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Title:  Serum-derived factor inducing cell differentiation and medical uses thereof

United States Patent:  6,372,262

Inventors:  Peled; Tony (Mevaseret, IL); Fibach; Eitan (Mevaseret Zion, IL); Rachmilewitz; Eliezer A. (Jerusalem, IL)

Assignee:  Hadasit Medical Research Services & Development Company Ltd. (Jerusalem, IL)

Appl. No.:  332254

Filed:  June 8, 1999

Abstract

The present invention relates to a biologically active serum-derived composition of matter (SDF), having a low molecular weight, being electrically charged at acidic pH and having absorption at 280 nm. The molecular weight of said SDF was determined by electron spray mass spectrometry and is of 316 Da. SDF of the present invention or its complex with ceruloplasmin (CP) have several therapeutic properties. For example, SDF or its complex with CP is capable of inducing terminal cell differentiation of leukemic cells, which as a result, may lose their ability to proliferate and their ability for self cell renewal. Further, SDF or the complex with CP is capable of stimulating the proliferation of early, normal progenitor cells and inhibiting enhanced angiogenesis. In addition, SDF or its complex with CP is capable of ex vivo expanding normal stem and progenitor cells. The invention also relates to pharmaceutical composition comprising as active ingredient SDF or its complex and optionally further comprising pharmaceutically acceptable additives. Such pharmaceutical compositions may be for inhibiting enhanced angiogenesis, for inducing remission of tumors, for maintaining tumor remission, and for expanding hematopoietic normal stem and progenitor bone marrow transplants.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a biologically active serum-derived composition of matter (SDF), having low molecular weight, being electrically charged at acidic pH, and having absorption at 280 nm. The molecular weight of SDF being 316, as determined by electron spray mass spectrometry.

More specifically, SDF is comprised of an aromatic moiety, and carries a double charge, as indicated by the fragmentation thereof on electron spray mass spectrometry.

The invention also relates to a method for the isolation and purification from plasma, of a low molecular weight composition of matter, comprising the steps of:--(a) transferring plasma through an affinity column to give an electrophoretically homogeneous fraction being the SDF-CP complex, which may optionally be concentrated by transferring through an anion exchange column and collecting the bound fraction, having an absorption at 280 nm; (b) isolating the SDF from its complex with the high MW CP protein by either (i) transferring the fraction obtained in step (a) through RP-HPLC "Resource".TM. column, elution buffer A, consisting of 0.05-0.1% TFA in water (pH 2.5), and elution buffer B, consisting of acetonitrile, are employed, the active fractions eluted at acetonitrile concentration of 0-2% and 13-17% being collected and combined, said CP fraction being eluted at acetonitrile concentration of about 40% and being devoid of activity; or (ii) extracting the fraction obtained in step (a) with an acidified solvent, wherein the active fraction is recovered from the organic phase whereas CP is recovered from the aqueous phase, in an inactive form. As will be shown hereafter, the two fractions obtained in step (b) were active. The difference in the retention time may be attributed to the state of ionization of the molecule and/or its association with some impurities, probably of small peptides (FIG. 2). The affinity chromatography column employed in step (a) is preferably a tentacle-agarose gel, derived using a reaction of Sepharose CL-6B or Sepharose 4B with chloroethylamine [Calabrese, L., Biochem. Int. 16:199-208 (1988)].

Another purification step is required in order to isolate the SDF from its impurities. The method of the invention thus further comprises (c) purifying the active fraction obtained in step (b) by RP-HPLC chromatography separation, using a C 18 column, wherein in a first optional separation step, elution buffer A, consisting of 0.1% TFA in water (pH 2.5), and elution buffer B, consisting of 0.1% TFA in acetonitrile, are employed, the fraction eluted at acetonitrile concentration of 0-2% being collected (void). This purification step at acidic pH (2.5) results in a partial disassociation of SDF from said impurities. The active fraction is eluted in correlation with the void volume, while most of the impurities are eluted with the gradient. The semi-purified SDF (in case the optional purification at acidic pH is employed) is then subjected to a subsequent separation step, by transferring the same through the same column, employing elution buffer A, consisting of 0.1% triethylamine in water, adjusted to pH 7.0, and elution buffer B, consisting of acetonitrile. The fraction appearing as a single, symmetrical peak, is eluted with acetonitrile concentration of 9-11% and collected. Optionally, the fraction obtained from step (b) is directly separated on the second separation step of (c).

A five-step purification procedure may be employed as an alternative to step (a). This five-step procedure comprises the steps of ammonium sulfate precipitation, anion exchange chromatography (DEAE), cation exchange chromatography (S-Sepharose), dye-ligand (Affigel blue) chromatography and hydrophobic chromatography (TSK-Phenil). Following the last purification step, the purified fraction may be further separated on an SDS-gel.

According to the invention the plasma from which SDF is obtained is human plasma. However, the inventors have found that an active fraction is also present in non-human plasma, in human urine and in bovine fetal serum. Since the full CP protein is too large to be present in the urine, it is assumed that the active fraction present therein is the SDF itself or in association with part of the CP protein or with other small peptides. In view of the above, SDF may be isolated and purified from human or non-human adult or fetal serum or urine, by any suitable method.

The invention also relates to a biologically active complex comprising CP and SDF.

Obviously, any biochemically pure SDF obtained by the method according to the invention are also within the scope of the present invention. The complex of SDF with CP can be obtained, for example, from step (v) of the five-step purification (Example 1B) procedure or after the one step affinity purification procedure of the method of the invention.

As shown in the description hereafter, the present inventors have found that normal serum, which sustains the growth and viability of cells in culture, contains a small molecular weight, natural product that exhibits dual activity on hematopoietic cells: on the one hand, it is extremely potent in stimulating development of a variety of normal blood cells, and, on the other hand, it inhibits leukemic cell growth by inducing terminal differentiation. Since this natural product was purified from serum it was termed "serum-derived factor" (SDF).

Effect of SDF on Normal Hemopoiesis

Use of Hemopoietic Growth Factors in Transplantation

Transplantation of hemopoietic cells, originally obtained from either autologous or allogeneic sources, has become the treatment of choice for a variety of inherited or malignant diseases. Recently, more defined populations, enriched for pluripotent hemopoietic stem cells (CD34+ cells) have been used in such treatments [Van Epps, D.E., et al., Blood Cells 20:411 (1994)]. In addition to bone marrow, stem cells could be derived from other sources, such as peripheral blood and neonatal umbilical cord blood [Emerson, S. G., Blood 87:3082 (1996)]. Compared to autologous bone marrow transplantation, transplantation with peripheral blood cells shortens the period of pancytopenia and reduces the risks of infection and bleeding [Brugger, W., et al., N. Engl. J. Med. 333:283 (1995); Williams, S. F., et al., Blood 87:1687 (1996); Zimmerman, R. M., et al., J. Heamatotherapy 5:247 (1996)]. An additional advantage of using peripheral blood for transplantation is its accessibility. However, the limiting factor in peripheral blood transplantation is the low number of circulating CD34+ cells. Therefore, in order to obtain enough cells for transplantation, peripheral blood derived stem cells are "harvested" by repeated leukophoreses, following their mobilization from the marrow into the circulation after treatment with colony stimulating factors and chemotherapy [Brugger et al. (1995), ibid.; Williams et al. (1996) ibid.].

Preliminary attempts have been made to enrich the CD34+ population by ex vivo expansion in tissue culture containing mixtures of growth factors [Koller, M. R., et al., Blood 82:378 (1993); Lebkowski, J. S., et al., Blood Cells 20:404 (1994)]. Such expansion of functional stem cells from a small number of CD34+ cells may have the following advantages:

It may reduce the volume of blood required for reconstitution of an adult hemopoietic system and may obviate the need for mobilization and leukophoresis [Brugger et al. (1995) ibid.].

It may enable storage of small number of peripheral blood, bone marrow or cord blood CD34+ cells for potential future use of the ex vivo expanded population.

In the case of autologous transplantation in patients with malignancies, decreasing the total volume of blood used and selecting CD34+ cells may reduce the load of tumor cells in the final transplant. Such contaminating tumor cells in autologous infusion can contribute to the recurrence of the disease [Brugger et al. (1995) ibid.].

The cultures may provide a significant depletion of T-lymphocytes, which may be useful in the allogeneic transplant setting for reducing graft-versus-host disease.

Clinical studies have indicated that transplantation of ex vivo expanded cells derived from a small number of peripheral blood CD34+ cells can restore hemopoiesis in patients treated with high doses of chemotherapeutical agents. Nevertheless, the up to date results do not allow for firm conclusions about the long term in vivo hemopoietic capabilities of such cultured cells [Brugger et al. (1995) ibid.: Williams et al. (1996) ibid.].

For successful transplantation, shortening the duration of the cytopenic phase, as well as long-term engraftment, is crucial. Inclusion of intermediate and late progenitor cells in the transplant could accelerate the production of donor-derived mature cells and shorten the cytopenic phase. It is important that ex vivo expanded cells will include, in addition to stem cells, more differentiated progenitors in order to optimize short-term recovery and long term restoration of hemopoiesis. For this purpose, expansion of intermediate and late progenitor cells, especially those committed to the neutrophilic and megakaryocytic lineages, concomitant with expansion of stem cells, is required [Sandstrom, C. E., et al., Blood 6:958 (1995)].

Regarding the autologous transplantation in patients with malignancies, it should be noted that different growth factors. e.g. G-CSF and GM-CSF. are currently used in BM transplantation. They have been shown to shorten the time of neutrophil recovery after transplantation (and chemotherapy), by stimulating myeloid progenitors. However, since myeloid leukemic cells have receptors for these factors, the proliferation of residual malignant cells is also stimulated. Since SDF itself, or its complex with CP, alone, or in combination with GM-CSF, potentiates the proliferation of normal progenitors, but inhibit "spontaneous" and GM-CSF stimulated proliferation of myeloid leukemic cells (Examples 2 to 6), it may have a dual effect: eradication of leukemic cells concomitantly with stimulation of the normal ones.

As exemplified hereafter, leukemic cells, treated with SDF lose their proliferation ability which indicates the cells have lost their leukemogenic potential. Therefore, it is believed that SDF may be of great therapeutical value.

Further, SDF or its complex with CP, is capable of stimulating proliferation of early, normal progenitor cells. Therefore SDF or its complex with CP may be used for ex vivo expansion of normal hematopoietic cells such as stem cells (CD34+) and myeloid and erythroid-committed progenitors as well as antigen-presenting dendritic cells. for BM transplantation treatment, or be utilized, for ex-vivo expansion of specific sub-populations that should be valuable in cell therapy (transplantation. and immuno- or gene therapy). In addition SDF or its complex with CP can be applied in vivo, where they may support the recovery of the hemopoietic tissue in aplastic states such as in aplastic anemia or following radio/chemotherapy.

In addition, although effective in inducing differentiation and inhibiting proliferation of leukemic cells, it was found by the inventors that neither SDF nor its complex with CP inhibit normal myeloid or erythroid development. Moreover, SDF or its complex with CP, alone or in combination with other growth or proliferation factors, was found to stimulate the proliferation of normal early progenitor cells. For example:

(a) In vitro stimulation of early hemopoietic stem and committed progenitor cells.

SDF was found to stimulate the amplification of early stem (CD34+) cells derived from PB or BM and therefore may be applied for ex-vivo expansion of pluripotent stem cells as well as lineage (granulocytic, erythroid and mega-karyocytic) committed progenitor cells. In combination with late growth factors (added to phase 2, described hereafter in the Examples) SDF increases the number of myeloid and erythroid colony forming cells. Such cultures are important in transplantation of CD34+ enriched populations derived from (immobilized) PB and neonatal cord blood and in gene therapy.

(b) In vivo stimulation of early stem and committed progenitor cells. SDF was found to stimulate in vitro proliferation of progenitor cells derived from the PB of patients with pure red cell aplasia. These results suggest that SDF or its complex with CP be administered, for recovery of normal hemopoiesis, to patients with aplastic states, such as aplastic anemia, Fanconi's anemia, myelodysplastic syndrome or following myeloablative radio/chemotherapy and BM transplantation.

(c) Ex vivo expansion of specific populations of subsets of lympho-hematopoietic cells with therapeutic potential such as the antigen presenting dendritic cells. Dendritic cells are "professional", immunostimulatory, antigen-presenting cells. Various studies have suggested the potential use of dendritic cells in immunotherapy. This modality involves infusion of dendritic cells pulsed in vitro with tumor antigens as therapeutic vaccines, as well as using dendritic cells for priming tumor antigen specific T cells in vitro for use in adoptive T cell therapy [Bernhard, H., et al., Cancer Res. 55:1099 (1995); Protti, M. P., et al., Cancer Res. 56:1210, (1996)]. According to the literature, the best "cocktail" for growing such cells is a mixture of cytokines (GM-CSF, SCF, IL-4, TNF.alpha.). When BM cells were cloned in the presence of such a cocktail, 40% of the total number of the developed colonies, contained dendritic cells [Moore, M. A., et al., J. Exp. Med. 182:1111 (1995)]. In order to obtain such colonies from PB progenitors, the cultured population should be enriched for CD34+ cells. SDF or its complex with CP induce commitment/expansion of PB CFU-dendritic. Using SDF or its complex with CP (without TNF or SCF), up to 80% dendritic colonies were obtained from non-enriched PB mononuclear cells.

As will be shown in Example 5, SDF has a potent inhibitory activity on the proliferation of endothelial cells from bovine aorta.

As mentioned above, in addition to BM transplantation, ex vivo expansion of hematopoietic stem cells using SDF or its complex with CP. may be used in gene therapy.

Effect of SDF on Leukemic Hemopoiesis

In addition, SDF or its complex with CP, whether obtained by the method of the invention or by any other suitable method, or synthesized by any suitable procedure, possess several therapeutical activities such as inducing differentiation and inhibiting proliferation of both human and murine established leukemic cell lines and of freshly explanted cells from acute and chronic human myeloid leukemias. In addition, SDF itself or its complex with CP are capable of inducing terminal cell differentiation of leukemic cells. Blast cells lose their leukemic phenotype and turn into functional, non-dividing macrophages. Further, either as a result of said terminal cell differentiation or independent therefrom, said leukemic cells, in the presence of SDF or its complex with CP, lose their ability to proliferate and their ability for self cell renewal. The effect of SDF, or its complex with CP, on leukemic cells makes it potentially useful in the treatment of myeloid leukemias in three clinical settings: (a) for induction of remission, optionally, in combination with other hemopoietic factors or low-dose chemotherapy, using "differentiation-inducing therapy" as the main modality; (b) for maintenance of remission state of tumors and (c) in autologous transplantation, for either in vitro or in vivo purging of residual leukemic cells.

In a different aspect, SDF or its complex with CP, may be utilized for regulating the proliferation and differentiation of hemopoietic cells, by modulating nuclear transcription factors.

Modulating the level of expression of specific genes is a prerequisite for controlling cellular growth and differentiation. Gene expression is controlled by sequence-specific DNA binding proteins (transcription factors) which in certain cases are targets for signal transduction from cell surface receptors. The importance of this process for growth control is emphasized by the finding that several proto-oncogenes, including c-Myc, c-Myb, c-Fos, c-Jun, etc. encode sequence-specific transcription factors [Xanthoudakis, S., EMBO J 11:3323 (1992); Ammendola, R., Eur. J. Biochem. 225:483 (1994)]. Although the activity of these factors can be modulated by phosphorylation, recent evidence has emerged for an additional form of regulation of DNA binding activity which is mediated by changes in reduction- oxidation (redox) status. It is suggested that redox status could provide a general mechanism for post-translational control of transcription factors in an analogous fashion to phosphorylation [Xanthoudakis (1992) ibid.; Ammendola (1994) ibid.]. For example, the binding of Fos-Jun hetero-dimers and Jun-Jun homo-dimers to DNA requires that these proteins be in a reduced state. This form of redox regulation may be widespread because the DNA binding activities of several other transcription factors, including Myb, Rel, and NF-kB are sensitive to changes in their oxidation state in a similar manner.

Recent disclosures suggest the contribution of small redox-potential molecules such as glutathion or large proteins such as thioredoxin [Walker, L. J., Mol. Cell. Biol. 13:5370 (1993)] to the regulation of DNA binding ability of several nuclear transcription factors like Sp-1. In human fibroblast cultures it was shown that small molecules with redox potential like pyrroloquinoline quinone (PQQ) stimulate proliferation [Naito, Y., Life Sciences 52:1909 (1993)]. The potency of PQQ was shown to be comparable to that of epidermal growth factor and is much higher than that of fibroblast growth factor or insulin growth factor. Pyrrolidine derivatives of dithiocarbamates trigger myeloid differentiation through AP-1 regulation [Aragones, J., J. Biol. Chem. 271:10924 (1996)]. Therefore, it may be concluded that small molecules with redox potential activity could modulate cell proliferation and differentiation via the regulation of transcription factors activity.

It was found by the inventors, that PQQ at high concentrations can induce differentiation of leukemic cells and stimulate proliferation of normal cells (Example 6). Nevertheless, in view of the high concentrations required, and compared to SDS, the efficiency of PQQ is low. Nevertheless. since SDF is a low molecular weight composition of matter, believed to be carrying a double negative charge, and in view of its potency in inducing differentiation. it is anticipated that, similarly to PQQ, SDF possesses a redox potential activity and consequently might modulates cell proliferation and differentiation via the regulation of transcription factors activity.

Effect of SDF on Angiogenesis

In a different aspect, SDF was found to have a potent inhibitory activity on endothelial cell proliferation, and therefore it might be applicable for inhibiting angiogenesis In certain pathological conditions angiogenesis is dramatically enhanced and is no longer self-limited. Pathological angiogenesis is seen during the development of many diseases, for example rheumatoid arthritis, psoriasis, retrolental fibroplasia, diabetic retinopathy and hemangiomas, during the rejection of organ transplants and most importantly in solid tumor malignancies. Well vascularized tumors expand both locally and by metastasis, while avascular tumors do not grow beyond a diameter of 1-2 mm. It has been suggested that this is the results of lack of balance between angiogeneic stimulators and inhibitors [Folkman, J., New Engl. J. Med. 285:1182-1186 (1971); Folkman, J., J. Natl. Cancer Inst. 82: 4-6 (1989)].

In another aspect, pharmaceutical composition comprising as active ingredient SDF or its complex with CP and optionally farther comprising pharmaceutically acceptable additives are within the scope of the invention. Such pharmaceutical compositions may be used for inhibiting enhanced angiogenesis in diseases where uncontrolled angiogenesis is associated with the pathological manifestations.

Other pharmaceutical composition of the invention may be for inducing remission of tumors comprising as active ingredient the SDF of the invention, and optionally further comprising pharmaceutically acceptable additives.

Alternatively, the pharmaceutical composition of the invention can be used for maintaining tumor remission state comprising as active ingredient the SDF of the invention, and optionally farther comprising pharmaceutically acceptable additives.

Further, pharmaceutical composition for expanding hematopoietic normal stem and progenitor bone marrow transplants comprising as active ingredient SDF, and optionally further comprising pharmaceutically acceptable additives are also with in the scope of the invention.

The magnitude of therapeutic dose of the SDF on the invention will of course vary with the group of patients (age, sex etc.), the nature of the condition to be treated and with the route administration and will be determined by the attending physician.

The pharmaceutical composition of the invention can be prepared in dosage units forms. The dosage forms may also include sustained release devices. The compositions may be prepared by any of the methods well-known in the art of pharmacy.

In the pharmaceutical compositions of the pharmaceutically acceptable additives may be any pharmaceutical acceptable carrier, excipient or stabilizer, and optionally other therapeutic constituents. Naturally, the acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed.

Finally, within the scope of the invention, is the use of SDF or its complex with CP, in the preparation of a variety of pharmaceutical compositions.

Claim 1 of 7 Claims

What is claimed is:

1. A biologically active serum-derived composition of matter (SDF), having a molecular weight of 316, as determined by electron spray mass spectrometry being electrically charged at acidic pH and having absorption at 280 nm.

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