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WO1994025067A1 - Anticorps diriges contre la selectine p et leurs utilisations - Google Patents

Anticorps diriges contre la selectine p et leurs utilisations Download PDF

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Publication number
WO1994025067A1
WO1994025067A1 PCT/US1994/004935 US9404935W WO9425067A1 WO 1994025067 A1 WO1994025067 A1 WO 1994025067A1 US 9404935 W US9404935 W US 9404935W WO 9425067 A1 WO9425067 A1 WO 9425067A1
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WO
WIPO (PCT)
Prior art keywords
selectin
antibody
humanized
variable region
immunoglobulin
Prior art date
Application number
PCT/US1994/004935
Other languages
English (en)
Inventor
Robert W. Chesnut
Margaret J. Polley
James C. Paulson
S. Tarran Jones
Jose W. Saldanha
Mary M. Bendig
Michael Kriegler
Carl Perez
Robert Bayer
Michael Nunn
Original Assignee
Cytel Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US1993/004274 external-priority patent/WO1993021956A1/fr
Priority claimed from US08/202,047 external-priority patent/US5800815A/en
Application filed by Cytel Corporation filed Critical Cytel Corporation
Priority to CA002162149A priority Critical patent/CA2162149C/fr
Priority to JP52465394A priority patent/JP3713045B2/ja
Priority to EP94917301A priority patent/EP0804235A4/fr
Priority to CN94192352A priority patent/CN1124928A/zh
Priority to AU69063/94A priority patent/AU684084B2/en
Priority to SK1377-95A priority patent/SK137795A3/sk
Publication of WO1994025067A1 publication Critical patent/WO1994025067A1/fr
Priority to FI955261A priority patent/FI955261A/fi
Priority to NO954403A priority patent/NO954403L/no

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C07K14/70564—Selectins, e.g. CD62
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • This application relates generally to novel immunoglobulins reactive with functional epitopes on a cell surface receptor P-selectin. This application also relates generally to diagnostic and therapeutic methods of using the antibodies. BACKGROUND OF THE INVENTION
  • adhesion molecules generally glycoproteins, expressed on cell membranes. Often, an adhesion molecule on one cell type will bind to another adhesion molecule expressed on a different cell type, forming a receptor counter-receptor pair.
  • adhesion molecules Three important classes of adhesion molecules are the integrins, selectins, and immunoglobulin (Ig) superfamily members (see Springer, Nature 346:425 (1990); Osborn, Cell 62:3 (1990);
  • P-selectin also known as CD62, granule membrane protein-140 (GMP-140) /platelet activation-dependent granule external membrane (PADGEM) /LECCAM-3 is a specialized cell- surface receptor on vascular endothelial cells and platelets that is involved in the recognition of various circulating cells.
  • P-selectin is a surface glycoprotein with a lectin-like domain, a region with homology to epidermal growth factor, and a region with homology to complement regulatory proteins (see McEver, Blood Cells 16:73-83 (1990)).
  • P-selectin is similar to that of two other vascular cell surface receptors, endothelial leukocyte adhesion molecule (ELAM-1) and lymphocyte homing receptor (LHR).
  • ELAM-1 endothelial leukocyte adhesion molecule
  • LHR lymphocyte homing receptor
  • P-selectin is present on the surface of platelets and endothelial cells in response to a variety of stimuli, where it mediates platelet-leukocyte and endothelium-leukocyte interactions.
  • ELAM-1 is expressed only on endothelial cells and LHR is expressed on a variety of leukocytes in endothelial venules of peripheral lymph nodes.
  • Expression of P-selectin is inducible and it is not expressed on unactivated endothelial cells or platelets. P-selectin expression does not require de novo synthesis since it is stored in secretory granules (or Weibel-Palade bodies) in both platelets and endothelial cells.
  • P-selectin is rapidly redistributed to the surface of the cell where it is
  • P-selectin in intercellular adhesion in vitro suggests that like other adhesion molecules, P-selectin might participate in cellular interactions that contribute to inflammatory diseases in vivo.
  • P-selectin might participate in cellular interactions that contribute to inflammatory diseases in vivo.
  • the specific inflammatory diseases in which P-selectin might play a major role have not yet been elucidated.
  • epitope-specificity of agents effective to abort particular P-selectin-mediated inflammatory diseases is also unclear.
  • HAMA human-antimouse antibody response
  • blocking antibodies that specifically bind to P-selectin are provided.
  • One such exemplified antibody is designated mu MAb PB1.3 (produced by a cell line having ATCC accession number HB11041).
  • Other antibodies competitively inhibit the binding of mu MAb PB1.3 to P-selectin measured by a competitive inhibition assay.
  • Preferred antibodies bind to P-selectin in the absence of Ca ++ .
  • the antibodies of the invention include fragments such as Fab, Fab' F(ab') 2 , Fabc, and Fv, as well as intact
  • compositions comprise a blocking P-selectin antibody as described above and a pharmaceutically acceptable carrier.
  • the invention also provides therapeutic methods for treating diseases of the immune system.
  • the methods comprise administering to a patient having such a disease a
  • reperfusion injury are examples of diseases treatable by the present invention.
  • the humanized immunoglobulins comprise a humanized heavy chain and a humanized light chain.
  • the humanized light chain comprises three complementarity
  • CDR1, CDR2 and CDR3 having amino acid sequences from the corresponding complementarity determining regions of a mouse PB1.3 immunoglobulin light chain, and a variable region framework from a human kappa light chain variable region framework sequence.
  • the humanized heavy chain comprises three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the
  • the humanized immunoglobulins specifically bind to P-selectin with a binding affinity havin ⁇ a lower limit of about 10 7 M -1 and an upper limit of about five-times the binding affinity of the mouse PB1.3 immunoglobulin.
  • the human kappa light chain variable region framework sequence is substituted in at least one position selected from a first group
  • the human heavy chain variable region framework sequence is substituted in at least one position selected from a second group consisting of H1, H2, H71 and H73, by an amino acid present in the equivalent position of the mouse PB1.3 heavy chain variable region framework sequence.
  • the humanized heavy chain variable region framework is a 21/28'CL heavy chain variable region framework sequence.
  • the humanized light chain variable region framework is a DEN light chain variable region framework sequence. Exemplary mature light chain variable region amino acid sequences are shown in
  • FIGs 14-17 Exemplary mature heavy chain variable region amino acid sequences are shown in Figures 11-13.
  • a preferred humanized immunoglobulin comprises the mature light chain variable region shown in Figure 17 and the mature heavy chain variable region shown in Figure 12.
  • the invention provides intact humanized
  • immunoglobulins and humanized immunoglobulin fragments such as Fab, Fab' F(ab') 2 , Fabc and Fv.
  • immunoglobulins further comprise a constant region, which may or may not have an effector function.
  • nucleic acids encoding the humanized immunoglobulins described above are provided. Some nucleic acid encode heavy chains and others encode light chains.
  • the invention also provides computers programmed to display a three dimensional representation of the
  • the invention further provides pharmaceutical compositions comprising the humanized immunoglobulins
  • the invention provides methods for detecting P-selectin using the humanized immunoglobulins described above.
  • a humanized immunoglobulin is administered to a patient or a tissue sample therefrom. Complexes formed by specific binding between the immunoglobulin and P-selectin present in the target sample are detected to indicate the presence of P-selectin.
  • the invention also provides methods of treating a patient suffering from a disease of the immune systems using humanized immunoglobulins.
  • the methods comprise administering to the patient a therapeutically effective dose of a
  • compositions as described above.
  • Diseases amenable to treatment include ischemia-reperfusion injury and acute lung injury.
  • the compositions are useful for treating patients suffering from epidermal, myocardial, renal, cerebral, splenic, hepatic, spinal, splanchnic, pulmonary, partial-body, or whole-body ischemia.
  • stable cell lines producing humanized immunoglobulins comprise two nucleic acids segments.
  • One nucleic acid segment encodes the heavy chain of a humanized immunoglobulin as described above, the segment operably linked to a promoter to allow expression of the heavy chain.
  • the second nucleic acid segment encodes the light chain of the humanized immunoglobulin, the second segment operably linked to a second promoter to allow expression of the light chain.
  • Preferred cell lines produce about 30 ⁇ g of the humanized immunoglobulin/10 6 cells/day.
  • fragments of P-selectin are provided. These fragments have up to 100 amino acids, the up to 100 amino acids comprising at least five contiguous amino acids from between positions 448 and 467 of the amino sequence shown in Fig. 34. In preferred fragments, the up to 100 amino acids comprise the entire contiguous segment of amino acids between positions 448 and 467.
  • Fig. 1 presents data showing that anti-inflammatory immunoglobulins of the invention inhibit binding of thrombin activated platelets to neutrophils.
  • Figs. 2A and 2B show that anti-inflammatory immunoglobulins of the invention effectively prevent lung injury induced by infusion of cobra venom factor (CVF) as measured by permeability (Fig. 2A) and hemorrhage (Fig 2B).
  • CVF cobra venom factor
  • Fig. 3A through 3H show that P-selectin expression content in lungs of animals is upregulated in response to cobra venom infusion visualized by horseradish proxidase stain of mu MAb PB1.3
  • panel (a)-(d) show expression of P-selectin in lung venules at various times after infusion of CVF; time 0 (a); 5, 10 and 15 min, panels b, c and d, respectively (Fig. 3A).
  • Fig. 3A show that P-selectin expression content in lungs of animals is upregulated in response to cobra venom infusion visualized by horseradish proxidase stain of mu MAb PB1.3
  • panel (a)-(d) show expression of P-selectin in lung venules at various times after infusion of CVF; time 0 (a); 5, 10 and 15 min, panels b, c and d, respectively (Fig. 3A).
  • Fig. 3A show that P-se
  • 3B are light and transmission electron micrographs of CVF-induced acute lung injury in rats treated with 200 ⁇ g non-blocking antibody (mu MAb PNB1.6) (frames a.c) or with blocking P-selectin antibody (mu MAb PB1.3) to P-selectin (frames b.d) .
  • vascular injury was indicated by extensive intra-alveolar hemorrhage (frame a), associated with intravascular aggregates of neutrophils (frame c. arrows) in close contrast with endothelial cells.
  • Fig. 4 shows the binding of the monoclonal antibody mu MAb PB1.3 to P-selectin: effect of chelating divalent cations with EDTA.
  • the open symbols (lower trace) is binding in the presence of Ca ++ and Mg ++
  • the filled symbols (upper trace) is binding in the presence of EDTA.
  • Antibody was diluted shown at 1.6 mg/ml.
  • Fig. 5 shows the effect of the peptide
  • Figs. 6A, 6B and 6C show the ability of monoclonal antibodies to cross-block binding to P-selectin. Blocking antibodies were present at 50 ⁇ g/ml. Dilutions of
  • biotinylated antibodies shown were from 1.6 mg/ml mu MAb
  • Fig. 7 shows that the extent of platelet aggregation induced by thrombin is equivalent under control conditions in the absence of antibody and in the presence of increasing concentrations (1-100 ⁇ g/ml) of purified mu MAb PB1.3
  • Fig. 8 shows that the extent of myocardial ischemia, expressed as a percentage of the area at risk, is
  • Fig. 9 shows the sequence and translation of PB1.3 heavy chain signal peptide and variable region.
  • Fig. 10 shows the sequence and translation of PB1.3 light chain signal peptide and variable region.
  • Fig. 11 shows the sequence and translation of CY1748 heavy chain-A signal peptide and variable region.
  • Fig. 12 shows the sequence and translation of CY1748 heavy chain-B signal peptide and variable region.
  • Fig. 13 shows the sequence and translation of CY1748 heavy chain-C signal peptide and variable region.
  • Fig. 14 shows the sequence and translation of CY1748 light chain-A signal peptide and variable region.
  • Fig. 15 shows the sequence and translation of CY1748 light chain-B signal peptide and variable region.
  • Fig. 16 shows the sequence and translation of CY1748 light chain-C signal peptide and variable region.
  • Fig. 17 shows the sequence and translation of CY1748 light chain-D signal peptide and variable region.
  • Fig. 18 shows physical map of the neo-plasmid expressing the human heavy chain (gamma-1 constant region).
  • Fig. 19 shows the physical map of the neo-plasmid expressing the human light chain (kappa constant region).
  • Fig. 20 shows the binding curves of the chimeric PB1.3 and reshaped versions of CY1748 to rsP-Selectin.
  • Fig. 21 shows the binding curves of the chimeric PB1.3 and reshaped versions of CY1748 to rsP-Selectin.
  • Fig. 22 shows the binding curves of the chimeric PB1.3 and reshaped versions of CY1748 to rsP-Selectin.
  • Fig. 23 shows the physical map of the dhfr-plasmid expressing the human heavy chain (gamma-1 constant region).
  • Fig. 24 shows the physical map of the dhfr-plasmid expressing the human heavy chain (gamma-4 constant region).
  • Fig. 25 shows the inhibition of biotinylated-PB1.3 binding to rsP-Selectin by PB1.3 and CY1748 RH A /RL A -IgG1.
  • Fig. 26 shows the inhibition of biotinylated-PB1.3 binding to rsP-Selectin by PB1.3 and CY1748 RH B /RL D -IgG4.
  • Fig. 27 shows that antibody PB1.3 reduces swelling associated with ischemia and reperfusion in the rabbit ear.
  • Fig. 28 shows that antibody PB1.3 prevents the development of necrosis following ischemia and reperfusion of the rabbit ear.
  • Fig. 29 shows that antibody PB1.3 inhibits leukocyte-endothelial cell interactions induced by the mast cell degranulating agent compound 48/80.
  • Fig. 30 shows the proposed folding pattern of the domains of P-selectin.
  • Fig. 31 shows a schematic of the deletion mutants of P-selectin used to map the epitope that PB1.3 recognizes.
  • Fig. 32 shows the binding of PB1.3 and rabbit polyclonal antibodies to P-selectin deletion mutants.
  • Fig. 33 shows the PB1.3 and P6H6 binding to P-selectin and to P-selectin ⁇ 5CRP which is a deletion mutant of P-selectin that lacks the fifth CRP.
  • Fig. 34 shows the sequences of synthetic peptides derived from the amino acid sequence of the human P-selectin 5th CRP and the PB1.3 reactivity to these immobilized
  • Stereoisomers e .g. , D-amino acids
  • conventional amino acids unnatural amino acids such as ⁇ , ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention.
  • Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ -N,N,N-trimethyllysine, ⁇ -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ⁇ -N-methylarginine, and other similar amino acids and imino acids (e . g. , 4-hydroxyproline).
  • amino acids may be modified by glycosylation, phosphorylation and the like.
  • lefthand direction is the amino terminal direction and the righthand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
  • the lefthand end of single-stranded polynucleotide sequences is the 5' end; the lefthand direction of double-stranded polynucleotide sequences is referred to as the 5' direction.
  • RNA transcripts The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".
  • polynucleotide sequence refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes self-replicating plasmids, infectious polymers of DNA or RNA and non-functional DNA or RNA.
  • a “reference sequence” is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, such as a polynucleotide sequence of Figs. 9-17, or may comprise a complete DNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides may each (1) comprise a sequence (i .e . , a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence
  • a “comparison window” refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a
  • polynucleotide sequence may be compared to a reference
  • sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i .e . , gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith & Waterman, Adv . Appl . Math . 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol . Biol . 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl . Acad . Sci .
  • sequence identity means that two polynucleotide
  • sequences are identical (i .e . , on a nucleotide-by-nucleotide basis) over the window of comparison.
  • the term "percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e .g. , A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i .e . , the window size), and multiplying the result by 100 to yield the percentage of sequence
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide
  • the reference sequence may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • the reference sequence may be a subset of a larger sequence, for example, the sequence shown in Figs. 10- 17.
  • sequence identity means peptides share identical amino acids at corresponding positions.
  • sequence similarity means peptides have identical or similar amino acids (i.e.,
  • the term "substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g. , 99 percent sequence identity). Preferably, residue positions which are not identical differ by conservative amino acid substitutions.
  • substantially similarity means that two peptide sequences share corresponding percentages of sequence similarity.
  • substantially pure means an object species is the predominant species present (i .e. , on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all
  • a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the
  • the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • substitutions as conservative or nonconservative, amino acids are grouped as follows: Group I (hydrophobic sidechains): norleucine, met, ala, val, leu, ile; Group II (neutral
  • hydrophilic side chains cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gin, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe.
  • Conservative substitutions involve
  • Non-conservative substitutions constitute exchanging a member of one of these classes for another.
  • Amino acids from the variable regions of the mature heavy and light chains of immunoglobulins are designated Hx and Lxx respectively, where x is a number designating the position of an amino acids according to the scheme of Kabat et al.. Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987) and (1991))
  • Kabat et al. list many amino acid sequences for antibodies for each subclass, and list the most commonly occurring amino acid for each residue position in that subclass. Kabat et al. use a method for assigning a residue number to each amino acid in a listed sequence, and this method for assigning residue numbers has become standard in the field. Kabat et al.'s scheme is extendible to other antibodies not included in the compendium by aligning the antibody in question with one of the consensus sequences in Kabat et al. The use of the Kabat et al. numbering system readily identifies amino acids at equivalent positions in different antibodies. For example, an amino acid at the L50 position of a human antibody occupies the equivalence position to an amino acid position L50 of a mouse antibody.
  • Immunoglobulin refers to an intact antibody or a binding fragment thereof that competes with the intact antibody for specific binding to P-selectin.
  • Substantial inhibition means at least about 50% inhibition, preferably about 60% to about 80%, and more usually about greater than 85% or more (as measured in an in vitro competitive binding assay).
  • Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin gene segments belonging to different species.
  • the variable (V) segments of the genes from a mouse monoclonal antibody may be joined to human constant (C) segments, such as ⁇ 1 and ⁇ 4 .
  • C constant
  • a typical therapeutic chimeric antibody is thus a hybrid protein
  • V or antigen-binding domain from a mouse antibody consisting of the V or antigen-binding domain from a mouse antibody and the C or effector domain from a human antibody, although other mammalian species may be used.
  • This invention provides compositions and methods for inhibiting diseases and conditions of the immune system mediated by P-selectin. Specifically, the invention provides immunoglobulins which have the ability to inhibit P-selectin-mediated adhesion of cells in vivo.
  • the basic antibody structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one
  • each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxyterminal portion of each chain defines a constant region primarily responsible for effector function.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids.
  • variable regions of each light/heavy chain pair form the antibody binding site.
  • the chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope.
  • CDR and FR residues are delineated according to the standard sequence definition of Kabat et al., supra .
  • An alternative structural definition has been proposed by Chothia et al., J. Mol . Biol . 196:901-917 (1987); Nature 342:878-883 (1989); and J. Mol. Biol . 186:651-663 (1989) (hereinafter collectively referred to as "Chothia et al.” and incorporated by reference in their entirety for all purposes).
  • the immunoglobulins (or antibodies) of the invention selectively bind a functional epitope on P-selectin associated with a response to tissue injury and inflammation.
  • binding of the antibodies to a functional epitope on P-selectin effectively inhibits adhesion of leukocytes to activated platelets and/or to the activated vascular endothelial cells
  • blocking antibodies impair the adhesion of leukocytes to the activated vascular endothelium to prevent or inhibit an inflammatory and/or thrombotic condition.
  • mu MAb PB1.3 an exemplary antibody designated mu MAb PB1.3 for specific binding to P-selectin.
  • a hybridoma producing the mu MAb PB1.3 antibody has been deposited with the American Type Culture Collection, Rockville, Maryland under the Budapest
  • Example 1 Other blocking antibodies of the invention compete with a second exemplified antibody designated 84/26.
  • Competition is determined by an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody (e.g., mu MAb PB1.3) to an antigenic determinant on a P-selectin molecule.
  • a reference antibody e.g., mu MAb PB1.3
  • sandwich competition assay see Stahli et al., Methods in Enzymology 9:242-253 (1983)
  • solid phase direct biotin-avidin EIA see Kirkland et al., J. Immunol. 137:3614-3619 (1986)
  • solid phase direct labeled assay solid phase direct labeled sandwich assay (see Harlow and Lane, "Antibodies, A Laboratory Manual, " Cold
  • solid phase direct label RIA using 1-125 label (see Morel et al., Molec . Immunol . 25(1):7- 15 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552 (1990)); and direct labeled RIA (Moldenhauer et al., Scand . J. Immunol . 32:77-82 (1990)).
  • such an assay involves the use of purified P-selectin or cells bearing P-selectin bound to a solid surface, an unlabelled test immunoglobulin and a labelled reference immunoglobulin.
  • Competitive inhibition is measured by
  • test immunoglobulin determining the amount of label bound to the solid surface in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess.
  • An example of a suitable competitive binding assay is presented in Example 8.
  • antibodies include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.
  • a competing antibody when present in excess, it will inhibit specific binding of a reference antibody to P-selectin by at least 10, 25, 50 or 75%.
  • the epitope bound by mu MAb PB1.3 has been mapped to an epitope between amino acids 448 and 467 of human P-selectin. See Figure 34 and Johnson et al.. Cell 56:1033-1044 (1989) (hereby incorporated by reference in its entirety for all purposes). The epitope occurs within the fifth CRP domain of P-selectin. See Figure 30. Antibodies that compete with mu MAb PB1.3 usually bind to a segment of amino acids between 448 and 467 or to another segment within the fifth CRP domain.
  • binding specificity of many blocking antibodies of the invention is further defined by their capacity to bind P-selectin in the complete or substantial absence of Ca ++ (e.g., in the presence of 25 mM EDTA (a calcium chelator) and the absence of Ca ++ in an in vitro assay).
  • Ca ++ e.g., in the presence of 25 mM EDTA (a calcium chelator) and the absence of Ca ++ in an in vitro assay.
  • Antibodies that compete with the mu MAb PB1.3 antibody are further characterized by the observation that their capacity to specifically bind to P-selectin is not inhibited by a fragment of P-selectin having the amino acid sequence CQNRYTDLVAIQNKNE. A molar excess of this fragment usually inhibits specific binding of such antibodies by less than 5%.
  • Mu MAb PB1.3 and competing antibodies are thereby distinguished from reported mouse antibodies designated G1, G2 and G3 (Geng et al . , The Journal of Biological Chemistry
  • some antibodies are capable of blocking specific binding of neutrophils to activated endothelial cells in vivo, without inhibiting platelet aggregation. Such antibodies are particularly useful for treating inflammatory disorders without impairing a patient's capacity to initiate a response to conditions (e.g., wounding) in which platelet aggregation is desirable.
  • the antibodies of the invention usually exhibit a specific binding affinity for P-selectin of at least 10 7 , 10 8 , 10 9 , or 10 10 M -1 .
  • the upper limit of binding affinity of the antibodies for P-selectin is within a factor of about three, five or ten of that of the mu MAb PB 1.3.
  • the lower limit of binding affinity is also within a factor of about three, five or ten of that of mu MAb PB1.3.
  • the term "about” encompasses the small degree of experimental error that may typically occur in the measurement of binding affinities.
  • non-human monoclonal antibodies e.g., murine, lagomorpha, equine
  • production of non-human monoclonal antibodies e.g., murine, lagomorpha, equine is well known and can be accomplished by, for example, immunizing an animal with a preparation containing cells bearing P-selectin (e.g., thrombin-activated platelets), isolated P-selectin molecules or fragments thereof, such as extracellular domains.
  • P-selectin e.g., thrombin-activated platelets
  • isolated P-selectin molecules or fragments thereof such as extracellular domains.
  • Antibody-producing cells obtained from the immunized animals are immortalized and screened, or screened first for the production of antibody which binds to P-selectin, and then, immortalized. See Harlow and Lane, supra. Alternatively, substantially monospecific antibody populations can be
  • human antibodies against P-selectin are provided. Some human antibodies are selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody, such as mu MAb PB1.3. Such antibodies are
  • Human antibodies to P-selectin can be produced by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246:1275-1281 (1989). Antibodies binding to P-selectin or a fragment thereof are selected. Sequences encoding such antibodies (or a binding fragments) are then cloned and amplified. The protocol described by Huse is rendered more efficient in combination with phage-display technology. See, e .g. , Dower et al., WO 91/17271 and
  • McCafferty et al. WO 92/01047 (each of which is incorporated by reference in its entirety for all purposes).
  • libraries of phage are produced in which members display different antibodies on their outersurfaces.
  • Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity are selected by affinity enrichment to an P-selectin polypeptide or fragment thereof.
  • human antibodies having the binding specificity of a selected murine antibody can be produced. See Winter, WO 92/20791.
  • either the heavy or light chain variable region of the selected murine antibody e .g. , mu MAb PB1.3
  • a phage library is constructed in which members displays the same light chain variable region (i.e., the murine starting material) and a different heavy chain variable region.
  • the heavy chain variable regions are obtained from a library of rearranged human heavy chain variable regions.
  • a phage showing strong specific binding for P-selectin e .g.
  • each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region.
  • the light chain variable regions are obtained from a library of rearranged human variable light chain regions. Again, phage showing strong specific binding for L-selectin are selected. These phage display the variable regions of completely human anti-P-selectin antibodies. These antibodies usually have the same or similar epitope specificity as the murine starting material (e.g., mu MAb PB1.3).
  • the invention provides humanized immunoglobulins having variable framework regions substantially from a human immunoglobulin (termed an acceptor immunoglobulin) and complementarity determining regions substantially from a mouse immunoglobulin termed mu MAb PB1.3 (referred to as the donor immunoglobulin).
  • the constant region(s), if present, are also substantially from a human immunoglobulin.
  • the humanized antibodies of the present invention offer several advantages over the mouse donor antibody, which has already shown to be effective in animals models:
  • the human immune system should not recognize the framework or constant region of the humanized antibody as foreign, and therefore the antibody response against such an injected antibody should be less than against a totally foreign mouse antibody or a partially foreign chimeric antibody.
  • the effector portion of the humanized antibody is human, it may interact better with other parts of the human immune system.
  • Injected mouse antibodies have been reported to have a half-life in the human circulation much shorter than the half-life of normal human antibodies (Shaw et al., J.
  • Example 10 The cloning and sequencing of cDNA encoding the mu MAb PB1.3 antibody heavy and light chain variable regions is described in Example 10, and the nucleotide and predicted amino acids sequences are shown in Figs. 11 and 12.
  • Tables 1 and 2 illustrate the subdivision of the amino acid coding sequence into framework and complementarity determining domains. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the numbering convention of Kabat et al., supra .
  • the substitution of mouse CDRs into a human variable domain framework is most likely to result in retention of their correct spatial orientation if the human variable domain framework adopts the same or similar conformation to the mouse variable framework from which the CDRs originated. This is achieved by obtaining the human variable domains from human antibodies whose framework sequences exhibit a high degree of sequence identity with the murine variable framework domains from which the CDRs were derived.
  • the heavy and light chain variable framework regions can be derived from the same or different human antibody sequences.
  • the human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See Kettleborough et al.. Protein Engineering 4:773 (1991); Kolbinger et al.. Protein Engineering 6:971 (1993).
  • Suitable human antibody sequences are identified by computer comparisons of the amino acid sequences of the mouse variable regions with the sequences of known human antibodies. The comparison is performed separately for heavy and light chains but the principles are similar for each. This
  • mu MAb PB1.3 light chain shows greatest sequence identity to human light chains of subtype kappa-1 and that the mu PB1.3 heavy chain shows greatest sequence identity to human heavy chains of subtype 1, as defined by Kabat et al., supra .
  • light and heavy human framework regions are usually derived from human antibodies of these subtypes, or from consensus sequences of such subtypes.
  • the preferred heavy and light chain human variable regions showing greatest sequence identity to the corresponding regions from mu MAb PB1.3 are from antibodies 21/28 'CL and DEN respectively.
  • substitution of certain amino acid residues lead to loss of binding affinity.
  • the selection of amino acid residues for substitution is determined, in part, by computer modelling.
  • Computer hardware and software for producing three-dimensional images of immunoglobulin molecules are widely available.
  • molecular models are produced starting from solved structures for immunoglobulin chains or domains thereof.
  • the chains to be modelled are compared for amino acid sequence similarity with chains or domains of solved three dimensional structures, and the chains or domains showing the greatest sequence similarity is/are selected as starting points for construction of the molecular model.
  • the starting point for modelling was the human light chain DEN.
  • the solved starting structures are modified to allow for differences between the actual amino acids in the immunoglobulin chains or domains being modelled, and those in the starting structure.
  • the modified structures are then assembled into a composite immunoglobulin.
  • the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from one another and that bond lengths and angles are within chemically acceptable limits.
  • Such a model can in turn serve as a starting point for predicting the three-dimensional structure of an antibody containing the mu MAb PB1.3 complementarity determining regions substituted in human framework structures. Additional models can be constructed representing the
  • humanized antibodies of the invention comprise variable framework regions substantially from a human immunoglobulin and complementarity determining regions substantially from a mouse immunoglobulin termed mu
  • MAb PB1.3 Having identified the complementarity determining regions of mu MAb PB1.3 and appropriate human acceptor
  • the next step is to determine which, if any, residues from these components should be substituted to optimize the properties of the resulting humanized antibody.
  • substitution of human amino acid residues with murine should be minimized, because introduction of murine residues increases the risk of the antibody eliciting a HAMA response in humans.
  • Amino acids are selected for substitution based on their possible influence on CDR conformation and/or binding to antigen. Investigation of such possible influences is by modelling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids.
  • the human framework amino acid should usually be substituted by the equivalent mouse amino acid if it is reasonably expected that the amino acid:
  • antigen directly (e.g., amino acids at positions H1, H2, and L60 of mu MAb PB1.3)
  • amino acids from the equivalent position of more typical human immunoglobulins can be substituted with amino acids from the equivalent position of more typical human immunoglobulins.
  • amino acids from equivalent positions in mu MAb PB1.3 can be substituted with amino acids from the equivalent positions in mu MAb PB1.3.
  • an exemplified humanized antibody designated reshaped MAb PB1.3 contains only one substitution of the human heavy-chain variable region framework residues and only two substitutions of the human light-chain variable region framework residues and yet exhibits substantially similar (within a factor of two) binding affinity to a murine or chimeric antibody having intact murine variable regions fused to a human constant region (designated chi MAb PB1.3).
  • Some humanized antibodies of the invention contain a substitution of a human light chain framework residue with a corresponding mu MAb PB1.3 residue in at least 1, 2 or 3, and sometimes 4 of the following positions: L21, L46, L60 and L70. (See Tables 2 and 4). Some humanized antibodies usually contain a substitution of a human heavy chain framework residue in at least 1, 2 or 3 and sometimes 4 of the following positions H1, H2, and H71 and H73. (See Tables 1 and 3) Many humanized antibodies contain substitutions of both light and heavy chain variable framework regions. Preferred antibodies contain a substitution of a human heavy chain framework residue at least at position H71, and substitutions of human light chain framework residues at least at positions 21 and 46.
  • CDR regions in humanized antibodies are substantially identical, and more usually, identical to the corresponding CDR regions in the mu MAb PB1.3 antibody.
  • immunoglobulins are usually substantially identical, and more usually, identical to the framework regions of the human antibodies from which they were derived. Of course, many of the amino acids in the framework region make little or no direct contribution to the specificity or affinity of an antibody. Thus, many individual conservative substitutions of framework residues can be tolerated without appreciable change of the specificity or affinity of the resulting humanized immunoglobulin. However, in general, such substitutions are undesirable.
  • the desired nucleic acid sequences will encode each immunoglobulin amino acid sequence.
  • the desired nucleic acid sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an earlier prepared variant of the desired polynucleotide.
  • Oligonucleotide-mediated mutagenesis is a preferred method for preparing substitution, deletion and insertion variants of target polypeptide DNA. See Adelman et al., DNA 2:183 (1983). Briefly, the target polypeptide DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a single-stranded DNA template. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that incorporates the oligonucleotide primer, and encodes the selected alteration in the target polypeptide DNA.
  • variable segments of humanized antibodies produced as described supra are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Human constant region DNA sequences can be isolated in accordance with well-known procedures from a variety of human cells, but preferably immortalized B-cells (see Kabat et al., supra, and WO87/02671) (each of which is incorporated by reference in its entirety for all purposes).
  • the antibody will contain both light chain and heavy chain constant regions.
  • the heavy chain constant region usually includes CH1, hinge, CH2, CH3, and CH4 regions.
  • the humanized antibodies include antibodies having all types of constant regions, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4. When it is desired that the humanized antibody exhibit
  • the constant domain is usually a
  • the complement-fixing constant domain and the class is typically IgG 1 .
  • the constant domain may be of the IgG 4 class.
  • the humanized antibody may comprise sequences from more than one class or isotype.
  • Nucleic acids encoding humanized light and heavy chain variable regions, optionally linked to constant regions, are inserted into expression vectors.
  • the light and heavy chains can be cloned in the same or different expression vectors.
  • the DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression
  • control sequences include a signal
  • Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly,
  • expression vectors will contain selection markers, e .g. , tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e .g. , U.S. Patent 4,704,362).
  • selection markers e .g. , tetracycline or neomycin
  • E. coli is one prokaryotic host useful particularly for cloning the polynucleotides of the present invention.
  • microbial hosts suitable for use include bacilli, such as Bacillus subtilus , and other enterobacteriaceae, such as Salmonella, Serratia , and various Pseudomonas species.
  • bacilli such as Bacillus subtilus
  • enterobacteriaceae such as Salmonella, Serratia , and various Pseudomonas species.
  • prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
  • Saccharomyces is a preferred host, with suitable vectors having expression control sequences, such as
  • promoters including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
  • mammalian tissue cell culture may also be used to express and produce the
  • polypeptides of the present invention see Winnacker, From Genes to Clones (VCH Publishers, N.Y., N.Y., 1987).
  • Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact
  • immunoglobulins have been developed.
  • immunoglobulins of the invention include: monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293) (Graham et al., J. Gen . Virol .
  • monkey kidney cells (CV1 ATCC CCL 70); african green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); and, TRI cells (Mather, et al., Annals N.Y. Acad. Sci . 383:44-46
  • the vectors containing the polynucleotide sequences of interest e .g. , the heavy and light chain encoding
  • sequences and expression control sequences can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for orokarvotic cells. whereas calcium phosphate treatment or electroporation may be used for other cellular hosts. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989) (incorporated by reference in its entirety for all purposes). When heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins. After introduction of recombinant DNA, cell lines expressing immunoglobulin products are cell selected. Cell lines capable of stable expression are preferred (i.e., undiminished levels of expression after fifty passages of the cell line).
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see generally Scopes, Protein Purification (Springer-Verlag, N.Y., 1982).
  • Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most
  • the recombinant techniques described above can also be used for expression of native sequences encoding human or murine antibodies. This approach is particularly advantageous for expression of human antibodies that are isolated as unstable cell lines.
  • fragments of the intact antibodies described above are provided.
  • Antibody fragments include separate heavy chains, light chains Fab, Fab' F(ab') 2 , Fabc, and Fv.
  • Fragments can be produced by enzymic or chemical separation of intact immunoglobulins.
  • a F(ab') 2 fragment can be obtained from an IgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 using standard methods such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Pubs., N.Y. (1988).
  • Fab fragments may be obtained from F(ab') 2 fragments by limited reduction, or from whole antibody by digestion with papain in the presence of reducing agents. (See id. ) Fragments can also be produced by recombinant DNA techniques. Segments of nucleic acids
  • fragments are produced by digestion of full-length coding sequences with restriction enzymes, or by de novo synthesis. Often fragments are expressed in the form of phage-coat fusion proteins. This manner of expression is advantageous for affinity-sharpening of antibodies as
  • immunoglobulins described above can undergo non-critical amino-acid substitutions, additions or deletions in both the variable and constant regions without loss of binding specificity or effector functions, or
  • immunoglobulins incorporating such antibodies are intolerable reduction of binding affinity (i.e., below about 10 7 M -1 ).
  • immunoglobulins incorporating such antibodies are intolerable reduction of binding affinity (i.e., below about 10 7 M -1 ).
  • a mutated immunoglobulin can be selected having the same specificity and increased affinity compared with a reference immunoglobulin from which it was derived.
  • Phage-display technology offers powerful techniques for selecting such immunoglobulins. See, e.g. , Dower et al., WO 91/17271 McCafferty et al., WO 92/01047; and Huse, WO 92/06204.
  • the invention also provides hybrid antibodies that share the specificity of blocking antibodies against P- selectin but are also capable of specific binding to a second moiety.
  • one heavy and light chain pair is usually from an anti-P-selectin antibody and the other pair from an antibody raised against another epitope. This results in the property of multi-functional valency, i.e., ability to bind at least two different epitopes simultaneously, where at least one epitope is the epitope to which the blocking P-selectin antibody binds.
  • Such hybrids can be formed by fusion of hybridomas producing the respective component antibodies, or by recombinant techniques.
  • Immunoglobulins can also be fused to functional regions from other genes (e.g., enzymes) to produce fusion proteins (e.g., immunotoxins) having novel properties.
  • nucleic acids The intact antibodies and antibody fragments described above are often produced by expression of nucleic acids. All nucleic acids encoding any antibody or antibody described in this application are expressly included in the invention. Modifications of nucleic acids are readily
  • nucleic acids of the invention show substantial sequence identity to nucleic acids encoding the heavy and light chains of mu MAb PB1.3 or the exemplified humanized derivatives thereof. VII. Computers
  • computers programmed to display three dimensional images of antibodies on a monitor or printer are provided.
  • a Silicon Graphics IRIS 4D workstation running under the UNIX operating system and using the molecular modelling package QUANTA
  • the three dimensional image will also identify many noncritical amino acids, which could be the subject of conservative substitutions without appreciable affecting the binding affinity of the antibody.
  • stable cells are produced containing multiple copies of the genes encoding heavy and light chain immunoglobulins in their chromosomes. Multiple copies are obtained by amplification of isolated region(s) of a cell's chromosomal DNA. Amplification is achieved using a selection agent, e.g., methotrexate (MTX), that inactivates a cellular enzyme (e.g., DHFR) that is essential under certain growth conditions. Amplification, i.e., the accumulation of multiple copies of the DHFR gene, results in greater amounts of DHFR being produced in response to greater amounts of MTX. Amplification pressure is applied notwithstanding the presence of endogenous DHFR, by adding ever greater amounts of MTX to the media. Amplification of desired genes (here genes encoding immunoglobulin heavy and light chains) is achieved by linking each gene to a copy of a DHFR gene on the same or separate plasmid(s), and
  • Amplification of the DHFR genes in response to MTX results in a concomitant increase in copy number of the desired immunoglobulin genes.
  • Increased copy number of the desired genes results in greater expression of the desired heterologous protein, stable cell lines expressing about 10-100, 20-50 or about 30 ⁇ g humanized immunoglobulin per 10 6 cells per day are preferred.
  • fragments of P-selectin comprise amino acids from the fifth C3b-C4b regulatory domain of P-selectin (fifth CRP).
  • the fragments contain the epitope bound by mu MAb PB 1.3 and/or proximal epitopes bound by competing antibodies.
  • the fragments usually contain up to 5, 10, 20, 25, 50, 75, 100 or 200 amino acids in total. Most fragments contain some or all of the amino acids between positions 448 and 467 of the human P-selectin sequence shown in Fig. 34. These amino acids define the epitope specifically bound by mu MAb PB1.3. In many fragments, at least five contiguous amino are from between positions 448 and 467 of the amino acid sequence shown in Fig.
  • fragments contain the entire contiguous segment of amino acids between positions 448 and 467. Some fragments consist essentially of this contiguous segment of amino acids. In these fragments, no other amino acids are present that contribute to the binding specificity or affinity of the fragment. Often, no other amino acids of any
  • the polypeptide fragments of the invention have a variety of uses.
  • the fragments are useful as immunogens for generating antibodies that compete with mu MAb PB 1.3 for specific binding to P-selectin.
  • the use of defined fragments as immunogens reduces the extent of screening required to isolate an antibody of desired specificity.
  • the polypeptide fragments are also useful in the diagnostic and therapeutic methods described infra . Some fragments compete with full-length P-selectin molecules for binding to activated
  • these fragments are useful for aborting diseases and conditions of the immune system mediate by interactions of P-selectin with its ligand(s) on activated neutrophils. Some fragments are also useful for monitoring the presence of activated neutrophils by binding to ligands to P-selectin present on the surface of these cells.
  • the small size of the fragments renders them particularly suitable for effective delivery following in vivo administration.
  • the exclusively human origin of the fragments reduces the risk of side-effects that might occur with some other agents.
  • the therapeutic methods employ the antibodies (whole and binding fragments), and fragments of P-selectin discussed above as therapeutic agents for treatment of various diseases.
  • the therapeutic agents are useful for prophylactic and
  • diseases and disorders include transplant rejection, graft versus host disease, autoimmune diseases such as insulin-dependent diabetes mellitus, multiple sclerosis, stiff man syndrome, rheumatoid arthritis, myasthenia gravis and lupus erythematosus, and inflammatory disorders.
  • the agents are also useful for preventing tumor metastasis inhibiting the adhesion of circulating cancer cells, such as carcinomas of the colon and melanoma.
  • Some therapeutic agents function by blocking or otherwise antagonizing the action of a P-selectin molecule with its ligand.
  • Other therapeutic agents function by killing cells bearing a polypeptide against which the agent is targeted.
  • the therapeutic agents are particularly suitable for treatment of inflammatory and thrombotic conditions including post-ischemic leukocyte-mediated tissue damage (reperfusion injury) arising from traumatic shock, stroke, myocardial infarction, acute transplantation rejection, frost-bite injury, compartment syndrome, and pathophysiologic conditions associated with cardio-pulmonary bypass, acute leukocyte-mediated lung injury (e.g., adult respiratory distress syndrome), septic shock, wound associated sepsis secondary to viral infection by e.g., herpes simplex virus, IgE-mediated allergic reactions such as acute phase asthmatic disease, and chronic inflammatory conditions, including rheumatoid
  • Ischemia/reperfusion injury is an inflammatory condition that occurs on restoring blood flow to organs suffering from an obstructed supply causing ischemia (oxygen deprivation). Unless rapidly relieved by reperfusion, ischemia causes death of surrounding cells, and eventually, death of a whole organ or patient. However, accumulating evidence suggests that reperfusion may itself exert
  • ischemia neutrophils in the restored blood flow.
  • Some patients have whole-body ischemia, whereas in other patients ischemia is confined to particular parts or organs of the body.
  • a patient may suffer from epidermal, myocardial, renal, cerebral, splenic, hepatic, spinal, splanchnic, pulmonary, partial-body, or whole-body ischemia.
  • therapeutic agents of the invention function by antagonizing the interaction of such lymphocytes with P-selectin.
  • mast cell degranulation includes a bronchoconstrictor response and also an inflammatory response characterized in part by leukocyte accumulation. Histamine, which is contained together with other inflammatory mediators in mast cells, can induce the expression of P-selectin on vascular endothelial cells, indicating that degranulation of mast cells may also result in P-selectin expression and subsequent leukocyte accumulation. Because mast cell degranulation is a key element in the pathogenesis of allergic conditions, administration of antibodies to P-selectin will be useful for the treatment of human allergic conditions.
  • the methods are particularly useful for humans, but may also be practiced on veterinary subjects.
  • the therapeutic methods are usually applied to organs present in living subjects. However, some methods, such as ischemia-reperfusion therapy, are equally applicable to dissected organs,
  • Therapeutic methods can also be performed ex vivo.
  • Therapeutic agents and compositions targeted against P-selectin can also be used in combination with agents
  • Suitable immunoglobulins include those specific for CD11a, CD11b, CD18, E-selectin, L-selectin and ICAM-1.
  • immunoglobulins should bind to epitopes of these adhesion molecules so as to inhibit binding of leukocytes, particularly neutrophils, to endothelial cells.
  • suitable antibodies for use in combination therapies are those specific for lymphokines, such as IL-1, IL-2 and IFN- ⁇ , and their
  • Such antibodies serve to block activation of endothelial cells, and thereby prevent their interaction with neutrophils in an inflammatory response.
  • the blocking P-selectin antibodies and pharmaceutical compositions of this invention are particularly useful for parenteral administration, i.e., subcutaneously, intramuscularly or intravenously.
  • parenteral administration i.e., subcutaneously, intramuscularly or intravenously.
  • pharmaceutical compositions of the present invention are suitable for parenteral administration, i.e., subcutaneously, intramuscularly or intravenously.
  • the blocking P-selectin antibodies can be directly or indirectly coupled to the chemotherapeutic agent.
  • the coupling which may be performed by means, generally known in the art, should not substantially inhibit the ability of the immunoglobulin to bind the receptor nor should it
  • chemotherapeutics can be coupled for targeting.
  • anti-inflammatory agents which may be coupled include immunomodulators, platelet activating factor (PAF) antagonists, cyclooxygenase inhibitors, lipoxygenase inhibitors, and leukotriene antagonists.
  • PAF platelet activating factor
  • Some preferred moieties include cyclosporin A, indomethacin, naproxen, FK-506, mycophenolic acid, and the like.
  • antioxidants e.g., superoxide dismutase, are useful in treating reperfusion injury.
  • anticancer agents such as daunomycin, doxorubicin, vinblastine, bleomycin, and the like can be targeted.
  • the P-selectin targeting may also be accomplished via amphipaths, or dual character molecules (polar:nonpolar) which exist as aggregates in aqueous solution.
  • Amphipaths include nonpolar lipids, polar lipids, mono- and diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids and salts. These molecules can exist as emulsions and foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions and lamellar layers. These are generically referred to herein as liposomes.
  • the drug to be delivered is incorporated as part of a liposome in which an anti-P-selectin immunoglobulin is embedded.
  • the immunoglobulin need not bind a functional epitope on the P-selectin molecule, so long as the
  • immunoglobulin effectively targets the liposome to P-selectin molecules.
  • the liposomes When the liposomes are brought into proximity of the affected cells, they deliver the selected therapeutic compositions.
  • Antibody targeted liposomes can be constructed using, for instance, liposomes which incorporate protein A (see Renneisen, et al., J. Biol . Chem . , 265:16337-16342 (1990) and Leonetti et al., Proc. Natl . Acad. Sci . (USA) 87:2448-2451 (1990).
  • compositions for parenteral administration usually comprise a solution of a therapeutic agent (e.g., an antibody against P-selectin (intact or binding fragment or a P-selectin fragment) or a cocktail of several such agents dissolved in an acceptable carrier, preferably an aqueous carrier.
  • a therapeutic agent e.g., an antibody against P-selectin (intact or binding fragment or a P-selectin fragment) or a cocktail of several such agents dissolved in an acceptable carrier, preferably an aqueous carrier.
  • aqueous carriers e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like.
  • These solutions are sterile and generally free of particulate matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH-adjusting and buffering agents, tonicity adjusting agents and the like, for example sodium a
  • formulations can vary widely, i.e., from less than about 0.5%, usually at or at least about 0.1% to as much as 1.5% or 2.0% by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and ate described in more detail in, for example, Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Company, Easton, Pennsylvania
  • the antibodies of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilization and reconstitution techniques can be employed. Lyophilization and reconstitution can lead to varying degrees of antibody activity loss (e.g., with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted to compensate.
  • compositions containing the present antibodies or a cocktail thereof can be administered for the prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient in an amount sufficient to cure or at least partially arrest the infection and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from about .05 mg/kg body weight to about 5 mg/kg body weight, preferably between about .2 mg/kg body weight to about 1.5 mg/kg body weight.
  • the materials of this invention may generally be employed in serious disease states, that is, life-threatening or potentially life-threatening situations. In such cases, in view of the minimization of extraneous substances and the lower probability of "foreign substance” rejections (e.g.,
  • HAMA which are achieved by human or humanized antibody forms of this invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these antibodies.
  • compositions containing the present antibodies or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance.
  • Such an amount is defined to be a "prophylactically effective dose.”
  • the precise amounts again depend upon the patient's state of health and general level of immunity, but is generally in the ranges described above.
  • compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • pharmaceutical formulations should provide a quantity of the immunoglobulins of this invention sufficient to treat the patient effectively.
  • the antibodies of invention can also be used for diagnostic purposes.
  • An amount sufficient for these purposes is defined to be a "diagnostically effective dose.” In diagnostic uses, the precise amounts will depend upon the patient's state of health, mode of administration, and the like.
  • the antibodies may either be labeled or unlabeled.
  • Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the antibody, such as antibodies specific for the particular immunoglobulin constant region. Alternatively, the antibodies can be directly labeled. A wide variety of labels may be employed, such as radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands
  • the antibodies are useful for detecting the presence of cells bearing the P-selectin receptor.
  • the presence of such cells is diagnostic of an inflammatory condition or disease and may signal the need for commencement of a therapeutic method discussed supra .
  • Diagnosis can be accomplished by removing a cellular sample from a patient. The amount of expressed P-selectin receptor in individual cells of the sample is then determined, e .g. , by immunohistochemical staining of fixed cells or by Western blotting of a cell extract with an antibody of the invention. Fragments of P-selectin are useful for monitoring the presence of activated neutrophils, a presence that can be indicate of an undesirable immune response.
  • Diagnosis can also be achieved by in vivo administration of a labelled antibody (preferably a humanized or human antibody) and detection by in vivo imaging.
  • concentration of MAb administered should be sufficient that the binding to cells having the target antigen is detectable compared to the background signal.
  • the diagnostic reagent can be labelled with a radioisotope for camera imaging, or a paramagnetic isotope for magnetic resonance or electron spin resonance imaging.
  • a change typically an increase in the level of P-selectin protein in a cellular sample or imaged from an individual, which is outside the range of clinically
  • established normal levels may indicate the presence of an undesirable inflammatory response reaction in the individual from whom the sample was obtained, and/or indicate a
  • P-selectin can also be employed as a differentiation marker to identify and type cells of certain lineages and developmental origins. Such cell-type specific detection can be used for histopathological diagnosis of undesired immune responses.
  • Kits can also be supplied for use with the subject antibodies.
  • the subject antibody composition of the present invention may be provided, usually in a lyophilized form in a container, either alone or in conjunction with additional antibodies specific for the desired cell type.
  • the antibodies which may be conjugated to a label or toxin, or unconjugated, are included in the kits with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., serum albumin, or the like, and a set of instructions for use.
  • buffers such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., serum albumin, or the like
  • these materials will be present in less than about 5% wt. based on the amount of active antibody, and usually present in total amount of at least about 0.001% wt. based again on the antibody
  • the second antibody capable of binding to an anti-P-selectin antibody is employed in an assay, the second antibody will usually be present in a separate vial.
  • the second antibody is typically conjugated to a label and formulated as described above.
  • Heparin-Agarose Chromatography The sample, in 20 mM Tris, pH 7.5, was applied to a 1 ml column of Heparin-Agarose, the column was washed with the same buffer, and eluted with 20 mM Tris, 1.5M NaCl, pH 7.5.
  • ACD Acid Citrate Dextrose anticoagulant
  • the needle was removed and the blood was transferred to two sterile 50 ml tubes.
  • the supernatant was centrifuged at 1200 rpm (approx.
  • the platelets were washed as follows: 2 ml Tyrode- Hepes Buffer pH 6.5, containing Prostaglandin, E 1 (PGE 1 at a final concentration of 100nM was added and the platelets were gently resuspended. A further 10 ml of the same buffer was added and the sample was centrifuged at 3000 rpm for 10 min.
  • Step #8 was repeated once more and then the
  • the platelets were counted in a Coulter Counter and diluted to 2 ⁇ 10 8 /ml.
  • the pH of the platelet suspension was adjusted to 7.2, and the amount of thrombin (Human thrombin- Sigma) required for maximal activation was
  • Maximal activation i.e.. maximal thrombin-induced aggregation appears to correspond to maximal expression of P-selectin.
  • thrombin The required amount of thrombin was added to the platelet preparation and the mixture was allowed to stand at room temperature for 20 min without stirring to prevent aggregation.
  • splenocytes Four days after the final boost the spleen was removed and 1.2 ⁇ 10 8 splenocytes were recovered. These were fused with FOX-NY myeloma cells using PEG 1500 (BMB) using the following protocol: The isolated splenocytes were washed twice in serum-free cell culture medium. Splenocytes and myeloma cells were combined at a ratio of 1:4.8 (myelomas to spleen cells). The combined cell pellet was washed twice in serum free medium, then aspirated to dryness. The cell pellet was resuspended by gentle tapping and heated in a waterbath at 37°C for 1 min. The pellet was distributed around the sides and bottom of a 50 ml conical centrifuge tube.
  • This example describes preparation of mAB PNB1.6 a monoclonal antibody to P-selectin that does not inhibit binding of thrombin-activated platelets to neutrophils.
  • the spleen was removed and the splenocytes were recovered. They were fused with FOX-NY myeloma cells using PEG 1500 (Sigma) generally as described in Oi et al., "Immunoglobulin-Producing Hybrid Cell Lines" in Selected Methods in Cellular Immunology, eds. Mishell and Shiigi, pp 351-372, 1980, which is incorporated herein by reference.
  • This example describes screening of supernatant media from fused cells produced in Examples 2 and 3.
  • Example 2 Supernatants from Example 2 were tested by ELISA assay against (a) thrombin-activated platelets, (b) purified P-selectin and (c) recombinant P-selectin. Supernatants from Example 2 were tested by ELISA assay against thrombin-activated platelets. Each of these assays is described below. Preparation of the reagents used in each of the assays is described in Example 1.
  • a 96 well flat bottom COSTAR plate was coated with 0.1% gelatin (2% gelatin-Sigma) by adding 100 ⁇ l/well and incubating at 37oC for 15 min.
  • activated platelets (10 8 /ml) were added to each well.
  • the plate was centrifuged at 800 rpm (90xg) for 2 min.
  • the plate was washed x7 with PBS.
  • the plate was allowed to develop at room temperature for up to 30 min.
  • the OD was measured at 414nm in a Titertek Multiskan MCC/340.
  • PBS Phosphate-buffered saline
  • the plate was washed once with Dulbecco's PBS (DPBS) and blocked with 200 ⁇ l DPBS containing 1% BSA for 30 min at room temperature.
  • DPBS Dulbecco's PBS
  • This example describes the detection of P-selectin by western blotting.
  • Samples containing P-selectin were mixed with an equal volume of SDS-PAGE sample buffer (non-reducing), heated at 100°C, run on a Novex 8-16% gradient gel, and transferred electrophoretically to nitrocellulose.
  • the nitrocellulose membrane was then blocked for at least 1 h in phosphate-buffered saline (PBS) +1% bovine serum albumin
  • This example provides data demonstrating the ability of blocking P-selectin antibody of the invention to treat acute lung injury.
  • systemic complement activation was produced by vascular infusion of the cobra venom factor (CVF) into rats.
  • CVF cobra venom factor
  • MPO myeloperoxidase activity
  • Fig. 3B endothelial cells
  • an additional group of rats was infused with CVF and animals sacrificed at times 0, 5, 10, 15, 20 and 60 min later.
  • Lungs were inflated with O.C.T., snap frozen, and sections obtained and examined for presence of Pyselectin by immunohistochemical techniques, using mu MAb PB1.3.
  • Little detectable reactivity in the pulmonary vasculature was found at time 0, whereas staining was clearly evident at 5 min and was increased at 15 and 20 min after infusion of CVF.
  • the pattern of staining involved pulmonary venules and septal areas in a pattern consistent with staining of interstitial capillaries. At 60 min.
  • activated platelets with mu MAb PNB1.6 did not affect the ability of mu MAb PB1.3 to block their P-Selectin mediated adhesion to neutrophils.
  • the monoclonal antibodies PNB1-6 and 84/26 were isolated from tissue culture supernatants by passage over a column of protein G Sepharose 4 Fast Flow (Pharmacia). The column was washed extensively with phosphate-buffered saline (PBS), and bound antibody was eluted with 0.1M glycine-HCl pH 2.7 into tubes containing 0.2 to 0.5 volume 1M Tris, pH 8.8. The procedure used for isolating mu MAb PB1.3 was identical except that the antibody bound to protein G was eluted with 0.1M acetate-HCl pH 2.5 into tubes containing 0.3 volumes 2M Tris, pH 10. Antibody containing
  • fractions were pooled, and dialyzed against several changes of PBS at 4°C.
  • DPBS Dulbecco's phosphate-buffered saline
  • Centriprep 30 spin concentrator (Amicon), aliquoted, and stored at -80oC.
  • Fmoc-Amide resin (Bachem Biosciences) was loaded into the peptide synthesis reaction vessel and washed one time with N-methylpyrolidone (NMP). The following operations were then sequentially performed:
  • Steps (3) and (4) were repeated two more times.
  • Steps 1-6 were repeated for each amino acid of the peptide.
  • the resin-bound peptide was deprotected by reaction with 25% piperidine in NMP, washed 7 times with NMP, and washed 2 times with dichloromethane.
  • the resin was dried in vacuo for 24 h.
  • the peptide was cleaved from the resin by treatment with trifluoroacetic acid containing 2.5% ethanedithiol, 5% thioanisole, 7.5% phenol, and 5% water.
  • the polystyrene resin was separated from the trifluoroacetic acid solution by filtration. Trifluoroacetic acid was removed by evaporation in vacuo.
  • microtiter plates (Falcon Microtest III) were coated overnight at 4°C with 50 ⁇ l/well of 2 ⁇ g/ml affinity purified P-selectin in DPBS.
  • mu MAbs PB1.3, PNB1.6, and 84-26 all bind to a single band of 140 KD when platelets are dissolved in SDS-PAGE running buffer (unreduced), subjected to SDS-PAGE, and transferred to nitrocellulose (data not shown).
  • mu MAbs PB1.3 and PNB1.6 no longer recognize P-selectin on western blots when the samples have been reduced with ⁇ - mercaptoethanol.
  • Plate l was blocked with 200 ⁇ l/well PBS+1% BSA, while plate 2 was blocked with 200 ⁇ l/well DPBS+1% BSA (DPBS contains Ca ++ and Mg ++ ).
  • DPBS contains Ca ++ and Mg ++
  • the plates were washed (all washes for plate 1 were PBS, for plate 2, with DPBS.
  • 25 ⁇ l of 25 mM EDTA in PBS+1% BSA was added to the wells of plate 1, and 25 ⁇ l of DPBS+1% BSA was added to plate 2.
  • 25 ⁇ l of dilutions of the appropriate antibody were added to the wells of either plate. Dilutions for plate 1 were made in PBS+1% BSA, for plate 2 in DPBS+1% BSA.
  • Fig. 4 shows the binding of the anti-P-selectin monoclonal antibodies to P-selectin in the buffer DPBS, which contains CA ++ and Mg ++ , and in PBS + 25 mM EDTA, a chelator of divalent metal cations.
  • concentration of divalent cations present in DPBS is more than sufficient to support neutrophil adhesion to P-selectin (Geng et al., 1991), while the chelator EDTA, at 25 mM, is more than sufficient to block neutrophil adhesion to P-selectin.
  • Mu MAb PB1.3 is a blocking antibody, that is, it is able to block the binding of neutrophils P-selectin, for example on activated platelets.
  • the PNB1.6 is a non-blocking antibody. The ability of 84/26 to block the binding of neutrophils to P-selectin has not yet been fully
  • microtiter plate and incubated for 1 hr.
  • the plate was washed with DPBS, and a 1 to 1000 dilution of sheep anti-mouse IgG horseradish peroxidase conjugate in DPBS+1% BSA was added. After 1 hr, the plate was washed, the substrate TMB was added, and the color development was stopped and the plates read as above.
  • Fig. 5 shows that the peptide CQNRYTDLVAIQNKNE, homologous to residues 19-34 of the lectin domain of P-selectin, has no effect on the binding of mu MAb PB1.3 to P-selectin when the peptide is present at a concentration of 0.35 mg/ml. This distinguishes this particular monoclonal antibody from the monoclonal antibodies G1, G2, and G3, whose binding to P-selectin is partially or completely blocked by this peptide.
  • Fig. 6 shows the ability of the mu MAbs PB1.3, PNB1.6, and 84/26 to interfere with the binding of each other to P-selectin.
  • Fig. 6A shows that only mu MAb PNB1.6 is able to block the binding of biotinylated mu MAb PNB1.6 to P-selectin, demonstrating that mu MAbs PB1.3 and 84/26 must recognize different epitopes from mu MAbs PNB1.6.
  • mu MAb PB1.3, but not mu MABs PNB1.6 or 84/26 is able to block the binding of biotinylated PB1.3 to selectin, demonstrating that the other two monoclonals must recognize different epitopes.
  • Fig. 6C both mu MAbs 84/26 and PB1.3, but not mu MAb PNB1.6, are able to block the binding of biotinylated mu MAb 84/26 to P-selectin.
  • transducer for the measurement of arterial blood pressure.
  • a midline thoracotomy was performed, the pericardium was opened and the heart was exposed.
  • a 2-0 silk suture was carefully placed around the left anterior descending artery (LAD) 10-12 mm from its origin. After a 30 min period of stabilization myocardial ischemia was initiated by complete ligation of the LAD for 1.5 h of ischemia followed by 4.5 h of reperfusion.
  • Blocking (mu MAb PB1.3) and non-blocking (mu MAb PNB1.6) anti- P-selectin antibodies were administered intravenously at a dose of 1 mg/kg 10 min prior to the initiation of reperfusion.
  • the ischemic myocardium was determined as that portion of tissue which did not stain with nitroblue
  • Area at risk was determined by reocclusion of the LAD at the end of the reperfusion period followed by the injection of Evans blue dye into the left atrium. The area of risk was, therefore, determined by negative staining.
  • Endothelial dependent relaxation of coronary artery rings was determined by measuring the acetylcholine-induced relaxation of rings previously contracted with the thromboxane A2 mimetic U46619. Data are expressed as percentage
  • Fig. 8 In cats treated with the non-blocking anti-P-selectin antibody mu MAb PNB1.6 the extent of myocardial ischemia was 33 ⁇ 5% of the area at risk. In contrast, the extent of ischemia in PB1.3 treated animals was significantly (P ⁇ 0.01) less at 15 ⁇ 3% of the area at risk. Endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries taken from cats treated with mu MAb PB1.3 compared to mu MAb PNB1.6 (67 ⁇ 6 vs 11 ⁇ 3%, P ⁇ 0.01).
  • Example 2 Total RNA was isolated from hybridoma cells producing mu MAb PB1.3.
  • the cDNAs encoding the variable regions of the heavy and light chains of mu MAb PB1.3 were amplified by polymerase chain reaction (PCR).
  • the entire mouse variable region was amplified using a mixture of
  • a molecular model of the V L and V H regions of mu MAb PB1.3 was built.
  • the model was built on a Silicon Graphics IRIS 4D workstation running under the UNIX operating system and using the molecular modelling package QUANTA (Polygen Corp., USA).
  • the amino acid sequence of the mu MAb PB1.3 variable regions were compared with the sequences of
  • variable regions were selected that have framework regions most similar to those found in the mu MAb PB1.3 variable regions.
  • variable region were selected as frameworks to join the corresponding complementarity determining regions (CDRs) of mu MAb PB1.3.
  • CDRs complementarity determining regions
  • CY1748RH A Three versions of the heavy chain variable region were modelled and were designated CY1748RH A, CY1748RH B , and CY1748RH C .
  • the nucleotide sequences and corresponding amino acid sequences of these variable regions and signal peptides are given in Figs. 11, 12, and 13, respectively.
  • CY1748RH B the first two mouse residues conserved in
  • version CY1748RH C substitutes the murine lysine at position 73 of CY1748RH A .
  • Version CY1748RH B contains the highest number of human amino acid residues. A summary of the amino acid differences among these three versions of the heavy chain is given in Table 3.
  • CY1748RL C and CY1748RL D .
  • the nucleotide sequences and corresponding amino acid sequences of these variable regions and signal peptides are given in Figs. 14, 15, 16 and 17, respectively.
  • Computer modelling suggested that the murine residue glutamic acid at position 60 might be important in P-selectin binding.
  • This residue was incorporated in version CY1748LR A but replaced with the original human framework residue serine in version CY1748LR B .
  • the murine aspartic acid residue might have a charge interaction with residue 24 in loop 1 (L1), therefore CY1748LR C is identical to CY1748LR A except for the replacement of the mouse residue aspartic acid at position 70 for the human framework amino acid glutamic acid.
  • CY1748LR D incorporates both these amino acid
  • the expression vector contains the human cytomegalovirus (HCMV) enhancer and promoter to drive
  • a HindIII-SacII fragment from a pUC8 vector containing the PstI-m fragment of HCMV (Boshart et al., Cell 41:521-530 (1985)) was converted to an EcoRI-HindIII fragment using appropriate adaptor oligonucleotides.
  • the 1.2 kb EcoRI-HindIII fragment containing the HCMV enhancer-promoter was ligated to a 5.05 kb BamH-EcoRI fragment from pSV2neo (Southern and Berg, J. Mol . App. Gener. 1:327-341 (1982)) and a 0.5 kb HindIII-BamHI fragment containing the V L lys kappa light variable region (Foote and Winter, J. Mol . Biol .
  • This vector began with the same three way ligation as described above for generation of pHCMV-V L lys-neo above save for the insertion of a 0.7 kb HindIII-BamHI fragment containing the V H lys heavy variable region
  • cDNA coding for human ⁇ 1 constant region was inserted into the BamHI site to produce a vector designated pHCMV-V H lys-neo.
  • cDNA coding for human ⁇ 1 constant region was cloned from a human cell line secreting a human ⁇ 1 antibody by PCR amplification. BamHI sites were created at each end of the cDNA, and a splice acceptor site and a 65bp intron sequence were introduced at the 5-end of the cDNA sequence.
  • the BamHI fragment (1176bp) containing the human ⁇ 1 cDNA plus the splice acceptor site and intron sequence was then cloned into the expression vector.
  • the BamHI site at the 3'-side of the human ⁇ 1 constant region was removed by filling-in with Klenow polymerase. with this vector,
  • the cDNAs encoding the heavy chain variable region of mu MAb PB1.3 and the three humanized versions were inserted into the expression vector HCMV ⁇ 1C-neo by standard techniques generating expression plasmids HCMV-1747CH- ⁇ 1C-neo, HCMV-1748RH A - ⁇ 1C-neo, HCMV-1748RH B - ⁇ 1C-neo, and HCMV-1748RH C - ⁇ 1C-neo.
  • the nucleotide sequence of the human IgG1 constant region cDNA and corresponding protein sequence is given in Ellison et al., Nuc. Acids Res . , 10:4071-4079 (1982).
  • CY1747(PB1.3) and the four humanized versions were inserted into the expression vector HCMV-KR-neo generating plasmids HCMV-1747CL-KR-neo, HCMV-1748RL A -KR-neo, etc.
  • chi MAb PB1.3 i.e., chimeric antibody comprising murine variable domains and human constant domains.
  • DNA was introduced into the COS cells by electroporation using the Gene Pulser apparatus (Biorad). COS cells were trypsinized and washed once in phosphate buffered saline (PBS). DNA (10 ⁇ g of each heavy chain plasmid and appropriate light chain plasmid) and a 0.8 ml aliquot of 1 ⁇ 10 7 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. After a 10 minute recovery period at ambient temperature, the
  • electroporated cells were added to 20 ml of DMEM media
  • ELISA to quantify levels of human antibody produced and analyze capacity to bind to recombinant soluble P-selectin (rsP-selectin).
  • Immulon microtiter plates (Dynatech, #011- 010-3355) were coated with 100 ⁇ l/well of a solution of goat anti-human IgG (whole molecule, Sigma #1-1886) to a final concentration of 12.5 ⁇ g/ml or a solution of rsP-selectin (1.2 mg/ml) diluted in Dulbecco's phosphate buffered saline (DPBS). Plates were coated overnight at 4oC or for 2 h at 37oC. The plates were then washed three times with DPBS. The plates were then blocked with 400 ⁇ l/well of DPBS + 1% bovine serum albumin (BSA, Sigma, #A-7888) for one h or longer at room temperature. The plates were then washed three times with DPBS.
  • BSA bo
  • substrate/H 2 O 2 (TMB, Kirkegaard and Perry Laboratories #50-76-00) was added to the plates, and the color was allowed to develop for an appropriate period of time (usually 3 to 15 min), and the reaction was quenched by the addition of 100 ⁇ l/well of 1-M phosphoric acid.
  • the plates were read at 450 nm in a Titertek Multiskan MCC/340.
  • the concentration of the antibodies produced in all COS cell supernatants was determined by comparing the IgG ELISA results to a human IgG1 antibody (kappa light chain, Sigma #1-3889) of known concentration. P-selectin binding was plotted as optical density against the concentration of IgG. Binding curves of chi PB1.3 and various reshaped versions of reshaped MAb PB1.3 are given in Figs. 20, 21, and 22. The objective was to select a humanized antibody showing substantially indistinguishable binding characteristics from chi MAb PB1.3.
  • the heavy chain variable regions CY1748RH A and CY1748RH B when coexpressed with light chain variable regions CY1748RL A and CY1748RL D also generated antibodies that bound to rsP-selectin to the same extent as chi MAb PB1.3 (Fig. 22).
  • the CY1748RH B heavy chain is preferable because it contains the most human amino acid residues.
  • a preferred humanized antibody is formed from light chain D and heavy chain B and is designated reshaped MAb PB1.3
  • This vector is identical to pHCMV-1748RH- ⁇ 1C-neo described in Example 11 except that the neo gene is replaced by the dihydrofolate reductase (dhrf) gene linked to a
  • pSV2-dhfr (Subramani et al., Mol . Cell . Biol . 1:854-864 (1981)) was digested with SphI and PvuII, filled-in with Klenow polymerase, and self-ligated to yield pSV2-dhfr- ⁇ E.
  • the BamHI site on the 3'-side of the human ⁇ 1 constant region was then removed by filling in with Klenow polymerase as in Example 11.
  • the resulting vector was designated pHCMV-174RH- ⁇ C-dhfr.
  • the HindIII-BamHI fragment of this vector containing the unwanted V H lys is easily replaced with the V H of a reshaped antibody.
  • This vector is identical to pHCMV-1748RH- ⁇ 1C-dhfr described above, but for the replacement of the cDNA clone of the human ⁇ 1 constant region with a genomic clone of the human 74 constant region.
  • a - 7.0 HindIII fragment of DNA containing the genomic clone of the human ⁇ 4 constant region was subcloned into the HindIII site of pUC19 to create a plasmid called 428D.
  • the subclone was inserted into pUC19 in the orientation that placed this site distal to the BamHI site in the polylinker of pUC19.
  • the 7.0 kb human 74 fragment was excised from 428D using BamHI and ligated into pHCMV-V H lys-neo to create the plasmid pHCMV-V H lys- ⁇ 4-neo.
  • the final plasmid was constructed by a three-way ligation of a 5.4 kb BamHI-HindIII fragment containing the HCMV enhancer-promoter and the pSV2-dhfr-AE plasmid sequence, a 0.5 kb HindIII-BamHI fragment containing the VH of reshaped human 1748 antibody and the 7.0 kb BamHI-BamHI fragment containing the genomic clone of the human ⁇ 4 constant region.
  • (2) Insertion of Immunoglobulin Coding Sequences The cDNA encoding 1748RH A heavy chain variable region was cloned into expression vector HCMV-1C-dhfr
  • HCMV-1748RH A -1C-dhfr The cDNA encoding 1748RH B was cloned into expression vectors HCMV-1C-dhfr and HCMV-4C-dhfr generating the plasmids HCMV-1748RH B -1C-dhfr and HCMV-1748RH B -4C-dhfr, respectively.
  • the nucleotide sequence of the human IgG4 constant region gene and corresponding amino acid sequence is given by Ellison et al., DNA, 1:11-18 (1981).
  • the vector HCMV-1C-dhfr expresses an IgG1 isotype antibody and vector HCMV- ⁇ 4C-dhfr expresses an IgG4 isotype antibody when they are each coexpressed with the kappa light chain vector.
  • the schematic maps of the dhfr-expression vectors are given in Figs. 23 and 24, respectively.
  • CHO dhfr-deficient cells were grown in ⁇ MEM (+ nucleosides, GIBCO/BRL) and 10% fetal bovine serum. Plasmid DNA was introduced into the COS cells by electroporation using the Gene Pulser apparatus. CHO cells were tryp ⁇ inized and washed once in phosphate buffered saline (PBS). DNA (10 ⁇ g of each heavy chain plasmid and appropriate light chain plasmid) and a 0.8 ml aliquot of 1 ⁇ 10 7 cells/ml in PBS were placed in a sterile Gene Pulser Cuvette (0.4 cm gap). A pulse was delivered at 1900 volts, 25 microfarads capacitance. After a 10 minute recovery period at room temperature, the
  • nucleosides 10% FBS.
  • FBS 500 ⁇ g/ml G418 (GIBCO/BRL, to select for the expression of the neo-containing plasmid).
  • Media was changed every 3-4 days until colonies emerged. Single clones were isolated via cloning cylinders, expanded and analyzed for IgG production via ELISA.
  • CHO cells were expanded and seeded into 10-chamber (6000 cm 2 total surface area) Nunc cell factories.
  • the medium was harvested at 72 h and passed over a protein-A Sepharose Fast Flow (Pharmacia) column.
  • the column was washed and bovine IgG was eluted at pH 4.5.
  • the humanized antibodies were eluted at pH 3.5, dialyzed in PBS or 20 mM acetate, 0.15 M NaCl, pH 5.5.
  • Antibody concentration was determined spectrophotometrically and confirmed by an ELISA for human IgG.
  • a purified human IgG4 antibody (kappa, Sigma #1-4639) was used as the control for mu MAb PB1.3 (H B /L D ) IgG4
  • the relative binding affinities of the various versions of reshaped MAb PB1.3 to P-selectin were determined by competitive ELISA assay.
  • the assay measures the
  • a 96-well plate (Microtest III, Falcon #3912) was coated with 50 ⁇ l/well of rsP-selectin (diluted to 2 ⁇ g/ml in DPBS) overnight at 4°C or 90 min at 37oC. The plate was inverted and residual fluid was tapped away.
  • the coated plate and an additional blank plate were blocked with 200 ⁇ l/well of DPBS + 1% bovine serum albumin
  • TMB tetramethylbenzidine peroxidase substrate/H 2 O 2
  • Apparent binding affinity is expressed as the concentration of antibody that binds rsP-selectin at one-half maximum optical density at 450 nm. The ratio of
  • CHO cells were plated into flasks and allowed to reach confluence before changing media (8 ml for a T-25 cm 2 flask or 20 ml for a T-75cm 2 flask). After incubating the cells for 48-72 h the media was harvested and assayed for IgG production by ELISA. The cells were trypsinized and counted. Antibody production rate was expressed as the amount of antibody in micrograms secreted by one million cells in a 24 h period.
  • the three to six highest-producing clones were amplified separately, and the remaining seven best clones were pooled and amplified.
  • Cells were plated at a density of 1 ⁇ 10 5 cells/100 mm dish in selection media.
  • 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 either 10, 20 or 50 nM methotrexate (Sigma #A-6770). Cultures were fed every four days. At 10-14 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 methotrexate concentrations 5-10 times the initial methotrexate concentration.
  • Table 5 provides a summary of the antibody production for the CHO cells expressing three humanized versions of PB1.3.
  • This clone was selected to be grown for large scale production of reshaped MAb PB1.3.
  • 160-g collagen-coated microcarrier beads (150-200 microns, JRH Biosciences #60142-100) were hydrated in 800-ml of sterile water for 30 min and the water decanted. The beads were resuspended in 800-ml water and autoclaved for 30 min. After cooling to room temperature the beads were washed twice with sterile serum free ⁇ MEM(-nucleosides) and resuspended in 1-1 complete media ⁇ MEM(-nucleosides)/5% dialyzed FBS.
  • the cell line CHO-48B4 which expresses reshaped MAb
  • PB1.3 H B L D (IgG4 isotype) at 33.0 ⁇ g/10 6 cells/day, was expanded into 36 T-225 cm 2 in ⁇ MEM(-nucleosides)/10% dialyzed FBS/500 nm methotrexate. When the cells reached confluence they were trypsinized and resuspended in 2-1 complete media (no methotrexate) at a final concentration of 5.0 - 8.0 ⁇ 10 4 cells/ml.
  • the bioreactor was programmed to replace complete media at 5-1/day utilizing a gravity filter attached to the harvest port (to leave microcarriers in the bioreactor).
  • the harvested media was stored at 4°C until processing.
  • the bioreactor was run for 5-7 days until
  • the media harvest was clarified by passing though a Millipak-60 0.45 micron filter (Millipore). 20-1 filtered supernatant was passed over a column (2.5 ⁇ 12 cm) of protein-A Sepharose Fast Flow (Pharmacia) at a flow rate of 0.55 1/hr. After all of the supernatant had passed through the column, the column was washed with 1.5-1 PBS (BioWhittaker, #17-516Y).
  • the column was pre-eluted with 8 ⁇ 40-ml 0.2-M sodium acetate, pH 4.5, and eluted with 8 ⁇ 40-ml 0.2-M glycine, 0.5-M NaCL, pH 3.5, both into tubes containing 10 ml 2-M Tris-HCl, pH 8.0.
  • the single fraction with the highest absorbance at 280 nm was dialyzed into 20-mM acetate, 0.15-M NaCl, pH 5.5, and stored at 4°C.
  • New Zealand White rabbits were anesthetized with a combination of ketamine and xylazine followed by infiltration of lidocaine into the base of the ear.
  • the left ear was partially amputated leaving the central artery, vein and a bridge of cartilage intact. All nerves were divided to render the ear totally anesthetic.
  • the ear was then reattached and a microvascular clip was placed across the artery to produce total ischemia. Rabbits were kept in a room maintained at 23.5°C for 6 h then the microvascular clamp was removed and the ear allowed to reperfuse.
  • Treatment was administered immediately prior to reperfusion with either mu MAb PB1.3(2 mg/kg) or with either saline or an isotype (IgG1) matched murine monoclonal antibody designated PNB1.6 (2 mg/kg). Ear volume was measured daily for 7 days by water displacement to quantify tissue edema. On day 7 necrosis was estimated as a percentage of total surface area.
  • tissue injury manifestations of tissue injury are substantially and significantly reduced in animals pre-treated with the anti-P-selectin antibody mu MAb PB1.3.
  • Microvascular patency was assessed by perfusion of the isolated gracilis muscle with contrast media (india ink) at the end of the experimental protocol.
  • Computerized video imaging was used to quantitate the number of ink-containing microvessels ( ⁇ 10 ⁇ m diameter) per muscle fiber in histologic sections obtained from isolated canine gracilis muscles subjected to 4.5 h of continuous perfusion, 4 h of ischemia followed by 0.5 h of reperfusion or ischemia reperfusion in the presence of the anti-P-selectin antibody mu MAb PB1.3 at a concentration of 40 ⁇ g/ml in the perfusate.
  • the ischemia and reperfusion protocol reduced the number of patent microvessels to 36 ⁇ 3 % of the value
  • the anti-P-selectin antibody, mu MAb PB1.3 completely prevented the development of the "no-reflow" phenomenon, determined by the number of patent microvessels, in the ischemic and reperfused canine gracilis muscle.
  • Surgical anaesthesia was induced by an intramuscular injection of ketamine/rompun/ acepromazine.
  • a catheter was placed in the left jugular vein, and a tracheostomy was performed to facilitate spontaneous breathing.
  • the abdomen was shaved and washed, and a 3/4 inch long incision was made through the midline into the peritoneal cavity, taking care to avoid any bleeding.
  • the rat was placed on a microscope stage specifically designed for the intravital microscopy procedure.
  • a well-vascularized posterior loop of the ileum was exteriorized, and the mesentery draped over a viewing pedestal heated to 37°C. Throughout the experiment, the mesentery was superfused at 2 ml/min with a heated
  • a high-resolution video system consisting of a video camera (Hitachi color CCD) clamped onto the microscope, a video timer (American Video Equipment), a VCR (Sony SVO-9500MD) and a monitor (Sony
  • Trinitron permitted video recordings of the microscope image.
  • 3 postcapillary venular segments of approximately 180 ⁇ m length were selected for repeated recordings according to the following criteria: (1) A true postcapillary venule receiving blood from confluent capillaries, diameter 15-30 ⁇ m; (2) Brisk blood flow, so that individual red blood cells could not be distinguished;
  • leukocytes present at any given time in the vascular segment), but no firmly adhering leukocytes.
  • a leukocyte was defined as being firmly adhering if it stayed immobile at the vessel wall for more than 30 seconds.
  • Each experimental group contained 5-6 animals with 2-3 venules from each animal. The mean and its standard error is given. Significant differences among group means were tested through analysis of variance followed by the Tukey- Kramer's HSD test. Differences before and after treatments in individual venules were studied by paired t-tests. All tests were run using JMP software running on a Macintosh Ilsi, and significance was accepted at p ⁇ 0.05.
  • Topical application of compound 48/80 to the rat mesentery resulted in visible degranulation of more than 90% of the mast cells, which took place within 3 min after application. Subsequently, an increase in leukocyte rolling and adhesion was seen. No significant difference in leukocyte accumulation was detected between the 20 and 30 minute timepoints post 48/80 application, the mean of these two timepoints was therefore used in the statistical analysis.
  • the venule-to venule variability in the model was quite high, with baseline interaction ranging from 0 to 4.8 leukocytes present in individual venules, and mast cell-induced
  • intravenous treatment with mu MAb PB1.3 inhibited the mast cell-induced intravascular leukocyte accumulation by 90% when compared with the
  • P-selectin is an integral membrane glycoprotein found in secretory granules of platelets and endothelial cells. After cellular activation, P-selectin is rapidly redistributed to the plasma membrane.
  • the mature molecule is a protein with multiple domains, including a "lectin” region, "EGF” region, nine tandem consensus "C3b-C4b Regulatory
  • CRP Protein
  • cytoplasmic domain See Johnston et al., Cell , 56:1033-1044 (1989) (herein incorporated by reference in its entirety for all purposes) for the cloning and description of P-selectin.
  • Fig. 30 shows a proposed folding pattern of the domains of P-selectin.
  • mutants of recombinant P-selectin were constructed by deleting consecutive CRP domains (Fig. 31). This site-specific mutagenesis was accomplished by PCR amplification using primers specific to each CRP domain (Table 6).
  • the template used for PCR-mutagenesis was a pUC DNA clone of P-selectin which was truncated and mutated at DNA sequence position 2354 (sequence positions are in accordance with the published sequence of P-selectin of Johnston et al., supra .
  • This template plasmid pG ⁇ l,T which was derived initially by in vitro mutagenesis, lacks the trans-membrane domain and the cytoplasmic carboxy-terminus of P-selectin, and encodes instead the carboxy-terminal 11 amino acid residues of SV40
  • T antigen This SV40-derived C-terminus is recognized by the monoclonal antibody KT-3. (See MacArthur et al., Journal of Virology 52:483-491 (1984.)) After PCR amplification, the deleted DNA molecules were purified, ligated, transformed into bacteria, cloned and sequenced as described below. DNAs with the appropriate linkage of CRP to the T-antigen tag sequence were subcloned into the pcDNA1 expression vector (Invitrogen) and transfected into COS cells.
  • pcDNA1 expression vector Invitrogen
  • PCR mutagenesis was performed on plasmid pGMP ⁇ 1,T. After phosphorylation at the 5'-end, the primers in Table 6 were added to individual PCR reactions at 1 ⁇ M, together with the T-antigen sequence primer lys-1. The PCR was performed in a Perkin Elmer-Cetus machine for 30 cycles at 95°C for 50 sec, 50°C for 1 min. and 73°C for 4 min. using Pfu polymerase
  • the pcDNA clones were transformed into E. coli strain MC1061 (Invitrogen) and purified plasmid DNAs used for transfection into COS.
  • COS cells were seeded at 1 ⁇ 10 6 cells/100 mm dish in DMEM (Biowhittaker), 10% fetal bovine serum (FBS). On day two, plasmid DNA was ethanol precipitated, and resuspended at a concentration of 20 ⁇ g/mL in sterile TE (10 mM Tris, pH 8.0, 1 mM EDTA).
  • 150 ⁇ L of DNA was mixed with 300 ⁇ l of sterile TBS (Tris Buffered Saline, 140 mM NaCl, 5 mM KCl, 1.4 mM Na 2 HPO 4 25 mM Tris-base, pH 7.5, 1.0 mM CaCl 2 , and 0.5 mM MgCl 2 ) and with 300 ⁇ l of sterile DEAE dextran (Sigma, #D-9885, 1 mg/ml in TBS). The growth media was aspirated, and the cell monolayers were washed once with PBS, and once with TBS. 750 ⁇ l of the DNA/DEAE dextran/TBS mixture was added to the monolayer.
  • TBS Tris Buffered Saline, 140 mM NaCl, 5 mM KCl, 1.4 mM Na 2 HPO 4 25 mM Tris-base, pH 7.5, 1.0 mM CaCl 2 , and 0.5 mM MgCl 2
  • the dish was incubated at ambient temperature inside a laminar flood hood rocking the dish every 5 min for 1 h. After the 1 h incubation, the DNA solution was aspirated and the cells were washed once with TBS and then once with PBS. The cells were incubated in a complete medium
  • a 96-well Costar plate was coated with KT-3 antibody (produced in ascites and purified by Protein G Sepharose, Pharmacia) 100 ⁇ l/well at 40 ⁇ g/ml at 4°C overnight.
  • the plate was washed 3x with DPBS, blocked for 1 h at ambient temperature with 250 ⁇ l/well of DPBS + 1% BSA.
  • the plate was washed 3x with DPBS, and incubated with COS cell supernatants 100 ⁇ l/well for 2 h at ambient temperature.
  • the bound P-selectin was detected by adding either 100 ⁇ l/well of (a) 1 ng/ml rabbit anti-P-selectin polyclonal Ig, or (b) biotinylated mu MAb PB1.3, or (c) biotinylated P6H6 diluted in DPBS/1% BSA for 1 h at ambient temperature.
  • the rabbit antibody was detected using 100 ⁇ l/well of a 1:1000 dilution of HRP-conjugated goat anti-rabbit antibody (Biorad), blocked with tissue culture media from the KT-3 cell line (10%) in DPBS/1% BSA for 30 min at ambient temperature.
  • HRP-conjugated goat anti-rabbit antibody Biorad
  • biotinylated antibodies were detected using 100 ⁇ l/well of a 1:1000 dilution of HRP-streptavidin (Pierce) for 30 min at ambient temperature.
  • the bound HRP was incubated with TMB and quenched with 1 M phosphoric acid.
  • Figure 32 shows the signal less the background from supernatants of mock transfected COS cells.
  • the "Mock" column is the supernatant from cells transfected with a full length form of P-selectin, which lacks the SV40 T-antigen tag, and therefore is not detected in this KT-3 capture ELISA.
  • the X-axis specifies the number of CRPs expressed by the various mutant P-selectin cDNAs, from all 9 CRPs in column 2 to one CRP in column 10.
  • the results show that, although P-selectin mutants lacking the carboxy-terminal 5 CRPs can be detected by the polyclonal rabbit Ig, these molecules do not react with PB1.3. This suggests that the binding site for PB1.3 lies within the fifth CRP repeat domain.
  • the mu MAb PB1.3 epitope was further characterized by synthesizing peptides spanning the 62 amino acid region of the fifth CRP of P-selectin (Fig. 34) and screening for mu MAb PB1.3 binding by ELISA.
  • the fifth CRP extends from amino acid sequence 407 to 468 according to the nomenclature of Johnston, et al.
  • the peptide sequences are 968.01, a.a. # 408-426; 968.02, a. a. #418-436; 968.04, a. a. #408-433; 968.05, an oxidized version of 968.04; 968.06, a.a. #428-447; and

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Abstract

Compositions et procédés destinés à traiter des inflammations et d'autres états pathologiques à l'aide de nouveaux anticorps de blocage dirigés contre la sélectine P, qui inhibent in vivo l'adhérence de leucocytes sur des plaquettes activées et/ou sur l'endothélium vasculaire activé. Des anticorps sous forme de murine et humanisés sont également décrits.
PCT/US1994/004935 1993-05-04 1994-05-04 Anticorps diriges contre la selectine p et leurs utilisations WO1994025067A1 (fr)

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CA002162149A CA2162149C (fr) 1993-05-04 1994-05-04 Anticorps pour la p-selectine et leurs utilisations
JP52465394A JP3713045B2 (ja) 1993-05-04 1994-05-04 Ｐ−セレクチンに対する抗体およびそれらの使用
EP94917301A EP0804235A4 (fr) 1993-05-04 1994-05-04 Anticorps diriges contre la selectine p et leurs utilisations
CN94192352A CN1124928A (zh) 1993-05-04 1994-05-04 P-选择蛋白抗体及其应用
AU69063/94A AU684084B2 (en) 1993-05-04 1994-05-04 Antibodies to P-selectin and their uses
SK1377-95A SK137795A3 (en) 1993-05-05 1994-05-04 Antibodies to p-selectin and their use
FI955261A FI955261A (fi) 1993-05-04 1995-11-02 P-selektiinin vasta-aineita ja niiden käyttö
NO954403A NO954403L (no) 1993-05-04 1995-11-03 Antistoffer mot P-selektin og deres anvendelse

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PCT/US1993/004274 WO1993021956A1 (fr) 1992-05-05 1993-05-04 Anticorps contre la p-selectine et leurs emplois
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US08/057,292 1993-05-05
US08/202,047 US5800815A (en) 1903-05-05 1994-02-25 Antibodies to P-selectin and their uses
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WO1996040942A1 (fr) * 1995-06-07 1996-12-19 Cytel Corporation Anticorps humanises anti-e-selectine
WO1997000956A1 (fr) * 1995-06-20 1997-01-09 Trustees Of Boston University Molecules d'adherence reagissant a l'hypoxemie, anticorps specifiques et leurs utilisations
WO1999018442A1 (fr) * 1997-10-07 1999-04-15 Centocor, Inc. Diagnostic d'accidents thrombotiques par detection de p-selectine
WO2005100402A1 (fr) 2004-04-13 2005-10-27 F.Hoffmann-La Roche Ag Anticorps anti-p-selectine
WO2009046978A1 (fr) * 2007-10-12 2009-04-16 F. Hoffmann-La Roche Ag Expression de protéine à partir d'acides nucléiques multiples
AU2012200852B2 (en) * 2004-04-13 2012-10-18 F. Hoffmann-La Roche Ag Anti-P-selectin antibodies
US8377440B2 (en) 2006-12-01 2013-02-19 Selexys Pharmaceuticals Corporation Anti-P-selectin antibodies and methods of using the same to treat inflammatory diseases
US8945565B2 (en) 2006-12-01 2015-02-03 Selexys Pharmaceuticals Corporation Methods of treating inflammatory or thrombotic conditions with anti-P-selectin antibodies
US9068001B2 (en) 2006-12-01 2015-06-30 Selexys Pharmaceuticals Anti-P-selectin antibodies
WO2019025847A1 (fr) 2017-08-04 2019-02-07 Novartis Ag Schémas de traitement
WO2019171326A1 (fr) 2018-03-08 2019-09-12 Novartis Ag Utilisation d'un anticorps anti-p-sélectine
WO2020247698A1 (fr) 2019-06-07 2020-12-10 Novartis Ag Utilisation d'un anticorps anti-p-sélectine
WO2021024220A1 (fr) 2019-08-08 2021-02-11 Novartis Ag Utilisation de l'anticorps anti-p-sélectine crizanlizumab pour le traitement de la drépanocytose et de la néphropathie chronique associée à la drépanocytose
EP4213878A4 (fr) * 2020-09-16 2024-07-03 Univ Ramot Méthodes de traitement du glioblastome

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WO1996040942A1 (fr) * 1995-06-07 1996-12-19 Cytel Corporation Anticorps humanises anti-e-selectine
WO1997000956A1 (fr) * 1995-06-20 1997-01-09 Trustees Of Boston University Molecules d'adherence reagissant a l'hypoxemie, anticorps specifiques et leurs utilisations
WO1999018442A1 (fr) * 1997-10-07 1999-04-15 Centocor, Inc. Diagnostic d'accidents thrombotiques par detection de p-selectine
CN1942483B (zh) * 2004-04-13 2012-09-26 弗·哈夫曼-拉罗切有限公司 抗p型选凝素抗体
USRE44389E1 (en) 2004-04-13 2013-07-23 Hoffman-La Roche Inc. Methods of inhibiting the binding of P-selectin to PSGL-1 with anti-P-selectin antibodies
KR100891620B1 (ko) * 2004-04-13 2009-04-02 에프. 호프만-라 로슈 아게 항-ｐ-셀렉틴 항체
NO340443B1 (no) * 2004-04-13 2017-04-24 Hoffmann La Roche Anti-P-selektinantistoffer, preparat omfattende samme, metode for fremstilling av slike samt anvendelser av disse for forebygging og behandling av sykdommer.
AU2012200852B2 (en) * 2004-04-13 2012-10-18 F. Hoffmann-La Roche Ag Anti-P-selectin antibodies
US7563441B2 (en) 2004-04-13 2009-07-21 Hoffman-La Roche Inc. Anti-P-selectin antibodies
US7754867B2 (en) 2004-04-13 2010-07-13 Hoffmann-La Roche Inc. Nucleic acid molecules encoding anti-P-selectin antibodies
JP2008508852A (ja) * 2004-04-13 2008-03-27 エフ．ホフマン−ラ ロシュ アーゲー 抗ｐ−セレクチン抗体
TWI473816B (zh) * 2004-04-13 2015-02-21 Hoffmann La Roche 抗ｐ-選擇素抗體
USRE44359E1 (en) 2004-04-13 2013-07-09 Hoffmann-La Roche Inc Nucleic acid molecules encoding anti-P-selectin antibodies
EP2357201A1 (fr) * 2004-04-13 2011-08-17 F. Hoffmann-La Roche AG Anticorps dirigés contre la sélectine P
EP2360186A1 (fr) * 2004-04-13 2011-08-24 F. Hoffmann-La Roche AG Anticorps dirigés contre la sélectine P
EP2374817A1 (fr) * 2004-04-13 2011-10-12 F. Hoffmann-La Roche AG Anticorps à sélection anti-P
AU2005233259B2 (en) * 2004-04-13 2011-12-15 F. Hoffmann-La Roche Ag Anti-P-selectin antibodies
USRE43568E1 (en) 2004-04-13 2012-08-07 Hoffmann-La Roche Inc. Anti-P-selectin antibodies
WO2005100402A1 (fr) 2004-04-13 2005-10-27 F.Hoffmann-La Roche Ag Anticorps anti-p-selectine
EP2067789A1 (fr) * 2004-04-13 2009-06-10 F. Hoffmann-La Roche Ag Anticorps dirigés contre la sélectine P
US7824684B2 (en) 2004-04-13 2010-11-02 Hoffmann-La Roche Inc. Methods of inhibiting the binding of P-selectin to PSGL-1 with anti-P-selectin antibodies
JP4633788B2 (ja) * 2004-04-13 2011-02-16 エフ．ホフマン−ラ ロシュ アーゲー 抗ｐ−セレクチン抗体
US9068001B2 (en) 2006-12-01 2015-06-30 Selexys Pharmaceuticals Anti-P-selectin antibodies
US8945565B2 (en) 2006-12-01 2015-02-03 Selexys Pharmaceuticals Corporation Methods of treating inflammatory or thrombotic conditions with anti-P-selectin antibodies
US9556266B2 (en) 2006-12-01 2017-01-31 Selexys Pharmaceuticals, Inc. Methods of treating sickle cell disease with anti-P-selectin antibodies
US8377440B2 (en) 2006-12-01 2013-02-19 Selexys Pharmaceuticals Corporation Anti-P-selectin antibodies and methods of using the same to treat inflammatory diseases
US9428766B2 (en) 2007-10-12 2016-08-30 Hoffmann-La Roche Inc. Protein expression from multiple nucleic acids
JP2010540583A (ja) * 2007-10-12 2010-12-24 エフ．ホフマン−ラ ロシュ アーゲー 複数の核酸からのタンパク質発現
US8771988B2 (en) 2007-10-12 2014-07-08 Hoffmann-La Roche Inc. Protein expression from multiple nucleic acids
KR101280704B1 (ko) * 2007-10-12 2013-07-01 에프. 호프만-라 로슈 아게 다수의 핵산으로부터의 단백질 발현
EP2592148A1 (fr) * 2007-10-12 2013-05-15 F. Hoffmann-La Roche AG Expression protéinique pour plusieurs acides nucléiques
WO2009046978A1 (fr) * 2007-10-12 2009-04-16 F. Hoffmann-La Roche Ag Expression de protéine à partir d'acides nucléiques multiples
WO2019025847A1 (fr) 2017-08-04 2019-02-07 Novartis Ag Schémas de traitement
WO2019171326A1 (fr) 2018-03-08 2019-09-12 Novartis Ag Utilisation d'un anticorps anti-p-sélectine
WO2020247698A1 (fr) 2019-06-07 2020-12-10 Novartis Ag Utilisation d'un anticorps anti-p-sélectine
WO2021024220A1 (fr) 2019-08-08 2021-02-11 Novartis Ag Utilisation de l'anticorps anti-p-sélectine crizanlizumab pour le traitement de la drépanocytose et de la néphropathie chronique associée à la drépanocytose
EP4213878A4 (fr) * 2020-09-16 2024-07-03 Univ Ramot Méthodes de traitement du glioblastome

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