such an antibody. For example, the humanized V^ (SEQ ID NO: 16) can be combined with a C_yl chain or a C_v4 chain and the humanized V^ (SEQ ID NO: 18) can be combined with a V_κ chain to produce a humanized anti-E-selectin IgGl or IgG4 antibody, respectively (see Example II). Thus, the present invention provides humanized anti-E-selectin antibodies of various isotypes, which can provide the related advantage conferred by the effector function of the particular immunoglobulin isotype.

As used herein, the term "humanized antibody" means an antibody that contains CDRs that correspond to those of an antibody produced by a species other than a human and contains FRs, and constant domains as desired, that correspond to those of a human antibody. For convenience of discussion, an antibody that contains the CDRs to be used in a humanized antibody is referred to as the "donor" antibody. Thus, u MAb CY1787 was used as the donor antibody to prepare the humanized anti-E-selectin antibodies disclosed herein. In addition, the term "host" is used in referring to an antibody that provides the FRs, and constant domains as desired. For example, human monoclonal antibodies NE M and FK-001 were the host antibodies used to prepare the humanized antibodies exemplified herein (see Example II). If desired, a humanized antibody can contain one or more amino acid substitutions in the host FRs, where the substituted amino acids correspond to the amino acids present in the donor antibody. Such a humanized anti-E-selectin antibody can specifically bind E-selectin in an appropriate host.

A humanized antibody comprising murine CDRs and human FRs as disclosed herein provides certain advantages, including, for example, that when administered to a human, the person's immune system should not recognize the antibody as foreign and, therefore, will not mount a human anti-murine antibody (HAMA) response against the humanized antibody. An additional advantage of humanized antibodies is that the effector portion of the humanized antibody, which generally is associated with the constant region domains of the antibody, can interact with the various components of the human immune system, including, for example, components of the complement pathway. Furthermore, the half-life a humanized antibody in a human is similar to the half-life of a naturally occurring human antibody, whereas the half-life of a non-human antibody in a human is significantly shorter (see, for example, Shaw et al., J. Immunol. 138:4534-4538 (1987)).

The skilled artisan will recognize that the methods disclosed herein for humanizing mu MAb CY1787 are generally applicable to other donor monoclonal anti-E-selectin antibodies, including, for example, ovine, bovine, equine or porcine monoclonal antibodies. An appropriate donor monoclonal anti-E-selectin antibody can be prepared using standard methods (see Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press 1988); see Chapters 6 and 7, each of which is incorporated herein by reference) or can be purchased from a commercial source.

The methods disclosed herein also can be used to reshape a donor monoclonal antibody such as a murine monoclonal antibody such that the reshaped antibody contains the FRs of a host of interest, other than a human host, and the CDRs, for example, of mu MAb CY1787. Such a reshaped antibody should not induce a host anti-donor antibody (HoADA) response in the host. Various host antibodies can be obtained using well known methods or can be purchased from a commercial source.

As used herein, the term "reshaped antibody" means an antibody that contains the V_H and V_L CDRs from a donor monoclonal antibody and the FRs from a host of interest. A humanized antibody is a particular example of a reshaped antibody. If desired, the reshaped antibody can contain one or more amino acid substitutions in the host FRs, where the substituted amino acids correspond to the amino acids present in the donor antibody. Such a reshaped anti-E-selectin antibody can specifically bind E-selectin in an appropriate host.

A humanized anti-E-selectin antibody of the invention is characterized by its ability to specifically bind E-selectin in vitro and in vivo. As used herein, the term "specifically binds" means that a humanized antibody of the invention can associate with E-selectin with an affinity constant of at least about 1 x 10⁵ liters/mole. In some circumstances, it can be desirable to have a humanized anti-E-selectin antibody that specifically binds E-selectin with an association constant of about 1 x 10⁷ to about 1 x 10⁸ liters/mole depending, for example, on whether the target E-selectin is concentrated in a region or is diffusely distributed in the subject and on whether the antibody is to be administered systemically or locally. Regardless of the particular application, however, the affinity of binding of a humanized anti-E-selectin antibody and E-selectin is sufficiently specific such that a bound complex can form in vivo and can form in vitro under appropriate conditions as described herein. The formation or dissociation of a bound complex can be identified as described in Examples III, IV and V or using other well known methods such as equilibrium dialysis, which can be useful for determining the affinity constant of a humanized antibody of the invention for E-selectin (see, for example, Kuby, supra , 1994; see Chapter 6, which is incorporated herein by reference) .

Specific binding of a humanized antibody of the invention with E-selectin was demonstrated by showing that the humanized antibody competitively inhibited the binding of the parental mu MAb CY1787 to a soluble form of E-selectin (see Example IV) . Under the conditions of this assay, the humanized anti-E-selectin antibodies of the invention have a relative binding efficiency of about 1% to about 65% the binding efficiency of mu MAb CY1787 for E-selectin. In addition, a humanized anti-E-selectin antibody inhibited the binding of human neutrophils to human umbilical vein endothelial cells in vitro as effectively as did the parental mu MAb CY1787 antibody (see Example V). These results demonstrate that the humanized antibodies of the invention specifically bind E-selectin.

The FRs and CDRs of the humanized antibodies of the invention can be modified to produce humanized antibodies having higher or lower binding affinities than that of mu MAb CY1787. Sequence comparisons, using protein sequence databases, were used to identify human antibody variable regions having a similar amino acid sequence to the mouse CY1787 variable regions. The computer model was used to identify amino acid residues in the FRs of the mouse CY1787 variable regions that can be involved in supporting the CDR loop, in binding directly to the antigen or in the actual packing of the heavy and light chain variable regions. When closely related human antibodies were identified, selected amino acid substitutions were made in the human variable chain FRs by replacing the amino acid in the human antibody with the corresponding amino acid in mu MAb CY1787. Criteria for selecting amino acid positions for substitution include the likely influence of the amino acid on CDR conformation or on antigen binding. In general, however, it is preferable to minimize the substitution of donor amino acid residues into the host antibody in order to minimize the likelihood of inducing a HoADA response. As disclosed herein, the criteria for selecting amino acid positions for substitution indicated that amino acid positions 20, 27, 29 and 30 (FR1) and 48 (FR2) and positions 67 to 71, 73, 76, 78, 82, 82c and 94 (FR3) were particularly suitable for substitution. Computer modeling indicated, for example, that the asparagine residue present in position 73 of the mu MAb 1787 ("murine Asn 73") could hydrogen bond with the murine Ser 28, which is part of the structural loop of CDR HI. However, the human NEWM antibody, which was used as the donor antibody for the humanized V_H chain, contains a Thr 73. Since the distance between a Thr 73 and Ser 28 is greater than the distance between an Asn 73 and Ser 28, it was considered that a hydrogen bond would not form in the human FR. Therefore, the human Thr 73 was substituted with the murine Asn 73 in some versions of a humanized antibody. Such substitutions were effected using recombinant DNA methods (see Example II; see, also. Protein Engineering: A practical approach, (ed. Rees et al.; IRL Press 1992), see Chapter 11, which is incorporated herein by reference) .

Another useful method for optimizing the binding affinity of an antibody utilizes a phage display library as described, for example, in United States Patent No. 5,223,409, which is incorporated herein by reference, and screening such a library with a soluble E-selectin containing the epitope recognized by the antibody (see, also, Rees et al., supra , 1992, Chapter 12; Huse, WO 92/06204; and Jespers et al.. Biotechnology 13:378-389 (1995), each of which is incorporated herein by reference). Using such phage display technology, amino acids in the FRs and CDRs can be varied so as to obtain humanized antibodies optimized for desired characteristics such as binding affinity. Various other changes also can be made to a humanized antibody of the invention, including, for example, introducing additional conservative amino acid substitutions such as the substitution of one hydrophobic amino acid for another. The skilled artisan will recognize that a humanized anti-E-selectin antibody as disclosed herein but having one or a few conservative amino acid substitutions are encompassed within the invention, provided the substituted humanized antibody can specifically bind E-selectin as determined, for example, using the methods disclosed in Examples IV and V or otherwise known in the art. Furthermore, it is well known that phage display technology is particularly useful for screening large numbers of mutated peptides, which contain one or a few amino acid changes relative to a starting peptide (see, for example, U.S. Patent No. 5,223,409). Accordingly, the skilled artisan would recognize that V_H and V_L chains similar to those disclosed herein but containing one or a few amino acid substitutions, deletions or additions can be prepared, that such mutated chains that specifically bind E-selectin can be identified and that the nucleic acid molecules encoding such mutated variable chains can be isolated. Humanized anti-E-selectin antibodies comprising such peptides are encompassed within the claimed invention.

The present invention also provides nucleic acid molecules encoding humanized anti-E-selectin antibodies. The invention provides, for example, nucleic acid molecules encoding various humanized V_H and V_L chains (see SEQ ID NOS: 5, 7, 9, 11, 13, 15, 17 and 19). It is recognized, however, that, due to the degeneracy of the genetic code, numerous other substantially similar nucleic acid sequences can encode the same amino acid sequences as shown in SEQ ID NOS: 6, 8, 10, 12, 14, 16, 18 and 20. Such nucleic acid molecules are considered to be encompassed within the present invention. The nucleic acids exemplified herein encode substantially the V_H FRs of human monoclonal antibody NEWM and substantially the V_L FRs of human monoclonal antibody FK-001 in combination with the appropriate V_H and V_L CDRs, respectively, of mu MAb CY1787. These nucleic acid molecules were produced by oligonucleotide directed mutagenesis such that the mu MAb CY1787 CDRs were "grafted" onto the corresponding human variable chain FRs (see Example II; see, also, Rees et al., supra , 1992), Chapter 11) . It is recognized, however, that the same or substantially similar nucleic acid molecules can be chemically synthesized using, for example, an automated DNA synthesizer.

The nucleic acid molecules of the invention are useful for producing humanized anti-E-selectin antibodies. Accordingly, the invention also provides vectors comprising the nucleic acid molecules of the invention and cells that contain the vectors. Cloning vectors and expression vectors as well as host cells for such vectors are well known in the art and can be constructed using well known methods or obtained from commercial sources (see, for example, Hodgson, Biotechnology 11:887-893 (1993); and Vectors: Essential Data (Gacesa and Ramji, ed.; John Wiley & Sons 1994), each of which is incorporated herein by reference) . In general, a vector useful for containing a nucleic acid molecule encoding a humanized anti-E-selectin antibody is an expression vector, which contains the appropriate regulatory elements for expression in a prokaryotic host cell or eukaryotic host cell as desired. Furthermore, a vector useful in the invention can be a plasmid vector or a viral vector, each of which has certain advantages and disadvantages well known to the skilled artisan. Methods for introducing a vector into an appropriate host cell are routine and well known in the art and include, for example, methods of transfection such as calcium phosphate precipitation, electroporation and lipid mediated transfection (see, for example, Sambrook et al.. Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989), which is incorporated herein by reference) and methods of viral infection using, for example, retrovirus, adenovirus, adenovirus- derived or baculovirus vectors.

An expression vector useful in the invention contains the nucleic acid molecule encoding the humanized antibody operably linked to an expressible element. As used herein, the term "operably linked to an expressible element" means that the nucleic acid molecule encoding the humanized antibody is properly positioned in relation to appropriate gene regulatory elements, such that the nucleic acid molecule can be transcribed, and appropriate translation regulatory elements, such that the resultant mRNA can be translated. Appropriate gene regulatory elements and translation regulatory elements are well known in the art and include, for example, promotors, enhancers, silencers, polyadenylation signals, ribosome recognition and binding sequences, splice signal sequences and leader/signal peptide-encoding sequences (see, for example, Meth. Enzymol. vol. 185 "Gene Expression Technology" (D.V. Goeddel, ed. ; Academic Press 1990); Meth. Enzymol. vol. 217 "Recombinant DNA, part H (R. Wu, ed. ; Academic Press 1993); see, also, Hippenmeyer and Highkin, Biotechnology 11:1037-1041 (1993), each of which is incorporated herein by reference) . Some or all of these regulatory elements can be present or absent in a vector as desired.

A gene promotor or enhancer element can be constitutively active, thereby permitting a steady-state level of transcription of a gene linked thereto when present in an appropriate cell type, or can be inducible, the expression from which can be modulated by exposure of the element to an appropriate regulatory factor. Furthermore, a gene regulatory element can be active in a variety of different cell types or can be tissue specific, in which case expression from the element occurs in only one or a few particular cell types. Examples of constitutive and inducible promotors and enhancers, including tissue specific promotors and enhancers, are well known in the art.

A particular vector must be propagated in an appropriate host cell. For example, a plasmid vector containing a bacterial origin of replication, is propagated in bacterial cells such as E. coli cells. Such a plasmid vector-bacterial host system is useful for producing large amounts of a nucleic acid molecule encoding a humanized anti-E-selectin antibody. In comparison, a vector such as a baculovirus vector, which is propagated in an insect cell, is useful for producing large amounts of a polypeptide encoded by the nucleic acid molecule present in the vector.

Particularly useful expression vectors, including plasmid vectors and viral vectors, are propagated in mammalian cells and provide the advantage that, upon expression, a polypeptide encoded by a nucleic acid molecule contained in the vector is modified post- translationally. For example, the vectors described in Examples II and IV are useful for producing a properly glycosylated humanized antibody upon expression of the nucleic acid molecule contained within the vector. In addition, such mammalian expression vectors can contain a nucleic acid sequence encoding dihydrofolate reductase (dhfr) . Such a vector, when propagated under suitable selection pressure, is amplified to a high copy number. Accordingly, a nucleic acid molecule encoding a humanized anti-E-selectin antibody contained in an amplified vector can be expressed at high levels and, upon translation. results in the production of high levels of the humanized antibody (see Example IV).

The invention also provides pharmaceutical compositions comprising a humanized anti-E-selectin antibody and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include aqueous solutions such as physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters.

A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize the humanized anti-E-selectin antibody. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the humanized antibody.

The invention further relates to methods of administering a humanized anti-E-selectin antibody to a subject in order to detect the presence of E-selectin or to reduce or inhibit E-selectin mediated cell adhesion to an endothelial cell. For example, a humanized antibody can be administered to a subject as a treatment for inflammation, septic shock, splanchnic occlusion shock, wound associated sepsis or acute respiratory distress syndrome, each of which is characterized, in part, by E- selectin mediated adhesion of leukocytes to endothelial cells. In addition, the composition can be administered to a cancer patient to reduce or inhibit E-selectin mediated adhesion of the patient's cancer cells to endothelial cells, thereby reducing the likelihood of metastasis in the subject.

One skilled in the art will know that a humanized anti-E-selectin antibody can be administered by various routes including, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intraσisternally. In addition, an antibody of the invention comprising, for example, an Fv fragment, can be administered by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively. Furthermore, the composition can be administered by injection, intubation or topically, the latter of which can be passive, for example, by direct application of an ointment or powder, or active, for example, using a nasal spray or inhalant.

The antibody also can be incorporated into liposomes, microspheres, foams, emulsions, micelles or other matrices (see, for example, Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton, FL 1984); see, also, U.S. Patent No. 4,837,028; each of which is incorporated herein by reference). Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. Incorporation of an anti-E-selectin antibody of the invention into a liposome provides the additional advantage that the antibody can target the liposome, for example, to an area of inflammation (see, for example, U.S. Patent No. 4,957,773 and No. 4,603,044, each of which is incorporated herein by reference), which can be particularly useful if the liposome is formed to contain an agent such as anti-inflammatory agent, which is targeted to the site of inflammation.

A pathological condition characterized, in part, by E-selectin mediated cell adhesion can be localized or systemic. Accordingly, one skilled in the art would select a particular route and method of administration of a humanized anti-E-selectin antibody depending on the particular condition. For example, in a subject suffering from a systemic condition such as bacterial endotoxin-induced sepsis, a pharmaceutical composition comprising an antibody of the invention can be administered intravenously, orally or by another method that distributes the antibody systemically. In comparison, in a subject suffering from a localized pathology such as psoriasis, a humanized anti-E-selectin antibody can be administered topically using, for example, a cream ointment. In a subject suffering from a localized pathology such as acute respiratory distress syndrome, an anti-E-selectin antibody can be suspended or dissolved in the appropriate pharmaceutically acceptable carrier and administered directly into the lungs using, for example, a nasal spray. It is recognized that administration to the lungs using a spray also can result in systemic distribution of a humanized antibody of the invention and is useful, therefore, for indications requiring systemic administration of an antibody.

An anti-E-selectin antibody of the invention can be used for diagnostic purposes to detect the presence of E-selectin in a subject or for therapeutic purposes to reduce or inhibit the severity of a condition characterized by E-selectin mediated adhesion of a cell to an endothelial cell. In order to effect such a diagnostic or therapeutic purpose, the humanized antibody must be administered in an effective amount, which generally is about 0.05 to about 20 mg/kg body weight. The total effective amount can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which the multiple doses are administered over a more prolonged period of time. One skilled in the art would know that the dose of a humanized anti-E-selectin antibody required to provide an effective amount to a subject depends on many factors including the age and general health of the subject as well as the route of administration, the number of treatments to be administered, and whether administration is localized or systemic. In view of these factors, the skilled artisan would adjust the particular dose so as to administer an effective amount of a humanized antibody of the invention to effect a particular purpose.

The invention provides methods of using a humanized anti-E-selectin antibody to detect the presence of E-selectin in a human subject. Such methods are useful, for example, to identify the site of an inflammatory response or of a pathology characterized by the involvement of E-selectin in the subject. In order to utilize an antibody of the invention to detect the presence of E-selectin in a subject, the humanized anti-E-selectin antibody can be labeled so as to be detectable. An anti-E-selectin antibody can be detectably labeled using methods well known in the art (see, for example, Harlow and Lane, supra , 1988; see chap. 9, which is incorporated herein by reference). For example, an antibody can be labeled with any of various detectable moieties including a radiolabel, an enzyme or enzyme substrate, biotin or a fluorochrome.

Although a humanized antibody is particularly valuable for use in vivo, the antibody also can be used in various in vitro assays, including, for example, in a prescreening assay to identify an antibody that can be used in vivo. For an in vitro assay, the humanized antibody can be detectably labeled, as above, or a labeled second antibody can be used to identify specific binding of an unlabeled humanized anti-E-selectin antibody. A second antibody generally will be specific for the particular class of the first antibody. For example, if an anti-E-selectin antibody is of the IgG class, a second antibody will be an anti-IgG antibody. Such second antibodies are readily available from commercial sources. If desired, the second antibody can be labeled using a detectable moiety as described herein or otherwise known in the art.

For detecting E-selectin in a subject, particularly useful labels are detectable external to the subject using an imaging device such as a scintillation detector, positron emission transaxial tomography (PETT) or nuclear magnetic resonance (NMR) imaging. A gamma ray emitting radionuclide such as indium-Ill or technitium-99, for example, can be conjugated to an antibody of the invention and can be detected using a solid scintillation detector. Alternatively, a positron emitting radionuclide such as carbon-11 or a paramagnetic spin label such as carbon-13 can be incorporated into a an antibody of the invention and, following administration to a subject, the localization of the antibody to a site where E-selectin is present can be detected using PETT or NMR imaging, respectively.

Such non-invasive imaging methods as described above are useful for detecting the presence of E-selectin in a subject. In particular, such methods can be useful for identifying a region of inflammation in a subject or can be diagnostic of a pathology characterized by the expression or accumulation of E-selectin. For example, inflammation is a normal physiological response that occurs due to tissue injury. Associated with the inflammatory response is the elicitation of cytokines, which have a variety of functions including inducing E-selectin expression by endothelial cells in the area of the inflammation. In some cases, however, a patient can present with generalized symptoms of inflammation, but the site of the inflammatory response in the subject may not be evident. Administration of a detectably labeled humanized anti-E-selectin antibody to a subject can be used to identify the location of the inflammation in the subject by binding to the endothelial cells that are expressing E-selectin in response to cytokine exposure.

The expression of E-selectin also is associated with various pathological conditions. For example, gross cystic breast disease can be a premalignant stage of breast cancer, depending, for example, on the type of cells involved in the cystic disease. Specifically, breast cysts composed of apocrine epithelium are associated with a much higher risk of developing into breast cancer than cysts lined by flattened epithelium (see Lai et la., Br. J. Surσ. 82:83-85 (1995)). Lai et al. have reported that significantly elevated levels of soluble E-selectin are present in the cystic fluid obtained from cysts containing apocrine epithelium. Thus, a labeled antibody of the invention can be administered to a women suspected of having a premalignant form of gross cystic breast disease, wherein the accumulation of the labeled humanized antibody in the cyst can be diagnostic of a premalignant breast tumor.

The invention also provides methods of reducing or inhibiting E-selectin mediated adhesion of a cell such as a leukocyte or a tumor cell to an endothelial cell in a human subject and methods of reducing or inhibiting the severity of an inflammatory response or of a pathology characterized by the involvement of E-selectin by administering a humanized anti-E-selectin antibody to the human subject. As used herein, the terms "reduce" and "inhibit" have their common meanings. The terms are used together to avoid any ambiguity as to the extent to which a humanized anti-E-selectin antibody acts. It is recognized, for example, that an antibody of the invention can decrease the level of E-selectin mediated adhesion of a cell below a level that is detectable using a particular assay. In this situation, one would be unable to determine whether cell adhesion was reduced to a lower level or was inhibited such that no adhesion was occurring. The use of these terms together precludes the need to distinguish these events.

E-selectin is involved in neutrophil adhesion to the vascular endothelium, which is an initial step in neutrophil migration to a site of inflammation. As discussed above, E-selectin expression is induced in endothelial cells in response to cytokines elicited at a site of inflammation. However, a systemic inflammatory response can be induced, for example, in a subject suffering from bacterial sepsis. In a patient with bacterial sepsis, the inflammatory response can be characterized as pathologic because the generalized neutrophil infiltration into tissues causes systemic tissue injury. Administration of a humanized anti-E-selectin antibody can reduce neutrophil adhesion to the endothelium and, therefore, can reduce or inhibit the severity of the tissue damage associated with bacterial sepsis. E-selectin mediated cell adhesion also can be involved in a localized pathologic inflammatory response as occurs, for example, in psoriasis or in acute respiratory distress syndrome. Administration of a humanized anti-E-selectin antibody can reduce or inhibit the severity of such localized pathologic conditions. Thus, the humanized anti-E-selectin antibodies of the invention can be used as medicaments to reduce or inhibit 24 the severity of a localized or systemic pathological condition characterized by the involvement of E-selectin mediated cell adhesion.

E-selectin expression also is induced due to ischemia or to low blood flow states as occurs, for example, in shock (see, for example, Altavilla et al., Eur. J. Pharmacol. 272:223-239 (1995)). In a subject suffering from shock, E-selectin expression may be induced by cytokines, resulting in leukocyte adhesion to the endothelium and migration into the tissues. As above, infiltration of leukocytes can contribute to the pathology associated with shock. Administration of a humanized anti-E-selectin antibody can reduce or inhibit the severity of shock by blocking E-selectin mediated leukocyte adhesion to vascular endothelium in the shock victim. In this regard, Altavilla et al. reported that administration of mouse anti-E-selectin antibody reduced the severity of splanchnic artery occlusion shock in rats (Altavilla et al., supra , 1995).

A number of tumor cells express carbohydrates, which can bind E-selectin and can mediate the binding of tumor cells to endothelium. E-selectin mediated adhesion of a tumor cell to endothelium can be involved in tumor metastasis (see Aruffo et al., Proc. Natl. Acad. Sci. _r USA 82:2292-2296 (1992); Rice and Bevilacqua, Science 249:1303-1306 (1989)). Administration of a humanized antibody of the invention can be used as means to minimize the likelihood of tumor metastasis in a subject by reducing or inhibiting E-selectin mediated tumor cell adhesion to the vascular endothelium.

Administration of a humanized anti-E-selectin antibody can be particularly useful as part of a combined modality treatment of a cancer patient. For example, surgical removal of a tumor is a common means for reducing a tumor burden in a cancer patient. As part of a combined modality treatment, a humanized anti-E-selectin can be administered to subject either systemically prior to surgery or at the site of the tumor during surgery for the purpose of reducing or inhibiting E-selectin mediated adhesion to the endothelium of any tumor cells remaining in the patient following surgery, thus reducing dissemination of the tumor cells in the subject.

The following examples are intended to illustrate but not limit the present invention.

EXAMPLE I

REAGENT PREPARATION

This example provides methods for preparing and isolating reagents used in the binding assays disclosed below.

A. Preparation of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Neutrophils (PMNs)

1. HUVEC preparation

Human umbilical cords were obtained following birth. Sections of umbilical cord 20 to 40 cm in length and free of clamp marks were used. Under sterile conditions, one end of the cord vein was cannulated and connected to a 3-way stopcock and syringe. The cord was flushed with Ca⁺/Mg⁺⁺ free phosphate buffered saline (PBS) to remove blood and clots. The opposite end of the cord was cannulated and connected to a 2-way stopcock and rinsed briefly with 1 mg/ml collagenase (Boehringer; Indianapolis IN) , then the 2-way stopcock was closed. The cord then was filled with collagenase, air was removed and the 3-way stopcock was closed. The cord was placed in a sterile chamber and incubated at 37°C for 15 to 20 min. Following incubation, the cord was flushed with 30 ml PBS and the eluted endothelial cells were collected in a sterile tube containing 5 ml EGM-UV medium (Clonetics; San Diego CA) containing 10% fetal bovine serum (FBS) to inhibit further collagenase activity. The cells were collected by centrifugation, then resuspended in EGM-UV medium containing 10% FBS, 10 ng/ml epidermal growth factor, 1 μg/ml hydrocortisone, "GENTAMYCIN" and "AMPHOTERCIN-B". A tissue culture flask was coated with 0.1% gelatin (endotoxin-free, isolated from bovine epidermis) for 15 min at RT, then the excess gelatin was removed. The endothelial cell suspension was plated in the flask and placed in a 5% C0₂ incubator. The medium was changed after 16 hr, when the cells appeared to be adherent. The cells were passaged when they attained about 75% confluency by removing the medium, rinsing the flask two times (2X) with 10 mM HEPES buffered saline, then adding 1 to 2 ml 0.025% trypsin-EDTA until the cells were in suspension (30 sec to 1 min). Fresh complete medium was added to the flask and the cells were split 1:2 or 1:3 into gelatin coated flasks.

2. Neutrophil preparation

Neutrophils were prepared from 50 ml whole blood drawn from a volunteer donor into heparinzed tubes. 25 ml blood was layered in a centrifuge tube over 15 ml "MONO-POLY RESOLVING MEDIUM" (Flow Laboratories). Tubes were centrifuged at 20 °C for 25 min at 2000 rpm in an RT 6000 centrifuge, then the speed was increased to 2500 rpm for an additional 25 min. The neutrophil layer, which was the lower of the two floating cell layers, was removed and placed in a clean 50 ml centrifuge tube. Thirty ml Hank's Balanced Salt Solution (HBSS; GIBCO/BRL) containing 20 mM HEPES and 0.2% glucose was added to the tube and the neutrophils were collected by centrifugation at 3000 rpm for 3 min at RT, then resuspended in HBSS/HEPES/glucose buffer and counted using a hemacytometer.

B. Expression and purification of human recombinant soluble E-selectin (sol-E-selectin)

1. Cells and plasmid DNA

The adenovirus transformed human kidney cell line 293 was obtained from the American Type Culture Collection (ATCC CRL 1573). These cells were grown as adherent cultures in DMEM ( Whittaker Bioproducts; Walkersville MD) , supplemented with 10% FBS (JRH Biochemical; Lenexa KS).

The plasmid pCDNAl was obtained from Invitrogen

(San Diego CA) . The plasmid cloning vector pBluescript II was obtained from Stratagene (San Diego CA) . The plasmid pSV2neo contains the E. coli gene encoding the aminoglycoside 3 '-phosphotransferase gene and confers resistance to the mammalian antibiotic G418 ("GENETICIN"; Sigma; St. Louis MO; see Southern and Berg, J. Mol. Appl. Gen. 1:327 (1982), which is incorporated herein by reference) .

2. Recombinant DNA

A plasmid directing the expression of a soluble form of E-selectin (sol-E-selectin) was engineered as described below. A 1.67 kb DNA fragment encoding a truncated E-selectin was isolated by PCR (polymerase chain reaction) amplification of cDNA derived from IL-1 activated human endothelial cells. The 5'-amplimer, 5 '-TCAAGATCGATCCTTTGGGTGAAAAG-3 ' (SEQ ID NO: 21), inserted a unique Cla I restriction site 28 nucleotides upstream from the authentic translation initiation codon of the E-selectin structural gene. The 3'-amplimer, δ'-TTGGACTCGAGTTATCATTCACAGGTAGGTAG-S' (SEQ ID NO: 22), inserted the termination codon TGA after amino acid 527 of the mature E-selectin sequence, followed by a unique Xho I restriction site. The carboxy terminus

(C-terminus) of the sol-E-selectin is located at the C-terminus of the sixth consensus repeat element (CRP) , thereby deleting the transmembrane domain. Following amplification, the 1.67 kb PCR fragment was digested with Cla I and Xho I and subcloned into Clal/XhoI-digested pBluescript II to produce pBSII-sol-E-selectin.

For expression of sol-E-selectin in mammalian cells, the 1.67 kbp DNA fragment encoding the sol-E-selectin cDNA fragment was isolated from pBSII- sol-E-selectin and subcloned into the Eco RV and Xho I sites of the expression vector pCDNAl. The resultant plasmid, designated pCDNAl-sol-E-selectin, enables the expression of a 527 amino acid soluble-E-selectin molecule containing 11 potential N-glycosylation sites (see Bevilacqua et al.. Science 243:1160-1165 (1989), which is incorporated herein by reference) .

3. Generation of a stable cell line secreting sol-E-selectin

A stable cell line secreting sol-E-selectin was generated by cotransfecting pCDNAl-sol-E-selectin and pSV2neo into 293 cells using the calcium phosphate technique (see, for example, Sambrook et al., supra, 1989). At 48 hr post-transfection, the transfected 293 cells were trypsinized and plated into DMEM containing 10% FBS and 600 mg/ml active G418 to initiate selection of stable cell lines. The selection medium was changed every 3 days until a stable G418-resistant population was established. Single clones of G418 resistant cells were isolated from the above transfection and screened for sol-E-selectin synthesis using an enzyme-linked immunoabsorbent assay (ELISA) . The murine anti-E-selectin monoclonal antibody CY1787, which is described in Example II, was used as the primary antibody in the ELISA screens. Clones that were positive for sol-E-selectin by ELISA were further assayed for sol-E-selectin synthesis by immunoprecipitation. For the immunoprecipitation analysis, positive clones were plated at 10⁶ cells/100 mm dish and 24 hr later were metabolically labeled with 100 μCi/ml ³⁵S-methionine (1000 Ci/mmol) for 5 hr. Labeled sol-E-selectin was immunoprecipitated from the culture medium with mu MAb CY1787 and analyzed by 10% polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. Clone 293 #3 (293-3) was selected as the stable cell line that produced the greatest amount of sol-E-selectin protein/cell and was used in all subsequent procedures. The apparent molecular weight of the recombinant protein secreted from this cell line was 110 kDa.

4. Large scale production of sol-E-selectin

A 10-chambered Nunc "CELL FACTORY" (6250 cm² total surface area; Nunc; Naperville IL) was seeded with 2.78 x 10⁸ 293-3 cells in 850 ml DMEM containing 5% FBS and incubated at 37°C for 72 hr. The medium was harvested and an additional 850 ml DMEM containing 5% FBS was added to the cells. The cells were further incubated at 37°C for 48 hr and the medium was harvested and again replaced with 850 ml DMEM containing 5% FBS. This procedure was repeated a third and final time. After each harvest, sodium azide was added to 0.02% to the collected medium. The medium was clarified by centrifugation at 5000 x g, passed through a 0.2 mm filter and stored at 4°C prior to further purification as described below.

5. Immunoaffinity column chromatography

The mu MAb CY1787 was conjugated to protein A-Sepharose essentially as described by Schneider et al. (J. Biol. Chem. 257:10766 (1982), which is incorporated herein by reference) . Briefly, 28 mg mu MAb CY1787 (5 mg/ml) in PBS was mixed with 5 ml protein A- Sepharose (Pharmacia; Uppsala, Sweden) beads for 30 min at room temperature (RT) . The beads were washed by centrifugation 4X using 25 ml 0.1 M borate buffer, pH 8.2, then 2X with 0.2 M triethanolamine, pH 8.2. The beads were suspended in 40 ml 0.2 M triethanolamine buffer, pH 8.2, containing 0.02 M dimethylpimeli idate. Conjugation proceeded for 45 min at RT on a rotator, then the beads were washed 2X with 0.02 M ethanolamine, pH 8.2, and 3X with 10 ml 0.1 M borate buffer, pH 8.2. Unbound antibody was removed by elution with 0.1 M sodium acetate buffer, pH 4.5. The reaction conjugated 89% of the antibody to the protein A-Sepharose beads.

6. Isolation of recombinant soluble E-selectin

Sol-E-selectin was purified from 2550 ml of the collected tissue culture medium isolated as described above. The medium was eluted through a 0.7 cm x 1.5 cm pre-column of protein A-Sepharose connected in series to a 1.5 cm x 3 cm CY1787-protein A-Sepharose affinity column. The flow rate was 20 l/hr, which allowed efficient binding. Following loading, the columns were disconnected and the CY1787 affinity column was washed with 20 mM Tris buffer, pH 7.5, containing 150 mM NaCl and 2 mM CaCl₂, until the A280 of the eluate approached zero. Bound sol-E-selectin was eluted by gravity flow using 0.1 M sodium acetate buffer, pH 3.5, containing 1 mM CaCl₂. One ml fractions were collected into 3 ml

2 M Tris, pH 10. Protein-containing fractions were pooled and dialyzed against Dulbecco's PBS (DPBS) . The samples were concentrated on an Amicon "CENTRIPREP 30" until the protein concentration was approximately 1 mg/ml as determined by spectrophotometry (Hitachi U-2000; Hitachi Instruments, Inc.; San Jose CA) . Ten mg E-selectin was purified from the 2550 ml cell culture medium. The purified E-selectin was aliquoted and stored at ^"80^° C. Purity was greater than 90% as determined by SDS-PAGE.

EXAMPLE II

PRODUCTION OF HUMANIZED ANTIBODIES TO E-SELECTIN

This example provides methods for preparing cDNA sequences encoding the V_H and V_L chains of mu MAb CY1787 and methods for humanizing mu MAb CY1787.

A. Cloning and Sequencing of Mu MAb CY1787 Variable Regions

The mu MAb CY1787 is produced by a hybridoma cell line designated as ATCC Accession # HB 10591 and was substantially purified from these hybridoma cells using standard methods (see, for example, Harlow and Lane, supra , 1988; see Chapter 8).

Nucleic acid molecules encoding the V_H (SEQ ID NO: 1) and V_L (SEQ ID NO: 3) chains of mu MAB CY1787 were obtained and used as the starting material to produce the humanized E-selectin-specific antibodies of the invention. Hybridoma cells producing mu MAb CY1787

(ATCC Accession # HB 10591) were adapted to growth in serum-free medium. RNA was isolated from the hybridoma cells and the cDNA molecules encoding the V_H and V_L regions of mu MAb CY1787 were obtained by PCR amplification (see Jones and Bendig (Biotechnology 9:88- 89 (1991); Biotechnology 9:579 (1991), each of which is incorporated herein by reference) .

The entire mouse variable region was amplified using a mixture of degenerate 5' primers, which hybridized within the leader sequence, and a single 3' primer, which hybridizes to the mouse constant region near the V-C junction. For the mouse kappa light chain, the degenerate 5'-primers consisted of a pool of 64 oligonucleotides having the general sequence,

5'-ACTAGTCGACATGGG(C/T)ATCAAGATGGAGTCACA(G/T) (A/T) (C/T) (C/T)C(A/T)GG-3' (SEQ ID NO: 23), wherein the nucleotides in parentheses indicate the degenerate sequences, and the 3'-primer (reverse primer) had the sequence, 5'-GGATCCCGGGTGGATGGTGGGAAGATG-3' (SEQ ID

NO: 24). For the mouse heavy chain variable region, the degenerate 5'-primers consisted of a pool of 16 oligonucleotides having the sequence, 5^•-ACTAGTCGACATGGCTGTC(C/T)T(A/G)G(G/C)GC T(A/G)CTCTTCTGC-3' (SEQ ID NO: 25), and the 3'-primer (reverse primer) had the sequence,

5'-GGAACCCGGGAAGGGATAGACAGATGG-3' (SEQ ID NO: 26). Following amplification, the PCR fragments were cloned and sequenced. The nucleotide sequences and corresponding amino acid sequences are shown for mu MAb CY1787 V_H (SEQ ID NOS: 1 and 2) and V_L (SEQ ID NOS: 3 and 4) chains.

B. Modeling of the mu MAb CY1787 Variable Regions and Design of Reshaped mu MAb CY1787 Variable Regions

A molecular model of the V_L and V_H regions of mu MAb CY1787 was generated to aid in the humanization procedures described below. The model was built on a Silicon Graphics IRIS 4D workstation running under the UNIX operating system and using the molecular modeling package QUANTA (Polygen Corp.). The amino acid sequence of the FRs of the mu MAb CY1787 variable regions was compared with the FR sequences of similar, structurally- solved V_H and V_L regions in order to select variable chains having structures that most closely matched the structures present in the mu MAb CY1787 variable regions.

By comparing amino acid sequences as described above, the mouse Dl.3 V_H region FRs (Fischmann et al., J. Biol. Chem. 266:12915 (1991), which is incorporated herein by reference) and the mouse MCPC/603 V_L region FRs (Satow et al., J. Mol. Biol. 190:593 (1987), which is incorporated herein by reference) were identified as the most suitable structurally-solved V_H and V_L regions upon which to base the model of mu MAb CY1787 variable regions. The heavy and light chain complementarity determining regions (CDRs) were grafted onto the above FRs using the canonical structures determined by Chothia (Nature 342:877 (1989), which is incorporated herein by reference) to model the loop structures.

The loops of CDRs 1-3 (L1-L3) of MCPC/603 fit the same canonical subgroups as the L1-L3 loops of mu MAb CY1787 V_L regions. Thus, light chain LI fit canonical subgroup 3 and was modeled on the LI loop of MCPC/603. Light chain L2 fit canonical subgroup 1 and was modeled on the L2 loop of MCPC/603. Light chain L3 fit canonical subgroup 1 and was modeled on the L3 loop of MCPC/603.

Similarly, the CDR1 (Hi) and CDR2 (H2) loops of Dl.3 fit the same canonical subgroups as the Hi and H2 loops of mu MAb CY1787 V_H regions. Thus, heavy chain HI fit canonical subgroup 1 and was modeled on the H2 loop of Dl.3 and heavy chain H2 fit canonical subgroup 1 and was modeled on the Hi loop of Dl.3. No canonical structure exists for CDR3 (H3) loops. Therefore, the H3 loop of the mu MAb CY1787 VH region was modeled on a loop from HFL as described by Sheriff et al. (Proc. Natl. Acad. Sci. , USA 84:8075 (1987), which is incorporated herein by reference), the amino acid sequence of which closely matched that of the H3 loop. The model was adjusted for readily apparent steric clashes, then subjected to energy minimization as implemented in QUANTA.

Based on amino acid sequence comparisons designed to select human FRs that most closely matched those found in the mu MAb CY1787 variable regions, the human MAb FK-001 light chain variable region (Nakatani et al.. Biotechnology 7:805 (1989), which is incorporated herein by reference) and the human MAb NEWM heavy chain region (Poljak et al.. Biochemistry 16:3412 (1977), which is incorporated herein by reference) were selected as FRs for the CY1787 V_L and V_H regions, respectively, in the humanized antibody. The CDRs of mu MAb CY1787 were grafted onto the human FRs to produce a CDR-grafted version of the antibody. The human FRs then were modified as described in Monoclonal Antibodies. 2: Applications in Clinical Oncology (ed. A. Epenetos;

Chapman and Hall, 1992); see therein, Bendig et al.. The Humanization of Mouse Monoclonal Antibodies by CDR Grafting: Examples with Anti-Viral and Anti-Tumor Cell Antibodies, each of which is incorporated herein by reference.

Six versions of the heavy chain variable region, designated CY1788V_HA, CY1788V_HB, CY1788V_HC, CY1788V_HD/ CY1788V_HE and CY1788V_H,, were modeled. The nucleotide and amino acid sequences of these variable regions, including the signal peptides, are shown in SEQ ID NOS: 5 to 16.

The computer model of mu MAb CY1787 indicated that seven changes should be made to the FRs of human NEWM in version CY1788V_HA (SEQ ID NO: 6) of the humanized heavy chain. Residues 26 to 30 of FR1 are part of the structural loop of HI (see Chothia, supra, 1989). Consequently, the mouse residues at these positions, i.e., phenylalanine at position 27 (mu Phe 27), mu Leu 29 and mu Thr 30 were conserved in CY1788V_HA. In addition, certain residues at specific locations within the FRs are important for maintaining the canonical structures of the CDR loops (see Chothia, supra, 1989). Position 71 in FR3, for example, is required for the canonical loop structure of HI. Thus, the Val residue at this position in FR3 of the donor human sequence (human Val 71) was substituted with mu Lys 71. Similarly, position 94 is required to maintain the canonical loop structure of HI. Thus, mu Thr 94 was substituted for human Arg 94 in the NEWM FRs. The computer model of mu MAb CY1787 also identified mu Ile 69, which is located at the edge of the binding site, can be involved in antigen binding. Accordingly, human Met 69 was replaced with mu Ile 69. Also, human Leu 70, which was present in FR3, rarely is found at this position; serine is more commonly found at this position. Since position 70 is buried in the structure and is located between two conserved mouse residues, human Leu 70 was replaced with mu Ser 70 in CY1788V_HA.

The computer model of mu MAb CY1787 indicated that mu Asn 73 is close to and in an appropriate position to hydrogen bound with mu Ser 28, which is an important residue in the structural loop of Hi. If present, a hydrogen bond between mu Ser 28 and mu Asn 73 can be involved in stabilizing the HI loop structure. In comparison, human NEWM contains a Thr 73. Thus, human Thr 73 was replaced with mu Asn 73 in MAb CY1788V_HB (SEQ ID NO: 8). The human Thr 73 also can bond with the mu Ser 28 in the humanized antibody. However, the greater distance between Thr and Ser ( ~ 3.5 A) as compared to Asn and Ser (« 3.0 A) can have two effects - either the distance between the Thr and Ser residues can be too great to allow hydrogen-bond formation, which reduces CDR1 loop support, or the bond can form, but its formation can slightly perturb the orientation of the HI loop. Consequently, CY1788V_HJ, (SEQ ID NO: 8) incorporated a mu Asn 73 substitution into CY1788V_HA (SEQ ID NO: 6).

To determine the effect of mu Ile 69, which is located on the surface of the antibody structure, on the antigen binding affinity of the humanized antibody, CY1788V_HC (SEQ ID NO: 10) was constructed. A surface residue such as mu Ile 69 can have an effect on antigen binding affinity. In addition, surface residues are important because, in a humanized antibody, substitution of a murine surface residue can render the antibody immunogenic. Thus, humanized version CY1788V_HC (SEQ ID

NO: 10), in which human Thr 69 was substituted for mu Ile 69 in CY1788V_Λ (SEQ ID NO: 6), was produced.

Version CY1788V_HD (SEQ ID NO: 12) is identical to CY1788V_HA (SEQ ID NO: 6), except that CY1788V_HD also contains mu Asn 73, as in CY1788V_Hfl(SEQ ID NO: 8) and mu Ile 69, as in CY1788V_HC (SEQ ID NO: 10). CY1788V_ro (SEQ ID NO: 12), in conjunction with CY1788V_HA, CY1788V_HB and CY1788V_HC( allows assessment of the two changes made at positions 68 and 73 in the various humanized antibodies.

CY1788V_HE (SEQ ID NO: 14) contained the same framework changes described in CY1788V_HB (SEQ ID NO: 8) and was further modified as described below. Computer analysis of the mu MAb CY1787 model did not indicate other potential modifications that should be made in the humanized antibody. However, a group of buried murine residues located close to each other or in the vicinity of H2 were selected for conservation in the humanized antibody. Specifically, mu Ile 20, mu Leu 48 and mu Leu 67, which are associated in mu MAb CY1787, were conserved in CY1788V_H-, (SEQ ID NO: 14). In addition, computer modeling suggested that mu Ser 76 could hydrogen bond with mu Val 24 between the main chains. Thus, mu Ser 76 was substituted for human Asn 76 in CY1788V_HE (SEQ ID NO: 14) .

CY1788V_HP (SEQ ID NO: 16) contained the same framework changes described in

(SEQ ID NO: 14) and was further modified as described below. Murine Ser 68 is located on the surface of the variable region near the edge of the binding site, though not as proximal to the binding site as mu Ile 69 (see above). Since the three dimensional shape of the target antigen is not known, it is not certain whether the antigen directly contacts mu Ser 68. However, this possibility cannot be dismissed. Thus, CY δβVg_j. (SEQ ID NO: 16) was constructed to substitute mu Ser 68 for human Thr 68. In addition, mu Val 78, mu Met 82 and Leu 82c, which are buried residues, the latter two in positions distal to the CDRs in CY1787V_H, replaced human Phe 78, human Leu 82 and human Val 82c, respectively, in CY1788V_2J, (SEQ ID NO: 16). These changes were made to minimize the likelihood that the orientation or conformation of the three heavy chain CDR loops would be altered by these residues.

Two versions of the light chain variable region, designated CY1788V_LA (SEQ ID NO: 18) and

(SEQ ID NO: 20) also were constructed based on the model of mu MAb CY1787. After the CDRs of mu MAb CY1787V_L was grafted onto the donor FRs of human FK-001, the model supported only one FR change at position 45 in FR2, which is located at the V_H/V_L interface. The murine MAb CY1787 contained Asn 45, which is relatively rare at this position, whereas the human FK-001 contained Lys 45, which is relatively more common at this position. The importance of this residue was compared by constructing two versions of the light chain, CY1788V_IA (SEQ ID NO: 18), which contained mu Asn 45, and CY1788V_LB (SEQ ID NO: 20), which contained human Lys 45.

C. Subcloning of cDNAs Encoding Heavy and Light Chain Variable Regions Into Expression Vectors

1. Construction of Expression Vectors

pHCMV-V_Llys-KR-neo

The pHCMV-V_Llys-KR-neo expression vector utilizes the human cytomegalovirus (HCMV) enhancer and promoter to drive transcription of the recombinant humanized light chain sequences. This vector also encodes the bacterial neo gene, which is useful as a selectable marker to obtain stable transfectants, and contains the SV40 origin of replication, which allows a high level of transient expression in COS cells.

pHCMV-V_Llys-KR-neo was constructed as described by Maeda et al.. Hum. Antibody Hybrid. 2:124-134 (1991), which is incorporated herein by reference. Briefly, a Hind III/Sac II fragment was obtained from a pUC8 vector containing the Pst I-m fragment of HCMV (Boshart et al., Cell 41:521-530 (1985), which is incorporated herein by reference) and was converted to an Eco RI/Hind III fragment using appropriate adaptor oligonucleotides. In a trimolecular ligation reaction, the 1.2 kb Eco RI/Hind III/Bam HI fragment containing the HCMV enhancer-promoter was ligated to a 5.05 kb Bam HI/Eco RI fragment from pSV2neo (Southern and Berg, J. Mol. App. Genet. 1:327-341 (1982), which is incorporated herein by reference) and a 0.5 kb Hind III/Bam HI fragment containing the V_Llys kappa light variable region (Foote and Winter, J. Mol. Biol. 224:487-499 (1992), which is incorporated herein by reference). The Hind III site in pSV2neo previously was removed by filling-in the recessed overhang with Klenow polymerase. The resultant vector was designated pHCMV- V_Llys-neo.

An approximately 2.6 kb Eco RI fragment of the genomic human kappa region (Rabbitts et al., Curr. Top. Microbiol. Immunol. 113:166-171 (1984), which is incorporated herein by reference) was cloned, then was subcloned into a pUC vector, pUCKRl7 (Mengle-Gaw, EMBO J. 6:1959-1965 (1987), which is incorporated herein by reference) to link the genomic sequence with the V_Llys sequence described above. For subcloning. Bam HI sites were added to both ends of the 2.6 kb fragment using Eco RI/Bam HI adapters. This fragment was inserted into the Bam HI site of pHCMV-V_Llys-neo in the appropriate orientation to produce pHCMV-V_Llys-K_R-neo. To facilitate insertion of reshaped human variable regions, the Bam HI site 3' to the human kappa constant region was removed by filling-in the end with Klenow polymerase. In the resulting vector, designated pHCMV-V_Llys-KR-neo, the Hind III/Bam HI fragment containing the V_Llys kappa region easily can be replaced with the V_L of a reshaped antibody.

pHCMV-Vnlys-ylC-neo

The pHCMV-V_Hlys-γlC-neo vector is similar to the light chain vector described above, except that the HCMV enhancer and promoter drives expression of humanized heavy chain sequences. The construction of this vector began with the three way ligation as described above, except that a 0.7 kb Hind III/Bam HI fragment containing the V_Hlys heavy variable region (Verhoeyen et al.. Science 239:1534-1536 (1988), which is incorporated herein by reference; see, also, Foote and Winter, supra, 1992) was incorporated instead of the 0.5 kb V_Llys kappa light chain fragment. This vector was designated pHCMV-V_Hlys-neo. The cDNA coding for human γl constant region then was inserted into the Bam HI site to produce pHCMV-V_Hlys-γl-neo.

Briefly, cDNA coding for human γl constant region was cloned by PCR amplification from a human cell line secreting a human γl antibody (see Takahashi et al. , Cell 29:671-679 (1982), which is incorporated herein by reference) . Bam HI sites were created at each end of the cDNA and a splice acceptor site and a 65 bp intron sequence were introduced at the 5-end of the cDNA sequence. The Bam HI fragment (1176 bp) containing the human γl cDNA, the splice acceptor site and intron sequence then was cloned into the expression vector. As with the light chain expression vector, the Bam HI site at the 3 '-side of the human γl constant region was removed by filling-in with Klenow polymerase. This vector, designated pHCMV-V_Hlys-γlC-neo, contained a Hind III/Bam HI fragment encoding V_Hlys, which readily can be replaced with the V_H of a reshaped antibody.

2. Insertion of Immunoglobulin Coding Sequences

A cDNA sequence encoding the heavy chain variable region of mu MAb CY1787 or one of the humanized versions of the murine antibody was inserted into the expression vector pHCMV-V_Hlys-γlC-neo by standard methods to produce the expression plasmids pHCMV-1787C_H-γlC-neo, pHCMV-1788V_HA-γlC-neo, pHCMV-1788V_HB-γlC-neo, pHCMV-1788V_HC- γlC-neo, pHCMV-1788V_BD-γlC-neo, pHCMV-1788V_HE-γlC-neo and pHCMV-1788V_HP-γlC-neo. The nucleotide sequence of the human IgGl constant region cDNA and corresponding protein sequence were reported by Ellison et al. (Nucl. Acids Res. _f 10:4071-4079 (1982), which is incorporated herein by reference) . A cDNA sequence encoding the CY1787 V_L or a humanized version of the antibody was inserted into the expression vector pHCMV-KR-neo to generate pHCMV-V_Llys-KR- neo, pHCMV-1788V_IA-KR-neo, and pHCMV-1788V_LB-KR-neo. The nucleotide sequence of the human kappa constant region gene and corresponding protein sequence was reported by Hieter et al. (Cell 22:197-207 (1980), which is incorporated herein by reference) .

By coexpressing appropriate combinations of heavy and light chains, various different functional antibodies were expressed and evaluated for binding activity. For example, coexpression of pHCMV-1788V_HA-γlC- neo and pHCMV-1788V_LA-KR-neo produced reshaped MAb CY1788 (^V _HA/^V _LA) • Coexpression of pHCMV-1787C_H-γlC-neo and pHCMV- 1787C_L-KR-neo gave rise to the chimeric MAb CY1787, which contained murine variable domains and human constant domains.

D. Expression of Humanized Antibodies

The nucleic acid molecules encoding the various antibody constructs were transfected into COS cells in order to transiently express a chimeric (mouse/human) MAb CY1787 or a humanized version of MAb CY1787 (designated "CY1788"). DNA was introduced into the COS cells by electroporation using the Gene Pulser apparatus (Biorad; Hercules CA) . COS cells were trypsinized and washed once in PBS.

Ten μg of a heavy chain plasmid and light chain plasmid and a 0.8 ml aliquot of 1 x 10⁷ cells/ ml in PBS were placed in a sterile Gene Pulser Cuvette (Biorad; 0.4 cm gap). A pulse was delivered at 1900 volts,

25 microfarads capacitance. The cells were allowed to recover for 10 min at RT, then were added to 20 ml DMEM containing 10% FBS. The cells were incubated for 48 hr. then the medium was collected, centrifuged to remove cellular debris, and stored under sterile conditions at 4°C. Medium that was not used within about two weeks was discarded.

EXAMPLE III

This example demonstrate that the recombinant humanized antibodies of the invention are functional.

A. Analysis of Humanized Antibodies

The various media collected from the transfected COS cells were assayed by ELISA to quantify levels of human antibody produced. The media also were analyzed to determine the capacity of an antibody to specifically bind recombinant soluble E-selectin (sol-E-selectin).

1. Quantification of antibody production

The wells of Immulon microtiter plates (Dynatech; Chantilly VA) were coated with 100 μl goat anti-human IgG (whole molecule; Sigma) diluted to a final concentration of 12.5 μg/ml in DPBS. Plates were coated overnight at 4°C or for 2 hr at 37°C. The coated plates were washed 3X with DPBS, blocked with 400 μl DPBS containing 1% bovine serum albumin (BSA; Sigma) for at least 1 hr at RT, then washed 3X with DPBS.

The monoclonal antibodies were serially diluted in DPBS containing 1% BSA (DPBS/BSA) and 100 μl was added to each well. The plates were incubated for 1 hr at RT, then washed 3X with DPBS. 100 μl second antibody (goat anti-human IgG peroxidase conjugate, diluted 1:1000 in DPBS/BSA) was added to each well and incubation was continued for 1 hr at RT. The plates were washed 3X with DPBS, then 100 μl tetramethylbenzidine peroxidase substrate/H₂0₂ (TMB; Kirkegaard and Perry Laboratories; Gaithersburg MD) was added to each well. The color was allowed to develop for about 3 to 15 min, then the reaction was quenched by adding 100 μl 1 M phosphoric acid to each well and the absorbance at 450 nm of each well was determined using a Titertek Multiscan MCC/340 (LABSYSTEMS; Basingstoke UK). The concentration of antibodies produced in each COS cell supernatant was determined by comparing the IgG ELISA results to a known amount of a human IgGl antibody (kappa light chain; Sigma) or a human IgG4 antibody (kappa light chain; Sigma) .

2. Antibody binding specificity

The wells of an Immulon-1 microtiter plate were coated with 50 μl sol-E-selectin (4 /g/ml) for 2 hr at RT. The wells were washed 3X with PBS, then blocked for 20 min at RT with 200 μl DPBS/BSA and washed 3X with PBS. Fifty μl of a CY1788 humanized antibody, serially diluted in DPBS/BSA, was added to each well and the plate was incubated for 30 min at RT, then washed 4X with DPBS/BSA. Fifty μl goat-anti human-horseradish peroxidase conjugate (1:1000; Sigma) was added to each well and the plate was incubated for 30 min at RT, then washed 3X with DPBS/BSA. 100 μl HRP substrate (24 mM citrate, 50 mM NaP0₄, 0.5 mg/ml O-phenylenediamine, 0.012 % H₂0₂) was added to each well and allowed to develop for approximately 6 min, then the reaction was quenched with 25 μl 4 N H₂S0₄ and the absorbance at 490 nm was determined for each well.

Sol-E-selectin binding was plotted as optical density against the concentration of IgG. The relative sol-E-selectin binding efficiency of reshaped MAb CY1788V_IA/V_HA was slightly greater than the binding of CY1788V_LB/V_HA. Based on these results, the versions of CY1788 used in later investigations used the CY1788V_1A (SEQ ID NO: 18) light chain variable region expressed from pHCMV-1788V_LA-KR-neo.

The heavy chain variable regions CY1788V_HC,

CY1788V_HO and CYπβδV∞ (SEQ ID NOS: 10, 12 and 14), when coexpressed with light chain variable region CY1788V_1A (SEQ ID NO: 18) generated antibodies CY1788(V_HC/V_IA) , CY1788(V_HD/V_1A), and CY1788(V_HE/V_LA), respectively. These antibodies bound sol-E-selectin less efficiently than antibodies generated using the CY1788V_IA in combination with CY1788V_HA (SEQ ID NO: 6), CY1788V_HB (SEQ ID NO: 8) or CY1788V_HT (SEQ ID NO: 16). Therefore, CY1788(V_B/V_U) , CY1788(V_HB/V_IA) and CY1788(V„/V_u) were stably transfected into CHO cells and generated sufficient quantities of purified antibody for analysis.

EXAMPLE IV

STABLE EXPRESSION of CY1788 MAbs in CHO CELLS

This example demonstrates that stably transfected CHO cells express high levels of humanized antibodies to E-selectin and provides a characterization of the binding characteristics of the recombinant humanized antibodies.

A. Introduction of Humanized Immunoglobulin Chains in CHO cells

1. Vector Constructions

pHCMV-V_πlys-vlC-dhfr

pHCMV-V_Hlys-γlC-dhfr is functionally identical to pHCMV-V_Hlys-γlC-neo (see Example III), except that the neo gene is replaced by a sequence encoding dihydrofolate reductase (dhfr) , which is useful as a selection marker for isolation of stable tranfectants and for amplification of the transfected sequence, which increases the gene copy number. The SV40 enhancer sequence was removed from the plasmid pSV2-dhfr (Subramani et al., Mol. Cell. Biol. 1:854-864 (1981), which is incorporated herein by reference) by digesting the plasmid with Sph I and Pvu II, filling-in the overhanging ends with Klenow polymerase and ligating the plasmid to yield pSV2-dhfr-ΔE.

An approximately 3.7 kb Eco RI fragment containing the HCMV promoter, the V_Hlys heavy chain variable region and a genomic DNA clone of the human γl constant region was excised from pHCMV-V_Hlys-γl-neo using Eco RI. This fragment was ligated to Eco Rl-digested pSV2-dhfr-ΔE to create pHCMV-V_Hlys-γl-dhfr. The genomic clone of the human γl constant region was replaced with a Bam HI fragment containing the cDNA clone of the human γl constant region (see Example III). The Bam HI site on the 3'-side of the human γl constant region was removed by filling-in the recessed overhang with Klenow polymerase to produce pHCMV-V_Hlys-γlC-dhfr. The Hind III/Bam HI fragment of this vector, which encodes the V_hlys sequence, readily can be replaced with the V_H of a reshaped antibody.

pHCMV-V_πlvs-v4-dhfr

pHCMV-V_Hlys-γ4-dhfr is identical to pHCMV-V_Hlys- γlC-dhfr as described above, except that the cDNA clone of the human γl constant region is replaced with a genomic clone of the human γ4 constant region. An approximately 7.0 kb Hind III fragment of DNA containing the genomic clone of the human γ4 constant region (Bruggeman et al., J. Exp Med. 166:1351-1361 (1987), which is incorporated herein by reference) was subcloned into the Hind III site of pUC19 to produce plasmid 428D. The human γ4 fragment contained a Bam HI site near its 3*-end and was inserted into pUCl9 in the orientation that placed this site distal to the Bam HI site in the polylinker of pUC19. The 7.0 kb human γ4 fragment was excised from plasmid 428D using Bam HI and ligated into pHCMV-V_Hlys-neo to produce pHCMV-V_Hlys-γ4-neo. The final plasmid was produced by a three-way ligation of a 5.4 kb Bam HI/Hind III fragment containing the HCMV enhancer- promoter linked to the pSV2-dhfr-ΔE plasmid sequence, a 0.5 kb Hind III/Bam HI fragment containing the V_H of mouse CY1787 or the reshaped human CY1788 antibody and the 7.0 kb Bam HI/Bam HI fragment containing the genomic clone of the human γ4 constant region.

Insertion of immunoglobulin coding sequences

Each of the cDNA sequences encoding the CY1788V_HA (SEQ ID NO: 6), CY1788V_HB (SEQ ID NO: 8) or CY1788V_HJ, (SEQ ID NO: 16) heavy chain variable regions was subcloned into pHCMV-γlC-dhfr to generate pHCMV-1788V_ω- γlC-dhfr, pHCMV-1788V_HB-γlC-dhfr and pHCMV-1788V_HF-γlC- dhfr. The cDNA encoding CY1788V_BJ. also was subcloned into pHCMV-γ4C-dhfr to generate pHCMV-1788V_HF-γ4C-dhfr. The nucleotide sequence of the human IgG4 constant region gene and corresponding amino acid sequence has been reported by Ellison et al., DNA. 1:11-18 (1981), which is incorporated herein by reference. Expression from pHCMV-γlC-dhfr produces an IgGl isotype antibody and vector pHCMV-γ4C-dhfr produces an IgG4 isotype antibody when coexpressed with the kappa light chain vector.

To generate stable cell lines co-expressing both the heavy and light chain sequences, CHO dhfr- deficient cells were grown in MEM (+ nucleosides; GIBCO/BRL) and 10% FBS. Plasmid DNA was introduced into the cells by electroporation as described above (see

Example II). Following electroporation, the cells were allowed to recover for 10 min at RT, then were added to 20 ml αMEM (+ nucleosides) /10% FBS and incubated for 24 to 48 hr.

Following incubation, the cells were trypsinized and plated into 100 mm dishes in αMEM (minus nucleosides) containing 10% dialyzed FBS to select for the expression of the dhfr-containing plasmid. The medium also was supplemented with 500 μg G418/ml (GIBCO/BRL) to select for the expression of the neo-containing plasmid. The medium was changed every 3 to 4 days until colonies appeared. Single colonies then were isolated using cloning cylinders and the cell populations were expanded and analyzed by ELISA for IgG production.

Four sets of heavy and light chain expression vectors were introduced into the CHO dhfr-deficient cells as follows: 1) pHCMV-1788V_HA-γlC-dhfr and pHCMV- 1788V_LA-KR-neo, expressing CY1788 (V-^/V^; IgGl isotype); 2) pHCMV-1788V_HB-γlC-dhfr and pHCMV-1788V_LA-KR-neo, expressing CY1788 (V^/V^; IgGl isotype); 3) PHCMV-1788V_H,,- γlC-dhfr and pHCMV-1788V_LA-KR-neo, expressing CY1788 (V_HF/V^; IgGl isotype); and 4) pHCMV-1788V_HF-γ4C-dhfr and pHCMV-1788V_LA-KR-neo, expressing CY1788 (V^/V^ IgG4 isotype.

Cell lines secreting the MAbs CY1788(V_ffi/V_u) and CY1788(V_HB/V_IA) were subjected to two rounds of methotrexate-induced dhfr-gene amplification. Cells were plated at a density of 1 x 10⁵ cells/100 mm dish in selection medium. For the first round of amplification, cells were plated in three sets of dishes containing αMEM (+ nucleosides)/10% dialyzed FBS supplemented with 500 μg/ml G418 and 10, 20 or 50 nM methotrexate (Sigma). Cultures were fed every four days. After 10 to 14 days, single colonies were isolated by cloning cylinders. expanded, and assayed for IgG production by ELISA. Additional rounds of amplification were performed on single clones or pools of clones at 5 to 10 times the initial methotrexate concentration.

To assess antibody production from the amplified lines, CHO cells were expanded and seeded into a 10-chamber (6000 cm² total surface area) Nunc "CELL FACTORY." After 72 hr incubation, the medium was harvested and passed over a protein A-Sepharose "FAST FLOW" column (Pharmacia; Piscataway NJ) . The column was washed and bovine IgG was eluted at pH 4.5. The humanized antibodies were eluted at pH 3.5 and dialyzed in PBS or in 20 mM acetate, 0.15 M NaCl, pH 5.5. Antibody concentration was determined using spectrophotometry and confirmed by an ELISA for human IgG. Purified human IgG4 antibody (kappa light chain, Sigma ) was used as the control for CY1788 (H_P/L_A) IgG4 isotype. Purity was confirmed by SDS-PAGE.

B. Competitive Binding Assay

The relative binding affinities for E-selectin of the various versions of CY1788 were determined by competitive ELISA assay. The concentration of unlabeled antibody that inhibited the binding of labeled mu MAb CY1787 to sol-E-selectin by 50% was determined.

Two ml purified mu MAb CY1787 (1 mg/ml) was mixed with 100.8 μl NHS-LC-biotin (1 mg/ml; Pierce) and incubated overnight at 4°C to biotinylate the antibody. Following incubation, the sample was loaded onto a NAP 25 size exclusion column (Pharmacia) . The column was eluted with PBS and 0.3 ml fractions were collected. The A280 was determined for each fraction and those containing the highest amounts of protein were pooled. The biotinylated-CYl787 was titered against sol-E-selectin to determine an appropriate concentration (approximately 0.67 mg/ml) for use in the competitive binding assays.

For titering the labeled antibodies, each well of a microtiter plate was coated with 50 μl sol-E-selectin (4 μg/ml) and incubated for 2 hr at RT. The plate was washed 3X with PBS, blocked with 200 μl DPBS/BSA/well for 20 min at RT, then washed 3X with PBS. Fifty μl unlabeled CY1787 or unlabeled CY1788 sample (serially diluted in DPBS/BSA) and 50 μl 1:500 dilution of biotinylated-CY1787 (undiluted stock was approximately 0.67 mg/ml) were added to each well and the plate was incubated for 30 min at RT. Following incubation, the plate was washed 4 times with DPBS/BSA, then 50 μl streptavidin horseradish peroxidase conjugate (1:1000 dilution in DPBS/BSA; Sigma) was added to each well and the plate was incubated for 30 min at RT. The plate was washed 3X with DPBS/BSA, then 100 μl HRP substrate (24 mM citrate, 50 mM NaP0₄, 0.5 mg/ml O-phenylenediamine, 0.012 % H₂0₂) was added to each well and the reaction was allowed to develop for approximately 6 min. The reaction was quenched with 25 μl 4 N H₂S0₄ and the absorbance at 492 nm was determined. Alternatively 50 μl HRP substrate tetramethylbenzidine (Kirkegaard and Perry Laboratories) was added to each well and allowed to develop for approximately 6 min, then the reaction was quenched with 50 μl 1 M phosphoric acid and the absorbance at 450 nm was determined.

Apparent binding affinity was expressed as the concentration of antibody that bound to sol-E-selectin at one-half maximum optical density. The ratio of concentrations at one-half maximum binding of the sample antibody to that of the control (unlabeled CY1787) antibody provided an estimate of the relative binding efficiency of a test antibody to sol-E-selectin. mu MAb CY1787 at a concentration of 2.5 μg/ml and CY1788(V_HF/V_IA)- IgG4 at a concentration of 3.85 μg/ml inhibited the binding of labeled CY1787 to 50% of maximum. These results demonstrate that the humanized CYl788 (V_HF/V_LA) -IgG4 antibody bound sol-E-selectin about 65% as well as CY1787. In addition, CYl788 (V_HF/V_LA) -IgG4 , CY1788 (V_HB/V_LA) - IgGl , CY1788 (V_HF/V_LA) -IgGl and CYl788 (V_HA/V_LA) -IgGl bound sol-E-selectin 65% , 22% , 8% and 1 .6% , respectively, as well as CY1787 . CY1788 (V_HF/V_IA) -IgG4 was selected for further studies .

EXAMPLE V

CYl788(V_πr/V_IJ -IαG4 INHIBITS HUMAN NEUTROPHIL ADHESION TO ACTIVATED HUVECS

This example demonstrates that the humanized anti-E-selectin antibody CY1788(V^/V^)-IgG4 blocks neutrophil adhesion to activated endothelial cells.

Neutrophil adhesion to activated HUVECs was determined in the presence or absence of the humanized E- selectin antibodies of the invention to assess the inhibitory activity of the humanized antibodies. 5 x 10⁴ HUVECs were plated into each well of a gelatin coated 96 well plate and were grown to confluence. Medium was changed one time following plating. E-selectin expression was induced by adding fresh medium containing 30 μg/ml recombinant IL-lβ (rIL-lβ; 2.5 x 10⁵ U/ml stock; Genzyme; Boston MA) and incubating the cells for 4 hr to obtain activated endothelial cells. Control wells received only fresh medium.

Following incubation, the adherent activated or control endothelial cells were washed 2X with HBSS/

HEPES/glucose buffer containing 5 mg/ml BSA, then 100 μl of the titrated CY1787 or CY1788(V_HF/V_LA)-IgG4 was added and the plates were incubated for 20 min. 2 x 10⁵ neutrophils in 50 μl were added to the wells and the plates were incubated for 6 min at RT. Non-adherent cells were removed from the wells by inverting the plates and washing them 4X with 200 μl medium. Following removal of the last wash, 50 μl solubilization buffer (24.3 ml 0.1 M citric acid, 25.7 ml 0.2 M dibasic sodium phosphate, 50 ml 0.2% NP-40) was added to each well and the plate was incubated with gentle mixing for 10 min. 50 μl OPDA (8 mg o-phenylenediamine, 8 μl 30% H₂0₂, 10 ml water) was added to each well to determine myeloperoxidase activity, the plates were incubated for 15 min at RT, then 15 μl 4 N H₂S0₄ was added to stop the reaction. A reagent blank was prepared by mixing 50 μl solubilization buffer, 50 μl OPDA and 15 μl 4 N H₂S0₄. 100 μl supernatant was removed from each well and transferred to a flexible ELISA assay plate (Falcon) and the plate was scanned at 492 nm within 30 min.

CY1788(V_HF/V_LA)-IgG4 inhibited the adhesion of human neutrophils to rIL-lβ activated HUVECs as effectively as did the parental mu MAb CY1787. The average optical density (OD) measured at 492 nm of 46 control wells incubated without a primary antibody but incubated with the secondary antibody was 2.373 ± 0.159. mu MAb CY1787 at a concentration of 0.25 μg/ml and CYl788(V_HP/V_IA)-IgG4 at a concentration of 0.25 μg/ml inhibited the binding of neutrophils to 50% of maximum, demonstrating that humanized CYl788(V_HF/V_LA)-IgG4 binds E-selectin as efficiently as mu MAb CY1787.

Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the claims. SEQUENCE LISTING

) GENERAL INFORMATION:

(I ) APPLICANT : CYTEL CORPORATION (II ) TITLE OF INVENTION : HUMANIZED ANTIBODIES TO E-SELECTIN

(iii) NUMBER OF SEQUENCES: 26

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Campbell & Flores LLP

(B) STREET: 4370 La Jolla Village Drive, Suite 700

(C) CITY: San Diego

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 92122

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 06 June 1996

(C) CLASSIFICATION:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Campbell, Cat ryn A.

(B) REGISTRATION NUMBER: 31,815

(C) REFERENCE/DOCKET NUMBER: FP-CY 2116

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (619) 535-9001

(B) TELEFAX: (619) 535-8949

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 427 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 16..415

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

AAGCTTGCCG CCACC ATG GCT GTC CTG GGG CTG CTC TTC TGC CTG GTG ACA 51

Met Ala Val Leu Gly Leu Leu Phe Cys Leu Val Thr 1 5 10

TTC CCA AGC TGT GTC CTA TCC CAG GTG CAG CTG GAG CAG TCA GGA CCT 99 Phe Pro Ser Cys Val Leu Ser Gin Val Gin Leu Glu Gin ser Gly pro 15 20 25

GGC CTA GTG CAG CCC TCA CAG AGC CTG TCC ATC ACC TGC ACA GTC TCT 147 Gly Leu Val Gin Pro ser Gin ser Leu Ser Ile Thr Cys Thr Val Ser 30 35 40

GGT TTC TCA TTA ACT AGC TAT GGT GTA CAC TGG GTT CGC CAG TCT CCA 195 Gly Phe Ser Leu Thr ser Tyr Gly Val His Trp Val Arg Gin Ser Pro 45 50 55 60

GGA GAG GGT CTG GAG TGG CTG GGA GTG ATA TGG AGT GAT GGA AGC ACA 243 Gly Glu Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Asp Gly Ser Thr 65 70 75

GAC TAT AAT GCA GCT TTC ATA TCC AGA CTG AGC ATC AGC AAG GAC AAT 291 Asp Tyr Asn Ala Ala Phe Ile Ser Arg Leu ser Ile Ser Lys Asp Asn 80 85 90

TCC AAG AGC CAA GTT TTC TTG AGA ATG AAC AGT CTG CAA GCT AAT GAC 339 Ser Lys ser Gin Val Phe Leu Arg Met Asn ser Leu Gin Ala Asn Asp 95 100 105

TCA GCC ATA TAT TAC TGT GCC ACC CTA TTA CTA CGG CTG GGG TGG GGC 387 Ser Ala Ile Tyr Tyr Cys Ala Thr Leu Leu Leu Arg Leu Gly Trp Gly 110 115 120

CAA GGG ACT CTG GTC ACT GTC TCT GCA G GTGAGTGGAT CC 427

Gin Gly Thr Leu Val Thr Val Ser Ala 125 130 (2) INFORMATION FOR SEQ ID Nθ:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 133 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:2:

Met Ala Val Leu Gly Leu Leu Phe Cys Leu Val Thr Phe Pro Ser Cys 1 5 10 15

Val Leu Ser Gin Val Gin Leu Glu Gin ser Gly Pro Gly Leu Val Gin 20 25 30

Pro Ser Gin Ser Leu ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45

Thr ser Tyr Gly Val His Trp Val Arg Gin Ser Pro Gly Glu Gly Leu 50 55 60

Glu Trp Leu Gly Val lie Trp Ser Asp Gly Ser Thr Asp Tyr Asn Ala 65 70 75 80

Ala Phe Ile Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gin 85 90 95

Val Phe Leu Arg Met Asn Ser Leu Gin Ala Asn Asp Ser Ala Ile Tyr 100 105 110

Tyr Cys Ala Thr Leu Leu Leu Arg Leu Gly Trp Gly Gin Gly Thr Leu

115 120 125

Val Thr Val Ser Ala 130

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 439 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 16..427

( i) SEQUENCE DESCRIPTION: SEQ ID NO:3:

AAGCTTGCCG CCACC ATG GGC ATC AAG ATG GAG TCA CAG ACT CAG GTC CTC 51 Met Gly Ile Lys Met Glu Ser Gin Thr Gin Val Leu 1 5 10

ATG TCC CTG CTG TTC TGG GTA TCT GGT ACC TGT GGG GAC ATT GTG ATG 99 Met Ser Leu Leu Phe Trp Val ser Gly Thr Cys Gly Asp Ile Val Met 15 20 25

ACA CAG TCT CCA TCC TCC CTG ACT GTG ACA GCA GGA GAG AAG GTC ACT 147 Thr Gin Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr 30 35 40

ATG AGC TGC AAG TCC AGT CAG AGT CTG TTA CAC AGT GGA AAT CAA AAG 195 Met ser Cys Lys Ser Ser Gin ser Leu Leu His Ser Gly Asn Gin Lys 45 50 55 60

AAC TAC TTG ACC TGG TAC CAG CAG AAA CCA GGG CAG CCT CCT AAT CTG 243 Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Asn Leu 65 70 75

TTG ATC TAC TGG GCA TCC ACT AGG GAA TCT GGG GTC CCT GAT CGC TTC 291 Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe 80 85 90

ACA GGC AGT GGA TCT GGA ACA GAT TTC ACT CTC ACC ATC AGC AGT CTG 339 Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 95 100 105

CAG GCT GAA GAC CTG GCA GTT TAT TAC TGT CAG AAT GAT TAT AGT TAT . 387 Gin Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gin Asn Asp Tyr ser Tyr 110 115 120

CCG CTC ACG TTC GGT GCT GGG ACC AAG GTG GAG CTG AAA C GTGAGTGGAT 437 Pro Leu Thr Phe Gly Ala Gly Thr Lys Val Glu Leu Lys 125 130 135

CC 439 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 137 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Gly Ile Lys Met Glu Ser Gin Thr Gin Val Leu Met Ser Leu Leu 1 5 10 15

Phe Trp Val Ser Gly Thr Cys Gly Asp Ile Val Met Thr Gin Ser Pro 20 25 30

Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys 35 40 45

Ser ser Gin Ser Leu Leu His Ser Gly Asn Gin Lys Asn Tyr Leu Thr 50 55 60

Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Asn Leu Leu Ile Tyr Trp 65 70 75 80

Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly 85 90 95

Ser Gly Thr Asp Phe Thr Leu Thr Ile ser Ser Leu Gin Ala Glu Asp 100 105 110

Leu Ala Val Tyr Tyr Cys Gin Asn Asp Tyr Ser Tyr Pro Leu Thr Phe

115 120 125

Gly Ala Gly Thr Lys Val Glu Leu Lys 130 135

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 423 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 12..411

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

AAGCTTCCAC C ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCA ACA 50

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr 1 5 10

GCT ACA GGT GTC CAC TCC CAG GTC CAA CTT GAG CAG AGC GGT CCA GGT 98 Ala Thr Gly Val His ser Gin Val Gin Leu Glu Gin Ser Gly Pro Gly 15 20 25

CTT GTG AGA CCT AGC CAG ACC CTG AGC CTG ACC TGC ACC GTG TCT GGA 146 Leu Val Arg Pro Ser Gin Thr Leu Ser Leu Thr cys Thr Val Ser Gly 30 35 40 45

TTC TCA CTC ACT AGT TAT GGT GTG CAC TGG GTT CGG CAG CCA CCT GGA 194 Phe Ser Leu Thr Ser Tyr Gly Val His Trp Val Arg Gin Pro Pro Gly 50 55 60

CGA GGT CTT GAG TGG ATT GGA GTA ATT TGG AGT GAT GGA AGT ACA GAC 242 Arg Gly Leu Glu Trp le Gly Val Ile Trp Ser Asp Gly ser Thr Asp 65 70 75

TAT AAT GCA GCG TTC ATA TCC AGA GTG ACA ATC TCC AAA GAC ACC AGC 290 Tyr Asn Ala Ala Phe Ile Ser Arg Val Thr Ile Ser Lys Asp Thr Ser 80 85 90

AAG AAC CAG TTC AGC CTG AGA CTC AGC AGC GTG ACA GCC GCC GAC ACC 338

Lys Asn Gin Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr 95 100 105

GCG GTT TAT TAT TGT GCA ACA TTA TTA CTA CGG CTC GGG TGG GGT CAA 386 Ala Val Tyr Tyr Cys Ala Thr Leu Leu Leu Arg Leu Gly Trp Gly Gin 110 115 120 125

GGC AGC CTC GTC ACA GTC TCC TCA G GTGAGTGGAT CC 423

Gly Ser Leu Val Thr val Ser Ser 130 (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 133 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

( i) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Met Gly Trp Ser cys Ile He Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15

Val His ser Gin Val Gin Leu Glu Gin Ser Gly Pro Gly Leu Val Arg 20 25 30

Pro Ser Gin Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45

Thr Ser Tyr Gly Val His Trp Val Arg Gin Pro Pro Gly Arg Gly Leu 50 55 60

Glu Trp Ile Gly Val Ile Trp Ser Asp Gly ser Thr Asp Tyr Asn Ala 65 70 75 80

Ala Phe Ile Ser Arg Val Thr Ile ser Lys Asp Thr Ser Lys Asn Gin 85 90 95

Phe Ser Leu Arg Leu Ser ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110

Tyr Cys Ala Thr Leu Leu Leu Arg Leu Gly Trp Gly Gin Gly Ser Leu

115 120 125

Val Thr Val Ser Ser 130

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 423 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 12..411

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

AAGCTTCCAC C ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCA ACA 50 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr 1 5 10

CGA GGT CTT GAG TGG ATT GGA GTA ATT TGG AGT GAT GGA AGT ACA GAC 242 Arg Gly Leu Glu Trp Ile Gly Val Ile Trp Ser Asp Gly ser Thr Asp 65 70 75

TAT AAT GCA GCG TTC ATA TCC AGA GTG ACA ATC TCC AAA GAC AAC AGC 290 Tyr Asn Ala Ala Phe Ile Ser Arg Val Thr Ile ser Lys Asp Asn Ser 80 85 90

AAG AAC CAG TTC AGC CTG AGA CTC AGC AGC GTG ACA GCC GCC GAC ACC 338 Lys Asn Gin Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr 95 100 105

GGC AGC CTC GTC ACA GTC TCC TCA G GTGAGTGGAT CC 423

Gly Ser Leu Val Thr Val Ser Ser 130 (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 133 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Met Gly Trp ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15

Val His Ser Gin Val Gin Leu Glu Gin Ser Gly Pro Gly Leu Val Arg 20 25 30

Pro Ser Gin Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe ser Leu 35 40 45

Thr Ser Tyr Gly Val His Trp Val Arg Gin Pro Pro Gly Arg Gly Leu 50 55 60

Glu Trp Ile Gly Val Ile Trp Ser Asp Gly Ser Thr Asp Tyr Asn Ala 65 70 75 80

Ala Phe Ile Ser Arg Val Thr Ile ser Lys Asp Asn Ser Lys Asn Gin 85 90 95

Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110

Tyr Cys Ala Thr Leu Leu Leu Arg Leu Gly Trp Gly Gin Gly Ser Leu 115 120 125

Val Thr Val Ser Ser 130

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 423 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 12.. 11

( i) SEQUENCE DESCRIPTION: SEQ ID NO:9:

CGA GGT CTT GAG TGG ATT GGA GTA ATT TGG AGT GAT GGA AGT ACA GAC 242 Arg Gly Leu Glu Trp lie Gly Val Ile Trp Ser Asp Gly Ser Thr Asp 65 70 75

TAT AAT GCA GCG TTC ATA TCC AGA GTG ACA ATG TCC AAA GAC ACC AGC 290 Tyr Asn Ala Ala Phe Ile Ser Arg Val Thr Met Ser Lys Asp Thr Ser 80 85 90

GGC AGC CTC GTC ACA GTC TCC TCA G GTGAGTGGAT CC 423

Gly Ser Leu Val Thr Val Ser Ser 130 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 133 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15

Val His ser Gin Val Gin Leu Glu Gin Ser Gly Pro Gly Leu Val Arg 20 25 30

Pro ser Gin Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45

Thr Ser Tyr Gly Val His Trp Val Arg Gin Pro Pro Gly Arg Gly Leu 50 55 60

Glu Trp Ile Gly Val Ile Trp Ser Asp Gly Ser Thr Asp Tyr Asn Ala 65 70 75 80

Ala Phe lie Ser Arg Val Thr Met Ser Lys Asp Thr Ser Lys Asn Gin 85 90 95

Phe ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110

Tyr Cys Ala Thr Leu Leu Leu Arg Leu Gly Trp Gly Gin Gly Ser Leu 115 120 125

Val Thr Val Ser Ser 130

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 423 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 12..411

( i) SEQUENCE DESCRIPTION: SEQ ID NO:11:

AAGCTTCCAC C ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCA ACA 50

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr 1 5 10

GCT ACA GGT GTC CAC TCC CAG GTC CAA CTT GAG CAG AGC GGT CCA GGT 98

Ala Thr Gly Val His ser Gin Val Gin Leu Glu Gin Ser Gly Pro Gly 15 20 25

CGA GGT CTT GAG TGG ATT GGA GTA ATT TGG AGT GAT GGA AGT ACA GAC 42 Arg Gly Leu Glu Trp Ile Gly Val Ile Trp Ser Asp Gly ser Thr Asp 65 70 75

TAT AAT GCA GCG TTC ATA TCC AGA GTG ACA ATG TCC AAA GAC AAC AGC 290 Tyr Asn Ala Ala Phe Ile Ser Arg Val Thr Met Ser Lys Asp Asn Ser 80 85 90

GGC AGC CTC GTC ACA GTC TCC TCA G GTGAGTGGAT CC 423

Gly ser Leu Val Thr val ser Ser 130 64

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 133 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Met Gly Trp Ser Cys lie Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15

Val His Ser Gin Val Gin Leu Glu Gin Ser Gly Pro Gly Leu Val Arg 20 25 30

Pro Ser Gin Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45

Thr ser Tyr Gly Val His Trp Val Arg Gin Pro Pro Gly Arg Gly Leu 50 55 60

Glu Trp Ile Gly Val Ile Trp Ser Asp Gly Ser Thr Asp Tyr Asn Ala 65 70 75 80

Ala Phe Ile Ser Arg Val Thr Met Ser Lys Asp Asn Ser Lys Asn Gin 85 90 95

Phe ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110

Tyr cys Ala Thr Leu Leu Leu Arg Leu Gly Trp Gly Gin Gly Ser Leu 115 120 125

Val Thr Val Ser ser 130

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 423 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 12..411

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

AAGCTTCCAC C ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCA ACA 50

Met Gly Trp Ser cys Ile Ile Leu Phe Leu Val Ala Thr 1 5 10

CTT GTG AGA CCT AGC CAG ACC CTG AGC ATC ACC TGC ACC GTG TCT GGA 146 Leu Val Arg Pro Ser Gin Thr Leu Ser Ile Thr cys Thr Val Ser Gly 30 35 40 45

CGA GGT CTT GAG TGG CTG GGA GTA ATT TGG AGT GAT GGA AGT ACA GAC 242 Arg Gly Leu Glu Trp Leu Gly Val Ile Trp ser Asp Gly ser Thr Asp 65 70 75

TAT AAT GCA GCG TTC ATA TCC AGA CTG ACA ATC TCC AAA GAC AAC AGC 290 Tyr Asn Ala Ala Phe Ile Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser 80 85 90

AAG AGC CAG TTC AGC CTG AGA CTC AGC AGC GTG ACA GCC GCC GAC ACC 338 Lys Ser Gin Phe ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr 95 100 105

GGC AGC CTC GTC ACA GTC TCC TCA G GTGAGTGGAT CC 423

Gly Ser Leu Val Thr Val Ser Ser 130 (2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 133 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

( i) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Met Gly Trp Ser Cys lie Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15

Val His ser Gin Val Gin Leu Glu Gin Ser Gly pro Gly Leu Val Arg 20 25 30

Pro ser Gin Thr Leu Ser lie Thr Cys Thr Val ser Gly Phe Ser Leu 35 40 45

Thr ser Tyr Gly Val His Trp Val Arg Gin Pro Pro Gly Arg Gly Leu 50 55 60

Glu Trp Leu Gly Val Ile Trp Ser Asp Gly Ser Thr Asp Tyr Asn Ala 65 70 75 80

Ala Phe le Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gin 85 90 95

Phe Ser Leu Arg Leu ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110

Tyr Cys Ala Thr Leu Leu Leu Arg Leu Gly Trp Gly Gin Gly Ser Leu 115 120 125

Val Thr Val Ser Ser 130

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 423 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 12..411

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

TAT AAT GCA GCG TTC ATA TCC AGA CTG AGC ATC TCC AAA GAC AAC AGC 290 Tyr Asn Ala Ala Phe Ile Ser Arg Leu Ser Ile ser Lys Asp Asn Ser 80 85 90

AAG AGC CAG GTT AGC CTG AGA ATG AGC AGC CTG ACA GCC GCC GAC ACC 338 Lys Ser Gin Val Ser Leu Arg Met ser Ser Leu Thr Ala Ala Asp Thr 95 100 105

GGC AGC CTC GTC ACA GTC TCC TCA G GTGAGTGGAT CC 423

Gly Ser Leu Val Thr Val Ser Ser 130 (2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 133 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15

Val His ser Gin Val Gin Leu Glu Gin Ser Gly Pro Gly Leu Val Arg 20 25 30

Pro ser Gin Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu 35 40 45

Thr ser Tyr Gly Val His Trp Val Arg Gin Pro Pro Gly Arg Gly Leu 50 55 60

Glu Trp Leu Gly Val Ile Trp Ser Asp Gly Ser Thr Asp Tyr Asn Ala 65 70 75 80

Ala Phe Ile Ser Arg Leu Ser Ile ser Lys Asp Asn Ser Lys Ser Gin 85 90 95

Val Ser Leu Arg Met Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110

Tyr cys Ala Thr Leu Leu Leu Arg Leu Gly Trp Gly Gin Gly Ser Leu 115 120 125

Val Thr Val Ser ser 130

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 439 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 16..427

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

AAGCTTGCCG CCACC ATG GGC ATC AAG ATG GAG TCA CAG TTC CAG GTC CTC 51

Met Gly Ile Lys Met Glu Ser Gin Phe Gin Val Leu 1 5 10

ACA CAG TCT CCA GAC TCC CTG GCT GTG TCT CTG GGC GAG AGG GCC ACC 147 Thr Gin Ser Pro Asp Ser Leu Ala val Ser Leu Gly Glu Arg Ala Thr 30 35 40

ATC AAC TGC AAG TCC AGT CAG AGT CTG TTA CAC AGT GGA AAT CAA AAG 195 He Asn Cys Lys Ser Ser Gin Ser Leu Leu His Ser Gly Asn Gin Lys 45 50 55 60

AGT GGC AGT GGA TCT GGA ACA GAT TTC ACT CTC ACC ATC AGC AGT CTG 339 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 95 100 105

CAG GCT GAA GAC GTG GCA GTT TAT TAC TGT CAG AAT GAT TAT AGT TAT 387 Gin Ala Glu Asp Val Ala Val Tyr Tyr cys Gin Asn Asp Tyr Ser Tyr 110 115 120

CCG CTC ACG TTC GGT CAA GGG ACC AAG GTG GAG ATA AAA C GTGAGTGGAT 437 Pro Leu Thr Phe Gly Gin Gly Thr Lys Val Glu Ile Lys 125 130 135

CC 439 (2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 137 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

( i) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Met Gly Ile Lys Met Glu Ser Gin Phe Gin Val Leu Met Ser Leu Leu 1 5 10 15

Phe Trp Val Ser Gly Thr Cys Gly Asp Ile Val Met Thr Gin Ser Pro 20 25 30

Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys 35 40 45 ser Ser Gin Ser Leu Leu His Ser Gly Asn Gin Lys Asn Tyr Leu Thr 50 55 60

Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Asn Leu Leu lie Tyr Trp 65 70 75 80

Ala Ser Thr Arg Glu ser Gly Val Pro Asp Arg Phe Ser Gly ser Gly 85 90 95

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Ala Glu Asp 100 105 110

Val Ala Val Tyr Tyr cys Gin Asn Asp Tyr Ser Tyr Pro Leu Thr Phe 115 120 125

Gly Gin Gly Thr Lys val Glu Ile Lys 130 135

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 439 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 16..427

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

AAGCTTGCCG CCACC ATG GGC ATC AAG ATG GAG TCA CAG TTC CAG GTC CTC 51 Met Gly Ile Lys Met Glu Ser Gin Phe Gin Val Leu 1 5 10

ATC AAC TGC AAG TCC AGT CAG AGT CTG TTA CAC AGT GGA AAT CAA AAG 195 Ile Asn Cys Lys Ser Ser Gin Ser Leu Leu His Ser Gly Asn Gin Lys 45 50 55 60

AAC TAC TTG ACC TGG TAC CAG CAG AAA CCA GGG CAG CCT CCT AAG CTG 243 Asn Tyr Leu Thr Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu 65 70 75

CC 439 (2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 137 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Met Gly Ile Lys Met Glu Ser Gin Phe Gin Val Leu Met Ser Leu Leu 1 5 10 15

Phe Trp val Ser Gly Thr cys Gly Asp lie Val Met Thr Gin Ser Pro 20 25 30

Asp Ser Leu Ala Val ser Leu Gly Glu Arg Ala Thr He Asn cys Lys 35 40 45

Ser Ser Gin Ser Leu Leu His Ser Gly Asn Gin Lys Asn Tyr Leu Thr 50 55 60

Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu Ile Tyr Trp 65 70 75 80

Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly 85 90 95

Ser Gly Thr Asp Phe Thr Leu Thr He ser ser Leu Gin Ala Glu Asp 100 105 110

Val Ala Val Tyr Tyr Cys Gin Asn Asp Tyr ser Tyr Pro Leu Thr Phe 115 120 125

Gly Gin Gly Thr Lys Val Glu He Lys 130 135

(2) INFORMATION FOR SEQ ID Nθ:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

( i) SEQUENCE DESCRIPTION: SEQ ID Nθ:21: TCAAGATCGA TCCTTTGGGT GAAAAG 26 (2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TTGGACTCGA GTTATCATTC ACAGGTAGGT AG 32

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

( i) SEQUENCE DESCRIPTION: SEQ ID NO:23: ACTAGTCGAC ATGGGYATCA AGATGGAGTC ACAKWYYCWG G 41

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GGATCCCGGG TGGATGGTGG GAAGATG 27

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

( i) SEQUENCE DESCRIPTION: SEQ ID NO:25: ACTAGTCGAC ATGGCTGTCY TRGSGCTRCT CTTCTGC 37 (2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GGAACCCGGG AAGGGATAGA CAGATGG 27

Claims

We claim:

1. A humanized antibody that specifically binds E-selectin, comprising:

a. a humanized variable heavy chain (V_H) having the amino acid sequence of human V_H framework regions 1, 2, 3 and 4 (FRs) and non-human V_H complementarity determining regions

1, 2 and 3 (CDRs) ; and

b. a humanized variable light chain (V_L) having the amino acid sequence of human V_L FRs and non-human V_L CDRs,

wherein said humanized antibody binds to E-selectin with at least one percent of the binding efficiency of murine monoclonal antibody CY1787, which is produced by the cell line designated as ATCC Accession # HB 10591.

2. The humanized antibody of claim 1, wherein said human V_H FRs correspond to the V_H FRs in human monoclonal antibody NEWM.

3. The humanized antibody of claim 2, wherein said human V_H FRs are substituted in at least one amino acid position selected from the group consisting of amino acid position 20, 27, 29, 30, 48, 67, 68, 69, 70, 71, 73, 76, 78, 82, 82c and 94.

4. The humanized antibody of claim 1, wherein said human V_L FRs correspond to the V_L FRs in human monoclonal antibody FK-001.

5. The humanized antibody of claim 4, wherein said human V_L FRs are substituted at amino acid position 45.

6. The humanized antibody of claim 1, wherein said non-human V_L and said non-human V_H correspond to the

V_t and V_H, respectively, of murine monoclonal antibody CY1787, which is produced by the cell line designated as ATCC Accession # HB 10591.

7. The humanized antibody of claim 1, further comprising a human constant heavy chain(C_H) and a human constant light chain(C_L) .

8. The humanized antibody of claim 7, wherein said human C_H is selected from the group consisting of human C_yl, C_y2, C_y3, C_y4, C_αl, C_α2, C_μ C_δ and C_e and said human C_ is selected from the group consisting of human C_κ and C_λ.

9. The humanized antibody of claim 7, wherein said human C_H is human C_yl and said human C_L is human C_κ.

10. The humanized antibody of claim 7, wherein s ■aaid human C_H is human C_y4 and said human C_L is human C -K„."

11. A humanized antibody that specifically binds E-selectin, comprising CY1788V_HA having the amino acid sequence shown in SEQ ID NO: 6.

12. A humanized antibody that specifically binds E-selectin, comprising CY1788V_HB having the amino acid sequence shown in SEQ ID NO: 8.

13. A humanized antibody that specifically binds E-selectin, comprising CY1788V_HC having the amino acid sequence shown in SEQ ID NO: 10.

14. A humanized antibody that specifically binds E-selectin, comprising

having the amino acid sequence shown in SEQ ID NO: 12.

15. A humanized antibody that specifically binds E-selectin, comprising CY1788V_HJ, having the amino acid sequence shown in SEQ ID NO: 14.

16. A humanized antibody that specifically binds E-selectin, comprising CY1788V_HF having the amino acid sequence shown in SEQ ID NO: 16.

17. A humanized antibody that specifically binds E-selectin, comprising CY1788V_IA having the amino acid sequence shown in SEQ ID NO: 18.

18. A humanized antibody that specifically binds E-selectin, comprising CY1788V_LB having the amino acid sequence shown in SEQ ID NO: 20.

19. A humanized antibody that specifically binds E-selectin, comprising:

a. a V_H selected from the group consisting of CY1788V_HA, C 1788V_H5, CY1788V_HC, CY1788V_JJ,,, CY1788V_HJ. and CY1788V_HF; and

b. a V_L selected from the group consisting of CY1788V_LA and CY1788V_LB.

20. A humanized antibody that specifically binds E-selectin, comprising CY1788V_HF and CY1788V_IA.

21. The humanized antibody of claim 20, wherein said CY1788V_HF further comprises a human C_y selected from the group consisting of human C_yl and human C_y4, and wherein said CY1788V_LΛ further comprises human C_κ.

22. A humanized antibody that binds

E-selectin, comprising non-human CDRs and substantially human FRs.

23. The humanized antibody of claim 22, wherein said non-human CDRs correspond to the CDRs in murine monoclonal antibody CY1787, which is produced by a cell line designated ATCC Accession # HB 10591.

24. A reshaped antibody that binds E-selectin, comprising non-murine FRs and murine CDRs, wherein said CDRs correspond to the CDRs in murine monoclonal antibody CY1787, which is produced by a cell line designated ATCC Accession # HB 10591.

25. A nucleic acid molecule encoding a humanized antibody that specifically binds E-selectin, said humanized antibody comprising:

a. a humanized variable heavy chain (V_H) having the amino acid sequence of human V_H framework regions 1, 2, 3 and 4 (FRs) , substituted in at least one amino acid position, and non-human V_H complementarity determining regions 1, 2 and 3 (CDRs); and

wherein said humanized antibody binds with at least one percent of the binding efficiency of murine monoclonal antibody CY1787, which is produced by the cell line designated as ATCC Accession # HB 10591.

26. The nucleic acid molecule of claim 25, wherein said non-human V_L and said non-human V_H correspond to the V_L and V_B, respectively, of murine monoclonal antibody CY1787, which is produced by the cell line designated as ATCC Accession # HB 10591.

27. The nucleic acid of claim 26, wherein said human V_H corresponds to the V_H in human monoclonal antibody NEWM and wherein said human V_L corresponds to the V_L in human monoclonal antibody FK-001.

28. The nucleic acid molecule of claim 27, wherein said V_H is substituted in at least one amino acid position selected from the group consisting of amino acid position 20, 27, 29, 30, 48, 67, 68, 69, 70, 71, 73, 76, 78, 82, 82c and 94, and wherein said V_L is substituted at position 45.

29. The nucleic acid molecule of claim 25,

wherein said humanized V_H has an amino acid sequence selected from the group consisting of:

CY1788V_HA as shown in SEQ ID NO 6;

CY1788V₃ as shown in SEQ ID NO 8;

CY1788V_HC as shown in SEQ ID NO 10;

CY1788V_HD as shown in SEQ ID NO 12;

CY1788V_HE as shown in SEQ ID NO 14; and

CY1788V_HP as shown in SEQ ID NO 16;

and wherein said humanized V_L has an amino acid sequence selected from the group consisting of:

CY1788V_LA as shown in SEQ ID NO: 18; and CY1788V_LB as shown in SEQ ID NO: 20.

30. The nucleic acid molecule of claim 29, wherein said humanized antibody contains a human C_H selected from the group consisting of human C_yl, C_γ2, C_y3, ^C _y4, C_Ql, C_α2, C_μι C_δ and C_e and contains a human C_L selected from the group consisting of human C_κ and C_λ.

31. The nucleic acid molecule of claim 25, wherein said humanized V_H has the amino acid of CY1788V_HF as shown in SEQ ID NO: 16 and wherein said humanized V_L has the amino acid sequence of CY1788V_LA as shown in SEQ ID NO: 18.

32. The nucleic acid molecule of claim 31, wherein said humanized antibody contains a human C_γl and a human C_κ.

33. The nucleic acid molecule of claim 31, wherein said humanized antibody contains a human C_y4 and a human C_κ.

34. A vector, comprising the nucleic acid molecule of claim 25.

35. The vector of claim 34, which is an expressible vector.

36. The vector of claim 35, wherein said vector is expressible in a prokaryotic cell.

37. The vector of claim 36, wherein said vector is expressible in a eukaryotic cell.

38. A prokaryotic cell containing the expressible vector of claim 36.

39. A eukaryotic cell containing the expressible vector of claim 37.

40. A pharmaceutical composition, comprising the humanized antibody of claim 1 and a pharmaceutically acceptable carrier.

41. A method for detecting the presence of E-selectin in a human subject, comprising the steps of:

a. administering an effective amount of the humanized antibody of claim 1 to said subject; and

b. detecting specific binding of said humanized antibody in said subject, wherein said specific binding indicates the presence of E-selectin in said subject.

42. The method of claim 41, wherein said humanized antibody is detectably labelled.

43. The method of claim 41, wherein the presence of E-selectin is diagnostic of an inflammatory response in said human subject.

44. The method of claim 41, wherein said detecting the presence of E-selectin is diagnostic of a pathology characterized by the involvement of E-selectin.

45. The method of claim 44, wherein said pathology is septic shock.

46. The method of claim 44, wherein said pathology is acute respiratory distress syndrome.

47. The method of claim 44, wherein said pathology is wound-associated sepsis.

48. The method of claim 44, wherein said pathology is gross cystic breast disease.

49. The method of claim 44, wherein said pathology is due to exposure to petroleum-based solvents.

50. A method of inhibiting E-selectin mediated cell adhesion in a human subject, comprising administering to the human subject an effective amount of the humanized antibody of claim 1, which inhibits E-selectin mediated cell adhesion.

51. The method of claim 50, wherein said cell is selected from the group consisting of a neutrophil, a monocyte and a lymphocyte cell.

52. The method of claim 50, wherein said cell is a tumor cell.

53. A method of reducing or inhibiting an inflammatory response in a human subject, comprising administering to the human subject an effective amount of the humanized antibody of claim 1.

54. A method of reducing or inhibiting the severity of a pathology characterized by the involvement of E-selectin in a human subject, comprising administering to the human subject an effective amount of the humanized antibody of claim 1.

55. The method of claim 54, wherein said pathology is septic shock.

56. The method of claim 54, wherein said pathology is acute respiratory distress syndrome.

57. The method of claim 54, wherein said pathology is wound-associated sepsis.

58. The method of claim 54, wherein said pathology is gross cystic breast disease.

59. The method of claim 54, wherein said pathology is cancer.