WO2006094402A1 - Novel hemoglobin scavenger receptor, cd163, and method of increasing cd163 expression on human stem cells and stimulation of erythroid progenitors - Google Patents

Abstract

A novel CD163 hemoglobin scavenger receptor is described. Methods and compositions for stimulating the growth, proliferation, differentiation and/or mobilization of stem and/or progenitor cells are also described. The method involves administering an effective amount of a substance which can activate the CD163 hemoglobin scavenger receptor signal transduction pathway. The methods can be used in cell culture, to obtain a source of CD163 receptor containing cells, assays, assays for identifying CD163 receptor ligands or modulators. The methods and compositions are useful in stimulating hematopoiesis and in treating a wide range of conditions including cytopenias, anemias and for use in preparing cells for transplantation.

Description

Title: NOVEL HEMOGLOBIN SCAVENGER RECEPTOR, CD163, AND METHOD OF INCREASING CD163 EXPRESSION ON HUMAN STEM CELLS AND STIMULATION OF ERYTHROID PROGENITORS

FIELD OF THE INVENTION

The present invention relates to a novel CD163 which is present on erythroid and stem cells but not on myeloid cells. The present invention also relates to methods and compositions for stimulating the growth, proliferation, differentiation and/or mobilization of stem cells and erythroid progenitors leading to the production of blood cells by increasing CD163 expression on human stem cells. BACKGROUND OF THE INVENTION A hemoglobin scavenger receptor has recently been identified on monocytes and macrophages (Kristiansen, 2001). This receptor scavenges hemoglobin by mediating endocytosis of haptoglobin-hemoglobin complexes. This receptor has also been identified as M130/CD163, an acute phase-regulated transmembrane protein that has been reported to be expressed exclusively on monocytes and macrophages. CD163 belongs to the group B scavenger receptor cysteine-rich superfamily, a family of receptors that includes CD5, CD6 and WC1 which are present on B, T and CD4^'8^" γδ T lymphocytes, respectively. Complexes of hemoglobin and multimeric haptoglobin exhibit higher functional affinity for CD163 than do complexes of hemoglobin and dimeric haptoglobin.

Human CD163 is composed of 17 exons and 16 introns and each of its' nine SRCR domains is encoded by a separate exon (Ritter et al, 1999). Two cytoplasmic variants have been described, both arising from the alternative splicing of intron 15. Similar isoforms has been described for CD6, another scavenger receptor (Robinson et al, 1995; Bowen et al, 1997) and it has been suggested that alternative splicing of the intracellular domains could modulate the signalling capacity of receptor proteins. However, it remains to be determined if the 3 intracellular variants of CD163 demonstrate a functional difference (Kristansen et al, 2001). For example SUDHL, a human myelomonocytic cells line, has been shown to express all three variants simultaneously (Law et al, 1993; Pulford et al, 1997). An extracellular variant has also been described which arises from the alternative splicing of intron 7 and a truncated form results from the alternative splicing of intron 5. The truncated form lacks a transmembrane domain and therefore may be secreted from the cell (Law et al, 1993) although the presence of a corresponding protein has yet to be demonstrated. A soluble plasma CD163 form, corresponding in electrophoretic mobility to the extracellular domain has been identified (Moller et al, 2002). Droste et al (1999) demonstrated a "shedding" of the CD163 with phorbol ester induction of monocytes. This is thought to be the result of cleavage of the membrane-bound form.

The mechanism which dictates which isoform of the CD163 receptor is expressed by various cells is unknown, however, there is evidence that tissue-specific isoforms of scavenger receptors exist. For example, M 160, a novel scavenger receptor recently described by Gronlund et al (2000), is a member of the group B SRCR superfamily. M160 contains twelve extracellular domains, a transmembrane domain and a cytoplasmic domain. It has been found to occur in two forms, a predominant form and an alternatively spliced variant. The predominant form M160-specific RNA was found in a variety of tissues while the splice variant was limited to spleen tissue.

Previous studies of antibody-mediated crosslinking of CD163 on cultured monocytes have demonstrated that ligation of surface CD163 induces tyrosine kinase - dependent signals resulting in the mobilization of intracellular calcium, inositol triphosphate production and increased secretion of anti-inflammatory cytokines, including interleukin 6 (IL-6) and granulocyte- macrophage colony stimulating factor (GM-CSF) (van den Heuvel et al, 1999). Hematopoiesis is defined as the production and development of blood cells, including erythrocytes, granulocytes, monocytes, macrophages, esoinophils, basophils, megakaryocytes, B cells and T cells (Wintrobe, 1999). Hematopoiesis occurs as the result of the proliferation and differentiation of hematopoietic stem cells. Hematopoietic stem cells are pluripotent cells which can give rise to the multiple cell lineages found in the blood. Hematopoietic stem cells reside in the bone marrow and their growth, proliferation and differentiation are influenced by both hematopoietic growth factors and the stromal cells within the bone marrow. Stem cells are believed to normally reside in a quiescent nondividing state until stimulated by specific growth factors whereupon they divide and give rise to highly proliferative progenitor cells committed to the production of blood cells of one or more lineages, such as the erythroid, myeloid or lymphoid lineages.

Certain clinical disorders, termed cytopenias, are characterized by the decreased level of a specific cell type in the circulating blood. For example neutropenia is a disorder whereby there is a diminished level of circulating neutrophils. This disorder can be treated by GM-CSF or G-CSF, two different hematopoietic growth factors. However, administration of these growth factors is often associated with a high incidence of adverse side effects. For example, the administration of G-CSF after allogeneic bone marrow transplantation may result in dyspnea, chest pain, nausea, hypoxemia, diaphoresis, anaphylaxis, syncope and flushing (Khoury et al, 2000).

Neutropenia is also associated with AIDS and is currently treated with growth factors (Dubreuil-Lemaire et al, 2000). There are also forms of severe congenital neutropenia (Dale et al, 2000) in which a small percentage of the patients are refractory to the administration of growth factors.

Anemia is the pathological consequence of insufficient hemoglobin to meet the oxygen transport requirements of the body. Historically, certain anemias have been treated with blood or red blood cell transfusions. A variety of complications associated with transfusions makes this treatment undesirable, including hemolytic, febrile and allergic reactions, along with the potential of the transmission of disease. Stimulating the growth and development of erythroid cells (erythropoiesis) is desirable in the W

- A - treatment of anemia. There are several causes of anemia, which include excessive blood loss, increased red blood cell destruction, decreased synthesis of red blood cells and abnormal production of hemoglobin. Decreased red blood cell production may result from an iron deficiency (either 5 dietary, maladsorption from the gastrointestinal tract, ineffective iron transport or iron utilization by developing red cells), insufficient erythropoietin (Epo) production (kidney dysfunction) or bone marrow failure. Since the erythropoietic activity of the bone marrow is intact in iron and Epo-dependent anemias, such anemias are amenable to iron or Epo therapy, respectively. 0 Anemia due to iron-deficiencies is typically treated by the oral or intravenous administration of iron. Patients with chronic renal failure typically suffer from Epo-dependent anemias due to the inability of the kidneys to produce Epo. These patients undergo dialysis and 90% are clinically anemic. The traditional treatment for anemia in dialysis patients consisting of multiple 5 blood transfusions has largely been replaced by the administration of Epo. Indeed, ~88% of all dialysis patients are treated with Epo. One third of patients on Epo therapy develop hypertension, which can generally be corrected using anti-hypertensive drugs. Erythroid progenitors are stimulated by Epo to differentiate into mature red blood cells and synthesize hemoglobin, 0 the main red blood cell protein.

A major limiting factor of Epo therapy is the cost of long term treatment. Typical Epo doses for patients with chronic renal failure are 225 Units/kg/week administered in three doses. Medicare reimbursement for Epo treatment in the U.S. is $10.00 per 1 ,000 Units, thus the typical cost for a 70 5 kg patient would be ~$8,000 yearly. In 1995, 175,000 US patients were on dialysis resulting in a market in excess of $883 million for this indication alone. Costs for this therapy are estimated to be ~$1.1 billion for 1996. Novel therapies which would reduce Epo requirements for the treatment of anemia would thus be beneficial to the patient and to the healthcare system. The 0 discovery of other agents capable of reducing Epo requirements for the treatment of Epo-dependent anemias would be advantageous. Furthermore, there are a variety of anemias which do not respond to Epo therapy. Examples of these types of anemia include chemotherapy-induced anemia and anemia of chronic disease, including malignancies. Patients with acquired immunodeficiency can also suffer from anemia, as do AIDS patients being treated with AZT. These types of anemia may be due to ineffective erythropoiesis as a result of either suppressed Epo production or a decreased response of the bone marrow to Epo. Treatment of these types of anemia involves treatment of the primary disorder; however, if the primary disorder cannot be readily treated, then the therapy for the anemia can include red blood cell transfusions. Adverse side-effects of transfusions include acute and delayed hemolytic reactions and the potential of transfusion of transmittable diseases.

Haemoglobin is one of the most abundant proteins of the human body serving a vital function in oxygen transport. Virtually every cell of the body has the potential to come into contact with haemoglobin either as a result of the natural turnover of senescent red blood cells, or as a result of a number pathological conditions causing haemolysis. It is, therefore, not unexpected that haemoglobin would have other important and wide ranging effects. Several of these are known, including the ability to bind the important regulatory molecule nitric oxide with high affinity (Gow et al, 1998), or to form potentially toxic reactive oxygen species (Winterbourn et al). Vasoactive peptides derived from the globin proteins chains are also believed to play an important role in the systemic response to cell-free haemoglobin (Lantz et al, 1991). The acute phase plasma protein haptoglobin is thought to be involved in promoting the clearance of circulating hemoglobin through the formation of a high affinity complex (Wada et al, 1970; Delanghe et al, 2002). The recent discovery that CD163 is the monocyte/macrophage scavenger receptor for haemoglobin/haptoglobin (Hb-Hp) complexes (Kristiansen, 2001) suggests a potential role for haemoglobin in the modulation of the inflammatory and immune responses (Gordon, 2001; Beuchler et al, 2002; Schaer, 2002). The additional finding that a membrane-derived, soluble form of the CD163 receptor suppresses T lymphocyte activation (Hogger et al, 2001 ; Frings et al, 2002; Moller et al, 2002) suggests that haemoglobin could play a central role in host defense.

Previous observations indicate that haemoglobin infusion into humans or rodents stimulates erythropoiesis (Amberson, 1937; Okamura et al, 1971; Feola et al, 1992; Hughes et al). However, the exact mechanism by which haemoglobin stimulates erythropoiesis is still not known. It has been suggested that the effect of haemoglobin on erythropoiesis may be mediated by its constituent haeme or iron, or via an increase in erythropoietin production; (Okamura et al, 1971; Brown et al, 1963) however, it has not been definitively established that these mechanisms are the only stimulatory activity of haemoglobin. Haemoglobin could also exert direct effects on erythroid progenitors. We have previously shown that highly purified haemoglobin increases the growth and differentiation of cultured hematopoietic progenitors (Mueller et al, 1997). Most notably, erythroid colonies were larger and "redder" in the presence of haemoglobin.

In view of the foregoing, there is a need in the art to develop improved methods for treating anemia in a patient through the stimulation of erythropoiesis. There is also a need in the art to develop improved methods for increasing the number of blood cells in a patient through the stimulation of hematopoiesis and/or erythropoiesis.

SUMMARY OF THE INVENTION

The present inventors have demonstrated that the CD163 expressed by primary erythroid cells is distinct from the CD163 expressed by monocytes and macrophages. The novel CD163 is a splice variant that varies in the intracellular region of CD163. The inventors have also shown that the novel receptor is present on CD34⁺ stem cells.

Accordingly, the present invention provides a novel CD163 receptor that is expressed on erythroid cells or CD34⁺ stem cells and has a molecular weight of approximately 135 kD. The receptor binds to the anti- CD163 antibody Mac-158 but not EdHu-1 on a Western blot. The present inventors have also found that primitive hematopoietic progenitors express CD163 thereby providing a mechanism whereby haemoglobin can exert direct stimulatory effects. CD163 has only previously been described on monocytes/macrophages (Morganelli et al, 1988; Pulford et al, 1998). Receptor occupation by cross-linking monoclonal antibodies activates a signal transduction pathway involving protein phosphorylation, intracellular inositol triphosphate production, Ca²⁺ mobilisation and the release of pro-inflammatory cytokines such as IL-6, IL-1β and GM-CSF (Van den Heuvel et al, 1999; Ritter et al, 2001). Interestingly, all of these cytokines have well-known haematopoietic activity. In the present study, activating anti- CD 163 monoclonal antibodies were found to mimic the pro-erythropoietic effects of haemoglobin on purified hematopoietic progenitors in vitro. These data support a role for haemoglobin in the receptor-mediated stimulation of hematopoietic stem cells (HSCs), and possibly the formation of blood cells. It is not entirely surprising that the presence of cell-free haemoglobin could provide a direct stimulus for increased red blood cell production.

The present inventors have determined that activation of the hemoglobin scavenger receptor, CD163, can stimulate the growth, proliferation and differentiation of erythroid progenitors, leading to increased blood cell production. The inventors have also demonstrated that CD163 is expressed by CD34⁺ hematopoietic stem cells.

Accordingly, the present invention provides a method of stimulating the growth, proliferation, differentiation and/or mobilization of a stem cell capable of expressing the CD163 receptor, or responding to the signal transduction pathway stimulated by the receptor, comprising administering an effective amount of a substance that can activate CD163 on the stem cell, to a cell or an animal in need thereof.

The present invention further provides a method of modulating, preferably stimulating, erythropoiesis comprising administering an effective amount of a substance that can activate CD163 to a cell or an animal in need thereof. The present invention also provides a method of modulating, preferably stimulating, hematopoiesis comprising administering an effective amount of a substance that can activate CD163 to a cell or an animal in need thereof. The present invention also includes pharmaceutical compositions comprising an effective amount of a substance that can activate CD163 in admixture with a suitable diluent or carrier.

The present invention further provides a cell culture additive useful for enhancing growth, proliferation, differentiation and/or mobilization of erythroid progenitor or stem cells comprising an effective amount of a substance that can activate CD 163.

The present invention also provides a method of selecting erythroid progenitor or stem cells in a sample comprising (a) contacting the sample with a substance that can bind the novel CD 163 and (b) selecting cells that are bound to the substance.

The present invention provides a method of delivering a substance to a stem cell or an erythroid cell comprising administering an effective amount of a conjugate comprising the substance coupled to a CD163 receptor ligand to an animal or cell in need thereof. In another embodiment, the invention provides a method for identifying CD163 receptor ligands. In one embodiment, it provides a method for identifying ligands of the novel CD163 receptor of the present invention. In another embodiment, it provides a method of identifying modulators of the CD163 receptor mediated pathways. The present invention also provides a method of increasing

CD163, preferably novel CD163, expression on human stem cells (HSCs) comprising administering to said stem cells or culturing said stem cells in an effective amount of EPO, IL-3, and one or more of the following factors: FeCb, haemin, or hemoglobin. In another embodiment, the present invention provides a method of stimulating erythroid progenitors comprising administering to said progenitors an effective amount of EPO, IL-3, and one or more of the following factors: FeCb, haemin, or hemoglobin.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art of this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings in which:

Figure 1 Western blot of CD34⁺ cells. Lysates from 1 x 10⁵ cells, CD34⁺ cells or SU-DHL-1 cells were electrophorosed through a polyacrylamide gel under non-reducing conditions and electroblotted onto nylon membranes. Membranes were probed with an anti-CD163 antibody (Mac-2-158) or an isotype control antibody (IgGi; not shown) as described in Materials and Methods. Antibody reactive proteins were visualized using enhanced chemiluminescence (ECL) detection. Lane 1: SU-DHL-1 cells, lane 2: empty, lane 3: ABM CD34⁺ cells.

Figure 2 is a Western blot showing that the CD 163 expressed on erythroid cells is distinct from that expressed by differentiated U937 cells of the monocytic/macrophage lineage. CD163 immunoreactivity in cell lysates prepared from erythroid progenitor colonies. Lysates from pooled BFU-E colonies were electrophoresed under non-reducing conditions through a polyacrylamide gel and electroblotted onto nylon membranes. Membranes were probed with either an anti-CD 163 specific antibody (Mac-2-158) or an isotype control antibody (IgGi). Antibody reactive proteins were visualized using enhanced chemiluminescence (ECL) detection. Lane 1 : Cell lysate from CFA with 0.5 U Epo/ml. Lane 2: Cell lysate from CFA with 2.0 U Epo/ml. Lane 3: U937 cell lysate..

Figure 3 illustrates the RT-PCR amplification of an ~ cDNA fragment of CD163 from ABM CD34⁺ cell mRNA. RT-PCR was conducted on mRNA isolated from ABM-derived CD34⁺ cells using CD163-specific primers. Five microlitres of product were electrophoresed through a 1% agarose gel in a

Tris-acetate buffer with 0.5 μg/ml ethidium bromide and the gel photographed using a UV light and the Kodak EDAS 290 system. Lane 1 : product from PCR reaction lacking cDNA template; lane 2: product from PCR reaction with

CD34⁺ cell cDNA as template; lane 3 (MWM): molecular weight markers (0.1 kb to 10 kb).

Figure 4 Relationship between K11 and other known human CD163 species. In Figure 4A solid bars show regions of sequence homology with gaps indicating deleted regions of CD163 cDNA. Amino acid residues corresponding to the deleted region of the K11 variant of CD163 are indicated by the single letter abbreviation. Figure 4B illustrates splice donor and acceptor sequences utilized by human CD163 variants (SEQ ID NOs. 1-2). Alternative sequences are underlined.

Figure 5 illustrates the splice donor and acceptor sequences utilized by human CD163 variants: predominant (A), variant 1 (B), variant 2 (D) and K11 CD163 (C). (SEQ ID NOs. 3-6) Alternative CD163 splice sequences are underlined.

Figure 6 CD163 is expressed on a subpopulation of CD34⁺ cells. In Figure 6A, ABM and UCB CD34⁺ cells were incubated with fluorescently labelled anti-CD163 antibodies (X-axis, fluorescein isothiocyanate (FITC) labelled) and anti-CD34 antibodies (Y-axis, phycoerythrin (PE) labelled) and analyzed by flow cytometry as described in Materials and Methods. Data are presented as dot plots . Cells were stained with isotypic antibodies (mouse IgGrFITC and IgGi-PE) as negative controls. In Figure 6B, the mean percentage of cells expressing extracellular CD163. ABM and UCB CD34⁺ cells were co-incubated with fluorescently labelled antibodies to CD34 and CD45, or to CD34 and CD163 and analyzed by flow cytometry as described in Materials and Methods. Data are presented as the percentage of cells that co-stain with both antibodies. The total number of CD34⁺ cells exceeded 97.0 % in all samples.

Figure 7 CD163 is present in the majority of CD34⁺ cells. ABM and UCB CD34⁺ cells were permeabilized and incubated with fluorescently labelled anti- CD163 or control (mouse IgGi) antibodies and analyzed by flow cytometry as described in Materials and Methods. Figure 7A Frequency histogram of cell number on the y-axis versus fluorescence intensity on a logarithmic scale on the x-axis. The erythroid leukemic K562 cell line did not stain positive for intracellular CD163 and served as a negative control cell (not shown). Figure 7B Mean percentage of ABM and UCB CD34⁺ cells positive for the presence of intracellular CD163.

Figure 8 illustrates the stimulation of erythroid colonies by activating anti-CD163 antibodies.

DETAILED DESCRIPTION OF THE INVENTION I. Novel CD163

The present inventors have demonstrated that the CD 163 expressed by primary erythroid cells is distinct from the CD163 expressed by monocytes and macrophages. The novel CD163 is a splice variant that varies in the intracellular region of CD163. The inventors have further shown that the novel CD 163 receptor is present on CD34⁺ stem cells.

It is herein demonstrated that novel CD 163 is expressed on the surface of a subpopulation of hematopoietic stem/progenitor cells. Flow cytometric analysis and indicates that 1.9 + 1.3 % (± SD, n =16) of adult bone marrow and 2.0 ± 1.8 % (n= 8) of umbilical cord blood CD34⁺ cells express cell surface CD163, and 69.1 ± 16.9 % (n=9) and 79.7 ± 22.4 % (n=6) of the respective cells contain the CD 163 protein intracellular^. This expression pattern is similar to that described in the initial characterization of CD163 expression on monocytes/macrophages (Pulford et al, 1992). The presence of CD163 in cell lysates was confirmed by Western blot analysis. RT-PCR and RACE-PCR of CD163-specific cDNA from CD34⁺-derived RNA identified two known CD163-specific transcripts (Law et al, 1993) with sequence differences in the 3' cytoplasmic domain. In addition, a new variant (K11) was identified in CD34⁺ cells with a minor deletion at the start of exon 15.

Accordingly, the present invention provides a novel CD 163 receptor that is expressed on erythroid cells or stem cells and has a molecular weight of approximately 135 kD as determined by Western blot. The receptor binds to the anti-CD163 antibody Mac-158 but not EdHu-1 on a Western blot.

The term "novel CD163" as used herein means a hemoglobin scavenger receptor, CD 163, or a CD 163 like-receptor, that is present on erythroid cells and on CD34⁺ stem cells but is distinct from the known CD163 present on monocytes and macrophages as described by Kristiansen (2001). The novel CD163 may result from alternate splicing or differential glycosylation of the monocyte/macrophage CD163 gene.

In one embodiment, the novel CD163 receptor (variant K11) is present on erythroid cells. In another embodiment it has a molecular weight of approximately 135 kD. In yet another embodiment, the CD163 receptor has a deletion at the start of exon 15. In one embodiment the deletion comprises a phosphorylation region and a sequence supporting efficient receptor endocytosis. K11 mRNA varies in the region encoding the cytoplasmic domain through alternative splicing of intron 15. Clone K11 is created through the combination of an alternative splice donor site with the splice acceptor site of variant 1. This alternative splicing results in a unique deletion of 93 nucleotides (Figure 4), and a loss of 31 amino acid residues from the predicted protein (Figure 5). In yet another embodiment said novel CD163 receptor has a splice donor and acceptor sequence of Figure 4B (SEQ ID NO: 1-2). In another embodiment, the novel CD163 receptor comprises the amino acid sequence of Figure 5C (SEQ ID NO.5) or a functional equivalent thereof. In yet another embodiment, the novel CD163 receptor is present on CD34⁺ stem cells and has a molecular weight of approximately 135 kD. The term "amino acid" as used herein includes all of the naturally occurring amino acids as well as modified amino acids.

The term "functional equivalent" as used herein includes modifications or chemical equivalents of the amino acid sequence of the present invention that perform substantially the same function as the proteins of the invention in substantially the same way. For example, functional equivalents of the invention include, without limitation, conservative amino acid substitutions. Functional equivalents of the invention also include additions and deletions, analogs and derivatives thereof.

A "conservative amino acid substitution", as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties.

The term "derivative", as used herein, refers to an amino acid sequence having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as derivatives are those proteins which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For examples: 4- hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. II. Therapeutic Methods

As mentioned previously, the inventors have shown that a novel CD163 receptor is on erythroid cells and stem cells. Accordingly, modulating the novel receptor may modulate erythropoiesis, as well as the growth, proliferation, differentiation and/or mobilization of stem cells.

Accordingly, in one embodiment, the present invention provides a method of modulating, preferably stimulating, erythropoiesis comprising administering an effective amount of a substance that can activate CD163 to a cell or an animal in need thereof. The present invention also provides a use of an effective amount of a substance that can activate CD163 to modulate, preferably stimulate, erythropoiesis. The present invention further provides a use of an effective amount of a substance that can activate CD 163 to prepare a medicament to modulate, preferably stimulate, erythropoiesis. In another embodiment, the present invention provides a method of modulating, preferably stimulating, the growth, proliferation, differentiation and/or mobilization of a stem cell comprising administering an effective amount of a substance that can activate CD163 on the stem cell to a cell or an animal in need thereof. The present invention also provides a use of an effective amount of a substance that can activate CD163 to modulate, preferably stimulate, the growth, proliferation, differentiation and/or mobilization of a stem cell. The present invention further provides a use of an effective amount of a substance that can activate CD 163 to prepare a medicament to modulate, preferably stimulate, the growth, proliferation, differentiation and/or mobilization of a stem cell.

The phrase "substance that can activate CD163" as used herein includes all substances that can bind, crosslink or ligate the novel CD163 hemoglobin scavenger receptor and result in the modulation of erythropoiesis or stem cell growth, proliferation, differentiation and/or mobilization. The term also includes any substance that can activate a signal transduction pathway that is activated in response to activation of CD163 or a CD163-related receptor. For example, the substance may activate the downstream signal transduction pathways that are activated in response to activation of CD163, including but not limited to tyrosine kinases, calcium mobilization and inositol triphosphate (IP3) and inositol tetrakisphosphate (IP4) mobilization. The phrase "substance that can activate CD 163 pathway" further includes any substance that blocks the inactivation of any signal transduction pathways that are activated in response to activation of CD163 or a CD163-related receptor.

The term "effective amount" as used herein means an amount effective and at dosages and for periods of time necessary to achieve the desired result (e.g., the stimulation of the growth, proliferation, differentiation and/or mobilization of stem cells and/or the stimulation of erythropoiesis).

The term "animal" as used herein includes all members of the animal kingdom and is preferably human. Administering a substance to an animal includes both in vivo and ex vivo administrations. The term "a cell" as used herein includes a single cell as well as a plurality or population of cells. Administering a substance to a cell includes both in vitro and in vivo administrations.

The term "stem cell" as used herein means a cell that is capable of differentiating into any cell including hematopoietic cells in an animal. The stem cell will express or will be capable of expressing the novel CD163 receptor or responding to the signal transduction pathway stimulated by the receptor.

Preferably, the stem cell is a CD34⁺ stem cell. CD34⁺ cells are traditionally considered "stem" cells in that they are capable of both self- renewal and re-populating an individual with cells from all hematopoietic lineages. CD34⁺ cells are further delineated into sub-populations by the co- expression of other markers, such as CD38, and the ability of these sub- populations to re-engraft and repopulate the hematopoietic system (eg., Henon et al., 2001). Stimulating stem cells may facilitate the mobilization of stem cells from extravascular marrow sites to circulating blood which is required for protocols using donor peripheral blood for autologous or heterologous transplantation. Recombinant human G-CSF is widely used for mobilizing CD34⁺ stem cells (for review, see Korbling, 1998). As the inventors have demonstrated the expression of CD163 by CD34⁺ cells, it may be possible that stimulation of the CD163 pathway on said cells will result in their growth and proliferation and possible mobilization to the peripheral blood. Use of CD163 stimulators (for example, a cross-linking antibody) instead of, or in conjunction with reduced amounts of, G-CSF may result in the mobilization of transplantable stem cells without the side effects that may be associated with cytokine administration. The phrase "stimulate the growth, proliferation, differentiation and/or mobilization of a stem cell" as used herein means that the substance can result in an increase in the growth proliferation, differentiation and/or mobilization of a stem cell as compared to the growth, proliferation, differentiation and/or mobilization of a stem cell in the absence of the substance.

Stimulating erythropoiesis is useful in treating anemia. Accordingly, in a specific embodiment, the present invention relates to a method of treating anemia comprising administering an effective amount of a substance that can activate the novel CD163 to a cell or animal in need thereof. The present invention also provides a use of an effective amount of a substance that can activate CD163 to treat anemia. The present invention further provides a use of an effective amount of a substance that can activate CD163 to prepare a medicament to treat anemia.

The phrase "stimulate erythropoiesis" as used herein means that the substance can stimulate or enhance the growth, proliferation, differentiation and/or mobilization of an erythroid cell or an erythroid progenitor cell or a stem cell as compared to the growth, proliferation, differentiation and/or mobilization of an erythroid cell or an erythroid progenitor or a stem cell in the absence of the substance. One skilled in the art can determine whether or not the substance that can activate CD163 can stimulate erythropoiesis. For example, the colony forming assay can be used. As used herein, and as well understood in the art, "to treat" or "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

III. Substances that Activate CD163

The present invention includes the use of any and ail substances that can activate the novel erythroid CD163 (as defined above) in the methods of the invention. The substance can be any type of substance, including but not limited to, proteins (such as antibodies), peptides, nucleic acids, carbohydrates, organic compounds, inorganic compounds, small molecules, drugs, CD163 ligands, soluble forms of the CD163 receptor, any and all CD163 agonists as well as any and all substances that inhibit CD163 antagonists. In a preferred embodiment, the substance that activates CD163 is a substance that binds the CD163 receptor on the stem cell or erythroid cell being treated. Examples of substances that bind CD163 include antibodies and CD163 ligands. (a) Antibodies In a specific embodiment, the substance that can activate

CD163 is an antibody that binds to the novel CD163 receptor. Antibodies to CD163 that bind erythroid CD163 can be from readily available commercial sources for example from Serotec Inc. or Maine Biotechnology Services. Further, one skilled in the art can readily prepare antibodies to CD163 using techniques known in the art such as those described by Kohler and Milstein, Nature 1975, 256: 495 and in U.S. Patent Nos. RE 32,011 ; 4,902,614; W

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4,543,439; and 4,411 ,993, which are incorporated herein by reference. (See also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring 5 Harbor Laboratory Press, 1988, which are also incorporated herein by reference).

For example, by using a peptide of the novel CD163, polyclonal antisera or monoclonal antibodies can be made using standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with an 0 immunogenic form of the peptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art. For example, the protein or peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of 5 antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.

To produce monoclonal antibodies, antibody producing cells 0 (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art, (e.g., the hybridoma technique originally developed by Kohler and Milstein (Nature 256, 495-497 (1975)) as well as other techniques such as the 5 human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy (1985) Allen R. Bliss, Inc., pages 77-96), and screening of combinatorial antibody libraries (Huse et al., Science 246, 1275 (1989)). Hybridoma cells can be screened 0 immunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated. The term "antibody" as used herein is intended to include fragments thereof which also specifically react with the novel CD163, or a peptide thereof. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.

Chimeric antibody derivatives, i.e., antibody molecules that combine a non-human animal variable region and a human constant region are also contemplated within the scope of the invention. Chimeric antibody molecules can include, for example, the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions. Conventional methods may be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes CD163 antigens. (See, for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81 ,6851 (1985); Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom Patent GB 2177096B). It is expected that chimeric antibodies would be less immunogenic in a human subject than the corresponding non- chimeric antibody.

Monoclonal or chimeric antibodies specifically reactive with a protein of the invention as described herein can be further humanized by producing human constant region chimeras, in which parts of the variable regions, particularly the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non- human origin. Such immunoglobulin molecules may be made by techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308- 7312 (1983); Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and PCT Publication WO92/06193 or EP 0239400). Humanized antibodies can also be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.) Specific antibodies, or antibody fragments, reactive against the novel CD163 may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with peptides produced from the nucleic acid molecules encoding CD163 or parts thereof. For example, complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries (See for example Ward et al., Nature 341 , 544-546: (1989); Huse et al., Science 246, 1275-1281 (1989); and McCafferty et al., Nature 348, 552-554 (1990)). Alternatively, a SCID-hu mouse, for example the model developed by Genpharm Inc, can be used to produce antibodies or fragments thereof.

The antibodies of the invention also include bifunctional antibodies comprising an antibody specific for CD163 linked directly to another antibody specific for another antigen on the surface of the stem cell. Bifunctional antibodies may be prepared by chemically coupling one antibody to the other, for example by using N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). The antibodies of the invention also include bispecific antibodies. Bispecific antibodies contain a variable region of an antibody specific for CD163 and a variable region specific for at least one antigen on the surface of the stem cells to be targeted. The bispecific antibodies may be prepared by forming hybrid hybridomas. The hybrid hybridomas may be prepared using the procedures known in the art such as those disclosed in Staerz & Bevan, (1986, PNAS (USA) 83: 1453) and Staerz & Bevan, (1986, Immunology Today, 7:241). Bispecific antibodies may also be constructed by chemical means using procedures such as those described by Staerz et al., (1985, Nature, 314:628) and Perez et al., (1985 Nature 316:354), or by expression of recombinant immunoglobulin gene constructs. (b) Other Substances

In addition to antibodies, other substances that can activate the novel CD163 can also be identified and used in the methods of the invention. For example, substances which can bind CD163 on stem cells or erythroid cells may be identified by reacting CD163 with a substance which potentially binds to CD163, then detecting if complexes between the CD163 and the substance have formed. Substances that bind CD 163 in this assay can be further assessed to determine if they are useful in the methods of the invention.

Accordingly, the present invention also includes a method of identifying substances which can bind to CD163 comprising the steps of:

(a) reacting novel CD 163 and a test substance, under conditions which allow for formation of a complex between the CD163 and the test substance, and

(b) assaying for complexes of CD163 and the test substance, for free substance or for non complexed CD163, wherein the presence of complexes indicates that the test substance is capable of binding CD163.

Conditions which permit the formation of substance and CD163 complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein.

The substance-CD 163 complex, free substance or non- complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof. To facilitate the assay of the components, antibody against CD163 or the substance, or labelled CD163, or a labelled substance may be utilized. The antibodies, CD163, or substances may be labelled with a detectable substance.

The CD163 or the test substance used in the method of the invention may be insolubilized. For example, the CD163 or substance may be bound to a suitable carrier. Examples of suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, silica, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc. The insolubilized CD163 or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.

The CD163 or test substance may also be expressed on the surface of a cell in the above assay. IV. Compositions

The present invention also includes pharmaceutical compositions containing the substances that can activate the novel CD 163 for use in stimulating erythropoiesis or the growth, proliferation, differentiation and/or mobilization of stem cells. Accordingly, the present invention provides a pharmaceutical composition comprising an effective amount of a substance which can activate CD163 in admixture with a suitable diluent or carrier.

For stimulating erythropoiesis, the pharmaceutical composition may additionally contain one or more hematopoietic growth factors such as erythropoietin.

Such pharmaceutical compositions can be for intralesional, intravenous, topical, rectal, parenteral, local, inhalant or subcutaneous, intradermal, intramuscular, intrathecal, transperitoneal, oral, and intracerebral use. The composition can be in liquid, solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets, solutions or suspensions.

The pharmaceutical compositions of the invention can be intended for administration to humans or animals. Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration.

The pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).

On this basis, the pharmaceutical compositions include, albeit not exclusively, the active compound or substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. The pharmaceutical compositions may additionally contain other agents such as other agents that can stimulate erythropoiesis, or can stimulate the growth, proliferation, differentiation and/or mobilization of stem cells and/or progenitor cells.

V. Culture Additives

The substances that activate CD 163 can be used as additives to culture medium for enhancing the growth, proliferation, differentiation and/or mobilization of stem cells, erythroid progenitor cells or for stimulating erythropoiesis. Accordingly, the present invention provides a cell culture additive useful for enhancing growth, proliferation, differentiation and/or mobilization of erythroid progenitor and/or stem cells comprising an effective amount of a substance that can activate CD163. Substances that activate CD163 can be added to a serum-free medium traditionally used for the expression of hematopoietic stem cells. For example, a serum-free medium with the addition of growth factors such as stem cell factor (SCF), interleukin 3 (IL-3), GM-CSF, Fit ligand (FL), thrombopoietin (TPO) and granulocyte-colony stimulating factor (G-CSF) (as described by Kobari et al., 2000) can be supplemented with a substance that can activate CD 163. The inventors have found that a culture comprising EPO, IL-3 and one or more of FeCI3, haemin or hemoglobin activate CD163, preferably novel CD163. Accordingly, in a particular embodiment, the present invention provides a method of increasing CD163 expression on human stem cells (HSCs) comprising administering to said stem cells or culturing said stem cells in an effective amount of EPO, IL-3, and one or more of the following factors: FeCI₃, haemin, or hemoglobin. The present invention also provides a use of an effective amount of EPO, IL-3 and one or more of the following factors: FeCb, haemin, or hemoglobin, to increase CD163 expression on human stem cells (HSCs) or culture of said stem cells. The present invention further provides a use of an effective amount of EPO, IL-3 and one or more of the following factors: FeCb, haemin, or hemoglobin to prepare a medicament to increase CD163 expression on human stem cells (HSCs) or culture of said stem cells.

In one embodiment, the hemoglobin is HbA₀. In another embodiment, the human stem cells are cultured under a 5% CO₂.

In a specific embodiment, the human stem cells are cultured in a media comprising 2 U/ml EPO₁ 10 ng/ml IL-3, and a factor selected from the group consisting of: 250 microM FeCI₃, 100 microM haemin and 1000 microgram/ml hemoglobin. In a particular embodiment, the cells are cultured in the presence of hemoglobin, preferably HbA₀.

In yet another particular embodiment, the invention provides a method of stimulating erythroid progenitors comprising administering to said progenitors an effective amount of EPO, IL-3, and one or more of the following factors: FeCb, haemin, or hemoglobin. The present invention also provides a use of an effective amount of EPO, IL-3 and one or more of the following factors: FeCb, haemin, or hemoglobin, to stimulate erythroid progenitors. The present invention further provides a use of an effective amount of EPO, IL-3 and one or more of the following factors: FeCb, haemin, or hemoglobin to prepare a medicament to stimulate erythroid progenitors.

In one embodiment, the hemoglobin is HbA₀. In another embodiment, the human stem cells are cultured under a 5% CO₂. In a specific embodiment, the cells are cultured in a media comprising 2 U/ml EPO, 10 ng/ml IL-3, and a factor selected from the group consisting of: 250 microM FeCI₃, 100 microM haemin and 1000 microgram/ml hemoglobin. In a particular embodiment, the cells are cultured in the presence of hemoglobin, preferably HbA₀. In another particular embodiment, the factor is haemin. The inventors have shown that activating the CD163 pathway may be useful in stimulating multi-lineage hematopoiesis as they have shown that both erythroid and myeloid progenitor cells can be stimulated by activating CD 163. Stimulating hematopoiesis is useful in generating both blood cells and cells of the immune system including erythrocytes, myeloid cells (such as monocytes, macrophages, eosinophils, neutrophils, basophils and megakaryocytes) and lymphoid cells (B cells, T cells and NK cells), as well as dendritic cells of both myeloid and lymphoid origin.

Accordingly, in yet another particular embodiment, the invention provides a method of stimulating hematopoietic progenitors comprising administering to said progenitors an effective amount of EPO, IL-3, and one or more of the following factors: FeCb, haemin, or hemoglobin. The present invention also provides a use of an effective amount of EPO, IL-3 and one or more of the following factors: FeCI₃, haemin, or hemoglobin, to stimulate hematopoietic progenitors. The present invention further provides a use of an effective amount of EPO, IL-3 and one or more of the following factors: FeCb, haemin, or hemoglobin to prepare a medicament to stimulate hematopoietic progenitors.

The inventors have also shown that activating CD163 increases the proliferation of myeloid cells. The phrase "stimulate myelopoiesis" as used herein means that the substance can stimulate or enhance the growth, proliferation, differentiation and/or mobilization of a myeloid cell or a myeloid progenitor cell or a stem cell as compared to the growth, proliferation, differentiation and/or mobilization of a myeloid cell or a myeloid progenitor cell or a stem cell in the absence of the substance. One skilled in the art can determine whether or not the substance that can activate CD163 can stimulate myeloid cells. For example, the colony forming assay described above and in the Examples can be used.

Accordingly, in yet another particular embodiment, the invention provides a method of stimulating myeloid progenitors comprising administering to said progenitors an effective amount of EPO, IL-3, and one or more of the following factors: FeCb, haemin, or hemoglobin. The present invention also provides a use of an effective amount of EPO, IL-3 and one or more of the following factors: FeCI₃, haemin, or hemoglobin, to stimulate myeloid progenitors. The present invention further provides a use of an effective amount of EPO, IL-3 and one or more of the following factors: FeCI₃, haemin, or hemoglobin to prepare a medicament to stimulate myeloid progenitors.

One skilled in the art can determine whether or not the substance that can activate CD163 can stimulate hematopoiesis. For example, the colony forming assay is a method to quantify hematopoietic stem cells and progenitors (McCulloch, 1984). In the colony forming assay, cells are plated into a semi-solid medium such as methylcellulose in the presence of various cytokines which support hematopoietic progenitor cell growth, survival and differentiation. The types of hematopoietic colonies which form include: burst forming unit - erythroid (BFU-E), colony forming unit - erythroid (CFU-E), colony forming unit - granulocyte macrophage (CFU- GM), colony forming unit - macrophage (CFU-M), colony forming units - megakaryocyte (CFU-Meg) and granulocyte, erythroid, monocyte and megakaryocyte (GEMM) colonies. In semi-solid assays, stem or progenitor cells respond to a variety of cytokines and various combinations of these cytokines have been optimized for the growth and differentiation of erythroid progenitors (for example, IL-3 in combination with EPO) or granulopoeitic/monocyte colonies (for example, IL-1β, IL-6 and SCF). Some combinations are required to enumerate both myeloid and erythroid colonies independently, in addition to the enumeration of more primitive "mixed" colonies (for example, those that include either G-CSF and GM-CSF, for review see Messner; 1991 , 7:18-22). Each stem cell or progenitor cell proliferates and differentiates to form a morphologically distinct colony. The two kinds of functionally distinct erythroid progenitors (BFU-E and CFU-E) are identified based on their abilities to form morphologically recognizable colonies when grown in semi-solid media. The BFU-E represents the most primitive erythroid progenitor and forms large multi-clustered, hemoglobinized colonies. The CFU-E is a more differentiated erythroid progenitor which forms smaller, hemoglobinized colonies. The BFU-E is the earliest identifiable progenitor fully committed to erythropoiesis and has a larger capacity for self-renewal that the more mature CFU-E. To develop, erythroid progenitor colonies typically require the presence of erythropoietin (Epo) in the media. Early on however, primitive erythroid progenitors proliferate in an Epo-independent fashion.

Along with providing a means to quantify hematopoietic stem cells and progenitors, the colony forming assay also provides a means to obtain information about factors affecting the proliferation and differentiation of the progeny of the stem and progenitor cells. For example, erythroid progenitor colonies arise from a single progenitor cell which divides and differentiates such that the mature colony is composed predominantly of hemoglobinized erythroblasts. Morphologically, the size of an erythroid colony may provide information on the rate or extent of proliferation of the progeny of the erythroid progenitor. Also, a redder erythroid colony would suggest a greater hemoglobin content, and possibly a greater degree of differentiation of the erythroblasts. Stimulating the growth, proliferation, differentiation and/or mobilization of myeloid cells can be used to treat neutropenias. Stimulation of myeloid cells through CD163 activation of the CD163 pathway may replace/augment the effectiveness of growth factors in overcoming neutropenias associated with bone marrow transplants with the added benefit of fewer side effects. The method can additionally be used to treat neutropenias associated with AIDS or severe congenital neutropenias. Accordingly, in a specific embodiment, the present invention provides a method of treating a neutropenia comprising administering an effective amount of a substance that can activate CD163 to a cell or animal in need thereof. The present invention also provides a use of an effective amount of a substance that can activate CD163 to treat a neutropenia. The present invention further provides a use of an effective amount of a substance that can activate CD163 to prepare a medicament to treat a neutropenia.

Vl. Cell Enrichment or Detection

As hereinbefore mentioned, the inventors have demonstrated the presence of the novel CD163 receptor on CD34⁺ cells derived from umbilical cord blood or adult bone marrow or peripheral blood. The presence of CD163 on these cells may provide an important research and clinical tool as the CD34⁺/CD163⁺ subpopulation of cells can be further examined for their ability to re-engraft and repopulate the various compartments of the hematopoietic system. In addition, "stem" cells derived from a variety of sources (bone marrow, mobilized peripheral blood or umbilical cord blood) may be enriched for, or sorted by, the expression of CD163.

Accordingly, the present invention provides a method of selecting erythroid progenitor or stem cells in a sample comprising (a) contacting the sample with a substance that can bind the novel CD 163 and (b) selecting cells that are bound to the substance.

In a preferred embodiment, the substance is an antibody that can bind the novel CD163 and the cells are selected using immunochemical techniques. For example erythroid cells expressing the CD163 receptor may be "sorted" or selected by employing an anti-CD163 antibody that does not bind to CD163 on other cell types, by complexing this antibody to a solid support, removing the CD163 positive cells from the other cells in the sample and subsequently removing the CD163 positive cells from the solid support. For example, anti-CD 163 antibodies can be complexed to magnetic beads. CD163 positive cells can be bound to the antibody and CD163 positive cells can be removed from other cells in the sample with a magnetic source. Upon separation from the non-CD163 positive cells, the CD163 positive cells can be detached from the magnetic beads. Alternatively, the expression of CD163 cells on stem cells may by used to sort these cells through fluorescence- activating cell sorting (FACS) by employing an anti-CD163 fluorescently- labelled Many other methods to purify the CD 163 positive cells would be obvious to one skilled in the art. The method can be used to select cells capable of forming colonies of the erythroid lineage or cells that are potentially capable of repopulating organisms with cells of the erythroid lineage. Accordingly, the present invention provides a method to select cells capable of forming colonies of the erythroid lineage comprising (a) contacting the sample with a substance that can bind CD163 on erythroid cells but not other cell types and (b) selecting cells that are bound to the substance, wherein the bound cells are capable of forming colonies of the erythroid lineage. The present invention also provides a method to select cells that are potentially capable of repopulating organisms with cells of the erythroid lineage comprising (a) contacting the sample with a substance that can bind CD163 on erythroid cells but not other cell types and (b) selecting cells that are bound to the substance, wherein the bound cells are potentially capable of repopulating organisms with cells of the erythroid lineage.

The invention also includes the use of a substance that binds to the novel CD163, such as an antibody, in a negative selection protocol to remove erythroid cells or stem cells from a sample. Accordingly, the present invention provides a method of removing erythroid cells or stem cells from a sample comprising (a) contacting the sample with a substance that can bind the novel CD163 and (b) removing the cells that bind to the substance from the sample.

VII. Targeted Delivery

The finding by the present inventors that a novel CD163 is on erythroid cells and stem cells allows the development of methods to target the delivery of substances directly to erythroid cells or to stem cells. Accordingly, the present invention provides a method of delivering a substance to a stem cell or an erythroid cell comprising administering an effective amount of a conjugate comprising the substance coupled to a CD 163 receptor ligand to an animal or cell in need thereof.

The substance can be any substance that one wishes to deliver, including therapeutics and diagnostics, to an erythroid cell or stem cell. In a specific embodiment, the substance is useful in treating anemia and is delivered to an erythroid cell.

The ligand can be any molecule that can bind the CD163 receptor on erythroid or stem cells. The substance may be coupled to the CD 163 receptor ligand either directly or indirectly. In direct coupling, the substance and ligand are physically linked such as by chemical or recombinant covalent binding or by physical forces such as van der Waals or hydrophobic or hydrophilic interactions. In indirect coupling, the substance and ligand are joined through another molecule or linker. As one example, the substance and ligand may be joined through a recombinant bispecific antibody that binds both the substance and linker.

Conjugates of the substance and the CD163 receptor ligand may be prepared using techniques known in the art. There are numerous approaches for the conjugation or chemical crosslinking of proteins and one skilled in the art can determine which method is appropriate for the substance to be conjugated. The method employed must be capable of joining the substance with the CD163 receptor ligand without interfering with the ability of the ligand to bind to the CD163 receptor and without significantly altering the activity of the substance. If the substance and ligand are both proteins, there are several hundred crosslinkers available in order to conjugate the substance with the ligand. (See for example "Chemistry of Protein Conjugation and Crosslinking". 1991 , Shans Wong, CRC Press, Ann Arbor). The crosslinker is generally chosen based on the reactive functional groups available or inserted on the substance. In addition, if there are no reactive groups a photoactivatible crosslinker can be used. In certain instances, it may be desirable to include a spacer between the substance and the ligand. In one example, the ligand and substance may be conjugated by the introduction of a sulfhydryl group on the ligand and the introduction of a cross-linker containing a reactive thiol group on to the substance through carboxyl groups (Wawizynczak, E.J. and Thorpe, P.E. in Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy of Cancer, CW. Vogel (Ed.) Oxford University Press, 1987, pp. 28-55.; and Blair, A.H. and T.I. Ghose, J. Immunol. Methods 59: 129 , 1983).

In another embodiment, the protein ligand and substance may be prepared as a fusion protein. Fusion proteins may be prepared using techniques known in the art. In such a case, a DNA molecule encoding the erythroid receptor ligand or antagonist is linked to a DNA molecule encoding the substance. The chimeric DNA construct, along with suitable regulatory elements can be cloned into an expression vector and expressed in a suitable host.

The conjugates of the invention may be tested for their ability to enter erythroid ceils and provide the desired pharmacological effect using in vitro and in vivo models.

The following non-limiting examples are illustrative of the present invention: EXAMPLES METHODS Isolation of LDMNC

UBC samples were obtained immediately post partum from normal human placentas scheduled for discard in accordance with established institutional practices. Blood was collected from the umbilical vein into 10 mL vacutainer tubes containing 143 USP units/mL sodium heparin (Fisher Scientific, Unionville, ON). Whole blood was layered onto an equal volume of 1.077 g/mL Ficoll-Hypaque (Histopaque-1077, Sigma-Aldrich, Oakville, ON) and centrifuged at 400 x g at room temperature for 30 minutes. LDMNC were removed from the interface, washed twice in AIM-V medium (Invitrogen, Burlington, ON) containing 10 μM 2-mercaptoethanol (Sigma-Aldrich) and 20 U/mL heparin (Organon Technicka, Toronto, ON), and resuspended in 10 mL AIM-V containing 10% fetal bovine serum (FBS, Intergen, Purchase, NY). Plastic-adherent cells were removed by incubating the LDMNC in a 25 cm² tissue culture flask (Corning-Costar; Cambridge, MA) overnight at 37⁰C and 5% CO₂. Non-adherent LDMNC were further purified with an additional density gradient separation as described above.

CD34⁺ cells

Cyropreserved, purified UCB or ABM CD34⁺ cells were purchased from Cambrex Corporation (East Rutherford, NJ) and StemGenix (Amherst, NY). Cells were thawed and prepared for use as proscribed by the suppliers. Colony forming assay

LDMNC were plated at a density of 1 x 10⁵ cells/mL into 0.9% methylcellulose in Iscove's Modified Dulbecco's Medium containing 30% FBS, 1% bovine serum albumin, 100 μM 2-mercaptoethanol and 2 mM L-glutamine (MethoCult™ H4320; StemCell Technologies Inc., Vancouver, BC) plus 10 ng/mL recombinant human IL-3 (R&D Systems, Minneapolis, MN) and 0, 0.2 or 2 U/mL recombinant human EPO (Ortho Biotech Inc., Raritan, NJ). Purified CD34⁺ cells were seeded at a density of 2.5 x 10³ cells/mL into a methylcellulose (StemGenix) containing IL-3 and EPO. Two 35 mm dishes (Corning-Costar) containing 1 mL of cells in methylcellulose were plated per condition. Plates were maintained at 37⁰C and 5% CO₂ at either ambient oxygen tension or 5% O2 in humidified chambers. Colonies were typed and enumerated after 14 days, and the results reported as the average of two plates. The following reagents were examined in the CFA: chromatographically purified (>99% pure) adult human haemoglobin Ao (Hemosol Inc., Mississauga, ON), hemin, (Porphyrin Products, Logan, UT) and Mac-2-158 (Maine Biotechnology Services, Inc., Portland, ME).

HPLC

Haemoglobin was extracted from erythroid colonies and analyzed by anion exchange high performance liquid chromatography (HPLC). Colonies were harvested from methylcellulose, pooled and washed three times in phosphate buffered saline (Life Technologies). Cell pellets were lysed in 50 mM

Tris (Tris[hydroxymethyl]aminomethane, pH 8.8, Sigma-Aldrich), and cellular debris removed by pelleting the lysate at 400 x g for 10 minutes Cell lysate supernatants were filtered through a 0.2 μm filter (Nalgene, Rochester, NY) and loaded onto a POROS^® HQ/H anion exchange HPLC column (PerSeptive

Biosystems, Framingham, MA). Haemoglobin was eluted with an increasing

NaCI gradient (0.08 - 0.5 M) and the optical density (O. D.) recorded at a wavelength of 414 nm. For haemoglobin quantification, a standard curve of the integrated peak area versus haemoglobin amount (μg) was derived using dilutions of purified HbA₀, prepared in 50 mM Tris, pH 8.8, or dilutions of fetal haemoglobin (HbF), prepared from UCB red blood cells lysed in 50 mM Tris, pH 8.8 as described above for colony-derived haemoglobin. The HbAo and HbF haemoglobin standards were quantified with a CO-Oximeter 482 (Instrumentation Laboratories, Lexington, MA).

Flow cytometry

Purified CD34⁺ cells from cryopreserved ABM and UCB were incubated in PBS/5% NBCS to prevent non-specific antibody staining then resuspended in PBS/2% newborn calf serm (NBCS) and stained with fluorescently labelled antibodies: anti-CD34 (581 -PE; Beckman Coulter, Miami, FL), anti-CD45 (H130-FITC; Caltag Laboratories, Burlingame, CA) and anti-CD163 (Mac-2- 48-FITC or Mac-2-158-FITC, Maine Biotechnology Services, Inc. for 30 minutes at 4⁰C. Aliquots of cells were also stained with labelled IgGi antibodies (IgGi-FITC and IgGrPE, Beckman Coulter) to serve as negative controls. Samples were analyzed using an EPICS XL flow cytometer and EXPO 32™ software (Beckman Coulter). Analysis of intracellular antigens by flow cytometry was performed by first permeabilizing CD34⁺ cells with Cytofix/Cytoperm ™ fixation/permeabilization solution (Pharmingen, Mississauga, ON) for 20 minutes at room temperature. Permeabilized cells were then incubated with either labelled anti-CD163 antibodies (Mac-2-158 FITC) or labelled mouse IgGi antibodies (IgGi-FITC) and washed with Perm/Wash™ solution (Pharmingen). Samples were analyzed using and EPICS XL flow cytometer as described above.

Reverse transcriptase - polymerase chain reaction (RT-PCR) amplification of CD163-specific mRNA

Total RNA was isolated from ABM and UCB CD34⁺ cells using the RNeasy Mini Kit (Qiagen Inc., Mississauga, ON) according to the manufacturer's instructions. First strand cDNA was prepared by incubating the RNA with M-MuLV reverse transcriptase (NEBiolabs, Mississauga, ON) and random primers for 90 mins at 42⁰C. PCR amplification of a CD163- specific sequence from this cDNA was conducted in a 50 μl_ reaction volume containing TITANIUM™ Taq DNA polymerase (BD Biosciences Clontech, Palo Alto, CA), TITANIUM™ Taq PCR buffer, 2 mM dNTPs, and 0.1 μg/ml of primer 1 [5' AGA GGC TGG GGA CTG AAA GAA 3'] (SEQ ID NO;7) and primer 2 [5' GCA GAT AAC TCC CGC ATC CTC CTT 3'] (SEQ ID NO:8). One sample contained all PCR reagents with the exception of the cDNA and served as a negative "template-less" control. Amplification was conducted under the following conditions: 5 cycles of 94⁰C for 30 seconds, 7O⁰C for 30 seconds, 72⁰C for 3 minutes, 5 cycles of 94⁰C for 30 sees, 68⁰C for 30 seconds, 72⁰C for 3 minutes, and 25 cycles of 94⁰C for 30 seconds, 65⁰C for 30 seconds, and finally 72⁰C for 3 minutes. PCR products were electrophoresed through a Tris-acetate gel containing ethidium bromide. PCR products of predicted size were sub-cloned into the pCR2.1 cloning vector (Invitrogen, Burlington, ON) or pDrive (Qiagen Inc.) and sequenced at the Core Molecular Biology Facility (York University, Toronto, ON) using a Perkin-Elmer/ABI 377 Sequencer.

PCR amplification of the variable 3' region was conducted on randomly primed cDNA as described above using with primer 1 and primer 4 (Ex16new)[5' CTC ACT GGG TTA ATT CCC ATT 3'] (SEQ ID NO:9) under the following conditions: 94⁰C for 2 mins, 29 cycles of 59.1⁰C for 45 sees, 72 ⁰C for 45 sees, and 95⁰C for 45 sees. The PCR product was then diluted 1 in 100 and "nested" with primer 5 (M 130) [5' AGG GAA TGA GTC TTC CTT GTG 3'] (SEQ ID NO: 10) and primer 4 under the same conditions. PCR products were electrophoresed through a Tris-acetate gel containing ethidium bromide. PCR products of predicted size were sub-cloned into the pDrive cloning vector (Qiagen) and sequenced as above. Sequence analysis was conducted using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST®). Western blot analysis

2 x 10⁵ treated U937 (cells were incubated for 24 hours with 10 μg/ml phorbol myristate acetate followed by 24 hours with 0.1 μM dexamethasone to induce CD163 expression prior to lysis) or SU-DHL-1 cells were washed in PBS, pelleted and frozen at -8O⁰C until analysis. Cell pellets were then solubized in CHAPS lysis buffer [50 mM Tris-HCI (pH 8.00), 150 mM NaCI, 10 mm CHAPS, and protease inhibitors (Roche, Laval, QC)], centrifuged at 1500 rpm for 10 minutes, and protein concentrations of the supematants were determined using the Coomassie Plus Protein (Pierce, Brockville, ON). Half the sample was loaded onto each of duplicate gels and subjected to SDS- PAGE under non-reducing conditions. Separated proteins were electro- blotted onto Immobilon-P nylon membrane (Millipore (Canada), Nepean, ON). Non-specific binding was blocked by incubating the blots for 1 hour in 5% (w/v) nonfat dry milk powder dissolved in PBS containing 0.05% Tween-20 (PBS-T). Blots were incubated for 1 hour with either unlabelled monoclonal antibody specific to CD163 (Mac-2-158, Maine Biotechnology Services, Inc.) or an isotype control (mouse IgGi, Serotec, Raleigh, NC) diluted 1 :2000 in 5% milk in PBS-T. Incubation was followed by four washes of 5 minutes each in PBS-T. Blots were then incubated with a secondary antibody (goat anti- mouse conjugated to horseradish peroxidase) for 60 minutes at 1 :5000 in 5% milk in PBS-T. Blots werewashed as described above. Antibody reactive proteins were detected using enhanced chemiluminescence (ECL) reagents (Amersham, Baie d'Urfe, QC).

Statistical analysis

Statistical comparisons in Table 2 (SA toxicity) are based on the

Student's t-test, using meaningful paired observations and a 1-tail rejection region. EXAMPLE 1

Co-Expression of Cell Surface CD163 and CD34 and Presence of

Intracellular CD163

The co-expression of cell surface CD 163 and CD34, as well as the presence of intracellular CD 163 as measured by flow cytometry, were confirmed by Western blot analysis of ABM CD34⁺ cells.

Figure 1 is a Western blot of CD34⁺ cells. Lysates from 1 x 10⁵ cells, CD34⁺ cells or SU-DHL-1 cells were electrophorosed through a polyacrylamide gel under non-reducing conditions and electroblotted onto nylon membranes. Membranes were probed with an anti-CD163 antibody (Mac-2-158) or an isotype control antibody (IgGi; not shown) as described in Materials and Methods. Antibody reactive proteins were visualized using enhanced chemiluminescence (ECL) detection. Lane 1: SU-DHL-1 cells, lane 2: empty, lane 3: ABM CD34⁺ cells.

Cell lysates prepared from CD34⁺ cells contained an ~125 - 135 kD protein (nonreduced) specifically recognized by a mouse anti-CD163 monoclonal antibody (Figure 1). The immunoreactive protein present in the CD34⁺ lysates co-migrated with the anti-CD163 reactive proteins expressed by SU-DHL-1 cells, a human histiocytic lymphoma cell line that constitutively expresses the two smallest known cytoplasmic domain variants of CD163 (Pulford et al, 1998; Pulford et al, 1992; Epstein et al, 1978; Law et al, 1993). No immunoreactivity was detected when normal mouse IgGi was used to probe the Western blot. The human myeloid progenitor cell line, KG-Ia, which expresses high levels of CD34 (Civin et al, 1984) has undetectable CD163, as assessed by both flow cytometry and Western analysis (data not shown).

EXAMPLE 2

CD163 Expressed by Erythroid Cells Within the BFU-E Colony is Distinct from CD163 Expressed by Monocytic Cells. Adult bone marrow CD34⁺ cells were seeded into methylcellulose (1 ,000 cells/mL) in the presence of 10 ng/mL IL-3 and either 0.5 or 2.0 U/mL Epo. Methylcellulose plates were incubated in a humidified incubator with 5% CO₂ and maintained at 37°C. After 14 days, cells from the BFU-E colonies were harvested from the plates, washed, enumerated and pelleted. Cell pellets were frozen at -8O⁰C. Cell lysates were prepared by resuspending frozen pellets in CHAPS buffer (0.5% CHAPS, 10 mM Tris, 1 mM MgCI2, 1 mM EDTA and 10% glycerol) with the addition of protease inhibitor cocktail and incubating on ice for 30 minutes. Lysates were then centrifuged and the supernatant was resuspended in non-reducing SDS- sample loading buffer. For comparison, U937 cells, a monocytic cell line that can be induced to express CD163, were also analyzed by Western blot (Figure 2). CD163 expression was induced in U937 cells by stimulating first with phorbol ester for 24 hours followed by a 24 hour treatment with 0.1 uM dexamethasome. Cells were harvested by gentle scraping in phosphate buffered saline and cell lysates were prepared as described above.

For Western analysis, lysates were electrophoresed through an 8% PAG and transferred to a nylon filter. The nylon filters were blocked in a skim milk-containing solution and then probed first with the MAC-158 anti- human CD163 mouse monoclonal antibody (MBS), followed by goat-anti- mouse-anti IgG antibody conjugated to HRP (BIO-RAD). Bound anti-lgG antibody was detected using ECL Kit from AP Biotech.

The CD163 expressed on erythroid cells is distinct from that expressed by the differentiated U937 cells, which represent monocytic/macrophage cells. The CD163 immunoreactivity detected in the differentiated U937 cells migrates as a doublet, at ~ 130 and 140 kD, whereas the immunoreactivity detected in the erythroid cells migrates at ~135. Furthermore, the CD163 immunoreactivity present in the differentiated U937 cells is readily detectable in Western blot by 2 different commercial anti- CD163 antibodies Mac-158 and EdHu-1 (Serotec) whereas the CD163 immunoreactivity present in the erythroid cells is only detected by the Mac- 158 antibody (data not shown). These differences suggest that erythroid cells express a different isoform of CD163 than that expressed by monocytes and macrophage.

The small difference in amino acid residues amongst the known CD 163 variants is typically not resolved on such gels. Shifts of this size may represent differences in CD163 glycosylation which are known to occur (Moller et al, 2002).

EXAMPLE 3

Isolating and Sequencing the Novel CD163. Sub-cloning CD163 and CD163-related sequences may be accomplished by using polymerase chain reaction (PCR) amplification and a variety of primer-pairs. Ribonucleic acid (RNA), either messenger RNA or total RNA, is extracted from cells that have been shown to express either CD 163 or a CD163-like protein. For example, CD34⁺ cells derived from human bone marrow (which comprise stem cells) or glyA+ erythroid cells may be used to isolate the novel form of the receptor. The first strand synthesis of complementary DNA (cDNA) is then accomplished by either the use of random hexamers or an oligo-dT primer and a reverse transcriptase. Amplification of the cDNA is then achieved using primer pairs based on known CD163 DNA sequence. Varying the stringency of the PCR by varying the annealing temperature allows for potential mismatches of the CD 163- specific primers to sequences that are similar to, but not exactly identical to, CD163. PCR products are then sub-cloned in vectors suitable for sequence analysis. This is most readily accomplished by utilizing residual dA added to the 3' end of the PCR product by Taq polymerases and a linearized vector created with an overhanging dT residue. Rapid amplification of cDNA ends (RACE) PCR is a technique commonly used to obtain the sequence data for the utmost 3' and 5' ends of the desired cDNA. Analysis of the utmost 3'ends of the CD163-like sequence can be accomplished using a primer specific to CD163 or CD163-like sequence and a "hybrid" primer that consists of numerous Ts and a unique sequence unrelated to CD163 to create a first- strand cDNA from the RNA sample. Amplification of this strand is achieved with the addition of a CD163 or CD163-like primer and a primer containing part of the unique sequence. These PCR products are then sub-cloned into a vector suitable for sequence analysis. The utmost 5' region of the CD163-like RNA transcript can be determined by using a reverse transcriptase which exhibit terminal transferase activity by adding 3-5 dC residues to the 3' end of the first strand cDNA. A primer is then designed with a terminal stretch of dG residues which then serves as an extended template for PCR in conjunction with either an oligo-dT primer or a gene-specific primer. PCR products generated in this manner can then be subcloned into a vector suitable for sequencing.

CD163 gene transcription was verified by RT-PCR amplification of CD34⁺ cell mRNA using CD163-specific primers. Primers were designed to amplify both the coding and the 5' and 3¹ untranslated regions using RACE technology.

In Figure 3 RT-PCR was conducted on mRNA isolated from ABM- derived CD34⁺ cells using CD163-specific primers. Five microlitres of product were electrophoresed through a 1% agarose gel in a Tris-acetate buffer with 0.5 μg/ml ethidium bromide and the gel photographed using a UV light and the Kodak EDAS 290 system. Lane 1 : product from PCR reaction lacking cDNA template; lane 2: product from PCR reaction with CD34⁺ cell cDNA as template; lane 3 (MWM): molecular weight markers (0.1 kb to 10 kb).

An ~1 ,400 bp cDNA fragment corresponding to the predicted size of a sequence within the extracellular domain of CD 163 was readily amplified from

ABM CD34⁺ cell-derived mRNA (Figure 3). Sequence analysis of this fragment and others confirmed the sequence identity of the cDNA as CD163.

Similar results were obtained using UCB CD34⁺ cell-derived mRNA (data not shown). Thus, some CD34⁺ cells express the CD163 haemoglobin/haptoglobin receptor mRNA. Two variant forms of CD163 mRNA have been described previously with insertions of 83 bp (variant 1) and 1 ,247 bp (variant 2) in the cytoplasmic domain of the receptor (Law et al, 1993; Ritter et al, 1999), corresponding to an additional 35 (variant 1) or 48 (variant 2) amino acid residues.

In order to determine if CD34⁺ cells express the same or other variant forms of CD163, PCR primers were designed to amplify CD163-specific DNA flanking the known variable regions. Sequence analysis of PCR products using these primers indicated that both the predominant and variant 1 , but not variant 2, forms of CD163 are expressed by HSCs. In addition, a previously undescribed variant designated K11 was found. Like the predominant form and variants 1 and 2, K11 mRNA varies in the region encoding the cytoplasmic domain through alternative splicing of intron 15. Clone K11 is created through the combination of an alternative splice donor site with the splice acceptor site of variant 1 (Figure 4). This alternative splicing results in a unique deletion of 93 nucleotides, and a loss of 31 amino acids residues from the predicted protein. The alternate donor site of the K11 clone has score of 0.68 using a predictive model for splice site detection (Reese et al, 1997), compared to a score of only 0.16 for the splice donor site normally used by both the previously described predominant and variant 1 forms.

In Figure 4 the relationship between K11 and other known human CD163 species is illustrated. In Figure 4A Solid bars show regions of sequence homology with gaps indicating deleted regions of CD163 cDNA. Amino acid residues corresponding to the deleted region of the K11 variant of CD163 are indicated by the single letter abbreviation. In Figure 4B splice donor and acceptor sequences utilized by human CD163 variants is illustrated. Alternative sequences are underlined.

EXAMPLE 4

Stimulation of Erythroid Progenitors by Haemoglobin Previously, we have used the colony forming assay (CFA) to compare the effects of iron haeme and haemoglobin on human erythroid progenitors in vitro. Highly purified haemoglobin Ao (HbAo), FeCI₃, or haemin was added directly to human umbilical cord blood (UCB) low density mononuclear cells (LDMNCs) in methylcellulose under standard conditions of 2 U/mL erythropoietin (EPO) and 10 ng/mL interleukin 3 (IL-3). The CFA culture conditions allow for the detection and quantification of early erythroid progenitors, particularly the primitive burst forming unit - erythroid (BFU-E) (Metcalf, 1977). Erythropoietin provides an essential growth and survival stimulus to differentiating erythroid progenitors while IL-3 provides a nonlineage-restricted, multipotential growth stimulus (Migliaccio et al, 1988). Both growth factors are all that is required by hematopoietic progenitors to form distinctive erythroid colonies, each reflective of a responding progenitor cell in the starting cell population. Typically 190 ± 169 BFU-E per 1 x 10⁵ LDMNC are present in cord blood (mean ± standard deviation, n=161; our unpublished data). At 1000 ug/mL, haemoglobin was found to increase the number of BFU-E to a greater extent than molar equivalents of both haeme and iron (Mueller et al, 1997).

In order to confirm that the increased size and redness of BFU- E that form in the presence of haemoglobin is due to the increased production of intracellular haemoglobin, the cells were harvested from the CFA plates after colony enumeration. Extracellular haemoglobin was removed through extensive cell washing, and the intracellular haemoglobin extracted for analysis and quantification by anion exchange chromatography. The total amount of haemoglobin that formed in the presence of the various culture additives was expressed as micrograms of haemoglobin per CFA plate as shown in Table 1. Data from three representative experiments indicate that

HbAo stimulates the greatest amount of haemoglobin production by the erythroid colonies. Haemin and FeCb also increased the amount of haemoglobin produced by erythroid colonies but this amount was consistently less than that observed in response to exogenous haemoglobin. The stimulation of increased haemoglobin production by erythroid colonies in response to HbAo is consistent with the observation that they are typically larger and redder than colonies that form in response to the other additives.

Table 1 illustrates haemoglobin production by erythroid colonies in response to iron, haeme and haemoglobin. UCB LDMNC were plated in methylcellulose containing 2 U/mL EPO and 10 ng/mL IL-3 in the presence or absence of FeCb, haemin or HbAo and maintained at 5% CO₂. Erythroid progenitors were harvested after 14 days, extensively washed free of extracellular material, and lysed as described in Materials and Methods. Cell lysates were analyzed by HPLC and the amount of haemoglobin quantified by comparison with purified adult and faetal haemoglobin standards.

Table 1 Haemoglobin production by erythroid colonies in response to iron, haeme and haemoglobin

Fold increase

Haemoglobin, μg over control

Culture additive Experiment Mean

1 2 3

8 19 3 1.0

250 μM FeCI₃ 15 22 7 1.8

100 μM haemin 28 38 3 2.2

1000 μg/mL HbA₀ 56 45 14 4.7

EXAMPLE 5

Stimulation of Erythroid Progenitors by Hemoglobin is More Than Just the Provision of Haeme One means by which haemoglobin could stimulate erythroid progenitor proliferation and/or differentiation is through the provision of its constituent haeme or iron. In order to determine the extent of the haeme- and iron- mediated activity of haemoglobin on erythroid progenitor proliferation, progenitors were plated into CFA in the presence of succinyl acetone (SA), an inhibitor of δ-aminolevulinic acid dehydratase (Ebert et al, 1979), the second enzyme in the haeme-synthesis pathway. Succinyl acetone is toxic to erythroid progenitors since it interferes with the requisite haeme biosynthesis in the developing cells (Muta et al, 1995). The uptake of exogenous haeme by cultured erythroid progenitors bypasses the requirement for endogenous haeme biosynthesis, thereby effectively counteracting the toxic effects of SA, whereas the uptake of free iron is not effective. To investigate the role of haemoglobin under these conditions, UCB LDMNC were plated into CFAs containing 1000 μg/mL HbAo, 100 μM haemin or 250 μM FeCi3 in the presence or absence of 500 μM SA. The data are expressed as the percentage of total erythroid progenitors enumerated in the presence of SA compared to the number in the absence of SA. SA treatment reduced the number of erythroid progenitors to 31% of the untreated controls (Table 2). As expected, haemin almost completely counteracted the inhibitory effect of SA while FeCb was unable to provide any protection to erythroid progenitors. A lower molar concentration of HbAo (64 μM heme/iron-equivalents) provided slightly greater protection against the toxicity of SA than did 250 M FeCb but less than that provided by 100 μM haemin, indicating that, although of comparable potency in stimulating eythroid progenitors, haemoglobin is a less efficient source of intracellular haeme, and suggesting that haemoglobin may have additional stimulatory activity.

Table 2 Ability of iron, haeme and haemoglobin to overcome SA toxicity to erythroid progenitors. UCB LDMNC were plated into methylcellulose (1x10⁵ cells/mL) containing 2 U/mL EPO and 10 ng/mL IL-3 in the presence of FeCb, haemin or HbAo with or without 500 μM SA and the total numbers erythroid progenitors were enumerated after 14 days. Data are expressed as the percentage of the total number of erythroid progenitors induced by each additive in the presence of SA relative to the total number or erythroid progenitors induced by each additive in the absence of SA. * p< 0.05; ** p< 0.01 as compared to no addition.

n corresponds to the number of independent experiments.

Table 2 Ability of iron, haeme and haemoglobin to overcome SA toxicity to erythroid progenitors.

Culture additive Total BFU-E: +SA / -SA n

% (± S.E.M.)

31 ± 5 12

250 _μM FeCI₃ 40 ± 5 11 100 μM haemin 90 ± 13** 12 1000 μg/mL HbA₀ 48 ± 7* 12

EXAMPLE 6

CD163 is Expressed by CD34* Cells

One means by which haemoglobin could stimulate erythroid progenitor cells is via direct cellular interaction mediated, at least in part, by a cell surface receptor for haemoglobin. CD163 is the recently described monocyte/macrophage haemoglobin scavenger receptor (Kristiansen et al,

2001), the expression of which has not been previously described on cells of other lineages (MorganeJli et al, 1988; Pulford et al, 1998; Van den Heuvel, 1999; Pulford et al, 1992). Since CD163, or a related receptor, could in principle mediate the stimulatory effects of haemoglobin on HSCs, CD34⁺ stem cells were analyzed for CD163 expression. Flow cytometric analysis of purified UCB and adult bone marrow (ABM) CD34⁺ cells revealed that a small percentage of CD34⁺ cells co-express CD163 (Figure 6). However, staining of permeabilized cells indicated that the majority of CD34⁺ cells contained intracellular CD163 (Figure 7). *

EXAMPLE 7 Co-Expression of Cell Surface CD163 and CD34, and Presence of Intracellular CD163

The co-expression of cell surface CD163 and CD34, as well as the presence of intracellular CD163 as measured by flow cytometry, were confirmed by Western blot analysis of ABM CD34⁺ cells. Cell lysates prepared from CD34⁺ cells contained an ~125 - 135 kD protein (nonreduced) specifically recognized by a mouse anti-CD163 monoclonal antibody (Figure 1). The immunoreactive protein present in the CD34⁺ lysates co-migrated with the anti-CD163 reactive proteins expressed by SU-DHL-1 cells, a human histiocytic lymphoma cell line that constitutively expresses the two smallest known cytoplasmic domain variants of CD163 (Pulford et al, 1998; Pulford et al, 1992; Epsteinet al, 1978; Law et al, 1993). No immunoreactivity was detected when normal mouse IgGi was used to probe the Western blot. The human myeloid progenitor cell line, KG-Ia, which expresses high levels of CD34 (Civin et al, 1984) has undetectable CD 163, as assessed by both flow cytometry and Western analysis (data not shown).

EXAMPLE 8

Stimulation of Erythroid Progenitors Through Activation of CD163

CD34⁺ cells express both the message and the protein corresponding to the CD163 haemoglobin/haptoglobin scavenger receptor. The presence of CD163 on CD34⁺ cells suggests that haemoglobin could mediate its erythropoietic effects on hematopoietic stem cells via this cell receptor. Furthermore, other non-haemoglobin-based ligands for CD163 could similarly stimulate CD34⁺ cells. Since monoclonal antibodies have been described that cross-link and activate CD163 (Van den Heuvel, 1999), the anti-CD163 monoclonal antibody, Mac-2-158 (Morganelli et al, 1998), was investigated for its ability to stimulate erythroid progenitors. ABM CD34⁺ cells were plated into methylcellulose with IL-3 and EPO in the presence and absence of Mac-2- 158. As previously described for haemoglobin, the erythroid colonies that formed in the presence of Mac-2-158 were larger and redder than those that formed in its absence (Figure 8). Similar results were obtained using UCB CD34⁺ cells and the monoclonal antibody, EdHU-1 , which is known to crosslink CD 163 on macrophages and activate the cells (Van den Heuvel, 1999; Ritter et al, 2001) (data not shown). These data support a role for CD163 in mediating the stimulatory effect of haemoglobin on erythroid progenitors.

Figure 8 illustrates the stimulation of erythroid colonies by activating anti- CD163 antibodies. Erythroid colonies that form in the presence of activating anti-CD163 antibodies are larger and more haemoglobinized that those that form in its absence. ABM CD34⁺ cells (2.5 x 10³) were plated into methylcellulose containing 2 U/mL EPO and 10 ng/mL IL-3 in the absence or presence of 50 μg/mL of Mac-2-158, an activating anti-CD163 antibody. Cultures were maintained at ambient O₂ for 14 days after which representative colonies were photographed (original magnification x 40). An IgGi isotype control antibody was added to colony-forming assays to serve as a control. The appearance of colonies in the presence of the isotype control antibody was similar to those without the addition of any antibodies (not shown).

The activating anti-CD163 antibodies maintain their stimulatory effect on erythroid progenitor colonies at reduced concentrations of EPO (0.2 or 0.5 U/mL EPO, as opposed to the 2 U/mL EPO used above). The erythroid colonies that formed under these conditions are bigger and redder (more haemoglobinized) than the colonies plated in the absence of the activating antibodies. Activating anti-CD163 antibodies, however, cannot replace EPO under these conditions. In the absence of EPO, erythroid progenitor colonies do not form, with or without the addition of the activating anti-CD163 antibodies.

As discussed above, Western blot analysis of harvested erythroid progenitor colonies reveals the presence of CD163 in the differentiating erythroid cells. The CD163 expressed on erythroid cells is distinct from that expressed by differentiated U937 cells, a monoblastoid cell line capable of phorbol myristate acetate-inducible CD163 expression (Pulford et al, 1998). The CD163 immunoreactivity detected in the differentiated U937 cells migrates as a doublet of ~130 and 140 kD, whereas the immunoreactivity detected in the erythroid cells migrates as a ~135 kD singlet (Figure 2). The small difference in amino acid residues amongst the known CD163 variants is typically not resolved on such gels. Shifts of this size may represent differences in CD163 glycosylation which are known to occur (Moller et al, 2002).

Interestingly, the level of CD163 expression in the erythroid colonies appears to be influenced by the amount of EPO present. On a per cell basis, there is a greater amount of CD163 detected in the cell lysates of erythroid colonies grown in the presence of 0.5 U/ml EPO as compared to 2.0 U/mL EPO, indicating that receptor expression is upregulated at low concentrations of EPO. These data suggest that the erythroblastic progeny of early progenitors such as the BFU-E also express CD163.

DISCUSSION

Although the extracellular SRCR domains of the CD163 receptor are well conserved across multiple species (Schaer, 2002; Sanchez et al, 1999), several isoforms of CD163 have been described, each varying within the intracellular domain. These isoforms are all thought to arise from a single CD163 gene through alternative splicing of intron 15 (Law et al, 1993). Cytoplasmic variant 1 uses the same splice donor site as the initially described predominant form of CD 163, but an alternative splice acceptor site located 83 bp upstream of exon 16 within intron 15 (Ritter et al, 1999). The resultant altered reading frame yields a cytoplasmic domain with a different C- terminal sequence. Cytoplasmic variant 2 has a 1 ,247 bp insertion between donor site 2 and acceptor site 1 as a result of an unspliced intron 15, resulting in an additional 89 aa residues and an 1 ,109 bp untranslated region. By using a PCR amplification strategy with primers flanking the known intracellular variable domains, it has been found that both the predominant and variant 1 forms of CD163 are expressed in Human Stem Cells (HSCs). In addition, it has been found that another cytoplasmic variant of CD163 in CD34⁺ cells. This variant, K11 , has a deletion of 32 amino acid residues (relative to the predominant, variant 1 and variant 2 forms) immediately adjacent to the transmembrane domain. As with the other cytoplasmic variants, the K11 variant arises as the result of alternative splicing of intron 15. Although cDNAs corresponding to all of the three previously described cytoplasmic variants have been found in monocyte/macrophages by RT-PCR, only a single CD163 mRNA species of approximately 3.8 kb has been detected by Northern blot analysis (Hogger et al, 2001 ; Law et al, 1993; Ritter et al, 1999). The predominant and variant 1 mRNA species of CD163 are close in size and are expected to comigrate on Northern blots but the reported absence of the larger mRNA species corresponding to variant 2 may indicate greater instability of the latter mRNA species.

The signal transduction pathway of CD163 activation on monocytes/macrophages has been partially characterized. Receptor cross- linking leads to casein kinase Il and protein kinase C dependent phosphorylation of the intracellular domain of CD163 (Van den Heuvel et al, 1999; Ritter et al, 2001). Sequence analysis suggests that serine/threonine (S/T) phosphorylation sites exist within the intracellular domain of CD163 and these may play a role in transducing activation signals to the cells. Fusion proteins comprised of the cytoplasmic domain of the three previously described CD163 variants interact specifically with β-subunit of casein kinase Il (Ritter et al, 2001), a constitutively active S/T protein kinase with a wide variety of substrates (Meggio et al, 2003). One of the predicted phosphorylation sites is found within the 32 residue region deleted from the K11 variant (Figure 5). In addition, a putative signal for highly efficient endocytosis of the receptor (YREM) is contained with the deleted region of K11. This sequence conforms to the consensus motif Y-X-X-φ (Bonifacino et al, 2003) and is similar to the YTRF motif described as essential for endocytosis of the transferrin receptor (Collawn et al, 1993).

We have shown that haemoglobin increases the growth and differentiation of cultured hematopoietic progenitors, and that primitive HSCs express CD163 thereby providing a mechanism whereby haemoglobin can exert its stimulatory effects. Although CD 163 can be detected on the surface of a small subpopulation of CD34⁺ cells, many more cells appear to express the protein intracellular^. CD163 has only been previously described on monocytes and macrophages (Pulford et al, 1992; Law et al, 1993) where it is believed to serve a function in scavenging cell-free haemoglobin and in modulating the inflammatory response (Gordon, 2001; Beuchler et al, 2002; Schaer, 2002; Pulford et al, 1998). As in the present study, the initial characterization of the monocyte/macrophage-associated CD163 protein (M130) revealed a low level of surface expression but a high percentage of cells with intracellular protein (Pulford et al, 1992). It has subsequently been found that macrophages shed a soluble form of CD 163 (Moller et al, 2002; Droste et al, 1999) and that the expression and trafficking of CD163 in monocytes/macrophages reflects the differentiation and/or activation state of these cells (Pulford et al, 1992). Although, the fate of intracellular stores of CD163 in HSC remains to be determined, the high frequency of cells expressing CD163 suggests an important role for this receptor in HSC development. The natural ligand for monocyte/macrophage CD163 is the complex of haemoglobin and the acute phase protein haptoglobin (Kristiansen et al, 2001). The high abundance of haptoglobin in blood plasma and its extremely high natural affinity for haemoglobin results in the rapid and essentially irreversible formation of haemoglobin-haptoglobin complexes under physiological conditions (Nagel et al, 1971). We have previously found that haemoglobin directly stimulates CD34⁺ progenitors in vitro. The colony forming assays were conducted in the presence of 30% fetal bovine serum, which should contain sufficient free haptoglobin to readily bind the exogenous haemoglobin added to the cultures. In general, we have found that the binding of haemoglobin by haptoglobin is preserved across many species, and that complexes of human haemoglobin and rat haptoglobin are internalized by mouse macrophages, for example (Levy et al, 2003). Thus, it is expected that the bovine haptoglobin-human haemoglobin complexes would interact with CD163 on HSCs to a similar extent to that of an entirely homologous protein complex.

The stimulatory activity of haemoglobin on hematopoietic progenitors and differentiating erythroid cells appears to be mediated in large part through interaction with CD163. The ability of activating monoclonal antibodies raised against the monocyte-derived CD163 antigen to mimic the effect of exogenous haemoglobin suggests that this is the prime mechanism for the stimulatory activity of haemoglobin on HSCs. That haemoglobin should provide an important and essentially immediate feedback loop to cells responsible for its own production is not entirely surprising given that the stimulatory effect of increased amounts of EPO in response to acute anemia would follow a slower time course. It is noteworthy that the activation of erythroid progenitors via CD163 requires the presence of EPO, suggesting an inter-dependehce of the two intracellular signalling pathways. This is born out by the observation that CD 163 expression is increased when EPO levels are reduced and that haemoglobin can largely maintain progenitor proliferation at very low doses of EPO. Receptor activation in monocytes/macrophages leads to the production of IL-6, IL-1 and GM-CSF ((Van den Heuvel, 1999). Further work is required to establish whether such cytokines are produced through activation of CD163 on HSCs, but autocrine stimulation through the production of such cytokines could explain some of the stimulatory effects of receptor activation. Interestingly, CD163 is a member of the group B scavenger receptor cysteine-rich (SRCR) family bearing 9 tandemly repeated extracellular cysteine- rich domains, a sequence motif commonly found in proteins not only involved in endocytosis but also in cellular adhesion (Gordon, 2001; Schaer, 2002; Resnick et al, 1994; Vila et al, 2000). The distribution of known SRCR family members is also closely associated with cells of the immune system and host defense. The role of cell adhesion events in controlling HSC proliferation, mobilization and homing during development, and under a variety of clinical conditions (Lapidot et al, 2002), suggests that adhesion mediated via CD163 could potentially play a role in regulating HSCs, and that this may be further modulated by the presence of cell-free haemoglobin.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

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3 Bonifacino, J. S. & Traub, L. M. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Ann. Rev. Biochem. 72, 395- 447 (2003).

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Claims

WHAT IS CLAIMED IS:

1. A CD163 receptor that is present on erythroid cells, has a molecular weight of approximately 135 kD and a deletion at the start of exon 15, wherein the deletion contains a phosphorylation region and a sequence supporting efficient receptor endocytosis, said CD163 receptor comprising an amino acid sequence as shown in Figure 5C (SEQ ID NO:5) or a functional equivalent thereof.

2. A novel CD163 receptor that is present on CD34⁺ stem cells and has a molecular weight of approximately 135 kD.

3. A method of modulating erythropoiesis comprising administering an effective amount of substance that can bind the CD 163 receptor according to claim 1 or 2 to a cell or animal in need thereof.

4. A method according to claim 3 for the stimulation of erythropoiesis.

5. A method according to claim 4 for the treatment of anemia.

6. A method according to claim 4 or 5 further comprising administering one or more hematopoietic growth factors.

7. A method according to claim 6 wherein the hematopoietic growth factor is erythropoietin.

8. A method of modulating the growth, proliferation, differentiation and/or mobilization of a stem cell comprising administering an effective amount of a substance that can bind the CD 163 receptor according to claim 2 to a cell or an animal in need thereof.

9. A method according to claim 8 for the stimulation of the growth, proliferation, differentiation and/or mobilization of a stem cell.

10. A method according to any one of claims 3 to 9 wherein the substance is an antibody that binds to CD163.

11. A use of an effective amount of a substance that can activate CD163 according to claim 1 or 2 to modulate erythropoiesis.

12. A use of an effective amount of a substance that can activate

CD163 according to claim 1 or 2 to prepare a medicament to modulate erythropoiesis.

13. A use according to claims 11 or 12 for the stimulation of erythropoiesis.

14. A use according to claims 11 or 12 for the treatment of anemia.

15. A use according to claims 11 or 12 further comprising one or more hematopoietic growth factors.

16. A use according to claim 15 wherein the hematopoietic growth factor is erythropoietin.

17. A use of an effective amount of a substance that can activate

CD163 according to claims 1 or 2 to modulate the growth, proliferation, differentiation and/or mobilization of a stem cell.

18. A use of an effective amount of a substance that can activate CD163 according to claims 1 or 2 to prepare a medicament to modulate the growth, proliferation, differentiation and/or mobilization of a stem cell.

19. A use according to claims 17 or 18, to stimulate the growth, proliferation, differentiation and/or mobilization of a stem cell.

20. A use according to any one of claims 11 to 19 wherein the substance is an antibody that binds to CD163.

21. A cell culture additive useful for modulating growth, proliferation differentiation and/or mobilization of an erythroid cell or CD34⁺ stem cell comprising an effective amount of a substance that can activate CD163 according to claim 1 or 2.

22. A cell culture additive according to claim 21 which is serum free.

23. A cell culture additive according to claim 22 which contains serum.

24. A cell culture additive according to claims 21-23 for use in enhancing growth, proliferation, differentiation and/or mobilization of stem cells or erythroid cells.

25. A cell culture additive according to claim 24 further comprising erythropoietin.

26. A method of selecting erythroid or stem cells in a sample comprising (a) contacting the sample with a substance that can bind CD163 according to claim 1 or 2 and (b) selecting cells that are bound to the substance.

27. A method to select cells capable of forming colonies of the erythroid lineage comprising (a) contacting the sample with a substance that can bind CD163 according to claim 1 or 2 and (b) selecting cells that are bound to the substance.

28. A method to select cells that are potentially capable of repopulating organisms with cells of the erythroid lineage comprising (a) contacting the sample with a substance that can bind CD163 according to claim 1 or 2 and (b) selecting cells that are bound to the substance.

29. A method of selecting erythroid cells or stem cells from a sample comprising (a) contacting the sample with a substance that can bind CD163 according to claim 1 or 2 and (b) removing the cells that bind to the substance from the sample.

30. A method according to any one of claims 26-29 wherein the substance that can bind CD 163 is an antibody.

31. A method of delivering a substance to an erythroid cell comprising administering an effective amount of a conjugate comprising the substance coupled to a ligand that binds to a CD 163 receptor according to claim 1 or 2 to a cell or animal in need thereof.

32. A method of delivering a substance to a stem cell comprising administering an effective amount of a conjugate comprising the substance coupled to a ligand that binds to a CD163 receptor according to claim 1 or 2 to a cell or animal in need thereof.

33. A method of identifying substances which can bind to CD163 comprising the steps of:

(b) reacting novel CD163 according to claim 1 or 2 and a test substance, under conditions which allow for formation of a complex between the CD163 and the test substance, and

(b) assaying for complexes of CD 163 and the test substance, for free substance or for non complexed CD163, wherein the presence of complexes indicates that the test substance is capable of binding CD163.

34. The method of claim 33, wherein the substance that can bind to the CD163 receptor is a ligand of the receptor.

35. A method of increasing CD163 expression on human stem cells

(HSCs) comprising administering to said stem cells or culturing said stem cells in an effective amount of EPO, IL-3, and one or more of the following factors: FeCb, haemin, or hemoglobin.

36. A method of claim 35 wherein the hemoglobin is HbA₀.

37. A method of claim 36 wherein the human stem cells are cultured under a 5% CO₂.

38. A method of claim 37 wherein the human stem cells are cultured in a media comprising 2 U/ml EPO, 10 ng/ml IL-3, and a factor selected from the group consisting of: 250 microM FeCb, 100 microM haemin and 1000 microgram/ml hemoglobin.

39. A method of claim 38 wherein the cells are cultured in the presence of hemoglobin.

40. A method of claim 39 wherein the hemoglobin is HbA₀.

41. A method of stimulating erythroid progenitors comprising administering to said progenitors an effective amount of EPO, IL-3, and one or more of the following factors: FeCb, haemin, or hemoglobin.

42. A method of claim 41 wherein the hemoglobin is HbA₀.

43. A method of claim 42 wherein the human stem cells are cultured under a 5% CO₂.

44. A method of claim 43 wherein the cells are cultured in a media comprising 2 U/ml EPO, 10 ng/ml IL-3, and a factor selected from the group consisting of: 250 microM FeCb, 100 microM haemin and 1000 microgram/ml hemoglobin.

45. A method of claim 44 wherein the cells are cultured in the presence of hemoglobin.

46. A method of claim 45 wherein the hemoglobin is HbA₀.

47. A method of claim 46, wherein the factor is haemin.