EP1328290A2 - Expression, preparation, utilisations et sequence de la proteine hla-g soluble derivee par recombinaison - Google Patents

Expression, preparation, utilisations et sequence de la proteine hla-g soluble derivee par recombinaison

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Publication number
EP1328290A2
EP1328290A2 EP01973167A EP01973167A EP1328290A2 EP 1328290 A2 EP1328290 A2 EP 1328290A2 EP 01973167 A EP01973167 A EP 01973167A EP 01973167 A EP01973167 A EP 01973167A EP 1328290 A2 EP1328290 A2 EP 1328290A2
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EP
European Patent Office
Prior art keywords
recombinant protein
seq
sequence
amino acid
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP01973167A
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German (de)
English (en)
Inventor
Joan S. Hunt
Pedro J. Morales
Margaret G. Petroff
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University of Kansas Medical Center
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University of Kansas Medical Center
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Publication of EP1328290A2 publication Critical patent/EP1328290A2/fr
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    • 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
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention is concerned with mechanisms affecting immune tolerance of organs, tissues, and cells having a genetically different origin than their host. More particularly, the present invention is concerned with immune tolerance of genetically different fetuses during pregnancy, genetically different organs which have been transplanted, and genetically different tissues or cells which have been grafted into an organism. More particularly, the present invention is concerned with recombinant proteins and methods of generating these recombinant proteins wherein the recombinant proteins affect tolerance of these foreign tissues, organs and cells. Still more particularly, the present invention is concerned with the use and production of recombinant soluble HLA-G protein including HLA-G 1 and HLA-G2.
  • the present invention is concerned with producing large amounts of recombinant soluble HLA-G 1 and recombinant soluble HLA-G2 protein and using this recombinant protein to alter immune-initiated responses to genetically different fetuses or genetically different organs, tissues, and cells which have been implanted or grafted into an individual.
  • HLA-G soluble substance
  • HLA-G is an MHC class lb protein that has been identified as a product of human trophoblast cells. Subpopulations of these cells, which comprise the fetal compartment of the maternal-fetal interface, are unique in their substitution of class lb antigens, HLA-G and HLA-E, which have few alleles, for the highly polymorphic HLA-A and HLA-B antigens. This substitution is believed to have a major role in the tolerance and accommodation of the genetically different embryo/fetus. This is because expression of class I genes such as HLA-E and HLA-G are unlikely to elicit an immune response due to a high degree of sequence conservation between all people which usually will not permit the immune system to distinguish between self and non-self antigens. This is contrast to genes encoding HLA class la antigens such as HLA-A and HLA-B. These genes express proteins which are highly polymorphic and which thereby invoke an immune response.
  • the trophoblasts also express HLA-C but the significance of this is as yet unknown.
  • the HLA-G message is differentially spliced to produce at least six different transcripts.
  • These membrane-bound isoforms of HLA-G are believed to protect fetal trophoblast cells from attack by maternal macrophages and or natural killer (NK) cells, both of which are present in substantial numbers in the specialized tissue derived from the uterine endometrium termed the decidua.
  • sHLA-Gl Soluble HLA-Gl
  • sHLA-Gl Soluble HLA-Gl
  • sHLA-G2 is identical to sHLA-Gl except that it lacks the ⁇ 2 domain.
  • HLA-G has been partially purified from placentas and trophoblast tumor cell lines.
  • purification of the natural protein does not result in an adequate supply of sHLA-G.
  • a method of producing unlimited quantities of pure recombinant proteins Preferably, these proteins would be produced in human cells in order to assure correct glycosylation. Still more preferably, the recombinant proteins will be secreted from the cells and into the surrounding culture medium.
  • methods of altering immune response in pregnant individuals as well as in individuals undergoing organ transplants, and tissue and/or cell grafts What is still further needed.
  • the present invention solves the problems inherent in the prior art and provides a distinct advance in the state of the art.
  • unlimited quantities of recombinant soluble HLA-G protein can be produced and used to modulate immune responses in individuals.
  • These recombinant proteins are produced in human cells thereby assuring proper glycosylation. Furthermore, these proteins give every evidence of identity with natural sHLA-Gl and sHLA-G2.
  • recombinant proteins provided by the present invention have differing effects on different target cells.
  • recombinant soluble HLA-Gl recombinant soluble HLA-Gl
  • rsHLA-G2 recombinant soluble HLA-G2
  • excess amounts of rsHLA- G may have adverse effects on certain individuals or under certain conditions.
  • HLA-G is present in men as well as women.
  • HLA-G modulates immune response and that the recombinant proteins of the present invention hold great promise in the treatment of immune disorders and in other areas where modulation of the immune response is important.
  • the present invention describes the first successful expression of rsHLA-Gl and rsHLA-G2 by eukaryotic cells. Such production is difficult in that the recombinant isoforms are difficult to prepare because they are derived from specific messages generated by alternative splicing of a single mRNA.
  • the splice variants must be isolated from one another, sequenced, spliced into appropriate plasmids and transfected into human cells for production. Such a sequence of events is schematically illustrated in Figs. 7, 8, and 9. The first of these figures, Fig.
  • FIG. 7 illustrates soluble HLA-Gl and soluble HLA-G2 cloning into the expression vector pcDNA3.1HisC and expression of sHLA-Gl and sHLA-G2 as N-tagged proteins to 6x-His andXpress.
  • Fig. 8 illustrates the sub-cloning of sHLA-Gl and sHLA-G2 into pRC/CMN vector containing BM40 signal peptide for the expression of soluble and secreted HLA-Gl and HLA-G2 ⁇ -tagged to FLAG peptide.
  • Fig. 8 illustrates the sub-cloning of sHLA-Gl and sHLA-G2 into pRC/CMN vector containing BM40 signal peptide for the expression of soluble and secreted HLA-Gl and HLA-G2 ⁇ -tagged to FLAG peptide.
  • FIG. 9 illustrates the stable transfection of apreferred cell line (HEK293 cells) with ssHLA-G constructs (secreted products) and protein purification using FLAG-M2 affinity gel.
  • a photograph of a Western Blot showing the proteins expressed by these transfected cells is given in Fig. 10.
  • sequences have been assigned the following SEQ ID NOS : the BM40 signal peptide is SEQ LO NO. 1; the Flag peptide is SEQ ID NO. 2; the ⁇ l domain is SEQ ID NO. 3; the ⁇ 2 domain is SEQ ID NO. 4; the ⁇ 3 domain is SEQ ID NO. 5; intron 4 is SEQ ID NO.
  • sequences including or having a sequence which has at least about 70% sequence identity or homology with any one of SEQ ID NOS. 1-8 and which exhibit similar desired properties are within the scope of the present invention. Preferably, such sequences will have at least about 79% sequence identity or homology with any one of SEQ ID NOS. 1-8 and still more preferably at least about 90% sequence identity or homology. Most preferably, such sequences will have at least about 95% sequence identity or sequence homology with any one of SEQ ID NOS 1-8.
  • sequences which differ from any one of SEQ ID Nos. 1-8 due to a mutation event or series of mutation events but which still exhibit similar properties are also embraced in the present invention.
  • Such mutation events include but are not limited to point mutations, deletions, insertions and rearrangements.
  • Sequence Identity refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are "identical” at a particular position if at that position, the nucleotides or amino acid residues are identical.
  • Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M.
  • Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN andFASTA (Altschul, S. F. et al., J. Molec.
  • BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVINLM NOT Bethesda, MD 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences.
  • nucleotide sequence having at least, for example, 95% "sequence identity" to a reference nucleotide sequence it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence, h other words, in a polynucleotide having a nucleotide sequence having at least 95% identity relative to the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence maybe deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a polypeptide having a given amino acid sequence having at least, for example, 95% sequence identity to a reference amino acid sequence it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence.
  • a given polypeptide sequence having at least 95% sequence identity with a reference amino acid sequence up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or the carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence.
  • residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.
  • sequence homology also refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned as described above, and gaps are introduced if necessary. However, in contrast to “sequence identity”, conservative amino acid substitutions are counted as a match when determining sequence homology.
  • 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence.
  • a “conservative substitution” refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, charge, hydrophobicity, etc., such that the overall functionality does not change significantly.
  • Isolated means altered “by the hand of man” from its natural state., i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a polynucleotide or polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated", as the term is employed herein.
  • Stable in terms of stable transfectants refers to the ability of a cell to continually produce or express a particular protein without damaging the cell. Stable transfectants will continue to express the protein indefinitely as long as the cells themselves are properly maintained.
  • a "signal peptide” refers to a peptide located at the N-terminal end of a protein which assist the protein in becoming incorporated into or transported and secreted through the plasma membrane of a cell. Post translational cleavage usually removes these signal sequences from the respective precursor protein.
  • a method of producing recombinant soluble HLA- G includes the steps of transfectiiig a cell with a recombinant expression vector wherein the recombinant expression vector includes a sequence encoding for intron 4 of HLA-G. Additionally, the recombinant expression vector includes at least one sequence which encodes for a domain of HLA-G.
  • HLA-G domains include the ⁇ l domain, the ⁇ 2 domain, the ⁇ 3 domain and combinations of these domains. When more than one of these domains is included, the sequences encoding for the domains preferably appear in numerical order.
  • the ⁇ l domain will preferably precede the ⁇ 2 domain which will preferably precede the ⁇ 3 domain.
  • the sequence of the recombinant expression vector is then expressed in the cell.
  • the recombinant expression vector includes a sequence encoding for a signal peptide
  • the recombinant protein can be secreted from the cell, into the culture medium, whereupon it may be isolated and purified.
  • the expressed recombinant protein preferably has a sequence having at least 70% sequence homology with a sequence selected from the group consisting of SEQ ID NOS. 7 and 8.
  • the recombinant protein has at least 79% sequence homology and still more preferably at least 90% sequence homology with a sequence selected from the group consisting of SEQ ID NOS. 7 and 8. Most preferably, the expressed recombinant protein has at least 95% sequence homology with either SEQ ID NO. 7 or SEQ ID NO. 8.
  • a method for obtaining recombinant soluble HLA-G protein includes the steps of transfecting a first cell containing the gene encoding HLA-G protein;isolating RNA from said transfected cell;preparing cDNA from said isolated RNA; amplifying said cDNA by performing PCR on said cDNA; selecting for amplified cDNA containing selected portions of the cDNA encoding for HLA-G; ligating said amplified cDNA containing said selected portions into a vector to produce a recombinant expression vector; transfecting a second cell with said recombinant expression vector; and expressing said selected portions using said transfected second cell.
  • the first cell comprises a mouse fibroblast cell line and a particularly preferred mouse fibroblast cell line is denominated SI 4/8.
  • reverse transcriptase with a particularly preferred reverse transcriptase being Moloney Murine Leukemia Virus Reverse Transcriptase.
  • a forward primer comprising a sequence having at least about 70% sequence homology with SEQ ID NO.9.
  • a preferred reverse primer comprises a sequence having at least about 70% sequence homology with SEQ ID NO. 10.
  • the selected portions of amplified cDNA preferably include sequences encoding for sequences having at least 70% sequence homology with SEQ ID NO.
  • the ligating step preferably utilizes T4 DNA ligase and the vector is preferably linearized.
  • a particularly preferred expression vector is denominated pcDNA3. lHisC.
  • the second cell is preferably a eukaryotic cell and still more preferably a mammalian cell.
  • One preferred group of mammalian cells includes HEK293 cells, human trophoblasts tumor cells, and Chinese hamster ovary cells.
  • the expressed selected portions preferably comprise recombinant soluble HLA-G protein.
  • This recombinant soluble protein is preferably selected from the group consisting of HLA-Gl and HLA-G2, and still more preferably has at least 70% sequence homology with either SEQ LO NO. 7 or SEQ ID NO. 8.
  • a method of promoting the acceptance of a fetus by the immune system of an individual comprises the steps of administering a recombinant protein to the individual and altering cytokine production in the individual by contacting a target cell within the individual with the recombinant protein.
  • the recombinant protein has at least 70% sequence homology with an amino acid sequence wherein the amino acid sequence comprises SEQ ID NO. 6 and at least one sequence selected from the group consisting of SEQ ID NOS. 3-5.
  • the recombinant protein may also include a sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, and combinations thereof.
  • the recombinant protein may comprise an amino acid sequence having at least 70% sequence homology with a sequence selected from the group consisting of SEQ ID NOS. 7 and 8. It is also preferably for the recombinant protein to be soluble in aqueous solutions. Such solutions should comprise at least 50% water and may include at least one member selected from the group consisting of salts, detergents, and buffers. Altering cytokine production can be accomplished in various ways including the alteration of signal transduction in the target cell.
  • the target cell is capable of binding soluble HLA-G and the target cell may be selected from the group consisting of leukocytes and decidual cells.
  • an individual's immune tolerance to foreign tissue is modulatable.
  • the tissue may be organs, cells, or a tissue graft.
  • the method generally comprises the steps of administering a recombinant protein to the individual wherein the recombinant protein binds with a target cell whereupon the target cell's gene expression is modulated. This modulation may result in an increase or a decrease in the gene expression, hi terms of the present invention, it is preferable that the target cell be capable of binding with soluble HLA-G.
  • Target cells include many cells including leukocytes and decidual cells .
  • the recombinant protein have at least 70% sequence homology with an amino acid sequence which comprises SEQ ID NO. 6 and at least one of SEQ ID NOS. 3-5. Such a sequence may further include SEQ ID NO. 1, SEQ ID NO. 2, or combinations thereof.
  • the recombinant protein may comprise an amino acid sequence having at least 70% sequence homology with either SEQ ID NO. 7 or SEQ ID NO. 8.
  • the recombinant protein is soluble in aqueous solutions.
  • aqueous solutions include solutions comprising at least 50% water and may include at least one member selected from the group consisting of salts, detergents, and buffers.
  • One of the preferred ways to modulate the gene expression of the target cell includes the step of altering signal transduction in the target cell.
  • the recombinant protein of the present invention may comprise a first amino acid sequence having at least 70% sequence homology with SEQ ID NO. 3, a second amino acid sequence linked with the first amino acid sequence and having at least 70% sequence homology with SEQ ID NO. 5, and a third amino acid sequence linked with the second amino acid sequence and having at least 70% sequence homology with SEQ ID NO. 6.
  • Preferred forms of this recombinant protein further include a signal peptide.
  • One preferred signal peptide has at least 70% sequence homology with SEQ ID NO. 1.
  • Another preferred signal peptide is the BM40 signal peptide. It is also preferred to have the signal peptide linked with the first amino acid sequence.
  • a fourth amino acid sequence may also be linked with the first amino acid sequence and it is preferred that this fourth amino acid sequence have at least 70% sequence homology with SEQ LO NO. 1.
  • the protein includes a fifth amino acid sequence which preferably has at least 70% sequence homology with SEQ ID NO. 2.
  • Another alternative form attaches a sixth amino acid sequence to the first amino acid sequence wherein this sixth amino acid sequence preferably has atleast70% sequence homology with SEQ ID NO.4. In all of the sequences listed in this paragraph, it is preferred for the sequences to have at least 79% sequence homology with its corresponding SEQ ID NO.
  • the recombinant protein should be soluble in aqueous solutions, as described above.
  • Effective recombinant proteins are also capable of altering activity in a cell and such cells include peripheral blood mononuclear cells and decidual cells.
  • a recombinant protein encoded by a gene transcript of a cell comprises, in succession from the 5' end to the 3' end, a first sequence encoding the ⁇ l domain of the protein, a second sequence encoding the ⁇ 3 domain of the protein and a third sequence encoding intron 4 of the protein.
  • the recombinant protein has at least 70% sequence homology with HLA-G.
  • the transcript further comprises a fourth sequence encoding the ⁇ 2 domain of the protein.
  • the transcript further comprises a fifth sequence encoding a signal peptide.
  • this signal peptide is cleaved from the recombinant protein in the cell and permits the recombinant protein to be secreted into the culture media.
  • the transcript further comprises a sixth sequence which encodes a purification-assisting peptide.
  • this purification-assisting peptide is cleaved from the recombinant protein in the cell. Sequences having at least 70% sequence homology with SEQ LO NO. 2 are embraced by the present invention.
  • An advantage of the present invention is that the recombinant protein maybe expressed in a eukaryotic cell.
  • This eukaryotic cell is preferably a transfected mammalian cell and particularly preferred transfected mammalian cells are transfected with an expression vector for soluble HLA-Gl (sHLA-Gl) and soluble HLA-G2 (sHLA-G2).
  • the expression vector preferably includes a sequence encoding a protein having at least 70% sequence homology with either SEQ ID NO. 7 or SEQ ID NO. 8.
  • the transfected cell may be from a variety of sources, however, particularly preferred cells include HEK293 cells, human trophoblast tumor cells, and Chinese hamster ovary cells. Of these, HEK293 cells are generally preferred due to their lack of a tendency to develop tumors.
  • the recombinant protein should also be soluble in aqueous solutions, such as those described above.
  • a recombinant protein is provided.
  • This recombinant protein has at least 70% sequence homology with an amino acid sequence wherein the amino acid sequence comprises SEQ ID NO. 6 and at least one sequence selected from the group consisting of SEQ ID NOS. 3-5. This coincides with HLA-G recombinant proteins which are encoded by a transcript which includes intron 4 and at least one ⁇ domain.
  • Other preferred recombinant proteins include an amino acid sequence selected from the group consisting of SEQ ID NO. 1, SEQ LD NO. 2, and combinations thereof.
  • Preferred forms of the recombinant protein have at least 70% sequence homology with a first amino acid sequence which comprises, in succession, SEQ ID NO. 3, SEQ ID NO. 5, and SEQ ID NO. 6.
  • Other forms of this preferred recombinant protein may include a second amino acid sequence which is linked to the first amino acid sequence and has at least 70% sequence homology with SEQ JD NO. 1.
  • Yet another preferred recombinant protein comprises, in succession, amino acid sequences having at least 70% sequence homology with SEQ ID NO.
  • Still another preferred recombinant protein comprises, in succession, amino acid sequences having at least 70% sequence homology with SEQ LD NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6.
  • Such a sequence corresponds to HLA-Gl and includes all of the portions of HLA-G2 and the ⁇ 2 domain.
  • the protein is expressed by transfected eukaryotic cells.
  • transfected eukaryotic cells are preferably mammalian cells including cells selected from the group consisting of HEK293 cells, human trophoblast tumor cells, and Chinese hamster ovary cells. Of these cells, it is preferred to use HEK293 cells transfected with an expression vector which includes a gene encoding HLA-G. More preferably, the expression vector includes sequences encoding for sHLA-Gl or sHLA-G2.
  • Other preferred recombinant proteins include amino acid sequences having at least 70% sequence homology with either SEQ ID NO. 7 or SEQ ID NO 8. Again, these recombinant proteins are preferably soluble in aqueous solutions with preferable aqueous solutions being described above.
  • the present invention also provides a recombinant expression vector wherein the vector expresses a recombinant protein having at least 70% sequence homology with an amino acid sequence which includes SEQ ID NO. 6 and at least one sequence selected from the group consisting of SEQ LD NOS. 3-5.
  • Preferred forms of this recombinant expression vector express recombinant proteins which include amino acid sequences having at least 70% sequence homology with SEQ ID NO. 1, SEQ ID NO. 2, or combinations thereof.
  • One particularly preferred recombinant protein expressed by the recombinant expression vector of the present invention has at least 70% sequence homology with a first amino acid sequence which comprises, in succession, SEQ JD NO. 3, SEQ ID NO. 5, and SEQ ID NO. 6.
  • such a recombinant protein has at least 70% sequence homology with HLA-G2.
  • Another particularly preferred recombinant protein expressed by the recombinant expression vector of the present invention has at least 70% sequence homology with a first amino acid sequence which comprises, in succession, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6.
  • Such a recombinant protein has at least 70% sequence homology with HLA-Gl . It is preferred for the recombinant expression vector to be transfected into eukaryotic cells and that the resultant transfected eukaryotic cells be stable.
  • Preferred eukaryotic cells include mammalian cells and preferred mammalian cells include HEK293 cells, human trophoblast tumor cells, and Chinese hamster ovary cells.
  • preferred mammalian cells include HEK293 cells, human trophoblast tumor cells, and Chinese hamster ovary cells.
  • amino acid sequences making up the recombinant protein are expressed in the transfected cells and still more preferable for the recombinant protein to be secreted from the transfected cells.
  • the cells may remain intact and continue producing additional recombinant protein.
  • the presence of SEQ ID NO. 6 renders the recombinant protein soluble in aqueous solutions.
  • Figure 1 is a schematic representation of a portion of the HLA-G gene (HLA 6.0) and the two alternatively spliced transcripts sHLA-Gl and sHLA-G2;
  • Fig. 2 is a schematic representation of the expected recombinant products and expected molecular weights
  • Fig. 3 is schematic representation of ELISA results confirming that recombinant sHLA- Gl and recombinant sHLA-G2 were produced and secreted by the HEK293 clones;
  • Fig. 4 is a photograph of an immunoblot detecting the recombinant proteins sHLA-Gl and sHLA-G2 using two different antibodies;
  • Fig. 5 is a photograph of an immunoblot and a schematic representation of the immunoblot results comparing enzymatic deglycosylation of the purified recombinant proteins under native and denaturing conditions;
  • Fig. 6 is a photograph of an immunoblot testing the recombinant proteins for binding to ⁇ 2m
  • Fig. 7 is a schematic representation of the cloning into the expression vector and expression of the soluble proteins
  • Fig. 8 is a schematic representation of the sub-cloning of the soluble proteins into the vector for the expression of the soluble and secreted proteins
  • Fig. 9 is a schematic representation of the stable transfection of HEK293 cells with the secreted products and the protein purification used in the present invention.
  • Fig. 10 is a Western Blot showing the purified proteins expressed by the transfected cells.
  • Fig. 11 is a graph showing IL-10 production in decidual macrophages treated with rsHLA-G.
  • Example 1 This example maintained the cell lines used to generate clones and also generated the stably transfected clones.
  • HEK293 cells (ATCC No. CRL-1573) were selected for transfection with the construct resulting from the transfection of the S 14/8.3 mouse fibrolblast cell line with the full length HLA 6.0 gene.
  • the HEK293 and S 14/8.3 cells were maintained at 37°C/5% CO 2 in Dulbecco's Modified Eagle Medium (D-MEM) supplemented with 10% fetal bovine serum (FBS) and penicillin (100U/ml)/streptomycin (O.lmg/ml).
  • D-MEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • penicillin 100U/ml
  • Streptomycin O.lmg/ml
  • Stable transfectants were selected using G418 (Life Technologies) and cloned by limiting dilution. Selection of positive sHLA-G producing clones was achieved by using an ELIS A to test for the presence of the secreted protein in the culture media supernatants.
  • Example 2 This example confirmed that the selected clones from Example 1 expressed the desired proteins.
  • wash buffer 0.05M Tris buffer pH 8.0, containing 0.15M NaCl and 0.05% Tween 20
  • each well was incubated with 50 ⁇ l of either 0.2 ⁇ g/ml of 16G1 or control mouse IgG (Vector Laboratories, Burlingame, CA) in blocking solution at 37C for 30 minutes.
  • each well was washed four times with 200 ⁇ l of wash buffer, 50 ⁇ l/per well of 2 ⁇ g/ml of the secondary antibody, peroxidase labeled horse anti-mouse IgG (Vector Laboratories), was added and incubated at 37C for 30 minutes.
  • Example 3 This example purified the isolated RNA and performed RT-PCR in order to obtain sHLA-G cDNAs.
  • MMLV Moloney Murine Leukemia Virus
  • Amplicons were separated by agarose gel electrophoresis, eluted from the gel and purified using Geneclean (Bio 101, Vista, CA). The presence of intron 4 in each amplicon was verified by PCR using previously described primers Gsi4 (SEQ ID NO. 15) and G1225 (SEQ ID NO. 10). Purified amplicons containing intron 4 were used as a templates in nested PCR using the 5'-phosphorylated cloning primers A-blunt: 5'-GGCTCC CAC TCC ATG AGG TAT TTC-3' (SEQ ID NO.
  • B-Xba 1 5'-GGG TCT AGA TTA AAG GTC TTC AGA-3' (SEQ JD No. 12), which includes the stop codon and part of intron 4.
  • Two amplicons of approximately 900 and 600 bp were obtained, which were then separated by agarose gel electrophoresis and purified by Geneclean.
  • Example 4 This example constructed the plasmid containing the expression vector.
  • the mammalian expression vector pcDNA3.1HisC (hivitrogen, Carlsbad, CA) was linearized using the restriction enzyme Kp ⁇ L and gel purified as described previously. To generate blunt ends the resulting 3 ' protuberant ends were digested using the 3 '->5 ' exonuclease activity of T4 DNA polymerase (Life Technologies) and subsequently the vector was dephosphorylated using calf alkaline phosphatase (Life Technologies) to reduce self re-ligation. Using T4 DNA ligase (Life Technologies), purified cDNA for sHLA-Gl and sHLA-G2 were ligated into pcDNA3.1HisC modified as described previously. The constructs, named pcDNA3.
  • IHisC-sGl and -sG2 generated are N-tagged to the XpressTM epitope and 6xHis tag and their products are not secreted.
  • the fragment HindJIL/Xbal was released from the constructs containing the intact cDNA for sHLA-Gl and sHLA-G2. PCR was performed using these purified fragments as a template with the modified primers A-NAelMTW 5'-CTA GCT AGC TAG AGG CTC CCA CTC CA-3' (SEQ ID NO. 13) and B- ⁇ baIMTW 5'- CTA GTC TAG ACT AGA TTA AAG GTC TTC AGA-3' (SEQ JD NO. 14).
  • Each generated amplicon containing Nbel and Xbal sites was gel purified by Geneclean and digested with the restriction enzymes then cloned into N/zel and Zbal sites of the modified vector pRc/CMV containing the BM40 signal peptide.
  • This expression vector derived from pRc/CMV (Invitrogen), contains a HindlJI-Apal D ⁇ A fragment encoding the signal peptide of the basal membrane protein (BM40) along with the Flag peptide and a site for enterokinase cleavage within the poly-linker site of the vector.
  • sequences of the expression constructs were determined by the flourescent dideoxy-terminator chemistry method, using an ABI PrismTM D ⁇ A sequencing kit (PE Applied Biosystems, Foster City, CA) following the manufacturer's protocol. The sequences were analyzed using an ABI automated sequencer (ABI 377 PrismTM, PE Applied Biosystems). Sequence alignments with the HLA-G 6.0 gene (GeneBank Ace .No. J03027) were performed with MegAlign Lasergene software (DNASTAR Inc., Madison, WI).
  • Example 5 This example used immuno-affinity purification to select for the Flag-tagged recombinant sHLA-Gl and sHLA-G2 using Flag M2 affinity gel chromatography.
  • Culture media supernatants from HEK293-sHLA-Gl and -sHLA-G2 clones were collected after 3 to 9 days in culture, centrifuged to pellet any cell debris and then filter-sterilized using a 0.22 micron polyethersulphone filter (Corning, New York, NY).
  • the filter-sterilized culture media supernatant was loaded into a 2 ml packed equilibrated Flag-M2 agarose bead affinity chromatography column (Sigma-Aldrich, St. Louis, MO). All devices and instrumentation were handled aseptically to avoid contamination, and endotoxin-free water (Baxter Healthcare Corporation, Deerfield LL) was used for all solutions.
  • the resin bed was washed with 3 bed volumes of TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) after the culture media supernatant entered the resin bed.
  • the recombinant proteins were subsequently eluted under native conditions by competition with the Flag peptide (SEQ ID NO. 2) (Sigma- Aldrich) in TBS at 100 ⁇ g/ml. Aliquots of 0.5 ml were collected and analyzed by SDS-PAGE. The aliquots containing the protein were pooled and subjected to ultra-filtration using Ultrafree®-4 (Millipore, Bedford, MA) to concentrate, exchange buffer for PBS and separate the purified protein from the Flag peptide.
  • TBS 50 mM Tris-HCl, 150 mM NaCl, pH 7.4
  • the recombinant proteins were filter sterilized by using 0.22 ⁇ m low protein binding filter devices (Millex-GV4, Millipore) and subjected to endotoxin detection by Pyrotell® gel-clot formulation Limulus amebocyte lysate assay, detection limit 0.03 EU/ml (Associates of Cape Cod Inc., Falmouth, MA) to verify the absence of bacterial contamination during the purification steps.
  • the purity was determined to be greater than 90% by Coomasie blue staining and densitometric analysis using a Gel-Pro Analyzer (version 3.0, Media Cybernetics).
  • Example 6 This example detected the soluble forms of HLA-G and ⁇ 2m using immunoblotting.
  • the purified recombinant proteins were resuspended in Laemmli buffer under reducing conditions and separated by electrophoresis in acrylamide gels (10% or 15% SDS- PAGE). The proteins were then electro-transferred onto nitrocellulose membranes (Schleider & Schuell, Keene, NH) and sHLA-G was detected using the monoclonal mouse antibody, 16G 1 or anti-Flag Ml antibody (Sigma- Aldrich, St. Louis MO). The membrane subsequently was incubated with a peroxidase conjugated anti-mouse IgG antibody (Jackson hnmuno Research hie, West Grove, PA).
  • the signal was developed using the chemiluminescent substrate SuperSignal West Pico (Pierce, Rockford, EL) and detected by exposing to Hyperfilm ECLTM (Amersham Pharmacia Biotech, Piscataway, NJ).
  • Hyperfilm ECLTM Amersham Pharmacia Biotech, Piscataway, NJ.
  • increasing amounts of the purified protein were loaded under reducing and denaturing conditions on a 15% SDS-PAGE gel, electro-transferred to nitrocellulose membranes and incubated with a mouse antibody specific for human ⁇ 2m (Amac Inc., Westbrook, ME) at 1 ⁇ g/ml, and detected as described above.
  • Example 7 This example determined the glycosylation patterns of the purified recombinant sHLA-Gl and sHLA-G2.
  • purified recombinant sHLA-Gl and sHLA-G2 were digested under native and denaturing conditions using an enzymatic deglycosylation kit that cleaves N-linked and sialic acid-substituted Gal ⁇ 1 -3 Gal ⁇ Ac ⁇ 1 O-linked oligosaccharides from glycoproteins (Bio-Rad, Hercules, CA). After enzymatic digestion, the samples were denatured in reducing Laemmli buffer, separated on SDS-PAGE acrylamide gels, transferred onto nitrocellulose membrane and detected with 16G1 antibody as described above.
  • Example 8 This example isolated peripheral blood mononuclear cells (PBMCs) which were later used to establish the biological activity of the recombinant HLA-G proteins.
  • PBMCs peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Complementary DNA was then synthesized using 200U/ml of MMLV reverse transcriptase from the Strip-EZ RT StripAble cDNA Probe Synthesis and Removal Kit (Ambion, Austin, TX) in the presence of 20mCi [ ⁇ - 33 P]-dATP (Perkin-Elmer Life Sciences, Boston, MA) according to the manufacture's instructions. Unincorporated nucleotides were removed from labeled cDNA using Bio-Spin 30 chromatography columns (Bio-Rad). A hand-held Geiger-Mueller counter was used to determine the percentage incorporation of the radioactive nucleotides into the cDNA.
  • incorporation of the radiolabeled nucleotide into the cDNA probes for the rsHLAGl array were control PBS, 40%, rsHLA-Gl treatment, 46%.
  • rsHLA-G2 array incorporations were, 47% and 32% for the control PBS probe and rsHLAG2 probe, respectively.
  • the cDNA probes had 75% (IFN- ⁇ ) and 45% incorporation (EFN- ⁇ + rsHLA-Gl) and 55% (IFN- ⁇ ) and 26% incorporation (JFN- ⁇ + rsHLA-G2) for experiments testing the effect of rsHLA-Gl and rsHLA-G2 respectively on JJFN- ⁇ -activated PBMC.
  • Example 10 This example determined gene expression using a cDNA array analysis performed on PBMCs after incubation with recombinant soluble HLA-G proteins.
  • PanoramaTM Human Cytokine Arrays enable the analysis of 375 differentially expressed genes including cytokines and related factors, their receptors and housekeeping genes.
  • Two Panorama Cytokine Array membranes (Sigma-Genosys) were pre- hybridized for at least 1 hour at 65C in Panorama hybridization solution (Sigma-Genosys) containing 0.1 mg/ml salmon testes DNA (Sigma).
  • the labeled cDNA synthesized from 1 ⁇ g of RNA from control, rsHLA-Gl, or rsHLA-G2-treated PBMC was added to hybridization solution containing 0.1 mg/ml salmon testes DNA (Sigma) and denatured at 90C to 95C for 10 minutes.
  • the arrays were hybridized overnight at 65C and subsequently washed three times at room temperature in 0.5% SSPE, 1% SDS, followed by two washes at 65C in 0.1% SSPE, 1% SDS.
  • the arrays were exposed to Cyclone Storage phosphor screens (Packard, Meriden, CT) for 3 to 4 days.
  • Optical densities (OD) were obtained using the Cyclone Storage Phosphor System and OptiQuant (version 3.0) acquisition and analysis software (Packard).
  • GPDH glyceraldehyde-3 -phosphate dehydrogenase
  • Example 10 This example demonstrates the effect of HLA-G on the immune response to grafted tissues.
  • Three groups of subjects undergoing a tissue graft will be tested for endogenous levels of soluble HLA-G prior to the graft. Once the graft is complete, the subjects will once again have endogenous levels of soluble HLA-G determined. Additionally, the first group subjects will be administered a first amount of rsHLA-G. The second group will receive a second amount of rsHLA-G and the third group of subjects will not receive any additional rsHLA-G. At predetermined times thereafter, each group which received the rsHLA-G will receive additional amounts coinciding with the first amount administered to each group. The subj ects will be tested periodically to determine the levels of rsHLA-G circulating in their bloodstream and subjects which reject the grafts will be recorded together with a current rsHLA-G measurement.
  • Example 11 This example measures EL- 10 production in decidual macrophages after treatment with rsHLA-G.
  • Decidual macrophages were isolated from extraplacental membranes by enzyme digestion and gradient centrifugation. Fresh placenta was obtained and decidual tissue was scraped away from the chorionic membrane, minced, rinsed of blood, and incubated in digestion solution (100X Penicillin/Streptomycin, 20mM HEPES, 30mM Sodium Bicarbonate, lOmg/ml BSA, 200U/ml collagenase, lmg/ml hyaluronidase, 150 ⁇ g/ml DNAse in HBSS) at 37°C for 1 hour. Cell digest was passed through cotton gauze and a 70 ⁇ m nylon mesh.
  • Cells were collected by centrifugation (1200 rpm for 10 min.), washed with freshmedium (RPMI 1640, 10% FCS, 2mM L-Gln, IX Penn/Strep), and resuspended. Cells were layered over an equivalent volume of Histopaque 1077 and centrifuged at 400xg for 40 min. at room temperature. Interface cells were collected, washed three times in culture medium, and counted by Trypan blue dye exclusion.
  • decidual cells were incubated overnight at 37°C/5% CO 2 for 18 hours in cell culture medium (RPMI 1640 containing 10% FCS, Penn/Strep, L-Gln, HEPES) to allow adherence of macrophages.
  • cell culture medium RPMI 1640 containing 10% FCS, Penn/Strep, L-Gln, HEPES
  • Non-adherent cells and supernatant was removed, adherent cells were washed once with HBSS, and fresh culture medium containing treatments was added (EFN ⁇ from Genzyme at 1 OOU/ml final well concentration, rsHLA-Gl and rsHLA-G2 at l ⁇ g/ml final well concentrations).
  • Cells were incubated for 24 hours at 37°C/5% CO 2 , and supernatants were collected for measurement of secreted IL-10.
  • JL-10 production was measured by enzyme-linked immunosorbant assay (ELIS A) using a pre-coated anti-human EL- 10 capture ELIS A kit from Pierce-Endogen. All kit procedures were followed, including the construction of a standard curve and duplicate sample measurements.
  • enzyme-linked immunosorbant assay (ELIS A) using a pre-coated anti-human EL- 10 capture ELIS A kit from Pierce-Endogen. All kit procedures were followed, including the construction of a standard curve and duplicate sample measurements.
  • Fig. 1 the sequences for both messages derived from the S14/8.3 cells encoding sHLA-Gl and sHLA-G2 aligned exactly with the expected sequences for the coding regions for the ⁇ l, ⁇ 2, ⁇ 3 domains and intron 4 retaining transcripts when compared with HLA 6.0.
  • the schematic representation of the HLA-G gene shown in Fig. 1 depicts the two alternatively spliced transcripts which retain intron 4, sHLA-Gl and sHLA-G2.
  • the exon 4 coding region reads into intron 4 a total of 21 amino acids, then the stop codon prevents further reading of the transmembrane domain, thereby rendering these proteins soluble.
  • the alternative splicing implies another difference between sHLA-Gl andsHLA-G2. This difference is depicted by the change in the first amino acid for the ⁇ 3 domain in sHLA-G2, GAC ⁇ AAC (resulting in a change from D to N).
  • the arrows in Fig. 1 indicate the position for the primers used in the second PCR to obtain the blunt ended cDNA fragment for cloning, as described above.
  • the overall sequences were exactly as expected from the splicing of the messages, with sHLA-Gl (SEQ ED NO. 7) containing ⁇ l (SEQ EO NO. 3), ⁇ 2 (SEQ ED NO. 4), ⁇ 3 (SEQ JD NO.
  • HEK293 cells After transfection of HEK293 cells with sHLA-G constructs derived from S 14/8.3 cells stably transfected clones were selected by resistance to G418. High-producing colonies were selected by limiting dilution analysis and evaluation of their production of rsHLA-G into the culture media was done using an ELISA as described in Example 2.
  • Fig. 3 shows the results of an ELISA for rsHLA-G using the anti-sHLA-G antibodyl6Gl.
  • 293-V gives the results for the culture media supematants from HEK293 cells stably transfected with the empty pRC/CMVBM40 vector
  • 293-sHLA-Gl and 293-sHLA-G2 give the results for the soluble constructs.
  • Each of these (293-V, 293-sHLA-Gl , and293-sHLA-G2) were used to coat a 96 well plate and an ELISA was performed as described above.
  • HEK293 cells stably transfected with the empty vector did not produce detectable reactivity for sHLA-G whereas clones carrying the construct for sHLA-Gland -G2 secreted high levels of soluble proteins into the culture media.
  • Human leukocyte antigens are heterodimeric molecules comprised of glycosylated heavy chains non-covalently associated with a 12 kDa non-polymorphic light chain called ⁇ 2m.
  • Purified rsHLA-Gl (10 tolOO ng) and rsHLA-G2 (10 to 1000 ng) were separated on 15% SDS- PAGE, transferred to nitrocellulose membranes and immuno-detected with anti-Flag Ml antibody or anti- ⁇ 2m, as shown in Fig. 6. OnlyrsHLA-Gl was positive for ⁇ 2m.
  • Recombinant sHLA-G2 failed to yield a positive signal for ⁇ 2m and was negative even when high amounts of the protein were used (up to 1 ⁇ g) (Fig. 6).
  • Recombinant soluble HLA-Gl and -G2 differentially regulate gene expression in resting and IFN- ⁇ -stimulated human blood mononuclear cells.
  • peripheral blood mononuclear cells were cultured for 6 hours in the presence or absence of JFN- ⁇ (100 U/ml) and rsHLA-Gl (1 ⁇ g/ml), rsHLA-G2 (1 ⁇ g/ml), or PBS (control).
  • Relative mRNA expression of 375 genes were analyzed by cytokine array, quantified by phosphorimage analysis, and normalized against a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. Results in the table are for the total number of messages detected (Total), and number (percent) of those messages with treatmen control ratios greater than 1.5 (Increased), 1.4 to 0.6 (Unchanged), or below 0.5 (Decreased).
  • rsHLA-G 1 and rsHLA-G2 had both overlapping and divergent effects on the cells.
  • EL-l ⁇ and Flt-3/Flt-2R were enhanced by both rsHLA-Gl and rsHLA-G2 in resting and stimulated cells, respectively, whereas the recombinant proteins differentially affected NGF Rand TRAIL message in JJFN- ⁇ -stimulated cells.
  • the effect on gene expression was dependent on the activation state of the cells. For example, expression of EL- 1 Rl, FGF 11 , and NGF R mRNAs were enhanced by rsHLA-G2 in resting cells, but decreased in stimulated cells; for caspase-1 and CAD12, the opposite was true.
  • HLA-G affecting tissue grafts
  • the presence of endogenous circulating HLA-G had a direct effect on the tolerance exhibited by the immune system of each individual.
  • tolerance of the graft will improve and therefore the percentage of successful grafts will rise.
  • HLA class I molecules are heterodimers composed of an alpha chain with three extracellular domains non-covalently associated with the invariant 12 kDa ⁇ 2m light chain. Ithas been suggested that four regions of the ⁇ chain, one in each ⁇ l and ⁇ 2 and two in ⁇ 3 were contact points between the a chain and ⁇ 2m. If one of these regions is not present one might predict that ⁇ 2m would not bind to the chain. Consistent with this prediction, the present invention determined that purified rsHLA-Gl bound ⁇ 2m, but, lacking the ⁇ 2 domain, rsHLA- G2 did not. These findings differ from earlier reports that membrane-bound HLA-G2 binds ⁇ 2m. One potential explanation for this discrepancy would be that ⁇ 2m became dissociated from the recombinant sHLA-G2 during the purification procedure. However, this is unlikely because all purification steps were performed with proteins in the native state.
  • the experiments indicate that the recombinant proteins exert differential biological activity depending on the activation state of the target cells; IFN- ⁇ -stimulated cells were much more susceptible to gene regulation by rsHLA-Gl and -G2 than resting cells.
  • the particular genes that were affected by the soluble proteins suggest that they function in regulation of chemokme and chemokine receptor expression, as well as expression of cell adhesion molecules.
  • Additional studies to determine which types of leukocytes respond to the rsHLA-G molecules and to define receptors for rsHLA-Gl and -G2 are in progress. Regarding the potential for signaling, receptors for HLA- Gl have been identified on monocytes, lymphocytes, and natural killer cells but receptors responsible for the actions of sHLA-G2have not been identified.
  • Fig. 11 illustrates EL- 10 production in decidual macrophages treated with rsHLA-G.
  • Decidual cells were isolated from extraplacental membranes and allowed to adhere on a plastic well-bottomed for 18 hours. Non-adherent cells and supernatant were removed and treatments were added as follows: control(PBS), rsHLA-Gl (l ⁇ g/ml), rsHLA-G2 (1 ⁇ g/ml). Cells incubated for 24 hours and supematants were collected for capture ELISA. The values shown are averages of duplicate wells.
  • the present invention reports the production of biologically active recombinant proteins derived from sequences encoding two soluble isoforms of HLA-G, i.e., rsHLA-Gl and rsHLA-G2. These proteins should be highly useful in defining the targets of the isoforms and determining their biological functions, which may include immunomodulation during pregnancy and other instances of tissue grafting and organ transplantation.

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Abstract

Cette invention se rapporte à des procédés pour produire et utiliser la protéine HLA-G soluble recombinée. Cette protéine HLA-G soluble recombinée modifie les réactions immunitaires contre les tissus, les organes, les foetus et les embryons qui sont génétiquement distincts de l'organisme recevant ou possédant un tel matériau antigénique. Des formes préférées de cette protéine contiennent des séquences possédant une homologie de séquence d'au moins 70 % avec des formes naturelles de HLA-G. Plus spécifiquement, chaque forme recombinée de HLA-G produite et utilisée par les procédés faisant l'objet de cette invention doit contenir une séquence possédant une homologie de séquence d'au moins 70 % avec l'intron 4 exprimé par le gène HLA-G et au moins une séquence possédant une homologie de séquence d'au moins 70 % avec l'un des domaines α exprimés par le gène HLA-G. Des formes préférées de cette invention contiennent une isoforme qui contient le domaine α1, le domaine α2, le domaine α3 et l'intron 4, et une seconde isoforme qui contient le domaine α1, le domaine α3 et l'intron 4. Plus préférablement, ces séquences contiennent une séquence peptidique d'aide à la purification et un peptide signal.
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