EP0643728A1 - Icam-related protein - Google Patents
Icam-related proteinInfo
- Publication number
- EP0643728A1 EP0643728A1 EP93919890A EP93919890A EP0643728A1 EP 0643728 A1 EP0643728 A1 EP 0643728A1 EP 93919890 A EP93919890 A EP 93919890A EP 93919890 A EP93919890 A EP 93919890A EP 0643728 A1 EP0643728 A1 EP 0643728A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- icam
- seq
- atcc
- cells
- binding
- 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- Withdrawn
Links
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- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus
Definitions
- the present invention relates generally to cellular adhesion molecules and more particularly to the cloning and expression of DNA encoding a heretofore unknown human polypeptide designated "ICAM-R" which possesses structural relatedness to the intercellular adhesion molecules ICAM-1 and -2.
- CAMs Cellular Adhesion Molecules
- CAMs single chain adhesion molecules
- CAMs single chain adhesion molecules
- VCAM-1 vascular adhesion molecule
- ICAM-1 and ICAM-2 are structurally homologous to other members of the immunoglobulin gene superfamily in that the extracellular portion of each is compriised of a series of domains sharing a similar carboxy terminal motif.
- a "typical" immunoglobulin-like domain cont ⁇ s a loop structure usually anchored by a disulfide bond between two cysteines at the extremity of each loop.
- ICAM-1 includes five immunoglobulin-like domains; ICAM-2, which differs from ICAM-1 in terms of cell distribution, includes two such domains; PECAM-1 includes six; VCAM includes six or seven, depending on splice variations, and so on.
- CAMs typically include a hydrophobic "transmembrane" region believed to participate in orientation of the molecule at the cell surface and a carboxy terminal "cytoplasmic" region.
- Graphic models of the operative disposition of CAMs generally show the molecule anchored in the cell membrane at the transmembrane region with the cytoplasmic "tail" extending into the cell cytoplasm and one or more immunoglobulin-like loops extending outward from the cell surface.
- WO91/16928 published November 14, 1991, for example, addresses humanized chimeric anti- ICAM-1 antibodies and their use in treatment of specific and non-specific inflammation, viral infection and asthma.
- Anti-ICAM-1 antibodies and fragments thereof are described as useful in treatment of endotoxic shock in WO92/04034, published March 19, 1992.
- Inhibition of ICAM-1 dependent inflammatory responses with anti-ICAM-1 anti-idiotypic antibodies and antibody fragments is addressed in WO92/06119, published April 16, 1992.
- Such seminal information would inter alia, provide for the large .scale production of the proteins, allow for the identification of cells naturally producing them, and permit the preparation of antibody substances or other novel binding proteins specifically reactive therewith and/or inhibitory of ligand/receptor binding reactions in which they are involved.
- the present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts thereof) encoding a novel human polypeptide, "ICAM-R," as well as polypeptide variants (including fragments and analogs) thereof which display one or more ligand/receptor binding biological activities and/or immunological properties specific to ICAM-R.
- ICAM-R-specific ligand/receptor binding biological activities encompass interactions of both the ICAM-R extracellular and cytoplasmic domains with other molecules (e.g., in processes of cell-cell adhesion and/or signal transduction).
- Preferred DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences.
- Bio replicas i.e., copies of isolated DNA sequences made in vivo or in vitro
- DNA sequences of the invention are contemplated.
- autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating ICAM-R sequences and especially vectors wherein DNA encoding ICAM-R or an ICAM-R variant is operatively linked to an endogenous or exogenous expression control DNA .sequence.
- host cells especially unicellular host cells such as procaryotic and eucaryotic cells, are stably transformed with DNA sequences of the invention in a manner allowing the desired polypeptides to be expressed therein.
- Host cells expressing such ICAM-R and ICAM-R variant products can serve a variety of useful purposes. To the extent that the expressed products are "displayed" on host cell surfaces, the cells may constitute a valuable immunogen for the development of antibody substances specifically immunoreactive with ICAM-R and ICAM-R variants.
- Host cells of the invention are conspicuously useful in methods for the large scale production of ICAM-R and ICAM-R variants wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown.
- Novel ICAM-R and ICAM-R variant products of the invention may be obtained as isolates from natural cell sources, but are preferably produced by recombinant procedures involving host cells of the invention. The products may be obtained in fully or partially glycosylated, partially or wholly de-glycosylated, or non-glycosylated forms, depending on the host cell .selected for recombinant production and/or post-isolation processing.
- Products of the invention include monomeric and multimeric polypeptides having the .sequence of amino acid residues numbered -29 through 518 as set out in SEQ ID NO: 1 herein.
- this .sequence includes a putative signal or leader sequence which precedes the "mature" protein sequence and spans residues -29 through -1, followed by the putative mature protein including, in order, five putative immunoglobulin-like domains (respectively spanning residues — 1 to 90, ⁇ 91 to 187, — 188 to 285, — 286 to 387, and —388 to 456), a hydrophobic "transmembrane” region extending from about residue 457 to about residue 481 and a "cytoplasmic" region constituting the balance of the polypeptide at its carboxy terminus.
- ICAM-R variants of the invention may comprise water soluble or insoluble monomeric, multimeric or cyclic ICAM-R fragments which include all or part of one or more of the domain regions specified above and having a biological or immunological property of ICAM-R including, e.g. , the ability to bind to a binding partner of ICAM-R and/or inhibit binding of ICAM-R to a natural binding partner.
- ICAM-R variants of the invention may also comprise polypeptide analogs wherein one or more of the specified amino acids is deleted or replaced: (1) without loss, and preferably with enhancement, of one or more biological activities or immunological characteristics specific for ICAM-R; or (2) with specific disablement of a particular ligand/receptor binding function.
- Analog polypeptides including additional amino acid (e.g. , lysine or cysteine) residues that facilitate multimer formation are contemplated.
- antibody substances e.g. , monoclonal and polyclonal antibodies, antibody fragments, single chain antibodies, chimeric antibodies, CDR-grafted antibodies and the like
- other binding proteins e.g. , polypeptides and peptides which are specific (i.e. , non-reactive with the ICAM-1 and ICAM-2 intercellular adhesion molecules to which ICAM-R is structurally related
- Antibody substances can be developed using isolated natural or recombinant ICAM-R or ICAM-R variants or cells expressing such products on their surfaces.
- 26I10E-2, 26H11C-2 which were deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, on June 2, 1992 as Accession Nos. HB 11053, HB 11054, HB 11055 and HB 11056, respectively, in support of U.S.S.N. 07/894,061; the hybridoma cell line designated 43H7C which was deposited with the ATCC on December 16, 1992 as Accession No. HB 11221 and the hybridoma cell lines designated 42C5H and 42D9B which were deposited with the ATCC on January 15, 1993 as Accession Nos. HB 11235 and HB 11236, respectively, in support of U.S.S.N.
- binding proteins of the invention are illustrated by these antibodies and are summarized in Table 11 of Example 21 herein. Such properties include the ability to modulate CD18-dependent and CD18-independent binding of ICAM-R to cells and cell surface molecules as well as the ability to modulate lymphocyte activation by SEA and/or alloantigen.
- Binding proteins of the invention are additionally susceptible to characterization in terms of binding site structure (e.g. , epitopes and/or sensitivity of binding properties to modifications in ICAM-R amino acid sequence).
- Binding proteins are useful, in turn, in compositions for immunization as well as for purifying polypeptides of the invention and identifying cells displaying the polypeptides on their surfaces. They are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) ligand/receptor binding biological activities involving ICAM-R, especi.ally tho.se ICAM-R effector functions involved in specific and non-specific immune system responses. Anti-idiotypic antibodies specific for anti-ICAM-R antibody substances and uses of such anti-idiotypic antibody substances in modulating immune responses are also contemplated. Assays for the detection and quantification of ICAM-R on cell surfaces and in fluids such as serum may involve, for example, a single antibody substance or multiple antibody substances in a "sandwich" assay format.
- DNA and amino acid sequences of the present invention are manifest.
- knowledge of the .sequence of a cDNA for ICAM-R makes possible the isolation by DNA/DNA hybridization of genomic DNA sequences encoding ICAM-R and specifying ICAM-R expression control regulatory sequences such as promoters, operators and the like.
- DNA DNA hybridization procedures carried out with DNA sequences of the invention and under stringent conditions are likewise expected to allow the isolation of DNAs encoding -allelic variants of ICAM-R, other structurally related proteins sharing one or more of the biological and/or immunological properties specific to ICAM- R, and non-human species proteins (e.g., rodent) homologous to ICAM-R.
- DNAs of the invention are useful in DNA/RNA hybridization assays to detect the capacity of cells to synthesize ICAM-R. Also made available by the invention are anti-sense polynucleotides relevant to regulating expression of ICAM-R by those cells which ordinarily express the same. As another series of examples, knowledge of the DNA and amino acid .sequences of ICAM-R makes possible the generation by recombinant means of ICAM-R variants such as hybrid fusion proteins (sometimes referred to as "immunoadhesions”) characterize by the presence of ICAM-R protein sequences and immunoglobulin heavy chain constant regions and/or hinge regions.
- hybrid fusion proteins sometimes referred to as "immunoadhesions”
- ICAM-R variant fusion proteins may also include, for example, .selected extracellular domains of ICAM-R and portions of other cell adhesion molecules.
- the DNA and amino acid .sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of ICAM-R and definition of those molecules with which it will interact on extracellular and intracellular levels.
- the idiotypes of anti-ICAM-R monoclonal antibodies of the invention are representative of such molecules and may mimic natural binding proteins (peptides -and polypeptides) through which ICAM-R intercellular and intracellular activities are modulated or by which ICAM-R modulates intercellular and intracellular events. Alternately, they may represent new classes of modulators of ICAM-R activities.
- Anti-idiotypic antibodies in turn, may represent new classes of biologically active ICAM-R equivalents.
- In vitro assays for identifying antibodies or other compounds that modulate the activity of ICAM-R may involve, for example, immobilizing ICAM- R or a natural ligand to which ICAM-R binds, detectably labelling the nonimmobilized binding partner, incubating the binding partners together and determining the effect of a test compound on the amount of label bound wherein a reduction in the label bound in the presence of the test compound compared to the amount of label bound in the absence of the test compound indicates that the test agent is an inhibitor of ICAM-R binding.
- the DNA sequence information provided by the present invention also makes possible the development, by homologous recombination or "knockout” strategies [see, e.g., Kapecchi, Science, 244: 1288-1292 (1989)], of rodents that fail to express a functional ICAM-R protein or that express a variant ICAM-R protein. Such rodents are u-seful as models for studying the activities of ICAM-R and ICAM-R modulators in vivo.
- FIGURE 1(A through G) depicts an isolated cDNA clone insert (SEQ ID NO: 2) derived from HL60 cells encoding ICAM-R and the deduced amino acid sequence (SEQ ID NO: 1) of an open reading frame therein;
- FIGURE 2(A through B) comprises bar graphs illustrating the results of Northern blot hybridization of transfected L cells using ICAM-R and ICAM-1 DNA probes;
- FIGURE 3(A through F) presents photomicrographs depicting the results of in situ hybridizations of transfected L cells using ICAM-R or ICAM-1 RNA probes;
- FIGURE 4A comprises bar graphs illustrating the results of assays for the adhesion of PMA-stimulated or unstimulated lymphoblastoid cells from patients with leukocyte adhesion deficiency to soluble ICAM-R in the presence and absence of anti-CD18 antibody
- FIGURE 4B comprir ⁇ s bar graphs illustrating the results of assays for the adhesion of various other PMA-stimulated or unstimulated cell lines to soluble ICAM-R in the presence and absence of anti- CD18 or anti-CDlla antibody;
- FIGURE 5 illustrates in histogram format the results of FACS analyses of indirect immunofluorescence staining of transfected L cells using monoclonal antibodies specific for ICAM-R, ICAM-1 or ICAM-2;
- FIGURE 6 is a diagram of three chimeric ICAM-R proteins utilized to map epitopes of anti-ICAM-R monoclonal antibodies of the invention.
- FIGURE 7(A through B) presents bar graphs depicting the results of actin-normalized Northern blot hybridization of human leukocyte cell lines .and umbilical cord endothelial cells using ICAM-R or ICAM-1 DNA probes;
- FIGURE 8(A through B) comprises photographs of Western blots of immunoprecipitations of lysates from human cells lines using ICAM-R specific monoclonal antibodies;
- FIGURE 9(A through G) presents photomicrographs of immunohistologic staining of various human tissues with an anti-ICAM-R monoclonal antibody
- FIGURE 10 is a bar graph depicting the effects of anti-ICAM-R monoclonal antibodies on the stimulation of lymphocyte proliferation by anti-CD3 antibodies
- FIGURE 11 (A through B) comprises bar graphs illustrating the effects of anti-ICAM-R monoclonal antibodies on superantigen-induced proliferation of human peripheral blood lymphocytes
- FIGURE 11C is a graph comprising logistic dose response curves of the effects of anti-IGAM-R monoclonal antibodies on superantigen-induced proliferation of human peripheral blood lymphocytes;
- FIGURE 12 is a bar graph depicting the effects of anti-ICAM-R monoclonal antibodies on alloantigen-induced T-cell proliferation.
- FIGURE 13 is a bar graph illustrating the effect of anti-ICAM-R monoclonal antibodies on superantigen-induced proliferation of "memory” T cells;
- FIGURE 14 compiles a bar graph depicting the effect of anti- ICAM-R monoclonal antibodies on superantigen-induced proliferation of "resting" T cells;
- FIGURE 15 complies a bar graph illustrating that crosslinking distinct ICAM-R epitopes differentially affects ICAM-R association with the cytoskeleton.
- Example 1 addresses the design and construction of oligonucleotide probes for PCR amplification of ICAM related DNAs.
- Example 2 addresses the use of the probes to amplify a genomic DNA fragment homologous to, but distinct from, DNAs encoding ICAM-1 and ICAM-2.
- Example 3 treats the screening of cDNA libraries with the genomic fragment to isolate additional ICAM-R coding •sequences.
- Example 4 refers to the further screening of cDNA libraries to isolate a full length human cDNA encoding ICAM-R.
- Example 5 provides a characterization of DNA and amino acid .sequence information for IGAM-R and relates the structures thereof to IGAM-1 -and ICAM-2.
- Example 6 describes the isolation of DNA sequences encoding rodent homologues of ICAM-R.
- Example 7 relates to the development of mammalian host cells expressing ICAM-R.
- Example 8 describes preliminary experiments indicative of ICAM-R participation in intercellular adhesion events involving CD18-dependent and CD18-idependent pathways.
- Example 9 presents experiments illustrating inhibition of cell adhesion to ICAM-R by ICAM-R derived peptides.
- Example 10 describes the construction and expression of a panel of rodent ICAM-R/glutathione S-transferase fusion proteins.
- Example 11 relates to the construction and expression of a soluble variant of ICAM-R.
- Example 12 describes the construction .and expression of ICAM-R variants having point mutations in their extracellular domains.
- Example 13 describes the preparation and preliminary characterization of anti-ICAM-R antibodies and the preparation of Fab' fragments thereof.
- Example 14 relates to mapping of the ICAM-R epitopes recognized by the anti-ICAM-R monoclonal antibodies of the invention.
- Examples 15, 16 and 17 relate to assessment of the distribution and biochemical characterization of ICAM-R polypeptide and RNA encoding the same in normal cells and tissues as well as in various cell lines.
- Example 19 describes assays for the involvement of ICAM-R in homotypic cell- cell adhesion.
- Example 20 addresses experiments indicating that ICAM-R is involved in immune cell activation/proliferation.
- Example 21 comprises a summary of characteristics of ICAM-R specific monoclonal antibodies of the invention.
- Example 22 describes experiments showing differential phosphorylation of and cytoskeletal associations with the cytoplasmic domain of ICAM-R.
- Nucleic acid and amino acid alignments of individual sets of CAMs did not manifest sufficient conservation between molecules to yield information useful in the design of consensus-type probes for isolating related novel genes.
- DNAs encoding cellular adhesion molecules therefore involved the development of degenerate con.sensus oligonucleotides representing putative spaced apart DNA sequences of various known molecules and the use of these oligonucleotides as primers for polymera chain reaction (PCR) amplification of DNA replicas of intermediate gene sequences which re.semble, but are not identical to, the known
- oligonucleotide primer design w-as the notation that the amino acids in regions surrounding cysteines which form immunoglobulin-like loops of certain CAMs are somewhat conserved.
- N-X-G-X-Y-X-C-X-(V or A) is typical.
- each individual CAM sequence was split into a domain of sub files defined by the cysteine motif termini described above.
- Subfiles were generated for each of the seven domains of human vascular adhesion molecule .(VCAM-1), the six domains of human platelet endothelial cell adhesion molecule (PECAM-1), the five domains of ICAM-1, the two domains of ICAM-2, three of the four domains of both human myeloglobin-related glycoprotein and human fibroblast growth factor receptor, and the five domains of mouse neural cell adhesion molecule (NCAM). All the subfiles were pooled and segregated independently from the CAM of origin using a multialignment homology computer algorithm designated "Multattn"
- Each of the primers included a PstI restriction endonuclease recognition site (CTGCAG) to facilitate cloning of amplified products.
- CGCAG PstI restriction endonuclease recognition site
- a total of 768 probes were designed as bottom strand primers as set out below in IUPAC nomenclature for amplification from a putative carboxy terminus of the motif.
- Each of the.se primers included an Xbal recognition site (TCTAGA) to facilitate cloning of amplified products.
- Oligonucleotides were synthesized with an automated Applied Biosystems, Inc. (Foster City, CA) Model 394 DNA synthesizer using an 0.2 micromolar .scale synthesis program and employing beta-cyanoethyl chemistry. Protective groups were then removed by heating at 55°C for in excess of six hours. Oligonucleotides were then lyophilized to dryness, rehydrated in TE (lOmM Tris, pH 7.0, 1mm EDTA) and desalted in TE by size exclusion chromatography with G25-150 Sephadex.
- TE lOmM Tris, pH 7.0, 1mm EDTA
- the two sets of probes whose design .and synthesis are described in Example 1 were employed in PCR amplification procedures applied to a human genomic DNA template. Briefly put, PCR-generated fragments of a size similar to that of the immunoglobulin-like loop regions of ICAM-1 and ICAM-2 were isolated, subcloned into Bluescript plasmid (Stratagene, La Jolla, CA) and screened both directly by sequencing and hybridization in arrays for homology to ICAM-2 DNA. Approximately 50% of the fragments were identical to ICAM-1 or ICAM-2 (except, of cour.se, in the regions of the degenerate primer).
- subclone 13-3C7 One subclone, designated 13-3C7, was found to have an open reading frame homologous to ICAM-1 and ICAM-2 in the region of their respective second domains. It did not correspond to .any known sequence present in the Genbank data base. The specific manipulations leading up to the isolation of subclone 13-
- the degenerate oligonucleotides were mixed to a final concentration of 10 ⁇ g/ml in a PCR reaction to amplify human genomic DNA obtained either from peripheral blood leukocytes or Hela cells.
- the DNA amplification was performed in PCR buffer (2mM MgCl 2 , 25mM KC1, lOmM Tris pH 8.3) with
- .sequences (clones 1.1, 1.3, 1.4, 1.6) were obtained which were 184-185 base pairs (bp) long and were 92-95% homologous to the second domain of ICAM-2.
- a 182 bp long DNA sequence (clone 1.5) was obtained which contained a frameshift in the open reading frame of an ICAM-1-like domain along with a 66 bp DNA (clone 1.2) corresponding to a truncated immunoglobulin-like domain.
- probe RM16 SEQ ID NO: 12
- ProbeRM12 (SEQID NO: 14) CCGACATGCTGGTAAGTGTGTCCAA
- Oligonucleotides RM16, RM15, RM12 were labelled by phosphorylation using [ ⁇ - 32 P]ATP.
- the nylon membranes were pre- hybridized in 20% formamide, 5X SSC, 5X Denhardt's solution and 0.5% SDS for 3 hours at 42°C then hybridize overnight with the different radiolabelled oligonucleotide probes under the same conditions.
- the membranes were then washed in 0.2X SSC, 0.5% SDS three times for 15 minutes each at room temperature then washed in the same buffer at 3TC for 15 minutes, rin.-'ed in 2X SSC and exposed.
- Each template that did not hybridize with either of the three oligonucleotide probes was further sequenced using the Sanger technique by DNA automatic sequencing and by sequenase kit. Using this technique, the 170 bp DNA sequence of a clone designated 13-3C7 was determined.
- Example 3 The cDNA insert of subclone 13-3C7 isolated in Example 2 was used as a hybridization probe to screen four different lambda phage cDNA libraries prepared from human spleen, human placenta (two libraries) and human leukocyte cell line U937 (ATCC CRL 1593). Briefly summarized, one hundred and twenty positive clones were picked (from among the approximately 1.6 million clones screened), subcloned, rescreened with the 13-3C7 probe, and the rescreening positive were size selected for in.serts of greater than approximately
- clone 19C A 1.3 kb clone derived from U937 cDNA, designated clone 19C, was sequenced and revealed DNA regions encoding two immunoglobulin-like domains separated by what appeared to be an intervening sequence (intron) resulting from improper or incomplete mRNA splicing prior to cDNA formation. The two regions displayed significant homology, but overall distinctness, in comparison to domains 2 and 3 of ICAM-1 and less homology to domains 1 and 2 of ICAM-2. The specific procedures leading up to isolation of clone 19C were as follows.
- the four libraries were constructed in lambda gtlO phage ( ⁇ gtlO) using cDNA obtained from the U937 cell line, from the spleen of a patient with chronic myelomonocytic leukemia and from human placenta.
- Exact match oligonucleotides designated 1 Hr-5' and lHr-3' were designed corresponding to the 5' and 3' sides of the domain-like region of subclone 13-3C7 (including bases attributable to incorporation of the original degenerate primer).
- the sequences of the 1 Hr-5' and 1 Hr-3' oligonucleotide primers are set out below.
- Primer 1 Hr-5' SEQ ID NO: 15
- Primer 1 Hr-3' (SEQ ID NO: 16) ATGGTCGTCTCTGCTGG Using these oligonucleotides in a PCR reaction with the 13-3C7 insert template and 32 P-dCTP, a 148 bp long DNA probe was generated.
- the cDNA libraries were plated and transferred to nylon membranes. The membranes were pre- hybridized in 40% formamide, 5X SSC, 5X Denhardt's, 0.5% SDS at 42°C for at least 15 minutes, then hybridized overnight with the probe in the .same buffer at 42°C. The membranes were washed several times at room temperature in 2X SSC and exposed. Most of the phage plaques that hybridized with the probe were derived from the U937 cDNA library.
- The.se phages were further purified and tested by PCR (using 1 Hr-5' and 1 Hr-3' as primers) for the presence of the domain inside the cDNA clones.
- the phage were also tested by PCR to determine the length of the clones and the location of the domain within the cDNA fragment (using a combination of 13-3C7 specific primers and primers homologous to flanking ⁇ gtlO vector .sequences). Two clones were selected.
- Clone IF was 0.7 kb long and clone 19C was 1.3 kb long. These cDNAs were digested with EcoRI .and subcloned in the Bluescript vector. In addition, the largest cDNA (clone 19C) was sonicated to obtain small pieces which were sub ⁇ cloned into Bluescript for sequencing.
- clone 19C cDNA contains 2 regions having homology to domains 2 and 3 of ICAM-1, respectively, with an intervening sequence of unrelated DNA. Hereinafter, these DNA regions are referred to as domains 2 and 3 of ICAM-R.
- Example 4 The 1.3 kb (clone 19C) DNA isolated in Example 3 and having regions encoding immunoglobulin-like loops resembling domains 2 and 3 of ICAM-1 was then employed to generate a probe for the screening of additional cDNA libraries in an attempt to isolate a full length cDNA clone. Briefly, the domain 2 and 3 regions within clone 19C were each amplified by PCR using unique probes designated to match respective amino (5 ') and carboxy (3 ') terminal portions of the domains.
- amplified DNAs provided probes for screening of cDNA libraries derived from: (1) the HL60 myelomonocytic cell line; (2) lipopolyaccharide-activated human monocytes; (3) HUT-78 T-cells (ATCC T1B161); and (4) activated peripheral blood leukocytes.
- the latter two libraries yielded no positive upon rescreening.
- Positives derived from HL60 and monocyte cDNA libraries were then .screened with a probe reprer ⁇ nting domain 2 of ICAM-1 DNA (GenBank, Accession No. 22634) in order to eliminate ICAM-1 clones.
- the DNA probe The approximately 1.7 kb insert from clone pVZ-147 was isolated and sequenced to provide the 1781 bp .sequence set out in SEQ ID NO: 2.
- the deduced .amino acid .sequence of the polypeptide encoded by this DNA is set out in SEQ ID NO: 1.
- the polypeptide was designated "ICAM-R" on the basis of its structural relatedness to ICAM-1 and ICAM-2.
- the DNA and deduced amino acid sequences of ICAM-R were published after the priority dates of this application in Vazeux et ⁇ l, Nature, 360. 485-488 (1992).
- Oligonucleotides for use in library .screening and rescreening had the following sequences.
- ProbeIcam 1-3 (SEQID NO: 22) GAAATTGGCTCCATGGTGA
- Probes IHr 2-5' and IHr 2-3' were employed in a PCR amplification u.sing 32 P- dCTP on the clone 19C template to generate a domain 2 specific probe for cDNA screening.
- probes IHr 3-5' and IHr 3-3' were employed to generate a domain 3 .specific probe.
- probes Icam 1-5 and Icam 1-3 were employed to amplify an ICAM-1 segment probe corresponding to b-ases 440 through 609 of the ICAM-1 cDNA .sequence (GenBank, Accession No. 22634), i.e., the ICAM-1 .second domain.
- the cDNA libraries were plated, transferred on nylon membranes, hybridized with the domain 2 probe (derived from clone 19C) in 40% formamide, 5X SSC, 5X Denhardt, 0.5% SDS and washed as described above. All the plaques that hybridized with the domain 2 probe were derived from the monocyte and HL60 libraries. These phage plaques were purified by dilution, plating, transfer and hybridization with the domain 2 probe.
- each plaque that had hybridized with the domain 2 probe was grown on an array in triplicate, transferred to a nylon membrane and hybridized under higher stringency conditions (50% formamide, 5X SSC, 5X Denhardt, 0.5 % SDS) with three different probes: the domain 2 probe; the domain 3 probe, and the ICAM-1 second domain probe.
- Five clones were found in the HL60 library and 2 clones in the monocyte library which hybridized with both domain
- a sixth clone from the HL60 library hybridized only with domain 2 probe and did not hybridize with either domain 3 or with ICAM-1 second domain.
- the cDNAs of the 6 clones from the HL60 library were further analyzed.
- the phages were tested by PCR for the pre.sence of properly .spliced cDNA using oligonucleotide primers corresponding to the 5' extremity (THr2-5') of domain 2 and to the 3' extremity (THr3-3') of domain 3.
- the clones were alr ⁇ tested by PCR for length and location of the domains inside the clones.
- Plasmid pVZ147 was determined to include the entire ICAM-R coding sequence in a single open reading frame.
- FIGURE 1(A through G) graphically illustrates the sequence of the human cDNA insert of the lambda phage clone pVZ 147 isolated in Example 4, above. The total of 1781 bp shown are as set out in SEQ ID NO: 2.
- the deduced -amino acid sequence of the ICAM-R polypeptide as set out in SEQ ID NO: 1 is graphically subdivided in FIGURE 1(A through G) into the following regions:
- a putative signal or leader sequence is illustrated preceding the sequence of the "mature" protein and spanning amino acids designated -29 through -1. Determination of whether the translation product is actually initiated at -29 or -26 will be provided by amino acid .sequencing of intercellular expression products. The designation of the first residue of the mature protein was based on generalized analogy to amino acids (and corresponding b-ases) for residues of secreted human proteins in the region of the junction of the mature protein and leader sequences. Confirmation of the actual initial residue of the mature protein awaits sequencing of a .secreted recombinant product or, e.g., an immunopurified natural product.
- Reference points in the FIGURE 1 (A through G) DNA having "historical" significance to the isolation of the ICAM-R gene include the following:
- bases 420 through 567 correspond to the subclone 13-3C7 ir ⁇ lated in Example 2;
- bases 373 through 663 correspond to the immunoglobulin-like domain 2 localized in clone 19C of Example 3 (with bases 418 through 435 and 561 through 578, respectively corresponding to probes IHr2-5' and IHr2-3' employed for PCR amplification of domain 2 to provide one of the oligonucleotide probes for use in Example 4);
- bases 664 through 957 correspond to the immunoglobulin-like domain 3 localized on clone 19C of Example 3 (with bases 699 through 717 and
- Example 6 DNA sequences encoding rodent homologues of human ICAM-R were isolated by low stringency hybridization using ICAM-R specific probes. Such DNAs can be employed in homologous recombination or "knockout" strategies to develop strains of rodents which lack ICAM-R expression. Additionally, the rodent ICAM-R DNA clones can be uised to produce recombinant rodent ICAM-R protein useful in the development of agents (e.g. , monoclonal antibodies) that can be tested in rodent models for modulation of the activities of ICAM-R in vivo.
- agents e.g. , monoclonal antibodies
- 32 P-radiolabelled probes were added at a concentration of 10 s - 10 6 cpm/ml of hybridization .solution. Following hybridization, filters were washed extensively at room temperature in 2X SSPE/0.1 % SDS and then exposed to X-ray film.
- Lambda DNA was prepared from lysates of each clone and digested with either HaelH or R ⁇ al. Fragments of the genomic DNA were cloned into a sequencing vector for analysis. Approximately 2.5 x 10° plaques from rodent cDNA libraries and
- the rat ICAM-R domain 2 DNA (SEQ ID NO:23) was used as a radiolabelled probe to screen rat macrophage, PBL and spleen ⁇ gtlO cDNA libraries (Clonetech, La Jolla, Ca). The library screening conditions were as described in Section A above. A single clone was isolated from the spleen cDNA library. Sequence analysis of the clone revealed an insert of 1294 bp (including EcoRI ends) with an open reading frame that spanned the entire insert.
- the DNA sequence of the partial rat ICAM-R cDNA is presented in SEQ ID NO: 25.
- ICAM-R sequences indicating that the ICAM-1 and ICAM-R genes are on the same chromosome.
- I llatyne et al Genomics, 3: 547 (1991) reports that the rat ICAM-1 gene is on rat chromosome 9.
- the DNA sequence of the clone determined to date is presented in IUPAC nomenclature in SEQ ID NO: 26 and includes exons corresponding to the partial rat ICAM-R cDNA clone as well as an additional 1340 bp upstream and 960 bp downstream of the coding sequences.
- a mouse genomic clone was isolated by the procedure described in Section A using the rat ICAM-R cDNA as a probe.
- ICAM-1 DNA construct pCDNA-neo-ICAM-1
- a cDNA fragment containing the complete ICAM-1 protein coding region was ligated into plasmid pCDNAl-neo and transfected into L cells by the calcium phosphate precipitation method.
- individual ICAM-R or ICAM-1 transfectants were subcloned using cloning cylinders (Bellco Glass Inc., Vineland, NJ).
- the clones expressing the highest level of ICAM-R and ICAM-1 protein were then .sorted on a cell-sorter. Constructs pCDNA-neo-ICAM-R and pCDNA-neo-ICAM-1 were also transfected into CV-1 cells by the calcium phosphate precipitation method. The clones expressing high levels of IGAM-R and ICAM-1 were selected as described above for L cell tranfectants. Based on FACs analysis with ICAM-R and ICAM-1 specific antibodies the level of protein expression was higher with CV-1 transfectants then with the mouse LTK transfectants.
- RNA samples were spun at 35 K (179,000 x g), 20" C, for 21 hours. All liquid was removed and the pelleted RNA was resuspended in 300 ⁇ l 0.3M .sodium acetate pH 5.2, then precipitated with 750 ⁇ l EtOH at -20 * C. The precipitate was resuspended in H 2 O, then treated with Proteinase K to remove My RNAses. After a phenol/chloroform extraction, the RNA was re-precipitated, resuspended in H 2 0 and the OD of the sample at 260 nm was measured.
- RNAs were electrophoresed in 1 % formaldehyde agarose gels, prepared with diethyl pyrocarbonate (DEPC) treated solutions. Ten ⁇ g of each total RNA sample was loaded per lane. RNA was electrophoresed at 30 V for approximately 18 hours with continuous circulation of buffers accomplished with a peristaltic pump. Each resulting gel was soaked two times in 20X SSPE for 20 minutes each at room temperature. Transfer of RNA to Hybond-C membranes (Amersham Corp., Arlington Heights, IL) was accomplished by capillary action overnight in 20X SSPE. Using a Stratagene stratalinker, RNA was stably crosslinked to each membrane by exposure to ultraviolet light.
- DEPC diethyl pyrocarbonate
- template DNA a 1.8 kb Xba/Kpn fragment incorporating the entire ICAM-1 coding s ⁇ uence
- H 2 0 and random hexamer boiled for 5 minutes, .and then incubated 5 minutes on ice.
- To the template DNA were added: 32 P-dCTP and 32 P-dTTP, lO ⁇ M dGTP/dATP, 10X Klenow Buffer (Boehringer Mannheim Biochemicals,
- the DNA probes were denatured with 5M NaOH, then neutralized with 1M Tris.
- the Hybond-C membranes were prehybridized at 50 "C for 30 minutes in a 50% formamide pre-hybridization mix.
- Probe was added to each membrane to a concentration of 1 x 10 6 cpm/ml hybridization mix (50% formamide, 5X Denhardt's solution, 5X SSPE, 1 % SDS), -and the membranes were incubated overnight at 42 * C. Each membrane was then washed 5 times in 2X SSPE/0.1 % SDS at room temperature for 10 minutes each wash.
- FIGURE 2 A illustrates specific hybridization of the ICAM-R probe with RNA extracted from ICAM-R transfectants, but not with RNA from ICAM-1 transfectants or untransfected L cells.
- FIGURE 2B indicates hybridization of the ICAM-1 probe with RNA extracted from ICAM-1 transfectants, but not with RNA from ICAM-R transfectants or parental L cells.
- L cells and L cells transfected as described above with either ICAM-R or ICAM-1 cDNAs were hybridize in situ with radiolabelled single- stranded RNA probes derived from ICAM-R or ICAM-1.
- Single-stranded RNA probes were generated from DNA templates corresponding to the first (i.e., N- terminal) immunoglobulin-like domain of ICAM-R or ICAM-1 by in vitro RNA transcription incorporating 3S S-UTP. Probes were chemically hydrolyzed to approximately 200 bp.
- Transfected and untransfected L cells were layered onto Vectabond
- Photomicrographs of the in situ hybridizations are set out in FIGURE 3(A through F) wherein photomicrograph 3A is of parental L cells probed with ICAM-R RNA; 3B is of ICAM-R transfected L cells probed with
- ICAM-R RNA 3C is of ICAM-1 transfected L cells probed with ICAM-R RNA; 3D is of parental L cells probed with ICAM-1 RNA; 3E is of ICAM-R transfected L cells probed with ICAM-1 RNA; .and 3F is of ICAM-1 transfected L cells probed with ICAM-1 RNA.
- the photomicrographs demonstrate specific hybridization of each RNA probe only with L cells transfected with a homologous cDNA.
- ICAM-R is a ligand/receptor for an adhesion molecule or molecules on leukocytes.
- SKW3 cells T lymphoblastoid cells
- phorbol ester to activate LFA-1 -dependent adhesion as described in Dustin et ⁇ l. , Nature,
- Untransfected L cells or L cells transfected with either ICAM-R or ICAM-1 were seeded in 24- well tissue culture plates (3 x 10 s cells per well) 24-48 hours prior to the adhesion assay.
- SKW3 cells were washed in serum-free RPMI (Gibco, Canada), labelled with Galcein-AM (Molecular Probes Inc., Eugene, OR), and stimulated with 10 ng/ml phorbol myristylacetate (PMA) for 20 minutes at 37" C.
- Selected stimulated SKW3 cells were then pretreated with anti-CD18 (TS1/18, ATCC HB203), anti-CDlla (TS1/22, ATCC HB202) hybridoma supernatant or control anti-CD2 (ATCC HB195) purified monoclonal antibody for 30 minutes at room temperature before incubation with adherent, transfected L cells.
- Antibody-treated -and non-antibody-treated, calcein- labelled SKW-3 cells were added (5 x 10 5 cells per well) to confluent monolayers of ICAM-R or ICAM-1 transfectants and incubated for 30 minutes at 37 "C in RPMI/1 % fetal calf serum (FCS, Hyclone Laboratories Inc., Logan, UT)
- Unbound cells were aspirated and wells were filled with RPMI-FCS. Plates were sealed, centrifuged in an inverted position at 200 rpm for 4 minutes .and aspirated. The plates were then washed with RPMI-FCS and scanned with an automatic fluorescence reader.
- ICAM-1 transfectants was inhibited by monoclonal antibodies against either the a (CD 1 la) or ⁇ (CD 18) chains of LFA- 1 indicating that ICAM-R may participate in intercellular adhesion events involving a ⁇ l integrin pathway. Intracellular adhesion was unaffected by the control anti-CD2 reagent.
- CD 18 negative lymphoblastoid cells from patients with leukocyte adhesion deficiency (LAD) bind to .soluble ICAM-R described in Example 11.
- Example 9 Human sequence ICAM-R peptides were used to inhibit SKW3 and
- ICAM-R peptides corresponding to potential integrin binding sites were synthesized by Macromolecular Resources (Colorado State University, Fort Collins, CO). Four ICAM-R sequences which lie between or at the border of predicted beta strands in domains 1 and 3 of were chosen. Similar but not identical jS-strand predictions for ICAM-1 -are set out in Staunton et al, Cell, 61: 243-254 (1990). Inhibition was assayed using a system involving cell adhesion to soluble ICAM-R coated plastic.
- Calcein-labeled cells were incubated with peptide at 1-2 mg/ml for 20 minutes at 25 "C and the cells were transfe ⁇ ed to wells of a 96-well plate previously coated with .soluble ICAM- R (see Example 11) and containing 10 ⁇ g/ml final concentration phorbol 12- myristate 13-acetate (PMA). After 50 minutes, the plate was inverted in PBS for 10 minutes to remove unbound cells. Bound cells were quantitated using a fluorescence concentration analyzer.
- NGSQI NGSQI corresponding to residues 72-76 of SEQ ID NO: 1 inhibited SKW3 binding to ICAM-R by 26% .
- the co ⁇ esponding ICAM-1 peptide DGQST, SEQ ID NO: 28
- did not inhibit binding in contrast, the ICAM-R domain 3 peptide (GDQML) corresponding to amino acids 230-234 of SEQ ID NO: 1 demonstrated the best inhibition (36%) of Jurkat binding to ICAM-R.
- the co ⁇ esponding ICAM-1 peptide DGQST, SEQ ID NO: 28
- GDQML ICAM-R domain 3 peptide
- ICAM-1 peptide (GDQRL, SEQ ID NO: 29) inhibited Jurkat binding by 22%.
- the tri-peptide RGD is a recognition sequence common to extracellular matrix components (e.g. , fibronectin and vitronectin) that are ligands of the beta-1 integrins. Cyclizing RGD-containing peptides has resulted in a ten- fold increase in efficiency of blocking integrin binding to vitronectin
- ICAM-R peptide sequences co ⁇ esponding to domain 1 residues 72-77 and domain 3 residues 230-234 are being cyclized using bromoacetic acid preparative to tesing in the assay outlined above.
- a panel of rat ICAM-R glutathione S-transferase (GST) fusion proteins was generated for use as immunogens using the bacterial expression vector pGEX-2T .(Pharmacia Biotech, Inc., .Alameda, CA).
- the plasmid vector contains .an IPTG inducible promoter adjacent to a multi-cloning site located upstream of GST encoding DNA sequences.
- the rat ICAM-R partial cDNA clone described in Example 6 was used to generate polynucleotides encoding ICAM-R fragments, the first composed of domains 2, 3 and the N terminal 36 amino acids of domain 4 (amino acids 1 to 240) of rat ICAM-R and the second including the remaining 104 amino acids of domain 4 and all of domain 5 (amino acid 240 to 430).
- the internal Ec ⁇ RI site at position 718 of the cDNA clone (SEQ ID NO: 25) was used to generate the polynucleotide fragments.
- Rat ICAM-R domain-specific fusion proteins were also constructed in pGEX-2T.
- the following primers that co ⁇ espond to the 5' and 3' ends of domains 1 and 2 were used to generate by PCR DNA fragments that res. pectively encoded IGAM-R domains 1 and 2.
- Domain 1 specific PCR primers were based on the sequence of the rat genomic clone and domain 2 specific primers were based on the sequence of the rat cDNA clone (.see Example 6).
- RRpGEX-Dl 5' SEQ ID NO: 30
- RRpGEX-Dl 3' SEQ ID NO: 31
- TGGAATTCGCTCACGGAAAGTTCGGAT RRpGEX-D25' (SEQIDNO: 32) GCGAATTCGGGTAGAGCTAGTGCCTCTG RRpGEX-D2 3' (SEQ ID NO: 33) TGGAATTCGAAACGTGCGGAGCTGTCT PCR was performed with 50 ⁇ l reaction mixtures consisting of domain 1 or domain 2 primer pairs (10 ⁇ g/ml), a mixture of all four dNTPs (0.2mM each), template DNA (1 ng of rat ICAM-R genomic clone DNA) and Taq polymerase
- Example 11 A soluble variant of ICAM-R was constructed .and expressed as follows.
- the human cDNA for ICAM-R was altered by standard procedures of site-directed mutagenesis [.see, e.g., Kunkel et al, Proc. Natl. Acad. Sci. USA, 82: 488-492 (1985)] in order to truncate the protein coding sequence at the predicted junction (amino acid 457) of its extracellular and transmembrane domains as determined by a computer algorithm that predicts hydropathy [Kyte et al. , J. Mol. BioL, 157: 105-132 (1982)].
- the DNA sequence of ICAM-R was cut from pVZ147 (Example 4) with restriction enzymes £all and Notl.
- the resulting fragment included the complete ICAM-R coding sequences beginning at the 5' end of the coding strand and also included at the 3' end a short segment of the multiple cloning sites.
- This fragment was subcloned into the M13 BM21 vector (Boehringer) linearized with Sail and Not! resulting in a molecule called M13 BM21ICAM-R.
- the oligonucleotide changes the phenylalanine at position 457 of ICAM-R to a stop codon.
- the oligonucleotide was utilize as described in Kunkel et ⁇ l , supra, to generate from M13 BM21ICAM-R six M13 phage isolates encoding a stop codon at position 457.
- An isolate designated BM21 ICAM-Rtl was chosen for further study.
- This single strand template was converted to a double strand DNA molecule by primer extension using Klenow DNA polymerase as follows. Ten ⁇ g of purified single strand M13 BM21ICAM-Rtl DNA was annealed to 50 ng Lac Z universal -20 primer (GTAAAACGACGGCCAGT, SEQ ID NO: 35) in IX
- Klenow DNA polymerase buffer (lOmM Tris-Cl pH 7.5, 5mM MgCl 2 , 7.5mM dithiothreitol) by incubating the mix at 65 "C for 5 minutes and then 25 * C for 5 minutes. The following mixture was then added to the annealing reaction: 33 ⁇ M final concentration dATP, dGTP, dCTP, dTTP; 4 units of Klenow DNA polymerase (Boehringer), and IX Klenow buffer. The primer extension reaction was allowed to incubate at 37 * C for 45 minutes prior to being stopped by a single phenol/chloroform (1:1) extraction and ethanol precipitation.
- a portion of the cDNA insert was released from the M13 BM21ICAM-Rtl phage by restriction digest using restriction enzymes EcoRV .and Ncol.
- the fragment of DNA released contained the complete coding .sequence for the truncated ICAM-R protein, the 3' untranslated region and a small segment of polylinker sequence from the M13 BM21 phage.
- linearized vector Bluebac III (Invitrogen Corp., San Diego, CA), a transfer vector containing genomic baculovirus sequences for homologous recombination that flank the ETL promoter driving expression of the E.coli beta- galactosidase gene and the polyhedron promoter driving expression of the gene of interest, in this case ICAM-Rtl.
- the Bluebac III vector had been prepared in the following way prior to ligation.
- Three ⁇ g of supercoiled plasmid DNA was digested with 20 units HinDIII endonuclease (Boehringer). After a phenol/chloroform extraction and ethanol precipitation the DNA pellet was resuspended in IX Klenow DNA polymerase buffer; 33 ⁇ M final concentration dATP, dGTP, dCTP, dTTP; 2 units of Klenow DNA polymerase (Boehringer) -and incubated at 37 * C for 60 minutes to fill in the termini of the molecule. The fill-in reaction was terminated by phenol/chloroform extraction and precipitation with ethanol. The blunt-ended DNA was resuspended in IX Ncol buffer, 20 units of Ncol endonuclease were added and incubated at 37 * C for 60 minutes.
- ICAM-Rtl DNA were maintained in spinner flasks in TNM-FH [Grace's medium (Gibco, Grand Island, NY) supplemented with 10% heat inactivated fetal bovine serum and gentamicin at 10 ⁇ g/ml] at 27 * C in a forced draft incubator.
- Spinner flask impellers were rotated at 60 rpm on -an insulated five place stir plate.
- Log phase Sf-9 cells (1.5-2.5xl ⁇ Vml) with greater than 90% viability were routinely subcultured twice weekly.
- Sf-9 cells at log growth phase were plated (2 x 10 6 cells/60 mm dish) in TNM-FH medium and allowed to attach for 1 hour at 27 * C. After this time the following mixture was made up in a sterile polystyrene tube and incubated at room temperature for 15 minutes: 1 ml TMN-FH medium, 1 ⁇ g linear Autographa calif ornica nuclear polyhidrosis virus (AcNPV, baculovirus) genomic DNA (Invitrogen), 3 ⁇ g of pBBIII.ICAM-Rtl DNA and 20 ⁇ l of a stock cationic liposome .solution (Invitrogen).
- AcNPV Autographa calif ornica nuclear polyhidrosis virus
- the transfection media containing virus was removed and there viral stocks were used to infect plates of Sf-9 cells for plaque identification.
- Sf-9 cells were seeded at 2x10 s cells/60 mm dish in TNM-FH medium -and -allowed to attach for approximately 1 hour at 27 * C. The media was removed.
- Several 10-fold serial dilutions were made from each viral stock and 1 ⁇ l of each dilution was added to a single dish of adherent Sf-9 cells and incubated for 1 hour at 27 * C.
- each dish of cells was overlayed with 3 ml of a mixture of TNM-FH medium, 0.625 % low melting point agaro.se (BRL, Gaithersburg, MD) and 300 ⁇ g/ml halogenated idolyl-beta-D-galactosidase (Bluo-gal, BRL) that had been previously equilibrated to about 30 * C and allowed to solidify at room temperature for 1 hour. The plates were then incubated until blue color developed (typically 4-5 days).
- plaques of recombinant viruses were transferred to individual wells of a 24-well cell culture plate that had been .seeded with 1 ml of Sf-9 cells (2 x lOVml) in TNM- FH. After 5 days at 27 * C the media was harvested, microfuged at 1,000 rpm for 5 minutes at 4°C and the resulting supernatant was transfe ⁇ ed to a fresh tube. These stocks were designated as BacR.Pl stocks with their respective isolate number.
- Example 13 was biotinylated as follows. A tenth volume of 1M NaCO 3 was added to monoclonal antibody 26I10E at 1 mg/ml. NHS-biotin (Sigma Chemical Co., St. Louis, MO) was dissolved into dimethyl sulfoxide (DMSO, Mallinckrodt, Paris, KY) at 1 mg/ml. One hundred eighty ⁇ l biotin solution was added to each 1 mg antibody and rotated at 4 * C overnight. The biotinylation reaction was terminated by dialysis against PBS for 16 hours with 3 ch-anges at 4 * C.
- DMSO dimethyl sulfoxide
- each well of a ninety-six well plate was coated with monoclonal antibody 26E3D (50 ⁇ l at 10 ⁇ g/ml) for either 2 hours at 37 * C or 16 hours at 4 * C. The coating was then aspirated and the wells were rinsed 2 times with PBS. Wells were blocked with 200 ⁇ l of 1 % BSA in PBS for 30 minutes at 37 * C. Two ten-fold serial dilutions of BacR.Pl stocks were made in PBS. Fifty ⁇ l from the BacR.Pl stocks (neat) or the dilutions were added to the wells and incubated for 30 minutes at 37 * C.
- the media was harvested by centrifugation at 1200 rpm for 5 minutes and 4 ml of the supernatant (designated BAC-R.P2 stock) was transferred to a 1 liter spinner flask containing 500 ml of TNM-FH seeded with 2 x 10 6 cells/ml.
- the infection media was harvested by centrifugation at 1000 rpm for 5 minutes. The supernatant was stored at 4 * C and was designated BAC-R.P3 stock.
- the BAC-R.P3 stock was titered by plating aliquots of ten fold .serial dilutions onto adherent Sf-9 cells and overlaying with 0.625% agaro.se in TNM-FH supplemented with 300 ⁇ g/ml Bluo-gal (BRL). After 4 days incubation at 27 * C, the number of plaques was counted -and a titer determined.
- ICAM-R protein was purified from the insect cell media as follows. Four ml IM Tris-Cl pH 7.5 was added to each 200 ml of insect cell supernatant and was pumped at about 35 ml/hour at 4 * C onto a —3.5 ml column of Lentil Lectin Sepharose (Pharmacia, Upp.sala, Sweden) previously equilibrated with 20 mM Tris-Cl pH 7.5/0. IM NaCl (equilibration buffer). After loading, the column was washed with 25 ml .equilibration buffer.
- the column was then eluted with 11 ml equilibration buffer containing 0.2M methyl ⁇ -D-mannopy ⁇ anoside.
- the eluate contained soluble ICAM-R.
- the partially purified soluble ICAM-R protein was assayed for binding to SKW3 cells that were pretreated with phorbol ester as described in Example 8 to activate LFA-1 -dependent adhesion.
- the ICAM-R protein was coated onto 96-well Immulon 4 (Dynatech) plates after adjusting the lectin eluate to 25mM carbonate pH 9.6 and incubated overnight at 4 * C.
- the plates were washed two times with PBS, blocked for 30 minutes at 37 * C with 200 ul/well PBS, 1 % BSA, and washed again with PBS before adding cells.
- SKW3 cells were washed in serum-free RPMI (Gibco), labelled with Calcein-AM (Molecular Probes), and stimulated with PMA. Cells were then added to the plates and incubated for 1 hour at 37 * C. The plates were inverted in prewarmed PBS, 1 %
- ICAM-R In vitro assays for identifying antibodies or other compounds which modulate the activity of ICAM-R may be developed that utilize soluble ICAM-R.
- an assay may involve immobilizing ICAM-R or a natural ligand to which ICAM-R binds, detectably labelling the nonimmobilized binding partner, incubating the binding partners together and determining the effect of a test compound on the amount of label bound wherein a reduction in the label bound in the presence of the test compound compared to the amount of label bound in the absence of the test compound indicates that the test agent is an inhibitor of ICAM-R binding.
- Functional ⁇ 7 leukointegrins that may be utilized in such assays are described in Dustin et al. , CSH Symp. Qual , 54: 753-765
- the following preliminary experiment shows that purified soluble ICAM-R can be bound to polystyrene beads and retain the ability to bind to purified leukointegrins coated on a plastic surface, thus providing the basis for development of an assay to identify modulators of ICAM-R binding.
- Purified soluble ICAM-R was used to coat 6 ⁇ m fluorescent polystyrene beads (Poly sciences, Inc., Warrington, PA) overnight according to the manufacturer's instructions .and then the beads were blocked with BSA. Replicate wells of a 96- well plate were coated with a diluted aliquot of purified LFA-1 (CD18/CDlla),
- Mac-1 (CD18/CDllb) or Gp 150,95 (CD18/CDllc).
- the plates were incubated in buffer alone or buffer including anti-CD 18 antibody (60.3).
- the ICAM-R-coated beads were aliquoted into the well and incubated for one hour at room temperature followed by inversion in a tank of PBS-D to remove unbound beads from the wells. Fluorescence remaining in the wells was detected using a Cytofluor 2300 (Millipore, Inc., Bedford, MA).
- leukointegrin preparations of LFA-1 or Mac-1 were coated on the fluorescent polystyrene beads and ICAM-R was immobilize.
- Example 12 To rapidly screen for the functional consequences (i.e., counter- receptor binding) of point mutations in ICAM-R extracellular immunoglobulin-like domains, a system was employed from which soluble IGAM-R molecules having point mutations can be expressed and purified. The system relies on the specific binding properties of a poly-histidinyl tract fused to the .amino or carboxyl terminus of a given protein [Hochuli et al , Bio/Technology, 6: 1321-1325
- Plasmids pCS57.1 and pCS65.10 [both are pcDNAlamp (Invitrogen) with the full length human ICAM-R cDNA inserted between EcoRV and Xhol sites, but pCS65.10 includes point mutations that encode Ala 37 and Ser 3g rather than the wild type Glu 37 and Thr 3g , respectively] were used for the initial studies. These DNAs were digested with SacI and EcoRI to release the entire extracellular domain of ICAM-R (amino acids -29 to +454) and the fragments were gel isolated.
- Two complimentary oligonucleotides were synthesized that encoded wild type residues Ser 454 and Ser 455 , and introduced a Gly 456 , Pro 457 and Gly 4Sg to encourage an alpha helical turn followed by a stretch of six His residues and a translational terminator codon.
- the sequences of the oligonucleotides were:
- the oligonucleotides which contain a SacI site and an Xbal site at the ends were ligated to the extracellular domain of ICAM-R and pcDNAlamp cut with EJSQRI and Xbal.
- One set of ligations contained 1/2 unit polynucleotide kinare to phosphorylate the 5' ends of the synthetic DNAs thus increasing the efficiency of ligation.
- a second set of ligation reactions contained pre- phoisphorylated oligonucleotides. Colonies were screened by either miniprep restriction enzyme digestion analysis and PCR with ICAM-R .specific oligonucleotide primers or PCR .alone. DNA sequence was obtained for several clones. The resulting plasmids were designated p57.1wtHis6 and p65.10E37T
- COS cells were seeded in 10 cm dishes and grown to about 50% confluency at which time they were transiently transfected by the method in serum free DMEM using 10 ug of purified plasmid DNA per dish or mock transfected. After a brief DMSO shock, the cells were incubated in DMEM supplemented with fetal bovine serum. After 24 hours, the medium was replaced and the cells allowed to reach confluency over the course of the next four days. The final medium harvest was removed from the cell monolayer and .spun at 1000 rpm to remove cells and stored at 4 * C until ready for column chromatography.
- Ni ++ -nitrilotriacetic acid (Ni ++ -NTA) agarose affinity column chromatography was performed essentially as described in Janknecht et al. , supra, except that the purification was from medium rather than from lysed cells. To the medium was added an equal volume of buffer A (830 mM NaCl, 34% glycerol, 1.6 mM imidazole) and the mixture was clarified by centrifugation at
- Peak fractions from wtHis ⁇ , E37His6 and mock transfectants were concentrated about 6.5 fold using Centricon 30 (Amicon) centrifugation units. The resultant concentrates were adjusted to equal vols. (0.34 ml) using PBS-D.
- Control soluble ICAM-R (15 ug/ml) (Example 11) in carbonate buffer pH 9.6 or in buffer D with 40mM imidazole were made up. Fifty ul of a protein .solution was aliquoted per well of a 96-well plate (Immulon 4, Dynatech) to coat the wells which were then assayed for binding of SKW3 cells as described in Example 11 using untreated, PMA-treated and anti-CD18 monoclonal antibody (60.3) treated cells.
- Example 13 Monoclonal antibodies specific for ICAM-R were generated from the fusion of NS-1 myeloma cells with spleen cells of Balb/c mice immunized with human cell lines that express ICAM-R. Monoclonal antibodies were generated from six different fusions designated fusions 26, 42, 43, 46, 56 and 63. A. Immunization of Mice For fusion 26, five 6 to 12- week old Balb/c mice (Charles River
- Biotechnical Services, Inc., Wilmington, MA, IACUC #901103) were immunized with HL-60 cells to generate anti-ICAM-R monoclonal antibodies.
- Two Balb/c mice were bled retro-orbitally for the collection of pre-immune serum on day 0. On day 2, each animal received a total of 6 x 10 s HL-60 cells in 0.5 ml PBS (0.1 ml s.c. and 0.4 ml i.p.).
- a second immunization with 9.5 x 10° HL-60 cells was administered on day 28 in the same manner.
- Immune serum was collected via retro-orbital bleeding on day 35 and tested by FACS (FACS .screening is described in detail in Section C below) to determine its reactivity to ICAM-R transfectants. Based on these results, both animals were immunized a third time on day 51 with 6.5 x 10° HL-60 cells .and a fusion was performed with spleen cells sterilely removed from one animal (#764) on day 54.
- mice For fusion 42, on day 0 each of five mice was prebled and then immunized i.p. with 5 x 10 6 SKW3 cells in 0.5 ml PBS containing 50 ⁇ g adjuvant peptide (Sigma). The mice were boosted in the same manner on days 21 and 42. Ten days after the third injection, the mice were bled and immune sera was tested by FACS. Mouse #843 was given a final boost of SKW3 cells on day 64. The spleen was sterilely removed three days later. For fusion 43, on day 0 each of five mice was prebled and then immunized i.v. with 5 x 10 6 cells from the erythroleukemic cell line K562.
- mice were given a daily i.p. injection of 1.5 mg cyclophosphamide in 150 ⁇ l for the next two days.
- SKW3 cells plus adjuvant peptide were injected as in Fusion 42.
- mice were given another cycle of K562 cells followed by cyclopho-sphamide.
- mice were boosted witi SKW3 cells with adjuvant peptide.
- Mice were bled on day 56 and immune sera was tested by FACS.
- Mouse #1021 was given a final boost of SKW3 cells and adjuvant peptide on day 78. The spleen was sterilely removed three days later.
- mice For fusion 46, a mouse (#900) w.as immunized as described for fusion 42. On day 128, the mouse was given a final boost of approximately 4 x
- 10° M ⁇ c ⁇ c ⁇ nemestrin ⁇ spleen cells The single cell suspension of monkey spleen was prepared as described below in the following paragraph.
- the monkey cells were pelleted and resuspended in erytiirocyte lysis buffer: 0.15M NHjCl, IM KHCO 3 , O.lmM Na 2 EDTA, pH 7.2-7.4. After lysing the erythrocytes, the splenocytes were washed twice in RPMI and once in PBS. Finally, die cells were resuspended in 400 ⁇ l PBS containing 50 ⁇ g adjuvant peptide and injected. The mou.se spleen was removed sterilely three days later.
- mice (#845 and #844) were immunized as described for fusion 42, except that no boost of SKW3 cells was given on day 64. Instead, these mice were given additional immunizations of SKW3 cells in PBS with adjuvant peptide on days 158 and 204 and were given i.p. injections of Macaca nemestrina spleen cells in 0.5 ml PBS containing 50 ⁇ g adjuvant peptide on days 128 and 177.
- mouse #845 was injected with 2.24 ⁇ g soluble ICAM-R (Example 11) in 700 ⁇ l PBS, 100 ⁇ l was given i.v. with the remainder given i.p.
- mice #844 was immunized on day 226 with Macaca nemestrina spleen cells as described for fusion 56 -and on day 248 with 50 ⁇ g soluble ICAM- R in 100 ⁇ l complete Freuds adjuv.ant given s.c.
- the mou.se received a final boost i.v. of 66 ⁇ g soluble ICAM-R in 100 ⁇ l PBS.
- the spleen was removed sterilely four days later.
- a single-cell suspension was formed from each mouse spleen by grinding die spleen between the frosted ends of two glass microscope slides submerged in serum free RPMI 1640 (Gibco), supplemented witi 2mM L- glutamine, ImM sodium pyruvate, 100 units/ml penicillin, and 100 ⁇ g/ml streptomycin (Gibco).
- the cell suspension was filtered through sterile 70 mesh Nitex cell strainer (Becton Dickinson, Parsippany, NJ), and washed twice by centrifuging at 200 g for 5 minutes and resuspending the pellet in 20 ml serum free RPMI.
- Thymocytes taken from three naive Balb/c mice were prepared in a similar manner.
- NS-1 myeloma cells kept in log phase in RPMI witii 11 % fetal bovine serum (FBS) or Fetalclone (Hyclone) for three days prior to fusion, were centrifuged at 200 g for 5 minutes, and the pellet was washed twice as described in the foregoing paragraph. After washing, each cell suspension was brought to a final volume of 10 ml in serum free RPMI, and 10 ⁇ l was diluted 1:100. Twenty ⁇ l of each dilution was removed, mixed witii 20 ⁇ l 0.4% trypan blue stain in 0.85% sziline (Gibco), loaded onto a hemacytometer (Baxter Healthcare Corp.
- FBS fetal bovine serum
- Hyclone Fetalclone
- Deerfield, IL Deerfield, IL
- a .sample of 2 x 10 8 spleen cells was combined with 4 xlO 7 NS-1 cells, centrifuged and the supernatant was aspirated.
- the cell pellet was dislodged by tapping the tube and 2 ml of 37 * C PEG 1500 (50% in 75mM Hepes, pH 8.0) (Boehringer) was added with stirring over the course of 1 minute, followed by adding 14 ml of .serum free RPMI over 7 minutes. An additional 16 ml RPMI was added and die cells were centrifuged at 200 g for 10 minutes.
- the pellet was resuspended in 200 ml RPMI containing 15% FBS or Fetalclone, 100 ⁇ M .sodium hypoxanthine, 0.4 ⁇ M aminopterin, 16 ⁇ M thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer) and 1.5 x 10 6 thymocytes/ml.
- the suspension was dispensed into ten 96- well flat bottom tissue culture plates at 200 ⁇ l/well.
- the assay plates were then washed 2 times with D-PBS containing NaN 3 only (i.e., no BSA) in the same manner as before and the last wash was replaced with 200 ⁇ l/well 1 % paraformaldehyde in D-PBS. Samples were then transfe ⁇ ed to polystyrene tubes with the aid of a multichannel pipet for flow cytometric analysis .(FACS) with a Becton Dickinson FACscan analyzer.
- Fusions 43 and 46 were screened initially by antibody capture ELISA, testing for the presence of mouse IgG in hybridoma supernatants.
- Immunlon 4 plates .(Dynatech, Cambridge, MA) were coated at 4 * C with 50 ⁇ l/well goat anti-mouse IgA, IgG or IgM (Organon Teknika Corp. , Durham, NC) diluted 1:5000 in 50mM carbonate buffer, pH 9.6. Plates were washed 3 times with PBS with 0.05% Tween 20 (PBST) and 50 ⁇ l culture supernatant was added. After incubation at 37 "C for 30 minutes, and washing as above, 50 ⁇ l of horseradish peroxidase conjugated goat anti-mouse IgG(fc) (Jackson
- Fusions 56 and 63 were screened initially by antigen capture ELISA. Immulon 4 plates (Dynatech) were coated at 4 * C overnight with 100 ng 26E3D Fab' (see Section F below) per well, diluted in 50mM carbonate buffer. The plates were blocked with 100 ⁇ l/well 2% BSA in PBS for 1 hour at ambient temperature. After the plates were aspirated, culture supernatant containing soluble ICAM-R was diluted 1:8 in PBST and added at 50 ⁇ l/well. After 1 hour incubation at ambient temperature, the wells were washed three times with PBST, hybridoma culture supernatant was added at 50 ⁇ l/well, and the plates were again incubated as above.
- L cells or L cells transfected with ICAM-R DNA were used for screening Fusion 26 antibodies and CV-1 cells or CV-1 cells transfected with ICAM-R DNA were u.sed for screening antibodies from Fusions 42, 43, 46, 56 and 63.
- Twenty-nine wells (designated 26E3D-1, 26E3E, 26H3G, 26H11C-2, 26I8F-2, 26I10E-2, 26I10F, 42C5H, 42D9B, 43H7C, 46D7E, 56D3E, 56I4E, 63A10E, 63C3F, 63C11A, 63E9G, 63E12C, 63G3G, 63H6H, 63H9H, 63I1C, 63I6G, 63I12F, 63G4D, 63E1 ID, 63H4C, showed preferential staining of the ICAM-R transfectants versus the control cells.
- the monoclonal antibodies produced by above hybridomas were isotyped in an ELISA assay.
- Immulon 4 plates (Dynatech) were coated at 4°C with 50 ⁇ l/well goat anti-mouse IgA, IgG or IgM (Organon Teknika) diluted
- FACS analysis of indirect immunofluorescence of Macaca fascicularis, porcine or canine peripheral blood leukocytes was performed using the -anti-ICAM-R monoclonal antibodies. Twenty ml of heparinized Macaca fascicularis blood or porcine blood was diluted with 280 ml of erythrocyte lysis buffer, incubated 3-5 minutes at room temperature, and centrifuged at 200 g for 5 minutes. The supernatant was discarded. The pellet was washed once in cold D-PBS containing 2% fetal bovine serum and the cells were counted by hemacytometer.
- heparinized canine blood was diluted in two volumes of Waymouth's medium (Gibco) plus 2% nonessential amino acids (NEAA). Each 5 ml of blood solution was layered over 4 ml of Histopaque (Sigma) and centrifuged at 1000 g for 20 minutes at room temperature. Cells were collected from the interface, washed once in Waymouth's medium plus 2%
- Hybridoma culture supernatants containing the anti-ICAM-R monoclonal antibodies listed in Table 11 in Example 21 were adjusted to 1.5M glycine, 3.0M NaCl, pH 8.9, and put over a 2 ml bed volume protein A column (Sigma). After washing with 1.5M glycine, 3M NaCl, pH 8.9, the column was eluted with lOOmM sodium citrate, pH 4.0. One ml fractions were collected into 100 ⁇ l of 1.5M Tris, pH 8.8. Fractions containing antibody as determined by A 280 were pooled and dialyzed against PBS. G. Affinity
- Fab' fragments were generated from the monoclonal antibodies produced by hybridomas 26E3D, 26I10E, 42D9B, 43H7C and 46D7E by the method described in Johnstone et al , p. 52 in Blackwell, Immunochemistry in Practice, Oxford Press (1982).
- Example 14
- the ICAM-R specific monoclonal antibodies listed in Table 11 in Example 21 were tested for their ability to inhibit binding of JY cells (CD18 + ) to recombinant .soluble human ICAM-R.
- Adhesion assays were performed as described in Example 12. Cells were treated with PMA and antibodies were then added at a final concentration of 10 ⁇ g/ml. Data was collected from triplicate wells during three independent experiments. Total CD18-dependent binding was determined as the amount of adhesion blocked by a control anti-CD 18 monoclonal antibody 60.3. The percentage of total CD18-dependent binding that was inhibited by each monoclonal antibody is shown below in Table 4 wherein the names assigned to monoclonal antibodies produced by each hybridoma are given. The monoclonal antibody names are used throughout the following examples instead of hybridoma designations.
- FACS-based competition assays utilizing human peripheral blood leukocytes or SKW3 cells indicate that monoclonal antibodies ICR-4.2 and ICR-1.1 are immunologically reactive with distinct epitopes of ICAM-R.
- human peripheral blood leukocytes obtained by Ficoll Hypaque centrifugation of normal peripheral blood were washed twice in ice cold FACS buffer (PBS containing 0.1 % .sodium .azide and 1 % bovine serum albumin) and 2 x 10 5 cells were incubated in triplicate polypropylene tubes with 5 ⁇ g of each of the following antibodies ICR-1.1, ICR-4.2, and control isotype IgG (Sigma). All tubes containing the first stage antibodies were then incubated for 30 minutes at 4 * C and washed twice in cold FACS buffer.
- PBS containing 0.1 % .sodium .azide and 1 % bovine serum albumin
- SKW3 cells were labelled with either 1 ⁇ g of antibody ICR-1.1 or ICR-4.2, washed in FACS buffer and incubated with 1 ⁇ g biotinylated-ICR-1.1 or biotinylated ICR-4.2. All tubes were then washed in FACS buffer, incubated with Strepavidin-phycoerythrin for an additional 30 minutes at 4 * C and analyzed by FACScan.
- an unlabelled antibody prevented the labelled .antibody from binding to ICAM-R
- a variation of the competition assay in which unlabelled antibody is used to "compete away" binding of a labelled antibody may also be utilized to determine if two antibodies recognize the same, sequential or sterically overlapping epitopes.
- the specific ICAM-R epitopes recognized by the v-arious monoclonal antibodies of die invention can be mapped by four different methods.
- the first method for mapping linear epitopes recognized by die ICAM-R specific antibodies of d e invention utilized d e Multipin Peptide Synthesis System (Chiron Mimotopes Pty. Ltd., Victoria, Australia) which places ten amino acid peptides representing overlapping segments of the protein of interest on the surface of a series of plastic pins.
- a modified ELISA test is performed to determine binding of a monoclonal antibody to each peptide.
- the ELISA to determine binding of the monoclonal antibodies to ICAM-R peptides was run as follows. The pins were placed in five 96-well plates containing 200 ⁇ l per well blocking buffer (2% weight/volume BSA, 0.1% volume/volume Tween 20, 0.01M PBS, pH 7.2) and incubated for one hour at
- the pins were then washed four times with 0.01M PBS, pH 7.2 (10 minutes/wash at 20 * C with agitation) and placed in plates containing 175 ⁇ l per well HRP-Goat anti-mouse IgG (H+L) (Kirkegaard and Perry Laboratory Inc., Gaithersburg, MD) diluted to an appropriate concentration in conjugate diluent (1 % volume/volume sheep serum, 0.1 % volume/volume Tween 20, 0.1 % weight/volume sodium caseinate and 0.01M PBS). The plates were agitated for one hour at 20" C, and washed four times with 0.0 IM PBS.
- the pins were transfe ⁇ ed to plates containing ABTS substrate solution [0.5 mg/ml ABTS, 0.01 % weight volume H 2 O 2 in substrate buffer (17.9 g/L Na 2 HPO 4 H 2 O, 16.8 g/L citric acid monohydrate, pH 4.0)] for 45 minutes at 20 "C with agitation and then the plates were read at 410/495 nm.
- Relative reactivity with individual pins was determined after normalizing results for differences in immunoglobulin concentrations in anti- ICAM-R and control hybridoma supernatants and reactivities of positive controls between assays.
- Mouse IgG levels for each supernatant had been determined by antibody capture ELISA as follows. Immulon 4 plates were coated -and washed as described in Example 13C. Fifty ⁇ l/well of culture supernatant diluted in
- PBST [or known concentrations in doubling dilutions in PBST of mouse IgG, and IgGz, (MOPC-21, and UPC-10) (Sigma)] was added to the plate. After incubating for 1 hour at room temperature and washing 3 times with PBST, horseradish peroxidase conjugated goat anti-mou.se IgG(fc) (Jackson ImmunoResearch, West Grove, Pennsylvania) was diluted 1:2000 for mouse IgG, and 1:1000 for IgG ⁇ , and added 50 ⁇ l/well. After the plate was incubated for 1 hour at room temperature and washed 4 times in PBST, the remainder of the assay was conducted as described in Example 13C. Antibody concentrations of culture supernatant were determined by fitting measured optical densities to the standard curve of the isotype matched control.
- LSAGGSLFVN Regions reactive with anti-ICAM-R antibodies can also be defined and/or verified using die following methodologies.
- Epitope mapping with the anti-ICAM-R antibodies was also performed using the Novatope Library Construction and Screening System (Novagen, Madison, WI). Using this method, a library of bacterial clones is generated wherein each clone expresses a polypeptide including a small peptide derived from the protein being examined. The library is then screened by standard colony lift methods using monoclonal antibodies as probes.
- Double-stranded DNA encoding the external domain of ICAM-R (amino acids 1 to 487) from pVZ147 (See Example 4) was cut with different amounts of DNAsel in the presence of lOmM manganese for 10 minutes at 21 * C.
- the reaction was stopped with EDTA and 1/10 of the reaction was electrophoresed on a 2% agarose gel with ethidium bromide and appropriate markers. Those reactions containing fragments in the 50-150 bp range were pooled and electrophoresed on anotiier 2% gel.
- the area of the gel between 50- 150 bp was excised, die fragments contained therein were electroeluted into dialysis tubing (SP Brand Spectra/Por 2, MWCO 12-14,000), and then phenol/chloroform extracted and ethanol precipitated.
- the dA tailed fragments are ligated into me pTOPE T-vector (Novagen) which is designed for the expression of inserts as stable fusion proteins driven by T7 RNA polymerase (die structural gene for which is carried on a replicon in the host cell).
- T7 RNA polymerase die structural gene for which is carried on a replicon in the host cell.
- the ligation reaction was run at 16 "C for 5 hours.
- NovaBlue(DE3) (Novagen) cells were transformed with 1 ⁇ l (1/10) of the reaction mix, and spread on LB agar (carbenicillin/tetracycline) plates to obtain an initial count of transformants. The remainder of the ligation reaction was put at 16" C for an additional 16 hours.
- Streaks were made from a stab of the isolated colony or colony -areas for re-sicreening.
- the streak from the isolated colony had positive reactive areas after a 20 minute incubation with substrate.
- the otiier three colony area samples were negative.
- a stab from the ICAM-R reactive area was re- streaked, incubated overnight at 37 "C and re-probed incubating with substrate for 10 minutes. Many ICR- 1.1 reactive colonies resulted.
- P.lasmid DNA recovered from tiiese colonies can be .sequenced and the amino acid .sequence corresponding to the ICR- 1.1 reactive epitope can be determined.
- Conformational epitopes of ICAM-R recognized by the monoclonal antibodies of the invention may be mapped by domain substitution experiments.
- chimeric variants of ICAM-R are generated in which selected immunoglobulin-like domains of ICAM-R are fused to portions of ICAM - 1 and assayed for binding to the monoclonal antibodies of the invention by FACS.
- FIGURE 7 is a diagram of the chimeric proteins whose construction is outlined below.
- Protein number 1 contains the ammo-terminal immunoglobulin-like domain of ICAM-R (residues 1 to 93) fused to ICAM-1
- Protein number 2 contains the first two amino terminal immunoglobulin-like domains of ICAM-R (residues 1 to 190) fused to ICAM-1 (residues 216 to 532).
- Protein number 3 contains the first three immunoglobulin- like domains of ICAM-R (residues 1 to 291) fused to ICAM-1 (residues 317 to 532).
- Protein number 1 was made by engineering a unique Nhe I site into the coding .sequences of ICAM-R and ICAM-1 at the junction of immunoglobulin- like domains 1 and 2 of each.
- the DNA .sequence of ICAM-R was subcloned into die Ml 3 BM21 vector (Boehringer) as described in Example 12 resulting in a molecule called M13 BM21ICAM-R.
- pBSSK(+) (Stratagene).
- the resulting plasmid, pBSSK(* )ICAM-1 was cut with Sail and Kpnl to release die ICAM-1 coding sequence along with a short segment of the multiple cloning sites and ligated to M13 BM21 cut with restriction enzymes SMI and Kpnl resulting in a molecule called M13 BM21ICAM-1.
- M13 phage isolates were verified by DNA sequence analysis.
- Nhel and nucleotide 15 of ICAMR.Dl .Nhel form mismatch base pairs when the oligos are annealed to their respective complementary DNA sequences. Both oligonucleotides introduce a recognition site for endonuclease Nhe I. Site-directed mutagenesis with the oligonucleotides was employed to introduce the sequences of these oligos into die respective
- ICAM-1 and ICAM-R target DNA sequences M13 BM21ICAM-1 and M13 BM21ICAM-R.
- Several phage isolates from each mutagenesis reaction were sequenced to verify that die co ⁇ ect DNA .sequence was present. These isolates were designated M13 BM21ICAM-R.NheI and M13 BM21ICAM-l.NheI.
- the coding region for the ICAM-R signal peptide and immunoglobulin-like domain 1 was isolated from M13 BM21ICAM-R.NheI by the following method. Ten ⁇ g of purified single strand M13 BM21 ICAM-R.
- Nhel phage DNA was annealed to 50 ng Lac Z universal -20 primer (SEQ ID NO: 35 in IX Klenow DNA polymerase buffer (lOmM Tris-Cl pH 7.5, 5mM MgCl 2 , 7.5mM ditiiiothreitol) by incubating the mix at 65 " C for 5 minutes and tiien 25 " C for 5 minutes. The following mixture was then added to the annealing reaction: 33 ⁇ M final concentration dATP, dGTP, dCTP, dTTP; 4 units of Klenow DNA polymerase (Boehringer), and IX Klenow buffer.
- IX Klenow DNA polymerase buffer lOmM Tris-Cl pH 7.5, 5mM MgCl 2 , 7.5mM ditiiiothreitol
- the primer extension reaction was allowed to incubate at 37 * C for 45 minutes prior to being stopped by a single phenol/chloroform (1 : 1) extraction and ethanol precipitation.
- the dried pellet was resuspended in IX EcoRI buffer and 20 units each of EcoRI and Nhel endonucleases were added prior to a 60 minute incubation at 37"C.
- a 412 bp fragment containing the coding sequence for ICAM-R signal peptide and immunoglobulin-like domain 1 was agarose gel purified.
- the DNA sequence of ICAM-1 containing die coding region for immunoglobulin-like domains 2 through 5, the transmembrane and cytoplasmic domains was isolated by restriction enzyme digest.
- a chimeric gene encoding protein number 1 was also generated by an alternative method as follows. An appoximately 375 bp EcoRI-Nhel fragment of ICAM-R containing domain 1 and an approximately 1500 bp Nhel-NotI fragment of ICAM- 1 containing the extracellular domains 2-5, die transmembrane domain and the cytoplasmic tail were gel purified after restriction enzyme digestion of the double stranded RF (replicative form) DNA from the M13BM21ICAM-R and M13 BM21ICAM-1 clones and agarose gel electrophoresis of the co ⁇ esponding double stranded plasmid DNAs.
- the resulting two DNA fragments were cloned by a three way ligation into an EcoRI and Notl digested and calf intestinal phosphatase-treated expression vector pcDNAI/Amp (Invitrogen).
- E. coli XL1 blue (Stratagene) strain was transformed with the ligation mixture and the transformants were selected on carbenicillin containing plate. Clones with the desired inserts were identified by restriction enzyme digestion of d e plasmid DNA minipreps.
- ICAM-R.D3.Af co ⁇ esponding to nucleotides 962 to 993 of ICAM-R) with the sequences set out below were synthesized for this purpose.
- ICAM-R.D3.Afjn (SEQ ID NO: 45) GACCCATTGTGAACTTAAGCGAGCCCACC
- the appropriate coding sequences of ICAM-R and ICAM-1 (.sequences encoding the first two amino terminal immunoglobulin-like domains of ICAM-R fused to sequences encoding ICAM-1 residues 118 to 532 for protein 2 and sequences encoding the first three immunoglobulin-like domains of ICAM-R fused to sequences encoding ICAM-1 residues 317 to 532 for protein 3) were then subcloned into expression plasmid pCDNAlAmp (Invitrogen) to generate isolates pCDNAlAmp.RDl-2.1D3-5 and pCDNAAmp.RDl-3.1D4-5 respectively encoding ICAM-R variant proteins 2 and 3.
- pCDNAlAmp Invitrogen
- Gene fusions encoding protein numbers 2 and 3 were also constructed by alternative methods as follows.
- an Nhel was introduced by oligonucleotide directed in vitro mutagenesis in between domains 2 and 3 in both ICAM-R and ICAM-1.
- ICAM-R domain deletion mutants were generated by similar oligonucleotide directed mutagenesis protocols as described above for chimeric protein numbers 1, 2 and 3.
- a domain 1 deletion mutant which lacks amino acids 2-90 of ICAM-R (SEQ ID NO: 1), a domain 1 and 2 deletion mutant which lacks amino acids 2-203, and a domain 3 deletion mutant lacking amino acids 188-285 were constructed.
- Control plasmids containing the full length ICAM-R or ICAM-1 cDNA sequences were generated by ligating gel-purified cDNA fragments to plasmid pCDNAlAmp.
- the two plasmids pCDNAlAmpICAM-1 and pCDNAlAmpICAM-R express the full length ICAM-1 and ICAM-R proteins, respectively, so that monoclonal antibody binding to native protein in equivalent cellular contexts can be assessed.
- COS cells were transfected with the plasmid DNA encoding the ICAM-R chimeric or deletion mutant proteins or with the plasmid DNA pCDNAlAmpICAM-1, pCDNAlAmpICAM-R or pCDNAlAmp by the DEAE- dextran method.
- the COS cells were seeded at a density of about 7.0 x 10 5 cells on a 10 cm diameter plate and grown overnight in Dulbecco's modified
- DMEM Eagles medium
- FBS fetal bovine serum
- COS cells transfected with constructs encoding d e ICAM-R chimeric proteins or control constructs were removed from die plates by EDTA treatment and aliquoted at 2.5 x 10 5 cells per well in a 96- well round bottom plate. Cells were washed 3 times with ice cold washing buffer (PBS containing 1 % BSA and 0.05 % sodium azide).
- Anti-ICAM-R monoclonal antibody was applied at 5.0 ug/ml in 50 ul final volume and incubated on ice for 30 minutes.
- results of the assay are given below in Table 5 as percent positive COS cell transfectants, wherein MOPC 21 (IgGl) and UPC 10 (IgG2a) are ir ⁇ type matched controls, 18E3D is an ICAM-1 specific monoclonal antibody and ICR-1.1 to ICR-9.2 are ICAM-R specific monoclonal antibodies.
- the reactivities of monoclonal antibodies ICR- 1.1 through ICR-9.2 were assayed in a different experiment than monoclonal antibodies ICR-12.1 through ICR-17.1.
- the results presented above show that the antibodies ICR- 1.1, 2.1, 3.1, 5.1, 7.1, 8.1, 12.1, 13.1, 14.1, 15.1, 16.1 and 17.1 recognize the hybrid molecule in which only the ICAM-1 domain 1 has been replaced witii the ICAM- R domain 1.
- the antibodies ICR-4.2, 6.2 and 9.2 recognize the molecule in which a minimum of 2 domains (domain 1 and 2) of ICAM-1 was replaced with the co ⁇ esponding domains of ICAM-R. Based on these results the antibodies have been categorized as either domain 1 or domain 2 specific.
- the ICAM-R chimeric and deletion mutant protein constructs can also be used to transfect rat L cells by a calcium phosphate co-precipitate protocol using 10 ⁇ g of 2X CsCl-banded plasmid DNA. In this protocol, forty-eight hours post-transfection the cells are released from the dishes by mild trypsinization. The cells are divided and incubated on ice with anti-ICAM-R monoclonal antibodies or a control isotype matched monoclonal antibody at a concentration of 10 ⁇ g/ml or no monoclonal antibody for 1 hour. The cells are then processed for FACS analysis as previously described in Example 13C. D.
- DNA synthesis and ligation reactions were carried out using T7 DNA polymerase and T4 DNA ligase, respectively.
- An aliquote of the synthesis reaction was used to transform E. coli XLl blue (Stratagene) strain and transformants were selected on carbenicillin containing plates. Growth of the uracil containing plasmid DNA in this strain markedly reduces the propagation of the uracil containing DNA (wild type) strand. Mutants were selected by plasmid DNA minipreps .and diagnostic restriction enzyme digestion. Sequences were further verified by DNA sequence analysis.
- Mutation "F21V/AS” indicates, for example, that the phenylalanine at position 21 of ICAM-R (SEQ ID NO: 1) and d e valine at position 22 were respectively changed to an alanine and a serine, while mutation
- T38/A indicates that the threonine at position 38 of ICAM-R (SEQ ID NO: 1) was changed to an alanine.
- Table 6 summarizes die results obtained, wherein a mutation with a "critical” effect was defined as 0-20% binding of an antibody in compari ⁇ n to binding to wild type ICAM-R, an "important” effect was defined as about 50% binding in comparison binding to wild type ICAM-R, and a minor effect was defined as about 75% binding in comparison to binding to wild type ICAM-R. Mutations that did not effect binding of an antibody are not listed in Table 6.
- ICAM-R protein and the expression of ICAM-R RNA in various cells and cell lines were respectively assayed by FACS analysis and Northern blot hybridization.
- ICAM-R is expressed on a wide variety of in vitro propagated cells lines representative of the major leukocyte lineages: T lymphocytes, B lymphocytes, and myeloid cells.
- Surface expression of ICAM-R was not detected on die primitive erythroleukemic line, K562.
- ICAM-R was not expressed detectably by cultured human umbilical vein endothelial cells (HUVECS) either before or .after stimulation with tumor necrosis factor which did upregulate expression of ICAM-1. This pattern of expression is also distinct from that ob.serv.ed for ICAM-2 which is expressed on endothelium.
- Table 7 below provides the mean fluorescence of each cell sample and the percent positive cells relative to a control in each cell sample (e.g., mean fluore.scence of 13 / 11 % positive cells).
- Example 13C FACS analyses performed as described in Example 13C on normal human and macaque peripheral blood leukocytes showed that the anti-ICAM-R monoclonal antibody ICR-2.1 reacted with the three major human leukocyte lineages: lymphoid, monocytoid and granulocytoid. See the final six entries of Table 7.
- monoclonal antibodies ICR-4.2 and ICR-9.2 cross-reacted with macaque leukocytes (Table 2 and Example 13E) indicating that tiiese monoclonal antibodies may be useful in monitoring the expression of ICAM-R in disease models executed in this animal.
- Example 7 HUVECS as described in Example 7, and was analy.zed by Northern blot hybridization (also as described in Example 7) by probing with either ICAM-R or ICAM-1 cDNA. After phosphorimaging of the initial hybridization, blots were stripped and reanalyzed using a human actin probe. The results of the actin normalized Northerns of ICAM-R and ICAM-1 probed blots are presented in
- FIGURE 7(A through B) as bar graphs.
- ICAM-R was expressed in a variety of leukocytic cell types. ICAM-R RNA expression was not necessarily concomitant with the expression of ICAM-1 RNA.
- unstimulated HUVECS express low levels of ICAM-1 and expression is upregulated following TNF stimulation (FIGURE 7B).
- detectable levels of ICAM-R message were not observed in unstimulated or stimulated HUVECS (FIGURE 7A).
- Example 17 Immunoprecipitations of detergent r ⁇ lubilized lysates of surface biotinylated human cell lines KGla, K562 and CEM were performed using the four anti-ICAM-R monoclonal antibodies: ICR-2.1 , ICR-1.1 , ICR-4.2, and ICR-
- the cells were pelleted by centrifugation, the supernatant was aspirated and die pellet was solubilized with 300 ul of lOmM Tris pH 8, 50mM NaCl, 1 % Triton X-100, ImM phenylmethylsulfonyl fluoride, ImM EDTA by incubating on ice for 15 minutes.
- the lysate was clarified by centrifugation and die supernatant was precleared by addition of 25 ul normal mou.se serum .and incubation for 1 hour at 4"C.
- sepharose beads were pelleted in a microcentrifuge and washed sequentially 2 times with 1 ml lOmM Hepes pH 7.3, 150mM NaCl, 1 % Triton X-100; lx with 0.1M Tris pH 8, 0.5M LiCl, 1 % beta mercaptoethanol; and lx with 20mM Tris pH 7.5, 50mM NaCl, 0.5% NP-40. Beads were then eluted with 50 ⁇ l 150mM Tris pH 6.8, bromphenol blue, 20% beta mercaptoethanol, 4% SDS and 20% glycerol; boiled for 5 minutes; and pelleted by centrifugation.
- FIGURE 8(A tiirough B) shows the resulting Western blots.
- a single specifically precipitated species of 120 kD was observed in immunoprecipitates with monoclonal antibody ICR-2.1 from KG1 cells, but not from K562 cells (See FIGURE 8A).
- a 120 kD band was alr ⁇ resolved in immunoprecipitates of the T cell line CEM (FIGURE 8B, wherein Lane A was reacted with monoclonal antibody ICR-2.1; Lane B, monoclonal antibody ICR-4.2; Lane C, monoclonal antibody ICR-3.1; Lane D, monoclonal antibody ICR-1.1; and Lane E, a negative control antibody).
- the size of the ICAM-R species resolved in other immunoprecipitations varied slightly depending on the cellular source. Species ranging from — 116 kD on some lymphoid cells to — 140 kD on some myeloid cells were observed.
- Immunohistologic staining with .anti-ICAM-R monoclonal antibodies ICR-4.2, ICR-1.1, -and ICR-2.1 and control .antibodies was carried out on various human tissues including tonsil, spleen, liver, lung, kidney, heart, digestive tract, skin, synovium, and brain (both normal and multiple sclerosis-afflicted brain tissue). Similar staining patterns were obtained using the different anti-ICAM-R antibodies as well as when using purified anti-ICAM-R monoclonal antibody ICR- 1.1 or hybridoma supernatant.
- Sections (6 ⁇ m) of various tissues were layered onto Vectabond (Vector) coated slides and stored at -70 * C (some sections were stored at -20 * C). Prior to use, slides were removed from -70 "C and placed at 55 * C for 5 minutes. Sections were then fixed in cold acetone for 10 minutes and air dried. Sections were blocked in a solution containing 1 % BSA, 60% normal human sera, and 6% normal horse sera for 30 minutes at room temperature. Primary antibody directed against ICAM-R, a negative control -antibody, anti-ICAM-1 monoclonal antibody or anti-ICAM-2 monoclonal antibody was applied to each section for 1 hour at room temperature.
- D.AB substrate (3 '3 diaminobenzidine-tetrahydrochloride, Sigma) (stock: 600 mg/ml DAB diluted 1: 10 in 0.05M Tris Buffer, pH 7.6, with 3% H 2 0 2 added to a final concentration of 1 %) was applied to each slide for 8 minutes at room temperature. Slides were washed in water for 5-10 minutes at room temperature and then 1 % osmic acid was added (to enhance color development) for one minute at room temperature. Slides were then washed in tap water for 5-10 minutes and counterstained in 1 % Nuclear Fast Red (NFR) for 30 .seconds at room temperature. Lastly, slides were alcohol dehydrated, treated with Histrocle * ⁇ r and mounted with coverslips using histomount.
- NFR Nuclear Fast Red
- FIGURE 9(A through G) A selection of results of staining with the monoclonal antibodies is presented in FIGURE 9(A through G) as photomicrographs wherein the tissue in 9A, 9B and 9E is human tonsil; in 9C and 9D is human liver; in 9F is brain from a human patient afflicted with multiple sclerosis; .and in 9G is normal human brain. Sections shown in 9A, 9C, 9F and 9G were stained with anti-ICAM-R monoclonal antibody ICR-4.2. Sections shown in 9B -and 9D were stained witii the negative control antibody, while the .section shown in 9E was stained with the anti-ICAM-1 antibody.
- IC.AM-R lymphoid tissues such -as tonsil (9A). Expression was also detected on tissue leukocytes in other nonlymphoid organs such as the liver wherein Kupfer cells (liver macrophages) were positively stained (9C).
- Kupfer cells liver macrophages
- ICAM-1 and ICAM-R expression are regulated distinctly in vivo is given by the staining pattern observed in tonsil and lymph node: ICAM-1 is strongly expressed on B cells in the germinal centers of secondary follicles and not expressed in primary follicles, whereas ICAM-R is expressed strongly in the primary follicles and weakly in the germinal centers (10A and 10E).
- ICAM-R expression was also detected on leukocytes infiltrating sites of inflammation.
- ICAM-R expression was observed on perivascular infiltrating leukocytes in the brain tissue of individuals afflicted with multiple sclerosis (9F). Similar staining was not observed in anatomically equivalent locations of brain tissue from normal individuals (9G). ICAM-R expression was also detected on leukocytes infiltrating synovia of arthritic joints. Also, whereas expression of ICAM-1 and ICAM-2 was detected on endothelia lining vessels, ICAM-R was not typically ob.served on vascular endothelium. Expression of ICAM-R was detected on cells in the aveoli of the lung.
- ICAM-R cells expressing ICAM-R were detected in .all normal and pathological tissues. These ICAM-R expressing cells could be identified morphologically and by comparison of serial immunological staining as leucocytes and antigen-presenting cells. All CD3 + T cells present in various tissues expressed high levels of ICAM-R. In contrast, only a subset of B cells (IgD- ) present in primary follicles and in the mantle zone of germinal centers expressed high levels of ICAM-R. Amongst antigen-presenting cells, Langerhans cells in the epithelium expressed high levels of ICAM-R while only a subset of other tissue macrophages expressed ICAM-R.
- ICAM-R monoclonal antibodies ICR- 1.1 and ICR-4.2 were -al-so used in procedures similar to those described above to stain biopsy tissue sections of both human mammary carcinoma (ductal and lobular) and melanomas. In both tumor types some sections exhibited specific patchy staining of the endothelia in a range of blood vessels (venular, arterioles and capillaries). Corresponding normal tissue showed no expression of ICAM-R on endothelium.
- ICAM-R is typically not expressed on endothelium of d e general vasculature, it is apparently expressed on a subset of vessels associated with two types of solid tumors.
- reagents e.g., monoclonal antibodies directed against ICAM-R may provide therapeutic vehicles which selectively target tumor versus normal vasculature.
- aggregation assays were performed with a panel of cell lines which express ICAM-R including T lymphoblastoid cell lines (SupTl, CEM, Molt 4, Hut 78, Jurkat, SKW3), B lymphoblastoid cells lines (Jijoye, Raji), monocytic cell lines (U937, HL60), a myelogenous cell line (KG-1) and die erytiiroleukemia cell line K562.
- T lymphoblastoid cell lines SupTl, CEM, Molt 4, Hut 78, Jurkat, SKW3
- B lymphoblastoid cells lines Jijoye, Raji
- monocytic cell lines U937, HL60
- KG-1 myelogenous cell line
- die erytiiroleukemia cell line K562 die erytiiroleukemia cell line K562.
- Anti-ICAM-R supernatants produced by hybridomas ICR-2.1, ICR-1.1, ICR-4.2, .and ICR-3.1 were u.sed as well as antibody preparations known to block aggregation through a ⁇ 2 integrin pathway: TS1/18 (ATCC HB203) specific for the CD18 molecule, the jS-subunit of LFA-1; TS1/22 (ATCC HB202) specific for the CDlla molecule, the ⁇ -chain of LFA-1; .and LM2/1 (ATCC HB204) .specific for the CDllb molecule, the ⁇ -subunit of MAC-1.
- TS1/18 ATCC HB203
- TS1/22 ATCC HB202
- TS1/22 ATCC HB202
- CDlla molecule the ⁇ -chain of LFA-1
- .and LM2/1 ATCC HB204
- Aggregation assays were done in duplicate, with and without addition of PMA (50 ng/ml). 3 x 10 5 cells in RPMI 1640 medium with 10% fetal calf serum were added in a flat-bottomed 96-well microtest plate. When one antibody was tested in an experiment, 50 ⁇ l of purified -antibody or hybridoma supernatant were added to d e wells (PMA was added at the same time to selected wells). When two antibodies were tested in the same experiment, the antibodies were added sequentially to the cells at room temperature and incubated for 30 minutes each (incubation for 15 minutes at 37 "C produced the same results), and then die cells were incubated at 37 * C.
- Table 8 sets out the results of one representative aggregation experiment wherein PMA was added. Aggregation scores are reported on a range from 0 to 5, wherein 0 indicates that no cells were in clusters; 1 indicates that less than 10% of the cells were in clusters; 2 indicates that 10 to 50% cells were aggregated; 3 indicates that 50 to 100% cells were in loose clusters; and 4 indicates tiiat almost 100% of the cells were in compact aggregates.
- Antibody Treatment 1 - - - ⁇ CD18 ⁇ CDlla ⁇ CDllb
- the fourth anti-ICAM-R monoclonal antibody did not stimulate cell aggregation but blocked the aggregation stimulated by the otiier anti- ICAM-R antibodies. At least a portion of the aggregation stimulated by anti- ICAM-R antibodies in PMA treated cells was blocked by pretreatment with monoclonal antibodies against CD 18 or CD 11a indicating that one or more leukointegrins may participate in this type of adhesion.
- Fab' purified from die same anti-ICAM-R monoclonal antibody (ICR- 1.1).
- the assays were performed with SKW3 T cells as described above using ICR-l.l-Ig and ICR-l.l-Fab' at a concentration of 1 ⁇ g/ml.
- Supernatants of anti-CD18 and anti-ICAM-R (CIR-l. l-sup and ICR-4.2-sup) hybridomas were used as controls. After four hours, the same increase in cell aggregation was found for whole immunoglobulin as for die Fab' fragments or the ICR- 1.1 supernatant (See Table 9 below).
- the process of activation and proliferation of cells of the immune system is marked by a continuum of cellular events.
- the upregulation of certain cell surface molecules e.g., CD69 and the transferrin receptor
- cell agglutination occurs early in the process of activation.
- the upregulation of the IL-2 receptor occurs at an intermediate to late stage and cell proliferation is a late event.
- Three types of experiments were performed to determine the extent to which ICAM-R is involved in immune cell activation/proliferation. In the first type, the capacity of ICAM-R presented on the surface of a transfected cell to stimulate proliferation of lymphocytes was examined.
- Mouse L cells transfected with either ICAM-R cDNA or ICAM-1 cDNA were assayed for tiieir ability to stimulate human peripheral blood mononuclear cell (PBMC) proliferation as measured by 3 H-ti ⁇ ymidine incorporation assays which indicate changes in the rate of DNA replication.
- Nontransfected mouse L cells or transfected L cells were obtained by trypsinization from tissue culture flasks and washed in RPMI- 1640 containing 10% fetal bovine serum.
- tissue culture media RPMI-1640 with 10% fetal bovine .serum
- the media was ti en removed in a sterile manner and 2 x 10 s freshly isolated PBMC in a total volume of 200 ⁇ l tissue culture media were added to individual wells containing either transfected or non-transfected mouse L cells.
- PBMC were also added to control wells containing no L cells. The PBMC were previously isolated from healthy donors by centrifugation on Histopaque gradients (Sigma).
- Fresh peripheral blood was mixed with an equal volume of PBS, layered onto Histopaque and centrifuged at 450 g for 20 minutes with no brake applied.
- PBMC-containing fractions were collected, washed in PBS and adjusted to 1 x 10 6 viable cells/ml prior to addition into wells.
- the tissue culture plates were tiien incubated for a total of 4 days either in the presence or absence of PMA at a final concentration of 5 ng/ml. Lymphocyte proliferation was then assessed after the addition of 1 uCi 3 H- tiiymidine (NEN, Boston, MA) to individual wells for the last 18-24 hours of culture.
- Anti-ICAM-R antibodies of the invention were also tested to determine their effect on immune cell activation and proliferation.
- Anti-ICAM-R monoclonal antibodies were preliminarily tested for the ability to affect early events in cell activation including upregulation of the cell surface molecules CD69, die transferrin receptor and die IL-2 receptor on die target cells as measured by flow cytometry analysis.
- Unstimulated lymphocytes express low levels of the transferrin and IL-2 receptors. Expression of the receptors increases dramatically when lymphocytes are activated.
- Anti-ICAM-R monoclonal antibodies ICR- 1.1 and ICR-4.2 were each tested for the ability to induce PMBC activation in the absence of other inducing stimuli.
- Monoclonal antibodies ICR-1.1 or ICR-42. (or control monoclonal antibodies) were added (10 ⁇ g/well in PBS) to individual wells of a 96- well flat bottom tissue culture plate and incubated for 3 hours at 37 * C in a 5% CO 2 incubator. The plates were washed 3 times with sterile PBS to remove unbound antibody and freshly isolated PBMC were immediately added to a final concentration of 2 x 10 5 cells/well in a volume of 200 ⁇ l media.
- the plates were then incubated for eitiier 1 or 3 days at which time me cells cultured in die presence of different antibodies were removed, washed as described above in PBS containing 0.01 % sodium azide and 1 % BSA (FACS buffer) and stained with either FITC (Becton Dickinson) -conjugated negative control antibodies or a panel of FITC-conjugated anti-CD69, anti-transferrin receptor and anti-IL-2 receptor antibodies. Results were obtained by FACScan analysis. Expression of CD69 and d e transferrin receptor but not the B -2 receptor increased after 1 day when PBMC were cultured on immobilize (i.e.
- Anti-ICAM-R monoclonal antibodies were also tested for their ability to alter early events in PMBC activation stimulated by immobilized anti-
- CD3 monoclonal antibody G19 [Ledbetter et al. J. Immunol, 135(4): 2331-2336 (1985)]. Monoclonal antibody G19 binds to die CD3 complex on T cells (the T cell receptor) and activates T cells.
- T cells the T cell receptor
- witii anti-CD3 antibody 0.05 ⁇ g/well
- CD69 expression was elevated after one day.
- cell surface expression of CD69, the transferrin receptor and the IL-2 receptor was dramatically elevated. Upregulation of these activation markers was correlated witii increases in cell size.
- Two x 10 5 cells were then resuspended in 50 ⁇ l ice cold FACS buffer, and 5 ⁇ l of FITC-conjugated anti-CD69, anti-transferrin receptor, anti-IL-2 receptor -antibody or anti-FITC conjugated control Ig was added.
- the cells were incubated at 4 * C for 30 minutes .and tiien washed 2 times in 0.5 ml ice cold FACS buffer. After the final wash the cells were resuspended in 0.5 ml FACS buffer and fluorescence determined by FACScan analysis.
- ICAM-R Monoclonal antibodies to ICAM-R were tested for tiieir ability to directly stimulate PMBC proliferation in either the presence or absence of human recombinant IL-2 which potentiates but does not induce cell proliferation.
- IgG 2 ) antibodies in PBS were added per well of 96-well flat bottom tissue culture plates and the plates were incubated for 3-4 hours at 37 * C in a 5% CO 2 incubator. After incubation, each well was rinsed 3 times with PBS and freshly obtained PBL were added to a final concentration of 2 x 10 5 cells/well in a volume of 200 ⁇ l.
- Ten units/ml human recombinant IL-2 (Genzyme, Boston,
- CD3 and anti-LFA-1 (60.3) monclonal antibodies monclonal antibodies. These results indicate tiiat while the immobilized anti-ICAM-R antibodies stimulate expression of activation markers such as CD69, etc., by them ⁇ lves they do not directly stimulate the entry of large numbers of PBMC into S phase of the cell cycle.
- anti-ICAM-R antibodies were tested for the ability to costimulate lymphocyte proliferation induced by immobilized anti-CD3 antibody.
- anti-ICAM-R antibodies were tested for their ability to costimulate proliferation of pure CD4 + T-lymphocytes, isolated using negative selection.
- tissue culture medium To isolate CD4 + cells PBMC were suspended in tissue culture medium, added to 75 ml tissue culture flasks (Corning) and incubated for 1 hour at 37 "C, 5% CO 2 .
- Plastic nonadherent cells were then removed from die flask by gently rinsing once with PBS.
- the nonadherent cell fraction was suspended (10 7 cells/ml) in an antibody cocktail containing 1 ⁇ g/ml anti-CD8 antibody (Pnarmingen, San Diego, CA), 1 ⁇ g/ml anti-CD19 (Becton Dickinson), 1 ⁇ g/ml anti-CD lib (Becton Dickinr ⁇ n) in 10% FBS-PBS (coating medium), and incubated for 1 hour at 4"C. Unbound antibody was removed by washing twice in coating medium.
- the plates were incubated for 3 hours at 37 * C in a 5 % CC*-- incubator and unbound antibody was removed by rinsing die wells 3 times in PBS. After die final PBS wash, monoclonal antibodies to ICAM-R (ICR-4.2 or ICR-1.1) or control antibodies were immediately added to a final concentration of 10 ⁇ g/well. The plates were then reincubated for an additional 3 hours at 37 * C. The wells were again washed tiiree times with PBS to remove unbound antibody and freshly isolated PBMC were immediately added to the wells (2 x 10 5 cells in a volume of 200 ⁇ l/well). The plates were then incubated for 3 days.
- Lymphocyte proliferation was measured by 3 H-thymidine incorporation by the PMBC or CD4 + cells.
- immobilized anti-ICAM-R monoclonal antibodies ICR- 1.1 and ICR-4.2 increased the PBMC and purified CD4 + cell response to anti-CD3.
- Effects of the immobilized anti-ICAM-R antibodies on PBMC aggregation (an earlier event than PBMC proliferation) induced by anti-CD3 monoclonal antibody were also examined in this experiment.
- Anti-CD3 stimulated aggregation was inhibited almost 100% by antibody ICR-1.1 but was unaffected by immobilized
- results of the assays for the ability of anti-ICAM-R antibodies to affect the proliferation of cells on which ICAM-R is expressed indicate that binding of the antibodies of the invention to ICAM-R transmits a direct intracellular signal to T lymphocytes which modulates cell proliferation.
- Soluble ICAM-R was assayed for die ability to costimulate human lymphocyte activation.
- Human peripheral blood lymphocytes (PBL) were obtained by Ficoll-Hypaque centrifugation .and 2 x 10 s cells per well were incubated in die presence of either media, plate bound soluble ICAM-R, plate bound anti-CD3 (OKT3) or a combination of plate bound anti-CD3 and soluble ICAM-R.
- PBL peripheral blood lymphocytes
- ICAM-R is involved in early events of qualitatively distinct types of cell-cell contact dependent T-lymphocyte activation (e.g. , responses to staph enterotoxin A and alloantigen).
- Lymphocyte fractions were collected and washed twice by .adding a fresh volume of RPMI supplemented witii 10% fetal bovine serum and centrifuging at 200 x g for 8 minutes. PBL were suspended in a final volume of 10 ml of RPMI-FBS. Viable PBL were counted using die metiiod of vital dye exclusion. Twenty ⁇ l of a dilution of cell suspension in 0.4% trypan blue stain (Gibco) was added to a hemacytometer chamber and dye-excluding cells were then counted using an inverted micro.scope.
- FIGURE 11C presents logistic dose response curves for monoclonal antibodies ICR-1.1 , ICR-2.1 , ICR-5.1 , ICR-6.2 and ICR-8.1 in terms of the percentage of proliferation ob.served compared to proliferation in the presence of control andibodies and Table 10 below sets out the ICso values obtained from d e curves.
- SEA induces cell aggregation.
- Effects of the monoclonal antibodies ICR-1.1 and ICR-4.2 on cell aggregation were measured using an inverted microscope. Plate-bound ICR- 1.1 also significantly inhibited cell aggregation at both SEA concentrations in comparison to plate-bound B-H9 and ICR-4.2 antibodies. Inhibition of aggregation by plate-bound ICR- 1.1 was almost complete. In contrast, plate- bound ICR-4.2 antibody only slightly inhibited aggregation in comparison to plate- bound B-H9. Aggregation of PBL induced by SEA was not affected by .soluble anti-ICAM-R antibodies ICR-1.1 or ICR-4.2 in comparison to soluble B-H9 antibody.
- ICAM-R tiiat binding of plate-bound ICR-1.1 or ICR-4.2 to ICAM-R transmits an intracellular signal capable of inhibiting proliferation even after cells have been removed from the immobilized antibodies.
- Plasmatic nonadherent PBMC (10 7 cells/ml) were incubated for 1 hour at 4 * C with a cocktail of antibodies (1 ⁇ g/ml each) containing anti-CD8, anti-CD 19, anti-CD lib, anti-HLA-DR (Becton Dickinson) and either anti-CD45RO (Amac)(to obtain CD45RA + CD4 + cells), or anti- CD45RA (Amac) (to obtain CD45RO + CD4 + cells) in coating medium.
- the cell suspension was washed twice with coating medium to remove unbound antibody and incubated with goat anti-mouse IgG coated magnetic beads. Cells bound to magnetic beads were then removed from d e suspension using a strong magnet.
- CD45RO + and CD45RA + populations obtained using this method were found to be >95% pure as determined by flow cytometric analysis. Two hundred thousand purified memory T cells, resting T cells or plastic adherent cells were incubated on immobilized ICR- 1.1, anti-ICAM-1 antibody LB-2 or anti-HLA-I antibody plO.l (10 ⁇ g/ml) (Gerald Nepom, Virginia Msison Research Center,
- Monoclonal antibodies to ICAM-R were also tested for the ability to alter lymphocyte proliferation (as measured by 3 H-thymidine incorporation) in response to alloantigenic irradiated stimulator cells.
- Responder cells were prep ⁇ Lred by obtaining PBMC from a normal donor uang Histopaque centrifugation as described above. To prepare stimulator cells, PBMC from a
- Immobilized monoclonal antibodies ICR-1.1, 2.1, 6.2 and 8.1 consistently reduced proliferation in comparison to control antibodies. ICR-8.1 also inhibited alloantigen-stimulated proliferation when administered in soluble form.
- Example 21 Table 11 below is a summary of various characteristics of ICAM-R specific monoclonal antibodies of the invention which have been specifically described in the foregoing examples.
- the abbreviation “NC” stands for “not conclusive” and die abbreviation “ND” stands for “not determined.”
- the antibodies marked with an asterisk in Table 11 enhanced activation at low concentrations.
- ICAM-R e.g., SEA and allogeneic cells
- ICAM-R specific signalling events are likely to involve the interaction of the cytoplasmic domain of ICAM-R with cellular enzymatic components (e.g., ltinases, phosphatases) of one or more second messenger pathways and/or with cytoskeletal components in a pattern unique to ICAM-R.
- cellular enzymatic components e.g., ltinases, phosphatases
- EDTA 1 % Triton X-100 (Pierce), 10 ug/ml pepstatin and leupeptin (Boehringer), 2mM PMSF for 1 hour on ice. Ly.sates were pelleted in a refrigerated microfuge at 14,000 rpm for 15 minutes and die resulting supernatant was applied to a DEAE sephacel column (Pharmacia) equilibrated in 20mM Tris pH 7.5, 0.5mM EDTA (Buffer A). The column was run at a rate of 0.25 ml minute and developed with a gradient of 0 to 0.35M NaCl in buffer A over 60 minutes.
- PKC protein kinase C
- Fractions enriched in PKC activity were pooled and u.sed .as a source of kinase(s) to test for differential pho.sphorylation of synthetic peptides of the complete cytoplasmic domains of ICAM- 1, ICAM-2.and ICAM-R (amino acids 481 to 518 of SEQ ID NO: 1).
- Assays were performed according to manufacture's instructions with peptides at 75 uM final concentration.
- ICAM-R but not ICAM-1.
- Two dimensional phosphoamino acid analysis on these phosphorylated peptides shows only serine phosphorylation on ICAM-R and ti reonine phosphorylation on ICAM-1.
- cytoplasmic domain of IC.AM-R differentially associates with cytoskeletal components. Binding of the non-competing monoclonal antibodies ICR-1.1 and ICR-4.2 to ICAM-R was examined to assess the potential influence of each antibody on die association of lymphocyte ICAM-R with the cytoskeleton.
- the antibodies may mimic distinct natural ICAM-R ligands which employ ICAM-R as a cell surface receptor through which regulated cellular responses may be elicited.
- T lymphocyte surface antigens which occur as cell surface transmembrane glycoproteins can be induced to associate with die cytoskeleton if cell surface- bound antibody specific for the.se antigens is crosslinked with secondary antibodies [Geppert et al, J. Immunol, 146: 3298 (1990)]. Many of the.se cell surface molecules are defined components of lymphocyte adhesion and/or activation pathways.
- the phenomenon of inducible association with die cytoskeleton is operationally defined as d e resistance of cell-surface immune complexes to detergent extraction under defined conditions. Inducible detergent resistance does not require metabolic energy and can be observed in cells maintained at 0-4 * C throughout the experiment.
- 26I10E-2 adjusted to saturating concentration in the same buffer. Antibody binding was permitted to proceed for 30 minutes on ice, afterwhich unbound antibody was removed by pelleting cells which had first been resuspended in 1 ml of PBS-5% FBS through an underlaid cushion (0.7 ml) of neat (undiluted) FBS. For groups stained with FITC-conjugated monoclonal antibody only, the 1 ml suspension was divided into two equal parts, each of which was separately underlaid with FBS, centrifuged, and the supernatant removed by aspiration.
- control buffer 13mM Tris pH 8.0, 150mM NaCl, 2mM MgCldon 2mM EGTA, 2% FBS, 2.5 ug/ml aprotinin, ImM PMSF, lOmM iodoacetamide
- detergent buffer 0.5% NP-40
- polynucleotides e.g., DNA and RNA
- RNA and RNA e.g., DNA and RNA
- Typical detection assays involving ICAM-R DNA include Northern blot hybridization, RNAse protection, and in sjtu hybridization cytological assays wherein the DNA or RNA (in suitably labelled, detectable form) hybridizes to RNA in the sample.
- ICAM-R encoding DNA (especially DNA encoding the first, fourth and fifth domains which have less homology to DNAs encoding ICAM-1 and ICAM-2 tiian the DNAs encoding domains 2 and 3) is expected to be useful in isolating genomic DNA encoding ICAM-R including genomic DNA specifying endogenous expression control DNA sequences for ICAM-R DNA.
- genomic DNA including genomic DNA specifying endogenous expression control DNA sequences for ICAM-R DNA.
- knowledge of polynucleotide sequences encoding ICAM-R and/or controlling expression of ICAM-R makes available a variety of antisense polynucleotides useful in regulating expression of ICAM-R.
- the present invention makes available the production of ICAM-R polypeptides and variants thereof, especially including soluble fragments thereof, such as fragments comprising one or more of the five immunoglobulin-like domains of ICAM-R in glycosylated, non-glycosylated, or de-glycosylated forms.
- Pharmaceutical compositions including the protein products of the invention have therapeutic potential in the modulation of immune cell activation/proliferation, e.g., as competitive inhibitors or stimulatory agents of intercellular and intracellular ligand/receptor binding reactions involving ICAM-R.
- Such tiierapeutic potential is especially projected for "immunoadhesin" type recombinant hybrid fusion proteins containing, at their amino terminal, one or more domains of ICAM-R and, at tiieir carboxy terminal, at least one constant domain of an immunoglobulin.
- hybrid fusion proteins are likely to be available in the form of homodimers wherein the Ig portion provides for longer serum half life and the ICAM-R portion has greater affinity for the ICAM-R binding partner than ICAM-R itself.
- Other multimeric forms of ICAM-R which may have enhanced avidity are also projected to have therapeutic potential.
- Antibody substances and binding proteins are made readily available by the present invention tiirough the u.se of immunogens comprising cells naturally expressing ICAM-R, recombinant host cells producing polypeptide products of the invention, the ICAM-R polypeptide products themselves, and polypeptide products of the invention bound to an ICAM-R specific antibody that stimulates cell-cell aggregation (i.e., polypeptide products tiiat may be in a "high .affinity" binding conformation).
- Such antibodies and other ICAM-R specific binding proteins can be employed for immunopurification of ICAM-R and variants and in pharmaceutical compositions for therapies premised on blocking and/or stimulating the ligand/receptor binding of ICAM-R and soluble fragments thereof.
- ICAM-R specific antibody and anti- idiotypic antibody substances may be humanized (e.g., CDR-grafted) by recombinant techniques well-known in the art.
- Antibodies specific for distinct regions of ICAM-R may be employed in ELISA systems involving immunological " sandwiches" for monitoring inflammatory processes characterized by increases in amounts of soluble ICAM-R polypeptides in body fluids such as serum.
- Inflammatory conditions which may be treated or monitored with ICAM-R related products include conditions resulting from a response of the non- specific immune system in a mammal (e.g., adult respiratory distress syndrome, multiple organ injury syndrome secondary to septicemia, multiple organ injury syndrome secondary to trauma, reperfusion injury of tissue, acute glomerulonephritis, reactive arthritis, dermatosis witii acute inflammatory components, stroke, thermal injury, hemodialysis, leukapheresis, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, granulocyte transfusion associated syndrome, and cytokine-induced toxicity) and conditions resulting from a response of the specific immune system in a mammal (e.g., psoriasis, organ/tissue transplant rejection and autoimmune diseases including Raynaud's syndrome, autoimmune thyroiditis, multiple sclerosis, rheumatoid arthritis and lupus erythematosus).
- ICAM-R products of the invention may
- ADDRESSEE Marshall, O'Toole, Gerstein, Murray &
- MOLECULE TYPE protein
- FEATURE :
- Lys lie Asp Arg Ala Thr Cys Pro Gin His Leu Lys Trp Lys Asp Lye 375 380 385
- GATTGTCCCA GCTCTGAGAA AATCGCCTTG GAGACGTCCC TATCAAAGGA GCTGGTGGCC 240 AGTGGCATGG GCTGGGCAGC CTTCAATCTC AGCAACGTGA CTGGCAACAG TCGGATCCTC 300 TGCTCAGTGT ACTGCAATGG CTCCCAGATA ACAGGCTCCT CTAACATCAC CGTGTACGGG 360 CTCCCGGAGC GTGTGGAGCT GGCACCCCTG CCTCCTTGGC AGCCGGTGGG CCAGAACTTC 420 ACCCTGCGCT GCCAAGTGGA GGGTGGGTCG CCCCGGACCA GCCTCACGGT GGTGCTGCTT 480 CGCTGGGAGG AGGAGCTGAG CCGGCAGCCC GCAGTGGAGG AGCCAGCGGA GGTCACTGCC 540 ACTGTGCTGG CCAGCAGAGA CGACCACGGA GCCCCTTTCT CATGCCGCAC AGAACTGGAC 600 ATGCAGCCCC AGGGGCTGGG
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- TTGTTAGTNA CAAGCGCCTA GGCTTGGGGA GCCATCTCGC CCGCTCCTCT GTATCTTTAG 2100
Abstract
Description
Claims
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US926693A | 1993-01-22 | 1993-01-22 | |
US9266 | 1993-01-22 | ||
PCT/US1993/000787 WO1993014776A1 (en) | 1992-01-27 | 1993-01-26 | Icam-related protein |
WOPCT/US93/00787 | 1993-01-26 | ||
PCT/US1993/007367 WO1994017100A1 (en) | 1993-01-22 | 1993-08-05 | Icam-related protein |
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US5891841A (en) * | 1991-06-11 | 1999-04-06 | The Center For Blood Research, Inc. | Methods of using intercellular adhesion molecule-3 (ICAM-3), antibodies thereto, and soluble fragments thereof |
US5811517A (en) * | 1992-01-27 | 1998-09-22 | Icos Corporation | ICAM-related protein variants |
US5837822A (en) * | 1992-01-27 | 1998-11-17 | Icos Corporation | Humanized antibodies specific for ICAM related protein |
US5470953A (en) * | 1993-12-23 | 1995-11-28 | Icos Corporation | Human β2 integrin α subunit |
JP2942496B2 (en) * | 1996-03-07 | 1999-08-30 | 株式会社椿本チエイン | Detent mechanism for screw type linear actuator |
AU1479600A (en) * | 1998-11-17 | 2000-06-05 | Fred Hutchinson Cancer Research Center | Anti-icam-r antibody-induced apoptosis |
US8980568B2 (en) | 2001-10-11 | 2015-03-17 | Aviva Biosciences Corporation | Methods and compositions for detecting non-hematopoietic cells from a blood sample |
US8986944B2 (en) | 2001-10-11 | 2015-03-24 | Aviva Biosciences Corporation | Methods and compositions for separating rare cells from fluid samples |
WO2008008515A2 (en) | 2006-07-14 | 2008-01-17 | Aviva Biosciences Corporation | Methods and compositions for detecting rare cells from a biological sample |
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- 1993-08-05 WO PCT/US1993/007367 patent/WO1994017100A1/en not_active Application Discontinuation
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WO1992022323A1 (en) * | 1991-06-11 | 1992-12-23 | Center For Blood Research, Inc. | Intercellular adhesion molecule-3 and its binding ligands |
Non-Patent Citations (2)
Title |
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ARTHRITIS AND RHEUMATISM, vol.37, no.6, June 1994, NEW YORK, NY, USA pages 846 - 854 H. EL-GABALAWY ET AL. 'Expression of ICAM-R (ICAM-3), a novel counter-receptor for LFA-1, in rheumatoid and nonrheumotoid synovium.' * |
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