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WO1993002698A1 - Anticorps monoclonaux contre la molecule-1 d'adhesion aux leucocytes - Google Patents

Anticorps monoclonaux contre la molecule-1 d'adhesion aux leucocytes Download PDF

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Publication number
WO1993002698A1
WO1993002698A1 PCT/US1992/006127 US9206127W WO9302698A1 WO 1993002698 A1 WO1993002698 A1 WO 1993002698A1 US 9206127 W US9206127 W US 9206127W WO 9302698 A1 WO9302698 A1 WO 9302698A1
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Prior art keywords
monoclonal antibody
lam
cells
animal
lam1
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PCT/US1992/006127
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English (en)
Inventor
Thomas F. Tedder
Olivier G. Spertini
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Dana-Farber Cancer Institute, Inc.
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Publication of WO1993002698A1 publication Critical patent/WO1993002698A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C07K14/70564—Selectins, e.g. CD62
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C07K16/2854—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72 against selectins, e.g. CD62
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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 human leukocyte-associated cell surface proteins, and monoclonal antibodies specific for such proteins.
  • ELAM-1 inducible endothelial-leukocyte adhesion molecule
  • GMP-140 granule membrane protein found in platelets and endothelial cells, termed GMP-140, has been cloned and is homologous with ELAM-1 (Johnston et al., Blood Suppl. 172:327A (1988)).
  • the invention generally features a leukocyte-associated cell surface protein LAM-1 (leukocyte adhesion molecule-1), which contains domains homologous with binding domains of animal lectins, growth factors, and C3/C4 binding proteins; the specific domains of the LAM-1 protein; and the genomic DNA sequences encoding the LAM-1 protein and the specific domains of LAM-1.
  • LAM-1 leukocyte adhesion molecule-1
  • Preferred embodiments of the invention include essentially purified proteins comprising sequences of amino acids having 90% or greater homology with, or identity with, the amino acid residues of specific domains of human
  • leukocyte-associated cell surface protein LAM-1 represented in Fig. 2, i.e., the lectin domain represented by residues 42-170, the EGF-like domain represented by residues 171- 206, the short consensus repeat unit I domain represented by residues 207-269, the short consensus repeat unit II domain represented by residues 270-331, the leader domain
  • residues 15-41 the transmembrane domain represented by residues 332-373
  • residues 374-380 the phosphorylation domain represented by residues 374-380.
  • a monoclonal antibody which recognizes (i.e., binds to, with immunologic
  • Each such epitope is defined by its binding to a particular anti-LAMl monoclonal antibody.
  • whether or not a given antibody is within the invention may be determined by comparison of its epitope to that of the stated anti-LAM1 mAb. This comparison may be done by any method or combination of methods for such determinations generally known to immunologists, including the following:
  • a recombinant gene encoding a chimeric protein composed of one of the LAM-1 functional domains (e.g., the lectin domain) fused to the other domains (e.g., the EGF-like and SCR domains) from another selectin could be used to assay for binding by the stated anti-LAM1 mAb and by the antibody to be tested. If one binds to the chimeric protein while the other does not, the two mAbs have different epitopes.
  • An antibody binding to its epitope can block the
  • LAM-1 functions are carried out by specific regions of the molecule, which are composed of individual epitopes.
  • an epitope involved in ligand binding can be identified by the ability of an antibody bound to that epitope to inhibit a given LAM-1 function, such as the binding of LAM-1 to PPME, fucoidin, HEV, or activated endothelial cells. Also, the binding of an antibody to its epitope may completely or only partially inhibit the given LAM-1 function, depending on the proximity of the epitope to the ligand binding site of the protein, so that the epitopes of two different antibodies may be
  • LAM-1 LAM-1
  • LAM1-1 or LAM1-5 epitope increases the level of PPME and fucoidin binding by LAM-1.
  • LAM-1 epitopes may be conformationally determined, and would therefore be expressed on the LAM-1 protein to different degrees under identical conditions.
  • the level of reactivity of two different antibodies for the same epitope would be expected to vary identically as the conditions of immunological assay are varied, thus providing a means for determining whether one antibody reacts to the same epitope as another.
  • epitopes are determined by primary amino acid sequence of the LAM-1 protein, subtle differences in amino acid sequence in specific regions of the protein, such as occurs during evolution of species, will alter structure of the epitope, and may therefore alter antibody binding to that epitope, without affecting binding of a different antibody to a different epitope on the same molecule.
  • antibodies to the LAM-1 proteins of different animal species can be used to identify distinct epitopes.
  • the invention includes anti-LAM1-1, -2, -4, -5, -6, -7, -8, -9, -10, -11, -14, and -15 and the hybridoma cells which produce any of the mAb of the invention.
  • Each of the mAb has been deposited with the American Type Culture Collection (ATCC).
  • the mAb of the invention may be used in a method of identifying cells expressing LAM-1 (i.e., bearing LAM-1 molecules on their surface), which method includes the steps of providing a sample of cells (e.g., from an animal or a cell line), at least some of which are suspected of expressing LAM-1; contacting the cells with a reagent which includes the mAb; and determining which cells form an immune complex with the reagent.
  • This reagent may be the mAb alone (e.g., in the form of a purified preparation or
  • unfractionated ascites fluid may be the mAb conjugated with a detectable label (such as a detectable enzyme, fluorophore, or radioactive moiety). Determination of which cells form an immune complex with the reagent may be
  • immunological techniques including immunoprecipitation, indirect or direct immunofluorescence with flow cytometry analysis, or immunosorbent assays.
  • the mAb of the invention may be used in a method of isolating cells expressing LAM-1, which method includes the steps of providing a sample of cells, at least some of which are suspected of expressing LAM-1; contacting the cells with the mAb or a reagent including the mAb; and separating those cells which have formed an immune complex with the mAb, from those cells which have not.
  • separation may be performed by any of a variety of standard immunological techniques, including fluorescence-based cell sorting, magnetic bead-based separation protocols, and the use of solid phase bound antibody.
  • the mAb of the invention is also useful in a diagnostic assay for detecting leukocyte activation in an animal, which activation may be attributable, for example, to inflammation, an autoimmune response, or rejection of an organ or tissue transplant.
  • This method is accomplished by obtaining a fluid sample from an animal (preferably a human), which sample may be, for example, blood, serum, plasma, saliva, tears, cerebral spinal fluid, or urine;
  • the mAb of the invention is particularly useful for blocking leukocyte interactions with endothelium of an animal, which method involves administering an effective amount of the mAb, or a therapeutic which includes the mAb or a LAM-1-blocking portion of the mAb, to the animal.
  • This LAM-1-blocking portion of the mAb may be, for example, a F(ab) fragment, and the mAb may be part of a chimerized antibody having a variable region derived from a mAb of the invention, and constant regions derived from a human
  • the animal (preferably a human) may be suffering from inflammation, an autoimmune response, rejection of an organ or tissue transplant, or any other condition involving leukocyte interactions with endothelium.
  • Also within the invention is a method of determining the degree of expression of LAm-1 in a sample of leukocytes (i.e., the amount of LAM-1 expressed by the cells,
  • a quantitative amount per cell or per sample as a percentage of a standard amount, as the percentage of cells which have over or under a certain amount, or perhaps just as relatively more or less than a standard sample of cells
  • which method includes the steps of contacting a sample of leukocytes with the mAb of the invention, and determining the level of immune complex formation in the sample, such level being indicative of the degree of expression of LAM-1 on the leukocytes.
  • the invention features methods of treating a patient suffering from a leukocyte-mobilizing condition (e.g., tissue damage, an autoimmune disease, or cancer), or who is an organ or tissue transplanted recipient, which method includes administering to the patient a therapeutic agent including a therapeutic amount of the LAM-1 protein or a domain thereof, or of an
  • the therapeutic agent is administered in a pharmaceutically acceptable carrier substance.
  • the invention features using the LAM-1 protein or domain thereof to identify a ligand that binds to the protein or to a molecule that is specifically associated with the protein, or fragment thereof, to
  • Ligands so identified can also be used in the methods of the invention described above.
  • the term "antagonist to LAM-1” includes any agent which interacts with LAM-1 and interferes with its function, e.g., antibody reactive with LAM-1 or any ligand which binds to LAM-1.
  • identify is any agent which interacts with LAM-1 and interferes with its function, e.g., antibody reactive with LAM-1 or any ligand which binds to LAM-1.
  • the term "essentially purified” refers to a protein or nucleic acid sequence that has been separated or isolated from the environment in which it naturally occurs.
  • Leukocyte-associated cell surface protein LAM-1 plays an important role in leukocyte-endothelial cell interactions, especially selective cell trafficking to sites of inflammation.
  • the LAM-1 protein or domains thereof, or other molecules that interfere with leukocyte adhesion and function, can be used therapeutically to inhibit the
  • Figs. 1A and 1B show the structure of LAM-1 cDNA clone.
  • Fig. 2 shows the cDNA nucleotide sequence and the predicted amino acid sequence of LAM-1.
  • Figs. 3A, 3B, and 3C show the homologies of LAM-1 with other proteins.
  • Fig. 4 depicts the immunoprecipitation of LAM-1 shed from a cell surface with anti-LAM1 antibodies or a control immunoprecipitation with an unreactive isotype-matched antibody with subsequent sodium dodecyl sulfate- polyacrylamide gel electrophoresis;
  • Fig. 5 depicts the percentage and reactivity of malignant cells being LAM-1 positive from patients having various forms of hematopoietic malignancies
  • Fig. 6 depicts the immunoprecipitation of LAM-1 from the surface of iodinated CLL cells using anti LAM-1
  • Fig. 7 depicts indirect immunofluorescence results obtained with the anti-LAM-3 antibody and PPME-FITC;
  • Fig. 8 depicts the modulation of cell surface LAM-1 by malignant cells and cDNA transfected cells after PMA exposure.
  • Fig. 9 depicts expression of LAM-1 epitopes by lymphocytes and neutrophils. Cells were examined by
  • Fig. 10 depicts modulation of PPME binding by anti- LAM-1 mAb.
  • PPME staining is completely inhibited by 5 mM
  • Fig. 11 depicts evolutionary conservation of the
  • LAM1-3 epitope Blood mononuclear cells from a human, cow, rhesus monkey, dog, cat and rabbit were examined in indirect immunofluorescence assays for expression of the LAM-1 epitope identified by the anti-LAM1-3 mAb (dark line) . The fluorescence histogram of cells treated with an unreactive murine IgG 1 mAb are also shown (thin line). Cells were examined by flow cytometry analysis and relative
  • fluorescence intensity of staining is shown on four or three decade scales as indicated. Cell samples were examined at different times with different flow cytometer settings so that individual histograms are not directly comparable.
  • Fig. 12 depicts conservation of the PPME binding receptor by human, tamarin, dog, and rabbit blood
  • Fig. 13 depicts hypothetical model for the
  • Leukocyte migration is regulated principally at the level of interactions between circulating leukocytes and the endothelium.
  • adhesion molecules that mediate leukocyte interactions with specialized endothelial cells have been identified (Stoolman Cell , 56:907, 1989;
  • Leukocyte Adhesion Molecule-1 (LAM-1, TQ-1, Leu-8, LEC-CAM-1), the human homologue of the mouse MEL-14 antigen, mediates the binding of blood
  • LAM-1 leuko ⁇ ytes to high endothelial venules (HEV) of peripheral lymph nodes.
  • LAM-1 is expressed on the surface of human peripheral lymphocytes, neutrophils, eosinophils, monocytes and hematopoietic progenitor cells (Pilarski et al., J.
  • lymphocytes and neutrophils express a single species of LAM- 1 protein (Kansas et al., Blood 76.:2483, 1990), but the Mr of cell-surface LAM-1 on lymphocytes is 74,000 (Tedder et al., Eur. J. Immunol. 20:1351, 1990) and that of neutrophil LAM-1 is 90-100,000 (Kansas et al., J. Immunol. 142:3058. 1989). This presumably results from different patterns of post-translational processing as occurs with many molecules expressed by both cell lineages.
  • LAM-1 affinity for ligand is transiently increased following leukocyte activation with lineage-specific agents, which may partially explain the differences in migration between neutrophils and lymphocytes (Spertini et al., Nature 349:691, 1991).
  • LAM-1 is expressed by the majority of circulating T and B cells, is lost following mitogen stimulation, but is found on some antigen-specific memory T cells (Tedder et al., Eur. J. Immunol. 20:1351, 1990; Kanof et al., J. Immunol.
  • LAM-1 is, a member of the selectin family of cellular adhesion/homing receptors, which play important roles in leukocyte-endothelial cell interactions, especially selective cell trafficking to sites of inflammation. Like other members of this family, LAM-1 contains an amino- terminal lectin-like domain, followed by an epidermal growth factor (EGF)-like domain and short consensus repeat units
  • EGF epidermal growth factor
  • HEV high endothelial venules
  • B cell-specific cDNAs were isolated from a human tonsil cDNA library (ATCC #37546) using differential
  • cDNAs derived from either B cell (RAJI) RNA or T cell (HSB-2) RNA (Tedder et al., Proc. Natl. Acad. Sci. USA 85: 208-212 (1988)). Positive plaques were isolated and cloned, and the cDNA inserts were subcloned into the plasmid pSP65 (Promega, Madison, WI). Nucleotide sequences were determined using the method of Maxam and
  • Fig. 1B a schematic model of the structure of the LAM-1 mRNA is shown. Thin lines indicate 5' and 3' untranslated sequences (UT), while the thick bar indicates the translated region. The boxes represent the lectin-like and epidermal growth factor (EGF)-like domains and the two short consensus repeat (SCR) units. The open box indicates the transmembrane (TM) region.
  • EGF epidermal growth factor
  • SCR short consensus repeat
  • pLAM-1 contains a 1,181 bp open reading frame that could encode a protein of 372 amino acids, as shown in
  • Fig. 2 The numbers shown above the amino acid sequence designate amino acid residue positions. The numbers to the right indicate nucleotide residue positions. Amino acids are designated by the single-letter code, and * indicates the termination codon. The boxed sequences identify
  • LAM-1 amino acid sequence of LAM-1 indicates a structure typical of a membrane glycoprotein.
  • the mature LAM-1 protein has an extracellular region of about 294 amino acids containing 7 potential N-linked carbohydrate
  • LAM-1 has a cytoplasmic tail of 17 amino acids containing 8 basic and 1 acidic residues.
  • the processed LAM-1 protein has a M r of at least 50,000 and can be isolated by conventional techniques, such as affinity column chromatography with antibody or ligand, from cell lines that normally express this receptor or from
  • the protein can be synthesized by in vitro translation of the LAM-1 cDNA.
  • LAM-1 combines domains homologous to domains found in three distinct families of molecules: animal lectins, growth factors, and C3/C4 binding proteins.
  • extracellular region of LAM-1 contains a high number of Cys residues (7%) with a general structure as diagrammed in Fig. 1B. As indicated in Fig. 3, segments of homologous proteins are shown with the amino acid residue numbers at each end. Homologous amino acids are shown in boxes. Gaps (-) have been inserted in the sequences to maximize
  • FIG. 3A are homologous with the low-affinity receptor for IgE (Kikutani et al., Cell 47:657 (1986)), the
  • the sequence homologies are less than 30%, all the invariant residues found in animal lectin carbohydrate- recognition domains are conserved (Drickamer, J. Biol. Chem. 263:9557 (1988)).
  • the lectin domain includes amino acid residues 42-170 given in Fig. 2.
  • the next domain of 36 amino acids, at residues 171- 206 shown in Fig. 2, is homologous (36-39%) with epidermal growth factor (EGF) (Gregory, Nature 257:325 (1975)) and the EGF-like repeat units found in Factor IX (Yoshitake et al., Biochem. 25:3736 (1985)) and fibroblast proteoglycan core protein (Krusius et al., supra) (Fig. 3B).
  • EGF epidermal growth factor
  • LAM-1 mRNA The expression of LAM-1 mRNA by cell lines of lymphoid and non-lymphoid origin was examined. Northern blot analysis revealed that LAM-1 cDNA hybridized strongly to a 2.6 kb RNA species and weakly to a 1.7 kb RNA species in poly(A) + RNA isolated from the B cell lines Raji, SB, Laz-509, and GK-5.
  • RNA isolated from two pre-B cell lines (Nalm-6, PB-697), three B cell lines (Namalwa, Daudi, BJAB), five T cell lines (CEM, Hut-78, HSB-2, Molt 15, Molt-3), a myelomonocytic cell line (U937 and U937 cultured with LPS) and erythroleukemic K-562 cell line did not hybridize with LAM-1 cDNA, suggesting that expression of this gene was preferentially associated with B lymphocytes.
  • Neutrophils expressed LAM-1 mRNA but had a relatively lower amount of transcript among total mRNA when compared with the Raji cell line or blood T lymphocytes.
  • LAM-1 cDNA has also been used to transfer expression of LAM-1 to cells that do not express the gene.
  • Anti-LAM-1 mAb produced by a total of 18 different hybridomas were analyzed.
  • the anti-LAM-1 mAb identified as anti-LAM1-1, anti-LAM1-2, anti-LAM1-3 and anti-TQ1 have been previously described (Tedder et al., J. Immunol. 144:532. 1990; Reinherz et al., J. Immunol.
  • the new anti- LAM-1 mAb (Table 7) were generated by the fusion of NS-1 myeloma cells with spleen cells from BALB/c mice that were repeatedly immunized with cells of the mouse pre-B cell line 300.19 stably transfected with a LAM-1 cDNA, as described (Tedder et al., J. Immunol. 144:532. 1990). Each hybridoma was cloned twice and used to generate ascites fluid. The isotypes of the mAb were determined by the immunostaining of cells preincubated with each anti-LAM-1 mAb with FITC- conjugated antibodies specific for the individual mouse H chain isotypes (Southern Biotechnology Associates,
  • FITC-conjugated anti-LAM-1 and Leu-8 mAbs were produced as described (Kansas et al., J. Immunol.
  • Phycoerythrin-labelled anti-TQ1 was from Coulter Immunology (Hialeah, FL). Cell samples and immunofluorescence analysis.
  • Mononuclear cells were isolated from human, rhesus monkey (Macaca mulatta), cotton-topped tamarin (Saguinus oedipus), dog (Canis familiar is), cat (Felis catus), sheep (Ovis aries) or rabbit (Oryctolagus cuniculus) blood by Ficoll- Hypaque density gradient centrifugation. Neutrophils were isolated from blood samples at 20°C by centrifugation for 20 min at 1000 x g on a cushion of Mono-Poly Resolving
  • Indirect immunofluorescence analysis was carried out after washing the cells three times. The cells were then incubated for 20 min on ice with each mAb as ascites fluid diluted to the optimal concentration for immunostaining. After washing, the cells were treated for 20 min at 4°C with FITC-conjugated goat anti-mouse Ig antibodies (Tago,
  • Tissue sections were isolated from thymus and mesenteric lymph nodes of rabbit, pig (Sus scrofa), goat (Capra hircus), rat (Rattus norvegicus), guinea pig (Cavia porcellus) and chicken (Gallus domesticus), and from rabbit appendix. These frozen sections were stained with a given anti-LAM-1 mAb at optimal concentrations, with subsequent development using immunohistological procedures as described (Mackay et al., J. Exp. Med. 167:1755, 1988). PPME binding assays. FITC-labeled PPME (PPME-FITC), was used for staining peripheral blood lymphocytes as described (Spertini et al., Leukemia (in press) 1991;
  • the anti-LAM-1 mAbs were added to the cell suspension for 10 min. After washing, the lymphocytes were incubated for 20 min with FITC-labeled goat anti-mouse Ig antibodies, with subsequent analysis by flow cytometry.
  • lymphocytes (4x10 6 ) were treated with neuraminidase (0.005 U/ml) for 30 min at room temperature, then incubated for 10 min on ice with the anti- LAM-1 mAbs as ascites fluid diluted at 1:100 in RPMI 1640 containing 5% fetal calf serum.
  • the anti-LAM1-3 and -4 mAb could be used at considerable dilution (1:5000) without a decrease in their ability to inhibit HEV binding.
  • the cells in a final volume of 100 ⁇ l, were then incubated under rotation (64 rpm) for 30 min at 4°C on four 12 ⁇ m frozen rat peripheral lymph node sections. After fixation overnight in PBS with 1% (w/v) glutaraldehyde, the number of HEVs per tissue section was determined and the number of lymphocytes adherent to HEV was quantitated.
  • Lymphocytes (5 x 10 5 ) were first incubated with 10-fold saturating concentrations of one anti-LAM-1 mAb as diluted ascites fluid for 20 min on ice followed by the addition of optimal concentrations of the second fluorochrome-labelled anti- LAM-1 mAb to be used for direct immunofluorescence analysis. After 20 min of further incubation, the cells were washed and mAb binding assessed immediately by flow cytometry, as described (Tedder et al., J. Immunol. 144:532. 1990).
  • Optimal concentrations for each antibody were determined by indirect immunofluorescence analysis.
  • MAb- producing hybridomas were generated by the fusion of NS-1 myeloma cells with spleen cells from mice immunized with LAM-1 cDNA-transfected 300.19 cells (Tedder et al., J.
  • erythroleukemia cells and NALM-6 pre-B cells transfected with the LAM-1 cDNA (Tedder et al., J. Immunol. 144:532, 1990), but not with those cells untransfected or transfected with CD19 or CD20 cDNAs (Tedder et al., Proc. Natl. Acad. Sci. USA 85:208, 1988; Tedder et al., J. Immunol. 143:712, 1989).
  • each of these mAb reacted with six human LAM-1+ lymphoblastoid cell lines, but were unreactive with four LAM-1-lymphoblastoid or myeloid cell lines.
  • neutrophils showed that the fluorescence intensity of staining with the anti-LAM-1 mAb ranged from low staining (+) to bright staining (++++) (Fig. 9, Table 7). No mAb were detected that stained lymphocytes or neutrophils preferentially. The level of staining observed was
  • Inhibition of Lymphocyte binding to HEV is blocked by some anti-LAM-1 mAb.
  • the anti-LAM-1 mAb were tested for their ability to inhibit lymphocyte binding to HEV of rat peripheral lymph node sections in the in vitro frozen section assay (Stamper et al., J. Exp. Med. 144:828. 1976).
  • the anti-LAM1-3, anti-LAM1-4 and anti-LAM1-6 mAb inhibited lymphocyte binding by 85 to 90% (Table 8).
  • the anti-LAM1-1 and anti-LAM1-2 mAb consistently inhibited binding to an intermediate degree ( ⁇ 65% and -45%, respectively).
  • anti-LAM-1 mAb did not inhibit PPME-FITC binding, as shown for the anti-LAM1-6 and -10 mAb (Fig. 10).
  • the enhanced binding of PPME induced by the anti- LAM1-1 mAb was further quantitated using iodinated PPME in a live cell binding assay, as described (Spertini et al..
  • This increase in PPME binding is comparable to that observed following lymphocyte activation through the T cell receptor complex or CD2 (Spertini et al.. Nature 349:691, 1991).
  • PPME binding was inhibited by the prior binding of the anti-LAM-3 mAb giving only 1160+ 104 and 1892 ⁇ 217 cpm bound, respectively, similarly,
  • Anti-LAM1-7 and -8 partially, and -9, -10, -11, -12 and -15, totally, inhibited anti-LAM1-1, -5 and Leu-8 binding, indicating that these epitopes are clustered closely together.
  • Anti-LAM1-6 appeared unique in this analysis, and therefore defines a spatially distant determinant.
  • anti-LAM1-14 binding partially inhibited binding of anti-LAM1-1 only. Therefore, while most mAb-defined epitopes appeared to be closely associated within regions involved in ligand
  • the anti-LAM1-1, -5 an -15 mAb recognized the fusion protein containing the lectin plus EGF domains of LAM-1, but not the fusion protein containing the lectin domain or lectin plus SCR domains only. Therefore, these mAb recognize epitopes within the EGF domain, or epitopes that are derived from the lectin plus EGF domains.
  • Anti-LAM1-14 recognized only the fusion protein containing the LAM-1 SCR domains, and therefore defines a SCR epitope.
  • Lymphocytes from a number of animal species were analyzed to determine if the functionally-defined epitopes described above were well conserved. In addition, we hypothesized that further distinctions between these
  • rhesus monkey mononuclear cells expressed this epitope at low levels, and tamarin mononuclear cells were not stained by LAM1-3, in contrast to animal species more phylogenetically distant. While the epitope identified by the anti-LAM1-4 mAb appears identical with the LAM1-3 epitope in all previous assays, its pattern of reactivity on animals was quite distinct. Similarly, within each group of mAb which previously were indistinguishable with respect to their functional, domain mapping and serological profile, differences in which animal species were recognized were detected (Table 11). The exceptions to this were the epitopes identified by the anti-LAM1-8 and -9 mAb and the epitopes identified by the anti-LAM1-10, -11 and -12 mAb, which recognized LAM-1 from the same species.
  • mononuclear cells with anti-LAM1-3 inhibited 53% and 88% of lymphocyte binding to HEV, while treatment of dog
  • LAM-1 mononuclear cells with anti-LAM1-3 inhibited 52% of the HEV binding.
  • LAM-1 epitopes which mediate or regulate HEV- and PPME- binding were characterized using a large panel of newly developed mAb (Table 7).
  • the physical proximites of the functional regions defined by these mAb were identified, indicating that LAM-1 can be divided into a number of overlapping regions associated with distinct functional properties.
  • Each mAb reacted with leukocytes with characteristic levels of immunofluorescence staining (Fig. 9, Table 7), suggesting that there may be heterogeneity in expression of LAM-1 epitopes.
  • many of the anti-LAM-1 mAb may resemble the FMC46 mAb that identifies a cell protrusion-associated epitope of LAM-1.
  • These mAb were also used to examine structural differences between LAM-1 of lymphocytes and neutrophils.
  • lymphocytes and neutrophils express a single species of LAM- 1 protein, but the Mr of cell-surface LAM-1 on lymphocytes is 74,000 and that of neutrophil LAM-1 is 90-100,000.
  • lymphocytes and neutrophils stained neutrophils and lymphocytes with the same fluorescence intensity (Fig. 9, Table 7). Because each of the functional domains identified by the different mAb were conserved between leukocyte types, the structural differences between LAM-1 isoforms may only play a minor role in the regulation of leukocyte trafficking.
  • LAM-1 One functionally dominant region of LAM-1 was defined by the binding of two mAb, anti-LAM1-3 and anti- LAM1-4, which strongly inhibited the ability of lymphocytes to bind both HEV and PPME.
  • the DREG-56 mAb also fits into this functionally defined group of mAb (Kishimoto et al., Proc. Natl. Acad. Sci. USA 87:2244, 1990).
  • the anti-LAM1-3 and -4 mAb bind the lectin domain of LAM-1 only (Table 10), and each completely blocked binding of the other (Table 9).
  • the anti-LAM1-3 and anti-LAM1-4 mAb also completely block neutrophil binding of PPME.
  • the anti-LAM1-15 mAb which blocks the binding of both LAM1-1 and -5, also defines an epitopes within the EGF domain, but failed to affect the binding of PPME or HEV. since the increase in PPME binding after binding of LAM1-1 or -5 occurred rapidly at 4oC, it is likely that mAb binding to LAM-1 in this region induces a ⁇ onformational change in the molecule that results in an increased functional activity of the receptor for PPME.
  • mAb binding to LAM-1 at these sites mimics a natural event or a component of the LAM-1 ligand, and that the region of the protein identified by LAM1-1 and -5 mAb serves a critical role in the regulation of receptor binding to ligand.
  • the epitope within the lectin domain identified by the anti-LAM106 mAb was also involved in ligand binding, but was distinct from the PPME binding site since this antibody has the ability to block HEV binding without modulating PPME binding.
  • Crossblocking studies indicate that the anti- LAM1-6 mAb binds a region that is spatially separated from the site defined by the anti-LAM1-3 mAb. However this domain is close to the region defined by the anti-LAM1-1 mAb since these two mAb crossblock each other.
  • the anti-LAM1-6 and anti-LAM101 binding sites are distinct since the former mAb binds the lectin domain of LAM-1 whereas the latter identifies the EGF-like domain.
  • LAM-1 has many advantages over molecular genetic analysis of the primary structure, since the highly organized and folded nature of the LAM-1 molecule does not readily allow conclusions based on linear sequences and the crystal structure of LAM-1 has yet to be determined.
  • Human lymphocytes bind HEV of human or rodent peripheral lympho nodes with a similar specificity. suggesting that the ligand for both murine and human
  • lymphocytes is well conserved through recent mammalian evolution (Stoolman et al., Blood 70:1842, 1987; Stamper et al., J. Exp. Med. 144:828. 1976; Butcher et al., J. Immunol. 134:2989. 1979; Butcher et al., Nature 280:496. 1979; Wu et al., J. Cell. Biol. 107:1845).
  • the demonstration that dog, tamarin and rabbit lymphocytes bind PPME in a calcium- dependent fashion (Fig. 12), in combination with the
  • each mammal species tested expressed one or more of the LAM- 1 epitopes (Table 11).
  • the reactivity of each mAb with numerous animal species also indicated that while several mAb were reactive with functionally identical regions of human LAM-1, most mAb identified unique epitopes. It is likely that subtle amino acid changes in LAM-1 between species accounts for the differences between reactivities of the anti-LAM-1 mAb.
  • LAM-1 lymphocyte-assocated cell surface protein LAM-1
  • LAM-1 leukocyte-adhesion molecule-1
  • Antagonists to LAM-1 were used in a method of treating a human patient suffering from a lymphocyte-mobilizing condition which involves administering a therapeutic amount of the antagonist in a non-toxic pharmaceutical carrier.
  • LAM-1 contains an amino-terminal, lectin-llke domain which may interact with specific glyco-conjugates expressed on high endothelial venules (HEV) of peripheral LN (lymph nodes) and activated endothelium [3-5].
  • HEV high endothelial venules
  • LAM-1 is expressed by human peripheral lymphocytes, neutrophils, eosinophils, monocytes and hematopoietic progenitor cells [5-8]. LAM-1 is
  • LAM-1 is shed from the cell surface within minutes of exposure of
  • lymphocytes and neutrophils express a single LAM-1 protein product, but the molecular weight (Mr) of cell-surface LAM-1 on lymphocytes is 74,000 and that of neutrophils is 90,000- 100,000 [6,9,12].
  • hematopoietlc malignant cells [1-3].
  • the mLHR has been implicated in the dissemination of lymphomas [14-16], and a calcium-dependent phosphomannosyl-binding site on human malignant lymphoblastold cell lines mediates peripheral LN HEV binding [17].
  • the structure, function and regulation of LAM-1 expression was examined on normal lymphocytes and compared to LAM-1 of malignant leukocytes.
  • the LAM-1 molecule is a member of a new family of cellular adhesion/homing molecules that contain a lectin-like domain at their amino-terminal end followed by an epidermal growth factor-like domain and short consensus repeat units like those found in C3/C4 binding proteins.
  • leukpocyte-adhesion molecule-1 itself and "LAM1-X" refers to an antibody x which binds to an epitope of LAM-1.
  • LAM1-1 and LAM1-2 were found to be reactive with the majority of blood lymphocytes, NK (Natural Killer) cells, neutrophils, monocytes and
  • LAM-1 hematopoeitic progenitor cells. Binding of LAM-1 may participate in the process of leukocyte extravasation into lytnphoid organs or sites of acute inflammation with subsequent loss of LAM-1 from the cell surface. LAM-1 is also recognized by the TQ1 and Leu-8 monoclonal antibodies that have been previously Identified.
  • LAM-1 in conjunction with other selectins and receptors, is involved in the extravasation of most leukocytes.
  • the expression of LAM-1 by different leukocytes sub-populations thus plays a key role in determining the characteristics and magnltude of local immune responses [5].
  • the present invention relates to the production of a new antibody to LAM-1.
  • the new monoclonal antibody, anti-LAM1-3 is useful in radioisotope or imaunofluorescent assays for the detection of LAM-1. For example, identifying species which have or do not have LAM-1.
  • the antibody is further useful for separating cells expressing LAM-1 from cells not expressing LAM-1 or visa versa. Furthermore, this monoclonal antibody also completely blocks leukocyte attachment to HEV or endothelium.
  • Neutrophil-mediated inflammation is involved in a number of human clinical manifestations, including the adult respiratory distress syndrome, multi-organ failure and reperfusion injury.
  • One way of inhibiting this type of inflammatory response would be to block competitively the adhesive interactions between neutrophils and the endothelium adjacent to the Inflamed region.
  • Anti-LAMl-3 reacts with LAM-1 on many animal species, but does not bind the mLHR.
  • Anti-LAM1-3 blocks completely lymphocytic traffic to lymph nodes and extravasation of neutrophils front blood to inflammatory sites. The administration of soluble forms of anti-LAM1-3 could be clinically effective for the inhibition of neutrophil-mediated inflammation.
  • Anti-LAM1-3 also blocks lymphocyte adhesion to human HEV and activated endothelium.
  • Antibodies are Y-shaped molecules consisting of two long "heavy” chains which define the stem and arms of the Y and two short "light” chains which are attached to the outside of the arms.
  • the amino-terminal ends of the arms of the antibody molecule contain the variable regions of the antibody.
  • the variable regions are specific for a particular antigen.
  • the stem of the molecule is the "constant" region which ends in a carboxylate function (COO-) and remains the same from molecule to molecule in antibodies of the same isotype in the same species.
  • the constant region of the mouse antibody has been found to be the primary source human immune reactions to mouse monoclonal antibodies.
  • mouse variable regions have been fused to human constant regions to generate "chimeric" (from chimera or chimaera, a monster of Greek mythology which had a lion's head, a goat's body and a serpent's tail) antibodies.
  • chimeric antibodies thus possess regions of different genetic origin and have been found to have a lower tenency to produce allergic reactions.
  • a hybrid cell line which produces the enti-LAM-1 monoclonal antibody anti-LAM1-3 embodying this invention was been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 on June 12, 1991 and is assigned A.T.C.C. Deposit No. HB 10771.
  • adhesion/homing receptor molecules identified by cDNA cloning.
  • Members of this family include the leukocyte adhesion molecule-1 (LAM-1) which is the human homolog of the mouse lymphocyte homing receptor (mLHR), the human granule-membrane protein (GMP-140, PAOGEM, CD62) which is expressed on activated platelets and endothelial cells, and the human endothelial leukocyte adhesion molecule- 1 (ELAM-1) expressed on activated endothelial cells.
  • LAM-1 leukocyte adhesion molecule-1
  • MMP-140 human granule-membrane protein
  • ELAM-1 human endothelial leukocyte adhesion molecule- 1
  • LN lymph node
  • LAM-1 leukocyte adhesion molecule-1
  • CLL chronic lymphocytic leukemia
  • NHL non-Hodgkin ' s lymphoma
  • PPME poly-phosphomonoester from the yeast HANSENULA
  • AML adult ⁇ yelogenous leukemia
  • CML chronic myelogenous leukemia
  • PBMC peripheral blood mononuclear cells
  • BM bone marrow
  • FSC follicular small cleaved cell lymphoma
  • CSF colony stimulating factor
  • DSC diffuse small cleaved cell lymphoma
  • LPS lipopolysaccharide
  • LAM-1 expression was examined on normal and neoplastic leukocytes to further understand the mechanisms that regulate leukocyte migration.
  • the immunoprecipitation of a large fragment of LAM-1 of molecular weight 69,000 from the supernatant liquid of normal lymphocytes cultured with PMA demonstrated that LAM-1 can rapidly be cleaved from the cell surface (Fig. 4). That the LAM-1 expression is down- modulated by shedding rather than by internalization suggests that a PMA-sensitive regulatory pathway which is distinct from that which regulates down-modulation of most other surface molecules, controls the expression of LAM-1. This regulatory pathway may specifically involve the activation of PKC (Table 3).
  • LAM-1 may also be continuously shed at a slow rate with its expression kept constant by the continuous synthesis of new receptors.
  • enzymatic cleavage of the cell-surface receptor may result from the specific activation of a membrane bound protease. This is a likely method since a soluble protease secreted by activated leukocytes was not detected in this work.
  • activation-induced changes in the comformation of the LAM-1 protein may expose nascent sites on LAM-1 that are then susceptible to cleavage by soluble proteases.
  • LAM-1 was most frequently expressed by CLL cells among the various hematologic malignancies studied (Table 1, Fig. 5). These results extend previous studies of LAM-1 expression to TQ1 and Leu-8 using CLL and NHL cells [35-37]. Since the expression of LAM-1 was somewhat restricted among hematologic malignancies, the expression, or absence of expression, may have a major impact on the trafficking of leukemlc cells and the dissemination of NHL. Immunoprecipitation of LAM-1 from CLL cells showed that it resembled the Mr 74,000 isoform of the glycoprotein
  • LAM-1 expressed by malignant cells was functional since LAM-1 on normal lymphocytes and CLL cells were both able to bind HEV and PPME (Table 2, Fig. 7). Both HEV and PPME binding was mediated by LAM-1 since the new monoclonal antibody, anti-LAM1-3, was able to completely block all HEV and PPME binding. The level of HEV binding was also proportional to the quantity of LAM-1 expressed, and LAM-1 negative cells were unable to bind HEV and PPME. LAM-1 was also shed from the surface of CLL cells following PMA exposure (Fig. 8).
  • the signalling pathway for shedding may be less active in some CLL cells since the time-course of LAM-1 shedding was slower than in normal lymphocytes. Malignant cells, therefore, express functional LAM-1 receptors that are indistinguishable from their normal counterparts on normal cells and the expression of LAM-1 by CLL cells correlated with the high tendency of these cells to localize into peripheral LN.
  • CD44 expression was found to be consistently expressd at high levels among the leukemias and NHL examined, while the expression of other adhesion
  • CD11/CD18, CD54 and CD58 was variable (Table 1). Expression of CD44 did not correlate with the ability of cells to bind to HEV since LAM-1 negative CLL cells that expressed high levels of CD44 did not bind to HEV in frozen section assays (Table 2) similar to what was shown by one
  • CD44 constitutes a broadly distributed family of glycoproteins expressed on virtually all hematopoietic cells, flbroblasts, epidermal, glial and melanocytic origin cells [21,38]. Although CD44 was initially regarded as the human homing receptor equivalent of the mLHR [28,29], it may be more generally involved in cell-cell or cell-matrix binding as a receptor for hyaluronate [39]. Previous studies have also suggested that CD44 is involved in the dissemination of NHL [40]. During the work resulting in the present invention, however, no clear relationship could be inferred from the results of CD44 expression alone.
  • LAM-1 is expressed on most neutrophils, monocytes, normal myeloid progenitor cells and early erythroid precursors in BM (bone marrow) [6].
  • the co-expression of this homing receptor and other adhesion molecules may control the physiological retention (homing) of these cells in BM.
  • the homing of intravenously transplanted hematopoietic stem cells is mediated by a recognition system with galactosyl and mannosyl specificities [41] which might also mimic the
  • LAM-1 ligand [42].
  • AML and CML cells were found to lack expression of LAM-1. Unlike the situation with lymphoid tumors, this is in sharp distinction with the high level expression of LAM-1 on normal myeloid cells. The absence of LAM-1 expression on most AML and CML cells might favor the passage of these cells into the bloodstream. Although overnight culture of CML cells did not result in the expression of LAM-1 on the cell surface, the overall lack of LAM-1 expression by these cells indicates that further investigations of the regulation of LAM-1 by leukemic myeloid cells is warranted.
  • LAM-1, on CLL and low-grade lymphoma cells may also contribute to the wide-spread dissemination of these malignant cells to LN as occurs with normal lymphocytes.
  • PBMC Peripheral blood mononuclear cells
  • BM bone marrow
  • BM bone marrow
  • Tumor type was classified according to conventional morphological, cytological and immunophenotype criteria.
  • Tumor cell lineage was determined by analysis of antigens (Ag) including surface and cytoplasmic immunoglobulin (Ig), HLA-DR Ag, CD1, CD2, CD3, CD4, CD5, CD6, CD8, CD9, CD10, CD11b, CD13, CD14, CD19, CD20 and CD33.
  • Ag antigens
  • Ig surface and cytoplasmic immunoglobulin
  • HLA-DR Ag CD1, CD2, CD3, CD4, CD5, CD6, CD8, CD9, CD10, CD11b, CD13, CD14, CD19, CD20 and CD33.
  • Cells were examined immediately after isolation or were immediately cryopreserved and kept frozen in liquid nitrogen until used. The frequency of malignant cells was always greater than 90% in every sample examined.
  • the anti-LAM-1 monoclonal antibodies anti-LAM1-1 and anti-LAM1-2 and the monoclonal antibody anti-TQ1 have been previously described [5,8].
  • IgG1 (IgG1) of the claimed invention was generated by the fusion of NS-1 myeloma cells with spleen cells from Balb/c mice that were repeatedly immunized with cells of the mouse pre-B cell line 300.19 transfected with a LAM-1 cDNA as described [5].
  • CDlla and 10F12 (CD18) [18] which were gifts from J. Ritz (Dana-Farber Cancer Inst., Boston MA); TS2/9 (CD58, anti- LFA-3) [19] and RR 1/1 (CD54, anti-ICAM-1) [20] which were gifts from T.A. Springer (Center for Blood Research, Boston, MA); 515 (CD44) [21] a gift from G.S. Kansas (Dana-Farber Cancer Inst.); and 904 (CD11b) [22].
  • Indirect immunofluorescence analysis was performed on viable cells isolated by Ficoll-Hypaque density gradient centrifugation.
  • the expression of LAM-1, CD11a, CD11b, the ⁇ subunit of CD11 complex (CD18), CD44, CD54, and CD58 was examined by indirect immunofluorescence with flow cytometry analysis (Coulter Epics C, Coulter Electronics, Hialeah FL). Isotype-matched murine antibodies that were unreactive with human leukocytes were used as negative controls. Cells were incubated with each monoclonal antibody for 20 minutes on ice, washed, and treated with FITC-conjugated goat anti- mouse Ig reagents (Southern Biotechnology Associates, Birmingham, AL).
  • fl-PPME fluorescein derivative of PPME
  • the in vitro HEV binding assay was performed using frozen tissue sections of human or rat peripheral LN using the methods of Stamper and Woodruff [25] and Butcher, et al.
  • RPMI 1640 medium Sigma, St. Louis, MO
  • neuraminidase 0.1 U/ml, Calbiochem, La Jolla, CA
  • LAM-1 was immunoprecipitated as described above from the supernatant fluid and the pellet of PBMC that had been cultured for 60 minutes at 37°C in RPMI 1640 medium alone or in RPMI medium containing PMA (100 ng/ml, Sigma, St. Louis, MO).
  • expression of LAM-1 was assessed after incubation of the cells with PMA (10 nM for 30 minutes) following the prior culture of the cells with sodium azide (Sigma) or the protein kinase inhibitors, 1-(5-Isoquinolinyl-sulfonyl)-2- methylpiperazine (H-7, Calbiochem) and staurosporine (Sigma) for 30 minutes at 37oC. Discussion of Figures 4-6.
  • LAM-1 is shed from the cell surface into the culture medium.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • the supernatant fluid and cells were harvested and immuno- precipitated with anti-LAM-1 antibodies or an unreactive isotype matched control antibody (Cont.).
  • Immuno- precipitated materials were electrophoresed on a 7.5% SDS aerylamide gel under reducing conditions followed by autoradlography. The migration of known molecular weight standards are shown in kilo-Daitons (kDa).
  • the frequency of LAM-1 expression by malignant cells Cells from 118 patients with various forms of hematopoietic malignancies were examined for surface LAM-1 expression using the anti-LAM1-1 monoclonal antibody in indirect immunofluorescence assays with flow cytometry analysis. In each instance, the background staining for each sample was determined using an unreactive isotype-matched monoclonal antibody and the level of background staining
  • Pre-B pre-B acute lymphoblastic leukemia
  • T-ALL T cell acute lymphoblastic leukemia
  • B-CLL B type chronic lymphocytic leukemia
  • FSC follicular small cleaved cell lymphoma
  • DSC diffuse small cleaved cell lymphoma
  • DLC diffuse large cell lymphoma
  • Burk. Burkitt's type lymphoma
  • M.M. multiple myeloma
  • AML acute myelogenous leukemia
  • CML chronle myelogenoua leukemia
  • the relative fluorescence staining intensity of the malignant cells is indicated where the positive population could be identified as a distinguishable peak from background fluorescence staining: ⁇ , where a shoulder of positively stained cells was evident, +, where a separata peak of positive cells was identified with weak fluorescence; ++, a definite separate peak of fluorescence positive cells of moderate fluorescence; +++, a peak of fluorescence positive cells of the same intensity as normal blood lymphocytes.
  • the tissue source of all malignant cells is also indicated.
  • LAM1-1 antibodies ( LAM-1 ) . or an unreactive isotype-matched antibody control ( Cont. ) . Immunoprecipitated materials were divided and analyzed under non-reducing and reducing conditions on a 12% SDS poly aery 1 amide gel followed by
  • Normal human lymphocytes and CLL cells are capable of binding PPME through LAM-1.
  • Cells were examined for LAM-1 expression by indirect immunofluorescence analysis after treatment with the anti-LAM1-3 monoclonal antibody ( dark line ) or with an unreactive isotype matched antibody ( thin line ) .
  • Cells were also reacted with FITC-conjugated PPME after treatment with the anti-LAM1-3 antibody (thin line ) or an unreactive control antibody ( dark line ) .
  • the fluorescence intensity of cell stsining was analyzed by flow cytometry.
  • LAM-1 Modulation of cell surface LAM-1 by malignant cells and cDNA transfected cells after PMA exposure.
  • Cells transfected with LAM-1 cDNA, K562-LAM1 and 300.19-LAM1, and malignant cells that expressed LAM-1 were either cultured for 90 minutes in media or in media containing 10 ng/ml PMA. Following culture, the cells were examined for LAM-1 expression using the anti-LAM1-1 antibody in indirect immunofluorescence assays with flow cytometry analysis. Cells were also stained with an unreactive control antibody and the level of background staining was always less than 5%. The frequency of cells expressing LAM-1 is shown with the number of background staining cells subtracted.
  • LAM-1 is released from the cell surface following PMA exposure.
  • Immunoprecipitation experiments were carried out to determine the fate of LAM-1 after modulation from the surface of PBMC exposed to PMA [5].
  • Surface iodinated cells were cultured for 60 minutes in RPM1 medium alone or medium containing PMA. After culture, the supernatant fluid and cells were separated, and the cells were lysed with detergent. The cell lysate and supernatant fluid were immunoprecipitated with a combination of anti-LAM1-1 and anti-TQ1 antibodies that bind to different epitopes of LAM-1 [5], and together are more efficient for immunoprecipitation.
  • the cells were also treated with neuraminidase prior to surface iodination since LAM-1 may be more readily immunoprecipitated after the removal of slallc acid residues.
  • LAM-1 and other cell surface molecules known to be involved in lymphocyte adhesion and migration were examined on malignant leukocytes from 118 patients by indirect immunofluorescence analysis. LAM-1 expression was most frequently demonstrated on CLL cells and among lymphomas classified as follicular (FSC) and diffuse small cleaved cell lymphoma (DSC) (Table 3). On the other hand, most acute myeloblastic leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelocytic leukemia (CML), diffuse large cell lymphomas (DLCT, Burkitt's lymphomas and multiple myelomas were LAM-1 negative.
  • FSC follicular
  • DSC diffuse small cleaved cell lymphoma
  • the level of cell surface LAM-1 expression was highest on CLL cells; and in 11 of 16 cases that expressed LAM-1, more than 50% of cells were L ⁇ M-1+ (FIG. 5).
  • the fluorescence intensity level of LAM-1 staining correlated with the frequency of LAM-1+ cells such that most malignant cell populations with less than 25% positive cells failed to express LAM-1 at easily detectable levels.
  • CLL cells rarely expressed adhesion molecules other than LAM-1, with the exception of CD44 (Table 3), which has also been associated with lymphocyte homing [28,29]. More than 90% of the cell samples were CD44+, consistent with its ubiquitous distribution on normal hematopoietic cells.
  • Anti-LAM-1 antibodies were used to immuno- precipitate LAM-1 from CLL cells.
  • LAM-1 migrated with a Mr of 68,000 under non-reducing conditions and at 73,000 after reduction (FIG. 6), similar to LAM-1 immunoprecipitated from normal lymphocytes (FIG. 4). Therefore, it appears that normal and malignant lymphocytes express the same cell- surface LAM-1 protein.
  • LAM-1 expression was examined using cells from normal circulating blood, three LAM-1 positive CLLs and one LAM-1 negative CLL. Cells were assessed for their ability to bind HEV of human peripheral LN using the frozen section assay of Stamper and Woodruff [25].
  • the LAM-1+ cells bound to HEV at levels which corresponded to the amount of LAM-1 expressed on their cell surface, while the LAM-1 CLL cells did not bind (Table 2).
  • CD44 expression was quite high on all of the cell samples examined and did not correlate with HEV adhesion. Additional studies examined the ability of anti-LAM-1 monoclonal antibody to block HEV binding.
  • a new antibody, anti- LAM1-3 was able to specifically block 92 to 95% of normal lymphocyte and LAM-1+ CLL cell binding (cells from Table 2) to rat peripheral LN HEV.
  • the binding of a different antibody, anti-LAM1-10, reactive with a different epitope of LAM-1 had no detectable effect on HBV binding. (See Table 4). Therefore, the levels of LAM-1 expression correlated with the ability of cells to bind HEV and antibodies reactive with LAM-1 specifically blocked binding.
  • the ligand for the mLHR is mimicked by the mannose 6 phosphate-rich polysaccharide PPME [24]. Therefore, the ability of normal human lymphocytes and CLL cells to bind fluoresceinated PPME was examined to further characterize the functional capacity of human LAM-1. Both normal blood lymphocytes and LAM-1+ CLL cells were able to bind PPME, while LAM-1- CLL cells did not bind PPME (FIG. 7). The specificity of PPME binding to LAM-1 was verified by the ability of anti-LAM1-3 antibody to completely block PPME binding to the cells (FIG. 7). Modulation of LAM-1 expresion.
  • FIG. 8 While it was only minimal in two cases (B-CLL #1 and #2, Fig. 8).
  • PMA Induced an almost complete modulation of LAM-1 expression after 90 minutes of stimulation (Fig. 8).
  • Fig. 8 In six LAM-1+ CLL cell samples tested further. PMA exposure lead to the complete loss of LAM-1 expression after 180 to 360 minutes of culture with PMA with similar kinetics to those of RAJI cells treated simultaneously.
  • LAM-1 cDNA transfected cells The erythro- leukemia cell line, K562, and the mouse pre-B cell line, 300.19, were transfected with LAM-1 cDNA as described [5], generating cells that express relatively high levels of cell surface LAM-1 (Fig. 8). In contrast to what was observed with RAJI cells and the majority of CLL cells, 90 minutes exposure of these cells to PMA induced an almost complete loss of LAM-1 from the cell surface.
  • PKC may regulate cell surface receptor expression through direct phosphorylation of LAM-1 which may signal for cleavage or through kinase reguatlon of protease activity. Shedding of LAM-1 does not result from the activation- induced secretion of a soluble protease.
  • lymphocytes (10 7 /ml) were activated with lineage-specific cytoklnes such as granulocyte/macrophage-CSF, to indue complete LAM-1 shedding [6].
  • lineage-specific cytoklnes such as granulocyte/macrophage-CSF
  • the supernatant fluid of these cultures was harvested and used as culture medium for lymphocytes or LAM-1 cDNA transfected cells for 120 minutes at 37°C. This treatment did not induce detectable LAM-1 shedding from the surface of lymphocytes as assessed by flow cytometry analysis.
  • the activation of neutrophils by lineage- specific stimuli in the presence of lymphocytes failed to induce detectable loss of lymphocyte LAM-1 while neutrophil shedding of LAM-1 was complete.
  • a membrane anchored protease cleaves LAM-1 from the cell surface or that cellular activation is required for cleavage to occur.
  • LAM-1 expression may be down-regulated during lymphocyte entry into tissues and this down-regulation is reversible in culture [5].
  • Malignant cells isolated from the highly infiltrated spleen of a CLL patient were found to express LAM-1 at a lower level than the CLL cells found in his peripheral blood (i.e 65% on blood cells and 25% on splenocytes).
  • the percentage of LAM-1 positive spleen cells was comparable to that of the patients peripheral B-CLL cells stimulated for 180 minutes with PMA. This suggests that LAM-1 expression was decreased with entry of the CLL cells into the spleen as occurs with normal lymphocytes.
  • LN cells from patients with NHL two patients with FSC, one with DLC and two with DSC
  • leukemia BM cells from two patiente with AML, one with ALL and one with CML
  • Table 5 lists a number of the properties of the monoclonal antibodies anti-LAM1-1, -2 and -3. Lines 1, 2, and 3 give the name of the antibody, the isotype and the differences in staining intensity. These properties are not necessaryily indicative of differences in epitope recognition.
  • Lymph nodes contain structures called high endothelial venules (HEV) which are utilized by lymphocytes to enter the lymph nodes (the site of Immune responses) from the blood stream. Emigration of lymphocytes into the node has been shown to be mediated by adhesion molecules which allow the cells to stick to and then traverse the venule. This process has been studied by incubating isolated lymphocytes with lymph node tissue sections. When the sections are incubated with lymphocytes alone, the cells will adhere to HEV, and the number of adherent cells can be counted.
  • Various monoclonal antibodies, including the LAM-1 antibodies have been used to block this binding, Line 4 gives the results of such studies for LAM1-1, -2 and -3.
  • the polysaccharide PPME mimics the natural ligand for the LAM-1 molecule. Since PPME can be directly fluores- ceinated, it is possible to study the effect of the various monoclonal antibodies on the interaction of LAM-1 and PPME.
  • Line 5 details the results using cells which were first incubated with a LAM-1 monoclonal antibody, followed by treat- ment with PPME-FITC.
  • Anti-TQ1, anti-LAM1-2 and anti-LAM1-3 blocked PPME binding and anti-LAM1-1 enhanced PPME binding.
  • Lines 7-10 detail the results of studies in which the ability of a given monoclonal antibody to block the subsequent binding of other monoclonal antibodies was analyzed. Blocking of one antibody by another provides evidence that the two antibodies in question recognize epitopes which are identical or close together on the molecule.
  • the results in Table 4 indicate that anti-LAM1-1 does not block anti-LAM1- 3, indicating that their epitopes are different.
  • Anti-LAM1- 2 does block anti-LAM1-3, indicating that these epitopes are at least close to each other.
  • There are differences between anti-LAM1-2 and anti-LAM1-3 however, because of the difference response they generate regarding Leu 8.
  • Anti-LAM1-2 does not at all block Leu 8, whereas anti- LAM1-3 strongly blocks it. Species cross-reactivity gives further indications of the diferencee which exist between the antibodies and the epitopes that they identify.
  • Line 11 gives the results of the domain mapping regarding the monoclonal antibodies.
  • the LAM-1 molecule contains three domains which are:
  • EGF epidermal growth factor-like domain
  • LAM-1 L plus SCR domains from LAM-1 (EGF from CD62). These cDNAs were transfected into cells which then produced the corresponding proteins. The pattern of reactivity of the various monoclonal antibodies was then determined as shown in Table 6, and the domain necessary for monoclonal antibody reactivity was assigned.
  • anti-LAM1-3 bound to cells expressing all the domains described with medium to very strong strength.
  • Anti-LAM1-1 did not bind to cells which contained LAM-1 (L + SCR) or LAM-1 (L) alone.
  • the epitope which is recognized by anti-LAM1-1 must, therefore, be composed of a site within the EGF, domain, or which contains part of the L and EGF domains, but not the SCR domain.
  • LAM1-3 on the other hand, must only contain the LAM-1(L) domain. The two antibodies are, therefore, distinguishable.
  • LAM-1 may be the most functionally and structurally conserved leukocyte adhesion molecule found in mammals, considering that many do not function reciprocally between man and mouse. Whether the evolutionary basis for this high level of conservation is fundamental to the function of the receptor or results from the unique nature of a carbohydrate-based ligand with limited potential for divergence will have to be determined after identification and characterization of the true ligand(s).
  • leukocyte-mediated inflammation is involved in a number of human clinical manifestations, including the adult respiratory distress syndrome, multi-organ failure and reperfusion injury.
  • One way of inhibiting this type of inflammatory response would be to block competitively the adhesive interactions between leukocytes and the endothelium adjacent to the inflamed region.
  • LAM-1 mediates the migration and adhesion of blood leukocytes, treatment of a patient in shock, e.g., from a serious injury, with an antagonist to cell surface LAM-1 function (such as the monoclonal
  • antibodies of the invention can result in the reduction of leukocyte migration to a level manageable by the target endothelial cells.
  • agents developed to block receptor function can inhibit the metastasis and homing of malignant cells which express the LAM-1 receptor protein.
  • the therapeutic agents may be administered orally, parenterally, or topically by routine methods in
  • Optimal dosage and modes of administration can readily be determined by conventional protocols.
  • the normal regulation of the lyam-1 gene (the name given to the human gene encoding LAM-1), as evidenced by the appearance and disappearance of the LAM-1 protein on the surface of a specific leukocyte subpopulation, can be monitored by use of the monoclonal antibodies of the
  • a The percentage of cells reactive with the anti- LAMl-1 monoclonal antibody was determined by indirect immunofluorescence analysis. The relative intensity of staining of the positive cells is indicated based on the mean fluorescence channel number (MFC No.) obtained with FACS analysis (256 channels, on a 3-decade log scale. Cells treated with an unreactive monoclonal antibody had 3% positive cells with a MFC no. of 40.
  • a The percentage of cells with the anti-LAM1-1 and 515 monoclonal antibodies were determined by indirect immunofluorescence analysis. The relative intensity of staining of the positive cells is Indicated based on e (-) being no reactivity and (++++) indicating the highest reactivity,
  • b Values represent the mean number of cells ( ⁇ SD) bound to each HEV. A total of 150 HEV were examined in each sample. The difference between the number of cells bound per HEV in LAM-1+ cases (1,2 and 4) and the
  • CD3 was crossllnked as shown by Spertini et al., Nature
  • the cells were Incubated with 125 I- labelled PPME 90.36 ⁇ g/ml, 2.2x10 5 c.p.m. per sample) at 4°C for 30 minutes.
  • Anti-LAM1-3 was added 1 minute before the eddltion of the test antibody and during all incubations. The calcium-independent binding of
  • [ 125 I]PPME was assessed in the presence of 5 mM EDTA. Cells were washed, resuspended in PBS-BSA and layered on a 750- ⁇ l cushion of 75% (v/v) calf serum. The cell pellet was Isolated end bound [ 125 I]PPME assessed by
  • EGF Epidermal Growth Factor-like
  • a Values represent the relative intensity of immunofluorescence staining of COS-7 cells transfected with the LAM-1 cDNA or recombinant cDNAs encoding the lectin, EGF-like, or two SCR domains of LAM-1 with the rest of the cDNAs encoding CD62.
  • SCR a domain of short consensus repeats
  • EGF epidermal growth factor-like domain.
  • a Human blood lymphocytes were assessed for their ability to bind to rat peripheral lymph node HEV as described in Materials and Methods. All HEV were counted regardless of lymphocyte binding with at least 150 HEV examined in each sample. The values represent the mean number of lymphocytes bound to each HEV ( ⁇ SD) obtained in the number of experiments indicated.
  • a Values represent the relative intensity of immunofiuorescence staining of COS-7 cells (- to +++ scale) transfected with the LAM-1 cDNA or chimeric cDNA encoding the lectin, EGF-like, or two SCR domains of LAM-1 with the rest of the cDNA encoding CD62. These results are representative of those obtained in at least three experiments.
  • LAM-1 may be the most functionally and structurally conserved leukocyte adhesion molecule found in mammals, considering that many do not function reciprocally between man and mouse. Whether the evolutionary basis for this high level of conservation is fundamental to the function of the receptor or results from the unique nature of a carbohydrate-based ligand with limited potential for divergence will have to be determined after identification and characterization of the true ligand(s).
  • leukocyte migration and infiltration into areas of tissue damage or injury or tissue transplant can cause or increase pathology
  • agents that impede these processes can be used for therapeutic treatment.
  • leukocyte- mediated inflammation is involved in a number of human clinical manifestations, including the adult respiratory distress syndrome, multi-organ failure and reperfusion injury, one way of inhibiting this type of inflammatory response would be to block competitively the adhesive interactions between leukocytes and the endothelium adjacent to the inflamed region.
  • LAM-1 mediates the migration and adhesion of blood leukocytes, treatment of a patient in shock, e.g., from a serious injury, with an antagonist to cell surface LAM-1 function (such as the monoclonal
  • antibodies of the invention can result in the reduction of leukocyte migration to a level manageable by the target endothelial cells.
  • agents developed to block receptor function can inhibit the metastasis and homing of malignant cells which express the LAM-1 receptor protein.
  • the therapeutic agents may be administered orally, parenterally, or topically by routine methods in
  • Optimal dosage and modes of administration can readily be determined by conventional protocols.
  • the normal regulation of the lyam-1 gene (the name given to the human gene encoding LAM-1), as evidenced by the appearance and disappearance of the LAM-1 protein on the surface of a specific leukocyte subpopulation, can be monitored by use of the monoclonal antibodies of the

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Abstract

Anticorps monoclonal reconnaissant l'épitope de LAM-1 reconnu par les anti-LAM1-1, -2, -4, -5, -6, -7, -8, -9, -10, -11, -14 ou -15; hybridome produisant un tel anticorps monoclonal; procédés d'utilisation dudit anticorps monoclonal.
PCT/US1992/006127 1991-07-29 1992-07-23 Anticorps monoclonaux contre la molecule-1 d'adhesion aux leucocytes WO1993002698A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5616352A (en) * 1994-09-19 1997-04-01 Skw Trostberg Aktiengesellschaft Process for the production of fat- and cholesterol-reduced powered products based on eggs which are characterized by a high phospholipid content
EP0770680A3 (fr) * 1989-02-21 1997-05-07 Dana-Farber Cancer Institute, Inc. Protéine de surface des cellules associée aux lymphocytes
EP0868197A4 (fr) * 1995-08-17 1998-11-18

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US5098833A (en) * 1989-02-23 1992-03-24 Genentech, Inc. DNA sequence encoding a functional domain of a lymphocyte homing receptor

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US5098833A (en) * 1989-02-23 1992-03-24 Genentech, Inc. DNA sequence encoding a functional domain of a lymphocyte homing receptor

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Title
CELL, Volume 56, issued 24 March 1989, STOOLMAN, "Adhesion Molecules Controlling Lymphocyte Migration", pages 907-910. *
CELL, Volume 62, issued 13 July 1990, OSBORN, "Leukocyte Adhesion to Endothelium in Inflammation", pages 3-6. *
JOURNAL OF CELL BIOLOGY, Volume 107, issued November 1988, N.W. WU et al., "Evolutionary Conservation of Tissue-Specific Lymphocyte-Endothelial Cell Recognition Mechanisms Involved in Lymphocyte Homing", pages 1845-1851. *
JOURNAL OF CELL BIOLOGY, Volume 109, issued July 1989, B.R. BOWEN et al., "Characterization of a Human Homologue of the Murine Peripheral Lymph Node Homing Receptor", pages 421-427. *
JOURNAL OF EXPERIMENTAL MEDICINE, Volume 170, issued 01 July 1989, T.F. TEDDER et al., "Isolation and Chromosomal Localization of cDNAs Encoding a Novel Human Lymphocyte Cell Surface Molecule, LAM-1", pages 123-133. *
JOURNAL OF IMMUNOLOGY, Volume 128, No. 1, issued 01 January 1982, E.L. REINHERZ et al., "Heterogeneity of Human T4 + Inducer T Cells Defined by a Monoclonal Antibody that Delineates Two Functional Subpopulations", pages 463-468. *
JOURNAL OF IMMUNOLOGY, Volume 144, issued 15 January 1990, T.F. TEDDER, "Expression of the Human Leukocyte Adhesion Molecule, LAM1: Identity with the TQ1 and Leu-8 Differentation Antigens", pages 532-540. *
PROC. NATL. ACAD. SCI. USA, Volume 87, issued March 1990, KISHIMOTO et al., "Identification of a Human Peripheral Lymph Node Homing Receptor: A Rapidly Down-Regulated Adhesion Molecule", pages 2244-2248. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0770680A3 (fr) * 1989-02-21 1997-05-07 Dana-Farber Cancer Institute, Inc. Protéine de surface des cellules associée aux lymphocytes
US5616352A (en) * 1994-09-19 1997-04-01 Skw Trostberg Aktiengesellschaft Process for the production of fat- and cholesterol-reduced powered products based on eggs which are characterized by a high phospholipid content
EP0868197A4 (fr) * 1995-08-17 1998-11-18

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