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WO1993007490A1 - Procedes et materiaux de diagnostic et de pronostic - Google Patents

Procedes et materiaux de diagnostic et de pronostic Download PDF

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Publication number
WO1993007490A1
WO1993007490A1 PCT/GB1992/001791 GB9201791W WO9307490A1 WO 1993007490 A1 WO1993007490 A1 WO 1993007490A1 GB 9201791 W GB9201791 W GB 9201791W WO 9307490 A1 WO9307490 A1 WO 9307490A1
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Prior art keywords
vcam
soluble
soluble vcam
assay
specific binding
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Inventor
Andrew John Hubert Gearing
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Vernalis R&D Ltd
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British Bio Technology Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70542—CD106
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • This invention relates to material useful in certain diagnostic and prognostic methods and to methods of diagnosis and prognosis using the material.
  • VCAM Vascular cell adhesion molecule
  • VCAM-1 which is 6 domains
  • VCAM-2 which is 7 domains
  • VCAM is basally expressed on endothelium but is dramatically upregulated by infective or immunological stimulation (Osborn L. et al., Cell, 59, 1203-1211 (1989) ) .
  • VCAM acts as an adhesive structure mediating the binding of cell types which express the integrin VLA-4 on their cell membranes (Elices M.J. et al., Cell 60 577-584 (1990)).
  • Two types of VCAM have been identified, a predominant HOkD form consisting of seven distinct extracellular immunoglobulin-li e domains, a transmembrane and intracytoplasmic domain, and a 90kD form which has a single immunoglobulin domain deletion.
  • WO-A-9013300 discloses cDNA sequences coding for certain soluble forms of VCAM.
  • VCAM is therefore a specific marker of endothelial activation. As access to endothelium would normally require some form of invasive sampling technique or biopsy, measurement of cell surface VCAM is not a feasible routine assay for endothelial activation. If VCAM expression could be more readily detected, there would be considerable potential for providing diagnostic and prognostic information on developing immune or inflammatory responses.
  • VCAM is naturally released by activated or damaged endothelium in soluble form into the blood or tissue fluid; it has also been found that the level of release is higher in activated or damaged endothelium; these observations provide the basis of a routine assay for VCAM.
  • the present invention provides the use of a soluble form of vascular cell adhesion molecule (VCAM) as an assay standard for soluble VCAM.
  • VCAM vascular cell adhesion molecule
  • VCAM soluble vascular cell adhesion molecule
  • Such a preparation can also enable antibodies, whether polyclonal or monoclonal, to be prepared against soluble VCAM.
  • a preparation of soluble VCAM may be regarded as substantially free of proteins with which it is naturally associated when at least 70%, 80%, 90%, 95%, 98% or 99% by weight, in increasing order of preference, of the total protein present is soluble VCAM.
  • the preparation of soluble VCAM may be as pure as is obtainable by affinity chromatography, as will be described below.
  • Other components such as water, salts and buffering materials and any other non-interfering materials, may be present in the preparation.
  • Soluble VCAM as used in the present invention may be in any (or any combination) of the forms in which it is naturally present in human serum and plasma, from which it can be isolated. Purification of soluble VCAM from plasma on a monoclonal antibody affinity column reveals three species with molecular weights of lOOkD, 80kD and 40kD which all reacted in western blotting with a polyclonal antiserum raised against recombinant soluble VCAM. N-terminal sequence analysis of the first 10 residues of the lOOkD form (FKIETTPESRYL - SEQ ID NO 1) identified the material as being identical to the N terminus of the published sequence of membrane bound VCAM.
  • soluble VCAM may correspond to membrane-bound VCAM but with the functional absence of that portion of the protein which effects binding to the membrane.
  • functional absence is meant that the relevant portion either is actually absent or is so modified or masked as to be ineffective for binding the protein to the membrane.
  • At least the transmembrane domain, and preferably the intracytoplasmic domain, of full length, membrane-bound VCAM is functionally absent in preferred soluble VCAM useful in this invention.
  • Soluble VCAM may be prepared in a variety of ways. First, it may be recovered from the supernatant of cells expressing (whether naturally or artificially) VCAM.
  • membrane-bound VCAM may be prepared either in cells which naturally produce VCAM (such as activated human umbilical vein endothelial cells (H ⁇ VEC) ) or in cells which are modified to produce VCAM (such as Chinese hamster ovary (CHO) or other suitable host cells transformed or transfected to express VCAM) .
  • VCAM activated human umbilical vein endothelial cells
  • CHO Chinese hamster ovary
  • soluble VCAM may be produced by proteolytic cleavage or other protein engineering of membrane-bound VCAM.
  • soluble human VCAM can also be found in human serum and plasma, from which it can be purified, for example by affinity chromatography.
  • soluble VCAM may also be prepared by recombinant DNA technology. This will involve expression of DNA coding for a VCAM molecule in which the transmembrane domain is functionally, and preferably actually, absent. The intracytoplasmic domain may also be functionally absent.
  • At least one immunoglobulin-like domain and preferably all the immunoglobulin-like domains present in the forms of natural membrane-bound VCAM, will generally be present.
  • the.N-terminus of VCAM up to and including Glu 606 , but nothing further, is present.
  • DNA useful in such a process will usually be recombinant or isolated and code for a VCAM molecule in which the transmembrane domain is functionally, and preferably actually, absent.
  • recombinant DNA may be in the form of a vector.
  • the vector may for example be a plasmid, cosmid or phage.
  • Vectors will frequently include one or more selectable markers to enable the selection of host cells transformed (or transfected: the terms are used interchangeably in 1 this specification) with them and, preferably, to enable selection of cells harbouring vectors incorporating heterologous DNA. Appropriate start and stop signals will generally be present. Additionally, if the vector is intended for expression, sufficient regulatory sequences to drive expression will be included. Vectors not including regulatory sequences are useful as cloning vectors.
  • Cloning vectors can be introduced into E. coli or any other suitable hosts which facilitate their manipulation.
  • Expression vectors can be introduced into host cells suitable for the expression of soluble VCAM.
  • prokaryotic cells may be used, eukaryotic and, particularly, mammalian cells such as CHO cells may be preferred for this purpose.
  • mammalian cells such as CHO cells may be preferred for this purpose.
  • DNA suitable for use in the invention can be prepared by any convenient method involving coupling together successive nucleotides, and/or ligating oligo- and /or poly-nucleotides, including in vitro processes, but recombinant DNA technology forms the method of choice.
  • soluble VCAM Purification of soluble VCAM, however prepared, can be achieved by any suitable method, but a particularly preferred technique is affinity chromatography, which may be embodied in the following method:
  • A providing a crude source of soluble VCAM (for example human serum or plasma or the supernatant of cells expressing a soluble VCAM protein) which may be crudely purified;
  • B introducing the crude source to an affinity matrix containing immobilised specific binding molecules for VCAM;
  • C permitting soluble VCAM to bind to the matrix;
  • D washing the matrix to remove unbound contaminants; and
  • E recovering soluble VCAM in substantially pure form by elution from the matrix.
  • Affinity columns can be prepared in a number of ways well known to those skilled in the art.
  • Specific binding molecules which have an affinity for soluble VCAM include anti-VCAM antibodies and natural ligands for VCAM such as VLA-4.
  • Cyanogen bromide-activated SEPHAROSE for example, can readily be used to couple antibodies or other specific binding molecules. (The word SEPHAROSE is a trade mark.)
  • a soluble VCAM preparation in accordance with the invention is useful in immunological and other assays for the detection of naturally released soluble forms of VCAM.
  • a method of assaying a sample of body fluid for the presence, and optionally the amount, of soluble VCAM comprising contacting the sample with a specific binding molecule for VCAM, detecting the amount of VCAM bound to the specific binding molecule and calibrating the assay with a soluble VCAM preparation as described above. Authentication of such assays is a critical issue.
  • the specific binding molecule may be a monoclonal or polyclonal antibody.
  • an VCAM enzyme-linked immunosorbant assay ELISA
  • ELISA VCAM enzyme-linked immunosorbant assay
  • Other antibody-based assay formats including but not restricted to radioimmunoassays, two site radiobinding assays and immunoblotting assays, can equally be applied to the detection of VCAM in samples. These assay formats are well known to those skilled in the art and are described by Johnstone and Thorpe in 'Immunochemistry in Practice', Blackwells Scientific, Oxford (1989).
  • VCAM The ligands for VCAM could also form the specific binding molecule of an assay system.
  • VCAM is known to bind to the integrin VLA-4 , which acts as the physiological ligand for VCAM.
  • a soluble form of VLA-4 could function in an analogous fashion to an anti-VCAM antibody in any of the assay formats described above, but would have the advantage of only binding to and therefore detecting biologically active VCAM.
  • the levels of soluble VCAM in biological samples such as serum, plasma, bronchiolar lavage fluid, ascites, cerebro-spinal fluid or synovial fluid can be determined quantitatively or semi-quantitatively using any of the assay formats described above.
  • kits for use in an assay for soluble VCAM comprising (a) means for detecting soluble VCAM and (b) .
  • an assay standard comprising a preparation of soluble VCAM as described above.
  • the means for detecting soluble VCAM will depend on the particular assay method used, but may include a detectably labelled specific binding molecule for VCAM.
  • Components (a) and (b) may be provided in separate containers within the kit.
  • Measurement of elevated levels of soluble VCAM in body fluids would provide an indication as to the state of activation or damage t ⁇ the endothelium and could provide diagnostic or prognostic information.
  • a method of assessing the state of activation of, and/or damage to, the endothelium of an individual comprising assaying body fluid from the individual for the presence of, and optionally the amount of, VCAM.
  • the amount of VCAM present need not be determined absolutely: it will often be sufficient to compare the amount of VCAM detected in the individual with that detected in a control (normally healthy) individual.
  • vasculitic disease such as systemic lupus erythematosis or systemic vasulitis
  • diabetes transplant rejection
  • septic shock in which local and systemic release of cytokines can activate endothelium
  • viral infections such as cytomegalovirus, which can infect endothelium.
  • Figure 1 shows the detection, by ELISA, of recombinant soluble VCAM which had been expressd in CHO cells and purified by monoclonal antibody affinity chromatography.
  • Figure 2 shows a western blot of soluble VCAM purified from human plasma by affinity chromatography: protein bands of 100, 80 and 40 kD are stained with a goat serum raised against recombinant soluble VCAM.
  • Figure 3 shows a plot of the serum levels of VCAM-1 and creatinine in a patient following renal allograft.
  • Figure 4 shows the detection of soluble VCAM in samples of serum taken from a patient at increasing times following a cardiac allograft.
  • the peak of soluble VCAM coincides with a cytomegalovirus infection.
  • VCAM VCAM
  • human umbilical vein endothelial cells (HUVEC) which have been activated to express VCAM using a mixture of interleukin-1, tumour necrosis factor and endotoxin.
  • Standard procedures were used to derive hybridoma lines from the spleen cells of immune mice, which were screened for reactivity with the activated HUVEC.
  • VCAM reactivity was confirmed by specific binding to COS cells transfected with the expression vector pCDM8 containing a full length human VCAM gene.
  • the methods used for derivation and characterisation of anti-VCAM monoclonal antibodies are identical to those described for ELAM by Piggott R. et al., Journal of Immunology, (1991) 147: 130-135.
  • Monoclonal antibodies could also be raised using the VCAM-transfected mammalian cells as immunogens, using VCAM which has been detergent extracted from activated HUVEC or VCAM-transfected mammalian cells, and using recombinant genetically engineered soluble forms of VCAM, or soluble VCAM from human serum or plasma.
  • Polyclonal antibodies can be raised in mammals or birds using standard procedures with purified VCAM as immunogen.
  • Example 1 Preparation of Purified Soluble VCAM by recombinant DNA technology
  • a recombinant soluble VCAM has been constructed by deletion of the transmembrane and intracytoplasmic domains of full length VCAM.
  • This construct can be expressed in mammalian cells using any of several expression vectors known to those skilled in the art.
  • this process is exemplified using CHO cells with the pGWIHG expression vector, disclosed in O-A-9109118, to give a supernatant containing soluble VCAM.
  • the full length VCAM gene was isolated by an antibody panning technique using anti-VCAM monoclonal antibodies to select COS cells expressing VCAM from a population of cells which had been transfected with a cDNA library from cytokine activated HUVEC in the expression vector pCDM ⁇ (method described in Pigott R. et al., J. Immunol. (1991) 147: 130-135).
  • VCAM-2 cDNA was isolated as a 3.0kb insert in the the BstXI site of pCDM8 (pCDM8/VCAM) - commercially available from the Laboratory Products Division of British Bio-technology Limited, Abingdon, Oxfordshire, UK.
  • a stop codon (TAG) at Glu 6 0 6 and a new Pstl restriction site 3' of the new stop codon to facilitate the removal of the remaining transmembrane region, cytoplasmic tail and 3' non-coding sequences.
  • TAG stop codon
  • From the mutated DNA a replicative form (double stranded DNA) was made and cut with the restriction enzymes EcoRI and Pstl and the soluble VCAM fragment sub-cloned into pCDM8/VCAM cut with EcoRI and Pstl to produce pCDM8/sVCAM.
  • the soluble VCAM was transferred to the expression vector pGWIHG.
  • a fragment encoding the soluble VCAM was excised from pCDM8/sVCAM with the restriction enzyme Xhol-Pstl and the protruding ends cut back with T4 DNA polymerase in the absence of dNTPs.
  • the resulting blunt ended fragment was cloned into pGWIHG also cut with Xbal and the 5' protruding ends filled in with T4 DNA polymerase in the presence of dNTPs.
  • Plasmids with sVCAM in the same orientation as the HMCV promoter were identified by the presence of a 8640bp and 617 bp fragments following digestion with EcoRI. A large scale preparation of this construct was made.
  • the pGWlHG/sVCAM DNA was linearised with the restriction enzyme Notl and electroporated into CHO cells for expression. Expression resulted in VCAM being released into the supernatant, as indicated in Figure 4.
  • the soluble VCAM produced by these cells is used as a laboratory unitage standard for VCAM.
  • soluble VCAM was prepared from a detergent extract of CHO cells which had been transfected with a full length VCAM gene in the expression vector pGWIHG.
  • Confluent T-75 flasks of CHO cells (approximately 5x10° cells) expressing the VCAM gene were drained and washed with room temperature phosphate buffered saline. The flasks were then placed on a bed of ice. 2 ml of lysis buffer (150mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50mM Tris, pH7.5), chilled to 4°C was added to each flask and left to incubate for 30 mins with occasional rocking.
  • lysis buffer 150mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50mM Tris, pH7.5
  • Soluble VCAM as prepared in Example 1 or 2 was purified by affinity chromatography, using cyanogen bromide-activated SEPHAROSE to couple antibodies. (The word SEPHAROSE is a trade mark.)
  • CNBr-activated Sepharose (lg giving about 3.5ml swollen gel) was washed thoroughly with ImM HC1.
  • the lyophylised capture antibody was dissolved in coupling buffer (0.1M NaHC0 3 , pH8.3 containing 0.5M NaCl), to give a 5mg/ml solution.
  • the antibody was mixed with the gel (1ml antibody per ml swollen gel) in a stoppered vessel and rotated end over end for 2 hours at room temperature. Excess antibody was washed away with coupling buffer and remaining active sites blocked with Tris-HCl buffer (O.IM, pH8.0) for 2 hours at room temperature.
  • the product was washed with 3 cycles of alternating pH.
  • Each cycle consisting of a wash with acetate buffer (O.IM, pH4) containing NaCl (0.5M) followed by a wash with Tris buffer (O.IM, pH8) containing NaCl (0.5M) .
  • the product was packed into glass chromatography columns and stored at 4°C.
  • Example 1 or 2 containing soluble VCAM were centrifuged at high speed to remove all cell debris. Buffering reagents were added to the sample solution to produce a solution containing O.IM Tris and 0.5M NaCl (pH8.5). This was loaded on to the appropriate column at a rate of 2ml/hr using a peristaltic pump. The column was washed with 20 column volumes of wash buffer (0.1M Tris, 0.5M NaCl, pH8.5) or until a baseline reading had been attained on a chart recorder. Bound proteins were eluted using glycine-HCl (O.IM, pH2.5) at lml/min using a peristaltic pump.
  • wash buffer 0.1M Tris, 0.5M NaCl, pH8.5
  • Antibodies can be used to construct assay systems for the detection of soluble VCAM in samples.
  • This example relates to a two site enzyme linked immunoassay (ELISA) for the detection of soluble VCAM.
  • ELISA enzyme linked immunoassay
  • antibody at lO ⁇ g/ml is allowed to bind in 50mM sodium carbonate buffer at pH 9.0 to the surface of wells in plastic culture plates. Unbound antibody is removed by washing with phosphate buffered saline containing 0.1% Tween (PBS-T) (the word Tween is a trade mark) , and any residual protein binding capacity of the plastic is blocked by an excess of another protein for example 1% casein in PBS. Samples containing VCAM are added to the wells where the VCAM specifically binds to the capture antibody.
  • PBS-T phosphate buffered saline containing 0.1% Tween
  • VCAM is washed off with PBS-T, and a second antibody to VCAM is added which binds to the captured VCAM.
  • This second antibody is directly chemically conjugated to NHS-LC biotin according to the manufacturers instruction (Pierce NHS-LC Biotin instructions 21335) . After 1 hour the unbound antibody is washed off with PBS-T and the biotinylated antibody is subsequently allowed to bind to a 1:1000 dilution of a streptavidin-horseradish peroxidase conjugate.
  • the enzyme After washing with PBS-T, the enzyme is revealed by conversion of a substrate, 2,2-azino-bis-(3-ethyl- benzthiazoline sulphonic acid at 1 mg/ml in phosphate buffer pH 9.0, to give a coloured reaction product.
  • the reaction is allowed to proceed for 30 minutes before stopping by the addition of 0.2M citric acid.
  • an ELISA is used to measure VCAM levels in a variety of serum samples.
  • the assay was calibrated using a standard preparation of recombinant soluble VCAM which has been aliquoted and stored at -20"C. This standard has been given a defined arbitrary unitage. VCAM can be detected using this assay in normal apparently healthy individuals.
  • Serum from two groups of individuals was examined.
  • One group of 34 were laboratory workers, with a mean level of 53.5 Units/ml; and the second group were 26 blood donors with a mean level of 58.0 Units/ml .
  • NBTS normals were normal blood donors
  • CAPD chronic ambulatory peritoneal dialysis
  • the source of the soluble VCAM is likely to be the vascular endothelium as soluble VCAM has been measured in the supernatant of HUVECs activated with cytokines.
  • Serum levels of VCAM were measured using an ELISA test as described in Example 5.
  • VCAM mean U/ml
  • VCAM was elevated during periods of disease activity, and did not fall when disease activity was reduced. Since this group of patients frequently relapse, measurement of continued elevated VCAM levels is an indication of the progression of this disease.
  • the levels of soluble E-selectin or ICAM-1 in patient sera provided no useful correlation with the presence of disease or its activity. However, measurement of levels of soluble VCAM in the serum of patients will help in the diagnosis and staging of vasculitic disease.
  • Serum levels of VCAM were measured using an ELISA test as described in Example 5. Levels of soluble VCAM were measured in the serum of 15 patients following renal allograft. In all cases, rejection episodes (defined by a rise in the level of creatinine) are at least coincident with and usually preceded by rise in the level of soluble VCAM. An example is shown in Figure 3. This test will therefore provide a useful advance warning of a rejection episode, allowing immunosuppressive treatment to be given earlier.
  • Serum levels of VCAM were measured using an ELISA test as described in Example 5. Levels of soluble VCAM were measured in a single patient with a cardiac allograft. Figure 4 shows that the peak of soluble VCAM coincides with a cytomegalovirus infection. This test can therefore be used to monitor patients at risk of rejection following viral infection.
  • soluble VCAM levels of soluble VCAM were measured in the serum of 15 patients with septic shock and multiple organ failure syndrome. In all cases, the levels of soluble VCAM were elevated above control levels.
  • Phe Lys lie Glu Thr Thr Pro Glu Ser Arg Tyr Leu 1 5 10

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Abstract

La présence de molécules d'adhésion de cellule vasculaire solubles (VCAM) dans des fluides corporels est un indicateur de l'état d'activation et/ou de détérioration de l'endothélium. Des préparations homogènes de VCAM soluble, qui sont utiles comme normes de dosage dans des dosages diagnostiques et pronostiques utilisés pour détecter des maladies affectant le système vasculaire sont obtenues selon différents procédés, y compris l'extraction détersive à partir de cellules exprimant les VCAM à fixation membranaire et l'expression d'ADN recombiné codant pour une forme tronquée des VCAM. La chromatographie par affinité constitue une étape de purification finale utile.
PCT/GB1992/001791 1991-09-30 1992-09-30 Procedes et materiaux de diagnostic et de pronostic Ceased WO1993007490A1 (fr)

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GB919120767A GB9120767D0 (en) 1991-09-30 1991-09-30 Diagnostic and prognostic materials and methods
GB9120767.0 1991-09-30

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990013300A1 (fr) * 1989-04-28 1990-11-15 Biogen, Inc. Molecules d'adhesion leucocytes-cellules endotheliales (elam) et molecules impliquees dans l'adhesion des leucocytes (mila)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990013300A1 (fr) * 1989-04-28 1990-11-15 Biogen, Inc. Molecules d'adhesion leucocytes-cellules endotheliales (elam) et molecules impliquees dans l'adhesion des leucocytes (mila)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS vol. 178, no. 3, 15 September 1991, DULUTH, MINNESOTA US pages 1498 - 1504 R. LOBB ET AL. 'EXPRESSION AND FUNCTIONAL CHARACTERIZATION OF A SOLUBLE FORM OF VASCULAR CELL ADHESION MOLECULE' *
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS vol. 187, no. 2, 16 September 1992, DULUTH, MINNESOTA US pages 585 - 589 R.PIGOTT ET AL. 'SOLUBLE FORMS OF E-SELECTIN,ICAM-1 AND VCAM-1 ARE PRESENT IN THE SUPERNATANTS OF CYTOKINE ACTIVATED CULTURED ENDOTHELIAL CELLS.' *
CELL. vol. 60, no. 4, 23 February 1990, CAMBRIDGE, NA US M. J. ELICES ET AL. 'VCAM-1 ON ACTIVATED ENDOTHELIUM INTERACTS WITH THE LEUKOCYTE INTEGRIN VLA-4 AT A SITE DISTINCT FROM THE VLA-4/FIBRONECTIN BINDING SITE.' cited in the application *

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