WO2004024746A9 - Anticorps anti-bgp(c-cam1) therapeutiques et leurs applications - Google Patents

Anticorps anti-bgp(c-cam1) therapeutiques et leurs applications

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Publication number
WO2004024746A9
WO2004024746A9 PCT/US2003/028416 US0328416W WO2004024746A9 WO 2004024746 A9 WO2004024746 A9 WO 2004024746A9 US 0328416 W US0328416 W US 0328416W WO 2004024746 A9 WO2004024746 A9 WO 2004024746A9
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WO
WIPO (PCT)
Prior art keywords
antibody
antibodies
cell
bgp
fragment
Prior art date
Application number
PCT/US2003/028416
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English (en)
Other versions
WO2004024746A2 (fr
WO2004024746A3 (fr
Inventor
Richard S Blumberg
Atul Bhan
Original Assignee
Brigham & Womens Hospital
Gen Hospital Corp
Richard S Blumberg
Atul Bhan
Priority date (The priority date 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 date listed.)
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Publication date
Priority claimed from US10/241,369 external-priority patent/US20040047858A1/en
Priority claimed from EP20020020334 external-priority patent/EP1400535A1/fr
Application filed by Brigham & Womens Hospital, Gen Hospital Corp, Richard S Blumberg, Atul Bhan filed Critical Brigham & Womens Hospital
Priority to AU2003270518A priority Critical patent/AU2003270518A1/en
Publication of WO2004024746A2 publication Critical patent/WO2004024746A2/fr
Publication of WO2004024746A9 publication Critical patent/WO2004024746A9/fr
Publication of WO2004024746A3 publication Critical patent/WO2004024746A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to antibodies which are capable of modulating T cell activity and uses thereof.
  • the antibodies of the invention and binding molecules derived thereof react with a specific domain of biliary glycoprotein (BGP), also known as CD66a, CEACAM1 and C-CAM1, and are capable of modulating T cell activity.
  • BGP biliary glycoprotein
  • the present invention relates to compositions comprising said antibodies and to methods of modulating immune cell proliferation, and treating immune response related diseases.
  • T-cell activation is a serial process involving multiple signaling pathways and sequential changes in gene expression resulting in differentiation of T-cells into distinct subpopulations, i.e., Thl and Th2, which are distinguishable by their pattern of cytokine production and characterize the mode of cellular immune response.
  • the T-cell response is initiated by the interaction of the antigen-specific T-cell receptor (TCR) with peptide presented by major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APCs).
  • TCR antigen-specific T-cell receptor
  • MHC major histocompatibility complex
  • Additional signals are provided by a network of receptor-ligand interactions mediated by a number of membrane proteins such as CD28/CTLA4 and B7, CD40/CD40L, LFA-1 and ICAM-1 (Lenschow, Science 257 (1992), 789-792; Linsley, Annu. Rev.
  • BGP biliary glycoprotein
  • CEACAMl a member of the carcinoembryonic antigen family
  • C-CAMl is expressed on T cells in the periphery (peripheral blood, etc.) outside the gut and intestinal epithelium in particular. Therefore, C-CAMl needs to be considered as a T cell activation antigen on T cells in general and not just limited to the gut. This therefore makes C-CAMl relevant to a wide variety of immune-mediated processes.
  • the technical problem of the present invention is to provide means and methods for modulation of the immune response in a subject.
  • the solution to said technical problem is achieved by providing the embodiments characterized in the claims, and described further below.
  • the present invention is directed to antibodies or antigen-binding f agments and similar BGP binding molecules which are capable of modulating T cell activity and binding to an epitope of the N-domain of BGP (C-CAMl).
  • a particularly preferred embodiment is a monoclonal antibody or antigen-binding fragment thereof which demonstrates the immunological binding characteristics of a monoclonal antibody selected from the group consisting of: 34B1 as produced by hybridoma 34B1, deposited with the American Type Culture Collection (ATCC) 10801 University Boulevard. Manassas, VA 20110-2209 U.S.A.
  • ATCC American Type Culture Collection
  • the antibody is a human, humanized, xenogeneic, or a chimeric human- murine antibody.
  • Therapeutic compositions including the antibody or active fragments thereof, or agonists and cognate molecules, or alternately, antagonists of the same, and methods of use of such compositions in the prevention, diagnosis or treatment of disease using these compositions are also included, wherein an effective amount of the composition is administered to a patient in need of such treatment.
  • the antigen-binding fragment of the monoclonal antibody can be a single chain Fv fragment, an F(ab') fragment, an F(ab) fragment, and an F(ab')2 fragment, or any other antigen-binding fragment.
  • the monoclonal antibody or fragment thereof is a murine IgG isotype antibody.
  • the invention extends to the hybridoma that produces monoclonal antibody 34B1, 26H7 or 5F4, which hybridoma is deposited with the ATCC as indicated hereinbefore.
  • the present invention also relates to polynucleotides encoding at least a variable region of an immunoglobulin chain of the antibody of the invention.
  • said variable region comprises at least one complementarity determining region (CDR) of the V H and/or V of the variable region of the antibody 34B1, 26H7 or 5F4 .
  • CDR complementarity determining region
  • the present invention also encompasses vectors comprising said polynucleotides and host cells transformed therewith as well as their use for the production of an antibody capable of modulating T cell activity or a functional fragment or immunoglobulin chain(s) thereof.
  • the antibody, immunoglobulin chain(s), binding fragments thereof and ligands other than BGP binding to said antibody can be used in pharmaceutical and diagnostic compositions for modulating and detecting an immune response, respectively.
  • the medicament is useful in the treatment of conditions related to immune system function, including autoimmune or immune mediated disorders and diseases.
  • Disorders would include autoimmune (e.g., inflammatory bowel disease, rheumatoid arthritis, psoriasis, multiple sclerosis), transplantation (e.g., kidney, heart) and cancer (e.g., colon, melanoma) wherein T cell activities would need to be increased or decreased where appropriate.
  • Figs. 1-3 show that 34B1 (Fig. 1), 26H7 (Fig. 2), and 5F4 (Fig. 3) anti-C-CAMl monoclonal antibodies inhibit Mixed Lymphocyte Reaction (MLR).
  • MLR Mixed Lymphocyte Reaction
  • the present invention relates to antibodies and binding molecules which are capable of modulating killer and other functional activities of T cells and which react with an epitope of the N-domain of BGP (C-CAMl).
  • the antibody or antigen-binding fragment thereof of the present invention demonstrate the immunological binding characteristics of monoclonal antibody 34B1 as produced by hybridoma 34B1, deposited with the American Type Culture Collection (ATCC) 10801 University Boulevard. Manassas, VA 20110-2209 U.S.A. on September 4, 2002, and assigned Accession Number PTA-4631 ; 26H7 as produced by hybridoma 26H7, deposited with the American Type Culture Collection (ATCC) on September 4, 2002, and assigned Accession Number PTA-4629; or 5F4 as produced by hybridoma 5F4, deposited with the American Type Culture Collection (ATCC) on September 4, 2002, and assigned Accession Number PTA-4630.
  • the term "immunological binding characteristics,” or other binding characteristics of an antibody with an antigen, in all of its grammatical forms refers to the specificity, affinity, cross-reactivity, and other binding characteristics of an antibody.
  • the invention extends to the hybridoma as well.
  • the invention advantageously provides an indefinitely prolonged cell source of a monoclonal antibody of the invention: the hybridoma.
  • the present invention also encompasses a method for producing anti-C-CAMl antibodies comprising culturing the hybridoma cell of the invention and isolating antibodies from the culture or cell or media.
  • the invention further relates to diagnostic assay methods and kits that comprise the monoclonal antibody of the invention and to therapeutic methods based thereon.
  • BGP human biliary glycoprotein
  • C-CAMl human biliary glycoprotein
  • Human BGP is a member of the CEA family of glycoproteins, part of the immunoglobulin supergene family, and encoded in a large cluster on cliromosome 19 (Watt et al, Blood 84 (1994), 200-210, Daniel et al, Int. J. Cancer 55 (1993), 303-310; Teixeira et al., Blood 84 (1994), 211-219; Barnett et al., Mol. Cell. Biol. 13 (1993), 1273-1282).
  • the CEA-cluster is highly related Jo the genetically linked, pregnancy specific gene cluster (Thompson et al., J. Clin. Lab. Anal. 5 (1991), 344-366; Obrink, Curr. Opin. Cell Biol. 9 (1997), 616-626).
  • the CEA-subgroup of this family is serologically defined as CD66a (BGP or C-CAMl), CD66b (CGM6), CD66c (NCA), CD66d (CGM1) and CD66e (CEA).
  • CD66a BGP or C-CAMl
  • CD66b CD66b
  • NCA CD66c
  • CD66d CD66d
  • CEA CD66e
  • CGM6 and NCA which are linked to the membrane by a glycosyl phosphatidyl-inositol anchor
  • CGM1 and BGP are type 1 transmembrane glycoproteins. Both of the latter exist as isoforms containing short or long cytoplasmic tails.
  • BGP and its mouse and rat homologues C-CAM (Rosenberg et al., Cancer Res. 53 (1993), 4938-4945; Lin et al., J. Biol. Chem. 264 (1989), 14408-14414; Obrink, BioEssays 13 (1991), 227-234,), have been regarded mainly as molecules which function in cell-cell adhesion that are expressed primarily by epithelial cells of the gastrointestinal tract and biliary tree, neutrophils and, more recently, B cells.
  • BGP also serves as a receptor for mouse hepatitis virus (Williams et al, Proc. Natl. Acad. Sci.
  • the present invention provides such molecules.
  • monoclonal antibodies and antigen-binding fragments are provided, which bind to an epitope of the N-domain of BGP (C-CAMl) and which are capable of modulating T cell proliferation.
  • C-CAMl BGP
  • international application WO99/52552 generally teaches the use of an anti-BGP monoclonal antibody for modulating T cell activity
  • the present application discloses three particular antibodies which distinguish themselves through different binding specificities for the different members of the CEA family and biological activities.
  • each of the three monoclonal antibodies exemplified in the present application is unique with respect to its immunological and biological activities.
  • hybridomas producing monoclonal antibodies 26H7, 5F4 and 34B1, respectively enables the persons skilled in the art to design and produce functionally equivalent antibodies, for example by adapting the antigen binding side of any one of the three mentioned antibodies, or to screen for functionally equivalent antibodies having the same or competitive binding specificities, for example by an antibody competitive binding experiment, as are well known in the art.
  • the monoclonal antibodies of the present invention may be distinguished from others by several means.
  • the instant antibodies do react with BGP(C-CAMl).
  • they react with the N-domain of BGP but do not substantially react with the Al, Bl and A2 domain of BGP.
  • they are capable of inhibiting T cell proliferation in an allogenic mixed lymphocyte reaction and, fourth, suppressing the cytolytic activity of intestinal intraepithelial lymphocytes (ilELs).
  • ilELs intestinal intraepithelial lymphocytes
  • antibodies 26H7 and 5F4 specifically recognize the N-domain of C-CAMl but not the N-domain of CEA (C-CAM5), NCA (C-CAM6), CGM6 (C-CAM8) or CGM1 (C- CAM3); see also publication by Morales et al. in J. Immunol. 1999. Please refer to Exp. Cell Res. 252:243 (1999), for the new nomenclature. There is no reason to believe that the 26H7 or 5F4 would recognize C-CAM 4 or 7 given their clear ability to distinguish C-CAMl from the others, however, it may not be excluded.
  • C-CAMl BGP
  • CGM6 C-CAM8
  • T-helper 1 cytokine production has been observed by the inventors as well (e.g., gamma-interferon); similar inhibition of Thl cytokine production is expected for the antibodies of the invention.
  • Monoclonal antibody 34B1 reacts with the native form of C-CAMl -FC protein and, to a lesser extent, with the denatured protein; see Watt et al., Blood 98, 1469-1479, 2001.
  • the monoclonal antibody 5F4 in particular binds to a particular domain within the N-domain of C-CAMl that is involved in homophilic interactions between the N-domains of different C-CAMl molecules (Watt et al., Blood 98 (2001), 1469-1479) and by the recent crystal structure of mouse C-CAMl (Tan et al. EMBO J.
  • the antibody of the present invention is any one of monoclonal antibody 34B1 as produced by hybridoma 34B1, deposited with the American Type Culture Collection (ATCC) 10801 University Boulevard. Manassas, VA 20110-2209 U.S.A.
  • ATCC American Type Culture Collection
  • the antibody of the present invention is a monoclonal antibody or antigen- binding fragment thereof, which competes for binding to C-CAMl with the antibodies produced by the above-described hybridomas.
  • Those antibodies may be murine as well, however, humanized, xenogeneic, or chimeric human-murine antibodies being preferred, in particular for therapeutic applications.
  • An antigen-binding fragment of the antibody can be, for example, a single chain Fv fragment, an F(ab') fragment, an F(ab) fragment, and an F(ab')2 fragment.
  • the present invention also relates to a hybridoma that produces any one of the above described monoclonal antibodies.
  • said hybridoma is selected from the group consisting of hybridoma 34B1, deposited with the American Type Culture Collection (ATCC) 10801 University Boulevard. Manassas, VA 20110-2209 U.S.A. on September 4, 2002, and assigned Accession Number PTA-4631; hybridoma 26H7, deposited with the ATCC on September 4, 2002, and assigned Accession Number PTA-4629; or hybridoma 5F4, deposited with the ATCC on September 4, 2002, and assigned Accession Number PTA-4630.
  • ATCC American Type Culture Collection
  • variable regions of the antibodies are required, which can be obtained by treating the monoclonal antibody isolated from the hybridoma with suitable reagents so as to generate Fab', Fab, or F(ab") 2 portions. Such fragments are sufficient for use, for example, in immunodiagnostic procedures involving coupling the immunospecific portions of immunoglobulins to detecting reagents such as radioisotopes.
  • the immortalized hybridoma cells can be used as a source of rearranged heavy chain and light chain loci for subsequent expression and/or genetic manipulation. Rearranged antibody genes can be reverse transcribed from appropriate mRNAs to produce cDNA.
  • variable regions can be linked to encode single chain Fv regions. Multiple Fv regions can be linked to confer binding ability to more than one target or chimeric heavy and light chain combinations can be employed.
  • the coding sequences can be inserted into expression systems contained on vectors which can be transfected into standard recombinant host cells.
  • a variety of such host cells may be used; for efficient processing, however, mammalian cells are preferred.
  • Typical mammalian cell lines useful for this purpose include CHO cells, 293 cells, orNSO cells.
  • the production of the antibody or analog is then undertaken by culturing the modified recombinant host under culture conditions appropriate for the growth of the host cells and the expression of the coding sequences.
  • the antibodies are then recovered by isolating them from the culture.
  • the expression systems are preferably designed to include signal peptides so that the resulting antibodies are secreted into the medium; however, intracellular production is also possible.
  • the present invention also relates to a polynucleotide encoding at least a variable region of an immunoglobulin chain of the antibody described above.
  • said variable region encoded by the polynucleotide comprises at least one complementarity determining region (CDR) of the VH and/or VL of the variable region of the antibody produced by any one of the above described hybridomas.
  • CDR complementarity determining region
  • each variable domain (the heavy chain VH and light chain VL) of an antibody comprises three hypervariable regions, sometimes called complementarity determining regions or "CDRs" flanked by four relatively conserved framework regions or "FRs".
  • variable regions of the antibody of the invention can be determined, e.g., according to Kabat, Sequences of Proteins of Immunological Interest (U.S. Department of Health and Human Services, third edition, 1983, fourth edition, 1987, fifth edition 1990).
  • variable domain of the antibody having the above-described variable domain can be used for the construction of other polypeptides or antibodies of desired specificity and biological function.
  • the present invention also encompasses polypeptides and antibodies comprising at least one CDR of the above-described variable domain and which advantageously have substantially the same or similar binding properties as the antibody described in the appended examples.
  • variable domains or CDRs described herein antibodies can be constructed according to methods known in the art, e.g., as described in EP-A1 0 451 216 and EP-A1 0 549 581.
  • binding affinity may be enhanced by making amino acid substitutions within the CDRs or within the hypervariable loops (Chothia and Lesk, J. Mol. Biol. 196 (1987), 901- 917) which partially overlap with the CDRs as defined by Kabat.
  • the present invention also relates to antibodies wherein one or more of the mentioned CDRs comprise one or more, preferably not more than two amino acid substitutions.
  • the antibody of the invention comprises in one or both of its immunoglobulin chains two or all three CDRs of the variable regions of antibodies 34B1, 26H7 or 5F4.
  • the polynucleotide of the invention encoding the above described antibody may be, e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination.
  • said polynucleotide is part of a vector.
  • Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • the polynucleotide of the invention is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells.
  • Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA.
  • Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly- A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally associated or heterologous promoter regions.
  • polynucleotides encoding at least the variable domain of the light and/or heavy chain may encode the variable domains of both immunoglobulin chains or only one.
  • said polynucleotides may be under the control of the same promoter or may be separately controlled for expression.
  • Possible regulatory elements pe ⁇ nitting expression in prokaryotic host cells comprise, e.g., the PL, lac, tip or tac promoter in E.
  • regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV- promoter, SV40-promoter, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40- enhancer or a globin intron in mammalian and other animal cells.
  • Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the S V40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • transcription termination signals such as the S V40-poly-A site or the tk-poly-A site
  • leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide of the invention and are well known in the art.
  • the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including a C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pCDM8, pRc/CMV, pcDNAl, pcDNA3 (Invitrogen), or pSPORTl (GLBCO BRL).
  • the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used.
  • the vector Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the immunoglobulin light chains, heavy chains, light/heavy chain dimers or intact antibodies, binding fragments or other immunoglobulin forms may follow; see, Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N.Y., (1979).
  • the polynucleotide of the invention can be used alone or as part of a vector to express the (poly)peptide of the invention in cells, for, e.g., gene therapy or diagnostics of diseases related to immune diseases.
  • the polynucleotides or vectors of the invention are introduced into the cells which in turn produce the antibody.
  • Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in- vivo techniques is one of the most important applications of gene transfer. Suitable vectors and methods for in- vitro or in- vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res.
  • the polynucleotides and vectors of the invention may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g., adenoviral, retroviral) into the cell.
  • said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom, most preferably said cell is a stem cell.
  • the present invention relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide encoding a variable domain of an immunoglobulin chain of an antibody of the invention; optionally in combination with a polynucleotide of the invention that encodes the variable domain of the other immunoglobulin chain of the antibody of the invention.
  • said vector is an expression vector and/or a gene transfer or targeting vector.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno- associated virus, herpes viruses, or bovine papilloma virus may be used for delivery of the polynucleotides or vector of the invention into targeted cell population.
  • viruses such as retroviruses, vaccinia virus, adeno- associated virus, herpes viruses, or bovine papilloma virus.
  • Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994).
  • the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
  • the vectors containing the polynucleotides of the invention can be transferred into the host cell by well known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts; see Sambrook, supra.
  • the present invention furthermore relates to host cells transformed with a polynucleotide or vector of the invention.
  • Said host cell may be a prokaryotic or eukaryotic cell.
  • the polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae.
  • prokaryotic is meant to include all bacteria which can be transformed or transfected with a DNA or RNA molecules for the expression of an antibody of the invention or the corresponding immunoglobulin chains.
  • Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis.
  • eukaryotic is meant to include yeast, higher plant, insect and preferably mammalian cells, most preferably NSO and CHO cells.
  • the antibodies or immunoglobulin chains encoded by the polynucleotide of the present invention may be glycosylated or may be non- glycosylated.
  • Antibodies of the invention or the corresponding immunoglobulin chains may also include an initial methionine amino acid residue.
  • a polynucleotide of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art.
  • methods for preparing fused, operably linked genes and expressing them in, e.g., mammalian cells and bacteria are well-known in the art (Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).
  • the genetic constructs and methods described therein can be utilized for expression of the antibody of the invention or the corresponding immunoglobulin chains in eukaryotic or prokaryotic hosts.
  • expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host.
  • the expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells.
  • Suitable source cells for the DNA sequences and host cells for immunoglobulin expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection ("Catalogue of Cell Lines and Hybridomas," Fifth edition (1985) Rockville, Maryland, U.S.A., which is incorporated herein by reference).
  • transgenic animals, preferably mammals, comprising cells of the invention may be used for the large scale production of the antibody of the invention.
  • the present invention relates to a method for the production of an antibody capable of modulating T cell proliferation in an allogenic mixed lymphocyte reaction and/or suppressing the cytolytic activity of intestinal intraepithelial lymphocytes (iLELs) or a functional fragment or immunoglobulin chain(s) thereof, said method comprising (a) culturing a cell described above; and
  • the transformed hosts can be grown in fermentors and cultured according to techniques l ⁇ iown in the art to achieve optimal cell growth.
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, "Protein Purification", Springer Verlag, N.Y. (1982).
  • the antibody or its corresponding immunoglobulin chain(s) of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions.
  • the isolation and purification of the, e.g., microbially expressed antibodies or immunoglobulin chains of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies directed, e.g., against the constant region of the antibody of the invention.
  • the antibodies of the invention can be further coupled to other moieties for, e.g., drug targeting and imaging applications.
  • Such coupling may be conducted chemically after expression of the antibody or antigen to site of attachment or the coupling product may be engineered into the antibody or antigen of the invention at the DNA level.
  • the DNAs are then expressed in a suitable host system, and the expressed proteins are collected and renatured, if necessary.
  • Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.
  • the antibodies Once purified, partially or to homogeneity as desired, the antibodies may then be used therapeutically (including extracorporeally) or in developing and performing assay procedures.
  • the present invention also involves a method for producing cells capable of expressing an antibody of the invention or its corresponding immunoglobulin chain(s) comprising genetically engineering cells with the polynucleotide or with the vector of the invention.
  • the cells obtainable by the method of the invention can be used, for example, to test the interaction of the antibody of the invention with its antigen.
  • the immunoglobulin or its encoding cDNAs may be further modified.
  • the method of the present invention comprises any one of the step(s) of producing a chimeric antibody, humanized antibody, single-chain antibody, Fab- fragment, bi-specific antibody, fusion antibody, labelled antibody or an analog of any one of those.
  • Corresponding methods are known to the person skilled in the art and are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.
  • xenogeneic antibodies such as human antibodies in mice
  • WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735 The general principle for the production of xenogeneic antibodies such as human antibodies in mice is described in, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735.
  • the antibody of the invention may exist in a variety of forms besides complete antibodies; including, for example, Fv, Fab and F(ab) 2 , as well as in single chains; see e.g., WO88/09344.
  • the antibodies of the present invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination.
  • Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g.,
  • Modifications of the antibody of the invention include chemical and/or enzymatic derivatizations at one or more constituent amino acids, including side chain modifications, backbone modifications, and N- and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like.
  • the present invention encompasses the production of chimeric proteins which comprise the described antibody or some fragment thereof at the amino terminus fused to heterologous molecule such as an immunostimulatory ligand at the carboxyl terminus; see, e.g., WO00/30680 for corresponding technical details.
  • the present invention encompasses small polypeptides including those containing a BGP binding fragment as described above, for example containing the CDR3 region of the variable region of any one of the mentioned monoclonal antibodies. Such peptides may easily be synthesized or produced by recombinant means to produce a BGP binding agent useful according to the invention. Such methods are well known to those of ordinary skill in the art.
  • Peptides can be synthesized for example, using automated peptide synthesizers which are commercially available.
  • the peptides can be produced by recombinant techniques by incorporating the DNA expressing the peptide into an expression vector and transforming cells with the expression vector to produce the peptide.
  • the sequence of the CDR regions, for use in synthesizing peptide BGP binding agents, may be determined by methods known in the art.
  • the heavy chain variable region is a peptide which generally ranges from 100 to 150 amino acids in length.
  • the light chain variable region is a peptide which generally ranges from 80 to 130 amino acids in length.
  • the CDR sequences within the heavy and light chain variable regions which include only approximately 3-25 amino acids may easily be sequenced by one of ordinary skill in the art.
  • the peptides may even be synthesized by commercial sources.
  • any known binding assay may be employed.
  • the peptide may be immobilized on a surface and then contacted with labeled BGP.
  • the amount of BGP which interacts with the peptide or the amount which does not bind to the peptide may then be quantitated to determine whether the peptide binds to BGP.
  • a surface having the aforementioned anti-BGP monoclonal antibodies immobilized thereto may serve as a positive control.
  • BGP binding agents also can be carried out utilizing a competition assay. If the BGP binding agent being tested competes with an anti-BGP monoclonal antibody, as shown by a decrease in binding of the monoclonal antibody, then it is likely that the agent and the anti-BGP monoclonal antibody bind to the same, or a closely related, epitope. Still another way to determine whether an agent has the specificity of the anti-BGP monoclonal antibodies described above is to pre-incubate the monoclonal antibody with BGP with which it is normally reactive (i.e., binds), and then add the agent being tested to determine if the agent being tested is inhibited in its ability to bind BGP. If the agent being tested is inhibited then, in all likelihood, it has the same or a functionally equivalent epitope and specificity as the anti-BGP monoclonal antibodies.
  • a BGP binding agent is useful according to the invention by determining whether the agent is one which modulates T cell proliferation or cytotoxicity in an in vitro assay such as measuring release of TNF from T cells or by 51 Cr release assay; see, e.g., Herin et al., Int. J. Cancer 39 (1987), 390-396. Other assays are described in the Examples and elsewhere herein.
  • polypeptides e. g., antibodies
  • BGP binding agents can also be used immunotherapeutically for T cell sensitive disorders in humans.
  • immunotherapeutically or “immunotherapy” as used herein in conjunction with the BGP binding agents denotes both prophylactic as well as therapeutic administration.
  • the peptides can be administered to high-risk subjects in order to lessen the likelihood and/or severity of a T cell sensitive disease, such as a tumor, transplant rejection or autoimmune disease, or administered to subjects already evidencing such diseases.
  • BGP binding agents which increase or decrease T cell activity can be selected using the assays described, for example, in WO99/52552 and according to standard T cell cytotoxicity and proliferation assays, such as mixed lymphocyte reactions, chromium release assays, TNF release assays, and thymidine incorporation assays.
  • a monovalent BGP binding agent for example derivatives of single chain antibodies which have only one binding domain of the original antibody, will inhibit the inhibitory signal of BGP by reducing cross-linking of BGP polypeptides expressed by T cells, and that a multivalent BGP binding agent (having two or more BGP binding sites), such as bi- and tri-specific antibodies, will increase the inhibitory signal of BGP in T cells by increasing cross-linking of BGP polypeptides expressed by T cells.
  • the present invention relates to any antibody and similar binding molecules, which preferably have substantially the same immunological binding characteristics as monoclonal antibodies 34B1, 26H7 or 5F4, i.e., which recognize and bind to the same epitope and with substantially the same affinity, or at least 1/10 of the affinity as the antibodies of the invention exemplified herein.
  • Such antibodies and binding molecules can be tested for their binding specificity and affinity by for example by using competitive assays with an antibody produced by a hybridoma of the invention.
  • the antibodies of the present invention will typically find use individually in treating substantially any disease susceptible to monoclonal antibody based therapy.
  • the immunoglobulins can be used as immunosuppressive agents.
  • typical disease states suitable for treatment include inflammatory symptoms.
  • the antibodies can be used therapeutically in, e.g., patients suffering an diseases related to immune response; see supra. Such therapy can be accomplished by, for example, the administration of antibodies of the invention.
  • Such administration can utilize unlabeled as well as labeled antibodies or antigens.
  • Labeling agents can be coupled either directly or indirectly to the antibodies or antigens of the invention.
  • One example of indirect coupling is by use of a spacer moiety.
  • the antibodies of the present invention can comprise a further domain, said domain being linked by covalent or non-covalent bonds.
  • the linkage can be based on genetic fusion according to the methods known in the art and described above or can be performed by, e.g., chemical cross-linking as described in, e.g., WO 94/04686.
  • the additional domain present in the fusion protein comprising the antibody of the invention may preferably be linked by a flexible linker, advantageously a polypeptide linker, wherein said polypeptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of said further domain and the N-terminal end of the antibody of the invention or vice versa.
  • the above described fusion protein may further comprise a cleavable linker or cleavage site for proteinases. These spacer moieties, in turn, can be either insoluble or soluble (Diener et al., Science 231 (1986), 148) and can be selected to enable drug release from the antigen at the target site.
  • Examples of therapeutic agents which can be coupled to the antibodies, antigens and epitopes of the invention for immunotherapy are drugs, radioisotopes, lectins, and toxins.
  • the drugs with which can be conjugated to the antibodies, antigens and epitopes of the invention include compounds which are classically referred to as drugs such as mitomycin C, daunorubicin, and vinblastine.
  • certain isotopes may be more preferable than others depending on such factors as leukocyte distribution as well as stability and emission. Depending on the autoimmune response, some emitters may be preferable to others.
  • a and b particle emitting radioisotopes are preferred in immunotherapy.
  • radioisotopes which can be bound to the antibodies, antigens or epitopes of the invention for therapeutic purposes are 125 1, 131 1, 90 Y, 67 Cu, 212 Bi, 212 At, 211 Pb, 47 Sc, 109 Pd and 188 Re.
  • Other therapeutic agents which can be coupled to the antibody, antigen or epitope of the invention, as well as ex vivo and in vivo therapeutic protocols, are known, or can be easily ascertained, by those of ordinary skill in the art. Wherever appropriate the person skilled in the art may use a polynucleotide of the invention encoding any one of the above described antibodies, antigens or the corresponding vectors instead of the proteinaceous material itself.
  • the present invention relates to compositions comprising the aforementioned antibody or antigen-binding fragment of the invention or chemical derivatives thereof, or the polynucleotide, vector or cell of the invention.
  • the composition of the present invention may further comprise a pharmaceutically acceptable carrier.
  • the term "chemical derivative" describes a molecule that contains additional chemical moieties that are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
  • Suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well l ⁇ iown conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. Aerosol formulations such as nasal spray formulations include purified aqueous or other solutions of the active agent with preservative agents and isotonic agents.
  • Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucous membranes, e.g., for intranasal administration.
  • Formulations for rectal or vaginal administration may be presented as a suppository with a suitable carrier.
  • the dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a typical dose can be, for example, in the range of 0.001 ⁇ g to 10 mg (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 0.01 ⁇ g to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 0.01 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 10 6 to 10 12 copies of the DNA molecule.
  • compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.
  • the pharmaceutical composition of the present invention comprises at least one second agent, preferably an agent which inhibits T-cell stimulation depending on the intended use.
  • Such agents include, for example, molecules that are capable of blocking or mimicking receptor/ligand interaction or the like which leads to T-cell suppression.
  • Such agents comprise those blocking the activity of, e.g., costimulatory molecules, such as anti-TIRC7 antibodies, anti-TNF-o! antibodies, integrins, Ig-superfamily molecules, selectins as well as drugs blocking chemokines and their respective receptor interactions, drugs blocking IL2/IL2 -receptor interaction and other conventional immunosuppressive drugs such as IL-2R monoclonal antibodies, IL-Toxins and IL-Muteins.
  • costimulatory molecules and their ligands are described in the prior art, e.g., in Schwartz, Cell 71 (1992), 1065-1068.
  • CD2 antigen and its ligand LFA-3 are described in Bagogui Li et al., review in Adhesion Molecules, Fusion proteins, Novel Peptides, and Monoclonal Antibodies, Recent Developments in Transplantation Medicine, Vol. II, 1995, Physicians&Scientists Publishing Co., Inc. and blocking of their interaction by using of monoclonal antibodies (anti-Leu- 5b, OKT11, Til) is reported in Bromberg, Transplantation 51 (1991) 219-225 or CD2.1gGl fusion protein. The use of monoclonal Abs against CD4 molecule is described in Cosimi, Surgery 108 (1990), 406-414. CD47 blockade by monoclonal antibodies is described by Rheinho Id, J.
  • Integrins and Ig-superfamily molecules include LFA-1 with its ligand ICAM-1, -2, -3, Mac-1 with its ligand ICAM-1, -3; ICAM-1 with its ligand LFA-1, Mac-1, CD43; ICAM-2 with its ligand LFA-1; ICAM-3 with its ligand LFA-1, Mac-1; VLA4 and VCAM-1 see, e.g., David, Adams, review in Adhesion Molecules, Fusion proteins, Novel Peptides, and Monoclonal Antibodies, Recent Developments in Transplantation Medicine, Vol.
  • VLA-4 monoclonal antibodies to the alpha4 integrin chain CD49d
  • betal integrin chain CD29
  • an activation-induced neo-epitope of VLA-4 as well as soluble VLA-4 ligands such as soluble fibronectin or its relevant peptide (GPEILDVPST), or soluble VCAM-1 or its relevant peptide.
  • More selectively blocking agents are antisense ohgonucleotides, designed to selectively hybridize with cytoplasmic alpha4, betal, or VCAM-1 mRNA; Fedoseyeva, J. Immunol. 57 (1994), 606-612.
  • Another example is the drug pentoxifylline (PTX) that is able to block expression of VCAM-1; Besler, J. Leukoc. Biol. 40 (1986), 747-754.
  • PTX drug pentoxifylline
  • VCAM-1 monoclonal antibody, M/K-2, anti-murine for example could prolong allograft survival, Orosz, Transplantation, 56 (1993), 453-460.
  • compositions of the invention are administered to an individual in a effective dose sufficient to treat or diagnose disorders in which modulation of BGP -related activity is indicated.
  • the effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
  • the pharmaceutical compositions may be provided to the individual by a variety of routes such as by intracoronary, intraperitoneal, subcutaneous, intravenous, transdermal, intrasynovial, intramuscular or oral routes. In addition, co-administration or sequential administration of other agents maybe desirable.
  • a therapeutically effective dose refers to that amount of antibodies, antigen-binding fragments, polynucleotides and vectors of the invention ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • ED50 the dose therapeutically effective in 50% of the population
  • LD50 the dose lethal to 50% of the population.
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • the present invention relates to the use of the antibody and the antigen of the invention for the preparation of a pharmaceutical composition for inhibition of an immune response, preferably for the treatment of graft versus host disease, autoimmune diseases, allergic diseases, infectious diseases, sepsis, for the treatment of tumors, for the improvement of wound healing or for inducing or maintaining immune unresponsiveness in a subject wherein in certain circumstances one would want to inhibit (e.g., GVHD, etc.) or enhance (e.g., anti- tumor response) T cell activity.
  • inhibit e.g., GVHD, etc.
  • enhance e.g., anti- tumor response
  • the present invention also relates to a method of modulating the immune response in a subject in need thereof, comprising administering the antibody or antigen- binding fragment of the invention.
  • Compositions comprising the antibody or the antigen- binding fragment of this invention can be added to cells in culture (in vitro) or used to treat patients, such as mammals (in vivo).
  • the polypeptide is preferably combined in a pharmaceutical composition with a pharmaceutically acceptable carrier such as a larger molecule to promote polypeptide stability or a pharmaceutically acceptable buffer that serves as a carrier for the antibodies that has more than one antibody coupled to a single entity.
  • the methods of the invention include administering to a patient, preferably a mammal, and more preferably a human, the composition of the invention in an amount effective to produce the desired effect.
  • the antibody or the antigen can be administered as a single dose or in multiple doses.
  • Useful dosages of the active agents can be determined by comparing their in vitro activity and the in vivo activity in animal models. Methods for extrapolation of effective dosages in mice, and other animals, to humans are known in the art.
  • the present invention also provides a method of modulating (e.g., activating or inhibiting) immune cell, preferably T lymphocytes, activation, proliferation, and/or differentiation that includes contacting an immune cell with an antibody or the antigen-binding fragment described above. Given the fact that C-CAMl is expressed by dendritic cells at least in mouse (and probably in human), the antibodies of the present invention may disrupt the initiation of an immune response in addition to arresting an ongoing immune response.
  • the present invention encompasses any use of a ligand binding molecule comprising at least one CDR of the above described antibody, in particular for diagnosing and/or treatment of a disorder related to the expression or malfunction of BGP(C-CAMl).
  • said ligand binding molecule is an antibody of the present invention or an immunoglobulin chain thereof.
  • the present invention relates to anti-idiotypic antibodies of the any one of the mentioned monoclonal antibodies described hereinbefore. These are antibodies or other binding molecules which bind to the unique antigenic peptide sequence located on an antibody's variable region near the antigen binding site.
  • the biological activity of the antibodies identified here suggests that they have sufficient affinity to make them potential candidates for drug localization to cells expressing the appropriate surface structures.
  • This targeting and binding to cells could be useful for the delivery of therapeutically or diagnostically active agents (including targeting drugs, DNA sequences, RNA sequences, lipids, proteins (e.g., human growth factors)) and gene therapy/gene delivery.
  • the therapeutically active agent is an anti- inflammatory agent.
  • Molecules/particles with an antibody of the invention would bind specifically to cells/tissues expressing BGP, and therefore could have diagnostic and therapeutic use.
  • the antibody or the antigen of the present invention can be labeled (e.g., fluorescent, radioactive, enzyme, nuclear magnetic) and used to detect specific targets in vivo or in vitro including "immunochemistry" like assays in vitro. In vivo they could be used in a manner similar to nuclear medicine imaging techniques to detect tissues, cells, or other material expressing BGP, in particular the N-domain thereof.
  • Another method involves delivering a therapeutically active agent to a patient.
  • the method includes administering at least one antibody or the antigen-binding fragment and the therapeutically active agent to a patient.
  • the therapeutically active agent is selected from drugs, DNA sequences, RNA sequences, proteins, lipids, and combinations thereof. More preferably, the therapeutically active agent is an antibacterial agent, anti-inflammatory agent, or antineoplastic agent.
  • the therapeutically or diagnostically active agent can be coupled to the antibody of the invention or an antigen-binding fragment thereof by various means.
  • variable regions of the antibody of the invention can be constructed into Fv molecules and coupled to alternative ligands such as those illustrated in the cited article.
  • Higgins, J. Infect Disease 166 (1992), 198-202 described a hetero-conjugate antibody composed of OKT3 cross-linked to an antibody directed to a specific sequence in the V3 region of GP120.
  • Such hetero-conjugate antibodies can also be constructed using at least the variable regions contained in the antibody of the invention methods. Additional examples of specific antibodies include those described by Fanger, Cancer Treat. Res. 68 (1993), 181-194 and by Fanger, Crit. Rev. Immunol. 12 (1992), 101-124.
  • Conjugates that are immunotoxins including conventional antibodies have been widely described in the art.
  • the toxins may be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin portions can be produced as fusion proteins.
  • the antibodies of the present invention can be used in a corresponding way to obtain such immunotoxins. Illustrative of such immunotoxins are those described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and by Fanger, Immunol. Today 12 (1991), 51-54.
  • the present invention relates to a diagnostic composition comprising any one of the above described antibodies, antigen-binding fragments, polynucleotides, vectors or cells of the invention and optionally suitable means for detection.
  • the antibodies of the invention are, for example, suited for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • immunoassays which can utilize the antibody of the invention are competitive and non-competitive immunoassays in either a direct or indirect format.
  • examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay), flow cytometry and the Western blot assay.
  • RIA radioimmunoassay
  • sandwich immunometric assay
  • flow cytometry flow cytometry
  • Western blot assay Western blot assay.
  • the antigens and antibodies of the invention can be bound to many different carriers and used to isolate cells specifically bound to said polypeptides.
  • Examples of well known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble or insoluble for the purposes of the invention.
  • the antibodies of the invention may also be used in a method for the diagnosis of BGP-related diseases in an individual by obtaining a body fluid sample from the tested individual which may be a blood sample, a lymph sample or any other body fluid sample and contacting the body fluid sample with an antibody of the invention under conditions enabling the formation of antibody-antigen complexes.
  • the level of such complexes is then determined by methods known in the art, a level significantly higher than that formed in a control sample indicating the disease in the tested individual.
  • the specific antigen bound by the antibodies of the invention may also be used.
  • the present invention relates to an in vitro immunoassay comprising the antibody or the antigen of the invention.
  • EXAMPLE 1 Hybridomas that secrete BGP(C-CAMl) specific antibodies
  • the 34B1, 26H7 and 5F4 monoclonal antibodies (niAbs) were produced by immunizing BALB/c mice with the activated human mucosal lymphocyte line, 191 E, as previously described (Russell et al., J. Immunol. 157 (1996), 3366-3374).
  • Three intraperitoneal injections and a final intravenous injection of 5 x 10 6 lymphocytes were given at two-week intervals.
  • Hybridomas were selected with aminopterin-containing medium, and hybridoma supernatants were screened by indirect immunoperoxidase staining of frozen intestinal and tonsillar tissue sections. Positive hybridomas were subcloned twice by limiting dilution, and ascites containing the antibody was produced by intraperitoneal injection of the hybridoma cells into pristane-treated BALB/c mice.
  • the isotypes of 34B1 (IgGl), 26H7 (IgGl) and 5F4 (IgGl) were determined by ELISA using murine isotype-specific mAb (Boehringer Mannheim, Indianapolis, IN).
  • W6/32 is a mouse IgG2a mAb specific for human MHC class I (kindly provided by Dr. Jack Strominger, Dana Farber Cancer Institute, Boston, MA).
  • OKT3 (IgG2a is a mouse anti-human CD3 mAb (kindly provided by Dr. Robert Finberg, Dana-Farber Cancer Institute, Boston, MA).
  • TS2/18 (kindly provided by Dr.
  • OKTl 1 (kindly provided by Dr. Ellis Reionherz, Dana- Farber Cancer Institute) is an anti-CD2 mAb (mouse IgG29).
  • OKT4 and OKT8 are mouse IgG2a mAbs specific for human CD4 and CD8-oc, respectively (obtained from American Type Culture Collection, Bethesda, MD).
  • UCHT1 directly conjugated to phycoerythrin, is a mouse IgGl mAb specific for human CD3-E (Dako, Denmark).
  • the MA22 (CD66a; clone YG-C94G7; IgGl), MA26 (CD66a; clone 4.3.17; IgGl), MA27 (CD66e; clone 26/5/1; IgG2a), MA28 (CD66e; clone 26/3/13; IgGl), MA30 (CD66c; clone 9A6; IgGl), MA41 (CD66b; clone BIRMA17c; IgGl), MA61 (CD66b; clone 80H3; IgGl), MA76 (CD66ae; clone 12-140-4; IgGl), MA79 (CD66b; clone B13.9; IgGl), MA81 (CD66b; clone G1OF5; IgGl), MA83 (CD66e; clone b7.8.5; IgGl), MA84 (CD66de; clone COL-1; IgG
  • the isotype matched mouse IgGl negative control mAb was purchased from Kakopatts (Copenhagen, Denmark) or Cappel (West Chester, PA). mAbs were purified by affinity purification and protein-A or G sepharose columns by standard methods.
  • Hybridoma 34B1 which produces monoclonal antibody 34B1
  • Hybridoma 26H7 which produces monoclonal antibody 26H7, was deposited with the ATCC on September 4, 2002, and assigned Accession Number PTA-4629;
  • Hybridoma 5F4 which produces monoclonal antibody 5F4, was deposited with the ATCC on September 4, 2002, and assigned Accession Number PTA-4630.
  • monoclonal antibodies 34B1, 26H7 and 5F4 produced by hybridomas ATCC PTA-4631, ATCC PTA-4629, and ATCC PTA-4630, inhibit T cell cytotoxicity and proliferation in an allogeneic mixed lymphocyte reaction in vitro (see below). Furthermore, they inhibit cytokine production (interferon - gamma) as well.
  • PBMC peripheral blood mononuclear cells
  • Phytohemagglutinin-A (Murex, Dartford, U.K.) or PBS was added to control wells. Cells were incubated at 37°C and 5% CO 2 for 5 days. At 18 h prior to harvest, 1 ⁇ Ci of 3 H-thymidine (New England Nuclear, Boston, MA) was added to each well. The cells were washed and harvested onto filters with a Tomtec Harvester 96 Mach II (Wallac, Gaithersburg, MD). After drying the filters, radioactivity was counted in the presence of scintillation fluid with a 2500 Betaplate liquid scintillation counter (Wallac). Each condition was performed in triplicate. Mean radioactivity incorporated by cells is reported Figs. 1-3 for various treatments including the antibodies 34B1 (Fig. 1), 26H7 (Fig. 2) and 5F4 (Fig. 3).

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Abstract

L'invention concerne des anticorps spécifiques capables de moduler l'activité des lymphocytes T et leurs applications. L'invention concerne plus particulièrement des anticorps et des fragments de liaison de ces derniers qui réagissent avec un domaine spécifique de la glycoprotéine biliaire (BGP), également désignée par CD66a, CEACAM1 et C-CAM1, et qui sont capables de supprimer l'activité cytolytique des lymphocytes intra-épithéliaux (LIE) intestinaux. L'invention concerne également des compositions contenant ces anticorps, ainsi que des méthodes de modulation de la prolifération de cellules immunitaires et de traitement de maladies liées à la réponse immunitaire.
PCT/US2003/028416 2002-09-11 2003-09-11 Anticorps anti-bgp(c-cam1) therapeutiques et leurs applications WO2004024746A2 (fr)

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