WO1996040942A1 - Anticorps humanises anti-e-selectine - Google Patents

Anticorps humanises anti-e-selectine Download PDF

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Publication number
WO1996040942A1
WO1996040942A1 PCT/US1996/009204 US9609204W WO9640942A1 WO 1996040942 A1 WO1996040942 A1 WO 1996040942A1 US 9609204 W US9609204 W US 9609204W WO 9640942 A1 WO9640942 A1 WO 9640942A1
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human
selectin
ser
antibody
cy1788v
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PCT/US1996/009204
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English (en)
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Mark A. Williams
Carl Perez
Mary M. Bendig
S. Tarran Jones
Jose W. Saldanha
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Cytel Corporation
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Priority to AU61558/96A priority Critical patent/AU6155896A/en
Publication of WO1996040942A1 publication Critical patent/WO1996040942A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C07K16/2854—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72 against selectins, e.g. CD62
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates generally to the fields of antibody engineering and immunology and more specifically to humanized antibodies that specifically bind E-selectin.
  • 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.
  • leukocytes white blood 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.
  • E-selectin mediated cell adhesion can produce or contribute to a pathological condition.
  • 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.
  • higher than normal levels of E-selectin, including a soluble form of E-selectin are associated with various pathological conditions.
  • 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.
  • 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.
  • 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.
  • the utility of non-human antibodies in humans is limited.
  • 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 ) .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • V L variable light chain
  • V H variable heavy chain
  • the human V H FRs correspond to those of human monoclonal NEWM
  • 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,
  • 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-sele ⁇ tin.
  • 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.
  • E-selectin can bind the sialyl
  • Lewis 35 determinant(sLe x ) which is present on neutrophils, sialyl Lewis 3 , 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.
  • E-selectin is transiently expressed on cytokine-activated endothelial cells and, therefore, is expressed on endothelial cells near regions of inflammation.
  • 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.
  • 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.
  • 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.
  • 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 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 ⁇ chain to produce a humanized anti-E-selectin IgGl or IgG4 antibody, respectively (see Example II).
  • 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.
  • 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.
  • an antibody that contains the CDRs to be used in a humanized antibody is referred to as the "donor” antibody.
  • u MAb CY1787 was used as the donor antibody to prepare the humanized anti-E-selectin antibodies disclosed herein.
  • host is used in referring to an antibody that provides the FRs, and constant domains as desired.
  • human monoclonal antibodies NE M and FK-001 were the host antibodies used to prepare the humanized antibodies exemplified herein (see Example II).
  • 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.
  • HAMA human anti-murine 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.
  • 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)).
  • 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.
  • a reshaped antibody should not induce a host anti-donor antibody (HoADA) response in the host.
  • HoADA host anti-donor antibody
  • Various host antibodies can be obtained using well known methods or can be purchased from a commercial source.
  • 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.
  • 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.
  • 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 10 5 liters/mole.
  • a humanized anti-E-selectin antibody that specifically binds E-selectin with an association constant of about 1 x 10 7 to about 1 x 10 8 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.
  • 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) .
  • 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.
  • 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.
  • 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.
  • 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).
  • amino acids in the FRs and CDRs can be varied so as to obtain humanized antibodies optimized for desired characteristics such as binding affinity.
  • 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.
  • additional conservative amino acid substitutions such as the substitution of one hydrophobic amino acid for another.
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a plasmid vector containing a bacterial origin of replication is propagated in bacterial cells such as E. coli cells.
  • 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.
  • 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 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.
  • 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.
  • such mammalian expression vectors can contain a nucleic acid sequence encoding dihydrofolate reductase (dhfr) .
  • dhfr dihydrofolate reductase
  • 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).
  • compositions comprising a humanized anti-E-selectin antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers 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.
  • 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.
  • carbohydrates such as glucose, sucrose or dextrans
  • antioxidants such as ascorbic acid or glutathione
  • chelating agents such as ascorbic acid or glutathione
  • low molecular weight proteins or other stabilizers or excipients such ascorbic acid or glutathione
  • 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.
  • 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.
  • 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.
  • 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, intra ⁇ isternally.
  • 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.
  • 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.
  • incorporación 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.
  • 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.
  • the humanized antibody 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.
  • 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.
  • 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).
  • an antibody can be labeled with any of various detectable moieties including a radiolabel, an enzyme or enzyme substrate, biotin or a fluorochrome.
  • 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.
  • 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.
  • the second antibody can be labeled using a detectable moiety as described herein or otherwise known in the art.
  • an imaging device such as a scintillation detector, positron emission transaxial tomography (PETT) or nuclear magnetic resonance (NMR) imaging.
  • PET positron emission transaxial tomography
  • NMR nuclear magnetic resonance
  • 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.
  • 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.
  • 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.
  • inflammation is a normal physiological response that occurs due to tissue injury.
  • cytokines which have a variety of functions including inducing E-selectin expression by endothelial cells in the area of the inflammation.
  • cytokines which have a variety of functions including inducing E-selectin expression by endothelial cells in the area of the inflammation.
  • cytokines which have a variety of functions including inducing E-selectin expression by endothelial cells in the area of the inflammation.
  • 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.
  • E-selectin also is associated with various pathological conditions.
  • 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.
  • 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. Sur ⁇ . 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.
  • 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.
  • a cell such as a leukocyte or a tumor cell to an endothelial cell in a human subject
  • 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 are administered to the humanized anti-E-selectin antibody to the human subject.
  • 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.
  • E-selectin expression is induced in endothelial cells in response to cytokines elicited at a site of inflammation.
  • 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.
  • 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.
  • 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)).
  • E-selectin expression may be induced by cytokines, resulting in leukocyte adhesion to the endothelium and migration into the tissues.
  • 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.
  • 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.
  • a humanized anti-E-selectin antibody can be particularly useful as part of a combined modality treatment of a cancer patient.
  • surgical removal of a tumor is a common means for reducing a tumor burden in a cancer patient.
  • 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.
  • This example provides methods for preparing and isolating reagents used in the binding assays disclosed below.
  • 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 ⁇ g/ml hydrocortisone, "GENTAMYCIN” and "AMPHOTERCIN-B".
  • EGM-UV medium Clonetics; San Diego CA
  • FBS fetal bovine serum
  • 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% C0 2 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.
  • 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.
  • HBSS Hank's Balanced Salt Solution
  • GIBCO/BRL Hank's Balanced Salt Solution
  • 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
  • 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) .
  • G418 mammalian antibiotic G418
  • a plasmid directing the expression of a soluble form of 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.
  • 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.
  • CRP sixth consensus repeat element
  • 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) .
  • 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).
  • 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) .
  • ELISA enzyme-linked immunoabsorbent assay
  • a 10-chambered Nunc "CELL FACTORY” (6250 cm 2 total surface area; Nunc; Naperville IL) was seeded with 2.78 x 10 8 293-3 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.
  • 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.
  • 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 CaCl 2 , 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 CaCl 2 . One ml fractions were collected into 3 ml
  • 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.
  • 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.
  • 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.
  • the degenerate 5'-primers consisted of a pool of 64 oligonucleotides having the general sequence,
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • heavy chain HI fit canonical subgroup 1 and was modeled on the H2 loop of Dl.3
  • heavy chain H2 fit canonical subgroup 1 and was modeled on the Hi loop of Dl.3.
  • 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;
  • CY1788V HA 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.
  • the Val residue at this position in FR3 of the donor human sequence (human Val 71) was substituted with mu Lys 71.
  • position 94 is required to maintain the canonical loop structure of HI.
  • 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.
  • 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 .
  • mu MAb CY1787 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.
  • 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.
  • CY1788V HC SEQ ID NO: 10.
  • a surface residue such as mu Ile 69 can have an effect on antigen binding affinity.
  • surface residues are important because, in a humanized antibody, substitution of a murine surface residue can render the antibody immunogenic.
  • 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 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).
  • 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 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 ⁇ Vg j . (SEQ ID NO: 16) was constructed to substitute mu Ser 68 for human Thr 68.
  • 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).
  • CY1788V LA SEQ ID NO: 18
  • SEQ ID NO: 20 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.
  • the pHCMV-V L lys-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 L lys-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.
  • 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 L lys 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 L lys-neo.
  • This fragment was inserted into the Bam HI site of pHCMV-V L lys-neo in the appropriate orientation to produce pHCMV-V L lys-K R -neo.
  • the Bam HI site 3' to the human kappa constant region was removed by filling-in the end with Klenow polymerase.
  • the Hind III/Bam HI fragment containing the V L lys kappa region easily can be replaced with the V L of a reshaped antibody.
  • the pHCMV-V H lys- ⁇ lC-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 H lys 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 L lys kappa light chain fragment.
  • This vector was designated pHCMV-V H lys-neo.
  • the cDNA coding for human ⁇ l constant region then was inserted into the Bam HI site to produce pHCMV-V H lys- ⁇ l-neo.
  • cDNA coding for human ⁇ l constant region was cloned by PCR amplification from a human cell line secreting a human ⁇ l 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 ⁇ l cDNA, the splice acceptor site and intron sequence then was cloned into the expression vector.
  • This vector designated pHCMV-V H lys- ⁇ lC-neo, contained a Hind III/Bam HI fragment encoding V H lys, which readily can be replaced with the V H of a reshaped antibody.
  • 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 H lys- ⁇ lC-neo by standard methods to produce the expression plasmids pHCMV-1787C H - ⁇ lC-neo, pHCMV-1788V HA - ⁇ lC-neo, pHCMV-1788V HB - ⁇ lC-neo, pHCMV-1788V HC - ⁇ lC-neo, pHCMV-1788V BD - ⁇ lC-neo, pHCMV-1788V HE - ⁇ lC-neo and pHCMV-1788V HP - ⁇ lC-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 L lys-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) .
  • 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.
  • 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.
  • 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).
  • Immulon microtiter plates (Dynatech; Chantilly VA) were coated with 100 ⁇ l goat anti-human IgG (whole molecule; Sigma) diluted to a final concentration of 12.5 ⁇ g/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 ⁇ l DPBS containing 1% bovine serum albumin (BSA; Sigma) for at least 1 hr at RT, then washed 3X with DPBS.
  • BSA bovine serum albumin
  • the monoclonal antibodies were serially diluted in DPBS containing 1% BSA (DPBS/BSA) and 100 ⁇ l was added to each well. The plates were incubated for 1 hr at RT, then washed 3X with DPBS. 100 ⁇ l 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 ⁇ l tetramethylbenzidine peroxidase substrate/H 2 0 2 (TMB; Kirkegaard and Perry Laboratories; Gaithersburg MD) was added to each well.
  • TMB tetramethylbenzidine peroxidase substrate/H 2 0 2
  • the color was allowed to develop for about 3 to 15 min, then the reaction was quenched by adding 100 ⁇ l 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) .
  • the wells of an Immulon-1 microtiter plate were coated with 50 ⁇ l 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 ⁇ l DPBS/BSA and washed 3X with PBS.
  • 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 .
  • 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.
  • CY1788V HO and CY ⁇ V ⁇ (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).
  • CY1788(V B /V U ) , CY1788(V HB /V IA ) and CY1788(V preparation/V u ) were stably transfected into CHO cells and generated sufficient quantities of purified antibody for analysis.
  • 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.
  • pHCMV-V H lys- ⁇ lC-dhfr is functionally identical to pHCMV-V H lys- ⁇ lC-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.
  • dhfr dihydrofolate reductase
  • the SV40 enhancer sequence was removed from the plasmid pSV2-dhfr (Subramani et al., Mol. Cell. Biol.
  • the Bam HI site on the 3'-side of the human ⁇ l constant region was removed by filling-in the recessed overhang with Klenow polymerase to produce pHCMV-V H lys- ⁇ lC-dhfr.
  • the Hind III/Bam HI fragment of this vector which encodes the V h lys sequence, readily can be replaced with the V H of a reshaped antibody.
  • pHCMV-V H lys- ⁇ 4-dhfr is identical to pHCMV-V H lys- ⁇ lC-dhfr as described above, except that the cDNA clone of the human ⁇ l constant region is replaced with a genomic clone of the human ⁇ 4 constant region.
  • An approximately 7.0 kb Hind III fragment of DNA containing the genomic clone of the human ⁇ 4 constant region was subcloned into the Hind III site of pUC19 to produce plasmid 428D.
  • the human ⁇ 4 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 ⁇ 4 fragment was excised from plasmid 428D using Bam HI and ligated into pHCMV-V H lys-neo to produce pHCMV-V H lys- ⁇ 4-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- ⁇ E 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 ⁇ 4 constant region.
  • 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- ⁇ lC-dhfr to generate pHCMV-1788V ⁇ - ⁇ lC-dhfr, pHCMV-1788V HB - ⁇ lC-dhfr and pHCMV-1788V HF - ⁇ lC- dhfr.
  • the cDNA encoding CY1788V BJ The cDNA encoding CY1788V BJ .
  • pHCMV- ⁇ 4C-dhfr also was subcloned into pHCMV-1788V HF - ⁇ 4C-dhfr to generate pHCMV-1788V HF - ⁇ 4C-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- ⁇ lC-dhfr produces an IgGl isotype antibody and vector pHCMV- ⁇ 4C-dhfr produces an IgG4 isotype antibody when coexpressed with the kappa light chain vector.
  • 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 ⁇ MEM (+ nucleosides) /10% FBS and incubated for 24 to 48 hr.
  • the cells were trypsinized and plated into 100 mm dishes in ⁇ MEM (minus nucleosides) containing 10% dialyzed FBS to select for the expression of the dhfr-containing plasmid.
  • the medium also was supplemented with 500 ⁇ g 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.
  • 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 10 5 cells/100 mm dish in selection medium.
  • For the first round of amplification cells were plated in three sets of dishes containing ⁇ MEM (+ nucleosides)/10% dialyzed FBS supplemented with 500 ⁇ g/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.
  • CHO cells were expanded and seeded into a 10-chamber (6000 cm 2 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.
  • 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.
  • each well of a microtiter plate was coated with 50 ⁇ l sol-E-selectin (4 ⁇ g/ml) and incubated for 2 hr at RT.
  • the plate was washed 3X with PBS, blocked with 200 ⁇ l DPBS/BSA/well for 20 min at RT, then washed 3X with PBS.
  • the plate was washed 3X with DPBS/BSA, then 100 ⁇ l HRP substrate (24 mM citrate, 50 mM NaP0 4 , 0.5 mg/ml O-phenylenediamine, 0.012 % H 2 0 2 ) was added to each well and the reaction was allowed to develop for approximately 6 min. The reaction was quenched with 25 ⁇ l 4 N H 2 S0 4 and the absorbance at 492 nm was determined. Alternatively 50 ⁇ l 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 ⁇ l 1 M phosphoric acid and the absorbance at 450 nm was determined.
  • HRP substrate 24 mM citrate, 50 mM NaP0 4 , 0.5 mg/ml O-phenylenediamine, 0.012 % H 2 0 2
  • 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 ⁇ g/ml and CY1788(V HF /V IA )- IgG4 at a concentration of 3.85 ⁇ g/ml inhibited the binding of labeled CY1787 to 50% of maximum.
  • 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 10 4 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 ⁇ g/ml recombinant IL-l ⁇ (rIL-l ⁇ ; 2.5 x 10 5 U/ml stock; Genzyme; Boston MA) and incubating the cells for 4 hr to obtain activated endothelial cells. Control wells received only fresh medium.
  • 50 ⁇ l 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 ⁇ l OPDA (8 mg o-phenylenediamine, 8 ⁇ l 30% H 2 0 2 , 10 ml water was added to each well to determine myeloperoxidase activity, the plates were incubated for 15 min at RT, then 15 ⁇ l 4 N H 2 S0 4 was added to stop the reaction.
  • a reagent blank was prepared by mixing 50 ⁇ l solubilization buffer, 50 ⁇ l OPDA and 15 ⁇ l 4 N H 2 S0 4 . 100 ⁇ l 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-l ⁇ 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.
  • NAME Campbell, Cat ryn A.
  • Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Asn Leu Leu Ile Tyr Trp 65 70 75 80
  • Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Asn Leu Leu lie Tyr Trp 65 70 75 80

Abstract

Cette invention se rapporte à des anticorps humanisés qui fixent spécifiquement l'E-sélectine, ces anticorps comprenant essentiellement des régions de détermination de complémentarité d'un anticorps non humain et essentiellement les régions de structure (FR) d'un anticorps humain, ces régions de structure pouvant être substituées dans une ou plusieurs positions des acides aminés. Cette invention concerne également des molécules d'acides nucléiques codant ces anticorps anti-E-sélectine humanisés, des vecteurs comprenant les molécules d'acides nucléiques, ainsi que des cellules contenant ces vecteurs. Cette invention concerne aussi des compositions pharmaceutiques comprenant un anticorps anti-E-sélectine humanisé, ainsi que des procédés d'utilisation d'un anticorps anti-E-sélectine humanisé, pour détecter la présence d'E-sélectine chez un sujet humain et des procédés pour diagnostiquer une pathologie se caractérisant par l'implication d'E-sélectine chez un sujet humain. En outre, cette invention se rapporte à des procédés pour réduire ou inhiber l'adhésion cellulaire induite par E-sélectine à une cellule endothéliale chez un sujet, et à des procédés pour traiter un sujet humain souffrant d'une pathologie se caractérisant par l'implication d'E-sélectine, en administrant à ce sujet un anticorps anti-E-sélectine humanisé.
PCT/US1996/009204 1995-06-07 1996-06-06 Anticorps humanises anti-e-selectine WO1996040942A1 (fr)

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US20110002881A1 (en) * 2007-12-10 2011-01-06 Mater Medical Research Institute Treatment and prophylaxis
WO2012054084A3 (fr) * 2010-10-20 2013-10-17 Oxford Biotherapeutics Ltd. Anticorps
US9181339B2 (en) 2009-04-20 2015-11-10 Oxford Bio Therapeutics Ltd. Antibodies specific to cadherin-17
US10519181B2 (en) 2014-12-03 2019-12-31 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectins and CXCR4 chemokine receptors
US11072625B2 (en) 2016-10-07 2021-07-27 Glycomimetics, Inc. Highly potent multimeric e-selectin antagonists
WO2021148983A1 (fr) * 2020-01-24 2021-07-29 Pfizer Inc. Anticorps anti-e-sélectine, compositions et procédés d'utilisation
US11197877B2 (en) 2017-03-15 2021-12-14 Glycomimetics. Inc. Galactopyranosyl-cyclohexyl derivauves as E-selectin antagonists
US11291678B2 (en) 2016-03-02 2022-04-05 Glycomimetics, Inc Methods for the treatment and/or prevention of cardiovascular disease by inhibition of E-selectin
US11433086B2 (en) 2016-08-08 2022-09-06 Glycomimetics, Inc. Combination of T-cell checkpoint inhibitors with inhibitors of e-selectin or CXCR4, or with heterobifunctional inhibitors of both E-selectin and CXCR4
US11548908B2 (en) 2017-12-29 2023-01-10 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3
US11707474B2 (en) 2018-03-05 2023-07-25 Glycomimetics, Inc. Methods for treating acute myeloid leukemia and related conditions
US11712446B2 (en) 2017-11-30 2023-08-01 Glycomimetics, Inc. Methods of mobilizing marrow infiltrating lymphocytes and uses thereof
RU2805176C1 (ru) * 2020-01-24 2023-10-11 Пфайзер Инк. Антитела против e-селектина, композиции и способы применения
US11845771B2 (en) 2018-12-27 2023-12-19 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3

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US9254322B2 (en) 2007-12-10 2016-02-09 The University Of Queensland Compositions comprising E-selectin antagonists and uses therefor
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US20110002881A1 (en) * 2007-12-10 2011-01-06 Mater Medical Research Institute Treatment and prophylaxis
US9181339B2 (en) 2009-04-20 2015-11-10 Oxford Bio Therapeutics Ltd. Antibodies specific to cadherin-17
WO2012054084A3 (fr) * 2010-10-20 2013-10-17 Oxford Biotherapeutics Ltd. Anticorps
US10519181B2 (en) 2014-12-03 2019-12-31 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectins and CXCR4 chemokine receptors
US11291678B2 (en) 2016-03-02 2022-04-05 Glycomimetics, Inc Methods for the treatment and/or prevention of cardiovascular disease by inhibition of E-selectin
US11433086B2 (en) 2016-08-08 2022-09-06 Glycomimetics, Inc. Combination of T-cell checkpoint inhibitors with inhibitors of e-selectin or CXCR4, or with heterobifunctional inhibitors of both E-selectin and CXCR4
US11072625B2 (en) 2016-10-07 2021-07-27 Glycomimetics, Inc. Highly potent multimeric e-selectin antagonists
US11780873B2 (en) 2016-10-07 2023-10-10 Glycomimetics, Inc. Highly potent multimeric e-selectin antagonists
US11197877B2 (en) 2017-03-15 2021-12-14 Glycomimetics. Inc. Galactopyranosyl-cyclohexyl derivauves as E-selectin antagonists
US11878026B2 (en) 2017-03-15 2024-01-23 Glycomimetics, Inc. Galactopyranosyl-cyclohexyl derivatives as e-selectin antagonists
US11712446B2 (en) 2017-11-30 2023-08-01 Glycomimetics, Inc. Methods of mobilizing marrow infiltrating lymphocytes and uses thereof
US11548908B2 (en) 2017-12-29 2023-01-10 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3
US11707474B2 (en) 2018-03-05 2023-07-25 Glycomimetics, Inc. Methods for treating acute myeloid leukemia and related conditions
US11845771B2 (en) 2018-12-27 2023-12-19 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3
US11597770B2 (en) 2020-01-24 2023-03-07 Pfizer Inc. Anti-E-selectin antibodies, compositions and methods of use
WO2021148983A1 (fr) * 2020-01-24 2021-07-29 Pfizer Inc. Anticorps anti-e-sélectine, compositions et procédés d'utilisation
RU2805176C1 (ru) * 2020-01-24 2023-10-11 Пфайзер Инк. Антитела против e-селектина, композиции и способы применения

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