WO2007102814A2

WO2007102814A2 - Recombinant mammalian molecules and method for production thereof

Info

Publication number: WO2007102814A2
Application number: PCT/US2006/008088
Authority: WO
Inventors: Cvlayton Parker; Donnie Rudd
Original assignee: Regenetech, Inc.
Priority date: 2006-03-07
Filing date: 2006-03-07
Publication date: 2007-09-13
Also published as: WO2007102814A3

Abstract

Disclosed are recombinant mammalian molecules produced by exposing the molecules to a square wave time varying electromagnetic force of from 0.05 gauss to 0.5 gauss during their production. The disclosure also includes a method for producing recombinant mammalian molecules by comprising exposing the molecules to a square wave time varying electromagnetic force of from 0.05 gauss to 0.5 gauss while producing them.

Description

RECOMBINANT MAMMALIAN MOLECULES AND METHOD FOR PRODUCTION THEREOF

The present invention pertains in general to expansion of recombinant mammalian cells and molecules from a relatively small number of starting cells while maintaining the cell geometry of the cells It also relates to use of specific factors to promote the growth of multipotential hematopoietic cells while maintaining their cell geometry.

Increasing numbers of recombinant products are being commercialized including recombinant versions of insulin (used in the treatment of diabetes), erythropoietin (used to treat renal disease, anemia and some cancers), streptokinase (used to treat cardiovascular disease), human growth hormone (used to treat growth deficiency), interferon- (used to treat cancers and viral infections), interferon- (used to treat pulmonary fibrosis), anti-T-cell antibody (used in organ transplants), hepatitis B antibody (used to treat hepatitis B), tissue plasminogen activator (used to treat cardiovascular disease) , 1 -antitrypsin (used to treat some forms of emphysema), interleukin-2 (used to treat cancers), granulocyte colony stimulating factor (used to treat neutropenia in cancer patients), granulocyte-macrophage colony stimulating factor (used to treat immunological deficiencies), coagulation factor VIII (used to treat hemophilia A), interleukin 10 (used to prevent thromobcytopenia), epidermal growth factor (used to treat burns and certain cancers), follicle stimulating hormone (used to treat infertility and glucagon (used to treat insulin-induced hypoglycemia).

Other promising recombinant products include mammalian pluripotent colony stimulating factors, specifically human pluripotent granulocyte colony- stimulating factor (hpG-CSF), to fragments and polypeptide analogs thereof and to polynucleotides encoding the same (described for example in US Patents 4,810,643, 5,582,823, 5,830 705, 6,004,548, 6,379,661, and 6,716,606 (all in the name of Lawrence M. Souza and assigned to Amgen Inc.) and for the production of stem cell factors of the type described in US Patent 6.218 148 (Zsebo et al. Assigned to Amgen Inc.)

Ex vivo expansion of hematopoietic progenitor cells for a variety of clinical uses including gene therapy, the augmentation of bone marrow transplantation (BMT) and the replacement of BMT has been described previously. Although there are previous reports on the expansion of cell numbers using various combinations of cytokines or stromal cells, the magnitude of expanded progenitor cells that has been achieved, especially multipotential progenitors, is typically low. This suggests that the differentiation and depletion of primitive cells occurs in the expansion cultures . It has been demonstrated that a combination of IL-6 and sIL-6R sustains self-renewal of the pluripotent embryonic stem (ES) cell through the activation of the gp 130 signaling process

The purification, cloning and use of IL-6 is known (EP 220 574, published May 6, 1987, Revel et al.; WO 88/00206, published Jan. 14, 1988, Clark et al.) The purification and cloning of sIL-6R has also been reported, as has its combined use with IL-6 in conditions such as bacterial infections, burns and trauma (EP 413 908, published Feb. 27, 1991, Novick et al.; JP 89271865; Yamasaki et al., Science, 241:825- 828, 1988). SCF is an early acting hematopoietic factor. The purification, cloning and use of SCF have been reported (see PCT WO 91/05795, entitled "Stem Cell Factor"). The use of SCF has been described for enhancing the engraftment of bone marrow and bone marrow recovery as well as for the treatment of leukopenia and thrombocytopenia. The use of SCF in combination with IL-6 has been described, but there are no previous reports on the combined use of SCF, IL-6 and sIL-6R for the expansion of hematopoietic progenitor cells.

US Patent 4,810,643 describes DNA sequences coding for all or part of hpG-CSF . Such sequences may include: the incorporation of codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences which facilitate construction of readily expressed vectors. US Patent 4810643 also describes DNA sequences coding for microbial expression of polypeptide analogs or derivatives of hpG-CSF which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (i.e., deletion analogs containing less than all of the residues specified for hpG-CSF; substitution analogs, such as [Ser¹⁷] hpG-CSF, wherein one or more residues specified are replaced by other residues; and addition analogs wherein one or more amino acid residues is added to a terminal or medial portion of the polypeptide) and which share some or all the properties of naturally-occurring forms.

Further described in US Patent 4810643 are DNA sequences which include sequences useful in securing expression in procaryotic or eucaryotic host cells of polypeptide products having at least a part of the primary structural conformation and one or more of the biological properties of naturally occurring pluripotent granulocyte colony-stimulating factor. DNA sequences of the invention are specifically seen to comprise numerous DNA sequences. Specifically comprehended are genomic DNA sequences encoding allelic variant forms of hpG-CSF and/or encoding other mammalian species of pluripotent granulocyte colony-stimulating factor. Specifically comprehended are manufactured DNA sequences encoding hpG-CSF, fragments of hpG-CSF and analogs of hpG-CSF which DNA sequences may incorporate codons facilitating translation messenger RNA in microbial hosts. Such manufactured sequences may readily be constructed according to the methods of Alton, et al., PCT published application WO 83/04053.

Also comprehended by the invention of US Patent 4810643 is that class of polypeptides coded for by portions of the DNA complement to the top strand human cDNA or genomic DNA sequences of FIGS. 2 or 3 herein, i.e., "complementary inverted proteins" as described by Tramontano, et al., Nucleic Acids Res., 12, 5049- 5059 (1984).

Polypeptide products of the invention of US Patent 4,810,643 may be useful, alone or in combination with other hematopoietic factors or drugs in the treatment of hematopoietic disorders, such as aplastic anemia. They may also be useful in the treatment of hematopoietic deficits arising from chemotherapy or from radiation therapy. The success of bone marrow transplantation, for example, may be enhanced by application of hpG-CSF. Wound healing burn treatment and the treatment of bacterial inflammation may also benefit from the application of hpG-CSF. In addition, hpG-CSF may also be useful in the treatment of leukemia based upon a reported ability to differentiate leukemic cells. Welte, et al., Proc. Natl. Acad. Sci. (USA), 82, 1526-1530 (1985) and Sachs, supra.

US Patent 5,861,315 demonstrates that the combination of the cytokines soluble IL-6 receptor (sIL-6R) and IL-6 together with stem cell factor (SCF) can support the ex vivo expansion of human hematopoietic multipotential cells. Neither sIL-6R nor IL-6, when singly combined with SCF, demonstrates this effect.

US Patent 6218148 describes factors, referred to herein as "stem cell factors" (SCF) having the ability to stimulate growth of primitive progenitors including early hematopoietic progenitor cells. These SCFs also are able to stimulate non- -A- hematopoietic stem cells such as neural stem cells and primordial germ stem cells. Such factors include purified naturally-occurring stem cell factors. US Patent 6218148 also describes non-naturally-occurring polypeptides having amino acid sequences sufficiently duplicative of that of naturally-occurring stem cell factor to allow possession of a hematopoietic biological activity of naturally occurring stem cell factor.

US Patent 6218148 also provides isolated DNA sequences for use in securing expression in procaryotic or eukaryotic host cells of polypeptide products having amino acid sequences sufficiently duplicative of that of naturally-occurring stem cell factor to allow possession of a hematopoietic biological activity of naturally occurring stem cell, factor.

US Patent 6218148 also describes vectors containing such DNA sequences, and host cells transformed or transfected with such vectors. Also comprehended by the invention are methods of producing SCF by recombinant techniques and the efficient recovery of stem cell factor from a material containing SCF, the process comprising the steps of ion exchange chromatographic separation and/or reverse phase liquid chromatographic separation.

There remains a need, however, for a means for production of recombinant mammalian molecules in good quantities. Most commercial production of recombinant molecules has been effected in bacteria such as E. coli or yeast such as S. cerevisiae or P pastoris. Production in procaryotic cells such as bacteria normally results in the production of polypeptides lacking glycosylation (which may be acceptable for insulin which is not glycosylated, but is unsatisfactory for other proteins) and although production in eucaryotic cells such as yeast results in glycosylation, the patterns of glycosylation may not be the same as those that result when a polypeptide is expressed in a mammalian cell.

From a first aspect, the present invention provides a means for production of recombinant mammalian products in good yields by expansion of recombinant mammalian cells while maintaining the cell geometry of the cells. The products may be either the cells themselves or polypeptides expressed by the cells or recombinant polynucleotide.

More particularly, from one aspect the present invention provides a means for expansion of recombinant cells, suitably of the types mentioned above, by growth of the cells in an environment in which they remain suspended, preferably under the influence of a time-varying electromagnetic force. In some cases, it may be possible to use the cells as produced. In others the cells may need to be lysed in order to release the desired recombinant molecules.

From a second aspect, the invention provides a means for expansion of multipotential hematopoietic cells by growth of the cells in an environment in which they remain suspended, preferably under the influence of a time- varying electromagnetic force in a reaction medium comprising soluble interleukin 6 receptor (s- IL-6R) , interleukin 6 (IL-6) and stem cell factor (SCF).that stimulate growth of hematopoietic progenitor cells. All of such factors may be produced by the method of the first embodiment if desired. Suitable cells for such a purpose are multipotential hematopoietic cells are progenitor and/or stem cells obtained by CD34 selection. This method can be used to generate differentiated blood cell colonies of the type described in US Patent 5,861315.

The invention further provides a kit, consisting of: soluble interleukin-6 receptor, interleukin-6 and stem cell factor, said soluble interleukin-6 receptor, interleukin-6 and stem cell factor, all of which have been exposed to a square wave time varying electromagnetic force of from 0.05 gauss to 6 gauss during their production, provided in individual containers or as a mixture in a single container in an amount effective for use in expansion of multipotential hematopoietic cells according to claim 16.

In the process of the present invention, the cells are expanded in such manner as to maintain, or have essentially the same, three-dimensional geometry and normally the same cell-to-cell support and cell-to-cell geometry as the cells prior to expansion. Typically this may be accomplished by maintaining the cells in which the recombinant molecules are being produced suspended in a culture medium and preventing them from contacting a solid surface during the cell expansion, for example by maintaining the cells in a bioreactor which rotates about a horizontal axis.

As used throughout this application, the term "carrier medium" is a fluid carrier such as cell culture media, cell growth media, buffer which provides sustenance to the cells and depending on the composition of the cell growth media allows the cells to replicate. The carrier medium can be refreshed and/or removed as needed. As used throughout this application, the term "three-dimensional geometry" refers to the geometry of cells in a three-dimensional state (same as or very similar to their natural state), as opposed to two-dimensional geometry for instance as found in cells grown in a Petri dish, where the cells become flattened and/or stretched.

As used throughout this application, the term "cell-to-cell support" refers to the support one cell provides to an adjacent cell. For instance, healthy tissue and cells maintain interactions such as chemical, hormonal, neural (where applicable/appropriate) with other cells in the body. In the present invention, these interactions are maintained typically within normal functioning parameters, meaning they do not for instance begin to send toxic or damaging signals to other cells (unless such would be done in the natural blood environment).

As used throughout this application, the term "cell-to-cell geometry" refers to the geometry of cells including the spacing, distance between, and physical relationship of the cells relative to one another. For instance, TVEMF-expanded stem cells of this invention stay in relation to each other as in the body. The expanded cells are within the bounds of natural spacing between cells, in contrast to for instance two-dimensional expansion containers, where such spacing is not kept.

For each of the above three definitions, relating to maintenance of cell-to-cell support and geometry and three dimensional geometry of stem cells of the present invention, the term "essentially the same" means that normal geometry and support are provided in TVEMF-expanded cells of this invention, so that the cells are not changed in such a way as to be for instance dysfunctional, unable to repair tissue, or toxic or harmful to other cells.

Among the types of reactors in which the present invention may be carried out are those described in U. S. Patents 5,153,133 (Schwarz et al., Method for culturing mammalian cells in a horizontally rotated bioreactor); 5,155,035 (Schwarz et al., Method for culturing mammalian cells in a perfused bioreactor); 5,155,034 (Wolf et al., Three-dimensional cell to tissue assembly process); 5,851,816 (Goodwin, Cultured high-fidelity three-dimensional human urogenital tract carcinomas and process); 6,485,963 (Wolf et al., Growth stimulation of biological cells and tissue by electromagnetic fields and uses thereof) and 6,673,597 (Wolf et al., Growth stimulation of biological cells and tissue by electromagnetic fields and uses thereof). BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 schematically illustrates an embodiment of a culture carrier flow loop of a bioreactor;

Figure 2 is an elevated side view of an embodiment of a "Time Varying Electromagnetic Force" ("TVEMF") bioreactor of use in the invention;

Figure 3 is a side perspective of an embodiment of the TVEMF- bioreactor of Figure 2;

Figure 4 is a vertical cross sectional view of an embodiment of a TVEMF- bioreactor;

Figure 5 is a vertical cross sectional view of a TVEMF- bioreactor;

Figure 6 is an elevated side view of a time varying electromagnetic force device that can house, and provide a time varying electromagnetic force to, a bioreactor;

Figure 7 is a front view of the device shown in Figure 6; and

Figure 8 is a front view of the device shown in Figure 6, further showing a bioreactor therein.

Numerous aspects and advantages of the invention will be apparent to those skilled in the art upon consideration of the following detailed description which provides illustrations of the practice of the invention in its presently-preferred embodiments.

Recombinant cells for which the present invention provides a particularly useful means of expansion include: recombinant hpG-CSF ; and recombinant cells in which stem cell factor or biologically active fragments thereof that stimulate growth of hematopoietic progenitor cells, said DNA being operatively linked to an expression control sequence are produced.

The method can also be used for producing recombinant erythropoietin, streptokinase, human growth hormone, interferon-, interferon- , anti-T-cell antibody, hepatitis B antibody, tissue plasminogen activator, 1 -antitrypsin, interleukin-2, interleukin-6, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, coagulation factor VIII, interleukin 10, epidermal growth factor, follicle stimulating hormone and glucagon..

The method can also be used for producing pluripotent granulocyte colony-stimulating factor.

Specific features of some of these will be discussed later.

Recombinant mammalian cells for use in the process of the present invention may be obtained by introducing the recombinant DNA into a host cell. Suitable host cells are known to those skilled in the art. They may include Chinese hamster ovary cells (CHO), mouse LTK^" cells, mouse myeloma , baby hamster kidney (BHK) and human retinal cells (PER-C6) or human blood or neuronal cells. The host cells used should normally be free of serum to avoid the risk of incorporating pathogens into the expansion medium.

Introduction of a desired recombinant nucleotide sequences into permissive mammalian host cells can be achieved by any method known in the art, including, but not limited to, transfection and transformation including, but not limited to, microinjection, electroporation, CaPO₄ precipitation, DEAE-dextran, liposomes, particle bombardment, and the like. Specific method for producing recombinant mammalian cells producing certain polypeptides are discussed for example in streptokinase: D Collen et al J Pharmacol Exp Ther. 1984; 231:146-152; hepatitis B surface antigen: U.S Patent 4,710,378; erythropoietin: Inoue N, et al Biotechnol Annu Rev. 1995; 1:297-313.

The growth medium used for the present invention may be a conventional one. However, it may be desirable to include a copper chelating agent in the medium. Such a chelating agent may be any non-toxic copper chelating agent, and is preferably Penicillamine or Trientine Hydrochloride. More preferably, the Penicillamine is D(-)-2-Ammo-3-Mercaptor-3-Methylbutanic Acid (Sigma-Aldrich), dissolved in DMSO and added to the peripheral blood mixture in an amount of about 10 ppm. The copper chelating agent may also be administered to a mammal, where peripheral blood will then be directly collected from the mammal. Preferably such administration is more than one day, more preferably more than two days, before collecting peripheral blood from the mammal. The purpose of the copper chelating agent, whether added to the peripheral blood mixture itself or administered to a blood donor mammal, or both, is to reduce the amount of copper in the peripheral blood prior to TVEMF-expansion. Not to be bound by theory, it is believed that the decrease in amount of available copper may enhance TVEMF-expansion. It has, however, been found that in some cases cell expansion may by improved by GP 130 signaling and the presence of suitable compounds for effecting such signaling, such as a combination of sIL-6R and IL-6 and preferably also stem cell factor may be useful in some cases.

The suspension environment for growing cells according to the present invention is preferably effected using a TVEMF bioreactor.

As used throughout this application, the term "cell culture chamber" and "chamber" or other similar words is meant to be a unit in which cells are free to live and preferably replicate, expand and grow including, but not limited to, such chambers as petri-dishes, bioreactor cell culture chambers or vessels, wells, and flasks.

As used throughout this application, the term "placed into a TVEMF- bioreactor" and "placed into a cell culture chamber" and similar words is not meant to be limiting. The cell mixture may be made entirely outside of the cell culture chamber, for instance of a bioreactor or a TVEMF-bioreactor, and then the cell mixture placed inside the cell culture chamber thereof. Also, the cell mixture may be entirely mixed inside the chamber. For instance, the cells (or a cellular portion thereof) may be placed in a chamber and supplemented with GTSF-2 medium and 10% fetal bovine serum or other serum, more preferably human serum albumin or other serum useful in cell culture, either already in the chamber, added simultaneously to the chamber, or added after the cells are already in the chamber.

As used throughout this application, the term "electromagnetic force" (EMF) is preferably measured in Gauss and more preferably is exposed to cells of this invention in an amount of about 0.05 to about 6 gauss.

As used throughout this application, the term "TVEMF" refers to "Time Varying Electromagnetic Force". The TVEMF of this invention is a square wave (following a Fourier curve). Preferably, the square wave has a frequency of about 10 cycles/second, and the conductor has an RMS value of about 1 to 1000 mA, preferably 1 to 6 mA. However, these parameters are not meant to be limiting to the TVEMF of the present invention, as such may vary based on other aspects of this invention. TVEMF may be measured for instance by standard equipment such as an ENl 31 Cell Sensor Gauss Meter.

As used throughout this application, the term "bioreactor" refers to a rotating bioreactor, when the cell culture chamber is closed and rotated about its longitudinal axis, as discussed throughout the application.

As used throughout this application, the term "TVEMF-bioreactor" refers to a rotating bioreactor to which TVEMF is applied, as described more fully in the Description of the Drawings, above. The TVEMF applied to a bioreactor is preferably in the range of 0.05 to 6.0 gauss, preferably 0.05-0.5 gauss. See for instance Figures 2, 3, 4 and 5 herein for examples (not meant to be limiting) of a TVEMF-bioreactor. In a simple embodiment, a TVEMF-bioreactor of the present invention provides for the rotation of an enclosed cell mixture at an appropriate gauss level (with TVEMF applied), and allows the cells therein to expand. Preferably, a TVEMF-bioreactor allows for the exchange of growth medium (preferably with additives) and for oxygenation of the recombinant cell mixture. The TVEMF-bioreactor provides a mechanism for growing cells for several days or more. The TVEMF-bioreactor subjects cells in the bioreactor to TVEMF, so that TVEMF is passed through the cells, thus undergoing TVEMF-expansion.

As used throughout this application, the term "TVEMF-expanding" refers to the step of mammalian cells in a chamber, preferably a two-dimensional tissue culture system and/or a TVEMF-bioreactor, living and preferably growing and replicating (splitting and growing) in the presence of TVEMF. The cells preferably replicate without undergoing further differentiation.

As used throughout this application, the term "expanding" and related terms refers to the step of cells in a culture chamber, preferably a bioreactor, replicating (splitting and growing). The mammalian cells preferably replicate without undergoing further differentiation.

As used throughout this application, the term "TVEMF-expansion" refers to the process of increasing the number of recombinant cells in a TVEMF-bioreactor by subjecting the cells to a TVEMF of about 0.05 to about 6.0 gauss. Preferably, the increase in number of recombinant cells is at least 7 times the number per volume (i.e. concentration) of the recombinant cell source. The increase in number of cells per volume is expressly not due to a simple reduction in volume of fluid, for instance, reducing the volume of growth medium from for example 70 ml to 10 ml and thereby increasing the number of cells per ml.

The expansion of recombinant cells in a TVEMF-bioreactor according to the present invention provides for cells that maintain, or have essentially the same, three-dimensional geometry and cell-to-cell support and cell-to-cell geometry as recombinant cells prior to TVEMF-expansion. Other aspects of TVEMF-expansion may also provide the exceptional characteristics of the cells of the present invention. Not to be bound by theory, TVEMF-expansion not only provides for high concentrations of cells that maintain their three-dimensional geometry and cell-to-cell support. Not to be bound by theory, TVEMF may affect some properties of cells during TVEMF-expansion, for instance up-regulation of genes promoting grown, or down regulation of genes preventing growth. Overall, TVEMF-expansion results in promoting growth but not differentiation overall.

As used throughout this application, the term "TVEMF-expanded cell" refers to a cell that has been subjected to the process of TVEMF-expansion.

In the simplest terms, a rotating TVEMF- bioreactor comprises a cell culture chamber and a time varying electromagnetic force source. In operation, a cell culture containing suitable recombinant cells is placed into the cell culture chamber. The cell culture chamber is rotated over a period of time during which a time varying electromagnetic force is generated in the chamber by the time varying electromagnetic force source. Upon completion of the time, the expanded cell mixture is removed from the chamber. In a more complex TVEMF- bioreactor system, the time varying electromagnetic force source can be integral to the TVEMF- bioreactor, as illustrated in Figures 2-5, but can also be adjacent to a bioreactor as in Figures 6-8. Furthermore, a fluid carrier, such as cell culture mixture or buffer , which provides sustenance to the cells, can be periodically refreshed and removed. Suitable TVEMF- bioreactors are described herein.

Referring now to Figure 1, illustrated is a preferred embodiment of a culture carrier flow loop 1 in an overall bioreactor culture system for growing mammalian cells having a cell culture chamber 19, preferably a rotating cell culture chamber, an oxygenator 21, an apparatus for facilitating the directional flow of the culture carrier, preferably by the use of a main pump 15, and a supply manifold 17 for the selective input of such culture carrier requirements as, but not limited to, nutrients 3, buffers 5, fresh medium 7, cytokines 9, growth factors 11, and hormones 13. In this embodiment, the main pump 15 provides fresh fluid carrier to the oxygenator 21 where the fluid carrier is oxygenated and passed through the cell culture chamber 19. The waste in the spent fluid carrier from the cell culture chamber 19 is removed and delivered to the waste 18 and the remaining cell culture carrier is returned to the manifold 17 where it receives a fresh charge, as necessary, before recycling by the pump 15 through the oxygenator 21 to the cell culture chamber 19.

In the culture carrier flow loop 1, the culture carrier is circulated through the living cell culture in the chamber 19 and around the culture carrier flow loop 1, as shown in Figure 1. In this loop 1, adjustments are made in response to chemical sensors (not shown) that maintain constant conditions within the cell culture reactor chamber 19. Controlling carbon dioxide pressures and introducing acids or bases corrects pH. Oxygen, nitrogen, and carbon dioxide are dissolved in a gas exchange system (not shown) in order to support cell respiration. The closed loop 1 adds oxygen and removes carbon dioxide from a circulating gas capacitance. Although Figure 1 is one embodiment of a culture carrier flow loop that may be used in the present invention, the invention is not intended to be so limited. The input of culture carrier requirements such as, but not limited to, oxygen, nutrients, buffers, fresh medium, cytokines, growth factors, and hormones into a bioreactor can also be performed manually, automatically, or by other control means, as can be the control and removal of waste and carbon dioxide.

Figures 2 and 3 illustrate an embodiment of a TVEMF- bioreactor 10 with an integral time varying electromagnetic force source. Figure 4 is a cross section of a rotatable TVEMF-bioreactor 10 for use in the present invention in a preferred form. The TVEMF- bioreactor 10 of Figure 4 is illustrated with an integral time varying electromagnetic force source. Figure 5 also illustrates a preferred embodiment of a TVEMF- bioreactor with an integral time varying electromagnetic force source. Figures 6-8 show a rotating bioreactor with an adjacent time varying electromagnetic force source.

Turning now to Figure 2, illustrated in Figure 2 is an elevated side view of a preferred embodiment of a TVEMF-bioreactor 10 of the present invention. Figure 2 comprises a motor housing 111 supported by a base 112. A motor 113 is attached inside the motor housing 111 and connected by a first wire 114 and a second wire 115 to a control box 116 that has a control means therein whereby the speed of the motor 113 can be incrementally controlled by turning the control knob 117. The motor housing 111 has a motor 113 inside set so that a motor shaft 118 extends through the housing 111 with the motor shaft 118 being longitudinal so that the center of the shaft 118 is parallel to the plane of the earth at the location of a longitudinal chamber 119, preferably made of a transparent material including, but not limited to, plastic. Examples of transparent materials that can be used are glass and plastics. In an embodiment of the invention, the transparent material is plastic.

In this preferred embodiment, the longitudinal chamber 119 has a horizontal longitudinal axis and is connected to the shaft 118 so that the chamber 119 rotates about its longitudinal axis horizontally. The chamber 119 is wound with a wire coil 120. The size of the wire coil 120 and number of times it is wound are such that when a square wave current preferably of from 0.1mA to 1000mA is supplied to the wire coil 120, a time varying electromagnetic force preferably of from 0.05 gauss to 6 gauss is generated within the chamber 119. The wire coil 120 is connected to a first ring 121 and a second ring 122 at the end of the shaft 118 by wires 123 and 124. These rings 121, 122 are then contacted by a first electromagnetic delivery wire 125 and a second electromagnetic delivery wire 128 in such a manner that the chamber 119 can rotate while the current is constantly supplied to the coil 120. An electromagnetic generating device 126 is connected to the wires 125, 128. The electromagnetic generating device 126 supplies a square wave to the wires 125, 128 and coil 120 by adjusting its output by turning an electromagnetic generating device knob 127.

Figure 3 is a side perspective view of the TVEMF-bioreactor 10 shown in Figure 2 that may be used in the present invention.

Turning now to the rotating TVEMF-bioreactor 10 illustrated in Figure 4 with a culture chamber 230 which is preferably transparent and adapted to contain a recombinant cell mixture therein, further comprising an outer housing 220 which includes a first 290 and second 291 cylindrically shaped transverse end cap member having facing first 228 and second 229 end surfaces arranged to receive an inner cylindrical tubular glass member 293 and an outer tubular glass member 294. Suitable pressure seals are provided. Between the inner 293 and outer 294 tubular members is an annular wire heater 296 which is utilized for obtaining the proper incubation temperatures for cell growth. The wire heater 296 can also be used as a time varying electromagnetic force device to supply a time varying electric field to the culture chamber 230 or, as depicted in Figure 5, a separate wire coil 144 can be used to supply a time varying electromagnetic force. The first end cap member 290 and second end cap member 291 have inner curved surfaces adjoining the end surfaces 228, 229 for promoting smoother flow of the mixture within the chamber 230. The first end cap member 290, and second end cap member 291 have a first central fluid transfer journal member 292 and second central fluid transfer journal member 295, respectively, that are rotatably received respectively on an input shaft 223 and an output shaft 225. Each transfer journal member 294, 295 has a flange to seat in a recessed counter bore in an end cap member 290, 291 and is attached by a first lock washer and ring 297, and second lock washer and ring 298 against longitudinal motion relative to a shaft 223, 225. Each journal member 294, 295 has an intermediate annular recess that is connected to longitudinally extending, circumferentially arranged passages. Each annular recess in a journal member 292, 295 is coupled by a first radially disposed passage 278 and second radially disposed passage 279 in an end cap member 290 and 291, respectively, to first input coupling 203 and second input coupling 204. Carrier in a radial passage 278 or 279 flows through a first annular recess and the longitudinal passages in a journal member 294 or 295 to permit access carrier through a journal member 292, 295 to each end of the journal 292, 295 where the access is circumferential about a shaft 223, 225.

Attached to the end cap members 290 and 291 are a first tubular bearing housing 205, and second tubular bearing housing 206 containing ball bearings which relatively support the outer housing 220 on the input 223 and output 225 shafts. The first bearing housing 205 has an attached first sprocket gear 210 for providing a rotative drive for the outer housing 220 in a rotative direction about the input 223 and output 225 shafts and the longitudinal axis 221. The first bearing housing 205, and second bearing housing 206 also have provisions for electrical take out of the wire heater 296 and any other sensor.

The inner filter assembly 235 includes inner 215 and outer 216 tubular members having perforations or apertures along their lengths and have a first 217 and second 218 inner filter assembly end cap member with perforations. The inner tubular member 215 is constructed in two pieces with an interlocking centrally located coupling section and each piece attached to an end cap 217 or 218. The outer tubular member 216 is mounted between the first 217 and second inner filter assembly end caps .

The end cap members 217, 218 are respectively rotatably supported on the input shaft 223 and the output shaft 225. The inner member 215 is rotatively attached to the output shaft 225 by a pin and an interfitting groove 219. A polyester cloth 224 with a ten-micron weave is disposed over the outer surface of the outer member 216 and attached to O-rings at either end. Because the inner member 215 is attached by a coupling pin to a slot in the output drive shaft 225, the output drive shaft 225 can rotate the inner member 215. The inner member 215 is coupled by the first 217 and second 218 end caps that support the outer member 216. The output shaft 225 is extended through bearings in a first stationary housing 240 and is coupled to a first sprocket gear 241. As illustrated, the output shaft 225 has a tubular bore 222 that extends from a first port or passageway 289 in the first stationary housing 240 located between seals to the inner member 215 so that a flow of fluid carrier can be exited from the inner member 215 through the stationary housing 240.

Between the first 217 and second 218 end caps for the inner member 235 and the journals 292, 295 in the outer housing 220, are a first 227 and second 226 hub for the blade members 50a and 50b. The second hub 226 on the input shaft 223 is coupled to the input shaft 223 by a pin 231 so that the second hub 226 rotates with the input shaft 223. Each hub 227, 226 has axially extending passageways for the transmittal of carrier through a hub.

The input shaft 223 extends through bearings in the second stationary housing 260 for rotatable support of the input shaft 223. A second longitudinal passageway 267 extends through the input shaft 223 to a location intermediate of retaining washers and rings that are disposed in a second annular recess 232 between the faceplate and the housing 260. A third radial passageway 272 in the second end cap member 291 permits fluid carrier in the recess to exit from the second end cap member 291. While not shown, the third passageway 272 connects through piping and a Y joint to each of the passages 278 and 279.

A sample port is shown in Figure 4, where a first bore 237 extending along a first axis intersects a corner 233 of the chamber 230 and forms a restricted opening 234. The bore 237 has a counter bore and a threaded ring at one end to threadedly receive a cylindrical valve member 236. The valve member 236 has a complimentarily formed tip to engage the opening 234 and protrude slightly into the interior of the chamber 230. An O-ring 243 on the valve member 236 provides a seal. A second bore 244 along a second axis intersects the first bore 237 at a location between the O-ring 243 and the opening 234. An elastomer or plastic stopper 245 closes the second bore 244 and can be entered with a hypodermic syringe for removing a sample. To remove a sample, the valve member 236 is backed off to access the opening 234 and the bore 244. A syringe can then be used to extract a sample and the opening 234 can be reclosed. No outside contamination reaches the interior of the TVEMF-bioreactor 10.

In operation, carrier medium input to the second port or passageway 266 to the shaft passageway and thence to the first radially disposed 278 and second radially disposed passageways 279 via the third radial passageway 272. When the carrier enters the chamber 230 via the longitudinal passages in the journals 292, 294 the carrier impinges on an end surface 228, 229 of the hubs 227, 226 and is dispersed radially as well as axially through the passageways in the hubs 227, 226. Carrier passing through the hubs 227, 226 impinges on the end cap members 217, 218 and is dispersed radially. The flow of entry fluid carrier is thus radially outward away from the longitudinal axis 221 and flows in a toroidal fashion from each end to exit through the polyester cloth 224 and openings in filter assembly 235 to exit via the passageways 266 and 289. By controlling the rotational speed and direction of rotation of the outer housing 220, chamber 230, and inner filter assembly 235 any desired type of carrier action can be obtained. Of major importance, however, is the fact that a clinostat operation can be obtained together with a continuous supply of fresh fluid carrier.

If a time varying electromagnetic force is not applied using the integral annular wire heater 296, it can be applied by another preferred time varying electromagnetic force source. For instance, Figures 6-8 illustrate a time varying electromagnetic force device 140 which provides an electromagnetic force to a cell culture in a bioreactor which does not have an integral time varying electromagnetic force, but rather has an adjacent time varying electromagnetic force device. Specifically, Figure 6 is a preferred embodiment of a time varying electromagnetic force device 140. Figure 6 is an elevated side perspective of the device 140 which comprises a support base 145, a cylinder coil support 146 supported on the base 145 with a wire coil 147 wrapped around the support 146. Figure 7 is a front perspective of the time varying electromagnetic force device 140 illustrated in Figure 6. Figure 8 is a front perspective of the time varying electromagnetic force device 140, which illustrates that in operation, an entire bioreactor 148 is inserted into a cylinder coil support 146 which is supported by a support base 145 and which is wound by a wire coil 147. Since the time varying electromagnetic force device 140 is adjacent to the bioreactor 148, the time varying electromagnetic force device 140 can be reused. In addition, since the time varying electromagnetic force device 140 is adjacent to the bioreactor 148, the device 140 can be used to generate an electromagnetic force in all types of bioreactors, preferably rotating.

Furthermore, in operation the present invention contemplates that an electromagnetic generating device is turned on and adjusted so that the square wave output generates the desired electromagnetic field in the mammalian cell mixture- containing chamber, preferably in a range of from 0.05 gauss to 6 gauss, more preferably in the range of 0.05 to 0.5 gauss. Preferably, the square wave has a frequency of about 2 to about 25 cycles/second, more preferably about 5 to about 20 cycles/second, and for example about 10 cycles/second, and the conductor has an RMS value of about 1 to about 1000 mA, preferably about 1 to about 6 mA. However, these parameters are not meant to be limiting to the TVEMF of the present invention, as such may vary based on other aspects of this invention. TVEMF may be measured for instance by standard equipment such as an ENl 31 Cell Sensor Gauss Meter.

In operation, during TVEMF- expansion, a TVEMF- bioreactor 10 of the present invention contains a recombinant cell mixture in the cell culture chamber. During TVEMF- expansion, the speed of the rotation of the recombinant cell-containing chamber may be assessed and adjusted so that the recombinant cell mixture remains substantially at or about the longitudinal axis. Increasing the rotational speed is warranted to prevent wall impact. For instance, an increase in the rotation is preferred if the cells in the mixture fall excessively inward and downward on the downward side of the rotation cycle and excessively outward and insufficiently upward on the upward side of the rotation cycle. When the recombinant cells increase in viscosity they may gravitate towards the wall of the chamber, with the medium surrounding the cluster of cells. To maintain their position in the center of the fluid filled chamber the rotational speed may be decreased so to maintain the cells in the center of the container and prevent the cells from colliding with the sides of the container. Thus, the cells are not damaged and are permitted to grow or expand at a significant rate. One method of monitoring the overall expansion of cells undergoing TVEMF-expansion is by visual inspection. Once the bioreactor begins to rotate and the TVEMF is applied, the cells preferably cluster in the center of the bioreactor chamber, with the medium surrounding the cluster of cells. Oxygenation and other nutrient additions often do not cloud the ability to visualize the cell cluster through a visualization (typically clear plastic) window built into the bioreactor. The cell mixture may preferably be visually assessed through the preferably transparent culture chamber and manually adjusted. The assessment and adjustment of the cell mixture may also be automated by a sensor (for instance, a laser), which monitors the location of the cells within a TVEMF- bioreactor 10. A sensor reading indicating too much cell movement will automatically cause a mechanism to adjust the rotational speed accordingly.

Formation of the cluster is important for helping the cells maintain their three-dimensional geometry and cell-to-cell support and cell-to-cell geometry; if the cluster appears to scatter and cells begin to contact the wall of the bioreactor chamber, the rotational speed is increased (manually or automatically) so that the centralized cluster of cells may form again. A measurement of the visualizable diameter of the cell cluster taken soon after formation may be compared with later cluster diameters, to indicate the approximate number increase in cells in the TVEMF-bioreactor. Measurement of the increase in the number of cells during TVEMF expansion may also be taken in a number of ways, as known in the art. An automatic sensor could also be included in the TVEMF-bioreactor to monitor and measure the increase in cluster size.

The TVEMF-expansion process may be carefully monitored, for instance by a person who will check cell cluster formation to ensure the cells remain clustered inside the bioreactor and will increase the rotation of the bioreactor when the cell cluster begins to scatter. An automatic system for monitoring the cell cluster and viscosity of the mammalian cell mixture inside the bioreactor may also monitor the cell clusters. A change in the viscosity of the cell cluster may become apparent about 2 days after beginning the TVEMF-expansion process, and the rotational speed of the TVEMF- bioreactor may be increased around that time. The TVEMF-bioreactor speed may vary throughout TVEMF-expansion. Optimally, the user is advised to preferably select a rotational rate that fosters minimal wall collision frequency and intensity so as to maintain the recombinant cell three-dimensional geometry and their cell-to-cell support and cell-to-cell geometry. The preferred speed of the present invention is of from 5 to 120 RPM preferably 10 - 40 RPM, and more preferably from 10 to 30 RPM. Furthermore, in operation the present invention contemplates that an electromagnetic generating device is turned on and adjusted so that the square wave output generates the desired electromagnetic field in the recombinant cell mixture- containing chamber, preferably in a range of from 0.05 gauss to 6 gauss.

During the time that the cells are in the TVEMF-bioreactor, they are preferably fed nutrients and fresh media (DMEM and 5% human serum albumin), and preferably exposed to hormones, cytokines, and/or growth factors (for example G-CSF and toxic materials are removed. The toxic materials removed from cells in a TVEMF- bioreactor may include the toxic granular material of dying cells and the toxic material of granulocytes and macrophages. The TVEMF-expansion of the cells is controlled so that the cells preferably expand (increase in number per volume, or concentration) at least seven times in a sufficient amount of time. Preferably, recombinant cells undergo TVEMF-expansion for at least 4 days, preferably about 7 to about 14 days, more preferably about 7 to about 10 days, even more preferably about 7 days. TVEMF- expansion may continue in a TVEMF-bioreactor for up to 160 days. While TVEMF- expansion may occur for even longer than 160 days, such a lengthy expansion is not a preferred embodiment of the present invention.

Preferably, the amount of cells used is about 1000 cells/ml to about 9 x 10⁹ cells/ml, more preferably about 10,000 cells to about 10⁷ cells/ml, even more preferably about 10⁵ to 10⁶ cells/ml, and for example 2.5 x 10⁵ cells/ml. Depending on the size and type of bioreactor or other cell culture chamber, the total volume of the cell mixture to be placed into the chamber is about 10 ml to about 1 IL.

Preferably, TVEMF-expansion is carried out in a TVEMF-bioreactor at a temperature of about 26°C to about 41⁰C, and more preferably, at a temperature of about 37⁰C + 2⁰C.

Also, the laboratory expert may, for instance once a day, or once every two days, manually (for instance with a syringe) insert fresh media and preferably other desired additives such as nutrients and growth factors, as discussed above, into the bioreactor, and draw off the old media containing cell wastes and toxins. Also, fresh media and other additives may be automatically pumped into the TVEMF-bioreactor during TVEMF-expansion, and wastes automatically removed. Cells may increase to at least seven times their original number about 7 to about 14 days after being placed in the TVEMF-bioreactor and TVEMF-expanded. Preferably, the TVEMF-expansion lasts about 7 to 10 days, and more preferably about 7 days.

As noted above, in some cases, for example when using microencapsulated cells in gene therapy, therapeutic benefit may be obtained by use of the cells themselves. In others, the active material may be expressed by the cells. When this is the case, the protein will naturally be released extracelluarly by transport across the cell wall. Use of a suitable leader sequence such as the Ig kappa leader sequence in the nucleotide sequence introduced into the host cell may facilitate expression of the desired final product. In others, lysis of the cells may be required. In cases where the desired protein is not released extracelluarly a variety of methods including mechanical methods such as direct mechanical description, for example by a homogenizer or a ball mill or use of ultrasonifϊcation may be used. Alternatively, the intracellular proteins may be removed from the cells by the techniques such as use of a freeze/thaw cycle, or chemical means using solvents, detergents or salts or by biological means using enzymes. In some cases, it may be desirable to add protease inhibitors to avoid degradation of the desired polypeptides. Irrespective of whether the desired protein is released from the cell by its own action or whether some external means is required, it is necessary to remove the desired protein from the resultant mixture. This can be accomplished by conventional means such as precipitation followed by gel filtration and ion exchange chromatography. However, other separation means known in the art may be used if desired, such as high pressure liquid chromatography, affinity chromatography, and size exclusion chromatography. Removal of other components of the mixture, for example DNA or RNA may be necessary, for example by use of chloroform or formaldehyde extractions.

When administered parenterally, proteins are often cleared rapidly from the circulation and may therefore elicit relatively short-lived pharmacological activity. Consequently, frequent injections of relatively large doses of bioactive proteins may be required to sustain therapeutic efficacy. Proteins modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified proteins [Abuchowski et al., In: "Enzymes as Drugs", Holcenberg et al., eds. Wiley-Interscience, New York, N. Y., 367-383 (1981), Newmark et al., J. Appl. Biochem. 4:185-189 (1982), and Katre et al., Proc. Natl. Acad. Sci. USA 84, 1487-1491 (1987)]. Polypeptides produced by the process of the present invention may be modified in this way where appropriate.

Numerous activated forms of PEG suitable for direct reaction with proteins have been described. Useful PEG reagents for reaction with protein amino groups include active esters of carboxylic acid or carbonate derivatives, particularly those in which the leaving groups are N-hydroxysuccinimide, p-nitrophenol, imidazole or l-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containing maleimido or haloacetyl groups are useful reagents for the modification of protein free sulfhydryl groups. Likewise, PEG reagents containing amino, hydrazine or hydrazide groups are useful for reaction with aldehydes generated by periodate oxidation of carbohydrate groups in proteins.

A composition of the present invention may include a pharmaceutically acceptable carrier that will allow the introduction of the recombinant molecule into a mammal, preferably a human. The term "introduction" of the composition to a mammal is meant to refer to "administration" of the composition to an animal. The pharmaceutical composition may be in a form selected from tablets, lozenges, capsules, powders, dragees, aqueous or oily suspensions, syrups, elixirs, and aqueous solutions. The nature of the composition employed will, of course, depend on the desired route of administration. The molecule mixture can be administered orally, topically, rectally, nasally, transdermally, or parenterally (i.e., intramuscular, intravenous, and subcutaneous) or in any other suitable way.

This invention also includes a method of therapeutically treating mammals comprising producing the recombinant molecule as described herein and thereafter administering a therapeutic amount of the natively recombinant molecule alone or in combination with another molecule, drug, or the like to a mammal to achieve a therapeutic affect.

Particular products for which the present invention is useful will now be considered.

Recombinant hpG-CSF DNA sequences encoding part or all of the polypeptide sequence of hpG- CSF have been isolated and characterized as described in US Patent 4,810,643. Suitable recombinant cells may be obtained by transforming or transfecting a host cell with a DNA molecule in a manner allowing expression of said polypeptide product, wherein said DNA molecule encodes a polypeptide product selected from the group consisting of:

[Met--¹] hpG-CSF;

[Ser¹⁷] hpG-CSF;

[Ser³⁶] hpG-CSF;

[Ser⁴²] hpG-CSF;

[Ser⁶⁴] hpG-CSF;

[Ser⁷⁴] hpG-CSF;

[Met^"1, Ser¹⁷] hpG-CSF;

[Mef¹, Ser.³⁶] hpG-CSF;

[Mef¹, Ser.⁴²] hpG-CSF;

[Mef¹, Ser⁶⁴] hpG-CSF; and

[Mef¹, Ser.⁷⁴] hpG-CSF.

Example 5 of U.S. Patent 4,810,643 is directed to the identification and sequencing of a genomic clone encoding hpG-CSF. This product may be introduced into a suitable mammalian host cell for use in the process of the present invention.

In addition to naturally-occurring allelic forms of hpG-CSF, the invention of US Patent 4,810,643 also embraced other hpG-CSF products such as polypeptide analogs of hpG-CSF and fragments of hpG-CSF. Following the procedures of the above-noted published application by Alton, et al. (WO/83/04053) one may readily design and manufacture genes coding for microbial expression of polypeptides having primary conformations which differ from that herein specified for in terms of the identity or location of one or more residues (e.g., substitutions, terminal and intermediate additions and deletions). Alternately, modifications of cDNA and genomic genes may be readily accomplished by well-known site-directed mutagenesis techniques and employed to generate analogs and derivatives. Such products would share at least one of the biological properties of hpG-CSF but may differ in others. As examples, projected products include those which are foreshortened by e.g., deletions; or those which are more stable to hydrolysis (and, therefore, may have more pronounced or longer lasting effects than naturally-occurring)or which have one or more tyrosine residues replaced by phenylalanine and may bind more or less readily to hpG-CSF receptors on target cells. Also comprehended are polypeptide fragments duplicating only a part of the continuous amino acid sequence or secondary conformations within hpG- CSF, which fragments may possess one activity of (e.g., receptor binding) and not others (e.g., colony growth stimulating activity). It is noteworthy that activity is not necessary for any one or more of the products of the invention to have therapeutic utility >see, Weiland, et al., Blut, 44, 173-175 (1982)! or utility in other contexts, such as in assays of hpG-CSF antagonism. Competitive antagonists may be quite useful in, for example, cases of overproduction of hpG-CSF

The recombinant mammalian cells obtained by using the DNA produced in the invention described in United States Patent No. 4,810,643 may be expanded by the process of the present invention to produce increasing amounts of the desired proteins.

The method of the present invention may also be used for the production of recombinant stem cell factor.

Human Stem Cell Factor Polypeptides

US Patent 6218148 describes DNA encoding for Stem Cell Factor.

The present invention may be emphasized for expansion of cells containing such DNA especially when in recombinant form.

The term "stem cell factor" or "SCF" as used herein refers to naturally- occurring SCF (e.g. natural human SCF) as well as non-naturally occurring (i.e., different from naturally occurring) polypeptides having amino acid sequences and glycosylation sufficiently duplicative of that of naturally-occurring stem cell factor to allow possession of a hematopoietic biological activity of naturally-occurring stem cell factor. Stem cell factor has the ability to stimulate growth of early hematopoietic progenitors which are capable of maturing to erythroid, megakaryocyte, granulocyte, lymphocyte, and macrophage cells. SCF treatment of mammals results in absolute increases in hematopoietic cells of both myeloid and lymphoid lineages. One of the hallmark characteristics of stem cells is their ability to differentiate into both myeloid and lymphoid cells [Weissman, Science, 241, 58-62 (1988)]. Treatment of Steel mice with recombinant rat SCF results in increases of granulocytes, monocytes, erythrocytes, lymphocytes, and platelets. Treatment of normal primates with recombinant human SCF results in increases in myeloid and lymphoid cells .

US Patent 6218148 provides DNA sequences which include: the incorporation of codons "preferred" for expression by selected nonmammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences which facilitate construction of readily-expressed vectors. US Patent 6218148 also provides DNA sequences coding for polypeptide analogs or derivatives of SCF which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (i.e., deletion analogs containing less than all of the residues specified for SCF; substitution analogs, wherein one or more residues specified are replaced by other residues; and addition analogs wherein one or more amino acid residues is added to a terminal or medial portion of the polypeptide) and which share some or all the properties of naturally-occurring forms. US Patent 6218148 specifically provides DNA sequences encoding the full length unprocessed amino acid sequence as well as DNA sequences encoding the processed form of SCF. Such DNA sequences include sequences useful in securing expression in procaryotic or eucaryotic host cells of polypeptide products having at least a part of the primary structural conformation and one or more of the biological properties of naturally-occurring SCF. The DNA sequences may incorporate codons facilitating transcription and translation of messenger RNA in microbial hosts. Such manufactured sequences may readily be constructed according to the methods of Alton et al., PCT published application WO 83/04053.

In particular, in US Patent 6,2181,48, FIG. 42 shows human SCF cDNA sequence obtained from the HTl 080 fibrosarcoma cell line and FIG. 44 shows human SCF cDNA sequence obtained from the 5637 bladder carcinoma cell line. These sequences are incorporated herein as SEQ ID No 1 and SEQ ID No 2, respectively.

The products of expression in vertebrate [e.g., non-human mammalian (e.g. COS or CHO) and avian] cells are free of association with any human proteins. Depending upon the host employed, polypeptides of the invention may be glycosylated with mammalian or other eucaryotic carbohydrates or may be non-glycosylated. The host cell can be altered using techniques such as those described in Lee et al. J. Biol. Chem. 264, 13848 (1989) hereby incorporated by reference. Polypeptides of the invention may also include an initial methionine amino acid residue (at position -1).

In addition to naturally-occurring allelic forms of SCF, US Patent 6,218,148 also embraces other SCF products such as polypeptide analogs of SCF. Such analogs include fragments of SCF. Following the procedures of the above-noted published application by Alton et al. (WO 83/04053), one can readily design and manufacture genes coding for microbial expression of polypeptides having primary conformations which differ from that herein specified for in terms of the identity or location of one or more residues (e.g., substitutions, terminal and intermediate additions and deletions). Alternately, modifications of cDNA and genomic genes can be readily accomplished by well-known site-directed mutagenesis techniques and employed to generate analogs and derivatives of SCF. Such products share at least one of the biological properties of SCF but may differ in others. As examples, products of the invention include those which are foreshortened by e.g., deletions; or those which are more stable to hydrolysis (and, therefore, may have more pronounced or longer-lasting effects than naturally-occurring); or which have been altered to delete or to add one or more potential sites for O-glycosylation and/or N-glycosylation or which have one or more cysteine residues deleted or replaced by, e.g., alanine or serine residues and are potentially more easily isolated in active form from microbial systems; or which have one or more tyrosine residues replaced by phenylalanine and bind more or less readily to target proteins or to receptors on target cells. Also comprehended are polypeptide fragments duplicating only a part of the continuous amino acid sequence or secondary conformations within SCF, which fragments may possess one property of SCF (e.g., receptor binding) and not others (e.g., early hematopoietic cell growth activity). It is noteworthy that activity is not necessary for any one or more of the products of the invention to have therapeutic utility [see, Weiland et al., Blut, 44, 173-175 (1982)] or utility in other contexts, such as in assays of SCF antagonism. Competitive antagonists may be quite useful in, for example, cases of overproduction of SCF or cases of human leukemias where the malignant cells over express receptors for SCF, as indicated by the overexpression of SCF receptors in leukemic blasts .

US Patent 6,218,148 also includes that class of polypeptides coded for by portions of the DNA complementary to the protein-coding strand of the human cDNA or genomic DNA sequences of SCF, i.e., "complementary inverted proteins" as described by Tramontano et al. [Nucleic Acid Res., 12, 5049-5059 (1984)].

SCF can be purified using techniques known to those skilled in the art. The subject invention comprises a method of purifying SCF from an SCF containing material such as conditioned media or human urine, serum, the method comprising one or more of steps such as the following: subjecting the SCF containing material to ion exchange chromatography (either cation or anion exchange chromatography); subjecting the SCF containing material to reverse phase liquid chromatographic separation involving, for example, an immobilized C₄ or C₆ resin; subjecting the fluid to immobilized-lectin chromatography, i.e., binding of SCF to the immobilized lectin, and eluting with the use of a sugar that competes for this binding. Details in the use of these methods will be apparent from the descriptions given in Examples 1, 10 and 11 of U.S. Patent 6218148 for the purification of SCF. The techniques described in Example 2 of the Lai et al. U.S. Pat. No. 4,667,016, hereby incorporated by reference are also useful in purifying stem cell factor.

Isoforms of SCF are isolated using standard techniques such as the techniques set forth in U.S Patent Application Serial No. 421,444, entitled Erythropoietin Isoforms, filed Oct. 13, 1989, now abandoned, but of which a continuation in part has issued as US Patent 5,856,298 hereby incorporated by reference.

The invention of U S Patent 6,218,148 also comprises compositions including one or more additional hematopoietic factors such as EPO, G-CSF, GM-CSF, CSF-I, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IGF-I, or LIF (Leukemic Inhibitory Factor). Expansion of cells producing all of these factors may be accomplished by the process of the present invention.

Examples of US Patent 6,218,148 that involve cell growth may be repeated using the method of the present invention subjecting the growth to time varying electromagnetic force. Specific Examples of US Patent 6,218,148 that may be replicated in this way and the desired expressed products obtained include:

Example 4 - growth of COS-I Cells transfected with vector Vl 9.1 coding for rat SCF ^1"162 and SCF ^M93; Example 5 - growth of CHO cells transformed with vector V 19.8 coding for SCF ^M62 or human SCF;

Example 11 - growth of CHO cells transformed with a vector coding for human SCF¹-¹⁶²;

Example 16 - growth of COS-7 cells transfected with a vector coding for human SCF^{1 "164};

Example 20 - growth of cell fusion hybrids of SCF^1"164 coding E coli cells and sp2/0 myeloma cells;

Example 28 - growth of E coli transformed with vectors coding for SCF^1" ¹⁸⁹, SCF^1"188, SCF¹-¹⁸⁵, SCF^1"180, SCF^1"156, SCF^1"141, SCF^1"137, SCF^1"130, SCF^2"164, SCF^5'

164_{5 SCF}11-164_{5 and} SCpI-IeO₅ ^1-157^ _SCF1-152_

Ex Vivo Expansion of Multipotential Hematopoetic Cells

produced by culturing the cells in a medium containing interleukin-6 receptor, interleukin-6, and stem cell factor

The present invention may also be used to increase production of multipotential hematopoetic cells. As discussed in US Patent 5861315 (Nakahata, assigned to Amgen Inc. and Toosh Corporation). GP 130 signaling involving sIL-6R and IL-6, in the presence of SCF, dramatically stimulates the ex vivo expansion of human primitive hematopoietic progenitor cells at a substantially increased level when the procedure utilizes time varying electromagnetic force.. Suitable multipotential cells may be obtained from inter alia cord blood, peripheral blood and bone marrow. Such cells may be expanded, and in some cases differentiate by cultivation under the influence of a square wave time varying electromagnetic force of from 0.05 gauss to 6 gauss, the cells being cultured in a medium comprising soluble interleukin-6 receptor, interleukin-6, and stem cell factor, said cells, interleukin-6 receptor, interleukin-6, and stem cell factor derived from a human species. Typically said interleukin-r receptor, interleukin-6, and stem cell factor will be present in amounts effective to produce a mature erythroid cell population in the absence of erythropoietin. Except for the nature of the cells being expanded and the presence of the interlekin-6, soluble interlekin 6 receptor and the stem cell factor in the cultivation medium, the comditions used are similar to those described above for the expansion of recombinant cells. If desired one or more of the interlekin-6, soluble interlekin 6 receptor and the stem cell factor may itself have been produced by expansion of recombinant cells.

GP 130, a signal transducing receptor component of IL-6, associates with a complex of IL-6 ligand and IL-6 receptor (IL-6R) and transduces signals. To examine the role of gpl30 signaling in the expansion of human hematopoietic progenitor cells, the effects of a recombinant soluble human IL-6 receptor (sIL-6R) and/or IL-6 were tested in combination with various cytokines on CD34⁺ cells.

US Patent 5861315 reports that a combination of sIL-6R and IL-6 (sIL- 6R/IL-6) was found to dramatically stimulate the expansion of hematopoietic progenitor/stem cells as well as CD34⁺ cells in the presence of stem cell factor (SCF). Neither the combination of sIL-6R and SCF nor the combination of IL-6, and SCF provided this result. A significant generation of multipotential hematopoietic progenitors occurred over a period of three weeks in suspension cultures receiving sIL- 6R/IL-6/SCF. The efficient formation of colonies, especially multilineage and blast cell colonies was observed in both serum-containing and serum-free cultures supplemented with the combined cytokines. The initial hematopoietic progenitor/stem cells may be obtained from any suitable cell source including the fetus, placenta, cord blood, peripheral blood, or bone marrow. These multipotential hematopoietic cells may include progenitor and/or stem cells obtained by CD34 selection. Optionally, the multipotential hematopoietic cells may be stem cells which are functionally selected or isolated by the removal of proliferating cells (Berardi et al., Science, 267:104-108, 1995).

US Patent 5,861,315 reports that two signal pathways, gρl30 signaling and c-Kit signaling are initiated by sIL-6R/IL-6 and SCF, respectively, synergistically promote the ex vivo expansion of human hematopoietic progenitor cells. sIL-6R has been reported to potentiate agonistic effects in the presence of IL-6 on some cell lines such as BAF-ml30 cells, gpl30 cDNA-transfected cells, and the murine osteoclasts (6- 8,25). SIL-6R/IL-6 in the presence SCF is a very potent stimulator for the proliferation of human primitive hematopoietic progenitors. Previous reports on ex vivo expansion had failed to demonstrate such a striking synergy as found with the sIL-6R/IL-6/SCF combination for the stimulation of human primitive hematopoietic cells.

Example 1 of US Patent 5861315 reports that a slight synergy between sIL-6R/IL-6 and either IL-3, GM-CSF or G-CSF exists in serum-containing cultures. No synergy exists between sIL-6R/IL-6 and those factors, however, in serum-free culture. Example 2 of US Patent 5861315 describes the effect of sIL-6R, IL-6 and SCF on expansion of hematopoietic multipotential cells in suspension culture.

The results revealed that sIL-6R/IL-6 acts synergistically with SCF in the expansion of hematopoietic progenitor cells. In subsequent experiments, sIL-6R/IL-6 was tested in combination with some early acting cytokines, including IL-3 and G-CSF, in the presence of SCF.

The study also compared the sIL-6R, IL-6 and SCF combination with an IL-3, IL-6 and SCF combination, which was the previous standard for use in expansion studies. The expansion of total progenitors using sIL-6R/IL-6/SCF was 1.5 -fold of that resulting from the use of the IL-3, IL-6 and SCF combination. The results demonstrated that the combination of sIL-6R, IL-6 and SCF is a more potent expansion combination, especially for the expansion of primitive progenitors.

Example 3 of US Patent 5,861,315 describes the effects of Anti-gpl30 mAb and Anti-IL-6R mAb on the expansion of multipotential cells . It was found that the addition of anti-gpl30 mAbs dose-dependently inhibited the expansion of total progenitor cells in the serum-containing suspension culture. The resulting expansion of total progenitor cells (.small circle.) and CFU-Mix (.cndot.) in cultures containing a combination of sIL-6R, IL-6 and SCF, and of total progenitor cells in cultures containing a combination of IL-3 and SCF (.DELTA.), when varying concentrations of anti-human gpl30 mAbs (A) and anti-human IL-6R mAb (B) are added to the cultures. The cultures without mAbs were estimated as control experiments. The expansion of CFU-Mix was completely blocked at an anti-gpl30 mAb concentration of 1 .mu.g/ml, whereas the mAbs appeared to have little or no effect on the expansion induced by the combination of IL-3 and SCF. The addition of anti-IL-6R mAb to cultures caused a similar display of inhibition, except at a slightly lower efficiency, with the complete abrogation of CFU-Mix expansion observed at a concentration of 10 .mu.g/ml. The anti- IL-6R antibody also failed to affect expansion stimulated by the combination of IL-3 and SCF. In contrast, an anti-EPO antibody inhibited the expansion induced by SCF and EPO but had no effect on that induced by sIL-6R, IL-6 and SCF. The same results were obtained in both serum-free suspension culture and methylcellulose culture.

U.S Patent 5861315 also describes erythroid cell production. Studies were performed to further examine the effects of gpl30 signaling on the generation of erythroid cells from human hematopoietic progenitor cells. The results clearly indicated that sIL-6R is functional and capable of transducing proliferative signals in CD34⁺ cells only in combination with IL-6. Dose response studies suggest that sIL-6R at 1280 ng/ml and IL-6 at 50 ng/ml may be an effective combination for the expansion of total cells from purified stem cells in serum-containing culture with SCF. The expansion of total cells by the combination of sIL-6R, IL-6 and SCF was also observed when CD34⁺ cells purified from human bone marrow were used.

The combination of sIL-6R, IL-6 and SCF stimulated the generation of not only total cell number but also total erythroid cells more significantly than did other combinations. At two weeks of culture, approximately 79% of the cells generated by the combination were erythroid cells (i.e., E-blasts and erythrocytes). At three weeks of culture, approximately 69% of the cells generated by the combination were erythroid cells. A small number of erythroid cells was also observed in cultures containing sIL-6R and IL-6 in combination with either IL-3 or GM-CSF, suggesting that gpl30 signaling plays a role in the generation of erythroid cells in vitro. No erythroid cells were detectable in cultures receiving other cytokine combinations, except those combinations which included erythropoietin. In comparison to erythropoietin alone, the total number of erythroid cells produced by the combination of sIL-6R, IL-6 and SCF was about 5- fold at 14 days of culture and about 115-fold at 21 days of culture. In comparison to a combination of erythropoietin, sIL-6R and IL-6, the total number of erythroid cells produced by the sIL-6R/IL-6/SCF combination was about 4.1 -fold at 14 days of culture and about 38.3-fold at 21 days of culture. The effect of the sIL-6R/IL-6/SCF combination on the generation of erythroid cells was observed in both serum-containing and serum-free culture of CD34 bone marrow cells.

Methylcellulose clonal culture of hematopoietic stem cells also indicated that the anti-gpl30 MAbs, but not the anti-erythropoietin antibody, completely blocked the development of both erythroid burst and mixed erythroid colony formation which was otherwise induced by the sIL-6R/IL-6/SCF combination. These results clearly demonstrate that the observed effects of the interleukin-6 ligand and soluble receptor result from the interaction of IL-6 and sIL-6R, and the association of the resulting IL- 6/sIL-6R complex with membrane-anchored gpl30 on the target progenitor cells. The results also indicated that the generation of erythroid cells from immature erythroid progenitors by gpl30 signaling in combination with SCF occurs independently from the presence of erythropoietin.

Claims

-35- C L A I M S

1. Recombinant mammalian molecules produced by exposing the molecules to a square wave time varying electromagnetic force of from 0.05 gauss to 6 gauss during their production.

2. Recombinant mammalian molecules as claimed in claim 1, wherein said molecules were exposed to a square wave time varying electromagnetic force of from 0.05 gauss to 0.5 gauss during their production.

3. Recombinant mammalian molecules as claimed in claim 1 , wherein said molecules are selected from human pluripotent granulocyte colony-stimulating factor (hpG-CSF) and stem cell factor.

4 A process for producing recombinant mammalian molecules by comprising culturing cells producing such molecules while exposing them to a square wave time varying electromagnetic force of from 0.05 gauss to 6 gauss during their production.

5. A process for producing recombinant mammalian molecules as claimed in claim 4, wherein said time varying electrmagnetic force is from 0.05 to 0.5 gauss.

6. A process as claimed in claim 4, wherein said cells are cultured in a reactor that rotates about a horizontal axis at a rotational speed such as to prevent the cells being cultured to contact the wall of the reactor.

7. A process as claimed in claim 4, wherein said cells are cultured in such manner as to maintain their three dimensional cell geometry and cell to cell support during cultivation.

8. A process as claimed in claim 6, wherein said reactor rotates at from 5 to 120 rpm.

9. A process as claimed in claim 7, wherein said reactor rotates at from 10 to 30 rpm.

10. A process as claimed in claim 4, wherein the square wave time-variable electromagnetic force cycles at from 2 to 25Hz.

-Soil . A process as claimed in any of claims 4 to 10, wherein the cells used to produce the recombinant molecules are mammalian cells.

12. A process as claimed in claim 11 , wherein said recombinant molecules are hpG-CSH or SCF polypeptides.

13. A process as claimed in claim 11 , wherein at least one of said recombinant molecules is selected from the group consisting of erythropoietin, streptokinase, human growth hormone, interferon-, interferon- , anti-T-cell antibody, hepatitis B antibody, tissue plasminogen activator, 1 -antitrypsin, interleukin-2, interleukin-6, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, coagulation factor VIII, interleukin 10, epidermal growth factor, follicle stimulating hormone and glucagon polypeptides.

14. In a process for producing recombinant mammalian molecules, the improvement comprising: increasing the production amount of the molecules by exposing the molecules to a square wave time varying electromagnetic force of from 0.05 gauss to 6 gauss during their production.

15. A process for the production of a human pluripotent granulocyte colony- stimulating factor (hpG-CSF) product having the in vivo granulocytopoietic biological property of naturally occurring hpG-CSF comprising the steps of:

(a) while exposing the molecules to a square wave time varying electromagnetic force of from 0.05 gauss to 6 gauss, culturing under suitable nutrient conditions, mammalian cells comprising promoter DNA, other than hpG-CSF promoter DNA, operatively linked to DNA encoding a hpG-CSF polypeptide; and (b) isolating said hpG-CSF expressed by said cells.

16. A method for the ex vivo expansion of multipotential hematopoietic cells, comprising: while exposing the multipotential hematopoietic cells to a square wave time varying electromagnetic force of from 0.05 gauss to 6 gauss, culturing the cells in a medium comprising soluble interleukin-6 receptor, interleukin-6, and stem cell factor, said cells, interleukin-6 receptor, interleukin-6, and stem cell factor derived from a human species, said interleukin-r receptor, interleukin-6, and stem cell factor being present in amounts effective to produce a mature erythroid cell population in the absence of erythropoietin. -37-

17. The method according to claim 16, wherein the cells to be expanded are obtained from cord blood, peripheral blood or bone marrow.

18. The method according to claim 16, wherein the multipotential hematopoietic cells are progenitor and/or stem cells obtained by CD34 selection.

19. A kit, consisting of: soluble interleukin-6 receptor, interleukin-6 and stem cell factor, said soluble interleukin-6 receptor, interleukin-6 and stem cell factor, all of which have been exposed to a square wave time varying electromagnetic force of from 0.05 gauss to 6 gauss during their production, provided in individual containers or as a mixture in a single container in an amount effective for use in expansion of multipotential hematopoietic cells according to claim 16.

20. A method generating differentiated blood cell colonies by ex vivo expansion of multipotential hematopoietic cells, comprising exposing the cell colonies to a square wave time varying electromagnetic force of from 0.05 gauss to 6 gauss while culturing the hematopoietic cells in a medium comprising soluble interleukin-6 receptor, interleukin-6, and stem cell factor, said hematopoietic cells, interleukin-6 receptor, interleukin-6, and stem cell factor derived from a human species, said interleukin-6 receptor, interleukin-6, and stem cell factor being present in amounts effective to produce a mature erythroid cell population in the absence of erythropoietin.

21. A process as claimed in claim 4, for the production of a polypeptide product having part or all of the primary structural conformation and the hematopoietic biological activity of naturally occurring pluripotent granulocyte colony-stimulating factor, said process comprising: growing, under suitable nutrient conditions, and while exposing the host cell colonies to a square wave time varying electromagnetic force of from 0.05 gauss to 6 gauss, host cells transformed or transfected with a DNA molecule in a manner allowing expression of said polypeptide product, wherein said DNA molecule encodes a polypeptide product selected from the group consisting of:

[Met-^"1] hpG-CSF;

[Ser¹⁷] hpG-CSF;

[Ser³⁶] hpG-CSF;

[Ser⁴²] hpG-CSF;

[Ser⁶⁴] hpG-CSF;

[Ser⁷⁴] hpG-CSF; -38-

[Mef¹, Ser¹⁷] hpG-CSF; [Met^'1, Ser.³⁶] hpG-CSF; [Mef¹, Ser.⁴²] hpG-CSF; [Met^"1, Ser⁶⁴] hpG-CSF; and [Mef¹, Ser.⁷⁴] hpG-CSF.

22. A method as claimed in claim 4, for preparing a human stem cell factor

(SCF) polypeptide, the method comprising the steps of: a) growing under suitable nutrient conditions, and while exposing the host cell colonies to a square wave time varying electromagnetic force of from 0.05 gauss to 0.5 gauss, host cells transformed or transfected with DNA encoding a stem cell factor (SCF) polypeptide having the amino acid sequence of SEQ ID NO:1, SEQ or SEQ ID NO: 2, or biologically active fragments thereof that stimulate growth of hematopoietic progenitor cells, said DNA being operatively linked to an expression control sequence; and b) isolating the polypeptide produced thereby.