WO1993014220A1 - Anticorps reconnaissant le quatrieme domaine de similitude a l'immunoglobuline de vcam1 - Google Patents

Anticorps reconnaissant le quatrieme domaine de similitude a l'immunoglobuline de vcam1 Download PDF

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WO1993014220A1
WO1993014220A1 PCT/US1993/000031 US9300031W WO9314220A1 WO 1993014220 A1 WO1993014220 A1 WO 1993014220A1 US 9300031 W US9300031 W US 9300031W WO 9314220 A1 WO9314220 A1 WO 9314220A1
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PCT/US1993/000031
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Laurelee Osborn
Christopher D. Benjamin
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Biogen, Inc.
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Priority to AU34318/93A priority Critical patent/AU664071B2/en
Priority to JP5512513A priority patent/JPH07506244A/ja
Priority to EP93902915A priority patent/EP0623173A1/fr
Publication of WO1993014220A1 publication Critical patent/WO1993014220A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—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 immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to monoclonal antibodies
  • MAbs specifically monoclonal antibodies that bind to the domain 4 of Vascular Cell Adhesion Molecule-1 (VCAM1).
  • VCAM1 (also known as INCAM-110) was first identified as an adhesion molecule induced on endothelial cells by inflammatory cytokines (TNF and IL-1) and LPS (Rice et al., 1989 [1]; Osborn et al., 1989 [2]). VCAM1 binds to cells exhibiting the integrin VLA-4 ( ⁇ 4 ⁇ 1 ), including T and B lymphocytes, monocytes, and eosinophils, but not neutrophils, and is thought to participate in recruitment of these cells from the bloodstream to areas of infection and inflammation (Elices et al, 1990 [3];
  • VCAM1/VLA-4 adhesion pathway has been associated with a number of physiological and
  • VCAM1 may participate in metastasis of such tumors (Rice et al., 1989 [1]).
  • VCAM1 is found on areas of arterial endothelium representing early atherosclerotic plaques in a rabbit model system (Cybulsky and Gimbrone, 1991 [5]).
  • VCAM1 is also found on follicular dendritic cells in human lymph nodes (Freedman et al., 1990 [6]). It is also present on bone marrow stromal cells in the mouse (Miyake et al., 1991 [7]), thus VCAM1 appears to play a role in B- cell development.
  • VCAM1 belongs to the immunoglobulin (Ig)
  • VCAM-7D has seven Ig homology units or domains; domains 4, 5 and 6 are similar in amino acid sequence to domains 1, 2 and 3, respectively, suggesting an intergenic duplication event in the evolutionary history of the gene (Osborn et al., 1989 [2]; Polte et al. 1990 [8]; Hession et al., 1991 [9]). There is also a minor 6-domain form (referred to as VCAM-6D herein) generated by alternative splicing, in which the fourth domain is deleted (Osborn et al., 1989 [2]; Hession et al. 1991 [9], Cybulsky et al., 1991 [10]). The biological significance of this alternate splicing is not known, however as shown below VCAM-6D can bind VLA-4-expressing cells and thus clearly has potential functionality in vivo.
  • VCAM1/VLA-4 adhesion pathway The apparent involvement of the VCAM1/VLA-4 adhesion pathway in infection, inflammation and possibly atherosclerosis has led to continuing intensive research to understand the mechanisms of cell-cell adhesion on a molecular level and has led investigators to propose intervention in this adhesion pathway as a treatment for diseases, particularly inflammation (Osborn et al., 1989 [2]).
  • Monoclonal antibodies that inhibit VCAM1 binding to VLA-4 are known.
  • anti-VLA-4 MAbs HP2/1 and HP1/3 have been shown to block attachment of VLA-4- expressing Ramos cells to human umbilical vein cells and VCAM1-transfected COS cells (Elices et al., 1990 [3]).
  • anti-VCAM1 antibodies such as the monoclonal
  • antibody 4B9 (Carlos et al., 1990 [11]) have been shown to inhibit adhesion of Ramos (B-cell-like), Jurkat (T-cell- like) and HL60 (granulocyte-like) cells to COS cells transfected to express VCAM-6D and VCAM-7D (Hession et al., 1991 [9]).
  • VCAM1 antibodies recognizing epitopes on other, non-binding domains of VCAM1 would be important research tools to map the portion(s) of VCAM1 essential for binding to VLA-4.
  • domain 4 of VCAM-7D contains a site involved in VLA-4 binding, which is in addition to the site recognized and blocked by MAb 4B9 (domain 1).
  • VCAM1 is identified as the first known adhesion molecule to have two binding sites for a single receptor (VLA-4); and the domain 4 monoclonal antibodies according to this invention are useful not only in mapping the molecular functionality of VCAM1 but also in at least partially inhibiting VCAM1 recognition by VLA-4.
  • VLA-4-expressing cells adhesion of VLA-4-expressing cells to VCAM1.
  • FIG. 1 is a schematic diagram of VCAM-7D, showing the linear arrangement of functional domains from the amino terminus to the carboxyl terminus. These are the signal peptide ("sig”), immunoglobulin-like domains 1 through 7, the transmembrane sequence ("tm”), and the cytoplasmic domain or “tail” ("cyt"). Also indicated are the relative positions of several restriction enzyme recognition sites, which were used to prepare additional VCAM1 constructs having one or more altered domains
  • FIG. 2 is a schematic diagram of VCAM-6D, showing the linear arrangement of functional domains from the amino terminus to the carboxyl terminus --the signal peptide ("sig”), immunoglobulin-like domains 1, 2, 3, 5, 6 and 7, the transmembrane sequence ("tm”), and the
  • cDNA encoding VCAM-6D having the diagrammed conformation was used to transfect C0S7 cells to produce COS7 cells exhibiting VCAM-6D on their surfaces.
  • FIG. 3 is a schematic diagram of a chimeric VCAM1, designated VCAM/ICAM-1, showing the linear
  • This polypeptide has the general structure of VCAM-7D, except the cDNA encoding VCAM-7D was modified so that a segment encoding most of domain 1 and part of domain 2 was excised and replaced by cDNA encoding analogous portions of Intercellular Adhesion Molecule-1 (ICAM1).
  • IAM1 Intercellular Adhesion Molecule-1
  • FIG. 4 is a schematic diagram of another chimeric VCAM1, designated VCAM/ICAM-2, showing the linear arrangement of functional domains.
  • This polypeptide has the general structure of VCAM-7D, except as indicated by the shaded area, the cDNA coding for most of domain 1 and a large part of domain 2 was excised and replaced with ICAM1 cDNA.
  • FIG. 5 is a schematic diagram of another chimeric VCAM1, designated VCAM/ICAM-3, showing the linear arrangement of functional domains.
  • This polypeptide has the general structure of VCAM-7D, except as indicated by the shaded area, the cDNA coding for part of domain 1, all of domains 2 and 3, and a small N-terminal part of domain 4 was excised and replaced with ICAM1 cDNA.
  • Figure 6 is a graph showing relative levels of expression in COS7 cells transfected with either cDNA encoding VCAM-6D or cDNA encoding VCAM-7D. Expression of either VCAM-6D or VCAM-7D was analyzed by flow
  • Figure 7 is a graph showing the extent of Ramos cell binding to COS cells separately tranfected with cDNA encoding VCAM-7D, VCAM-6D, VCAM/ICAM-1, VCAM/ICAM-2, and VCAM/ICAM-3.
  • the effects on Ramos binding of preincubation of the transfected COS cells with 20 ⁇ g/ml of MAb 4B9 (anti-VCAM1) and 10 ⁇ g/ml of MAb HP1/2 (anti- VLA-4) are shown.
  • the negligible Ramos binding to cells transfected with pCDM8 (expression vector only; negative control) and cells transfected to exhibit surface ICAM1 is shown. Binding is expressed as Ramos cells bound per square millimeter ("Ramos cells bound/mm" in the figures).
  • FIG. 8 is a schematic diagram of another chimeric VCAM1, designated VCAM6D/ICAM-1, showing the linear arrangement of functional domains. This
  • polypeptide has the general structure of VCAM-6D (no domain 4), however, as indicated by the shaded area, the cDNA coding for most of domain 1 and a small part of domain 2 was excised and replaced with ICAM1 cDNA.
  • FIG. 9 is a schematic diagram of another chimeric VCAM1, designated VCAM6D/ICAM-2, showing the linear arrangement of functional domains. This
  • polypeptide has the general structure of VCAM-6D (no domain 4), however, as indicated by the shaded area, the cDNA coding for most of domain 1 and a large part of domain 2 was excised and replaced with ICAM1 cDNA.
  • Figure 10 is a graph depicting the extent of Ramos cell binding to COS cells separately tranfected with CDNA encoding VCAM/ICAM-1, VCAM6D/ICAM-1, VCAM/ICAM-2, and VCAM6D/ICAM-2. In addition, the negligible Ramos binding to cells transfected with pCDM8 (expression vector only; negative control) and cells transfected to exhibit surface ICAM1 is shown.
  • FIG 11 is a schematic diagram of another chimeric VCAM1, designated VCAM6D/VCAM4-1, showing the linear arrangement of functional domains.
  • polypeptide has the general structure of VCAM-6D (no domain 4), however, as indicated by the shaded area, the cDNA coding for domain 1 and a small part of domain 2 was excised and replaced with cDNA encoding domain 4 (and one amino acid of domain 5) of VCAM-7D.
  • Figure 12 is a graph depicting the extent of Ramos cell binding of COS cells separately tranfected with cDNA encoding VCAM/ICAM-1, VCAM6D/ICAM-1, and
  • VCAM6D/VCAM4-1 Negligible Ramos binding to ICAM1 and negative (pCDM8) controls is also shown.
  • Figure 13 is a graph depicting the extent of Ramos cell binding to transfectants expressing various chimeric VCAM1 constructs in the absence of any
  • Figure 14 is a graph depicting the extent of Ramos cell binding to transfectants expressing various chimeric VCAM1 constructs in the absence of any interfering antibody or in the presence of MAb 4B9 or MAb
  • Figure 15 is a graph depicting the extent of
  • anti-VCAM1 MAb (anti-VCAM1 MAb, this invention) is compared.
  • an immortal cell line typically murine myeloma cells
  • lymphocytes typically murine myeloma cells
  • splenocytes from a mammal immunized with whole cells expressing a given antigen, e.g., VCAM1, and/or with purified antigen, and the culture supernatants of the resulting hybridoma cells are screened for antibodies against the antigen.
  • a given antigen e.g., VCAM1
  • purified antigen e.g., VCAM1
  • the culture supernatants of the resulting hybridoma cells are screened for antibodies against the antigen. See, generally, Kohler and Milstein,
  • Immunization may be accomplished using standard procedures.
  • the unit dose and immunization regimen depend on the species of mammal immunized, its immune status, the body weight of the mammal, etc.
  • the immunized mammals are bled and the serum from each blood sample is assayed for particular antibodies using appropriate screening assays.
  • anti-VCAM1 antibodies according to the present invention were identified by testing the ability of the immune serum to block Ramos binding to plates coated with a recombinant soluble form of VCAM-7D (rsVCAM-7D).
  • the lymphocytes used in the production of hybridoma cells typically are isolated from immunized mammals whose sera have already tested positive for the presence of anti-VCAM1 antibodies using such screening assays.
  • the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridomas producing a desired antibody are detected by screening the hybridoma culture supernatants using assays which detect MAbs having the desired
  • hybridomas according to the invention were identified by their ability to bind to VCAM-7D-expressing cells but not to VCAM-6D-expressing cells. (See Example 1, supra.)
  • hybridoma cells that tested positive in such screening assays were cultured in a nutrient medium under conditions and for a time sufficient to allow the hybridoma cells to secrete the monoclonal antibodies into the culture medium. Tissue culture techniques and culture media suitable for
  • hybridoma cells are well known.
  • the conditioned hybridoma culture supernatant may be collected and the anti-VCAM1 antibodies optionally further purified by well-known methods.
  • the desired antibody may be produced by injecting the hybridoma cells into the desired antibody.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the antibody may be any substance that accumulates as ascites fluid.
  • the invention recognize epitopes dependent on the fourth Ig domain of the seven-Ig-domain form of VCAM1, i.e., VCAM- 7D. That is, the MAbs of the present invention bind to epitopes that either are contained within the fourth Ig domain of VCAM-7D or are partly contained within the fourth Ig domain, such that elimination of the fourth Ig domain eliminates their ability to recognize the VCAM1 structure.
  • domain 4 MAbs are referred to as "domain 4 MAbs" herein.
  • the domain 4 MAbs bind to VCAM-7D but do not bind to VCAM-6D.
  • All of the antibodies of this invention are distinct from known VCAM1/VLA-4 blocking antibodies such as 4B9.
  • 4B9 recognizes an epitope in domain 1 of VCAM1, and thus recognizes both VCAM-7D and VCAM-6D.
  • the monoclonal antibodies of the present invention recognize VCAM-7D but do not cross-block MAb 4B9. Some of the monoclonal antibodies described herein are effective to inhibit adhesion of Ramos and other VLA-4-expressing cells to VCAM1-expressing cells.
  • Some of the domain 4 monoclonal antibodies described herein bind to epitopes of VCAM1 which are not involved in the adhesion pathway between mononuclear leukocytes and VCAM1-expressing cells that is mediated by VCAM1.
  • This latter type of antibody may be useful, for example, to detect the presence of VCAM1, in particular VCAM-7D, in vitro or in vivo without interference with the binding of VCAM1.
  • inventions may be produced naturally as outlined above or may be synthesized using recombinant DNA techniques.
  • Suitable recombinant antibodies include antibodies
  • suitable expression vector containing DNA encoding the light and heavy immunoglobulin chains of the desired antibody, and recombinant chimeric antibodies, wherein some or all of the hinge, constant and/or variable regions of the heavy and/or the light chains of the anti-VCAM1 antibody have been substituted with corresponding regions of an immunoglobulin light or heavy chain of a different species.
  • This includes so-called "humanized” antibodies. See, e.g., Jones et al., 1986 [13], Ward et al., 1989 [14], and U.S. Patent 4,816,397 (Boss et al.) [15], all incorporated herein by reference.)
  • domain 4-binding fragments of anti- VCAM1 antibodies such as Fab, Fab', F(ab) 2 , and F(v) fragments; heavy chain monomers or dimers; light chain monomers or dimers; and dimers consisting of one heavy chain and one light chain are also contemplated herein.
  • antibody fragments may be produced by chemical methods, e.g., by cleaving an intact antibody with a protease, such as papain or pepsin, or via recombinant DNA techniques, e.g., by using host cells transformed with truncated heavy and/or light chain genes.
  • Heavy and light chain monomers may similarly be produced by treating an intact antibody with a reducing agent such as
  • antibody fragments having similar specificities may be isolated by phage cloning methods. (See, e.g., Clackson et al. (1991) [16].)
  • Monoclonal antibodies of the present invention may be used in any application where antibody recognition of domain 4 of VCAM1 is advantageous, including
  • monoclonal antibodies of the present invention may be used in the treatment of inflammation or diseases characterized by leukocyte binding to endothelium, such as post-reperfusion injury, microbial infections or other cell migration
  • the present monoclonal antibodies may be used in combination with 4B9 or other anti-VCAM1 antibodies in the treatment of disorders characterized by VCAM1 expression in endothelium.
  • the present monoclonal antibodies may be used in combination with antibodies recognizing other endothelial cell
  • the antibodies of the present invention, or VCAM1- recognizing fragments thereof may be combined or linked to cytotoxic molecules such as TNF, ricin or the A chain of diphtheria toxin, in order to provide antibody/toxin conjugates capable of targeting cytotoxic elements to VCAM1-expressing cells.
  • the antibodies of the present invention may also be immobilized on a chromatographic substrate (e.g., Protein A-sepharose) to provide an affinity chromatography resin useful, for example, for separating or purifying VCAM-7D preferentially over VCAM- 6D.
  • a chromatographic substrate e.g., Protein A-sepharose
  • an affinity chromatography resin useful, for example, for separating or purifying VCAM-7D preferentially over VCAM- 6D.
  • Detectably labeled antibodies according to the present invention may also be used in screening methods or diagnostic methods for detecting VCAM1 protein in a sample or VCAM1-expressing endothelial cells in vitro or in vivo.
  • samples may be screened for the presence of VCAM1 or VCAM1-expressing cells by contacting the sample with a labeled antibody according to the present invention and detecting whether a VCAM1/anti-VCAM1 antibody complex is formed.
  • Suitable labels can be radioactive, enzymatic, fluorescent, magnetic or chemiluminescent.
  • Radiolabeled antibodies are prepared in known ways by coupling a radioactive isotope such as 3 H, 32 P, 35 S, 59 Fe, 125 I, which can then be detected by gamma counter, scintillation counter or by autoradiography.
  • Antibodies of this invention may be suitably labeled with enzymes such as yeast alcohol dehydrogenase, horseradish peroxidase, alkaline
  • Suitable fluorescent labels include fluorescein isothiocyanate, fluorescamine, rhodamine, and the like.
  • Suitable chemiluminescent labels include luminol, imidazole, oxalate ester, luciferin, and the like.
  • the monoclonal antibodies of the present invention may be formulated as a
  • composition comprising an effective amount of the antibody admixed with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier typically, the antibodies of the present
  • compositions may alternatively be formulated to control release of the active ingredients or to prolong their presence in a patient's system.
  • suitable drug delivery systems include, e.g., hydrogels,
  • microemulsions are microspheres, and the like.
  • compositions contemplated herein may be administered by any suitable means such as orally, intranasally, subcutaneously, intramuscularly, intravenously, intra-arterially, or parenterally.
  • VCAM-7D Monoclonal antibodies according to the invention that recognize domain 4 of VCAM-7D were prepared in the following manner.
  • VCAM- 6D/CHO whole CHO cells that express VCAM-6D on their surfaces
  • the CHO culture was a Chinese Hamster Ovary cell line stably transfected with cDNA encoding VCAM-6D and was obtained from Biogen, Inc. (Cambridge, MA). Initially, each mouse received approximately 10 7 cells in PBS intraperitoneally (i.p.).
  • i.p. a Chinese Hamster Ovary cell line stably transfected with cDNA encoding VCAM-6D and was obtained from Biogen, Inc. (Cambridge, MA).
  • each mouse received approximately 10 7 cells in PBS intraperitoneally (i.p.).
  • rsVCAM-7D a recombinant soluble form of VCAM- 7D (rsVCAM-7D, consisting of the N-terminal 674 amino acids of mature VCAM-7D and lacking the transmembrane and cytoplasmic regions; obtained from Biogen, Inc.,
  • PBS buffered saline
  • FCS fetal calf serum
  • the cells in positive culture wells were:
  • the COS7 cells transfected with either VCAM-6D or VCAM-7D cDNA, were washed and suspended in suspension buffer (PBS/5mM EDTA/2% FCS/0.05% sodium azide) and incubated 45 min. at 4° C with supernatant from hybridoma cultures. The transfected cells were then washed with suspension buffer and incubated with a fluoresceinated goat anti-mouse Ig (Jackson Immunoresearch, West Grove PA) for 45 min. at 4° C. The cells were washed, fixed with 0.2% paraformaldehyde in PBS and evaluated using a FACStar (Becton Dickinson, Mountain View CA).
  • FACStar Becton Dickinson, Mountain View CA.
  • Ramos is a B-lymphoblastoid cell line that expresses VLA-4 and thus binds to VCAM1 but does not bind to other induced endothelial cell molecules such as ICAM-1 or ELAM-1 (Osborn et al., 1989 [2]). Ramos is therefore a convenient model cell line with which to assay VLA-4- dependent binding. Ramos cells bind to both recombinant VCAM-6D and recombinant VCAM-7D when these molecules are expressed on COS cells.
  • VCAM-7D binding to VCAM-7D was somewhat (less than 2 fold) greater than to VCAM-6D in transient transfection assays of a single preparation of each plasmid (Hession et al., 1991 [9]). To determine if this quantitative difference in binding activity was reproducible, several preparations of each plasmid were made and electroporated in parallel into COS cells. Repeated transfections revealed that although expression of the two plasmids is similar, VCAM-7D on average binds significantly more cells per square
  • VCAM-6D millimeter than does VCAM-6D.
  • Binding to both six- and seven-domain forms of VCAM1 was inhibited by the anti-VLA-4 MAb HP1/2 or by anti-VCAM MAb 4B9 ( Figure 7).
  • the binding of Ramos cells was measured as follows: Confluent monolayers of the target cells (e.g., COS transfectants or induced HUVECs) in 48-well plastic tissue culture plates were washed with RPMI/10% fetal calf serum (FCS), and an amount (e.g., 10 ⁇ g/ml, varying per MAb and per experiment) of the test MAb (e.g., 4B9, HP1/2, GH12, etc.) in a volume of 0.15 ml was added.
  • FCS RPMI/10% fetal calf serum
  • the cells were preincubated 4, 24, 48 or 72 hours at 37° C with 20 ng/ml recombinant human TNF (Biogen, Inc.) to induce expression of VCAM1.
  • Ramos cells were fluorescently labeled by adding 2',7'-bis(2-carboxyethyl)- 5(and 6)-carboxy-fluorescein ("BCECF", Molecular Probes, Eugene OR) according to the manufacturer's instructions.
  • BCECF 2',7'-bis(2-carboxyethyl)- 5(and 6)-carboxy-fluorescein
  • the cells were first centrifuged and then resuspended in RPMI/10% fetal calf serum (FCS) to a concentration of 5 x 10 6 cells ml.
  • FCS fetal calf serum
  • VCAM-7D VCAM-7D
  • VCAM-6D various VCAM/ICAM constructs exhibiting domain 4-dependent cell binding
  • COS7 cells transfected with a particular VCAM, ICAM (control) or chimeric VCAM/ICAM (or VCAM/VCAM, see Fig. 11) cDNA construct were removed from 100 mm culture dishes by incubation in HBSS/5 mM EDTA for 15 min. at 37° C, followed by vigorous pipetting.
  • VCAM1/ICAM chimeras were constructed by excising portions of the VCAM-7D and VCAM-6D cDNAs and replacing them with analogous regions of ICAM-1, using unique restriction endonuclease sites in the VCAM1 cDNA (see Fig. 1) and polymerase chain reaction (PCR)-generated fragments of ICAM1. Substitution of the excised VCAM1 regions was done to eliminate as much as possible the structural distortions that would accompany deletion of one or more domains of the VCAM1 molecules. ICAM1 was selected as a source of substituent structures because it is also a member of the Ig superfamily and has the highest degree of amino acid homology to VCAM1 among Ig
  • VCAM1/ICAM1 chimeric polypeptides are shown in Figures 1-5, 8, 9 and 11.
  • the cDNA constructs coding for each of these were prepared as follows:
  • ICAM1 inserts were made by PCR from an ICAM1/CDM8 plasmid isolated from an endothelial cDNA library previously described (Osborn et al., 1989 [2]), using oligonucleotide probes based on the published sequence of ICAM1 (Simmons et al., 1988 [19]; Staunton et al., 1988 [20]).
  • the PCR primers were designed as follows: Primer P-1 (SEQ ID NO: 1)
  • the first twelve nucleotides correspond to the coding region for VCAM1 amino acid residues 9-12; the following twenty-three nucleotides correspond to the coding region for ICAM1 amino acid residues 11-18.
  • Primer P-2 (SEQ ID NO: 2 )
  • the first thirteen nucleotides correspond to the coding region for VCAM1 amino acid residues 86-89 ; the following twenty-one nucleotides correspond to the coding region for
  • Primer P-3 (SEQ ID NO: 3)
  • the first twelve nucleotides correspond to the coding region for VCAM1 amino acid residues -3-1; the following twenty nucleotides correspond to the coding region for
  • Primer P-4 (SEO ID NO: 4)
  • the first fourteen nucleotides correspond to the coding region for VCAM1 amino acid residues 92-96; the following twenty-two nucleotides correspond to the coding region for
  • Primer P-5 (SEQ ID NO: 5)
  • the first eleven nucleotides correspond to the coding region for VCAM1 amino acid residues 134-137; the following twenty-four nucleotides correspond to the coding region for ICAM1 amino acid residues 121-128.
  • Primer P-6 (SEO ID NO: 6)
  • the first eleven nucleotides correspond to the coding region for VCAM1 amino acid residues 302-305; the
  • nucleotides correspond to the coding region for ICAM1 amino acid residues 279-286.
  • Primer P-7 (SEO ID NO: 7)
  • the first thirteen nucleotides correspond to the coding region for VCAM1 amino acid residues 92-96; the following eighteen nucleotides correspond to the coding region for VCAM1 amino acid residues 385-394.
  • VCAM/ICAM-1 (Fig. 3) was primed with P-1 and P-4; for construct VCAM/ICAM-2 (Fig. 4), P-1 and P-5; for construct VCAM/ICAM-3 (Fig. 5), P-2 and P-6; for construct
  • VCAM6D/VCAM4-1 (Fig. 11), P-3 and P-7.
  • VCAM6D/ICAM-1 and VCAM6D/ICAM-2 (Figs. 8 and 9) were made using the same inserts as for VCAM/ICAM-1 and VCAM/ICAM-2, respectively, cloned into the VCAM-6D expression vector (VCAM41/CDM8) instead of the VCAM-7D expression vector (VCAM1E11/CDM8).
  • VCAM41/CDM8 VCAM-6D expression vector
  • VCAM7D expression vector VCAM1E11/CDM8
  • VCAM/ICAM-3 VCAM6D/ICAM-1, VCAM6D/ICAM-2, and
  • VCAM6D/VCAM4-1 are set forth in the Sequence Listing below (in order, SEQ ID NOS: 8 through 15).
  • COS7 cells found to be well expressed when tested by FACS analysis after staining with polyclonal antiserum to VCAM1.
  • VCAM/ICAM-1 (Fig. 3) and VCAM/ICAM-2 (Fig. 4)
  • ICAM1 sequence see shaded areas, Figs. 3 and 4,
  • VCAM/ICAM-3 (Fig. 5)
  • VCAM domains 2 and 3 and small portions of domains 1 and 4 were replaced by ICAM1 sequence (see shaded area, Fig. 5).
  • VCAM1 significantly inhibits binding of VLA-4-bearing cells to induced HUVECs or VCAM1 transfectants (Carlos et al., 1990 [22]). These results suggested that the binding site of VCAM1 recognized by VLA-4 and obstructed by 4B9 is in the N-terminal portion of the VCAM1 molecule.
  • VCAM/ICAM-1 and VCAM/ICAM-2 did not bind MAb 4B9, both bound Ramos cells at about 30% of the level seen with intact VCAM-7D ( Figure 7).
  • This binding was mediated by VLA-4/VCAM1, since anti-VLA-4 mAb HP1/2 completely inhibited the interaction, while a blocking antibody to the ICAM-1 pathway (MAb 60.3) did not affect binding (data not shown).
  • Construct VCAM/ICAM-3 bound Ramos cells weakly but measurably, at about 10% of the level seen with intact VCAM-7D ( Figure 7).
  • VCAM/ICAM-1 and VCAM/ICAM-2 were made, except that cDNA coding for VCAM-6D (no domain 4) instead of VCAM-7D was used as the substrate or backbone for the substituted ICAM1 inserts (see. Figs. 8 and 9). These constructs, designated VCAM6D/ICAM-1 and
  • VCAM6D/ICAM-2 were well expressed in COS7 cells as measured by anti-VCAM1 polyclonal antiserum staining, but were unable to bind Ramos cells (Figure 10). These results indicated that the binding of Ramos cells to constructs VCAM/ICAM-1 and VCAM/ICAM-2 appears to be due to the presence of domain 4.
  • VCAM6D/VCAM4-1 in which domain 4 of VCAM-7D was
  • MAb GH12 bound to COS7 transfectants expressing VCAM-7D, VCAM/ICAM-1 and 2, and VCAM6D/VCAM4-1 but did not bind to transfectants expressing VCAM-6D or VCAM/ICAM-3.
  • Ramos binding experiments showed that MAb GH12 at 10 ⁇ g/ml inhibited Ramos binding to COS7 transfectants expressing the VCAM/ICAM-1, VCAM/ICAM-2 and VCAM6D/VCAM4-1 constructs but did not bind to the
  • VCAM/ICAM-3-expressing tranfectants indicating that the small portion of domain 4 eliminated from the VCAM/ICAM-3 construct was important to GH12 binding.
  • MAb GH12 did not block Ramos cell binding to VCAM-6D or VCAM-7D.
  • Binding experiments showed that MAb ED11 bound to COS7 cells expressing VCAM-7D, VCAM/ICAM-l and
  • VCAM/ICAM-2 but not to cells expressing VCAM-6D
  • VCAM/ICAM-3 or VCAM6D/VCAM4-1 VCAM/ICAM-3 or VCAM6D/VCAM4-1.
  • MAb ED11 at 5 ⁇ g/ml inhibited Ramos binding to COS7 transfectants expressing the VCAM/ICAM-1 and VCAM/ICAM-2 constructs but did not inhibit VCAM-6D-expressing, VCAM/ICAM-3-expressing or VCAM6D/VCAM4-1-expressing tranfectants.
  • MAb ED11 did partially block Ramos cell binding to VCAM-7D- expressing COS7 cells.
  • VCAM/ICAM-1 and VCAM/ICAM-2 did not block Ramos adhesion to transfectants expressing VCAM6D/VCAM4-1
  • VCAM/ICAM-3 or VCAM-6D did not inhibit Ramos binding to VCAM-7D-expressing COS7 cells at the single concentration tested.
  • EDll or GH12 were capable of blocking adhesion to VCAM1 on endothelial cells (which is predominantly VCAM-7D), human umbilical vein endothelial cells (HUVECs) were treated with 20 ng/ml human recombinant TNF for 4, 24, 48, or 72 hours, and binding of Ramos cells and the ability of the antibodies to block binding was measured. All MAbs were added at a concentration of 10 ⁇ g/ml. The results are shown for each activation period in Figures 16-19.
  • VCAM-7D is the predominantly expressed (RNA and protein) form of VCAM-1
  • VCAM-6D may be the predominant species of VCAM-1 in bone marrow stromal cells and tonsillar follicular dendritic cells. This suggests a potential therapeutic advantage of domain 4 specific anti-VCAM1 antibodies over domain 1 specific anti-VCAM1 antibodies, in that domain 4 antibodies
  • lymphopoiesis which is the role that VCAM-1 plays in bone marrow (Miyake et al., 1977 [7]). Such an inhibition of B-cell lymphopoiesis may be undesirable in longer term therapy with anti-VCAM1 antibodies.
  • VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site," Cell, 60, 577-584.
  • VCAM1 vascular cell adhesion molecule-1
  • Vascular cell adhesion molecule-1 mediates lymphocyte adherence to cytokine- activated cultured human endothelial cells. Blood, 76 , 965-970.
  • ICAM an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM," Nature, 331, 624-627.
  • AGT CCA CTG AAT GGG AAG GTG ACG AAT GAG GGG ACC ACA TCT ACG CTG 240 Ser Pro Leu Asn Gly Lys Val Thr Asn Glu Gly Thr Thr Ser Thr Leu
  • AGA AAA GCC AAC ATG AAG GGG TCA TAT AGT CIT GTA GAA GCA CAG AAA 2208 Arg Lys Ala Asn Met Lys Gly Ser Tyr Ser Leu Val Glu Ala Gln Lys
  • GCT CCG AAA ATC CTG TGG AGC AGG CAG CTC CCT AAC GGG GAG CTA CAG 1392 Ala Pro Lys lle Leu Trp Ser Arg Gln Leu Pro Asn Gly Glu Leu Gln
  • AAA AAA GCG GAG ACA GGA GAC ACA GTA CTA AAA TCT ATA GAT GGC GCC 1680 Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala
  • Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser lle Asp Gly Ala
  • GCT AAA TTA CAC ATT GAT GAA ATG GAT TCT GTG CCC ACA GTA AGG CAG 624 Ala Lys Leu His lle Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln
  • GCT GTA AAA GAA TTG CAA GTC TAC ATA TCA CCC AAG AAT ACA GTT ATT 672 Ala Val Lys Glu Leu Gln Val Tyr lle Ser Pro Lys Asn Thr Val lle
  • CAA GAG AAA CCA TTT ACT CTT GAG ATC TCC CCT GGA CCC CGG ATT GCT 960 Gln Glu Lys Pro Phe Thr Val Glu lle Ser Pro Gly Pro Arg lle Ala
  • GTC ACT GTA AGC TGC AAG GTT CCT AGC GTG TAC CCC CTT GAC CGG CTG 1296 Val Thr Val Ser Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg Leu
  • GTA CTA AAA TCT ATA GAT GGC GCC TAT ACC ATC CGA AAG GCC CAG TTG 1968 Val Leu Lys Ser lle Asp Gly Ala Tyr Thr lle Arg Lys Ala Gln Leu

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Abstract

L'invention décrit des anticorps monoclonaux reconnaissant le quatrième domaine de l'immunoglobuline de la molécule 1 d'adhésion aux cellules vasculaires (VCAM1). Lesdits anticorps sont efficaces, par exemple, dans le traitement d'inflammations aiguës et de maladies caractérisées par une adhésion de leucocytes provoquées par VCAM1, ainsi que dans des procédés de purification ou de diagnostic et dans des kits de diagnostic, particulièrement quand la distinction entre les formes de sixième domaine et de septième domaine de VCAM1 présente un avantage.
PCT/US1993/000031 1992-01-13 1993-01-12 Anticorps reconnaissant le quatrieme domaine de similitude a l'immunoglobuline de vcam1 WO1993014220A1 (fr)

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AU34318/93A AU664071B2 (en) 1992-01-13 1993-01-12 Antibodies recognizing the fourth immunoglobulin-like domain of VCAM1
JP5512513A JPH07506244A (ja) 1992-01-13 1993-01-12 Ｖｃａｍ１の第４免疫グロブリン様ドメインを認識する抗体
EP93902915A EP0623173A1 (fr) 1992-01-13 1993-01-12 Anticorps reconnaissant le quatrieme domaine de similitude a l'immunoglobuline de vcam1

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Cited By (6)

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Publication number Priority date Publication date Assignee Title
WO2007139359A1 (fr) 2006-05-31 2007-12-06 Hanwha Chemical Corporation Anticorps monoclonal vcam-1 spécifique
WO2010114312A3 (fr) * 2009-03-31 2011-03-31 Hanwha Chemical Corporation Anticorps monoclonal humain qui se lie spécifiquement à vcam-1 et composition pour traiter une maladie inflammatoire ou un cancer comprenant celui-ci
WO2013160676A1 (fr) * 2012-04-24 2013-10-31 Isis Innovation Limited Anticorps anti-cd106 (vcam-1)
WO2017205560A1 (fr) * 2016-05-27 2017-11-30 Albert Einstein College Of Medicine, Inc. Méthodes de traitement du cancer par ciblage de vcam1 et de maea
US10947311B2 (en) 2015-11-20 2021-03-16 The Board Of Trustees Of The Leland Stanford Junior University VCAM-1 mediated methods and compositions for treating aging-associated impairments
US11560433B2 (en) 2016-05-27 2023-01-24 Albert Einstein College Of Medicine Methods of treatment by targeting VCAM1 and MAEA

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Title
CELL REGULATION vol. 2, no. 5, May 1991, BETHESDA MD, US pages 347 - 355 D. TAICHMAN ET AL. 'Tumor cell surface alpha 4 beta 1 integrin mediates adhesion to vascular endothelium: Demonstration of an interaction with the N-terminal domains of INCAM-110/VCAM-1.' *
CELLULAR AND MOLECULAR MECHANISMS OF INFLAMMATION vol. 2, 1991, SAN DIEGO CA, US pages 151 - 169 R. LOBB ET AL. 'Vascular cell adhesion molecule-1.' *
JOURNAL OF EXPERIMENTAL MEDICINE vol. 173, no. 6, 1 June 1991, NEW YORK, US pages 1553 - 1556 B. BOCHNER ET AL. 'Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: Contributions of endothelial cell adhesion molecules.' *
JOURNAL OF EXPERIMENTAL MEDICINE vol. 175, no. 6, 1 June 1992, NEW YORK, US pages 1433 - 1442 R. VONDERHEIDE ET AL. 'Lymphocyte adhesion through very late antigen 4: Evidence for a novel binding site in the alternatively spliced domain of vascular cell adhesion molecule 1 and an additional alpha 4 integrin counter-receptor on stimulated endothelium.' *
JOURNAL OF EXPERIMENTAL MEDICINE vol. 176, no. 1, 1 July 1992, NEW YORK, US pages 99 - 107 L. OSBORN ET AL. 'Activated endothelium binds lymphocytes through a novel binding site in the alternately spliced domain of vascular cell adhesion molecule-1.' *
THE FASEB JOURNAL vol. 5, no. 6, 19 March 1991, BETHESDA MD, US page A1602 P. DICORLETO ET AL. 'Monoclonal antibodies (Mab) to ELAM-1 and VCAM-1 that inhibit monocytic cell adhesion to activated and sparse endothelial cells (EC).' *
THE JOURNAL OF IMMUNOLOGY vol. 146, no. 2, 15 January 1991, BALTIMORE MD, US pages 592 - 598 M. THORNHILL ET AL. 'Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells.' *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7655417B2 (en) * 2006-05-31 2010-02-02 Hanwha Chemical Corporation VCAM-1 specific monoclonal antibody
WO2007139359A1 (fr) 2006-05-31 2007-12-06 Hanwha Chemical Corporation Anticorps monoclonal vcam-1 spécifique
US8715670B2 (en) 2009-03-31 2014-05-06 Hanwha Chemical Corporation Human monoclonal antibody that specifically binds to VCAM-1 and a composition for treating an inflammatory disease or a cancer comprising the same
WO2010114312A3 (fr) * 2009-03-31 2011-03-31 Hanwha Chemical Corporation Anticorps monoclonal humain qui se lie spécifiquement à vcam-1 et composition pour traiter une maladie inflammatoire ou un cancer comprenant celui-ci
EP2414392A2 (fr) * 2009-03-31 2012-02-08 Hanwha Chemical Corporation Anticorps monoclonal humain qui se lie spécifiquement à vcam-1 et composition pour traiter une maladie inflammatoire ou un cancer comprenant celui-ci
CN102369216A (zh) * 2009-03-31 2012-03-07 韩华石油化学株式会社 特异性结合vcam-1的人单克隆抗体以及用于治疗炎性疾病或癌症、包含该人单克隆抗体的组合物
EP2414392A4 (fr) * 2009-03-31 2013-03-13 Hanwha Chemical Corp Anticorps monoclonal humain qui se lie spécifiquement à vcam-1 et composition pour traiter une maladie inflammatoire ou un cancer comprenant celui-ci
WO2013160676A1 (fr) * 2012-04-24 2013-10-31 Isis Innovation Limited Anticorps anti-cd106 (vcam-1)
US10040854B2 (en) 2012-04-24 2018-08-07 Oxford University Innovation Limited Antibodies against CD106 (VCAM-1)
US10947311B2 (en) 2015-11-20 2021-03-16 The Board Of Trustees Of The Leland Stanford Junior University VCAM-1 mediated methods and compositions for treating aging-associated impairments
WO2017205560A1 (fr) * 2016-05-27 2017-11-30 Albert Einstein College Of Medicine, Inc. Méthodes de traitement du cancer par ciblage de vcam1 et de maea
US11560433B2 (en) 2016-05-27 2023-01-24 Albert Einstein College Of Medicine Methods of treatment by targeting VCAM1 and MAEA
US11732053B2 (en) 2016-05-27 2023-08-22 Albert Einstein College Of Medicine Methods of treatment by targeting VCAM1 and MAEA

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AU664071B2 (en) 1995-11-02
AU3431893A (en) 1993-08-03

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