Patent Application
20060025570
Not applicable.
Not applicable.
The present invention relates generally to a family of novel protein factors which share homologous sequences and biological activities with megakaryocyte colony-stimulating factor (meg-CSF) and which participate in the differentiation or maturation of megakaryocyte progenitors. Also provided are processes for obtaining and producing the factors from natural sources or by artificial means, e.g., chemical synthesis or recombinant genetic engineering techniques.
Megakaryocytes are the hematopoietic cells, largely found in the bone marrow, but also in peripheral blood and perhaps other tissues as well, which produce platelets (also known as thrombocytes) and subsequently release them into circulation. Megakaryocytes, like all of the hematopoietic cells of the human hematopoietic system, ultimately are derived from a primitive stem cell after passing through a complex pathway comprising many cellular divisions and considerable differentiation and maturation.
The platelets derived from these megakaryocytic cells are critical for maintaining hemostasis and for initiating blood clot formation at sites of injury. Platelets also release growth factors at the site of clot formation that speed the process of wound healing and may serve other functions. However, in patients suffering from depressed levels of platelets (thrombocytopenia) the inability to form clots is the most immediate and serious consequence, a potentially fatal complication of many therapies for cancer. Such cancer patients are generally treated for this problem with platelet transfusions. Other patients frequently requiring platelet transfusions are those undergoing bone marrow transplantation or patients with aplastic anemia.
Platelets for such procedures are obtained by plateletphoresis from normal donors. Like most human blood products, platelets for transfusion have a relatively short shelf-life and also expose the patients to considerable risk of exposure to dangerous viruses, such as the human immunodeficiency virus (HIV) or a hepatitis virus.
Clearly the ability to stimulate endogenous platelet formation in thrombocytopenic patients with a concomitant reduction in their dependence on platelet transfusion would be of great benefit. In addition, the ability to correct or prevent thrombocytopenia in patients undergoing radiation therapy or chemotherapy for cancer would make such treatments safer and possibly permit increases in the intensity of the therapy thereby yielding greater anti-cancer effects.
For these reasons considerable research has been devoted to the identification and purification of factors involved in the regulation of megakaryocyte and platelet production. Although there is considerable controversy on this subject, the factors regulating the growth and differentiation of hematopoietic cells into mature megakaryocytes and the subsequent production of platelets by these cells are believed to fall into two classes: (1) megakaryocyte colony-stimulating factors (meg-CSFs) which support the proliferation and differentiation of megakaryocytic progenitors in culture, and (2) thrombopoietic (TPO) factors which support the differentiation and maturation of megakaryocytes in vivo, resulting in the production and release of platelets. [See, e.g., E. Mazur, Exp. Hematol., 15:340-350 (1987).]
Each class of factors can be defined by bioassay. Factors with meg-CSF activity support megakaryocyte colony formation, while factors with TPO activity elicit an elevation in the numbers of circulating platelets when administered to animals. It is not clear how many species of factors exist that have either one or both of these activities. For example, human IL-3 supports human megakaryocyte colony formation and, at least in monkeys, frequently elicits an elevation in platelet count. However, IL-3 influences hematopoietic cell development in all of the hematopoietic lineages and can be distinguished from specific regulators of megakaryocytopoiesis and platelet formation which interact selectively with cells of the megakaryocytic lineage.
Many different reports in the literature describe factors which interact with cells of the megakaryocytic lineage. Several putative meg-CSF compositions have been derived from serum [See, e.g., R. Hoffman et al, J. Clin. Invest., 75:1174-1182 (1985); J. E. Straneva et al, Exp. Hematol., 15:657-663 (1987); E. Mazur et al, Exp. Hematol., 13:1164-1172 (1985]. A large number of reports of a TPO factor are in the art. [See, e.g., T. P. McDonald, Exp. Hematol., 16:201-205 (1988); T. P. McDonald et al, Biochem. Med. Metab. Biol., 37:335-343 (1987); T. Tayrien et al, J. Biol. Chem., 262: 3262-3268 (1987) and others]. From the studies reported to date, it is not clear whether factors having activities identified as meg-CSF also have TPO activity or vice versa.
Although there have been numerous additional reports tentatively identifying these regulatory factors, the biochemical and biological identification and characterization of meg-CSF and TPO factors have been hampered by the small quantities of the naturally occurring factors which are present in natural sources, e.g., blood and urine.
The present inventors have previously identified a purified meg-CSF factor from urine described in PCT application No. WO91/02001, published Feb. 21, 1991, and further described in pending U.S. patent application Ser. No. 07/643,502. This homogeneous meg-CSF, purified from urine and which may be produced via recombinant or synthetic techniques, is characterized by a specific activity in the murine fibrin clot assay of greater than 5×107 dilution units per mg and preferably, 2×108 dilution units per mg protein. This meg-CSF was processed from a precursor protein encoded by an approximately 18.2 kb genomic clone which contains ten exons. Two additional exons were subsequently identified outside this 18.2 kb segment. cDNAs made from the full length cDNA and from partial cDNAs have been expressed in COS-1 cells and CHO cells. These references are incorporated herein by reference for the disclosure of that meg-CSF molecule.
There remains a need in the art for additional proteins either isolated from association with other proteins or substances from their natural sources or otherwise produced in homogeneous form, which are capable of stimulating or enhancing the production of platelets in vivo, to replace presently employed platelet transfusions and to stimulate the production of other cells of the lymphohematopoietic system.
In one aspect the present invention provides a family of human megakaryocyte stimulating factors (hereafter called MSFs) or biologically active peptide fragments thereof that are substantially free from association with proteins or substances with which they occur in nature. The MSF family includes non-naturally occurring MSF proteins which are characterized by sharing similar structure, DNA and amino acid sequences, and/or biological activity with naturally-occurring MSFs. Naturally-occurring MSFs may be isolated and purified from natural sources. Non-naturally occurring MSFs may be prepared by chemical synthesis and/or recombinant genetic engineering techniques.
These MSF proteins comprise DNA sequences encoding one or more of the exon sequences described below in
Another aspect of the present invention includes a mixture of different MSFs, and active fragments thereof.
Another aspect of the present invention includes DNA sequences that encode the expression of these naturally occurring and non-naturally occurring MSFs.
Still another aspect of the present invention are recombinant DNA molecules comprising vector DNAs and DNA sequences encoding the MSFs of this invention. Each DNA molecule provides the MSF DNA in operative association with a regulatory sequence capable of directing the replication and expression of each MSF in a selected host cell. Host cells transformed with such DNA molecules for use in expressing recombinant MSF proteins are also provided by the present invention.
The DNA molecules and transformed cells of the invention may be employed in a novel process for producing recombinant MSFs, or biologically active peptide fragments thereof. A cell line, transformed with a DNA sequence encoding expression of individual MSFs or fragments thereof (or a recombinant DNA molecule as described above) in operative association with a suitable regulatory or expression control sequence capable of controlling expression of the protein, is cultured under appropriate conditions permitting expression of the recombinant DNA. The expressed MSF protein is then harvested from the host cell, cell lysate or culture medium by suitable conventional means.
Still a further aspect of the present invention is a process for isolating and purifying an MSF composition of the present invention or a fragment thereof, or a mixture of MSFs and fragments, from natural sources, e.g. urine. or peripheral blood leukocytes. Alternatively, the MSFs may be isolated and purified from conditioned medium or cell lysate of cells expressing recombinant MSF protein.
Another aspect of this invention provides pharmaceutical compositions containing a therapeutically effective amount of one or more MSFs of the present invention. These pharmaceutical compositions may be employed in methods for treating disease states or disorders, for example, diseases characterized by a deficiency of platelets and other disorders referred to herein.
A further aspect of the invention, therefore, is a method for treating such disease states by administering to a patient a therapeutically effective amount of at least one MSF in a suitable pharmaceutical carrier. Alternatively, several MSFs may be administered in combination. These therapeutic methods may include administering in combination, simultaneously, or sequentially with an MSF, an effective amount of at least one other TPO-like factor, meg-CSF or other cytokine, e.g, IL-3 or steel factor, hematopoietin, interleukin, growth factor, or antibody.
Still another aspect of the present invention provides antibodies directed against an MSF of this invention. As part of this aspect, therefore, the invention includes cell lines capable of secreting such antibodies and methods for their production and use in diagnostic or therapeutic procedures.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description of preferred embodiments thereof.
The novel family of human megakaryocyte stimulating factors (MSFs) provided by the present invention are protein or proteinaceous compositions substantially free of association with other human proteinaceous materials, contaminants or other substances with which the factors occur in nature. An MSF may be purified from natural sources as a homogeneous protein, or from a selected cell line secreting or expressing it. Mixtures of naturally occurring MSFs may be obtained from natural sources, or from selected cell lines by similar purification techniques. Another class of MSFs are “recombinant or genetically engineered proteins” which are defined herein as naturally occurring and non-naturally occurring proteins prepared by chemical synthesis and/or recombinant genetic engineering techniques, and/or a combination of both techniques. These MSFs may also be provided in optional association with amino acid residues and other substances with which they occur by virtue of expression of the factors in various expression systems. Recombinant or genetically-engineered MSFs of this invention may further be defined as including a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin with sequences from the meg-CSF DNA of
The MSFs of the present invention include active fragments and alternatively spliced sequences derived from the DNA [SEQ ID NO: 1] and amino acid [SEQ ID NO: 2] sequences reported in
The exons of the meg-CSF gene [SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26] were identified by analysis of cDNA clones from COS cells transfected with the gene or pieces of the gene or from cDNAs isolated from stimulated human peripheral blood lymphocytes. The first exon [SEQ ID NO: 4], containing the initiating methionine, encodes a classical mammalian protein secretion signal sequence. Exons II [SEQ ID NO: 6] through IV [SEQ ID NO: 8] contain the amino acid sequences of the original mature urinary meg-CSF protein which most likely terminates in a region between amino acid residues 134 and 205 of
Analysis of the nucleotide and amino acid sequences of all of the exons of the meg-CSF precursor gene [SEQ ID NOS: 3-26] have revealed some interesting relationships to other genes. Both Exons II [SEQ ID NO: 5 and 6] and III [SEQ ID NO: 7 and 8], which are related to each other, share significant sequence similarity with the somatomedin B domain of vitronectin and of PC-1 (a surface membrane glycoprotein found on plasma cells). The Cys-rich regions of Exons II [SEQ ID NO: 5 and 6] and III [SEQ ID NO: 7 and 8] are also related to similar sequences in placental protein 11, a putative serine protease. A sequence containing approximately 75 repeats of the sequence Lys-Pro-Thr which is present in Exon VI [SEQ ID NO: 13 and 14], resembles similar repeat sequences which have been found in membrane-bound glycoproteins and precursor proteins, such as spasmolysin.
Exons VI [SEQ ID NO: 13 and 14] through XII [SEQ ID NO: 25 and 26], in particular Exons VII [SEQ I) NO: 15 and 16], VIII [SEQ ID NO: 17 and 18] and IX [SEQ ID NO: 19 and 20], also contain sequences which are found in vitronectin distinct from the somatomedin B domain. These exons encode a sequence found in vitronectin and members of the collagenase family, e.g., human stromelysin. Another functional domain of vitronectin including the RGD adhesion sequence known to bind integrins is not found in any of the exons of the meg-CSF gene. The functions of the amino acid sequences from Exons V [SEQ ID NO: 11 and 12] through XII [SEQ ID NO: 25 and 26] have not yet been determined, but may play roles which effect the three dimensional structure, i.e., folding of the molecule.
During the course of the analysis of the structure of the 18.2 kb “meg-CSF” gene, it was discovered that the primary RNA transcript is spliced in a variety of ways to yield a family of mRNAs each encoding different MSF proteins. In addition, these precursor proteins can be processed in different ways to yield different mature MSF proteins. Thus, a family of MSF's exist in nature, including the meg-CSF which was isolated from urine from the bone marrow transplant patients. All members of this family are believed to be derived from the 18.2 kb meg-CSF gene plus a few additional exons, found in the peripheral blood leukocyte cDNA located just downstream from the 3′ end of the 18.2 kb gene.
The entire 18.2 kb genomic sequence inserted as a NotI fragment in bacteriophage lambda DNA was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, USA under accession # ATCC 40856. The 3′ PBL partial cDNA clone beginning one third of the way from the 5′ end of Exon VI [SEQ ID NOS: 13 and 14] (containing the SnaB site) extending through Exon XII [SEQ ID NOS: 25 and 26] is stored at Genetics Institute, Inc. and can be readily made available without restrictions by deposit at the ATCC upon an indication of allowance of this application.
This invention also contemplates the construction of “recombinant or genetically-engineered” classes of MSFs, which may be generated using different combinations of the amino acid sequences [SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26] of the exons of
Without being bound by theory, and based on analysis of the naturally occurring meg-CSF sequence of
Both naturally occurring MSFs and non-naturally-occurring MSFs may be characterized by various combinations of alternatively spliced exons of
Naturally-occurring MSFs may possess at least Exon I [SEQ ID NO: 3 and 4], which contains both an initiating methionine necessary for translation and a secretory leader for secretion of the factor from mammalian cells, and one or more additional exons of
Other naturally occurring MSFs may possess both Exon I [SEQ ID NO: 3 and 4] and Exon XII [SEQ ID NO: 25 and 26], which latter exon contains a termination codon for translation, and at least one additional exon selected from Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof. It is speculated that both the initiating Met of Exon I [SEQ ID NO: 3 and 4] and the termination codon of Exon XII [SEQ ID NO: 25 and 26] are required to produce an active, properly folded, naturally-occurring MSF in a eukaryotic cell. Thus naturally-occurring MSFs may contain at least Exons I [SEQ ID NO: 3 and 4] and XII [SEQ ID NO: 25 and 26] and another exon. An exemplary MSF [SEQ ID NO: 61] of this class includes a protein [SEQ ID NO: 62] represented by the spliced-together arrangement of exons selected from Exons I through XII of
Another class of naturally occurring MSFs may be characterized by the presence of Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], or a biologically active fragment thereof, and all of Exons VI through XII [SEQ ID NOS: 13-26]. An exemplary MSF [SEQ ID NO: 39 and 40] of this class includes spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8], IV [SEQ ID NO: 9 and 10], and VI through XII [SEQ ID NO: 13-26]. Another MSF of this class [SEQ ID NO: 41 and 42] is formed by spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8], and VI through XII [SEQ ID NO: 13-26]. Still another MSF sequence [SEQ ID NO: 43 and 44] is formed from spliced together Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8], and VI through XII [SEQ ID NO: 13-26]. Another MSF sequence [SEQ ID NO: 45 and 46] of this class includes spliced together Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8], IV and VI through XII [SEQ ID NO: 13-26].
Still another class of naturally occurring MSFs may be characterized by the presence of Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQ ID NO: 5 and 6]; III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof; and Exons V through XII [SEQ ID NO: 11-26]. An exemplary MSF [SEQ ID NO: 47 and 48] of this class includes spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8], and V through XII [SEQ ID NO: 11-26]. Another MSF [SEQ ID NO: 141 and 142] of this class is formed by spliced together Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8], and V through XII [SEQ ID NO: 11-26]. Still another MSF [SEQ ID NO: 49 and 50] sequence is formed from spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], and V through XII [SEQ ID NO: 11-26]. Another MSF sequence [SEQ ID NO: 51 and 52] of this class includes spliced together Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8], IV [SEQ ID NO: 9 and 10] and V through XII [SEQ ID NO: 11-26].
Another class of naturally occurring MSFs may be characterized by the presence of Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof; Exon V [SEQ ID NO: 11 and 12], and Exons VII through XII [SEQ ID NO: 15-26]. An exemplary MSF [SEQ ID NO: 53 and 54] of this class includes spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8], IV [SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12] and VII through XII [SEQ ID NO: 15-26]. Another MSF of this class [SEQ ID NO: 63 and 64] is formed by Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8], V [SEQ ID NO: 11 and 12] and VII through XII [SEQ ID NO: 15-26] in a spliced together form. Still another MSF sequence [SEQ ID NO: 65 and 66] is formed from spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], IV [SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12] and VII through XII [SEQ ID NO: 15-26]. Another MSF sequence [SEQ ID NO: 67 and 68] of this class includes Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8], IV [SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12] and VII through XII [SEQ ID NO: 15-26] spliced together.
Yet another class of naturally occurring MSFs may be characterized by the presence of Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10] and a biologically active fragment thereof, at least one of Exons V through XI [SEQ ID NO: 11-24]; and Exon XII [SEQ ID NO: 25 and 26]. An exemplary MSF [SEQ ID NO: 69 and 70] of this class includes spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8], IV [SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12], X [SEQ ID NO: 21 and 22] and XII [SEQ ID NO: 25 and 26]. Another MSF [SEQ ID NO: 71 and 72] of this class is formed by spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8], VIII [SEQ ID NO: 17 and 18], IX [SEQ ID NO: 19 and 20] and XIII [SEQ ID NO: 25 and 26]. Still another MSF sequence [SEQ ID NO: 73 and 74] is formed from spliced together Exons I [SEQ ID NO: 3 and 4], III [SEQ ID NO: 7 and 8], VI [SEQ ID NO: 13 and 14] and XII [SEQ ID NO: 25 and 26]. Another MSF sequence [SEQ ID NO: 75 and 76] of this class includes spliced together Exons I [SEQ ID NO: 3 and 4], II [SEQ ID. NO: 5 and 6], IV [SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12], VII [SEQ ID NO: 15 and 16] and XII [SEQ ID NO: 25 and 26].
For recombinant or genetically engineered MSFs, Exon I [SEQ ID NO: 3 and 4] may be replaced by a synthetic or heterologous sequence containing an initiating Met and a selected secretory leader designed for use in a selected expression system (hereafter referred to for simplicity as “artificial Exon I”). The natural Exon I [SEQ ID NO: 3 and 4] may be completely absent for intracellular expression in a bacterial host, such as E. coli. A secretory leader may be selected from among known sequences for secretion of proteins from a variety of host cells. A number of secretory leaders are known for bacterial cells, yeast cells, mammalian cells, insect cells and fungi which may be useful as host cells for expression of a recombinant or genetically-engineered MSF. The construction of an appropriate genetically engineered Exon I sequence containing a secretory leader and initiating Met is within the skill of the art with resort to known sequences and techniques. Thus, one class of recombinant MSFs may be characterized by a genetically-engineered Exon I in place of the naturally occurring Exon I [SEQ ID NO: 3 and 4] of
Additionally, the termination codon supplied by Exon XII [SEQ ID NO: 25 and 26] to naturally occurring MSFs may be replaced by inserting into, or after, a selected exon of
One class of recombinant MSFs include a naturally-occurring Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof; and an artificial termination codon. An example of such an MSF is MSF-K130 [SEQ ID NO: 77 and 78] and MSF-N141 [SEQ ID NO: 79 and 80], among others described in detail below.
Another class of recombinant MSFs include an artificial Exon I, at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof; and Exon XII [SEQ ID NO: 25 and 26].
Still another class of recombinant MSFs include an artificial Exon I, at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof; and an artificial termination codon.
Another class of recombinant, genetically-engineered MSFs include genetically-engineered Exon I, at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof; and all of Exons V through XII [SEQ ID NO: 11-26].
Still another class of recombinant MSFs may be characterized by the presence of genetically-engineered Exon I, at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof; and Exons VI through XII [SEQ ID NO: 13-26].
Another class of recombinant MSFs may be characterized by the presence of genetically-engineered Exon I, at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof; Exon V [SEQ ID NO: 11 and 12], and Exons VII through XII [SEQ ID NO: 15-26].
Yet another class of recombinant MSFs may be characterized by the presence of genetically-engineered Exon I, at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10] and a biologically active fragment thereof; at least one of Exons V through XI [SEQ ID NO: 11-24]; and an artificial termination codon.
Another class of recombinant MSFs is characterized by genetically-engineered Exon I, at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof; all of Exons V through XI [SEQ ID NO: 11-24], with an artificial termination codon either inserted into, or added onto a selected last exon of the sequence.
Another class of recombinant MSFs is characterized by genetically-engineered Exon I, at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof; all of Exons VI through XI [SEQ ID NO: 13-24], with an artificial termination codon.
Another class of recombinant MSFs may be characterized by the presence of native Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof, and all of Exons V through XI [SEQ ID NO: 11-24], with an artificial termination codon.
Still another class of recombinant MSFs may be characterized by the presence of Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10], and a biologically active fragment thereof; and all of Exons VI through XI [SEQ ID NO: 13-24], With an artificial termination codon.
Yet another class of recombinant MSFs may be characterized by the presence of Exon I [SEQ ID NO: 3 and 4], at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10] and a biologically active fragment thereof; at least one of Exons V through XI [SEQ ID NO: 11-24]; and an artificial termination codon.
A further class of recombinant, genetically-engineered MSFs is characterized by the complete absence of an Exon I. Such MSFs are useful for intracellular expression in bacterial cells, such as E. coli. These MSFs may comprise at least one of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10] and a biologically active fragment thereof; optionally one or more exons from Exons V through XII [SEQ ID NO: 11-26]. In the absence of Exon XII [SEQ ID NO: 25 and 26], an artificial termination codon may be inserted into or after the last preferred carboxy terminal exon. Exemplary MSFs of this invention are MSF-234 [SEQ ID NO: 81 and 82] and MSF 236 [SEQ ID NO: 83 and 84 described below in detail.
In another class of naturally-occurring or non-naturally occurring MSFs, either the sequences of Exon VIII [SEQ ID NO: 17 and 18] and Exon IX [SEQ ID NO: 19 and 20] will be present together, or neither of these two exons will be present. This is primarily due to frame shifts between these exons and the remaining MSF exons.
Finally an MSF [SEQ ID NO: 61 and 62] exists which contains all twelve exons [SEQ ID NO: 3-26].
While the above described MSF sequence structures will provide for precursor MSFs capable of being processed naturally, or by a host cell expression system, into mature MSF proteins, it is considered that mature, processed form of the proteins produced in eukaryotic systems will be missing all or part of Exon I [SEQ ID NO: 3 and 4]. Perhaps the mature proteins may be missing a portion of Exon II [SEQ ID NO: 5 and 6] as well, in order to remove the leader sequence from the processed form. The processed forms of MSF proteins may also be missing substantial sequences from the carboxy terminus. For example, sequences from Exons V through XII [SEQ ID NO: 11-26] may be absent in mature, processed MSF proteins. As another example, sequences from Exons VI through XII [SEQ ID NO: 13-26] may be absent in mature, processed MSF proteins. As still another example, sequences from Exons VII through XII [SEQ ID NO: 15-26] may be absent in mature, processed MSF proteins. In such manner human urinary meg-CSF, an illustrative naturally-occurring MSF [SEQ ID NO: 81 and 82], has a mature protein sequence characterized by the presence of Exons II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8] and IV [SEQ ID NO: 9 and 10] in a predominantly homodimeric form.
Selected examples of artificial MSFs were prepared by the following methods. During the analysis of the meg-CSF gene, a contiguous cDNA was constructed containing Exons I through VI [SEQ ID NO: 3-14], in which the primary translation product was artificially interrupted by inserting artificial termination codons at different positions in Exons IV [SEQ ID NO: 9 and 10], V [SEQ ID NO: 11 and 12], and VI [SEQ ID NO: 13 and 14] near the point at which the original meg-CSF was believed to be processed, i.e. the region between amino acid residues 134 and 209. These cDNAs were transfected into COS cells and the resulting supernatants were tested for meg-CSF activity. Through this process, several different biologically active MSFs were identified.
One MSF of the present invention is characterized by the DNA sequence [SEQ ID NO: 77] extending from nucleotide number 1 of Exon I through nucleotide number 390 of Exon IV, encoding an amino acid sequence [SEQ ID NO: 78] which is a continuous fusion in frame extending from amino acid I of Exon I [SEQ ID NO: 3 and 4] through amino acid 130 of Exon IV [SEQ ID NO: 9 and 10] of the sequence of
Under SDS-PAGE non-reducing conditions, the molecular weight ranged from about 20 to about 50 kD. Upon expression in COS-1 cells, this MSF cDNA sequence [SEQ ID NO: 77] produces a mixture of monomeric and homo-dimeric species. The homodimer has exhibited activity in the fibrin clot assay of Example 8. In the inventors' hands, this is one of the most easily expressed MSF [SEQ ID NO: 77 and 78] which is also highly active in the fibrin clot assay. The MSF expressed by this sequence [SEQ ID NO: 77 and 78] in mammalian cells approximates the structure and properties of the native human urinary meg-CSF.
Another MSF of the present invention, called MSF-N141 [SEQ ID NO: 79 and 80], is characterized by a nucleotide sequence [SEQ ID NO: 79] extending from nucleotide number 1 of Exon I [SEQ ID NO: 3] through nucleotide number 423 of Exon IV [SEQ ID NO: 9], encoding an amino acid sequence [SEQ ID NO: 80] extending from amino acid 1 through amino acid 141 of the sequence of
Still another MSF of the present invention, MSF-S172 [SEQ ID NO: 87 and 88], is characterized by a nucleotide sequence [SEQ ID NO: 87] extending from nucleotide numbers 1 of Exon I through 516 of Exon V, encoding an amino acid sequence [SEQ ID NO: 88] extending from amino acid 1 through amino acid 172 of the sequence of
A further MSF of the present invention, MSF-R192 [SEQ ID NO: 89 and 90], is characterized by a nucleotide sequence [SEQ ID NO.: 89] extending from nucleotide number 1 of Exon I through 576 of Exon V, encoding an amino acid sequence [SEQ ID NO: 90] extending from amino acid 1 through amino acid 192 of the sequence of
Yet another MSF of the present invention, called MSF-K204 [SEQ ID NO: 91 and 92], is characterized by a nucleotide sequence [SEQ ID NO: 91] extending from nucleotide numbers 1 of Exon I through 612 of Exon VI, encoding an amino acid sequence [SEQ ID NO: 92] extending from amino acid 1 through amino acid 204 of the sequence of
Still a further MSF of the present invention, called MSF-K209 [SEQ ID NO: 93 and 94], is characterized by a nucleotide sequence [SEQ ID NO: 93] extending from nucleotide numbers 1 of Exon I through 627 of Exon VI, encoding an amino acid sequence [SEQ ID NO: 94] extending from amino acid 1 through amino acid 209 of the sequence of
Another MSF of the present invention, MSF-D220 [SEQ ID NO: 95 and 96], is characterized by a nucleotide sequence [SEQ ID NO: 95] extending from nucleotide numbers 1 of Exon I through 660 of Exon VI, encoding an amino acid sequence [SEQ ID NO: 96] extending from amino acid 1 through amino acid 220 of the sequence of
Although in all of the above-described MSFs, the amino and carboxy termini of each MSF is defined precisely, it is to be understood that addition or deletion of one or several amino acids (and consequent DNA coding region) from either end of any of the MSFs (or from either end of any of the exons forming the spliced MSFs) is not likely to significantly alter the properties of the particular MSF. Such truncated MSFs which also retain MSF biological activities are also encompassed by this disclosure. The deliberate insertion of artificial termination codons at other positions in the MSF sequences can provide other members of the MSF family.
The alternatively spliced MSFs of the present invention are characterized by amino acid sequences containing at least two exons and less than twelve exons of
In the PCR technique, the primers extended across exon junctions between Exons I through VI [SEQ ID NO: 3-14]. Primers for use between Exons VI and XII [SEQ ID NO: 13-26]; are currently being designed. Therefore, these seven exons may all be present in these MSFs. Alternatively, no exon from Exon VI through XII [SEQ ID NO: 13-26] may be present. Still alternatively one or more of Exon VI through XII [SEQ ID NO: 13-26] may be present in these representative alterrately spliced MSFs.
For example, the 5′ end of one such MSF, called MSF-136 [SEQ ID NO: 97 and 98], identified by PCR is characterized by a contiguous amino acid sequence containing amino acid 1 to 25 of Exon I [SEQ ID NO: 4] (nucleotides 1 through 76 of
Another PCR-identified 5′ MSF sequence, called MSF-1236 [SEQ ID NO: 101 and 102], is characterized by a contiguous amino acid sequence [SEQ ID NO: 102] containing amino acid 1 to 25 [SEQ ID NO: 4] (nucleotides 1 through 76 [SEQ ID NO: 3]) of Exon I joined in frame to amino acid 26 to 66 [SEQ ID NO: 6] (nucleotides 77 through 199 [SEQ ID NO: 5]) of Exon II, joined in frame to amino acid 67 to 106 [SEQ ID NO: 8] (nucleotides 200 through 319 [SEQ ID NO: 7]) of Exon III, joined in frame to amino acid 200 to about 250 [SEQ ID NO: 100] (nucleotides 598 through about 748 [SEQ ID NO: 99]) of Exon VI. This 5′ MSF sequence [SEQ ID NO: 101 and 102] has been detected by PCR analysis of the following cell lines: U20S, H128, SK-N-SH, SK-N-MC, the neuroglioma epithelial-like cell line H4 (ATCC No. HTB148), OCIM1, K562, K562 in the presence of PMA, the erythroleukemia cell line HEL (ATCC No. TIB180) in the presence of PMA, OCIM2, HEPG2 and stimulated PBLs. The presence of this MSF-1236 [SEQ ID NO: 101 and 102] indicates that a naturally-occurring alternately spliced MSF may comprise Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8], VI [SEQ ID NO: 13 and 14] and optionally one or more of Exons VII through XII [SEQ ID NO: 15-26]. A recombinant MSF mimicking this structure may have an artificial termination codon inserted within or after Exon VI [SEQ ID NO: 13 and 14].
Still another MSF [SEQ ID NO: 103 and 104] according to this invention is characterized by a contiguous amino acid sequence containing amino acid 1 to 25 [SEQ ID NO: 4] (nucleotides 1 through 76 [SEQ ID NO: 3]) of Exon I joined in frame to amino acid 26 to 66 [SEQ ID NO: 6] (nucleotides 77 through 199 [SEQ ID NO: 5]) of Exon II, joined in frame to amino acid 67 to 106 [SEQ. ID NO: 8] (nucleotides 200 through 319 [SEQ ID NO: 7]) of Exon III, joined in frame to amino acid 107 to 156 [SEQ ID NO: 10] (nucleotides 320 through 469 [SEQ ID NO: 9]) of Exon IV, joined in frame to amino acid 200 to 1140 [SEQ ID NO: 14] (nucleotides 598 through 3421 [SEQ ID NO: 13]) of Exon VI. This MSF-12346 [SEQ ID NO: 103 and 104] has been detected by PCR analysis of the following cell lines: U20S, SK-N-SH, SK-N-MC, OCIM1 in the presence of PMA, K562 in the presence of PMA, HEPG2 and stimulated PBLs. The presence of this MSF-12346 [SEQ ID NO: 103 and 104] indicates that a naturally-occurring alternately spliced MSF may comprise Exons I [SEQ ID NO: 3 and 4], II [SEQ ID NO: 5 and 6], III [SEQ ID NO: 7 and 8], IV [SEQ ID NO: 9 and 10], VI [SEQ ID NO: 13 and 14] and optionally one ormore of Exons VII through XII [SEQ ID NO: 15-26]. A recombinant MSF mimicking this structure may have an artificial termination codon inserted within or after Exon VI [SEQ ID NO: 13 and 14].
Another MSF sequence of this invention may include MSF-1234 [SEQ ID. NO: 105 and 106], characterized by a contiguous amino acid sequence containing amino acid 1 to 25 [SEQ ID NO: 4] (nucleotides 1 through 76 [SEQ ID NO: 3]) of Exon I (signal peptide) joined in frame to amino acid 26 to 66 [SEQ ID NO: 6] (nucleotides 77 through 199 [SEQ ID NO: 5]) of Exon II, joined in frame to amino acid 67 to 106 [SEQ ID NO: 8] (nucleotides 200 through 319 [SEQ ID NO: 7]) of Exon III, joined in frame to amino acid 107 to 156 [SEQ ID NO: 10] (nucleotides 320 through 469 [SEQ ID NO: 9]) of Exon IV. This sequence optionally has a 3′ sequence comprising one or more of Exons V through XII [SEQ ID NO: 11-26]. This sequence may also contain an artificial termination codon inserted within or after any selected C-terminal exon.
Still another MSF sequence, MSF-134 [SEQ ID NO: 107 and 108], is characterized by a contiguous amino acid sequence containing amino acid I to 25 [SEQ ID NO: 4] (nucleotides 1 through 76 [SEQ ID NO: 3]) of Exon I (signal peptide) joined in frame to amino acid 67 to 106 [SEQ ID NO: 8] (nucleotides 200 through 319 [SEQ ID NO: 7]) of Exon III, joined in frame to amino acid 107 to 156 [SEQ ID NO: 10] (nucleotides 320 through 469 [SEQ ID NO: 9]) of Exon IV. This sequence [SEQ ID NO: 107 and 108] optionally has a 3′ sequence comprising one or more of Exons V through XII [SEQ ID NO: 11-26). This sequence [SEQ ID NO: 107 and 108] may also contain an artificial termination codon inserted within or after any selected C-terminal exon.
Two examples of MSFs that may be useful for bacterial intracellular expression include MSF-234 [SEQ ID NO: 81 and 82], characterized by a contiguous amino acid sequence containing amino acid 26 to 66 [SEQ ID NO: 6] (nucleotides 77 through 199 [SEQ ID NO: 5]) of Exon II joined in frame to amino acid 67 to 106 [SEQ ID NO: 8] (nucleotides 200 through 319 [SEQ ID NO: 7]) of Exon III, joined in frame to amino acid 107 to 156 [SEQ ID NO: 10] (nucleotides 320 through 469 [SEQ ID NO: 9]) of Exon IV; and MSF-236 [SEQ ID NO: 83 and 84], characterized by a contiguous amino acid sequence containing amino acid 26 to 66 [SEQ ID NO: 6] (nucleotides 77 through 199 [SEQ ID NO: 5]) of Exon II joined in frame to amino acid 67 to 106 [SEQ ID NO: 8] (nucleotides 200 through 319 [SEQ ID NO: 7]) of Exon III, joined in frame to amino acid 200 to 1140 [SEQ ID NO: 14] (nucleotides 598 through 3421 [SEQ ID NO: 13]) of Exon VI. These sequences [SEQ ID NOS: 81 and 82; SEQ ID NOS: 83 and 84] each optionally may have a 3′ sequence comprising one or more of Exons V through XI [SEQ ID NO: 11-26]. These sequences [SEQ ID NOS: 81 and 82; SEQ ID NOS: 83 and 84] may also contain an artificial termination codon inserted within or after any selected C terminal exon.
It is further contemplated by the present invention that other MSFs which may be characterized by having MSF biological activities and which may be useful as research agents, diagnostic agents or as therapeutic agents, include factors having other combinations and arrangements of two or more of the exons identified in
Additionally, analogs of MSFs are included within the scope of this invention. An MSF analog may be a mutant or modified protein or polypeptide that retains MSF activity and preferably has a homology of at least about 50%, more preferably about 70%, and most preferably between about 90 to about 95% to human urinary meg-CSF. Still other MSF analogs are mutants that retain MSF activity and preferably have a homology of at least about 50%, more preferably about 80%, and most preferably between 90 to 95% to MSF-K130 [SEQ ID NO: 77 and 78] and the other truncated MSFs. Typically, such analogs differ by only 1, 2, 3, or 4 codon changes. Examples include MSFs with minor amino acid variations from the amino acid sequences of native or recombinant meg-CSF, or any of the above-described MSFs, in particular, conservative amino acid replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into four families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on the MSF activity, especially if the replacement does not involve an amino acid at the active site of the MSF.
The MSFs of this invention may form monomers or homo- or hetero-dimers when expressed in suitable expression systems, due to the presence of cysteine-rich sequences in the exons. As indicated above, two specific homodimeric forms have been identified, namely the MSF-K130 [SEQ ID NO: 77 and 78] characterized by the sequence of amino acid 1 through 130 of
Other MSFs of this invention are predominantly monomers rather than mixtures, such as the MSFs characterized by the sequence of amino acids 1 through 209 of
MSFs of the present invention may act directly on megakaryocyte progenitor cells and/or megakaryocytes. MSFs may act indirectly on accessory cells, such as macrophages and T cells, to produce cytokines which stimulate megakaryocyte colony functions. Specifically, MSFs display megakaryocyte colony stimulating activity. Another MSF activity is the promotion of megakaryocyte maturation. The active MSF compositions of the present invention have biological activity in the murine fibrin clot megakaryocyte colony formation assay. For example, the MSF characterized by the amino acid sequence amino acid 1 of Exon I through amino acid 130 of Exon IV (MSF-K130) [SEQ ID NO: 77 and 78] has a specific activity of greater than approximately 1×107 dilution units/mg protein.
MSFs may also be used in synergy with other cytokines. For example, MSFs also display enhancement of IL-3-dependent megakaryocyte colony formation. MSFs may display enhancement of steel factor-dependent megakaryocyte colony formation [See, K. M. Zsebo et al, Cell, 63:195-201 (1990) for the identification of steel factor, also known as SCF. This document is incorporated by reference herein]. Together, these cytokines, IL-3 and steel factor, have been shown to stimulate increased megakaryocyte colony formation in vitro. In addition, IL-3 has been shown to elevate the level of platelets in non-human primates in vivo.
It is contemplated that all MSFs encoded by the combinations of sequences selected from Exons I through XII [SEQ ID NO: 61 and 62] as reported above will have MSF biological activity, for example, activity in the murine fibrin clot assay, either alone or in combination with other cytokines. All modified or mutant MSF peptides or polypeptides of this invention, including the spliced forms of MSF, may be readily tested for activity in the megakaryocyte fibrin clot assay, either alone or in combination with other known cytokines including IL-3, steel factor or GM-CSF. Other cytokines which may be useful in combination with MSF include G-CSF, CSF-1, GM-CSF, IL-1, IL-4, erythropoietin, IL-6, TPO, M-CSF1, CSF-11, meg-CSF and IL-7.
These MSFs may also have biological or physiological activities in addition to the ability to stimulate the growth and development of colonies consisting of intermediate and large size megakaryocytes in culture in the assay using murine bone marrow target cells. In the murine fibrin clot megakaryocyte colony formation assay, an MSF composition of the present invention stimulates the growth of multiple colony types, but at least 50% of the colonies are pure megakaryocytic or mixed lineage colonies having significant numbers of megakaryocytes. The exact composition of colony types may vary with different assay conditions (fetal calf serum lots, etc). Among the megakaryocyte-containing colonies, typically 50-70% are pure megakaryocytic in composition.
In the murine agar megakaryocyte colony formation assay, an MSF of the present invention may stimulate colonies of megakaryocytes. In some cases, the particular MSF may not by itself stimulate megakaryocyte colony formation, but rather may enhance megakaryocyte colony formation supported by other factors, such as IL-3 or steel factor; or it may synergize with other factors, such as IL-11, which alone is not capable of supporting megakaryocyte colony formation in the fibrin clot assay.
It is presently anticipated that maximal biological activities of these MSFs in vitro may be achieved by activating the factors with acid, or denaturing conditions in SDS-PAGE, or by reverse phase high pressure chromatography (RP-HPLC). With both the native urinary protein and the recombinant MSF-K130 [SEQ ID NO: 77 and 78], an increase in the number of units of activity has been routinely detected after SDS-PAGE and RP-HPLC.
The present invention also encompasses MSF-encoding DNA sequences, free of association with sequences and substances with which the DNA occurs in natural sources. These DNA sequences, including the sequences reported in
Examples of MSF DNA sequences may include a DNA sequence comprising nucleotides 1 through 390 of
Other MSF DNA sequences include the 5′ sequences of certain alternately spliced MSFs, such as a sequence [SEQ ID NO: 109] comprising nucleotides 1-76 of
Other MSF DNA sequences which encode homo- or heterodimers of the above-described MSF DNA sequences or DNA sequences encoding a biologically active fragment of such sequences are also included in this invention. Similarly an allelic variation of the MSF DNA sequences, and a DNA sequence capable of hybridizing to any of MSF DNA sequences, which encodes a peptide or polypeptide having activity in the fibrin clot assay are also encompassed by this invention.
It is understood that the DNA sequences of this invention which encode biologically active human MSFs may also comprise DNA sequences capable of hybridizing under appropriate conditions, or which would be capable of hybridizing under said conditions, but for the degeneracy of the genetic code, to an isolated DNA sequence of
DNA sequences which hybridize to the sequences for an MSF under relaxed hybridization conditions and which code for the expression of MSF peptides having MSF biological properties also encode novel MSF polypeptides. Examples of such non-stringent hybridization conditions are 4×SSC at 50° C. or hybridization with 30-40% formamide at 42° C. For example, a DNA sequence which shares regions of significant homology, e.g., Exons II [SEQ ID NO: 5], III [SEQ ID NO: 7] or IV [SEQ ID NO: 9], and/or glycosylation sites or disulfide linkages, with the sequences of MSF and encodes a protein having one or more MSF biological property clearly encodes an MSF polypeptide even if such a DNA sequence would not stringently hybridize to the MSF sequences.
The DNA sequences of this invention may include or contain modifications in the non-coding sequences, signal sequences or coding sequences based on allelic variation among species, degeneracies of the genetic code or deliberate modification. Allelic variations are naturally-occurring base changes in the species population which may or may not result in an amino acid change. Degeneracies in the genetic code can result in DNA sequences which code for MSF polypeptides but which differ in codon sequence. Deliberate modifications can include variations in the DNA sequence of MSF which are caused by point mutations or by induced modifications to enhance the activity, half-life or production of the polypeptides encoded thereby. All such sequences are encompassed in the invention.
Utilizing the sequence data in
Modifications of interest in the MSF sequences may include the replacement, insertion or deletion of a selected nucleotide or amino acid residue in the coding sequences. For example, the structural gene may be manipulated by varying individual nucleotides, while retaining the correct amino acid(s), or the nucleotides may be varied, so as to change the amino acids, without loss of biological activity. Mutagenic techniques for such replacement, insertion or deletion, e.g., in vitro mutagenesis and primer repair, are well known to one skilled in the art [See, e.g., U.S. Pat. No. 4,518,584]. The encoding DNA of a naturally occurring MSF may be truncated at its 3′-terminus while retaining its biological activity. A recombinant, genetically-engineered MSF DNA sequence may be altered or truncated at both its 3′ and 5′ termini while retaining biological activity. It also may be desirable to remove the region encoding the signal sequence, and/or to replace it with a heterologous sequence. It may also be desirable to ligate a portion of the MSF sequence to a heterologous coding sequence, and thus to create a fusion peptide with the biological activity of MSF.
Specific mutations of the sequences of an MSF polypeptide may involve modifications of a glycosylation site. The absence of glycosylation or only partial glycosylation results from amino acid substitution or deletion at any asparagine-linked glycosylation recognition site or at any site of the molecule that is modified by addition of O-linked carbohydrate. An asparagine-linked glycosylation recognition site comprises a tripeptide sequence which is specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are either Asn-X-Thr or Asn-X-Ser, where X can be any amino acid except proline. For example, such a site can be found in the cDNA illustrated in
Other analogs and derivatives of the sequence of an MSF which would be expected to retain MSF activity in whole or in part may also be easily made by one of skill in the art given the disclosures herein. One such modification may be the attachment of polyethylene glycol (PEG) onto existing lysine residues in an MSF sequence, as taught in U.S. Pat. No. 4,904,584, which is incorporated herein by reference. Alternatively, the insertion of one or more lysine residues or other amino acid residues that can react with PEG or PEG derivatives into the sequence by conventional techniques may enable the attachment of PEG moieties. Existing cysteines may be used according to techniques taught in published PCT Patent Application US90/02144, which is incorporated herein by reference. Such modifications are believed to be encompassed by this invention.
In addition to the above, other open reading frames (ORFs) or structural genes encoding MSFs may be obtained and/or created from cDNA libraries from other animal cell sources. For example, a murine MSF genomic clone and several partial MSF cDNA clones have been isolated by the inventors.
A naturally occurring MSF of this invention may be obtained as a single homogeneous protein or mixture of various alternately spliced MSF proteins and purified from natural sources. Among such natural sources are human urine or stimulated PBLs, other mammalian cell sources producing the factors naturally or upon induction with other factors from cell lines. The DNA of such MSFs may also be obtained and purified from natural sources.
To isolate and purify the naturally-occurring MSFs from natural sources, the purification technique comprises the following steps which are described in more detail in Example 1 below. The example. and the following summary illustrate the purification for an exemplary naturally-occurring MSF, human urinary meg-CSF, which is isolated from human urine. For the urinary meg-CSF, the purification includes concentrating pooled bone marrow transplant patient urine through an Amicon YM-10 filter. The concentrated urine is passed through an anion exchange chromatographic column and the flow-through is bound onto a cation exchange chromatographic column. The urinary protein eluate is then subjected to pooling, dialyzing and heating and is applied to a lectin affinity chromatographic column. This eluate is then dialyzed and applied to a cation exchange fine performance liquid chromatography (FPLC) column. Finally this eluate is applied through three cycles of reverse phase high pressure liquid chromatography (HPLC) using different solvent systems for each HPLC run.
According to this purification scheme, batches with the highest levels of MSF in the murine fibrin clot assay, described below, are selected for further purification at the semi-preparative scale (between 30 and 100 liters urine equivalent) to maximize recovery and yield. Thus a homogeneous MSF, native meg-CSF, may be obtained by applying the purification procedures described in Example 1 to human urine or other sources of human MSF, e.g., activated PBLs.
Other tissue sources and cell lines from which natuially occurring MSFs may be isolated include HeLa cell lines, e.g. ATCC #098-AH2, and bone marrow cell lines, such as murine bone marrow cell line, FCM-1 [Genetics Institute, Inc., Cambridge, Mass.], osteosarcoma cell line U20S, small cell lung carcinoma H128, neuroblastoma SK-N-SH, neuroblastoma SK-N-MC, neuroglioma epithelial-like cell line H4, erythroleukemia cell line OCIM1 and OCIM2, erythroleukemia cell line K562 in the presence of PMA, erythroleukemia cell line HEL in the presence of PMA, and hepatoma cell line HEPG2. Procedures for culturing a cell source which may be found to produce an MSF are known to those of skill in the art.
The MSF proteins and the DNA sequences encoding MSFs of this invention can be produced via recombinant genetic engineering techniques and purified from a mammalian cell line which has been designed to secrete or express the MSF to enable large quantity production of pure, active MSFs useful for therapeutic applications. The proteins may also be expressed in bacterial cells, e.g., E. coli, and purified therefrom. The proteins may also be expressed and purified in yeast cells or in baculovirus or insect cells. Alternatively, an MSF or active fragments thereof may be chemically synthesized. An MSF may also be synthesized by a combination of the above-listed techniques. Suitable techniques for these different expression systems are known to those of skill in the art.
To produce a recombinant MSF, the DNA sequence encoding the factor can be introduced into any one of a variety of expression vectors to make an expression system capable of producing an MSF or one or more fragments thereof in a selected host cell.
The DNA sequences of the individual exons may be obtained by chemical synthesis or may be obtained from the following two deposits by standard restriction enzyme subcloning techniques or by the polymerase chain reaction (PCR) using synthetic primers for each exon based on the nucleotide sequences of
An approximately 12 kb genomic fragment (referred to as Meg Kpn-SnaBI) containing the sequences spanning Exon I through part of Exon VI [See
The MSF DNA obtained as described above or modified as described above may be introduced into a selected expression vector to make a recombinant molecule or vector for use in the method of expressing novel MSF polypeptides. These vectors contain the novel MSF DNA sequences recited herein, which alone or in combination with other sequences, code for MSF polypeptides of the invention or active fragments thereof. The vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the invention. Regulatory sequences preferably present in the selected vector include promoter fragments, terminator fragments and other suitable sequences which direct the expression of the protein in an appropriate host cell. The resulting vector is capable of directing the replication and expression of an MSF in selected host cells. The transformation of these vectors into appropriate host cells can result in expression of the MSF polypeptides.
Numerous types of appropriate expression vectors are known in the art for mammalian (including human) expression, as well as insect, yeast, fungal and bacterial expression, by standard molecular biology techniques. Mammalian cell expression vectors are desirable for expression. Bacterial cells, e.g., E. coli, are also desirable for expression of MSFs.
Mammalian cell expression vectors described herein may be synthesized by techniques well known to those skilled in this art. The components of the vectors, e.g. replicons, selection genes, enhancers, promoters, and the like, may be obtained from natural sources or synthesized by known procedures. See, Kaufman et al, J. Mol. Biol., 159:511-521 (1982); and Kaufman, Proc. Natl. Acad. Sci., USA, 82:689-693 (1985). Alternatively, the vector DNA may include all or part of the bovine papilloma virus genome [Lusky et al, Cell, 36:391401 (1984)] and be carried in cell lines such as C127 mouse cells as a stable episomal element.
One such vector for mammalian cells is pXM [Y. C. Yang et al, Cell, 47:3-10 (1986)]. This vector contains the SV40 origin of replication and enhancer, the adenovirus major late promoter, a cDNA copy of the adenovirus tripartite leader sequence, a small hybrid intervening sequence, an SV40 polyadenylation signal and the adenovirus VA I gene, in appropriate relationships to direct the high level expression of the desired cDNA in mammalian cells [See, e.g., Kaufman, Proc. Natl. Acad. Sci. USA, 82:689-693 (1985)]. To generate constructs for expression of MSF, the pXM vector is linearized with an appropriate restriction endonuclease enzyme and separately ligated to the cDNA encoding MSF which has been appropriately prepared by restriction endonuclease digestion, for example.
Another similar vector is pMT21. pMT21 is prepared by linearizing pMT2pc (which has been deposited with the ATCC under Accession Number 40348) by digestion with PstI. The DNA is then blunted using T4 DNA polymerase. An oligonucleotide [SEQ ID NO: 123]:
is then ligated into the DNA, recreating the PstI site at the 5′ end and adding an EcoRI site and XhoI site before the ATG of the DHFR cDNA. This plasmid is called pMT21. Preferably a desired polylinker with restriction sites for NotL KpnI, SalI and SnabI is introduced into this vector for ready insertion of the MSF coding sequence.
Still another vector which may be employed to express MSF in CHO cells is pED4DPC-1. This vector is prepared from pED4, also known as pEMC2B 1. As does pXM, described above, this vector contains the SV40 origin of replication and enhancer, the adenovirus major late promoter, a cDNA copy of the majority of the adenovirus tripartite leader sequence, a small hybrid intervening sequence, an SV40 polyadenylation signal and the adenovirus VA I gene, in appropriate relationships to direct the high level expression of the desired cDNA in mammalian cells. In addition, it contains DHFR and β-lactamase markers and an EMC sequence which pXM does not contain. To made pED4DPC-1, the sequence 1075 through 1096 is removed from pED4 to remove a stretch of cytosines. A new polylinker is added to introduce the restriction sites NotI, SalI and SnabI to the plasmid. The vector is linearized with an appropriate endonuclease enzyme and subsequently ligated separately to the cDNA encoding MSF.
These above-described vectors do not limit this invention; because one skilled in the art can also construct other useful mammalian expression vectors by, e.g., inserting the DNA sequence of the MSF from the plasmid with appropriate enzymes and employing well-known recombinant genetic engineering techniques and other known vectors, such as pJL3 and pJL4 [Gough et al., EMBO J., 4:645-653 (1985)] and pMT2 (starting with pMT2-VWF, ATCC #67122; see PCT application PCT/US87/00033).
Once the vector is prepared, a selected host cell is transformed by conventional techniques with the vector containing MSF. The method of this present invention therefore comprises culturing a suitable cell or cell line, which has been transformed with a DNA sequence coding for expression of an MSF polypeptide under the control of known regulatory sequences.
Suitable cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO) or the monkey COS-1 cell line. CHO cells are preferred as a mammalian host cell of choice for stable integration of the vector DNA, and for subsequent amplification of the integrated vector DNA, both by conventional methods. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al, U.S. Pat. No. 4,419,446. Another suitable mammalian cell line is the CV-1 cell line. Further exemplary mammalian host cells include particularly primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable. Candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene. Other suitable mammalian cell lines include, but are not limited to, HeLa, mouse L-929 cells, 3T3 or 293 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.
Similarly useful as host cells suitable for the present invention are bacterial cells. For example, the various strains of E. coli (e.g., HB101, MC1061 and strains used in the following examples) are well-known as host cells in the field of biotechnology. When used as host cells, E. coli permits the expression of the MSF protein as a single protein. MSF may also be expressed in bacterial cells as a fusion protein with thioredoxin, as disclosed in detail in U.S. patent application Ser. No. 07/652,53 1, which is incorporated herein by reference. Various strains of B. subtilis, Pseudomonas, other bacilli and the like may also be employed in this method.
Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. Additionally, where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g. Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) and references cited therein. Fungal cells may also be employed as expression systems.
Once the MSF is expressed by the transformed and cultured cells, it is then recovered, isolated and purified from the culture medium (or from the cell, if expressed intracellularly) by appropriate means known to one of skill in the art.
A preferred purification procedure to isolate a recombinant or synthetic MSF from serum free mammalian cell (COS-1) conditioned medium is characterized by steps are similar to those for the purification of native meg-CSF from urine and are described in detail in Example 7. The recombinant MSF is concentrated from COS-1 cell supernatant through an Amicon YM- 10 filter with a 10,000 Dalton molecular weight cut-off. The concentrate is dialyzed into 20mm sodium acetate, pH4.5, and then applied to an S Toyopearl cation exchange FPLC column equilibrated in 20mM sodium acetate, pH4.5. The bound material is then eluted from the column and applied through a cycle of C4 reverse phase HPLC using 0.1% TFA/acetonitrile as the solvent system. In the case of MSF-K130, the protein elutes between 20-30% of a buffer containing 0.1% TFA, 95% acetonitrile. Other non-naturally occurring MSFs described above may be obtained by applying this purification scheme described in detail in Example 7 for MSF-K130 [SEQ ID NO: 77 and 78].
MSF polypeptides may also be produced by known conventional chemical synthesis, e.g., by Merrifield synthesis or modifications thereof. Methods for constructing the polypeptides of the present invention by synthetic means are known to those of skill in the art. The synthetically-constructed MSF polypeptide sequences, by virtue of sharing primary, secondary, or tertiary structural and conformational characteristics with native MSF polypeptides may possess MSF biological properties in common therewith. Thus, they may be employed as biologically active or immunological substitutes for natural, purified MSF polypeptides in therapeutic and immunological processes.
One or more MSFs or active fragments thereof, purified in a homogeneous form or as a mixture of different MSFs from natural sources or produced recombinantly or synthetically, may be used in a pharmaceutical preparation or formulation. The MSF pharmaceutical compositions of the present invention or pharmaceutically effective fragments thereof may be employed in the treatment of immune deficiencies or disorders. MSFs may also be employed to treat deficiencies in hematopoietic progenitor or stem cells, or disorders relating thereto. MSFs may be employed in methods for treating cancer and other pathological states resulting from disease, exposure to radiation or drugs, and including for example, leukopenia, bacterial and viral infections, anemia, B cell or T cell deficiencies, including immune cell or hematopoietic cell deficiency following a bone marrow transplantation. MSFs may also be used to potentiate the immune response to a variety of vaccines creating longer lasting and more effective immunity. MSFs may be employed to stimulate development of B cells, as well as megakaryocytes.
The MSFs of the present invention may also have utility in stimulating platelet production, stimulating platelet recovery following chemotherapy or bone marrow transplantation, treating thrombocytopenia, aplastic anemia and other platelet disorders, preserving and extending the lifetime of platelets in storage, and stimulating platelet production in vitro for use in platelet transfusions. MSFs may also be employed to stimulate the growth and development of other colonies of hematopoietic and non-hematopoietic cells. Similarly, these factors may be useful in cell-targeting. MSF may also be useful in the treatment of wounds or bums, alone or with other wound-healing agents, such as fibroblast growth factor (FGF). Adhesion related therapeutic uses are also contemplated for MSFs of this invention. MSF compositions may be used as an adjunctive therapy for bone marrow transplant patients.
Therapeutic treatment of such platelet disorders cr deficiencies with these MSF polypeptide compositions may avoid undesirable side effects caused by treatment with presently available serum-derived factors or transfusions of human platelets. It may also be possible to employ one or more active peptide fragments of MSF in such pharmaceutical formulations.
Therefore, as yet another aspect of the invention are therapeutic compositions for treating the conditions referred to above. Such compositions comprise a therapeutically effective amount of a MSF protein, a therapeutically effective fragment thereof, or a mixture of variously spliced or otherwise modified MSFs in admixture with a pharmaceutically acceptable carrier. This composition can be systemically administered parenterally. Alternatively, the composition may be administered intravenously. If desired, the composition may be administered subcutaneously. When systemically administered, the therapeutic composition for use in this invention is in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such pharmaceutically acceptable protein solutions, having due regard to pH, isotonicity, stability and the like, is within the skill of the art.
The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician considering various factors which modify the action of drugs, e.g. the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of about 1 to about 1000 micrograms of MSF protein, mixture of MSF proteins or fragments thereof. Alternatively about 50 to about 50,000 units (i.e., one unit being the minimum concentration of MSF protein, or MSF protein mixture, which yields the maximal number of colonies in the murine fibrin clot megakaryocyte colony formation assay) of MSF protein per kilogram of body weight may be a desirable dosage range.
The therapeutic method, compositions, purified proteins and polypeptides of the present invention may also be employed, alone or in combination with other cytokines, hematopoietins, interleukins, growth factors or antibodies in the treatment of disease states characterized by other symptoms as well as platelet deficiencies. It is anticipated that an MSF, if it does not itself have TPO activity, will prove useful in treating some forms of thrombocytopenia in combination with general stimulators of hematopoiesis, such as IL-3, IL-6, GM-CSF, steel factor, IL-11 (described in co-owned copending U.S. patent application Ser. No. 07/526,474 and in published PCT Patent Application No. WO91/07495, both incorporated herein by reference) or with other megakaryocytic stimulatory factors or molecules with TPO-like activity. Additional exemplary cytokines or hematopoietins for such co-administration include TPO, G-CSF, the CSF-1s (including M-CSF), IL-1, IL-4, IL-7, erythropoietin, and variants of all of these cytokines, or a combination of multiple cytokines. The dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition. For example, the MSF may be administered in amounts from 1 to 1000 μg/kg body weight and the other cytokine may be administered in the same amounts in such a co-administration protocol. Alternatively, co-administration may permit lesser amounts of each therapeutic agent to be administered. Progress of the treated patient can be monitored by conventional methods.
Other uses for these novel proteins and recombinant polypeptides are in the development of antibodies generated by standard methods for in vivo or in vitro diagnostic or therapeutic use. As diagnostic or research reagents, antibodies generated against these MSFs may also be useful in affinity columns and the like to further purify and identify the complete meg-CSF protein. Such antibodies may include both monoclonal and polyclonal antibodies, as well as chimeric antibodies or “recombinant” antibodies generated by known techniques.
The antibodies of the present invention may be utilized for in vivo and in vitro diagnostic purposes, such as by associating the antibodies with detectable labels or label systems. Alternatively these antibodies may be employed for in vivo and in vitro therapeutic purposes, such as by association with certain toxic or therapeutic compounds or moities known to those of skill in this art. These antibodies also have utility as research agents.
Also provided by this invention are the cell lines generated by presenting MSF or a fragment thereof as an antigen to a selected mammal, followed by fusing cells of the animal with certain cancer cells to create immortalized cell lines by known techniques. The methods employed to generate such cell lines and antibodies directed against all or portions of a human MSF protein or recombinant polypeptide of the present invention are also encompassed by this invention.
The following examples illustratively describe the purification and characteristics of homogeneous human MSF and other methods and products of the present invention. These examples are for illustration and are not intended to limit the scope of the present invention.
The following procedures are employed to obtain native meg-CSF protein from urine of human bone marrow transplant patients. The same or similar procedure may be employed to purify other MSFs from natural sources. Urine from patients with aplastic anemia or thrombocytopenia accompanying other disease states may also be used as the source of the factor employing this purification.
STEP 1: Urine was collected from bone marrow transplant patients between days 5 and 18 following transplant. One hundred liters of pooled urine were treated with protease inhibitors phenylmethyl-sulfonylfluoride (PMSF) and ehtylenediaminetetraacetic acid (EDTA). This pooled urine was concentrated on an Amicon YM-10 filter (10,000 molecular weight cut-off) to remove excess pigments and reduce the volume. A cocktail of protease inhibitors [leupeptin, aprotinin, ethylene glycol-bis-tetraacetic acid (EGTA) and N-ethylmaleimide (NEM)] was added to the urine at this step and the next three steps to minimize proteolysis. The pH of the urine concentrate was adjusted to 8.0 and diluted to a conductivity of 7mS/cm.
STEP 2: The retentate from this first step of the purification was then subjected to anion exchange column chromatography on a QAE Zetaprep [Cuno] at pH 8.0. The QAE flow-through was adjusted to a pH4.5 with 1M acetic acid.
STEP 3: The flow-through from the second purification step was bound to a cation exchange chromatographic column, an SP-Zetaprep column [Cuno] at pH 4.5. Bound protein containing Meg-CSF was eluted with 1M NaCl at a pH of 4.5. The eluate was pooled, protease inhibitors were added as above and the bound protein was either neutralized to pH7 and stored at −80° C. until further chromatography was performed or dialyzed into a Tris-buffered saline (TBS) solution, with the addition of the protease inhibitors described in Step 1. This dialyzate was heated at 56° C. for 30 minutes. Addition of the protease inhibitors, while not essential for recovery of protein, enabled greater amount of protein to be recovered from this step, not denatured by the proteases in the system. Pools from this step were also analyzed for the presence of megakaryocyte-specific growth factors. These pools were found to contain Meg-CSF activity.
STEP 4: The resulting material was added to a lectin affinity chromatographic column, a Wheat Germ Sepharose column [Pharmacia]. Urinary Meg-CSF was found to bind to this column. This protein was then eluted with 0.25 M N-acetyl glucosamine (N-acglcNH2) in TBS, and dialyzed against 20 mM sodium acetate, pH 4.5 in the presence of the protease inhibitors of Step 1.
STEP 5: This dialysate was applied to a 10 ml S-Toyopearl FPLC cation exchange column and eluted using a linear gradient of 0 to 1M NaCl in 20mM sodium acetate at pH 4.5. The protein eluted from this step was tested for Meg-CSF activity in the fibrin clot assay described below. The Meg-CSF activity was observed to elute in two discrete peaks. The major activity eluted between 0.1M and 0.25M NaCl. A minor, but reproducible activity eluted between 0.3M and 0.5N NaCl. The two activities may be due to protein or carbohydrate modification of a single protein; however the data presented further herein refers to the major protein.
STEP 6: The eluate from this fifth purification step was then purified on a reverse phase HPLC (C4) column [Vydac; 1cm×25cm] which was eluted with a linear gradient of between 23-33% acetonitrile in 0.1% trifluoroacetic acid (TFA). This step removes an abundant 30Kd protein contaminant. Recombinant MSF elutes at a slightly lower gradient of about 20 to about 30% of the buffer.
STEP 7: The HPLC step was repeated in a different solvent system, after the eluate of Step 6 was diluted with two parts acetic acid and pyridine. The purified material eluted between 6-15% n-propanol in pyridine and acetic acid on a C18 reverse phase HPLC column (0.46×25 cm). The material produced after this step, when assayed gave the specific activity of greater than 5×107 dilution units reported in the murine assay. This optional step removes the bulk of urinary ribonuclease, a major contaminant, from the preparation.
STEP 8: The HPLC step was repeated once more on a C4 column (Vydac; 0.46×25 cm) using 0.15% HFBA in acetonitrile. The material eluted between 27-37% acetonitrile. The last HPLC step removed substantially all remaining ribonuclease and proteinaceous contaminants present after Step 7.
This purified meg-CSF material was then analyzed by. SDS-PAGE, bioassayed and labelled with 125I. Homogenous protein is obtained from this procedure, omitting step 7, having a specific activity ranging from about 5×107 to about 2-5×108 dilution units per mg protein in the murine megakaryocyte colony assay described below. A unit of activity is defined as the reciprocal of the maximum dilution which stimulates the maximum number of megakaryocyte colonies per ml.
This process is preferably used to purify any naturally occurring MSF protein, or mixture thereof, from a natural source.
A preliminary analysis of COS supernatant expressing the Kpn-SnaB1 12 kb genomic subclone was performed previously, and indicated that a protein which reacted with MSF-specific antibodies was expressed by COS cells and was secreted into the culture medium. Dialysed, concentrated cell supernatant was variably active in the murine meg-colony assay.
Analysis by Northern and Western indicated that the level of MSF mRNA and protein was very low. A Western immunoblot of the protein from COS supernatant expressed in conditioned medium revealed the presence of three heterogenous species which specifically bind anti-meg CSF peptide antibodies and the binding can be competed with excess peptide. The molecular weights of these species, 200 kD, 30 kD, and 14 kD, are different from the partially purified meg-CSF from the BMT urine which has an apparent molecular weight ranging between about 18-about 28 kD under reducing conditions.
Recombinant MSFs were obtained by the following techniques. Six MSF cDNA clones, truncated at putative MSF protein processing sites in Exons V and VI, were constructed by using the polymerase chain reaction. A seventh clone, MSF-K130 [SEQ ID NO: 77 and 78], was isolated as a consequence during the PCR reaction by the inadvertent insertion of an artificial termination codon in Exon IV after amino acid 130. Seven oligonucleotide primers were synthesized as follows:
Primer (1) was designed to hybridize to the cDNA flanking the initiating methionine codon and contains nine MSF-homologous nucleotides, a NotI restriction endonuclease site and three additional nucleotides to enhance.restriction endonuclease recognition (as suggested in the New England Biolabs catalog).
The remaining oligonucleotide primers were designed to hybridize to the 3′ regions of the cDNA and to flank the putative MSF protein processing site codons for [SEQ ID NO: 87] MSF-S172 (2), [SEQ ID NO: 89] MSF-R192 (3), [SEQ ID NO: 91] MSF-K204 (4), [SEQ ID NO: 77] MSF-K130 and [SEQ ID NO: 95] D220 (5), [SEQ ID NO: 79] N141 (6) and [SEQ ID NO: 131] T208 (7). The 3′ primers contain twelve nucleotides of MSF-homologous sequence, a translation termination codon, an XhoI restriction endonuclease site and three additional nucleotides to enhance restriction endonuclease recognition.
Six PCR reactions were performed in duplicate, using the conditions recommended by Perkin-Elmer Cetus Corp. Each of the six duplicate reactions contained the 5′ primer (No. 1, 1.0 μM), one of the 3′ primers (1.0 μM) and 1 ng of MSF cDNA as template. The reactions were carried through two rounds of eighteen cycles each. One cycle consisted of a two minute denaturation of 95° C. followed by three minutes of annealing/extension of 72° C. After the first round of eighteen cycles, 10 μl of the first reaction was transferred to a fresh reaction mixture (100 μl total), and the amplification cycles were repeated. The PCR products generated by the second round of amplification reactions (twelve in all) were digested with NotI and XhoI, using conditions described by the vendor, and fractionated by agarose gel electrophoresis.
To obtain expression of these truncated MSFs in mammalian host cells, the appropriate DNA bands were then excised and ligated into a NotI and XhoI digested pMT21-2 vector. The vector pMT21-2 is identical to the vector pMT21 except for the polylinker region, containing PstI, NotI, KpnI, Apal, EcoRV, EcoRI and XhoI sites, which was changed to facilitate cloning of MSF DNA fragments. Competent DH5 cells were transformed with the recombinant plasmid and selected for resistance to ampicillin. Plasmid DNA was prepared from transformed cells and sequenced with selected internal oligonucleotides across the MSF insert. All the above techniques are conventional and described in Sambrook et al, cited above.
The clones listed above were identified as having the correct nucleotide sequence to encode the desired MSF proteins. For example, S172 [SEQ ID NO: 87] encodes MSF amino acids 1-172 [SEQ ID NO: 88], terminating with a serine residue. Position 173 encodes a translation termination codon. The exception was one of the two reactions designed to synthesize D220 [SEQ ID NO: 95 and 96]. During this PCR reaction, a serendipitous deletion of nucleotide 392 of the cDNA sequence resulted in clone MSF-K130 [SEQ ID NO: 77], which encodes MSF amino acids 1-130 [SEQ ID NO: 78] and terminates in a lysine followed by a TAA stop codon. Clone MSF-K130 [SEQ ID NO: 77 and 78] may readily be deliberately synthesized by a PCR reaction designed for this purpose. This would require using an oligonucleotide primer similar in design to the other 3′ primer oligonucleotides, i.e., an oligonucleotide containing twelve nucleotides of MSF-homologous sequence, a translation termination codon, an XhoI restriction site and a few additional nucleotides to enhance restriction endonuclease recognition. An example of a suitable 3′ primer would be the following sequence: SEQ ID NO: 133 GCGCTCGAGCTAATTTGATGGTTT.
The pMT21-2 plasmid, containing the MSF DNA sequence is transfected onto COS cells. The conditioned medium from the transfected COS cells contains MSF biological activity as measured in the murine assays. Similarly the modified pED4DPC-1 construct containing the cDNA for MSF is transfected into CHO cells.
The vector pED4DPC-1 may be derived from pMT21 vector. pMT21 is cut with EcoRI and XhoI which cleaves the plasmid at two adjacent cloning sites. An EMCV fragment of 508 base pairs is cut from pMT2ECAT1 [S. K. Jong et al, J. Virol., 63:1651-1660 (1989)] with the restriction enzymes EcoRI and TaqαI. A pair of oligonucleotides 68 nucleotides in length are synthesized to duplicate the EMCV sequence up to the ATG. The ATG is changed to an ATT, and a C is added, creating a XhoI site at the 3′ end. A TagαI site is situated at the 5′ end. The sequences of the oligonucleotides are [SEQ ID NO: 134]:
and the respective complementary strands.
Ligation of the pMT21 EcoRI-to-XhoI fragment to the EMCV EcoRI-to-TaqαI fragment and to the TaqαI/XhoI oligonucleotides produces the vector pED4. A polylinker, containing PstI, NotI, SalI, SnaBI and EcoRI sites, is inserted into this vector as described above to create pED4PC-1.
Stable transformants are then screened for expression of the product by standard immunological, biological or enzymatic assays, such as those described below in Example 8. The presence of the DNA and mRNA encoding the MSF polypeptides is detected by standard procedures such as Southern and Northern blotting. Transient expression of the DNA encoding the polypeptides during the several days after introduction of the expression vector DNA into suitable host cells is measured without selection by activity or immunologic assay, e.g., the murine fibrin clot assay, of the proteins in the culture medium.
One skilled in the art could manipulate the sequences encoding the MSF polypeptide by eliminating any human regulatory sequences flanking the coding sequences, eliminating the mammalian secretory sequence of Exon I, and inserting bacterial regulatory sequences to create bacterial vectors for intracellular or extracellular expression of the MSF polypeptide of the invention by bacterial cells. The DNA encoding the polypeptides may be further modified to contain different codons to optimize bacterial expression as is known in the art.
The sequences encoding the mature MSF are operatively linked in-frame to nucleotide sequences encoding a secretory leader polypeptide permitting bacterial expression, secretion and processing of the mature MSF polypeptides, also by methods known in the art. The expression of MSF in E. coli using such secretion systems is expected to result in the secretion of the active polypeptide. This approach has yielded active chimeric antibody fragments [See, e.g., Bitter et al, Science, 240:1041-1043 (1983)].
Alternatively, the MSF may be expressed as a cytoplasmic protein in E. coli, either directly or as a carboxy terminal fusion to proteins, such as thioredoxin, which can maintain many peptides in soluble form in E. coli. The fusion proteins can be cleaved and the free MSF isolated using enzymatic cleavage (enterokinase, Factor Xa) or chemical cleavage (hydroxylamine) depending on the amino acid sequence used to fuse the molecules.
If the cytoplasmic MSF or MSF fusion protein is expressed in inclusion bodies, then either molecule would most likely have to be refolded after complete denaturation with guanidine hydrochloride and a reducing agent a process also known in the art. For procedures for isolation and refolding of intracellularly expressed proteins, see, for example, U.S. Pat. No. 4,512,922. If either MSF protein or MSF-fusion protein remain in solution after expression in E. coli, they are likely to not require denaturation but only mild oxidation to generate the correct disulfide bridges.
The compounds expressed through either route in bacterial host cells may then be recovered, purified, and/or characterized with respect to physicochemical, biochemical and/or clinical parameters, all by known methods.
An MSF can be expressed at high levels in E. coli as a thioredoxin fusion protein using the modified expression vector similar to pALtrxA/EK/IL11ΔPro-581 described in pending U.S. patent application Ser. No. 07/652,351.
As one such example, MSF-K130 [SEQ ID NO: 77 and 78] was employed. For expression in E. coli, the first 25 amino acids of Exon I [SEQ ID NO: 3 and 4] which encode the secretory leader, were removed from the MSF-K130 sequence [SEQ ID NO: 77 and 78]. An enterokinase site, which is [SEQ ID NO: 143] Asp Asp Asp Asp Lys, was inserted at the 5′ end of Exon II [SEQ ID NO: 5 and 6] of MSF-K130. Additionally, the N-terminal Asp of MSF was deleted and replaced with the dipeptide Asn-Gly, encoded by the sequence AACGGT, which encodes a hydroxylamine cleavage site. The sequence of MSF-K130 [SEQ ID NO: 77 and 78] which was added as a fusion to thioredoxin, and which contained certain codon changes for preferred expression in E. coli is shown in
The plasmid expression vector used for expression is a modified pALtrxA/EK/IL11ΔPro-581, illustrated in
Nucleotides 1-2060 contain DNA sequences originating from the plasmid pUC-18 [Norrander et al, Gene, 26: 101-106 (1983)] including sequences containing the gene for β-lactamase which confers resistance to the antibiotic ampicillin in host E. coli strains, and a colE1-derived origin of replication. Nucleotides 2061-2221 contain DNA sequences for the major leftward promoter (pL) of bacteriophage λ [Sanger et al, J. Mol. Biol., 162:729-773 (1982)], including three operator sequences, OL1, OL2 and OL3. The operators are the binding sites for λcI repressor protein, intracellular levels of which control the amount of transcription initiation from pL. Nucleotides 2222-2241 contain a strong ribosome binding sequence derived from that of gene 10 of bacteriophage T7 [Dunn and Studier J. Mol. Biol., 166:477-535 (1983)].
Nucleotides 2242-2568 contain a DNA sequence encoding the E. coli thioredoxin protein [Lim et al, J. Bacteriol., 163:311-316 (1985)]. There is no translation termination codon at the end of the thioredoxin coding sequence in this plasmid.
Nucleotides 2569-2583 contain DNA sequence encoding the amino acid sequence for a short, hydrophilic, flexible spacer peptide “—GSGSG—”. Nucleotides 2584-2598 provide DNA sequence encoding the amino acid sequence for the cleavage recognition site of enterokinase (EC 3.4.4.8), “—DDDDK—” [Maroux et al, J. Biol. Chem., 246:5031-5039 (1971)].
Nucleotides 2599-3132 contain DNA sequence encoding the amino acid sequence of a modified form of mature human IL11 [Paul et al, Proc. Natl. Acad. Sci. USA, 87:7512-7516 (1990)], deleted for the N-terminal prolyl-residue normally found in the natural protein. The sequence includes a translation termination codon at the 3′-end of the IL11 sequence.
Nucleotides 3133-3159 provide a “Linker” DNA sequence containing restriction endonuclease sites. Nucleotides 3160-3232 provide a transcription termination sequence based on that of the E. coli aspA gene [Takagi et al, Nucl. Acids Res., 13:2063-2074 (1985)]. Nucleotides 3233-3632 are DNA sequences derived from pUC-18.
This plasmid is modified in the following manner to replace the ribosome binding site of bacteriophage T7 with that of λCII. In the above-described plasmid, nucleotides 2222 and 2241 were removed by conventional means. Inserted in place of those nucleotides was a sequence of nucleotides formed by nucleotides 35566 to 35472 and 38137 to 38361 from bacteriophage lambda as described in Sanger et al (1982) cited above. This reference is incorporated by reference for the purpose of disclosing this sequence.
The DNA sequence encoding human IL11 in modified pALtrxA/EK/IL11ΔPro-581 (nucleotides 2599-3135) is replaced by the sequence shown in
The resulting plasmid was transformed into the E. coli host strain GI724 (F−, lacIq, lacPL8, ampC::λcI+) by the procedure of Dagert and Ehrlich, Gene, 6: 23 (1979). The untransformed host strain E. coli G1724 was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. on Jan. 31, 1991 under ATCC No. 55151 for patent purposes pursuant to applicable laws and regulations. Transformants were selected on 1.5% w/v agar plates containing IMC medium, which is composed of M9 medium [Miller, “Experiments in Molecular Genetics”, Cold Spring Harbor Laboratory, New York (1972)] supplemented with 0.5% w/v glucose, 0.2% w/v casamino acids and 100 μg/ml ampicillin.
G1724 contains a copy of the wild-type λcI repressor gene stably integrated into the chromosome at the ampC locus, where it has been placed under the transcriptional control of Salmonella typhimurium trp promoter/operator sequences. In G1724, λcI protein is made only during growth in tryptophan-free media, such as minimal media or a minimal medium supplemented with casamino acids such as IMC, described above. Addition of tryptophan to a culture of GI724 will repress the trp promoter and turn off synthesis of λcI, gradually causing the induction of transcription from pL promoters if they are present in the cell.
GI724 transformed with the MSF containing plasmid was grown at 37° C. to an A550 of 0.5 in IMC medium. Tryptophan was added to a final concentration of 100 μg/ml and the culture incubated for a further 4 hours.
All of the thioredoxin-MSF fusion protein was found in the soluble cellular fraction, representing up to 10% of the total protein. The fusion protein was heat stable, remaining soluble after treatment at 80 degrees Celsius for fifteen minutes. Preliminary testing with the fusion protein has shown activity in the fibrin clot assay described below.
Manipulations can be performed for the construction of an insect vector for expression of MSF polypeptides in insect cells [See, e.g., procedures described in published European patent application 155,476].
Similarly yeast vectors. may be constructed employing yeast regulatory sequences to express cDNA encoding the precursor, in yeast cells to yield secreted extracellular active MSF. Alternatively the polypeptide may be expressed intracellularly in yeast, the polypeptide isolated and refolded to yield active MSF. [See, e.g., procedures described in published PCT application WO 86/00639 and European patent application EP 123,289.]
An initial 3 L batch of serum-free conditioned medium from COS-1 cells transfected with MSF-K130 yielded 140 ug of purified, active MSF protein using a three step purification process. COS-1 cell conditioned medium harvested under serum free conditions was concentrated on an Amicon YM10 membrane with a molecular weight cutoff of 10,000 daltons. The concentrate was centrifuged at 10,000 rpm in an SS34 rotor at 4° C. to remove cellular debris and precipitate. The supernatant was dialyzed against 20mM sodium acetate pH 4.5 overnight at 4° C. The dialyzed protein solution was centrifuged again at low speed to remove residual precipitate.
The dialyzed MSF-K130 containing solution was applied to an S Toyopearl cation exchange FPLC column, equilibrated in 20mM sodium acetate pH 4.5. Bound protein was eluted with a gradient of 0 to 1M NaCl in 20mM sodium acetate, pH 4.5, at room temperature. Typically, MSF-K130 [SEQ ID NO: 77 and 78] eluted between 0.1 to 0.2 M NaCl. The active MSF peak was observed to have a molecular weight on SDS-PAGE 10-20% gradient polyacrylamide gels of between 20-50 kD under non-reducing conditions and 18-21 kD under reducing conditions. This was determined by probing a Western Immunoblot with rabbit antisera raised against a peptide in Exon III of the MSF gene of
The pool of active MSF-K130 [SEQ ID NO: 77 and 78] was divided into three aliquots based on the molecular weights under non-reducing conditions. Pool A consisted of mostly high molecular weight dimer, 35-50 kD. Pool B consisted of intermediate molecular weight dimer species ranging from 20-45 kD; and pool C comprised predominantly monomer species of molecular weight range 14-25 kD. MSF from all three pools had a molecular weight of 18-21 kD under reducing conditions.
The final purification step was one cycle of reverse phase-HPLC. Protein from the three pools were acidified with 10% TFA to 0.1% TFA (v/v), filtered through a 0.45 urn PVDF membrane and injected at 1 ml/min onto a 25cm×4.6mm C4 (Vydec) reverse phase HPLC column equilibrated in 0.1% TFA at room temperature. Bound protein was eluted with a gradient of 0-95% acetonitrile in 0.1% TFA. Typically, MSF-K130 activity eluted between 15-30% buffer B (95% acetonitrile in 0.1% TFA).
The following assays were performed using the purified native urinary meg-CSF described in Example 1, and in some cases, crude or highly purified preparations of recombinant MSF-K130 [SEQ ID NO: 77 and 78]. The other recombinant or naturally occurring MSFs may exhibit MSF biological properties in these same assays or other assays.
A. Murine Fibrin Clot Assay
The meg-CSF obtained from Step 7 of the purification techniques of Example 1 was tested for activity in the megakaryocyte colony formation assay performed substantially as described in S. Kuriya et al, Exp. Hematol., 15:896-901 (1987). A fibrin clot was formed containing 2.5×105 mouse bone marrow cells in a 6-well plate. The diluted sample was layered around the clot and incubated for 6 days. Thereafter, cells were fixed and megakaryocytes were stained for acetylcholinesterase, a specific marker for murine megakaryocytes. A colony was defined as three or more megakaryocytes per unit area.
A mixture of pure and mixed colonies containing megakaryocyte colonies were routinely observed: 70% pure megakaryocyte colonies containing no additional cell types: 30 % mixed megakaryocyte colonies containing additional non-megakaryocyte cell types to 50% pure: 50% mixed type depending on the assay. The pure colonies typically contain on average 4 to 5 cells per colony, ranging from 3 to 8 cells per colony. The cells within the colony are variable in size and appear to contain both mature and immature megakaryocytes. The megakaryocytes are fairly dispersed within the colony. A typical mixed megakaryocyte colony is composed on average of 10 cells per colony ranging from 7 to 17 cells. The cells appear more clustered than the megakaryocytes in pure megakaryocyte colonies.
The following control samples were included in every assay. A positive control was WEHI conditioned medium (murine IL-3), which produced between 7-25 (average 12) megakaryocyte colonies per clot, approximately 50% pure and 50% mixed megakaryocyte colonies. Another positive control was serum taken from lethally irradiated dogs at the nadir or low point of the platelet count [see Mazur et al, Exp Hematol., 13:1164-1172 (1985)], which produced between 6-22 (average 15) megakaryocyte colonies per clot, of which approximately 70% were pure and 30% were mixed megakaryocyte colonies. The negative control was Iscoves Medium, which produced 2-4 megakaryocyte colonies per clot.
In the assay, the purified urinary meg-CSF has a specific activity of greater than approximately 5×107 dilution units/mg of protein. A unit of activity is defined as described in Example 1.
The major meg-CSF obtained from bone marrow transplant urine eluted from the S-Toyopearl cation exchange column chromatography step in the purification of Example 1 has been analyzed in this assay alone, together, and in combination with other cytokines. In the fibrin clot assay, it produced between 6-16 (average 13) megakaryocyte colonies, with 50-70% pure megakaryocyte colonies. The urinary meg-CSF has been shown to have variable synergy with murine IL-3 and does not synergize with human IL-6 or murine IL4 in the fibrin clot culture system.
Megakaryocyte colony formation was observed in response to recombinant MSF-K130 [SEQ ID NO: 77 and 78] in the murine bone marrow fibrin clot assay. Murine megakaryocytes were identified as acetylcholinesterase positive cells and a megakaryocyte colony was defined as greater than three megakaryocyte cells per unit area in a fibrin clot culture. Recombinant MSF typically stimulated megakaryocyte colonies of three to six cells/unit area and averaged between 6 to 15 colonies/2.5×105 murine bone marrow cells.
Two types of megakaryocyte colonies were observed in the assay, pure megakaryocyte colonies and megakaryocyte cells with other cell types, termed mixed megakaryocyte colonies. In one fibrin clot, the two colony types were.at a ratio between 1:1 to 7:3 pure colonies to mixed megakaryocyte colonies. This ratio was consistent throughout the purification of recombinant MSF-K130 [SEQ ID NO: 77 and 78]. The number of megakaryocyte cells/colony and size of megakaryocytes were about the same for both pure and mixed colonies, some megakaryocytes were smaller in the mixed megakaryocyte colonies.
An increase in bioactivity was usually observed from active MSF fractions obtained from the C4 RP-HPLC column. All three pools from the S Toyopearl cation exchange column gave rise to bioactive MSF protein on RP-HPLC. The final specific activity of MSF after the RP-HPLC step was greater than 1×107 units/mg in all three pools. The active peaks were also positive on the MSF Western Blot.
When RP-HPLC-purified MSF-K130 [SEQ ID NO: 77 and 78] from the A pool was subjected to SDS-PAGE under non-reducing conditions, bioactive protein was extracted from gel slices corresponding to 35-50 kD molecular weight species. A silver stain gel and Western immunoblot data showed that 95% of the 35-50 kD recombinant MSF protein reduced to 18-21 kD and 5% did not shift upon reduction on a 10-20% acrylamide gradient SDS-PAGE.
The supernatant from COS-1 cells transfected with MSF-K130 cDNA [SEQ ID NO: 77] was variably active on the fibrin clot assay. In each assay the samples were tested in duplicate and in three dilutions.
B. Human Plasma Clot Megakaryocyte Colony Formation
The human urinary meg-CSF of this invention was also tested for human activity on the plasma clot MSF assay described in E. Mazur et al, Blood, 57:277-286 (1981) with modifications. Non-adherent peripheral blood cells were isolated from Leukopacs and frozen in aliquots. The test sample was mixed with platelet-poor human AB plasma and 1.25×105 cells in 24-well plates and allowed to clot by the addition of calcium. After a 12 day incubation, megakaryocytes were identified using a monoclonal antibody directed against platelet glycoproteins IIb/IIIa and a horseradish peroxidase/anti-peroxidase chromogenic detection system. Recombinant human IL-3 [Genetics Institute, Inc.] was used as a positive control, producing 12-30 megakaryocyte colonies per clot with approximately 60% pure and 40% mixed megakaryocyte colonies. As in the murine assay, the aplastic dog serum was also used as a positive control, which produced between 5-10 megakaryocyte colonies per clot, of which approximately 50% were pure megakaryocyte colonies containing less than 10 cells, and 50% were mixed megakaryocyte colonies containing more than 40 megakaryocytes. The negative control was Alpha Medium, which produced 0-1 megakaryocyte colonies per clot.
The human urinary meg-CSF product from Step 6 of the purification scheme of Example 1 had variable activity in this assay. MSF-K130 [SEQ ID NO: 77 and 78] has shown variable activity in the human plasma clot megakaryocyte colony assay.
C. Synergistic Effects
Recombinant MSF-K130 Cos-1 cell supernatant and purified recombinant MSF were assayed alone and in combination with other cytokines in the various CFU-MEG assay systems, fibrin clot, agar and the human CFU-MEG plasma clot assays.
Variable synergy with IL-3 was observed in the murine bone marrow fibrin clot assay. The stimulation of megakaryocyte colonies increased above either protein alone when both murine IL-3 and MSF-K130 were cultured with bone marrow cells progenitors in the fibrin clot assay. A suboptimal level of murine IL-3 (15 units/ml) and an optimal level of MSF-K130 each stimulate an average of 6-15 CFU-meg/2.5×105 murine bone marrow cells in the fibrin clot assay. In combination, increased megakaryocyte colony stimulation of over 35 megakaryocyte colonies have been observed. The ratio of pure megakaryocyte colonies to mixed megakaryocyte colonies and the size of the megakaryocyte colonies were the same for the combination cultures as for the individual MSF cultures.
D. E. Coli Expressed MSF Activity
MSF expressed in Escherichia coli as a thioredoxin-MSF-K130 fusion protein was soluble and active in the fibrin clot assay. E. coli expressed MSF-K130 stimulated the same range of CFU-meg/2.5×105 murine bone marrow cells as COS derived MSF-K130. This activity was not neutralized by the addition of anti-IL-3 antibody at a level that did neutralize CFU-Meg formation by IL-3. Megakaryocyte colony formation activity of the MSF-K130 thioredoxin fusion protein from E. coli lysate was 5×106 dilution units/ml. The specific activity of the MSF-K130 thioredoxin fusion protein in E. coli lysate was greater than 1×106 U/mg. Thioredoxin was not active in the assay.
One method for producing high levels of the MSF protein of the invention from mammalian cells involves the construction of cells containing multiple copies of the cDNA encoding the MSF.
The cDNA is co-transfected with an amplifiable marker, e.g., the DHFR gene for which cells containing increasing concentrations of methotrexate (MTX) according to the procedures of Kaufman and Sharp, J. Mol. Biol., (1982) supra. This approach can be employed with a number of different cell types. Alternatively, the MSF cDNA and drug resistance selection gene (e.g., DHFR) may be introduced into the same vector. One desirable vector for this approach is pED4DPC-1. MSF-K130 [SEQ ID NO: 77 and 78] and MSF-N141 [SEQ ID NO: 79 and 80] are being expressed in vector pEMC3-1, a vector identical to pEO4DPC-1, but in which the polylinker has been changed (PstI, NotI, SalI, SnaBI, EcoRI, PacI) as described above per pMT21.
For example, the pMT21 vector containing the MSF gene in operative association with other plasmid sequences enabling expression thereof is introduced into DHFR-deficient CHO cells, DUKX-BII, along with a DHFR expgression plasmid such as pAdD26SVpA3 [Kaufman, Proc. Natl. Acad. Sci. USA, 82:689-693 (1985)] by calcium phosphate coprecipitation and transfection. Alternatively, the pED4DPC-1 vector containing the MSF gene in operative association with other plasmid sequences enabling expression thereof is introduced into DHFR-deficient CHO cells, DUKX-BII, by protoplast fusion or transfection. The MSF gene and DHFR marker gene are both efficiently expressed when MSF is introduced into pEMC2B1.
DHFR expressing transformants are selected for growth in alpha media with dialyzed fetal calf serum. Transformants are checked for expression of MSF by Westem blotting, bioassay, or RNA blotting and positive pools are subsequently selected for amplification by growth in increasing concentrations of MTX (sequential steps in 0.02, 0.2, 1.0 and 5uM MTX) as described in Kaufman et al., Mol. Cell Biol., 5:1750 (1983). The amplified lines are cloned, and MSF protein expression is monitored by the fibrin clot assay. MSF expression is expected to increase with increasing levels of MTX resistance.
In any of the expression systems described above, the resulting cell lines can be further amplified by appropriate drug selection, resulting cell lines recloned and the level of expression assessed using the murine fibrin clot assay described in Example 4.
The MSF expressing CHO cell lines can be adapted to growth in serum-free medium. MSF expressed in CHO cells is purified from serum-free conditioned medium using the same purification scheme as COS-1 cell supernatant. Homogeneous MSF can be isolated from conditioned medium from the cell line using methods familiar in the art, including techniques such as lectin-affinity chromatography, reverse phase HPLC, FPLC and the like.
The foregoing descriptions detail presently preferred embodiments of the invention. Numerous modifications and variations in practice of this invention are expected to occur to those skilled in the art. Such modifications and variations are encompassed within the following claims.
This application is a continuation application which claims priority to pending U.S. patent application Ser. No. 07/757,022, filed Sep. 10, 1991, which is a continuation-in-part of U.S. patent application Ser. No. 07/643,502, filed Jan. 18, 1991, which is a continuation-in-part of U.S. patent application Ser. No. 07/546,114, filed Jun. 29, 1990, now U.S. Pat. No. 5,326,558, which is a continuation-in-part of U.S. patent application Ser. No. 07/457,196, filed Dec. 28, 1989, which is a continuation-in-part of U.S. patent application Ser. No. 07/390,901, filed Aug. 8, 1989; these applications are each incorporated by reference herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10124557 | Apr 2002 | US |
| Child | 11169232 | Jun 2005 | US |
| Parent | 07757022 | Sep 1991 | US |
| Child | 10124557 | Apr 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 07643502 | Jan 1991 | US |
| Child | 07757022 | Sep 1991 | US |
| Parent | 07546114 | Jun 1990 | US |
| Child | 07643502 | Jan 1991 | US |
| Parent | 07457196 | Dec 1989 | US |
| Child | 07546114 | Jun 1990 | US |
| Parent | 07390901 | Aug 1989 | US |
| Child | 07457196 | Dec 1989 | US |
