Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-03-21
2003-09-02
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210, C435S070210, C435S452000, C435S330000, C435S334000, C435S344000, C436S518000, C436S548000, C436S906000, C530S387700, C530S388220, C530S388800, C530S391100, C530S853000, C600S033000, C600S034000
Reexamination Certificate
active
06613538
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the detection of HLA-G. The present invention provides antigens for generating specific antibodies to both soluble and membrane bound HLA-G, as well as exemplary antibodies.
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.
Implantation and placental development physically connect the mammalian embryo to the maternal uterus. Establishing this connection is essential for subsequent development. The initial developmental events which occur in the embryo set aside unique extraembryonic cellular lineages which are the precursors of the placenta. The first differentiation event gives rise to trophoblasts, which are specialized epithelial cells of the placenta that physically connect the embryo and the uterus, see for example Cross et al. (1994),
Science
266: 1508 for a review of the events surrounding implantation and formation of the placenta.
After fertilization in the oviduct, a series of cell divisions create a mass of totipotent cells called 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. In primates, 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. During implantation, 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 chances occur. For example, 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.
After implantation in humans, distinct populations of differentiated trophoblasts form. Proliferative cytotrophoblast stem cells are anchored to basement membranes surrounding a stromal core in two types of chorionic villi. In floating villi, cytotrophoblast stem cells detach from the underlying basement membrane and fuse to form a syncytium, a polynucleate cell, which covers the villus and is in direct contact with maternal blood. In anchoring villi, cytotrophoblast stem cells differentiate, by detaching from their basement membrane and aggregating to form columns of mononuclear cells which attach to and invade the uterine decidua (interstitial invasion) and its arterial system (endovascular invasion). Interstitial invasion puts cytotrophoblasts in direct contact with the highly specialized subset of leukocytes that are home to the uterus during pregnancy. Endovascular invasion puts cytotrophoblasts, like the syncytiotrophoblasts covering the anchoring villi, in direct contact with maternal blood. Thus, antigen presentation by trophoblasts at the maternal-fetal interface is an important component of maternal immunological responses during pregnancy.
MHC class I molecules and the peptides they present regulate alloreactivity, see for example Sherman, at al. (1993),
Annu. Rev. Immunol.
11: 385. Thus, one key to understanding maternal tolerance of the fetal semi-allograft lies in studying trophoblast expression of class I molecules. 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, see for example Geraghty et al. (1987)
Proc. Natl. Acad. Sci. U.S.A.
84: 9145. The gene has an intron/exon organization identical to that of the class 1a genes (HLA-A, -B and -C), and the HLA-G protein product has 86% sequence identity to the class I consensus sequence, see for example Parham et al. (1988)
Proc. Natl. Acad. Sci U.S.A.
85: 4005. HLA-G has a lower molecular mass (37-39 kDa) then class 1a molecules due to a stop codon in exon 6 that results in the deletion of all but 6 amino acids in the cytoplasmic tail, see for example Shimizu et al. (1988)
Proc. Natl. Acad. Sci. U.S.A.
85: 227. With regard to the 5′ flanking region of the gene, the HLA-G promoter has elements (e.g., AP-1, NFkB) similar to sequences found in class 1a genes, but lacks an interferon response element, suggesting novel transcriptional regulatory mechanisms. 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 (see for example Ishitani and Geraghty (1992)
Proc, Natl, Acad. Sci. U.S.A.
85: 3947), or exon four (see for example Kirszenbaum et al. (1994)
Proc. Natl. Acad. Sci; U.S.A.
91:4209). To what extent these alternatively spliced mRNAs are translated is unclear. A soluble form of HLA-G encoded by an mRNA containing intron 4 was described by Fujii et al. (1994)
J. Immunol.
153: 5516.
HLA-A, -B and -C are highly polymorphic, but 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 sequence data also indicate that HLA-G has relatively limited polymorphism. However, there is suggestion that at least in some populations, i.e., African Americans, HLA-G exhibits substantial polymorphism, see for example 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 and monitoring of pregnancy, and for detecting cytotrophoblasts from biological fluids. In addition, HLA-G levels in the maternal blood are indicative of the vigor of cytotrophoblast invasion and the corresponding health of the placental-maternal interface. Prior to the work of Fisher et al., as described in International Publication No. WO 96/31604, suitable antibodies to HLA-G had not been obtained, due to the high similarity of HLA-G to class 1a molecules which are expressed in adults.
One complication of pregnancy in which an abnormal maternal immune response to the fetus has been implicated is pre-eclampsia. This condition is characterized by development of the classic triad of hypertension, edema, and proteinuria, usually in the third trimester of pregnancy, and is usually associated with signs of neurologic hyperirritability, which may eventually result in grand mal seizures. This pregnancy complication remains one of the major causes of maternal and perinatal mortality in both North American and the developing world, see for example Rochat R W, et al. (1988) Maternal mortality in the United States: report from the Maternal Mortality Collaborative,
Obstet Gynecol,
72(1):91-7, and AbouZahr C, et al. (1996) Maternal mortality,
World Health Stat Q,
49(2):77-87.
Despite extensive research into pre-eclampsia, the underlying cause or causes remain unknown. Evidence accumulated thus far strongly suggests that the causative agent is the placenta, see for example Redman (1991)
Placenta,
12(4):301-8.
Pre-eclampsia can occur with hydatidiform mole or choriocarcinoma, see fo
Librach Clifford L.
Yie Shang-mian
Chin Christopher L.
Grun James L.
Price Heneveld Cooper DeWitt & Litton
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