EP0462111A1 - Sequences de guidage et leurs emplois - Google Patents

Sequences de guidage et leurs emplois

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Publication number
EP0462111A1
EP0462111A1 EP90902104A EP90902104A EP0462111A1 EP 0462111 A1 EP0462111 A1 EP 0462111A1 EP 90902104 A EP90902104 A EP 90902104A EP 90902104 A EP90902104 A EP 90902104A EP 0462111 A1 EP0462111 A1 EP 0462111A1
Authority
EP
European Patent Office
Prior art keywords
lpam
binding
mel
dna sequence
domain
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP90902104A
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German (de)
English (en)
Other versions
EP0462111A4 (en
Inventor
Irving L. Weissman
Bernard Holzmann
Mark H. Siegelman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leland Stanford Junior University
Original Assignee
Leland Stanford Junior University
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Filing date
Publication date
Application filed by Leland Stanford Junior University filed Critical Leland Stanford Junior University
Publication of EP0462111A1 publication Critical patent/EP0462111A1/fr
Publication of EP0462111A4 publication Critical patent/EP0462111A4/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K16/2839—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 integrin superfamily
    • C07K16/2842—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 integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the technical field of the subject invention concerns physiologically active proteins associated with cellular homing to target anatomical sites.
  • the immune system unlike most organ systems which are consolidated in one anatomical location, is dispersed over an entire organism. It exists as circulating elements in the blood, through which it gains access to nearly all body tissues, and as
  • the immune system is placed under a special constraint, which is managed by substituting extensive cell-cell recognition and interactive events.
  • lymphoid system is relieved by scattered solid collections of lymphoid elements, such as thymus, lymph nodes, Peyer's patches, and spleen, which
  • lymphocytes Perpetual percolation of lymphocytes through lymphoid organs efficiently arms each of these organs with the entire repertoire of antigen-reactive cells; lymphocytes recirculate from blood to lymphoid organs and back to blood, generally passing the efferent lymphatic vessels and their collecting ducts.
  • the specific portal of entry of lymphocytes from lymphoid organs efficiently arms each of these organs with the entire repertoire of antigen-reactive cells
  • HEV's high endothelial venules
  • lymphocyte homing appears to be regulated by the expression of complementary adhesion molecules on each of the two participants, the recirculating lymphocyte and the specialized lymphoid organ HEV's.
  • the homing phenomenon is an important aspect of many systems, both for the benefit and detriment of the host.
  • the ability to home specific cells to particular organs can be of great benefit in the defense of disease, particularly where the cells may be introduced adjacent to the particular organ of
  • the homing receptor may serve to enhance metastases, so as to spread the neoplasia throughout the immune system.
  • Homing may be an aspect of the inflammatory response, which may result in autoimmune diseases.
  • the ability to diminish the inflammatory response or attack on native tissue may serve as a therapy in the case of such diseases as rheumatoid arthritis.
  • lymphoid organs e.g., lymph nodes and mucosal lymphoid organs, e.g., Peyer's patches
  • methods for modulating homing to peripheral lymphoid organs employing antibodies to the homing receptor core proteins, nucleic acid compositions for the expression of core proteins, methods of transfecting cells to provide homing capability, and the use of the various lymphoid organs, e.g., lymph nodes and mucosal lymphoid organs, e.g., Peyer's patches, are provided, employing antibodies to the homing receptor core proteins, nucleic acid compositions for the expression of core proteins, methods of transfecting cells to provide homing capability, and the use of the various
  • compositions in diagnosis and therapy are described.
  • mouse and human alpha and beta subunits of the integrin family used for homing to mucosal lymphoid organs and lymph node homing receptors are described.
  • VLA-4 is a member of the integrin family associated with homing to the high endothelial venules (HEV's) associated with Peyer's patches, while a ubiquinated protein which is highly glycosylated is shown to be associated with a lymph node homing receptor.
  • HEV's high endothelial venules
  • nucleic acids encoding the core proteins or physiologically active fragments thereof the use of such nucleic sequences for transfection of cells to provide homing to the particular sites or produce peptides which may be used as antagonists, the proteins and fragments thereof which may be used as antagonists, antibodies to the proteins, and antidiotypes are described.
  • compositions may be used in a variety of ways: In diagnosis, to define the presence or absence of cells, tissue or bodily fluids containing and/or expressing the homing receptors or the
  • compositions may be used to direct various compositions to
  • the nucleic acid sequences may be used for producing the subject peptides, or fragments thereof, in accordance with genetic techniques or may be joined to other nucleic acid sequences, under conditions involving a replicating species, where the conditions provide for expression of the subject peptides jointly with other proteins, thus directing the replicating species to the target sites.
  • Second will be considered the mucosal lymphoid tissue and organs, including Peyer's patch, homing receptors, associated with the integrin family, where both the mouse and human proteins will be described. It is understood that the mouse and human proteins find analogy, one with the other, in that these proteins are immunologically cross reactive, and that there is substantial conservation of these sequences in the two species. However, the proteins of the two species are given different names and until a common nomenclature is provided, the different names and their analogues will be considered.
  • LPAM-1 and 2 The mouse proteins which are described are referred to as LPAM-1 and 2, where LPAM stands for lymphocyte Peyer's patch HEV adhesion molecule, while VLA stands for Very Late Antigens (of lymphocytes).
  • LPAM-1 and -2 share a common alpha unit referred to as ⁇ 4m , which binds to two different beta subunits, where the beta subunit of LPAM-1 is referred to as ⁇ P which does not find analogy with the heretofore reported beta subunits of the human integrin VLA proteins, ⁇ 1 , ⁇ 2 , and ⁇ 3 .
  • the LPAM-2 beta unit finds analogy with integrin ⁇ 1 .
  • the LPAM proteins are further provided to as ⁇ 4m , which binds to two different beta subunits, where the beta subunit of LPAM-1 is referred to as ⁇ P which does not find analogy with the heretofore reported beta subunits of the human integrin VLA proteins, ⁇ 1 , ⁇ 2 , and ⁇ 3 .
  • the LPAM-2 beta unit finds analogy with integrin ⁇ 1 .
  • the LPAM proteins are further provided
  • LPAM-1 is a heterodimer of ⁇ and ⁇ subunits of about 160kd and 130kd M r respectively, that the association requires the presence of calcium ions, and that proteins of 84kd M r and 62kd M r present in LPAM-1 precipitates appear to be products of
  • LPAM-1 is virtually identical of that of the human integrin receptor VLA-4, with cross-reactivity of monospecific antisera between the alpha units of the VLA-4 and LPAM-1 proteins being observed.
  • the LPAM-1 and -2 proteins and their subunits are provided in purified form, generally being at least 50 wt.%, usually at least about 90 wt.% preferably at least about 99 wt.%, particularly as to the presence of other proteins.
  • the compositions may be present in lyophilized form, in solution, or formulated with other components, as desired.
  • the alpha and beta subunits are transmembrane glycoproteins with large extracellular and short cytoplasmic domains.
  • the human beta subunits show 4048% identity to each other. In amino acid sequence, their extracellular domains contain 56 cysteine residues, all of which are conserved.
  • the alpha subunits of integrins contain a series of sites capable of divalent cation binding, show substantial amino acid sequence similarities between the various alpha
  • subunits include 4 repeats of an 8 cysteine motif.
  • the lymph node homing receptor binds to the antibody MEL-14 which may also recognize a ubiquitin epitope.
  • the lymph node receptor is characterized by being a highly glycosylated protein which is also ubitiquinated and has a core structure as described in the experimental section.
  • the precursor protein has an unusually long signal sequence, which has the normal hydrophobic region, which in turn is followed by a hydrophilic domain.
  • the molecular weight of the glycosylated protein is about 90 kD, while the
  • ubiquitin-free core protein is about 35-40 kD.
  • the mature protein has a pi of about 4-4.5 (See Siegelman and Weissman, Ubiquitin, ed. Martin Rechsteiner, Plenum Publishing Corp., 1988, chapter 9, pp. 239-69).
  • Murine and human lymph node homing receptors have the nucleic acid coding and flanking sequences and related amino acid sequence as described in the
  • Experimental section has a 54 bp 5' untranslated region followed by an initiator ATG codon, which begins an uninterrupted open reading frame of 1,116 bp.
  • the reading frame encodes a protein with a hydrophobic leader sequence 38 amino acids in length, before reaching the initial tryptophan residue of the mature protein.
  • the leader sequence has a length unusual for a signal sequence.
  • the mature protein possess 10 potential asparagine-linked glycosylation sites consistent with protein characterization studies which show extensive glycosylation in endoglycosidase F digestion. These are contained within an identical repeat unit
  • the mature protein contains 22 cysteine residues, where 12 of the cysteines are present in a complement regulatory repeat structure and an
  • cysteines are concentrated in the 60 amino acids just preceding the repeat units involving the EGF-like domain. This results in a highly cysteinerich pre-transmembrane region of 180-190 amino acids.
  • the deduced mature protein is 334 amino acids in length with a calculated molecular weight of 37,600.
  • the hydrophobic transmembrane regions encompassing amino acids from about 295-317 is followed by a cluster of positively charged residues and a hydrophilic cytoplasmic tail of 18 amino acids.
  • a hydropathy plot further shows distinct regions of relative hydrohilicity, concentrated in the amino-terminal 150 acids and in the membrane proximal approximate 20 amino acids.
  • the intervening extracytoplasmic portion is comprised of a relatively electrically neutral stretch which is characterized by repeat units, identical at nucleotide as well as protein level.
  • the extracytoplasmic portion of the receptor is made of three separate extracytoplasmic domains, defined by their homology to three disparate protein motifs.
  • EGF epidermal growth factor
  • the individual domains may serve for their respective purposes as separate and distinct entities.
  • the lectin domain may be used for binding to a complementary sugar or identifying sugars with the particular domain.
  • the EGF domain may be used to bind to the EGF receptor, competing with natural EGF for binding to the receptor.
  • the complement regulatory repeat units may be used in regulating complement, by being combined with the members of the complement cascade to modulate complement formation and lysis.
  • the EGF-like domain preserves many of the cys-gly residues characteristic of the EGF repeat unit, with six consensus cysteines present, as well as glycines at 147 and 150, and tyrosine at 148.
  • the relationship of these conserved residues is identical to that of human and bovine blood clotting factors IX and X, and the Drosophila Notch gene product, and similar to other molecules containing EGF-like
  • the EGF-like domain further shares homology with a portion of one of the cysteinerich repeat units of the ⁇ -chain of the integrin LFA-1 B 2 -chain in the human.
  • the duplicated repeat unit has 62 amino acids in length and spans positions 156-217 and 218-279.
  • a known protein exhibiting significant homology to this sequence is the murine complement factor H, a serum protein with complement regulatory activity.
  • the same homologous repeat motif exists in a number of
  • complement regulatory proteins which bind C3/C4, and in other proteins such as 11-2 receptor, the ⁇ 2 -glycoprotein serum protein and factor XIII.
  • the lymph node homing receptor will be substantially conserved among the various mammalian species. Thus, the receptor will have a signal
  • sequences may be used to inhibit binding of the homing receptor to the HEV.
  • sequences may be modified, where a sequence of only about 8 amino acids may be employed coming within one of the sequences of the various domains.
  • the sequences may be mutated, by changing up to 20% of the amino acids, more usually not more than about 10%, where deletions and insertions may involve from about 1 to 10, usually from about 1 to 5 amino acids.
  • DNA sequences corresponding to the various domains may be used as probes for finding other
  • homing will be primarily to mucosal tissue, which includes Peyer's patches, appendix, tonsils, adenoids, bronchial mucosa, mesenteric lymph nodes, or the like.
  • mucosal tissue which includes Peyer's patches, appendix, tonsils, adenoids, bronchial mucosa, mesenteric lymph nodes, or the like.
  • peripheral lymphoid organ homing receptor all peripheral lymph nodes, and potentionally the spleen, will be the primary targets.
  • the subject proteins, nucleic acid sequences encoding the proteins, or chemically, biologically or physiologically active or useful fragments thereof may find a variety of applications.
  • the proteins or fragments thereof may be used to produce antisera or monoclonal antibodies specific for one or more epitopes of the subject proteins.
  • the antibodies may be used to produce anti-idiotype antibodies which may directly compete with the homing receptor for binding to the complementary ligand. These antibodies find use in inhibiting the complex formation between the homing receptor and its complementary ligand.
  • the antibodies may be used to prevent homing of cells to mucosal sites or lymph nodes.
  • the inhibition of homing may find use in the treatment of inflammatory bowel diseases such as regional ileitis, ulcerative colitis, severe lymphadenitides, histiocytic disorders of lymph nodes or other inflammatory conditions.
  • the antibodies may be used to inhibit metastases, where a neoplastic condition is associated with transport to mucosal sites or lymph nodes.
  • proteins or fragments thereof, capable of binding to the complementary ligand may also be used as antagonists for complex formation.
  • the protein may serve to home to the complementary ligand and inhibit the binding of the homing receptor associated with the target cells.
  • the proteins, fragments thereof, or anti-idiotypes Rather than acting as inhibitors to prevent complex formation between lymphocytes and HEV's, the proteins, fragments thereof, or anti-idiotype
  • antibodies may serve to direct a wide variety of molecules to the homing site.
  • neoplastic tissue by administering one of the subject compounds or compositions bound to a therapeutic drug, one can direct the binding of the therapeutic drug to the desired site for retention and concentration at the desired site.
  • cytotoxic drugs either directly or in the lumen of liposomes, where the subject protein would direct the cytotoxic drug to the homing site.
  • nucleic acid sequences encoding the proteins of the subject invention will usually be at least 12nt, more usually at least 16nt, and may be 50nt or more , providing for a sequence different from the members of the homing receptor proteins having
  • the DNA sequences will be present as other than a mammalian chromosome, generally present as less than 50knt, particularly during
  • the sequence may be integrated in the chromosome, but may be at other than its natural site in the genome.
  • the sequence may be a genomic sequence, comprising all or part of the structural gene or a cDNA comprising all or part of the coding
  • sequences may be identical to the sequence of the gene or be different, including transitions, transversions, deletions or insertions.
  • related sequences may have as little as 30% homology, usually at least about 40% homology.
  • mutant sequences or closely related proteins there will usually be at least about 95% identity with the wild-type sequence, particularly conservative
  • substitutions although there may be substitutions which result in fewer than 5% changes in amino acids, usually not more than a total of 10 amino acids, preferably not more than about 5 amino acids.
  • the nucleic acid sequence may be modified by being labeled with a label capable of providing a detectable signal, either directly or indirectly, such as a radioisotope, biotin, fluorescer, etc.
  • the nucleic acid sequences encoding the subject proteins or fragments thereof may be used for expression of the peptides.
  • vectors may be prepared which provide for expression of a peptide of interest, which may then be harvested for use as described above.
  • a large number of expression vectors are commercially available or have been described in the literature for expression in a variety of
  • prokaryotic and eukaryotic hosts include E. coli, B. subtilis, yeast, such as
  • Saccharomyces, Kluyveromyces, etc. filamentous fungi, such as Neurospora, mammalian cells, such as CHO, COS, HeLa cells, L cells, immortalized T- or B-cells, e.g., EBV immortalized B-cells, etc.
  • Replication systems include ColEl, simian virus 40, baculovirus, lambda, 2m ⁇ plasmid, bovine papilloma virus, etc.
  • a large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. The literature. is replete with examples of these regions, methods for isolating them, and their manner of
  • Vectors may be prepared which will usually include one or more replication systems for cloning or expression, one or more markers for selection in the cellular host, e.g., antibiotic resistance, and one or more expression cassettes for expression of the subject proteins. Desirably when expressing the subject proteins in a cell to be used for homing to a target site, regions other than the wild-type transcription initiation region will be used, where the initiation may be constitutive or inducible, but not subject to the wild-type regulation.
  • the coding sequences may be synthesized, isolated from natural sources, may be prepared as hybrids, or the like. Joining of the coding sequences to the transcriptional regulatory sequences may be achieved by restriction, ligation, use of adaptors or polylinkers, or the like.
  • the particular method of preparing the expression vector, introducing the vector into an appropriate host, growing the host, whereby the subject peptide is expressed, and then isolating the subject peptide is not critical to this invention and any convenient technique or protocol may be employed.
  • stem cells usually syngeneic or allogeneic, and cultivate the stem cells to produce stem cells of a particular lineage or subset, such as natural killer cells, tumor
  • lymphocytes infiltrating lymphocytes, cytotoxic T-lymphocytes, B-cells, or the like.
  • cytotoxic T-lymphocytes cytotoxic T-lymphocytes
  • B-cells or the like.
  • CD4-' CD8- precursor cells
  • mature cells e.g., CD4 + or CD8 +
  • ubiquinated protein Using microorganism hosts or other eukaryotic hosts which do not have the processing capability to ubiquinate the core protein will result in a product which is unprocessed. By contrast, by using an appropriate host, the ubiquinated product will be obtained.
  • the signal sequence of the lymph node homing receptor may also be used for transport of a wide variety of proteins along particular pathways of intracellular trafficking to result in special posttranslational modifications for placement in various intracellular compartments or into the nutrient medium.
  • the subject signal sequence provides an additional signal sequence which may find preferred application with certain proteins.
  • the ⁇ 4m or b P protein may be used to obtain the gene encoding the a 4m or b P protein, either as the genomic gene or as cDNA.
  • a probe based on an amino acid sequence of the a 4m or b_ protein of at least about 6, preferably 8, amino acids using the redundancy of the codons to prepare all possible variations, one can identify sequences in a library comprising either cDNA or genomic DNA.
  • the cDNA library may be prepared in accordance with conventional ways from cytoplasmic RNA from a homing Peyer's patch HEV binding lymphoma, e.g., TK1, and then subtracted with a T-cell lymphoma which does not home to Peyer's patches. The subtracted library may then be probed with the probe indicated above. Positive clones may then be sequenced to identify the presence of a nucleic acid sequence encoding the correct amino acid
  • the truncated sequence may be used as a probe to identify a clone having a complete sequence or, if necessary, to use the
  • sequence as a primer for reverse transcription of mRNA from the original source.
  • the DNA may be used to provide conjugates for specific binding to complementary sequences in a host cell. In this way one may identify cells comprising mRNA for the homing receptor proteins. Furthermore, such sequences may be used as therapeutic agents to destroy expression of homing receptor in cells expressing the homing receptor, by linking such sequences to agents capable of cleaving nucleic acid sequences, such as ribozymes, metal chelates, etc.
  • the subject proteins may also be used to provide vaccines, by introducing a sequence coding for the subject proteins in place of a gene in a virus encoding the envelope protein.
  • the viruses would then be transported to a site having a large lymphocyte population, where the virus could be endocytosed resulting in a strong immune response.
  • the subject proteins or fragments thereof may find use as conjugates to various compounds, aggregations, cells or the like, for directing specific compositions to the target site.
  • the epitopic binding site of the homing receptor may be radiolabelled for specifically directing a radioisotope for diagnosis or therapy to high endothelial venules of Peyer's patches or other mucosal sites or lymph nodes. In this way the radiolabel may be concentrated at sites of interest for diagnosis of neoplasia, treatment of aberrant cells, etc.
  • the subject epitopic site may be used for directing cytotoxic compounds to specific sites, such as natural toxins, antibiotics, enzyme inhibitors, or the like.
  • the subject compounds may be bound to liposomes by conventional ways for directing a liposome to a particular site.
  • the lumen of the liposome may serve to carry drugs or other compounds of interest to the site for diagnosis or therapy. Examples of conjugation of proteins to lipids finds extensive exemplification in the literature.
  • the subject proteins may be used to direct specific subtypes of antibodies or cells producing particular antibodies to target sites, providing protective antibody at the target sites.
  • IgG, IgA, IgM, IgE or IgD may find particular use.
  • variable regions of antibodies have been cloned and shown to be effective in binding to
  • fusion protein products may be produced which will provide the desired binding capability at the target site.
  • the subject nucleic acid gene sequences may also be used to transform cells in order to direct the cells to particular target sites.
  • DNA constructs may be introduced In vitro into a target cell to provide homing capability to the cell.
  • cells e.g., lymphocytes, may be transformed with expression
  • casettes comprising a transcriptional and translational initiation region functional in the host cell, a gene encoding one or the other homing receptors or of the subunits of a homing receptor, and a functional
  • the activated lymphocytes would have the homing receptor on the surface and be directed to the target site.
  • proteins will be administered in an appropriate physiologically acceptable medium, e.g., water, saline, phosphate buffered saline, or the like. Administration will normally be parenteral, particularly intravascularly. For the reasons given above, the course of treatment will also vary. For therapeutic use of cells, the number of cells will also vary as indicated above.
  • the subject compositions may be used in diagnostic assays for the proteins or the nucleic acids.
  • the proteins may be used as standards, conjugated to labels as reagents, or the like to determine the presence of the subject protein on a cell.
  • Cells may then be segregated in accordance with their target by using a FACS, the number of cells for a particular target determined as an indication of the health status of an individual, or the like.
  • the nucleic acids may be used as probes to detect transcription of the gene encoding the subject peptides as indicative of the state of the cell, e.g. activated or not activated, the nature of the integrin, or the like.
  • Conventional assay techniques may be used to determine the various events.
  • the major index cell line utilized for these studies was EL-4/MEL-14 hi , a variant of the continuous T cell lymphoma cell line, EL-4, selected by
  • Immunoprecipitation of cell surface 125 I- iodinated EL4/MEL-14 hi by MEL-14 antibody was performed as follows. 2 X 10 7 cells were surface radioiodinated using lactoperoxidase, then solubilized in 2 ml PBS containing 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 0.1M NaCl, 0.01 M Na phosphate, pH 7.5, and 5mM PMSF according to the method of Witte, et al. Proc. Natl. Acad. Sci. USA 75:2488 (1978) and clarified by ultracentrifugation (30 minutes at 30,000 rpm).
  • the lysate was incubated with a 20X concentrated MEL-14 hybridoma supernatant (Gallatin et al. Nature 304:30-34 (1983)), equivalent to 10-20 ug of monoclonal antibody, for 3-4 hours, at 4oC, followed by the addition of a four-fold excess over first stage of affinity purified goat anti-rat IgG, and incubated overnight at 4oC to effect formation of a solid precipitate.
  • a 20X concentrated MEL-14 hybridoma supernatant Giblatin et al. Nature 304:30-34 (1983)
  • EL4/MEL-14 hi cells metabolically labelled with 3 H-Leucine was performed as follows. 2 X 10 8 cells were labeled with 10 mCi of 3 H-Leucine. Briefly, cells were harvested in rapid growth phase and placed in culture at 10 7 cells/ml for 4-6 hours in Spinner balanced saline solution (Gibco), 10% fetal calf serum,
  • phosphorylase b 97,400; bovine serum albumin, 68,000; ovalbumin, 43,000.
  • NEPHGE non-equilibrium pH gradient electrophoresis
  • CNBr digestion was performed basically as described. Briefly, the glass fiber filter containing the sample is removed from the sequenator, acylated with trifloroacetic anhydride to block remaining free amino groups, and digested by wetting with 25 yl of CNBr solution (100mg/ml in 60% trifloroacetic acid) in a closed container at room temperature for 20 hours. The filter is then returned to the gas phase sequenator for resumption of
  • Sepharose 4B conjugated to R7D4 an isotype matched rat monoclonal antibody negative control which recognizes the immunoglobulin idiotype on 381C13 cells (R. Levy, Stanford University). After four washes with lysis buffer, the samples were eluted with buffer containing 1% SDS, 1% 2-mercaptoethanol, and 1% NP40 by heating to 90°C for 3 minutes.
  • Endoglycosidase F Endo F
  • the gel was dried and fluorographed on Kodak XAR-5 film for 7d.
  • Staphylococcus aureus (IgGsorb, The Enzyme Center, Inc.) and 0.5 ml Sepharose 4B prior to immunoprecipitations.
  • CNBr activated Sepharose 4B (Pharmacia) was conjugated to affinity purified Goat-anti-mouse IgG (Pelgreeze, 2mg antibody/ml gel bed) and this
  • Sepharose Sepharose
  • antibody-coated Sepharose was incubated overnight in an ice-water bath with labeled cell lysate (25 ⁇ l Sepharose/ml lysate). Samples were washed four times in lysis buffer, eluted in Laemmli sample buffer, and subjected to SDS-PAGE analysis on a 9% SDS polysacrylamide gel under reducing conditions.
  • oligonucleotide corresponding to the amino-terminal five amino acids of the mature protein, determined by single tritiated-amino acid metabolic labeling was synthesized on an Applied Biosystems nucleotide
  • Total RNA was prepared by the guanidinethiocyanate RNA extraction procedure. Briefly, cells or tissues were homogenized in 8-16 volumes of 5M guanidine-thiocyanate in a Polytron homogenizer.
  • total cytoplasmic RNA was prepared as previously described, briefly recounted as follows.
  • Cells or tissue homogenates were centrifuged at 1500 rpm 4°C, 10', and resuspended in 20 ml of icecold isotonic high pH buffer (IHB) (140mM NaCl, 10mM Tris-HCl, pH p.4, and 1.5 mM MgCl 2 ).
  • IHB icecold isotonic high pH buffer
  • An additional 20 ml IHB, 1.0% NP40 were added and the lysate allowed to sit 5'.
  • Nuclei were centrifuged at 4300 rpm, 10' and supernatant was removed and treated with 1/10 volume Proteinase K, and SDS added to 0.5%. Digestion was allowed to proceed at room temperature for 30'.
  • EDTA was added to a final concentration of 5mM, and the mixture extracted with phenol:chloroform, then again with chloroform, and precipitated with Et
  • Poly-A containing mRNA was isolated on oligodT cellulose as described, from either total or total cytoplasmic RNA as follows. Approximately 0.25g of oligo-dT cellulose were placed in a sterile column and washed with 10 column volume 0.1N NaOH in ETS buffer ( ImM EDTA, 10mM Tris-HCl, pH 7.2, 0.25% SDS) and equilibrated in High Salt Buffer (HSB) (0.5M NaCl, 10mM Tris-HCl, pH 7.4, 50mM MgCl 2 ).
  • ETS buffer ImM EDTA, 10mM Tris-HCl, pH 7.2, 0.25% SDS
  • HBS High Salt Buffer
  • RNA was applied to the column after heating to 65°C for 5', allowed to slowly run through, and the eluate reapplied 3X, with subsequent wash of the column with another 15-20 ml HSB. Bound material was eluted with 4ml ETA, the solumn was reequilibrated with HSB, and eluted material was reapplied after again heating to 65°C, and
  • Poly-dT primed cDNA was synthesized from 4 ⁇ g p-A selected mRNA following the basic RNAse H procedure of Gubler and Hoffman. Double-stranded CDNA was modified by placing Xhol adapters on the ends of the cDNA species and the population of cDNA molecules was ligated into the Xhol site of lambdaZAP gt10 vector (Stratagene, Inc.).
  • oligonucleotides were labeled with 32 P ATP utilizing polynucleotide kinase. Hybridization was performed in 5X SSPE, 5X Denhardt's, 0.5% SDS, at 25°C. for 18 hours. Filters were subsequently washed in several changes of 5X SSPE, 0.2% SDS. Probing of filters with one pool of oligonucleotides of eight-fold degeneracy, constructed and deduced from the protein sequence obtained (5' TGG AC(A/G) TA(T/C) CA(T/C) TAT 3'), resulted in the identification of 58 independent isolates which reproducibly hybridized with this set of oligonucleotides. These purified clones were excised using helper phage and recircularized to generate sublones in the phagemid vector pBluescript SK(-)
  • Fragments of clones or entire clones were sequenced either in the pBluescript SK(-) excised from original lambdaZAP isolates, Bluescript KS(-), or versions of phage M13, mp18 and mp19, modified to include a Not I site for convenient directional
  • oligonucleotide sequencing primers were synthesized to obtain the remaining sequence of the full-length clone and to derive second strand sequence where needed. mRNA blot hybridization analysis
  • Northern blot analysis was performed on a variety of poly A-selected RNA species isolated from a variety of tissue and cell line sources by the
  • Hybridization to isolated insert DNA was performed to 18 hours at 42°C, 50% formamide, 5X Denhardt's, 5X SSPE.
  • Nylon filters were washed at high stringency with rinses of 2X SSPE, 0.2% SDS, room temperature, followed by 0.1X SSPE, 65°C, for 30' 2X. Autoradiographs were developed after exposure to XAR-5 film.
  • the amino terminal protein sequence obtained by automated sequence analysis of material purified from extracts of MEL-14 positive cells was compared to the protein sequence encoded by the mLHR c cDNA clone.
  • Purification of gp90 MEL-14 from EL-4/MEL-14hi cells metabolically labelled with radiolabelled amino acids was performed as described ( 24 ) using the monoclonal MEL-14 antibody.
  • 2 X 10 8 cells were labelled with 10 mCi of a single 3 H- or 35 S-amino acid ( 84 ) for 4-6 hours in Spinner balanced salt solution (Gibco), 10% fetal calf serum was supplemented with all amino acids except the radiolabelled one which was added at 200 Ci/ml.
  • polyacrylamide tube gels using the Laemmli discontinuous gel system ( 85 ), as modified by Cullen ( 86 ). Gel fractions were incubated in 0.1% SDS overnight at 4°C to elute the protein. Radiolabelled fractions were monitored in Biofluor scintillation fluid (New England Nuclear) in a Beckman LS counter (Model LS-230).
  • the index cell line utilized for these studies was EL-4/MEL-14hi, a variant of the continuous T-cell lymphoma cell line, EL-4, selected by fluorescence activated flow cytometry for high level expression of the MEL-14 antigen, a property which cosegregated with the capacity to bind peripheral node venules.
  • C6V1 and VL3 are both radiation-induced leukemia virus thymoma clonal cell lines.
  • Northern blot analysis was performed by the formaldehyde procedure as described ( 88 ), on a variety of poly A-selected RNA species isolated from a variety of tissues and cell lines.
  • RNA was applied to each gel lane, and after electrophoresis RNA was transferred to Genetran nylon filter.
  • Hybridization to probe labelled with 32 P-dCTP using the random primer procedure ( 89 ) was performed for 18 hrs at 42°C, 50% formamide, 5X Denhardt's, 5X SSPE.
  • Nylon filters were washed with 2X SSPE, 0.2% SDS, at room temperature, followed by 0.1X SSPE, 65°C, for 30', twice. Autoradiographs were developed after X hour exposure to XAR-5 film.
  • mLHRc murine lymph node homing receptor core peptide
  • EL-4/MEL-14lo lane F, VL3; lane G, C6V1; lane H, thymus; lane I, spleen; lane J, mesenteric lymph node; lane K, liver; lane L, kidney; lane M, testes; lane N, brain; b) Hybridization using 32 P-labelled actin.
  • EL-4/MEL-14Xhi, EL-4/MEL-14hi and EL-4/MEL-141o show transcript abundance paralleling cell surface
  • the filter was stripped and rehybridized with sequences of a relatively ubiquitous transcript, the beta-actin gene. Hybridization was reasonably homogenous between lanes, indicating that the differences observed for the transcript were related to abundance.
  • lymphoid distribution in normal tissues shows a predominant lymphoid distribution, paralleling tissue staining patterns for MEL-14. Thymus, spleen and masenteric lymph nodes are positive for the same size transcript found in cell lines, while liver, kidney and brain show no detectable transcripts.
  • Fluorescence activated cell sorter analysis of cell lines varying with respect to expression of gp90 MEL-14
  • staining pattern containing two discrete populations of cell expression - a predominant negative population and a relatively small population, about 5% of cells expressing gp90 HEL-14 .
  • the 3% highest and lowest intensity staining cells were sorted, immediately grown, and mRNA extracted.
  • transcript in Northern blot is present in the high population and absent in the negative population, thereby showing, in combination with the variants described above, cosegregation of transcript and cell surface antigen expression in variants derived from the same clonal cell line.
  • FACS analysis of Cos-7 cells transfected with mLHR c DNA The full length cDNA clone was transferred to the expression vector CDM8, a plasma with
  • Plasmid DNA was transfected into confluent Cos-7 cells using the DEAE-dextran transfection procedure as described
  • the results of analysis of the transfected cells show a population of positive cells when stained with MEL-14 compared to staining with an isotype matched control antibody. Identical backgrounds were obtained staining mock transfected or Thy-1 transfected Cos-7 cells with MEL-14. Immunoprecipitation of MEL-14 reactive cell surface determinant (s) from enriched mLHR c transfected Cos-7 cells
  • Non reducing gel A:
  • transfectants isotype control
  • B transfectants , MEL-14 antibody
  • C EL-4/MEL-14hi , MEL-14 antibody.
  • Reducing gel A: transfectants, MEL-14 antibody
  • B transfectants, isotype control
  • C EL-4/MEL-14hi, MEL-14 antibody.
  • the nucleotide sequence of the cDNA was determined by the dideoxy chain termination method of Sanger and Coulsen, employing the engineered T7 DNA polymerase Sequenase system (U.S. Biochemical Corp.). Single stranded template DNA's were derived from either pBluescript SK(-) (excised from original lambdaZAP isolates), Bluescript KS(-), or versions of
  • oligonucleotide primers were synthesized to obtain the remaining sequence of the full-length clone and to obtain second strand sequence where needed.
  • the predicted protein sequence is indicated below beginning with the initiator methionine at nucleotide position 54; numbering to the right indicates the nucleotide and protein positions.
  • Cysteine residues in the mature protein are marked with an asterisk (*) above, and canonical N-linked carbohydrate recognition sites (Asn-X-Ser/Thr) are overlined with arrow bars.
  • the 15 nucleotides encoding the amino terminal five amino acids and hybridizing to the oligonucleotide probe used for screening are underlined in bold. Poly-A splice and common polyadenylation recognition sequences are double underscored.
  • the cDNA clone has a 54 bp 5' untranslated region followed by an initiator ATG codon, which begins an uninterrupted open reading frame of 1,116 bp.
  • the TGA stop codon at position 1169 is followed by 327 bp of 3' untranslated region.
  • the reading frame encodes a protein with a hydrophobic leader sequence 38 amino acids in length before reaching the initial tryptophan residue of the mature protein. Hydropathy analysis confirms a
  • the signal sequence includes 3 positively charged residues, 4 cysteine residues, and 3 histidine residues, clustered in the 12 residues preceding the mature protein.
  • the mature protein possesses 10 potential asparagine-linked glycosylation sites, with 6 of these contained within an identical repeat unit structure.
  • the mature protein contains 22 cysteine residues, where 12 of the cysteines are present in the complement regulatory protein repeat structures, and an additional 9 cysteines are concentrated in the 60 amino acids preceding the repeat units involving the EGF-like domain, resulting in a highly cysteine-rich pretransmembrane region of 180-190 amino acids.
  • the deduced mature protein is 334 amino acids in length with a calculated molecular weight of
  • a hydropathy plot shows distinct regions of relative hydrophilicity, concentrated in the amino terminal 150 amino acids and in the membrane proximal approximate 20 amino acids.
  • the intervening extracytoplasmic portion is comprised of a relatively electrically neutral stretch which includes the presence of the aforementioned repeat units, identical at both the nucleotide and protein level.
  • amino-terminal domain shows homology to the carbohydrate binding domains of animal lectins (position 74-118); the succeeding 37 amino acids
  • positions 119-155 occupy the region between the lectin domain and the complement regulatory repeat units, exhibit similarity to the epidermal growth factor (EGF) cysteine-rich repeat unit; and the third region is comprised of 2 identical repeat units
  • the mLHR c is homologous over a stretch of 45 amino acids equivalent to the 50 carboxy-terminal residues of the binding domain in animal lectins.
  • the region includes three invariant cysteines at 90, 109, and 116 in mLHR c and -W at 75-76, a characteristic E-T-N (80-82), an E at 88, C-V at 90-91, and the conserved G-WND at 102-106.
  • HEV addressin is inactivated by treatment with neuraminidase, but not alkaline phosphatase, and an as yet unidentified, nonphosphorylated sialic-acid dependent molecule is indicated as the ligand for mLHR c .
  • the EGF-like domain in mLHR c consists of a single copy homolog of the EGF repeat unit, which preserves many of the C/G residues characteristic of the structure. All 6 consensus C's are present as well as G's at 147 and 150, and tyrosine at 148 of mLHR c . The relationship of these conserved residues is
  • EGF-like domain shares homology with a portion of one of the cysteine-rich repeat units of the beta chain of the integrin LFA-1 ⁇ 2 chain in the human (positions 449-483).
  • a 12 amino acid region comprising mLHR c 142-154 aligns directly with 480-492 of the LFA-1 ⁇ 2 subunit, retaining the conserved spacing of 3 cysteines, with identity of 7 residues.
  • the next domain is a precisely duplicated repeat unit, with each unit of 62 amino acids in length, spanning positions 156-217 and 218-279.
  • Murine complement factor H a serum protein with complement regulatory activity, exhibits significant homology. In factor H, there are 20 contiguous, homologous, though not identical, repeat units having approximately 10-31% homology with the mLHR c receptor. The same homologous repeat motif exists in a number of complement
  • the consensus sequence position is represented in the homing receptor repeat unit sequence T-4, P-7, F-30, C-32, G-35, C-46, G-50, W-52, P-57, and C-59.
  • the consensus sequence and an insertion of 3 residues between P-7 and F-30 relative spacing of the remaining residues of the consensus sequence is completely preserved in the homing receptor sequence.
  • parentheses may or may not be present in a sequence conforming to the motif. Dashes indicate positions that must be occupied by an amino acid, while spaces demarcate regions of variable length. A. mLHR c
  • R-MBPC rat mannose binding protein C
  • R-MBP-A rat mannose binding protein A
  • H-MBP-H human mannose binding protein H
  • CPSa canine pulmonary surfactant a
  • RASGPR rat asialoglycoprotein receptor
  • HASGPR human immunosemiconductor receptor
  • asialoglycoprotein receptor HFc e R, human Fc epsilon receptor
  • CHL chicken hepatic lectin
  • ISL sarcophaga peregrina hemolymph lectin
  • Ech echinoidin, lectin from sea urchin coelemic fluid
  • EGF epidermal growth factor
  • TGF transforming growth factor
  • tPAhu human tissue plasminogen activator
  • LDL low density lipoprotein
  • CRl complement receptor 1
  • H factor H
  • C 4 bp C 4 binding protein
  • Ba factor Ba
  • ⁇ GPI ⁇ -glycoprotein I
  • Il-2R interleukin-2 receptor.
  • Poly-dT primed cDNA was synthesized from 4 ⁇ g p-A selected mRNA following the basic RNase H procedure of Gubler and Hoffman. Double-stranded cDNA was synthesized and ligated into the EcoRI site of lambda gtll. Approximately 1.0 x 10 6 phage plaques in E. coli strain LE 392 were plated onto 150 mm agar plates at about 20,000 plaques per plate, lifted onto nitrocellulose filters in duplicate, denatured in base, neutralized, and baked for 2 hours at 80°C.
  • the fulllength mouse lymph node homing receptor cDNA clone (mLHR c ) was excised as a Notl/Notl restriction fragment of about 1500 bp or as an approximate 1200 bp fragment excised with Xhol emcompassing all but the 5' 300 bp of the full-length clone.
  • These inserts were purified and labeled with 32P alpha-dCTP by the standard hexamer priming method.
  • Hybridization was performed in 5X SSPE, 5X Denhardt's, 0.5% SDS, at 25°C for 18 hours, with duplicate filters, with 45% formamide in one probe mixture and 35% formamide in the other.
  • the Notl fulllength probe was placed in the 45% formamide set and the Xhol excised probe in the 35% set. Filters were hybridized for 18 hours and subsequently washed in several changes of 5X SSPE, 0.2% SDS at room
  • Lambda gtll inserts were isolated and subcloned into the EcoRI site of M13mp19 for sequence analysis by the dideoxy-sequencing method described above. Human Lymph Node Homing Receptor Sequence
  • rat monoclonal antibody Rl-2 (IgG2b) recognizing the o chain of the LPAM-1 molecule was prepared as follows. Spleen cells from Fisher rats immunized 3X i.p. with the Peyer's patch HEV binding lymphoma line TK1 were fused with a non-secreting mouse myeloma P3x63AG8.653 using standard procedures (Galfre et al (1977) Nature 266:550-552). Hybridomas producing antibodies reactive in immunofluorescence assays with TK1 cells but not HEV binding lymphoma TK5 were cloned by limiting dilution.
  • Hybridomas M17/4.3 and M18/20 secreting rat monoclonal antibodies specific for the a and ⁇ chain of the murine LFA-1 antigen were obtained from Dr. T.A. Springer, Dana-Farber Cancer Institute, Boston.
  • a polyvalent rabbit antiserum raised against a synthetic peptide corresponding to the COOH-terminal domain of the chicken integrin B 1 - subunit was obtained from Drs. E.E. Marcantonio and R.O. Hynes, Massachusetts Institute of Technology, Cambridge. This anti- ⁇ 1 -peptide antiserum was shown to be monospecific for integrin ⁇ 1 and reacts with ⁇ chains from a variety of vertebrates (Marcantonio and Hynes (1988) J. Cell Biol. 106:1765-1772).
  • the rabbit anti-VLA- ⁇ antiserum was obtained from Dr. M.E. Hemler, Dana-Farber Cancer Institute, Boston.
  • the polyvalent rabbit antiserum specific for platelet glycoprotein IIIa was obtained from Dr. L.L.K. Leung, Stanford University, Medical School.
  • T cell lymphomas TK23, TK40, and TK50 were passaged by subcutaneous injections of 10 4 - 10 7 cells into syngeneic AKR/cum recipients. All other cell lines were maintained in tissue culture using RPMI 1640 with 7% fetal calf serum.
  • HBSS balanced salt solution
  • calf serum 5% calf serum
  • 20mM HEPES pH7.4 were incubated with mild rotation for 30 minutes at 7°C on freshly cut frozen sections of murine peripheral (axillary, brachial, inguinal and cervical) nodes or Peyer's patches. After incubation, adherent cells were fixed to the tissue section in cold 1.25x PBS containing 2% formaldehyde (J.T. Baker
  • chymostatin at 10 ⁇ g/ml
  • soybean trypsin inhibitor at 20 ⁇ g/ml
  • 1mM phenylmethylsulfonylfluoride were included as protease inhibitors. Lysates were
  • Immunosorbents were incubated with lysates for 3 hr at 4°C and washed in lysis buffer. Immunoprecipitates were analyzed by SDS-PAGE on 6% or 7% polyacrylamide gels. Molecular weight standards were myosin (M r )
  • RNA was pelleted through a cushion of 5.7M CsCl (Chirgwin et al., (1979) Biochemistry 18:5294-5299).
  • Poly(A + ) RNA was isolated by chromatography on oligo(dT) cellulose (type III, Collaborative Research). For each cell line, 4 ⁇ g of denatured poly(A + ) RNA was separated on a 0.8%
  • Probes were labeled to a specific activity of 2-4X10 8 cpm/ ⁇ g of DNA using the hexamer primer labeling procedure (Feinberg and Vogelstgein, (1983) Anal. Biochem. 132:6-13).
  • the filters were hybridized at 42°C for 16hr in 3x SSPE, 50% formamide, lx Denhardt's, 1% SDS and 100 ⁇ g/ml herring testis DNA and washed in 0.2x SSPE, 0.1% SDS at 65°C.
  • the ⁇ subunit of LPAM-1 (hereafter called ⁇ 4m ) has been shown to be analogous to the ⁇ chain of the human integrin molecule VLA-4 as indicated below.
  • the VLA-4 ⁇ chain is noncovalently associated with the integrin ⁇ 1 subunit. Whether the LPAM-1 ⁇ chain
  • ⁇ P is analogous to ⁇ 1 was tested. Different rabbit antisera specific for ⁇ 1 did not recognize ⁇ P or other proteins in lysates of surface labeled LPAM-1 + TK1 lymphoma cells.
  • the analogy between the alpha subunit of LPAM-1 (hereafter called ⁇ 4m ) and the alpha chain of the human integrin molecule VLA-4 was established as follows. A rabbit polyclonal antiserum specific for the alpha chain of human VLA-4 was tested for its ability to recognize the P160 subunit of LPAM-1.
  • Immunoprecipitated SDS-denatured LPAM-1 was diluted in a buffer containing excess Triton X-100 and reanalyzed with different rabbit polyclonal antiserum.
  • the rabbit anti VLA-4 serum was obtained by immunization with purified alpha chains and does not cross react with other integrin alpha
  • the anti VLA-4 antibodies immunoprecipitate a cell surface heterodimer of M r 150,000 and 130,000, as well as two proteins of M r 80,000 and 70,000, which were shown to be fragments of M r 150,000 a chain protein. This is analogous to the alpha chain of the
  • LPAM-1 antigen which upon reducing conditions produces four proteins of apparent molecular weights of 160,000 (P160), 130,000 (P 130), 84,000 (P84), and 62,000
  • TK1 lymphoma cells, 3T3 fibroblasts or murine platelets were cell surface iodinated and immunoprecipitates were analyzed by SDS-page.
  • the antibody used was Rl-2.
  • Immunoprecipitated material was treated with 10 mM EDTA in 50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100 and eluted material was analyzed using the LPAM-1 heteroantiserum.
  • ⁇ P subunits a polyvalent rat antiserum specific for LPAM-1 was obtained by immunization with immunoaffinity-isolated protein. As the assocation of LPAM-1 ⁇ and ⁇ subunits is dependent on the presence of Ca ++ ions, ⁇ subunits can be
  • LFA-1 and LPAM-1 were both isolated from TK1 cells and their subunits compared by SDS-polyacrylamide gel electrophoresis. It was found that the ⁇ chain of LFA-1 (integrin ⁇ 2 ) and ⁇ P could be clearly distinguished based on their molecular
  • LPAM-1 heteroantiserum did not crossreact with or coprecipitate LFA-1 subunits.
  • the ⁇ P subunit was also compared to integrin ⁇ 3 which is identical to glycoprotein IIIa.
  • Proteins analyzed were LPAM-1 subunit ⁇ P , integrin ⁇ 1 , integrin ⁇ 2 , and integrin ⁇ 3 .
  • the cellular sources of the various beta subunits were TK1 lymphoma cells, RAW112 lymphoma cells, 3T3 fiberblasts and murine platelets.
  • the mapping showed that the digestion of ⁇ P yielded peptide patterns clearly distinct from those of ⁇ 1 , ⁇ 2 , and ⁇ 3 .
  • digestion of B 1 , ⁇ 2 , and ⁇ 3 each gave a unique peptide pattern. Therefore, these results support the concept that ⁇ P represents a unique ⁇ subunit.
  • the LPAM-1 subunit ⁇ P was further compared to ⁇ 1 by Northern blot analysis. Consistent with the absence of ⁇ 1 protein from TK1 cells a cDNA clone coding for an N-terminal fragment of murine ⁇ 1 did not hydridize with RNA from TK1 cells. The comparison was performed by isolating poly (A + ) RNA from ⁇ p + ⁇ 1 - TK1 cells or ⁇ P - ⁇ 1 + RAW 112 cells and hybridizing with cDNA clone pMINT ⁇ encoding amino acids 1-333 of the murine integrin ⁇ 1 subunit or with a ⁇ -actin probe. Filters were hybridized and washed under low stringency or high stringency conditions.
  • the subunits associated with ⁇ 4m were analyzed following immunoprecipitation from a panel of lymphoma cell lines using antibody Rl-2 covalently linked to Affigel 15.
  • a panel of lymphoma cell lines was cell surface iodinated and immunoprecipatated using the ⁇ 4m specific antibody Rl-2 covalently linked to Affigel 15.
  • Bound proteins were eluted with 100 ml of glycine pH 2.5, 1% Triton X-100 and eluates were diluted 1:5 with 50 mM Tris pH 8.8, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100.
  • ⁇ P and the M r 115,000 protein were isolated from cell lines TK23 and TK40.
  • the ⁇ 4m subunits were isolated from cell lines TK1, TK23, TK40, and TK50. The identity of the ⁇ subunits
  • VLA-4-like LPAM-1 ⁇ chain is the common subunit of two distinct cell surface
  • LPAM-1 composed of ⁇ 4m associated with ⁇ P
  • LPAM-2 consisting of ⁇ 4m and integrin ⁇ 1 .
  • LPAM-1 and LPAM-2 are involved in lymphocytePeyer's patch HEV interactions
  • LPAM-1 and LPAM-2 heterodimers were investigated. The presence of LPAM-1 and LPAM-2 was determined by
  • novel proteins may be employed for specific binding to particular anatomical sites.
  • the different proteins may be used in a variety of ways to prevent cells from binding or to direct compositions to the desired sites.
  • the immune system may be modulated by increasing or decreasing lymphocyte populations at specific sites.
  • the ability to control the lymphocyte population at particular sites may be used to protect against autoimmune diseases, reduce the inflammatory response, to localize specific cells or drugs for diagnosis or therapy for neoplastic
  • lymphocytes or monocytes for presentation to T-cells.

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Abstract

Des protéines sont identifiées comme récepteurs de guidage pour les plaques de Peyer et les ganglions lymphatiques, ces protéines pouvant s'utiliser pour inhiber le guidage des lymphocytes ou pour permettre le guidage de médicaments ou d'autres compositions pour le diagnostic ou la thérapie in vivo. En outre, des compositions d'acide nucléique peuvent s'utiliser pour l'expression des protéines ou de leurs fragments ou bien pour transformer des cellules afin de permettre une capacité de guidage accrue ou encore pour inhiber ou moduler un tel guidage.
EP19900902104 1988-12-23 1989-11-10 Homing sequences and their uses Withdrawn EP0462111A4 (en)

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US5776775A (en) * 1989-02-21 1998-07-07 Dana-Farber Cancer Institute Anti-LAM 1-3 antibody and hybridoma
US7238668B1 (en) 1989-09-01 2007-07-03 Fred Hutchinson Cancer Research Center Inhibition of lymphocyte adherence with CS-1-peptides and fragments thereof
US6551593B1 (en) 1995-02-10 2003-04-22 Millennium Pharmaceuticals, Inc. Treatment of Inflammatory bowel disease by inhibiting binding and/or signalling through α 4 β 7 and its ligands and madcam
US7750137B2 (en) 1995-09-01 2010-07-06 Millennium Pharmaceuticals, Inc. Mucosal vascular addressins
US7803904B2 (en) 1995-09-01 2010-09-28 Millennium Pharmaceuticals, Inc. Mucosal vascular addressing and uses thereof
US7147851B1 (en) 1996-08-15 2006-12-12 Millennium Pharmaceuticals, Inc. Humanized immunoglobulin reactive with α4β7 integrin
US6111090A (en) * 1996-08-16 2000-08-29 Schering Corporation Mammalian cell surface antigens; related reagents
EP1924685B1 (fr) 2005-08-25 2014-08-27 The Arizona Board of Regents on behalf of the University of Arizona Modèle de carcinogenèse par fusion de cellules souches
EP2704742B1 (fr) 2011-05-02 2017-07-12 Millennium Pharmaceuticals, Inc. Formulation pour un anticorps anti- 4 7
UA116189C2 (uk) 2011-05-02 2018-02-26 Мілленніум Фармасьютікалз, Інк. КОМПОЗИЦІЯ АНТИ-α4β7 АНТИТІЛА

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EP0303463A2 (fr) * 1987-08-11 1989-02-15 The Board Of Trustees Of The Leland Stanford Junior University Méthode pour contrôler l'extravasation des leucocytes

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EP0303463A2 (fr) * 1987-08-11 1989-02-15 The Board Of Trustees Of The Leland Stanford Junior University Méthode pour contrôler l'extravasation des leucocytes

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Title
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