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WO1996031604A1 - Anticorps pour la detection de hla-g - Google Patents

Anticorps pour la detection de hla-g Download PDF

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Publication number
WO1996031604A1
WO1996031604A1 PCT/US1996/003765 US9603765W WO9631604A1 WO 1996031604 A1 WO1996031604 A1 WO 1996031604A1 US 9603765 W US9603765 W US 9603765W WO 9631604 A1 WO9631604 A1 WO 9631604A1
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WIPO (PCT)
Prior art keywords
hla
antibody
polypeptide
seq
antibodies
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PCT/US1996/003765
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English (en)
Inventor
Susan J. Fisher
Clifford Librach
Caroline Damsky
Michael T. Mcmaster
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The Regents Of The University Of California
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Priority to AU52568/96A priority Critical patent/AU696118B2/en
Priority to JP8530319A priority patent/JPH11503320A/ja
Priority to EP96908869A priority patent/EP0819171A1/fr
Publication of WO1996031604A1 publication Critical patent/WO1996031604A1/fr

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    • 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
    • C07K16/2833—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 against MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • BACKGROUND OF THE INVENTION A central question in pregnancy is how the fetal-placental unit avoids maternal immune rejection. Although fetal and maternal cells interact throughout pregnancy, the fetus typically remains a privileged site, not subject to rejection. It is likely that the particular nature of the cells at the fetal-maternal interface and their products help prevent rejection of the fetus by the maternal immune system.
  • the morula After fertilization in the oviduct, a series of cell divisions create a mass of totipotent cells (the morula).
  • the first differentiation event occurs after compaction of the morula, leading to formation of the blastocyst.
  • Cells of the trophoblast lineage are formed based upon their position in the morula in a complex cascade of inter- and intra-cell signaling events.
  • implantation of the blastocyst occurs shortly after the blastocyst hatches from the zona pellucida.
  • the uterus is made receptive to implantation as a result of events controlled largely by production of estrogen and progesterone from the ovaries.
  • trophoblasts attach to the receptive uterine epithelium initiating several changes in the endometrium.
  • Vascular changes occur, such as increased permeability of uterine blood vessels, and inflammatory cells are recruited to the implantation site.
  • Proinflammatory cytokines are produced in the uterus and several cellular changes occur.
  • the uterine epithelium is lost and decidual cells undergo an epithelioid transition and proliferate, producing a massively thickened uterine wall.
  • the decidua also contains abundant macrophages, lymphocytes and other bone-marrow derived cells with unusual properties such as reduced alloreactivity, and responsiveness to stimulation by CD3 antibody.
  • HLA-G The molecule HLA-G, which is expressed by placental cells, was cloned in a search for novel class I genes encoded by the human MHC (Geraghty et al. (1987) Proc. Nat/. Acad. Sci. U. S. A. 84: 9145).
  • the gene has an intron/exon organization identical to that of the class la genes (HLA-A, -B and -C), and the HLA-G protein product has 86% sequence identity to the class I consensus sequence (Parham et al. (1988) Proc. Nat/. Acad. Sci. U. S. A. 85: 4005).
  • the primary HLA-G RNA transcript is also differentially spliced; in addition to the full length mRNA, transcripts are produced that lack either exon two, both exons two and three (Ishitani and Geraghty (1992) Proc. Natl. Acad. Sci. U. S. A. 89: 3947), or exon four (Kirszenbaum etal. (1994) Proc. Natl. Acad. Sci. U. S. A, 91 : 4209). To what extent these alternatively spliced mRNAs are translated is unclear. Recently, a soluble form of HLA-G encoded by an mRNA containing intron 4 was described (Fujii et al. (1994) J. Immunol. 153: 5516).
  • HLA-G appears to exhibit relatively less polymorphism.
  • Immunoprecipitation of HLA-G from 13 individuals and a human choriocarcinoma (malignant trophoblast) cell line showed identical two-dimensional electrophoretic profiles, suggesting reduced polymorphism at this locus.
  • Genomic and cDNA sequehce data also indicate that HLA-G has relatively limited polymorphism.
  • a recent study suggests that at least in some populations ⁇ i.e., African Americans), HLA-G exhibits substantial polymorphism (van der Ven and Ober (1994) J. Immunol. 153: 5628). Whether HLA-G is complexed with endogenous trophoblast peptides and how this repertoire is affected by its degree of polymorphism remains to be determined.
  • HLA-G is not generally expressed in non-pregnant adults, making it a suitable marker for the diagnosis of pregnancy.
  • the present invention provides antigens for generating specific antibodies to both soluble and membrane bound HLA-G, as well as exemplar antibodies.
  • HLA-G levels in the maternal blood are indicative of the vigor of cytotrophoblast invasion and the corresponding health of the placental-matemal interface.
  • HLA-G is not generally expressed in adults, it is an ideal marker for diagnosing and monitoring pregnancy and for detecting cytotrophoblasts from biological fluids.
  • designing and obtaining suitable antibodies to HLA-G was not previously feasible, due to the high similarity of HLA-G to class la molecules which are expressed in adults. This invention overcomes these problems by providing specific epitopes for generating HLA-G-specific antibodies, exemplar antibodies, and methods for their use.
  • the present invention provides polypeptides which can be used to elicit antibodies which specifically bind to HLA-G. These antibodies are used, for example, to monitor soluble HLA-G levels in maternal blood. Soluble HLA-G levels in the maternal blood can be used to diagnose pregnancy, or to monitor the health of the fetal maternal interface.
  • this invention provides purified polypeptides, comprising a sequence of at least 5 contiguous amino acids selected from an amino acid sequence consisting essentially of amino acid residues 61 to 83 of the ⁇ 1 domain of the human HLA-G protein, wherein said polypeptide, when presented as an immunogen, elicits the production of an antibody which specifically binds to HLA-G, and wherein said peptide does not bind to antisera raised against HLA-G which has been fully immunosorbed with the peptide of Seq. Id. No. 1 .
  • An exemplar polypeptide is the polypeptide which consists or essentially of the sequence EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. No. 1 ).
  • polypeptides are useful as components of an immunogenic composition.
  • the peptide(s) are covalently linked to additional polypeptides such as an immunogenic carrier (e.g., keyhole limpet hemocyanin).
  • an immunogenic carrier e.g., keyhole limpet hemocyanin.
  • the present invention provides nucleic acids encoding the polypeptides described above which comprise a sequence of at least 5 contiguous amino acids selected from an amino acid sequence consisting essentially of amino acid residues 61 to 83 of the ⁇ 1 domain of the human HLA-G protein, wherein said polypeptide, when presented as an immunogen, elicits the production of an antibody which specifically binds to HLA-G, and wherein said peptide does not bind to antisera raised against HLA-G which has been fully immunosorbed with the peptide of Seq. Id. No. 1.
  • the present invention encodes a polypeptide substantially identical to the sequence EEETRNTKAHAQTDRMNLQTLRG (S
  • Antibodies which specifically bind to a subsequence in the ⁇ 1 domain of HLA-G, and methods for making these antibodies are provided.
  • the antibodies specifically bind t o s u b s e q u e n c e s o f t h e a m i n o a c i d s e q u e n c e EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. No. 1 ).
  • Exemplar antibodies include 1 B8 and 3F6, described herein. Monoclonal as well as polyclonal antibodies are provided.
  • Recombinant cells which include nucleic acids encoding the polypeptides and antibodies described above are also provided.
  • Exemplar cell lines include 1 B8 and 3F6.
  • detection formats include ELISA, RIA, western blot and other immunoassays.
  • the invention further provides for kits comprising the various elements described above.
  • antibody refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • An exemplar immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
  • Antibodies exist e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2 a dimer of Fab which itself is a light chain joined to V H -C H 1 by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hmge region (see, Fundamental Immunology, Third Edition, W.E.
  • antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology.
  • antibody as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies.
  • biological sample or fluid refers to material derived from a living organism, including, e.g., blood, cervicovaginal secretions, amniotic fluid, cord blood, urine, tissues, bones and cells.
  • blood sample as used herein includes whole blood or derivatives of whole blood well known to those of skill in the art.
  • a blood sample includes the various fractionated forms of blood such as plasma or serum and whole or fractionated blood which additionally comprises various diluents as may be added to facilitate storage or processing in a particular assay.
  • diluents are well known to those of skill in the art and include various buffers, anticoagulants, preservatives and the like.
  • HLA-G refers to human leukocyte antigen G and unless otherwise stated includes both the soluble and insoluble forms.
  • the term may in appropriate context refer to either the antigen or the genetic locus.
  • immunoassay is an assay that utilizes an antibody to specifically bind an analyte.
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, or quantify the analyte.
  • isolated refers to material which is substantially or essentially free from components which normally accompany it as found in its native state.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
  • nucleic acid probe refers to a molecule which binds to a specific sequence or subsequence of a nucleic acid.
  • a probe is preferably a nucleic acid which binds through complementary base pairing to the full sequence or to a subsequence of a target nucleic acid.
  • probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
  • the probes are preferably directly labelled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labelled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
  • polypeptide polypeptide
  • peptide protein
  • the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • a “label” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal a n t i b o d i e s a r e a v a i l a b l e ( e . g . , t h e p e p t i d e EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. No. 1 ) can be made detectible, e.g., by incorporating a radiolabel into the peptide, and used to detect antibodies specifically reactive with the peptide).
  • a "labeled nucleic acid probe” is a nucleic acid probe that is bound, either covalently, through a linker, or through ionic, van der
  • recombinant when used with reference to a cell indicates that the cell contains nucleic acid with an origin exogenous to the cell. Thus, for example, recombinant cells replicate and/or express genes that are not found within the native (non-recombinant) form of the cell.
  • nucleic acid or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman Adv. AppI. Math. 2: 482 (1981 ), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci.
  • polypeptide comprises a sequence with at least 80% sequence identity to a reference sequence, or preferably 90%, or more preferably 95% sequence identity to the reference sequence, over a comparison window of about 20 amino acid residues.
  • An indication that two polypeptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
  • a polypeptide is substantially identical to a second polypeptide where the two peptides differ only by a conservative substitution.
  • nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • nucleic acid sequences are substantially identical.
  • Stringent conditions are sequence dependent and will be different with different environmental parameters. Generally, stringent conditions are selected to be about 5° C to 20 °C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical.
  • the phrases “specifically binds to” or “specifically hybridizes to” or “specifically immunoreactive with”, when referring to an antibody refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologies.
  • the specified antibodies bind preferentially to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein.
  • antibodies can be raised to the peptide EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. No. 1 ) which specifically bind to proteins comprising the sequence EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. No. 1 ) (such as HLA-G) and not to other proteins present in a blood sample.
  • EEETRNTKAHAQTDRMNLQTLRG Seq. Id. No. 1
  • solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • a particular antibody and the cell which produces the antibody are often referred to by the same designation.
  • the monoclonal antibody 1 B8 is produced by the immortalized cell line 1 B8.
  • Human placental trophoblasts lie at the maternal-fetal interface, mediating maternal tolerance of the fetus. Central to this mediation is their unusual MHC class I expression; they suppress class la production while expressing HLA-G, a class lb molecule.
  • a synthetic peptide corresponding to the region from amino acids 61 to 83 of the ⁇ 1 domain of the HLA-G protein was used to produce monoclonal antibodies which specifically bound HLA-G.
  • the epitope has the advantage of being a linear epitope rather than a conformational epitope. It is available to the antibody without denaturation. It is highly antigenic and produces high titer, high avidity antibodies that detect all forms of HLA-G. Prior to the present invention, there was no way of knowing whether the synthetic peptide would be specific to HLA-G, or whether the conformation of native HLA-G would permit antibody binding to this region.
  • Antibody specificity was demonstrated by immunoaffinity purification of HLA-G from choriocarcinoma cells. These antibodies were incubated with tissue sections of the maternal-fetal interface containing cytotrophoblasts in all stages of differentiation, demonstrating that HLA-G is expressed only by cytotrophoblasts which invade the uterus. In vitro studies presented herein show that when early-gestation cytotrophoblast stem cells are cultured they differentiate rapidly along the invasive pathway, upregulating HLA-G production. Cytotrophoblasts from term placentas, which have reduced invasive capacity in vitro, also had decreased ability to upregulate HLA-G protein expression in vitro.
  • HLA-G mRNA High levels of HLA-G mRNA were detected in cytotrophoblasts isolated from first and second trimester placentas that had high invasive capacity. In comparison, term cells contained a greatly reduced level of HLA-G mRNA. Taken together, these results show that HLA-G production is a component of cytotrophoblast differentiation along the invasive pathway.
  • This invention provides methods and compositions for the generation and use of antibodies which specifically recognize HLA-G.
  • the present invention can be used in conjunction with other techniques such as PCR, TAS, 3SR, QB amplification and cloning, to amplify a any nucleic acid in a biological sample.
  • the nucleic acids of the present invention encode the region of the ⁇ 1 domain of HLA-G which is specific to HLA-G, i.e., that region of the ⁇ 1 domain which exhibits the least similarity to other class I molecules.
  • the flanking regions of nucleic acid which encodes amino acid residues 61 to 83 of the ⁇ 1 domain of the HLA-G protein can be used as a primer binding site for amplification.
  • the products can be used in combination with conventional expression systems to generate the peptide of Seq. ID. No. 1 for purposes of obtaining antigen for antibody production.
  • the nucleic acids of the present invention may be cloned, or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription- based amplification system (TAS), the self-sustained sequence replication system (3SR) and the Q ⁇ replicase amplification system (QB).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription- based amplification system
  • 3SR self-sustained sequence replication system
  • QB Q ⁇ replicase amplification system
  • Seq. ID. No. 1 are also well known. Automatic peptide synthesizers are available commercially from a variety of sources.
  • a number of immunogens may be used to produce antibodies specifically reactive with HLA-G polypeptides.
  • Recombinant or synthetic polypeptides comprising the region from amino acid residues 61 to 83 of the ⁇ 1 domain of the human HLA-G protein (EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. No. 1)) are the preferred polypeptide immunogen for the production of monoclonal or polyclonal antibodies.
  • EEETRNTKAHAQTDRMNLQTLRG Seq. Id. No. 1
  • the entire HLA-G moiety, or a subsequence thereof especially the ⁇ 1 domain of HLA-G; see, Geraghty etal. (1987) Proc. Natl. Acad. Sci.
  • Patent No. 4,722,848 is mixed with an adjuvant and animals are immunized with the mixture.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the polypeptide of interest.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the polypeptide is performed where desired. See, e.g. , Coligan (1991 ) Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane ( 1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY, which are incorporated herein by reference, and the examples below.
  • Antibodies, including binding fragments and single chain recombinant versions thereof, against predetermined fragments of polypeptides can be raised by immunization of animals with conjugates of the fragments with carrier proteins as described above.
  • the immunogen of interest is a peptide of at least about 3 amino acids, and more typically the peptide is 5 amino acids in length or greater.
  • the peptides are typically coupled to a carrier protein, or are recombinantly expressed in an immunization vector.
  • Antigenic determinants on peptides to which antibodies bind are typically 3 to 10 amino acids in length.
  • Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or modified polypeptides, or screened for agonistic or antagonistic activity, e.g. , activity mediated through membrane-bound HLA-G. Specific monoclonal and polyclonal antibodies will usually bind with a K D of at least about 500 ⁇ M, and most preferably at least about 1 M or better.
  • monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, etc.
  • Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane, Supra; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, NY; and Kohler and Milstein (1975) Nature 256: 495-497. Summarized briefly, this method involves injecting an animal with an immunogen.
  • hybridomas The animal is then sacrificed and cells are taken from its spleen, which are then fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro.
  • the population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
  • Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells is enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.
  • the polypeptides and antibodies of the present invention are used with or without modification, and include chimeric antibodies such as humanized murine antibodies.
  • Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al.
  • the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275, 149; and 4,366,241. Also, recombinant immunoglobulins may be produced. See, Cabilly, U.S. Patent No. 4,816,567; and Queen et al. (1989) Proc. Nat'/ Acad. Sci. USA 86: 10029-10033.
  • the antibodies of this invention can also be used for affinity chromatography to isolate HLA-G polypeptides.
  • Columns can be prepared, e.g., with the antibodies linked to a solid support, e.g. , particles, such as agarose, Sephadex, or the like, where a cell lysate is passed through the column, washed, and treated with increasing concentrations of a mild denaturant, whereby purified HLA-G polypeptides are released.
  • the antibodies can be used to screen expression libraries for particular expression products such as HLA-G or for histology studies to locate HLA-G expressing cells. Usually the antibodies in such a procedure will be labeled with a moiety allowing easy detection of presence of antigen by antibody binding. b. Immunoassays
  • Concentration of HLA-G in a biological sample can be measured by a variety of immunoassay methods.
  • immunological and immunoassay procedures in general, see Stites and Terr (eds.) 1991 Basic and Clinical Immunology (7th ed.).
  • the immunoassays of the present invention can be performed in any of several configurations, e.g., those reviewed in Maggio (ed.) (1980) Enzyme Immunoassay CRC Press, Boca Raton, Florida; Tijan (1985) " Practice and Theory of Enzyme Immunoassays, " Laboratory Techniques in Biochemistry and Molecular Biology , Elsevier Science Publishers B.V., Amsterdam; Harlow and Lane, supra; Chan (ed.) (1987) Immunoassay: A Practical Guide Academic Press, Orlando, FL; Price and Newman (eds.) (1991) Principles and Practice of Immunoassays Stockton Press, NY; and Ngo (ed.) (1988) Non-isotopic Immunoassays Plenum Press, NY.
  • Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte.
  • the labeling agent may itself be one of the moieties comprising the antibody/analyte complex.
  • the labeling agent may be a labeled HLA-G peptide or a labeled anti-HLA-G antibody.
  • the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/HLA-G complex, or to a modified capture group (e.g., biotin) which is covalently linked to the HLA-G peptide or anti-HLA-G antibody.
  • the labeling agent is an antibody that specifically binds to the capture agent (anti-HLA-G).
  • anti-HLA-G antibodies that specifically bind antibodies of the particular animal species from which the capture agent is derived.
  • the label agent may be a goat anti-mouse IgG, i.e., an antibody specific to the constant region of the mouse antibody.
  • proteins capable of specifically binding immunoglobulin constant regions such as streptococcal protein A or protein G may also be used as the label agent. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species. See, generally Kronval, et al., J. Immunol., 1 1 1 : 1401-1406 (1973), and Akerstrom, et al., J. Immunol. , 135:2589-2542 (1985).
  • Immunoassays for detecting HLA-G may be either competitive or noncompetitive.
  • Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case HLA-G) is directly measured.
  • the capture agent anti-HLA-G antibodies
  • these immobilized antibodies then capture HLA-G present in the test sample.
  • the HLA-G thus immobilized is then bound by a labeling agent, such as a second HLA-G antibody bearing a label.
  • the second HLA-G antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • Sandwich assays for detecting and/or quantitating HLA-G may be constructed. As described above, immobilized anti-HLA-G specifically binds to HLA-G present in the sample via the epitope defined by Seq. ID. No. 1. Then the second labeled anti-HLA-G (not necessarily HLA-G specific) binds to the bound HLA-G molecule. Free labeled antibody is then washed away and the remaining bound labeled anti- HLA-G is detected ⁇ e.g., using a gamma detector where the label is radioactive).
  • the amount of analyte (HLA-G) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte displaced (or competed away) from a capture agent (anti HLA-G antibody) by the analyte present in the sample.
  • a capture agent in this case an antibody that specifically binds the analyte.
  • the amount of analyte bound to the antibody is inversely proportional to the concentration of analyte present in the sample.
  • the capture agent is immobilized on a solid substrate.
  • the amount of HLA-G bound to the capture agent may be determined either by measuring the amount of HLA-G present in an HLA-G/antibody complex, or alternatively by measuring the amount of remaining uncomplexed HLA-G.
  • the amount of HLA-G may be detected by providing a labeled HLA-G.
  • a hapten inhibition assay is another preferred competitive assay.
  • a known analyte in this case HLA-G is immobilized on a solid substrate.
  • a known amount of anti-HLA-G antibody is added to the sample, and the sample is then contacted with the immobilized HLA-G.
  • the amount of anti-HLA-G antibody bound to the immobilized HLA-G is proportional to the amount of HLA-G present in the sample.
  • the amount of immobilized antibody may be measured by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above.
  • HLA-G peptides may be defined by their binding properties to antibodies and antisera. As described in the definitions section herein, one indication that two peptides are substantially similar is that they both specifically bind to the same antibody or antibodies. Conversely, if peptides are substantially similar, one peptide is not specifically bound by an antibody which does not specifically bind the second peptide. Thus, the peptides of the present invention can be defined by their binding properties in the various immunoassays described herein. For instance, antisera raised against HLA-G which is immunosorbed with the peptide of Seq. Id . No. 1 (EEETRNTKAHAQTDRMNLQTLRG (Seq. Id. No.
  • HLA-G is used to immunize mice from an inbred strain, such as BALB/c, using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see Harlow and Lane, supra and the procedures described herein).
  • a synthetic peptide derived from HLA-G and conjugated to a carrier protein or a peptide vector containing the sequence can be used an immunogen.
  • Polyclonal sera are collected and titered against the immunogen protein in an immunoassay as described herein, for example, a solid phase immunoassay with the immunogen ⁇ e.g., HLA-G synthetic peptide) immobilized on a solid support.
  • Polyclonal antisera with a titer of 10 4 or greater are selected and tested for their ability to bind HLA-G, using a competitive binding immunoassay as described above, e.g., using generic competitors which are unrelated to HLA-G (e.g., bovine serum albumin).
  • Antisera which bind HLA-G are then selected for characterization of a peptide of interest, i.e., one which may be substantially identical to the peptide of SEQ Id. No. 1.
  • No. 1 have an affinity for both the immunoabsorbed polyclonal antisera (the antisera which does not specifically bind to the peptide of Seq. Id. No. 1 ) and the antibodies isolated during the immunoabsorbtion process (the antisera which does bind the peptide of Seq. Id. No. 1 ) which is the same within the experimental error of the system, plus or minus 10%, the polypeptides are substantially identical.
  • the experimental error of the system is monitored by using identical polypeptides as controls from one experiment to the next, i.e., identical polypeptides have the same antibody binding properties, and any discrepancies reflect the experimental error in the system.
  • Soluble HLA-G is preferably quantified in a biological sample derived from a patient.
  • biological samples include blood and cervicovaginal secretions.
  • U.S. Patent No. 5,096,830 describes cervicovaginal secretions as diagnostic assay samples, and provides means for taking such samples.
  • HLA-G is quantified in whole blood or blood derivatives such as blood plasma or blood serum.
  • Blood samples are isolated from a patient according to standard methods well known to those of skill in the art, most typically by venipuncture. The sample may be pretreated as necessary by dilution in an appropriate buffer solution or concentrated, if desired. Any of a number of standard aqueous buffer solutions, employing one of a variety of buffers, such as phosphate, Tris, or the like, at physiological pH can be used.
  • HLA-G may be detected and quantified by any of a number of means well known to those of skill in the art. These include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.
  • analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like
  • immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIA), enzyme
  • the biological sample is enriched for soluble HLA-G, e.g., by removing non-HLA-G components prior to performing the assay. It is often desirable to fractionate the biological sample prior to performing the above-described techniques to increase the sensitivity of any of the assays above for the detection of soluble HLA-G.
  • an initial rough separation of soluble HLA- G from other biological components can be performed by appropriate centrifugation, filtration, column chromatography, or isotonic washing of the biological sample.
  • certain non-HLA-G components of the biological sample can be specifically removed from the sample using the techniques described herein, where capture agents specific for non- HLA-G components are used to remove those non-HLA-G components from the sample.
  • erythrocytes can be specifically removed from a biological sample comprising blood by, e.g, immunoadsorbtion or affinity chromatography.
  • Western blot analysis can also be used to detect and quantify the presence of HLA-G in the sample.
  • the technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind HLA-G.
  • a suitable solid support such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter
  • the anti-HLA-G antibodies specifically bind to HLA-G on the solid support.
  • These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies where the antibody to HLA-G is a murine antibody) that specifically bind to the anti-HLA-G.
  • LISA liposome immunoassays
  • liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see, Monroe et al., Amer. Clin. Prod. Rev. 5:34-41 ( 1986)), which is incorporated herein by reference.
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the antibody used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • detectable labels have been well-developed in the field of immunoassays and, in general, most labels useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g. DynabeadsTM), fluorescent dyes (e.g.
  • radiolabels e. ⁇ -, 3 H, 125 l, 35 S, 4 C, or 32 P
  • enzymes e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA
  • colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation of the compound, stability requirements, available instrumentation, and disposal provisions.
  • Non-radioactive labels are often attached by indirect means.
  • a ligand molecule e.g., biotin
  • the ligand then binds to an anti-ligand ⁇ e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a signal system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
  • a number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
  • Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
  • Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
  • Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence, e.g., by microscopy, visual inspection, via photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected simply by observing the color associated with the label.
  • conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • Some assay formats do not require the use of labeled components.
  • agglutination assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
  • the solid surface may be a membrane (e.g. , nitrocellulose), a microtiter dish ⁇ e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick ⁇ e.g. glass, PVC, polypropylene, polystyrene, latex, and the like), a microcentrifuge tube, or a glass, silica, plastic, metallic or polymer bead.
  • the desired component may be covalently bound, or noncovalently attached through nonspecific bonding.
  • organic and inorganic polymers both natural and synthetic may be employed as the material for the solid surface.
  • Illustrative polymers include polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and the like.
  • Other materials which may be employed, include paper, glasses, ceramics, metals, metalloids, semiconductive materials, cements or the like.
  • substances that form gels such as proteins ⁇ e.g., gelatins), lipopolysaccharides, silicates, agarose and poiyacrylamides can be used.
  • Polymers which form several aqueous phases such as dextrans, polyalkylene glycols or surfactants, such as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like are also suitable. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.
  • a plurality of different materials may be employed, e.g., as laminates, to obtain various properties.
  • protein coatings such as gelatin can be used to avoid non ⁇ specific binding, simplify covalent conjugation, enhance signal detection or the like.
  • the surface will usually be polyfunctional or be capable of being polyfunctionalized.
  • Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like.
  • the manner of linking a wide variety of compounds to various surfaces is well known and is amply illustrated in the literature. See, for example, Immobilized Enzymes, Ichiro Chibata, Halsted Press, New York, 1978, and Cuatrecasas, J. Biol. Chem. 245 3059 (1970) which are incorporated herein by reference.
  • Noncovalent binding is typically nonspecific absorption of a compound to the surface.
  • the surface is blocked with a second compound to prevent nonspecific binding of labeled assay components.
  • the surface is designed such that it nonspecifically binds one component but does not significantly bind another.
  • a surface bearing a lectin such as Concanavalin A will bind a carbohydrate containing compound but not a labeled protein that lacks glycosylation.
  • Various solid surfaces for use in noncovalent attachment of assay components are reviewed in U.S. Patent Nos. 4,447,576 and 4,254,082, which are incorporated herein by reference.
  • the assays of the present invention are used to quantify average soluble HLA-G levels in a reference population of pregnant women for comparison to, e.g., a particular pregnant patient.
  • Any of the techniques described herein for quantitating an analyte are used to quantify HLA-G levels in biological samples from a particular population such that an average level of HLA- G in, e.g., the maternal blood is derived for the population.
  • the population is selected such that all members of the population are in the same term of their pregnancy, are all apparently healthy with apparently normal pregnancies, and such that all members of the population are of a similar genetic background (i.e., all members of a single racial sub ⁇ type, e.g., Northern Europeans).
  • This population is termed a "reference population.” Individual patients with the same general genetic background and in the same gestational age of their pregnancy are then compared to the reference population to determine whether their HLA-G levels are normal ⁇ i.e., the same as the reference population).
  • HLA-G e.g., in the blood indicates that a patient is pregnant.
  • the presence of HLA-G in maternal blood may be determined by the methods described above.
  • the level of HLA-G in the maternal blood may be a general indicator of the health of the fetal-maternal interface. Because HLA-G is expressed largely by invasive trophoblasts, an elevated or reduced level of HLA-G in the maternal blood of a patient compared to a reference population could indicate that trophoblast invasion is proceeding abnormally.
  • kits for the diagnosis of pregnancy and disease states related to abnormal levels of circulating HLA-G levels preferably include an antibody that specifically binds to HLA-G.
  • the antibody may be free or immobilized on a solid support as described above.
  • the kit may also contain, e.g., instructional materials teaching the use of the antibody in an assay for the detection of pregnancy, appropriate diluents, chemical reagents and the like.
  • nucleic acid probes used in previous studies resulted in artifacts from cross reaction of the probes with class la mRNAs present in the villus core.
  • northern hybridization using probes corresponding to the 450 bp Pvu II fragment from the 3' untranslated region of the HLA-G cDNA cross-reacted with class la mRNAs under all but the most stringent conditions See, Examples below.
  • Cytotrophoblast stem cells isolated from early-gestation placentas, rapidly invade extracellular matrices on which they are plated. During this process they switch their repertoire of integrin extracellular matrix receptors, (as do cytotrophoblasts invading the uterus in vivo) and upregulate 92 kDa type IV collagenase synthesis/ activation (Librach et al. (1994) J. Biol. Chem. 269: 17125). Perturbing either adhesion molecule or proteinase function inhibits invasion, suggesting that these molecules play key roles in acquisition of cytotrophoblast invasiveness.
  • Cytotrophoblast proteinase and integrin expression in vitro also varies dramatically as gestation proceeds.
  • Term cytotrophoblasts which have lost their invasive capacity, produce little of the 92 kDa type IV collagenase, fail to undergo integrin switching and contain no detectable mRNA encoding these molecules.
  • first and second trimester cytotrophoblasts upregulated HLA-G expression in culture. Immunolocalization studies performed on tissue sections showed that villus cytotrophoblasts do not react with anti-HLA-G antibodies. Thus, as would be expected, only a small percentage of isolated villus cells produce HLA-G immediately after plating. After 12 h in culture, 62% of first trimester cytotrophoblasts expressed HLA-G. However, it is clear that cytotrophoblast HLA-G expression is regulated differently from that of integrins and proteinases. HLA-G mRNA levels remained constant throughout the culture period. There are two possible explanations for this phenomenon.
  • cytotrophoblast stem cells may contain high levels of HLA-G mRNA; isolating them does not change these levels, but promotes translation.
  • HLA-G mRNA production could be induced to maximal levels during the isolation procedure.
  • the time course of cytotrophoblast HLA-G mRNA production in vitro differs from that of proteinases and adhesion molecules.
  • a significant percentage (42%) of term cells which no longer express antigens involved in invasion, upregulated HLA- G expression in culture even though they contained much lower HLA-G mRNA levels than early gestation cells.
  • Enhanced metalloproteinase expression and integrin switching are characteristics of the early gestation cytotrophoblasts that mediate uterine invasion. Downregulating the expression of these genes is one mechanism that controls cytotrophoblast invasiveness. In contrast, extravillus cytotrophoblasts of all gestational ages retain the ability to upregulate HLA-G expression in vitro.
  • the peptide was coupled to maleimide-activated keyhole limpet hemocyanin (KLH; Imject; Pierce Chemical Co., Rockford, IL) via a C-terminal cysteine (added for this purpose) according to the manufacturer's instructions.
  • KLH keyhole limpet hemocyanin
  • Two-month-old female BALB/c mice (Charles River) were given i.p. injections of 100 ⁇ g peptide-KLH conjugate emulsified in Freund's complete adjuvant and were boosted after 14 days with 50 ⁇ g antigen in incomplete adjuvant. Test bleeds were obtained 10 days later from tail veins, and sera were screened by ELISA (described below) for reactivity against the peptide immunogen.
  • mice exhibiting the best humoral responses were given a final intravenous boost (50 ⁇ g) and sacrificed three days later.
  • Spleen cells were isolated and fused with SP2/0 myeloma cells according to published procedures (Kohler and Milstein ( 1975) Nature 256: 495; and Harlow and Lane (1988), Supra). Cultures were selected in HAT medium (UCSF Cell Culture Facility) and cloned by limiting dilution.
  • the HLA-G antibodies isolated using the above procedure belonged to the IgM class. At least two factors probably contributed to this outcome. First, the mice were immunized for a relatively short period of time before the hybridomas were produced, and early antigenic responses result primarily in IgM-class antibody production. Second, the reporter antibodies used in the hybridoma screening react with IgM as well as IgG. Using the above method, one can reproducibly obtain HLA- G specific antibody producing hybridomas at at least 1 % or greater.
  • HLA-A, -B, -C Two methods to determine whether 1 B8 antibody reacted with class la molecules. Flow cytometry experiments (flow cytometry techniques are described below) indicated that peripheral blood leukocytes isolated from the blood of 12 different individuals did not bind the antibody. In contrast, JEG-3 cells and a B- lymphoblastoid HLA-null cell line (LCL 721.221 ) stably transfected with a vector expressing HLA-G gave strong positive signals when stained with 1 B8 or W6/32, an antibody that recognizes monomorphic determinants of all class I heavy chain/ ?2-microglobulin complexes (Barnstable et al. (1978) Cell 14: 9). However, the parental (untransfected) lymphoblastoid cells reacted with neither antibody. An isotype-matched irrelevant IgM (Sigma) did not react with any of the cells.
  • the placenta and placental bed contain many cells that express class la antigens.
  • a hallmark of human pregnancy is leukocyte infiltration of the decidua (Ferry et al. (1990) Immunology 70: 446) .
  • Floating chorionic villi composed entirely of fetal cells, contain abundant stromal cells and macrophages (Hofbauer cells) which also express class la proteins (Nakamura et al (1990) Hum. Pathol. 21 : 936).
  • immunolocalization studies performed on placental tissue from 26 individuals showed that none of these class la- expressing cell populations stained with HLA-G antibodies; the 1 B8 and 3F6 mAbs reacted only with specific populations of cytotrophoblasts.
  • Hybridomas were screened for reactivity against the peptide immunogen by antibody-capture ELISA according to standard methods (see, Harlow and Lane, supra). Microtiter plates were coated with 50 ⁇ of PBS containing 10 //g/ml of the synthetic peptide, washed three times with PBS and blocked for 1 hour at room temperature with PBS containing 0.02% (v/v) Tween 20, 0.25% BSA (w/v) and 0.02% sodium azide (w/v; blocking buffer).
  • test fluids test sera or hybridoma supernatants
  • test fluids test sera or hybridoma supernatants
  • rabbit anti-mouse IgG conjugated to alkaline phosphatase Jackson Immuno Research Labs., Inc., West Grove PA
  • Reactivity was assessed by adding 50 ⁇ p-nitrophenyl phosphate substrate solution (3 mM PNPP, 0.05 M NaCO 3 , 0.5 mM MgCI 2 , pH 9.5) and measuring the absorbance at 405 nm with a microplate reader (Molecular Devices Inc., Menlo Park, CA).
  • Hybridomas were also screened for their ability to react with cytotrophoblasts in tissue sections of early second trimester (16-18 week) placentas that contained anchoring villi. Double indirect immunofluorescence was performed using hybridoma supernatants and a rat monoclonal IgG against cytokeratin (7D3) .
  • the cytokeratin antigen is present in differentiated epithelial cells, but not on connective tissue, endothelium, muscle or blood cells.
  • Placental tissues were fixed for 30 min with 3% paraformaldehyde in calcium-containing PBS, pH 7.2. They were then incubated in 10 mM glycine to quench unreacted aldehyde groups, infiltrated with 15% sucrose, embedded in OCT (Miles Scientific, Naperville, IL), and frozen in liquid nitrogen. Sections (5 ⁇ m) were cut using a Slee HR cryostat and collected on 22-mm 2 coverslips. Before staining, the sections were washed for 10 min each in PBS and in PBS containing 0.2% BSA. Primary antibodies were applied to the tissue sections as undiluted hybridoma supernatants for 1 h at room temperature.
  • the class of the antibodies selected for further characterization was determined using an ImmunoType Kit (Sigma, St. Louis, MO) according to the manufacturer's instructions.
  • Several antibodies were isolated that bound HLA-G peptide using the ELISA and stained cytotrophoblasts in the first trimester placenta. Two of these antibodies (1 B8 and 3F6), both IgM class antibodies, were further characterized.
  • Example 3 Affinity purification of HLA-G
  • the immunoaff inity column was constructed using the 1 B8 antibody as follows. First, the peptide immunogen was coupled to thiopropyl-activated Sepharose 6B (Pharmacia, Piscataway, NJ) according to the manufacturer's instructions. Antibody was then bound to the matrix by passing 1 B8 ascites fluid, diluted 1 : 10 in PBS, through the column and washing with 20 bed volumes of PBS. That the antibody was specifically bound was demonstrated by eluting the column with 100 mM glycine, pH 2.5. This fraction contained pure IgM as assessed by silver staining of SDS-polyacrylamide gels.
  • the antibody column was used to purify HLA-G from 35 S- labeled JEG-3 choriocarcinoma cell extracts as follows. 1 x 10 7 JEG-3 cells were metabolically labeled by overnight incubation in methionine- and cysteine-free DMEM (Gibco, Gaithersburg, MD) containing 0.5 mCi/ml 35 S-protein labeling mix (EXPRE 35 S 35 S Protein Labeling Mix, > 1000 Ci/mmol, New England Nuclear, Boston, MA). After labeling, cells were washed once with cold PBS and lysed with cold buffer containing 50 mM Tris and 1 % NP-40, pH 8.0.
  • Lysates were cleared by centrifugation at 16,000 x g for 10 min at 4° C, and supernatants were applied to the column.
  • the column was washed with 20 bed volumes of PBS and eluted with 100 mM glycine, pH 2.5. Eluates and flow- through fractions (800 cpm per sample) were separated by 10% SDS- PAGE, processed for fluorography, and exposed to x-ray film at -80°C.
  • PBLs Peripheral blood lymphocytes
  • PBLs or JEG-3 cells were washed in PBS containing 1 mg/ml BSA and incubated with either 1 B8 (hybridoma supernatant diluted 1 : 10) or W6/32 (1 //g/ml) followed by fluorescein-conjugated anti-mouse IgM or IgG, respectively (diluted 1 : 100; Jackson Immuno Research). Cells were then washed, fixed in 0.2% paraformaldehyde, and analyzed on a FACScan cytometer (Becton Dickinson, Mountain View, CA).
  • Example 5 Immunofluorescent localization of HLA-G in the placenta and placental bed; HLA-G is expressed by cytotrophoblasts that differentiate along the invasive pathway in vivo.
  • Reactivity of the 1 B8 and 3F6 antibodies was detected using fluorescein-conjugated goat anti-mouse IgM, diluted 1 :200 (Jackson Immuno Research). Control experiments included incubation of tissues with primary or secondary antibodies alone, non- immune mouse serum or normal mouse IgG.
  • Sections contained floating chorionic villi and anchoring villi (including cytotrophoblast cell columns), as well as decidualized endometrium and myometrium. Thus, cytotrophoblast stem cells, as well as differentiated trophoblasts (syncytiotrophoblasts and invasive cytotrophoblasts), were evident. Sections were double-stained with an anti-cytokeratin antibody (7D3), which in the placental bed is specific for trophoblast cells, and either the 1 B8 or 3F6 anti-HLA-G mAbs (described supra).
  • 7D3 anti-cytokeratin antibody
  • the 3F6 mAb gave an identical staining pattern to that of 1 B8. None of the components of floating villi, including undifferentiated cytotrophoblasts anchored to the villus basement membrane and fetal elements within the villus core, reacted with the anti-HLA-G antibodies. In contrast, . invasive cytotrophoblasts within the cell columns of anchoring villi stained brightly (Fig. 2, panel B). Antibody reactivity was first detected in the distal part of the cell columns as the cytotrophoblasts made contact with the uterine wall. Cytotrophoblasts participating in interstitial invasion also stained brightly.
  • cytotrophoblasts within blood vessels also showed intense reactivity with the anti-HLA-G antibodies.
  • Cytotrophoblast staining in first trimester samples was nearly identical to that in second trimester tissue. None of the floating villi components reacted with either antibody. The only exception was occasional syncytial brush border staining.
  • Third trimester tissue exhibited the same pattern; floating villi (including the syncytial brush border) did not stain whereas interstitial and endovascular cytotrophoblasts reacted with the antibodies.
  • the third trimester cytotrophoblasts within uterine wall stained less brightly than cells in a comparable location earlier in gestation.
  • Example 6 HLA-G is expressed by cytotrophoblasts that differentiate along the invasive pathway in vitro
  • cytotrophoblasts were prepared from first, second and third trimester chorionic villi as previously described (Fisher et al. (1989) J. Cell Biol. 109: 891 ; and Librach, et al. (1991 ) J. Cell Biol. 1 13: 437).
  • Cells were plated on the laminin-rich extracellular matrix preparation Matrigel (Collaborative Research, Bedford, MA), in MEM (UCSF Cell Culture Facility) containing 2% Nutridoma (Boehringer Mannheim Biochemicals, Indianapolis, IN). To promote the formation of large aggregates, 2.5 x 10 5 cells were plated on plugs of Matrigel formed in capsules (6.5 mm diameter).
  • the Matrigel plugs and cytotrophoblast aggregates were fixed in 3% paraformaldehyde, sectioned and processed for immunostaining. Sections of aggregates that had invaded Matrigel plugs were stained essentially as described above, exceptthat reactivity was detected using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions.
  • the second culture condition was used to determine the time course of HLA-G protein production in vitro.
  • Cytotrophoblasts isolated from placentas of different gestational ages were cultured on a thin layer of Matrigel. Under these conditions the cells differentiate along the invasive pathway, as they do when cultured on Matrigel plugs (Damsky et al. (1994) Development 120: 3657). However, they form smaller aggregates, which permits determination of the staining pattern of individual cells without sectioning.
  • Cytotrophoblasts of all gestational ages upregulated HLA-G production in culture. For example, immediately after plating approximately 25% of first trimester cells expressed HLA-G, but by 12 h in culture nearly 60% reacted with the antibody. This level remained constant throughout the 48 h assay period. With increasing gestational age there was a decrease in the percentage of immunopositive cells at most time points. For example, significantly fewer third than first trimester cells expressed HLA-G after
  • Example 7 Northern hybridization shows that Cytotrophoblast HLA-G mRNA production in vitro is gestationally regulated
  • Northern hybridization was used to quantify mRNA levels in cultured first, second and third trimester cytotrophoblasts. High levels of HLA-G mRNA were detected in first and second trimester cytotrophoblasts immediately after isolation, and the level of mRNA expression did not change over time in culture. In comparison, term cells contained a greatly reduced level of HLA-G mRNA throughout the culture period.
  • the northern analysis was performed as described below. Total RNA was extracted from cultured cytotrophoblasts according to published methods (Chomczynski and Sacchi (1987) Anal. Biochem. 162: 156).
  • HLA-G-specific cDNA probe was synthesized by random priming of the 450 bp Pvu II fragment from the 3' untranslated region of HLA-G using 32 P-CTP and the Klenow fragment of DNA polymerase I according to standard methods (Tabor et al. (1993) In Current Protocols in Molecular Biology, vol. 1 . K. Jannssen, ed. John Wiley and Sons, New York p. 3.0.1 ). Probes had a specific activity of 2 x 10 9 dpm///g.
  • RNA (10/ g) was separated by formaldehyde-agarose gel electrophoresis, transferred to Nytran membranes (Schleicher and Schuell, Keene, NH) and analyzed by Northern blot hybridization as previously described (Lehrach et al. (1977) Biochemistry 16: 4743; and De et al. (1990) J. Biol. Chem. 265: 15267)).
  • gels were stained with acridine orange prior to transfer to ensure integrity of the RNA samples, and to confirm that equal amounts of RNA had been loaded onto each lane.
  • the final post-hybridization washes were carried out in 0.3 X SSC (150 mM NaCl, 15 mM sodium citrate, pH 7.4) and 0.1 % SDS at 68° C.

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Abstract

La molécule HLA-G de classe I est spécifiquement exprimée par des cytotrophoblastes invasifs. L'invention décrit des antigènes qui élicitent des anticorps spécifiques de HLA-G et des anticorps qui fixent ces antigènes. L'invention décrit également des procédés liés à la mesure des taux de HLA-G dans les liquides biologiques.
PCT/US1996/003765 1995-04-07 1996-03-21 Anticorps pour la detection de hla-g WO1996031604A1 (fr)

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WO1999042128A1 (fr) * 1998-02-20 1999-08-26 Commissariat A L'energie Atomique Methode de selection de tumeurs exprimant hla-g, sensibles a un traitement anticancereux et ses applications
FR2775294A1 (fr) * 1998-02-20 1999-08-27 Commissariat Energie Atomique Methode de selection de tumeurs sensibles a un traitement anticancereux et ses applications
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GB2425129A (en) * 2005-04-16 2006-10-18 Axordia Ltd Cytotrophoblast stem cells
WO2017207775A1 (fr) * 2016-06-03 2017-12-07 Invectys Anticorps spécifiques anti-hla-g
US11117971B2 (en) 2018-08-31 2021-09-14 Invectys SA Chimeric antigen receptors against multiple HLA-G isoforms
US11208487B2 (en) 2018-09-27 2021-12-28 Tizona Therapeutics Anti-HLA-G antibodies, compositions comprising anti-HLA-G antibodies and methods of using anti-HLA-G antibodies
US11492387B2 (en) * 2018-05-09 2022-11-08 Tianjin Dongya Biological Technology Co., Ltd. Broad spectrum vaccine, preparing method and application thereof

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FR2760023A1 (fr) * 1997-02-21 1998-08-28 Commissariat Energie Atomique Cellules eucaryotes exprimant a leur surface au moins une isoforme d'hla-g et leurs applications
WO1998037098A1 (fr) * 1997-02-21 1998-08-27 Commissariat A L'energie Atomique Cellules eucaryotes exprimant a leur surface au moins une isoforme d'hla-g et leurs applications
WO1999042128A1 (fr) * 1998-02-20 1999-08-26 Commissariat A L'energie Atomique Methode de selection de tumeurs exprimant hla-g, sensibles a un traitement anticancereux et ses applications
FR2775294A1 (fr) * 1998-02-20 1999-08-27 Commissariat Energie Atomique Methode de selection de tumeurs sensibles a un traitement anticancereux et ses applications
US6790638B1 (en) 1998-02-20 2004-09-14 Commissariat A L'energie Atomique Method for selecting tumours expressing HLA-G, sensitive to anticancer treatment and uses
WO1999043851A1 (fr) * 1998-02-25 1999-09-02 National University Of Ireland, Cork Gene de susceptibilite a la pre-eclampsie et aux fausses-couches lie au hla
WO2000078337A1 (fr) * 1999-06-18 2000-12-28 Commissariat A L'energie Atomique Compositions contenant des formes solubles d'hla-g dans le traitement de pathologies inflammatoires de la peau
FR2794977A1 (fr) * 1999-06-18 2000-12-22 Commissariat Energie Atomique Utilisation de compositions contenant des formes solubles d'hla-g dans le traitement de pathologies inflammatoires de la peau, et leur procede d'obtention
WO2001023893A3 (fr) * 1999-09-27 2001-05-17 Clifford L Librach Detection de hla-g
WO2001023893A2 (fr) * 1999-09-27 2001-04-05 Librach Clifford L Detection de hla-g
US6613538B2 (en) 1999-09-27 2003-09-02 Clifford L. Librach Detection of HLA-G
FR2810047A1 (fr) * 2000-06-13 2001-12-14 Commissariat Energie Atomique Nouvelle isoforme d'hla-g et ses applications
WO2001096564A2 (fr) * 2000-06-13 2001-12-20 Commissariat A L'energie Atomique Isoforme d'hla-g (hla-g7) et ses applications
WO2001096564A3 (fr) * 2000-06-13 2002-03-14 Commissariat Energie Atomique Isoforme d'hla-g (hla-g7) et ses applications
GB2425129B (en) * 2005-04-16 2008-11-05 Axordia Ltd Cytotrophoblast stem cell
GB2425129A (en) * 2005-04-16 2006-10-18 Axordia Ltd Cytotrophoblast stem cells
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WO2017207775A1 (fr) * 2016-06-03 2017-12-07 Invectys Anticorps spécifiques anti-hla-g
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CN109563169B (zh) * 2016-06-03 2022-07-01 英韦克泰斯公司 抗hla-g特异性抗体
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US11492387B2 (en) * 2018-05-09 2022-11-08 Tianjin Dongya Biological Technology Co., Ltd. Broad spectrum vaccine, preparing method and application thereof
US11505608B2 (en) 2018-08-31 2022-11-22 Invectys SA Chimeric antigen receptors against multiple HLA-G isoforms
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US11208487B2 (en) 2018-09-27 2021-12-28 Tizona Therapeutics Anti-HLA-G antibodies, compositions comprising anti-HLA-G antibodies and methods of using anti-HLA-G antibodies

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