SUMMARY OF THE INVENTION According to the present invention, pharmaceutical compositions that induce stem cell proliferation and uses thereof as medicaments are provided. The present invention describes uses of pharmaceutical compositions comprising CTLA2 in the treatment of depletion of a cellular population, as well as the implications for use in transplantation and in gene therapy.

BACKGROUND Embryogenesis is the fundamental process of differentiation of all tissues from a fertilized egg.

During this process, the cells of the developing embryo differentiate and raise their level of commitment, starting out as pluripotent cells, and ending up as fully differentiated mature cells. In mammals, the property of pluripotency is restricted to cells of the early embryo and to tissue- specific stem cells (for review see Smith A. (1998) Curr. Biol. 8 (22): R802-R804).

Stem cells are characterized by two main traits: (Morrison, S. J. , Shah, N. M. and Anderson, D. J.

Regulatory Mechanisms in Stem Cell Biology. Cell 1997,88 : 287-298., Weissman, I. L. Stem Cells : Units of Development, Units of Regeneration, and Units in Evolution. Cell 2000,100 : 157- 168. ).

1. They are pluripotent (they produce daughter cells that can differentiate and give rise to several different cell types).

2. They are self-renewing (they have the capacity to produce daughter cells that maintain the characteristics of the mother stem cell).

Over the past few years, the importance of stem cells for therapy in injury and disease states has been widely recognized. For example, these cells are used to compensate for loss or death of cells or to replace cells with impaired function. The ability to stimulate proliferation and differentiation of stem cells in vivo is crucial for their use in medical and/or therapeutic procedures. Another

possibility is to isolate or generate stem cells in culture, and use them for transplantation, such as bone marrow transplantation. In this case, the expansion of the stem cell fraction in the bone marrow, and the induction of proliferation and differentiation after transplantation, can contribute to successful recovery.

Hematopoietic stem cells Hematopoietic stem cells (HSC) are the best characterized stem cells (reviewed in Morrison, S. J. et al. The Biology of Hematopoietic Stem Cells. Annu. Rev. Cell Dev. Biol. 1995, 11: 35-71). They persist during lifetime and display the two main characteristics of real stem cells: they are self- renewing, and they are pluripotent. Functionally, HSC are defined by their capacity to repopulate all hematopoietic lineages in marrow ablated animals.

Embryonic stem cells Embryonic stem cells are derived from the totipotent cells of the inner cell mass (ICM) in the early mammalian embryo and are capable of unlimited, undifferentiated proliferation in vitro (Robertson E. J. Embryo-derived Stem Cell Lines. In: Robertson E. J. (ed. ) Teratocarcinoma and Embryonic Stem Cells: A Practical Approach. Oxford, JRL Press 1987,72-112). ES cells are true pluripotent cells as they can differentiate to all cell types.

The ability to obtain fully differentiated cells from the undifferentiated ES cells suggests that in vitro the cells progress through the commitment steps that result in morphological and molecular modifications.

In vitro differentiation of ES cells is extensively used as a model for early mammalian embryogenesis, gene function and development (for reviews see Wobus A. M. and Boheler K. R.

(ed) In : Cell Tissue Organs 1999,165 : 131-245).

Accumulated evidence indicates that ES cells (before and after differentiation) produce growth factors able to induce expansion of hematopoietic stem cells.

Clinical importance of HSCs Hematopoietic stem cells are of increasing importance in clinical applications, and are of great importance in bone marrow transplantations. Today, HSC transplantations are performed in a growing spectrum of diseases (Armitage J. O. (1999) in Harrison's Principles of Internal Medicine, 1999) ; transplantations have been used for many years in cases of leukemia or after the treatment of solid tumors with high-dose chemotherapy or irradiation. There are also reports of

the use of HSC transplantations in the treatment of serious blood disorders (Mol Ther 2001 Jan ; 3 (1) : 14-23 ; Int JHematol 2001 Feb ; 73 (2) : 162-9 ; JIntern Med 2001 Apr ; 249 (4) : 379-90 ; Br J Hae7natol 2000 Mar ; 108 (4) : 666-78 ; Haematologica 2000 Jan ; 85 (1) : 59-62 ; J Pediatr Surg.

1993 Oct,-28 (10) : 1232-7), or in autoimmune diseases (Int J Hematol 2001 Feb ; 73 (2) : 162-9 ; J Intern Med 2001 Apr ; 249 (4) : 379-90). In all these cases transplantations are either autologous or from MHC (Major Histocompatibility Complex) matched donors. There is now accumulating evidence that the transplantation of very large doses of HSC can overcome the MHC barrier, the main obstacle for a much larger use of HSC and other transplantations in a variety of additional diseases (Biol Blood Marrow Transplant 1996 ;2:3-14; Leuk Lymphoma 2001 Mar;41(1-2):19- 34), and induce donor-type tolerance of the host.

Several reports have shown that HSC exhibit a much higher degree of cell plasticity than previously believed. If HSCs are injected into damaged tissue or even to the blood stream of animals with tissue damage, they are able to differentiate into skeletal muscle (Ferrari, G. et al.

Science 1998,279 : 1528-1530), hepatocytes (Lagasse, E. et al. Nature Med. 2000,6 : 1229-1234.), myocardium (Orlic, D. et al. Nature 2001,410 : 701-705. ), neurons (Mezey, E. et al. Science 2000, 290: 1779-1782.; Six, I. et al. Eur J Pharmacol 2003,458 : 327-328) and participate in the neovascularisation of ischemic myocardium (Kocher, A. A. et al. Nature Med. 2001,7 : 430-436.) retina (Grant, B. et al. Nature Med. 2002,8 : 607-612). Thus, there may be many possible clinical uses of HSCs.

The available sources of human HSC are bone marrow, mobilized peripheral blood (MPB), umbilical cord blood and fetal liver. For most transplantations MPB is used. In this case, the donor is treated with G-CSF which induces the mobilization of the HSC from the bone marrow.

White cells are then collected from the peripheral blood and the HSC are purified. Umbilical cord blood is a very good source of multipotent HSC, but the cell number is very limited and can only be used to repopulate in children. Fetal liver 10-14 weeks after gestation is an excellent source of HSC, but rarely available.

The growing need for HSC for transplantation procedures, which often demand higher cell numbers than are actually available, led to intensified efforts to expand HSC in vitro. Expansion protocols leading to cell proliferation without differentiation will not only produce larger stem cell numbers for transplantations, but also improve gene transfer into human HSC.

The main obstacles for HSC transplantation are the lack of MHC-matched donors, the quantity of HSC available and the slow recovery (low neutrophil and platelet counts) of the patients after

myeloablative treatment (chemotherapy, irradiation).

The first two impediments may be overcome by the ex vivo expansion of HSC, as the transplantation of megadoses of HSC might overcome the graft rejection, and the growing number of frozen umbilical cord blood samples will be a more and more available source of HSC for autologous or allogeneic transplantation.

Ex vivo maintenance and expansion of HSC are also of growing importance for gene therapy protocols. Congenital hematologic diseases may be corrected by the introduction of the intact gene into HSC. The durable expression of the transgene in the differentiated daughter cells has proved to be very low, due to the low gene transfer efficiency to the most primitive repopulating HSC. The ex vivo expansion of HSC without induction of differentiation can overcome this main obstacle to gene therapy in HSC.

CTLA2 (cytotoxic T-lymphocyte antigen 2) CTLA2alpha was isolated from a subtracted cDNA library (T-versus B-lymphocytes). Its expression is restricted to T-lymphocytes, where it is inducible upon activation (in mast cells). It was isolated together with the homologous cDNA encoding CTLA2beta. Both are homologous to the pro-region of cystein proteinase precursors, and map to the Cl band of mouse chromosome 13. (Denizot F, Brunet JF, Roustan P, Harper K, Suzan M, Luciani MF, Mattei MG, Golstein P.: Novel structures CTLA2alpha and CTLA2beta expressed in mouse activated T cells and mast cells and homologous to cysteine proteinase proregions. Eur JImmunol 1989 Apr ; 19 (4) : 631-5).

CTLA2alpha was also isolated from a mouse differential display library of pregnant versus non- pregnant uterus. (Kasik J, Rice E.: A novel complementary deoxyribonucleic acid is abundantly and specifically expressed in the uterus during pregnancy. Am J Obstet Gynecol 1997 Feb ; 176 (2) : 452-6). CTLA2beta (the recombinant protein without the putatively cleaved leader sequence a. a. 1-30) is a competitive inhibitor of certain cystein proteases including papain, cathepsins L and H but not B. CTLA2alpha is also a potent inhibitor of Bombyx Cystein Proteinase (BCP) and cathepsinL. (Ki=0. 23 and 0.38 nM, respectively) and a slightly weaker inhibitor of Cathepsin H and papain (Ki=86 and 560 nM, respectively, Kurata, M. et al. (2003) Protein Expr. Purif. 32: 119-125). Cystein protease inhibitors could prove to be useful in the treatment of chronic degenerative diseases, and breast carcinoma, as cystein proteases are expressed at increased levels in these diseases (Delaria K, Fiorentino L, Wallace L, Tamburini P, Brownell E, Muller D.: Inhibition of cathepsin L-like cysteine proteases by cytotoxic T- lymphocyte antigen-2 beta. J Biol Chem 1994 Oct 7 ; 269 (40). 25172-7). The subcellular localization of CTLA2alpha/beta is probably membranal (contains transmembranal region

according SMART) or maybe secreted (contains cleavable signal peptide). The significant homology to the proregions of cystein proteinase precursors, which are preferentially lysosomally located, and the absence of N-glycolysation sites strongly suggest the CTLA2 proteins to be intracellular, non-cytoplasmic (maybe granular) (Denizot F, Brunet JF, Roustan P, Harper K, Suzan M, Luciani MF, Mattei MG, Golstein P.: Novel structures CTLA2alpha and CTLA2beta expressed in mouse activated T cells and mast cells and homologous to cysteine proteinase proregions. Eur JImmunol 1989 Apr ; 19 (4) : 631-5). CTLA2alpha was also found to be amongst many genes that are expressed in stem cells (Terskikh et al.,"From hematopoiesis to neuropoiesis: Evidence of overlapping genetic programs", PNAS 98 (14): 7934-7939).

WO 94/02504 and US patent No. 5,962, 633 both disclose CTLA2beta, its use in the inhibition of cysteine proteases, and alleged possible applications in the treatment of ostoporosis, arthritis, Alzheimer's disease and additional conditions.

None of the aforementioned references relate to the use of CTLA2 in the context of stem cell maintenance, expansion or proliferation.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is the nucleotide (mRNA) and deduced amino acid sequence of CTLA2alpha.

Figure 2 is the nucleotide (mRNA) and deduced amino acid sequence of CTLA2beta.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods for renewing or expanding populations of Hematopoietic Stem Cells (HSCs) for treating patients suffering from depletion of hematopoietic cell populations, as discussed above. As shown in the Examples below, the inventors of the present invention have found that the gene CTLA2alpha is up-regulated in the long-and short-term repopulating HSC cell fraction compared to less primitive cells and to the very-long term repopulating cells. CTLA2alpha (or beta) is also up-regulated in the HSC-supporting stromal cell line MS-5 and, to a lesser extent, in FBMD- 1, when they were treated with horse serum + FCS or with hydrocortisone, both treatments strongly improving the potency of these stromal lines to support in vitro maintenance of HSC. CTLA2alpha (or beta) expression was not changed under the same treatments in the supporting 14F1. 1 and the non-supporting MBA13 stromal cell lines.

The term"CTLA2alpha", as used herein refers to the polypeptide of the CTLA2alpha gene, and is understood to include, for the purposes of the instant invention, the term"BCPI" (Bombyx Cystein Protease Inhibitor from silkmoth Bombyx mori, which also exists in two forms, BCPIa and BCPIb : Kurata M. et al. , (2001) J. Biochem. 130: 857-863), derived from any organism, optionally mouse, splice variants and fragments thereof retaining CTLA2alpha biological activity, and homologs thereof, preferably having at least 70%, more preferably at least 80%, even more preferably at least 90% or 95% homology thereto. While a human homolog has not yet been isolated, this term is understood to encompass such a homolog or homologs. In addition, this term is understood to encompass polypeptides resulting from minor alterations in the CTLA2alpha coding sequence, such as, inter alia, point mutations, substitutions deletions and insertions which may cause a difference in a few amino acids between the resultant polypeptide and the naturally occurring CTLA2alpha. Polypeptides encoded by nucleic acid sequences which bind to the CTLA2alpha coding sequence or genomic sequence under conditions of highly stringent hybridization, which are well-known in the art (for example Ausubel et al. , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1988), updated in 1995 and 1998), are also encompassed by this term. Chemically modified CTLA2alpha or chemically modified fragments of CTLA2alpha are also included in the term, so long as the biological activity is retained. The polypeptide sequence of CTLA2alpha is depicted in Figure 1 (SEQ ID No: 2). Particular fragments of the CTLA2alpha polypeptide include amino acids 1-20,21-40, 41- 60,61-80, 81-100,101-120 and 121-136 of SEQ ID No: 2. Further particular fragments of the CTLA2alpha polypeptide include amino acids 10-30,31-50, 51-70,71-90, 91-110 and 111-136 of SEQ ID No: 2.

The term"CTLA2beta", as used herein refers to the polypeptide of the CTLA2beta gene, derived from any organism, optionally mouse, splice variants and fragments thereof retaining CTLA2beta biological activity, and homologs thereof, preferably having at least 70%, more preferably at least 80%, even more preferably at least 90% or 95% homology thereto. While a human homolog has not yet been isolated, this term is understood to encompass such a homolog or homologs. In addition, this term is understood to encompass polypeptides resulting from minor alterations in the CTLA2beta coding sequence, such as, inter alia, point mutations, substitutions deletions and insertions which may cause a difference in a few amino acids between the resultant polypeptide and the naturally occurring CTLA2beta. Polypeptides encoded by nucleic acid sequences which bind to the CTLA2beta coding sequence or genomic sequence under conditions of highly stringent hybridization, which are well-known in the art (for example Ausubel et al. , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1988), updated in

1995 and 1998), are also encompassed by this term. Chemically modified CTLA2beta or chemically modified fragments of CTLA2beta are also included in the term, so long as the biological activity is retained. The polypeptide sequence of CTLA2beta is depicted in Figure 2 (SEQ ID No: 4). Particular fragments of the CTLA2beta polypeptide include amino acids 1-20, 21-40,41-60, 61-80,81-100, 101-120 and 121-141 of SEQ ID No: 4. Further particular fragments of the CTLA2beta polypeptide include amino acids 10-30,31-50, 51-70,71-90, 91-110 and 111- 141 of SEQ ID No : 4.

While the term CTLA2alpha is mostly used herein, it is to be understood that for all exemplary embodiments, CTLA2beta can effectively replace CTLA2alpha, or act in conjunction with or in addition to CTLA2alpha.

The term"CTLA2alpha gene"refers to the CTLA2alpha coding sequence open reading frame, as shown in Figure 1, or any homologous sequence thereof preferably having at least 70% identity.

This term is understood to encompass any sequence which encodes CTLA2alpha, as defined above.

The term"CTLA2beta gene"refers to the CTLA2beta coding sequence open reading frame, as shown in Figure 2, or any homologous sequence thereof preferably having at least 70% identity.

This term is understood to encompass any sequence which encodes CTLA2beta, as defined above.

The term"CTLA2"as used herein is understood to refer to the polypeptide expressed by either the CTLA2alpha or the CTLA2beta gene, or both ; the term"CTLA2 gene"refers to nucleotide sequence which encodes either CTLA2alpha or CTLA2beta, or both, encompasses the terms "CTLA2alpha gene"and"CTLA2beta gene", and is used interchangeably with the term"CTLA2 polypeptide coding sequence".

By"biological effect of CTLA2"or"CTLA2 biological activity"is meant the effect of CTLA2 on stem cells, which may be direct or indirect, and includes, without being bound by theory, the promotion of cell proliferation, be it ex vivo or in vivo, and the ability of CTLA2 to bind to stem cells. The indirect effect includes, but is not limited to, CTLA2 binding to or having an effect on one or several molecules which are involved in a signal transduction cascade resulting in proliferation of stem cells.

By"homolog/homology", as utilized in the present invention, is meant at least about 70%, preferably at least about 75% homology, advantageously at least about 80% homology, more advantageously at least about 90% homology, even more advantageously at least about 95%, e. g., at least about 97%, about 98%, about 99% or even about 100% homology. The invention also comprehends that these polynucleotides and polypeptides can be used in the same fashion as the herein or aforementioned polynucleotides and polypeptides.

Alternatively or additionally, "homology", with respect to sequences, can refer to the number of positions with identical nucleotides or amino acid residues, divided by the number of nucleotides or amino acid residues in the shorter of the two sequences, wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm ( (1983) Proc. Natl. Acad. Sci.

USA 80: 726), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data, including alignment can be conveniently performed using commercially available programs (e. g., IntelligeneticsTM Suite, Intelligenetics Inc., CA). When RNA sequences are said to be similar, or to have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. RNA sequences within the scope of the invention can be derived from DNA sequences or their complements, by substituting thymidine (T) in the DNA sequence with uracil (U). Additionally or alternatively, amino acid sequence similarity or homology can be determined, for instance, using the BlastP program (Altschul et al., Nucl. Acids Res. 25: 3389-3402) and available at NCBI. The following references provide algorithms for comparing the relative identity or homology of amino acid residues of two polypeptides, and additionally, or alternatively, with respect to the foregoing, the teachings in these references can be used for determining percent homology: Smith et al., (1981) Adv. Appl. Math. 2: 482-489; Smith et al., (1983) Nucl. Acids Res. 11: 2205-2220; Devereux et al., (1984) Nucl. Acids Res. 12: 387-395; Feng et al., (1987) J. Molec. Evol. 25: 351-360; Higgins et al., (1989) CABIOS 5: 151-153; and Thompson et al., (1994) Nucl. Acids Res. 22: 4673-4680.

By"substitution"is meant replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. As regards amino acid sequences the substitution may be conservative or non-conservative.

"Conservative substitution"refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous polypeptides found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix.

Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gln, or Glu, is a conservative substitution.

"Non-conservative substitution"refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gln.

"Deletion"is a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.

"Insertion"or"addition"is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring sequence.

By"polypeptide"is meant a molecule composed of amino acids and the term includes peptides, polypeptides, proteins and peptidomimetics, A peptidomimetic is a compound containing non-peptidic structural elements that is capable of mimicking the biological action (s) of a natural parent peptide. Some of the classical peptide characteristics such as enzymatically scissille peptidic bonds are normally not present in a peptidomimetic.

The term"amino acid"refers to a molecule which consists of any one of the 20 naturally occurring amino acids, amino acids which have been chemically modified (see below), or synthetic amino acids.

By"chemically modified"-when referring to the product of the invention, is meant a product (polypeptide) where at least one of its amino acid residues is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art. Among the numerous known modifications typical, but not exclusive examples include: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.

The term"expression vector"-refers to vectors that have the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are known and/or commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.

The term"alleviation"in the context of disease or illness refers to lessening of symptoms or amelioration of inability to function.

The present invention, in some of its embodiments, provides the possibility of maintaining and/or expanding HSCs and transplanting them to patients suffering from depletion of the hematopoietic cell compartment, so as to alleviate the detrimental effects of depleted cellular populations.

One embodiment of this invention relates to the expansion of cultured stem cells. According to the claimed process, a CTLA2 modulator is administered to cultured stem cells, in a sufficient amount so as to cause proliferation of the stem cells. Said modulator may be an enhancer, which may be a chemical compound. In addition, the modulator may be an expression vector comprising a CTLA2 gene/polypeptide coding sequence, or a CTLA2 polypeptide.

The stem cells which are being induced to proliferate can be, but are not limited to, hematopoietic stem cells or embryonic stem cells.

In the context of stem cell proliferation, the term proliferation refers to growth or multiplication, and is understood to include expansion and renewal of the stem cells (defined, inter alia, by potency to repopulate irradiated mice or humans).

A"modulator"is any molecule that is capable of modulation, i. e. that either increases (promotes) or decreases (prevents). The term is understood to include partial or full inhibition, stimulation and enhancement. In the case of a modulator of a polypeptide, such as a CTLA2 modulator, the modulator may be a direct modulator of the biological activity of CTLA2, or it may be a modulator of either, or both CTLA2 genes; in the latter case, the biological activity of CTLA2 is indirectly modulated by a modulator that affects the transcription or translation of the gene (and does not directly act on the polypeptide).

In the context of the present invention, by"inhibitor"and"enhancer"is meant any molecule that can inhibit or enhance the biological activity of CTLA2, respectively.

The terms"chemical compound", "small molecule", "chemical molecule""small chemical molecule"and"small chemical compound"are used interchangeably herein and are understood to refer to chemical moieties of any particular type which may be synthetically produced or obtained from natural sources and typically have a molecular weight of less than 2000 daltons, optionally less than 1000 daltons or even less than 600 daltons.

The term"antibody"-refers to IgG, IgM, IgD, IgA, and IgE antibody, inter alia. The definition includes polyclonal antibodies or monoclonal antibodies. This term refers to whole antibodies or fragments of the antibodies comprising the antigen-binding domain of the anti-GPCRV product antibodies, e. g. antibodies without the Fc portion, single chain antibodies, fragments consisting of essentially only the variable, antigen-binding domain of the antibody, etc. The term"antibody"may also refer to antibodies against nucleic acid sequences obtained by cDNA vaccination.

Antibody fragments retain the ability to selectively bind with their antigen or receptor and are exemplified as follows, inter alia: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield a light chain and a portion of the heavy chain; (2) (Fab') 2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F (ab'2) is a dimer of two Fab fragments held together by two disulfide bonds; (3) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (4) Single chain antibody (SCA), defined as a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Disclosure on how to prepare all types of antibodies is provided in Example 8.

An additional embodiment of the present invention concerns a process for inducing proliferation of stem cells comprising administering to cultured stem cells CTLA2 in a sufficient amount to cause proliferation of the stem cells.

The stem cells which are being induced to proliferate can be, but are not limited to, hematopoietic stem cells or embryonic stem cells.

In another embodiment, the present invention provides a process for inducing proliferation of stem cells comprising culturing the stem cells with a second type of cells wherein the second type

of cells express or overexpress CTLA2; in addition, CTLA2 may be administered to the stem cells.

By"culturing with"is meant growing the stem cells in culture with the second type of cells, preferably on the second type of cells.

The second type of cells may over-express CTLA2 (methods for causing cells to over-express the polypeptide of a certain gene are well known in the art), and are preferably stromal cells.

Stromal cells provide a bed for closely associated hematopoietic cells in the bone marrow (reviewed in Dorshkind:"Regulation of hemopoiesis by bone marrow stromal cells and their products", Annu Rev Immunol. 1990; 8: 111-37), where they are in close contact with hematopoietic cells through adhesion molecules which transmit them together with secreted molecules proliferation and differentiation signals.

Stromal cells can be, but are not limited to, non-hematopoietic bone marrow cells of mesenchymal origin lacking the general leukocyte marker CD45. They may be of variable morphologic types including preadipocytes, adipocytes, smooth-muscle-like, fibroblastoid, endotheloid and epitheloid. Additionally, they may express specific markers like stro-1 and adhesion molecules directly involved in the binding of hematopoietic cells, such as VLA-4, N- CAM and V-CAM.

Another embodiment of the present invention concerns a method for treating a patient suffering from depletion of a cellular population, comprising administering to the patient stem cells that have been expanded (i. e. , induced to proliferate) according to the methods of the present invention. The patient may be suffering from depletion of a cellular population as a result of a disease or a disease treatment; the disease may be, inter alia, cancer, a blood disorder or an auto- immune disease; the treatment may comprise chemotherapy or radiotherapy.

In addition, the present invention provides a method for treating a patient suffering from depletion of a cellular population comprising administering to the patient a pharmaceutical composition comprising a CTLA2 modulator, further comprising a pharmaceutically acceptable carrier, in a dosage sufficient to induce proliferation of a cellular population. Said modulator may be an enhancer, which may be a chemical compound. In addition, the modulator may optionally be an expression vector comprising a CTLA2 polypeptide coding sequence, or a CTLA2 polypeptide.

The present invention also provides a method for treating a patient suffering from depletion of a cellular population comprising administering to the patient a pharmaceutical composition comprising CTLA2, further comprising a pharmaceutically acceptable carrier, in a dosage sufficient to induce proliferation of a cellular population.

The above methods may be employed to treat a patient who is suffering from depletion of a cellular population as a result of a disease or a disease treatment; the disease may be, inter alia, cancer, a blood disorder or an auto-immune disease; the treatment may comprise chemotherapy or radiotherapy.

By"depletion of a cellular population"is meant that the patient, as a consequence of disease or adverse effects of certain disease treatment, no longer has a sufficient amount of a certain type of cells in order to function as before the onset of the disease or without pain. The stem cells administered to the patient may have the capacity to replace the type of cells which have been depleted in the patient, and in so doing, alleviate the symptoms associated with the depletion of a cellular population.

The term"cellular population"refers to a population of more then one cell, wherein all the cells are of the same type. Cellular populations include, but are not limited to, populations of skeletal muscle cells, myocardial cells, bone marrow cells, nervous cells, blood cells, hematopoietic stem cells, embryonic stem cells and stromal cells.

By"chemotherapy"is meant treatment with a chemotherapeutic drug, such as, inter alia : etoposide, 5-FU (5-fluorouracil), cis-platinum, doxorubicin, a vinca alkaloid, vincristine, vinblastine, vinorelbine, taxol, cyclophosphamide, ifosfamide, chlorambucil, busulfan, mechlorethamine, mitomycin, dacarbazine, carboplatinum, thiotepa, daunorubicin, idarubicin, mitoxantrone, bleomycin, esperamicin Al, dactinomycin, plicamycin, carmustine,. lomustine, tauromustine, streptozocin, melphalan, dactinomycin, procarbazine, dexamethasone, prednisone, 2- chlorodeoxyadenosine, cytarabine, docetaxel, fludarabine, gemcitabine, herceptin, hydroxyurea, irinotecan, methotrexate, oxaliplatin, rituxin, semustine, tomudex and topotecan, and chemotherapeutically active analogs of these drugs.

Depletion of a cellular population as a. direct result or indirect result (through side-effects of treatment methods for said illness, for example) of any illness can also be treated by

administration to the patient of stem cells that have been expanded using the aforementioned claimed methods.

Some of the claimed methods of treatment disclosed herein offer the possibility of transplanting very large numbers of stem cells to the patient. The large number of stem cells can overcome host- rejection, and the stem cells can differentiate into the cell type in which the patient has been depleted as a result of the disease.

The present invention further provides a pharmaceutical composition comprising a CTLA2 modulator further comprising a pharmaceutically acceptable carrier. Said modulator may be an enhancer, which may be a chemical compound. In addition, the modulator may be an expression vector comprising a CTLA2 polypeptide coding sequence, or a CTLA2 polypeptide.

In addition, the present invention provides a pharmaceutical composition comprising CTLA2 further comprising a pharmaceutically acceptable carrier.

In addition, the above pharmaceutical compositions may be formulated in a dosage sufficient to induce proliferation of a cellular population. For additional disclosure on dosage and formulation see Example 5.

An additional embodiment of the present invention provides for the use of any one of the above pharmaceutical compositions as a medicament. In particular, this embodiment provides for the use of a pharmaceutical composition comprising CTLA2 or a CTLA2 modulator as a medicament.

Said modulator may be an enhancer, which may be a chemical compound. In addition, the modulator may optionally be an expression vector comprising a CTLA2 gene. In an exemplary embodiment, the pharmaceutical composition being used as a medicament causes proliferation of a cellular population. An additional embodiment provides for CTLA2 for use as a medicament, and for the use of CTLA2 for expansion of stem cells/induction of proliferation of stem cells, or for use of CTLA2 in the preparation of a composition for expansion of stem cells/induction of proliferation of stem cells.

It is understood that, in the context of the present invention, it may be beneficial to treat a patient with a CTLA2 inhibitor. Said inhibitor may be, inter alia, a protein such as an antibody or a dominant negative or dominant positive peptide, an antisense molecule or a vector encoding an

antisense molecule, an siRNA molecule or a vector encoding an siRNA molecule, other RNA molecules which may act as inhibitors such as, inter alia, ribozymes, or a chemical compound.

By"silencing RNA" (siRNA) is meant an RNA molecule which decreases or silences the expression of a gene/mRNA of its endogenous or cellular counterpart. The term is understood to encompass"RNA interference" (RNAi), and"double-stranded RNA" (dsRNA). For recent information on these terms and proposed mechanisms, see Bernstein E., Denli AM. , Hannon GJ: The rest is silence. RNA. 2001 Nov; 7 (11) : 1509-21; Nishikura K.: A short primer on RNAi : RNA- directed RNA polymerase acts as a key catalyst. Cell. 2001 Nov 16; 107 (4): 415-8 ; and PCT publication WO 01/36646 (Glover et al)..

An additional embodiment of the present invention provides a process for obtaining a compound which induces stem cell proliferation by modulation of CTLA2 comprising: (a) measuring the proliferative activity of CTLA2/a CTLA2 polypeptide; (b) contacting CTLA2 with said compound; and (c) determining whether the activity of CTLA2 is affected by said compound.

This embodiment further provides a process of preparing a pharmaceutical composition which comprises the steps of : (a) obtaining a compound by the above method; and (b) admixing said compound with a pharmaceutically acceptable excipient.

In addition, a process for obtaining a compound which induces stem cell proliferation by modulation of CTLA2/a CTLA2 polypeptide is provided, comprising: a) measuring the binding of CTLA2/a CTLA2 polypeptide to a species with which it interacts in vivo ; b) contacting the CTLA2 polypeptide with said compound; and c) determining whether the activity of the CTLA2 polypeptide is affected by said compound.

A kit for obtaining a compound which induces stem cell proliferation is also provided, comprising : (a) CTLA2/a CTLA2 polypeptide; (b) a species with which the CTLA2 polypeptide interacts in vivo ; (c) means for measuring said interaction; and

(d) means for determining whether the binding of the CTLA2 polypeptide to the species is affected by said compound.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention can be practiced otherwise than as specifically described.

Throughout this application, various publications, including United States patents and applications, are referenced by author and year and patents and applications by number. The disclosures of these publications and patents and patent applications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

EXAMPLES Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the claimed invention in any way.

Example 1 Methods cDNA micro-array design Mouse General Development (MGD) microarray The MGD microarray was imprinted with cDNAs derived from Embryoid Bodies at late spontaneous differentiation stages, teratocarcinomas and mouse embryos.

Differentiated ES cells (Embryoid Bodies-EBs 1. Differentiation in suspension : Large populations of undifferentiated mouse ES cells (129/Sv) are synchronously induced to start differentiation in culture by growing the cells in suspension, using bacteriological petri dishes.

The cells adhere to each other and form small three-dimensional structures within 24 hours. Four to five days later, almost 100% of the aggregates exhibit endoderm formation (termed simple embryonic bodies-SEBs). SEBs occur after an additional incubation period of 8-10 days. By this time, a high percentage of the EBs develop fluid filled cavities accompanied by formation of ectoderm-like cells.

2. Differentiation on a substrate SEBs formed after 4 days in suspension culture are passed on gelatinized tissue culture plates. The EBs attach to the plate surface by the outgrowth of endodermal cells. Continued culture of these aggregates gives rise to an array of cell phenotypes like nerve, muscle, cartilage, hematopoietic cells and more.

Teratocarcinomas When ES cells are injected subcutanously into nude mice, tumors are readily formed. These tumors contain differentiated tissues of all kinds, similar to those in the developing embryo, but in an unorganized pattern.

Mouse embryos Different days of development in the mouse represent different differentiation stages. First HSCs can be detected at 10.5 dpc (days post coitus).

Library preparation cDNA was prepared from RNA collected from differentiated embryonic bodies (EBs) at the indicated time points, mouse teratocarcinomas 16,32 and 40 days after injection and mouse embryos at 9.5, 11.5, 13.5, 15.5 and 17.5 dpc (table 1).

Table 1: MGD Chip design Cells Days after Time points (total) differentiation induction EBs, Differentiation 11-14 Once in 2 days in suspension 15-21 Once in 3 days EBs, Attached 11-14 Once in 2 days Differentiation 15-21 Once in 3 days Mouse 16,32 40 days after Teratocarcinomas injection Mouse Embryos Days 9.5, 11.5, 13. 5,15. 5,17. 5

Three libraries were prepared according to the methods disclosed in co-assigned PCT publication WO 02/45472 ("Prime and Kill"), as follows: - from the spontaneously differentiated EBs collected at the time points indicated in table 1 (MLE), - from the teratocarcinomas collected at the indicated time points (MTC) and - from the mouse embryos collected at the indicated time points (MEB).

In addition, cDNAs representing regulated genes known form the literature (up-or down- regulated and indifferent during ES cells differentiation) were printed on the microarray as controls.

Mouse Hematopoietic (MHB) microarray This microarray was imprinted with cDNAs from different sub-populations of primitive hematopoietic cells.

The hematopoieitc system is organized in a pyramide-like manner, with the most primitive, pluripotent and self-renewing stem cells on the top. These cells are functionally defined by their ability to long-term repopulate (for at least six months) the bone marrow of lethally irradiated recipient mice and to produce all the cell lineages in the blood (LTR-HSC).

The less primitive short-term-repopulating cells are still pluripotent, but do not self-renew. They repopulate irradiated mice up to two months.

Short term repopulating cells differentiate into more multipotent progenitors with more restricted differentiation capacities. These further differentiate to committed progenitors and finally differentiated, mature cells.

Lin-neg. cell fraction (fraction 1) The lin-neg. cell fraction contains all bone marrow cells not expressing any differentiation marker (i. e. lineage marker) of hematopoietic cells. This cell fraction represents a mixed cell population containing all the primitive hematopoietic stem cells (long-and short term repopulating cells as well as very early progenitors).

Lin-neg. cell fraction"on stroma" (fraction 2) In the bone marrow, HSC are in close interaction with stromal cells. Stromal cells regulate HSC proliferation and differentiation through growth factors as well as direct cell-cell contact. They

therefore influence the gene expression pattern of HSCs. To mimic this type of interaction, HSC were incubated for 12 hours on a bone-marrow derived stromal cell line (pre-adipocyes, FBMD- 1) and thereafter re-separated from the stromal cells by fluorescence-activated cell sorting (FACS).

"LTR-HSC" (*action 3 ! The long term repopulating HSC cell fraction was defined by the expression of the following antigens: Stem cell antigen-1 (Sca-1)-positive/stem cell factor-receptor (c-kit) -positive, lineage- negative/CD34-negative (Sca-1+/linlc-kit+/CD34~, Osawa M. et al. , (1996) Science 273: 342-345).

This population was isolated from total mouse bone marrow by four-color-FACS.

Library preparation The array was imprinted with three different types of libraries ; some were prepared according to the methods disclosed in co-assigned PCT publication WO 01/75180 ("SDGI"). The libraries were the following: 1. From each of the fractions a full-length library was produced.

2. Two SDGI libraries were created: from a pool of fractions land 2 and from fraction 3.

3. A gene expression Fingerprint (GEF) differential library enriched in cDNA fragments characterizing the LTR-HSC cell population (Zinovyeva M. V. et al. , (2000) Exp. Hematol.

28: 318-334).

In addition, control cDNAs of known genes from the literature were printed on the microarray.

Mouse Stroma (MST) chip This chip was imprinted with cDNAs derived from stromal cell lines subjected to different treatments, primary total bone marrow cultures subjected to different treatments and fetal livers.

Fetal livers The fetal liver is the main hematopoietic organ in the fetus from E13 until close to birth, when the HSC migrate to the bone marrow. HSC can be detected in the fetal liver already from E11, when they start migrating from the AGM and the yolk sac (Dzierzak E et al. (1998) Immunol. Today 19: 228-236). The fetal liver must therefore present an ideal environment containing HSC supporting stroma. Several HSC supporting cell lines were deduced from fetal liver (Charbord P. et al. , (2002) Exp. Hematol. 30: 1202-1210).

Primary bone marrow cultures

Total bone marrow grown at high fetal calf and horse serum concentrations produces an adherent "stromal"cell layer and long term hematopoiesis. The adherent cell layer plays a role in the maintenance of the hematopoietic stem cells, which can stay for months in a quiescent state under the stromal cell layer, until they start proliferating and differentiating. Such"Dexter-type" cultures produce preferentially myeloid cells and can be maintained for months (Dexter T. M. et al. , (1976) J. Cell. Physio. 91: 335-344). The use of low fetal calf serum concentration and poor medium in such total bone marrow cultures leads also to the development of a stromal feeder layer, which supports preferentially the development of pre-B and B cells (Whitlock C. A. and Witte O. N. , (1982) Proc. Natl. Acad. Sci. USA 79: 3608-3612).

Stromal cell lines Many stromal cell lines were isolated from primary"Dexter-type"bone marrow cultures. These lines support maintenance of HSC for several weeks. MS-5, FBMD-1 and 14F1. 1 are pre- adipocyte-type stromal cell lines well defined in the literature (Itoh K. et al. , (1989) Exp.

Hematol. 17: 145-153; Breems D. A. et al. , (1994) Leukemia 8: 1095-1104; Zipori D. et al. , (1985) Blood 66: 447-455). The maintenance potential of stromal cell lines is strongly dependent on the presence of high fetal calf and horse serum concentrations. Hydrocortisone and LIF and, in certain cases, bFGF and hypoxia, improve the maintenance potential of stromal cell lines. G-CSF and cyclophosphamide induce in vivo the mobilization of HSC from the bone marrow to the blood and influence therefore the cell-cell interaction between HSC and stroma. y-irradiation is used for myeloablation of hosts to be repopulated and was shown to substantially increase the homing efficiency of injected HSC, in correlation with the up-regulation of several genes in the stroma of the bone marrow (for example, SDF).

Library preparation The array was imprinted with three different types of libraries, all prepared by the"prime and kill"method: - MFL : Mouse fetal livers at developmental days 9.5, 10.5, 11.5 and 12.5. The redundancy of this library was initially relatively high (42%) and was further reduced by screening it with probes for the three most redundant genes, and only negative clones were selected.

- MBC : Dexter-type primary bone marrow cultures grown with or without hydrocortisone, LIF, G- CSF, subjected to hypoxia or gamma-irradiated, and Whitlock-Witte cultures.

- MSL : Mouse stromal cell lines (MS-5, FBMD-1 and 14F1. 1 subjected to treatments of hydrocortisone, LIF, FCS+horse serum, cyclophosphamide, G-CSF for 1 or 7 days and after hypoxia or gamma-irradiation.

Probe design and hybridizations Common normalizing probe In order to achieve meaningful differentials of expression profiles it is of importance to normalize the signals obtained in each hybridization. Therefore, in each hybridization, in addition to the probe of interest which is labeled with one fluorescent dye, a second reference probe is added, labeled with a second dye. While the test probe varies from one hybridization to another, the reference probe, also called"common normalizing probe", is invariant and permits the normalization of the hybridization signals and herewith the comparison between two independent hybridizations.

For the hybridizations on the MHB microarray using different HSC subtractions as specific probes, the lin-cell fraction was used as the common normalizing probe (biologically relevant common probe).

For the hybridizations on the MGD microarray, the common normalizing probe used was a mixture of RNA from undifferentiated ES cells (6 parts), and one part of each of the libraries printed on the microarray (Embryoid Bodies at late differentiation stages, teratocarcinomas, mouse embryos; biologically irrelevant common probe).

For the MST microarray, the common probe used was a 1: 1 mix of RNA derived from the three different stromal lines after different treatments, and rat brain RNA (biologically irrelevant common probe).

Specific probes One set was prepared from MS-5, FBMD-1 and MBA14F1. 1 cells (hematopoiesis supporting adipocyte-like stromal cell lines) and MBA13 cells (non-supporting cell line). The cell lines were treated with hydrocortisone, horse serum, hypoxia and, for MBA13 and MBA14F1. 1, were collected at subconfluent state. This set was used for the hybridizations on the MGD and the MST microarrays.

A second set of probes was prepared from different sub-fractions of the lin-negative cell population: Sca-1+/lin/c-kit-/CD34-, Sca-1+/lin~/c-kit+/CD34~, Sca-1+/linlc-kit+/CD34+, Sca-1+/lin~ /c-kit% CD34+, and the total lin-fraction itself. This set was used for the hybridizations on the MHB microarray.

In addition,"membrane-bound"mRNA probes, in which mRNA for membranal and secreted proteins are enriched, were prepared from undifferentiated ES cells as well as MS-5 and FBMD-1 cells. This is in order to identify a maximum of cDNAs coding for membranal and secreted proteins on all the three microarrays.

Statistical and bioinformatic analysis of the hybridization results Statistical algorithms adapted by the inventors for the study of gene expression patterns along the time course of a specific treatment were used. This method enabled the inventors to build clusters of genes, the expression pattern of which correlates with the regulation of the potency of the stromal cell line to support hematopoiesis.

Following sequencing, sequence analysis tools were used for annotation and extension mining of sequence information. Those tools include Phred phrap, BLAST, and other public databases, along with proprietary applications.

Validation of selected candidate genes Initial verification of the hybridization results was carried out by RT-PCR on mRNA extracted from MS-5, FBMD-1 and 14F1. 1 cells after different treatments. For candidates confirming the hybridization results and/or with an interesting expression profile in the different cell lines after different treatments (i. e. up- or down-regulated in a line or treatment known to support in vitro hematopoiesis), in situ hybridization on mouse embryos at early stages was performed to find expression in the AGM, expression specificity in this region and possible expression in the liver and yolk sac. Furthermore, adult bone marrow, thymus and spleen were analyzed.

Candidate genes were then expressed in stromal cell lines and the effect of the transgene on stem cell maintenance and expansion tested in LTC-IC and CFAC assays in vitro. Then, the effect of the over-expressed protein was tested by injection of HSC cultured on stroma over-expressing the candidate gene to irradiated animals for repopulation assays.

Candidate genes showing a positive effect on the in vitro maintenance of HSC in these assays, are produced as recombinant proteins and tested in vitro directly on the HSC and in vivo by injection to transplanted animals.

CTLA2 was identified by several of the above methods, as described in Example 3.

Example 2 Analysis of HSCs Several different in vivo and in-vitro assays were utilized in order to estimate the presence, amount and differentiation state of the HSCs (for review see Domen J. & Weissman I. L. Mol.

Med. Today 1999,5 : 201-208).

In vivo assays : 1. Long/short term reconstitution of mice-A defined number of cells of interest, which can be un-separated, e. g. , bone marrow cells or any purified cell population, is injected to irradiated mice. The contribution of the donor cells to the total peripheral blood or bone marrow of the host is assayed after a period of 2 months (STR-HSC) or after a period of over 6 months (LTR-HSC). To quantify more precisely the amount of stem cells, competitive repopulation units (CRU) are estimated by injecting limiting numbers of donor-type cells together with a standard number of host-type total bone marrow cells. (Szilvassy, S. J. et al. Proc. Natl. Acad.

Sci. USA 1990,87 : 8736-8740). The frequency of CRU is then calculated from the proportion of negative mice (showing less than 3% donor-type cells in the bone-marrow) by the method of maximal likelihood for each injected cell number (Taswell, C. J. Immunol. 1981,126 : 1614-1619).

In vitro assays : 1. Long-term-culture-Initiating cells (LTC-IC) -The cell population to be tested is plated on stroma feeder cells and hematopoietic progenitor cells are quantified after 5 weeks. This in vitro assay reveals a cell population closest to the stem cells identified in repopulation assays.

(Taking into consideration the higher frequency and the relative facility of LTC-ICs transduced with viral vectors, they seem to be less primitive cells than LTR-HSCs).

2. Cobblestone area forming cells (CAFC) -The cell population of interest is plated on stroma cells and the cultures scored for light-dense colonies called"cobble-stone areas"7-35 days after plating. This assay reveals a population resembling LTC-IC, but is much less quantitative.

3. Cobblestone area forming cell-limiting dilution (CAFC-LD) The cell population to be tested is plated on stromal cells in a limiting dilution mode by varying the number of inoculated cells per culture over a wide range. Cultures containing no"cobblestone area"were counted each week and from the percentage of negative cultures in relation to the number of plated cells the number of CAFC can were accurately determined. CAFC producing late appearing cobblestone areas (28-35 days) are considered to represent the more primitive stem cells, whereas early appearing cobblestone areas (7-14 days) reflect the presence of less primitive stem and progenitor cells. (Breems D. A. , et al. Leukemia 1994,8 : 1095-1104).

Isolation and Culturing of HSCs ex vivo Isolation: Sca-1+ cells were separated form total bone marrow cells by Ficoll gradient and Sca-1+- cell labeling by anti-Sca-1-PE antibodies. Cells were then incubated with anti-PE antibodies

coupled to microbeads and passaged through a magnetic column. The positive population remaining in the column was collected. Cells were then labeled for lineage markers, Sca-1 and c- kit, and Sca-1+/lini/c-kit+ cells were sorted by FACS.

Culturing: isolated cells were cultured in the presence of stromal cells, in the following medium: alpha-MEM medium containing 12.5% FCS (Stem Cell Inc. ), 12.5% HS (Stem Cell Inc.), 100U/ml Penicillin, 0. 1 mM ß-mercaptoethanol. Half the medium was changed once weekly.

Example 3 Experimental results Expression pattern Hybridization on the MHB DNA array using probes from different hematopoietic organs in the mouse embryo (as detailed in Example 1) was performed in three independent experiments, and the following results were obtained: CTLA2alpha was found to be up-regulated in the lin-/sca-1+/c-kit+/CD34-long-term repopulating (LTR) cell and strongly up-regulated in the linlsca-1+/c-kit+/CD34+ short-term repopulating (STR) cell population as compared with the more primitive lin% sca-1+/c-kit-/CD34- population ; it was also found to be down-regulated in the more mature lin/sca-1+/c-kit/CD34+cell population.

Hybridization of the microsomal probe MBR on the MHB array (MS-5 stromal cells) indicates that CTLA2alpha is expressed in this stromal cell line and that it is membranal or secreted. The microsomal probe derived from ES cells showed no differential. 34-K-34-K-34-K-34-K+ 34-K+ 34-K+ 34+K+ 34+K+ 34+K+ 34+K-34+K-34+K-lin-MBR MBR (1) (2). (3) (1) (2) (3) (1) (2) (3) (1) (2) (3) ES MS-5 1. 3 1 1. 5 2.2 2. 7 2.6 3.2 7 6.1 1.7 2 1.6 2. 3 1. 4 5 Table 2: hybridization results on the MHB array.

In addition, hybridizations were conducted on the MGD microarray. CTLA2alpha was found to be strongly up-regulated in MS-5 cells treated with hydrocortisone, (this treatment has a strong effect on maintenance of HSC). CTLA2alpha is also strongly expressed in the HSC supporting FBMD-1 cell line in the presence of hydrocortisone. The cell line 14 F1. 1 does not regulate CTLA2alpha expression. MS-5 FBMD-1 14F1. 1 MBA13 cont HS-HS-HC-HC-Hyp cont HS-HS-HC-HC-Hyp cont HS-HS-HC-HC-hypo sub cont HS-HC-sub ld 7d ld 7d ox ld 7d ld 7d ox ld 7d ld 7d x 7d 7d 1 -1.2 1.3 3.3 3.7 -1.1 7.6 3.2 7.4 3.4 5.4 4.3 -1.3 -1.1 -1. 4-1. 1 -1.1 1.1 -1.5 -1.6 -1.6 -1.8 -1.9

Table 3: results of hybridization to the MGD array.

The same hybridization series was subsequently repeated on the MST microarray. The same clone was printed several times on this chip. Although these hybridizations were conducted with an independently obtained series of probes, the results proved highly similar to those obtained on the MGD array. MS-5 FBMD-1 14F1. 1 MBA13 cont HS-HS-HC-HC-Hyp cont HS-HS-HC-HC-Hyp cont HS-HS-HC-HC-hypo sub cont HS-HC-sub ld 7d ld 7d ox ld 7d ld 7d ox ld 7d ld 7d x 7d 7d 1 1.2 2.4 5.4 12.8 -1.0 16.9 4.6 15.3 9.2 17. 8 9.5-1. 5-1. 1-1. 4 1. 1-1. 7 1. 2-1. 6-2. 3-2. 1-2. 1-2.4 Table 4: results of hybridization to the MST array.

RT-PCR RT-PCR was performed on MS-5 and FBMD-1 stromal cell lines after different treatments.

Specific primers were used to determine CTLA2alpha and CTLA2beta expression separately. All results are normalized to a 7 day-old control of the same sample.

MS-5 FBMD-1 14F1. 1 CTLA2a CTLA2 CTLA20t CTLA2 CTLA2a CTLA2p Number of cycles 30 35 25 30 30 35 Control 7d 0 BFGF I d 0-----0-- BFGF 7d Hydrocortisone 1d + - 0 0 ++++ - Hydrocortisone 7d + + 0 0 ++++ 0 LIF ld ++++ LIF 7d ++++ Horse Serum 1d 0 ++ 0 - +++ -- Horse Serum 7d 0 +++ + +++ N.D. ++++ Cyclophosph. ld 0 1 0 N. D. N. D. Cyclophosph. 7d G-CSF ld G-CSF 7d Hypoxia (16 hrs) 0 0 0 0 ++ - Irradiation (300 rad) Table 5: CTLA-2a and CTLA-2ß expression in stromal cell lines after different treatments.

The expression levels for each gene are compared to"control 7d"in the same stromal cell line.

+ up-regulated-down-regulated 0 unchanged A large differential was observed in the expression level of CTLA2 in different stromal cell lines:

No. of MS-5 FBMD-1 14F1. 1 MBA13 cycles CTLA2a300+++++++'--co CTLA2 (3 35 0 +-H-++--------o0 + up-regulated-down-regulated 0 unchanged Table 6: levels of expression of CTLA2 in different stromal lines (RT-PCR).

The expression levels for each gene are compared to MS-5 (control-7 days).

The RT-PCR results confirm the results obtained in the hybridizations. As the mRNA of CTLA2 alpha/beta is expressed at a very low level in the 14F1. 1 cell line, the regulation of the gene was not measurable in the hybridizations. As CTLA2alpha and CTLA2beta are highly homologous, it is likely that the 2 proteins possess largely overlapping biological functions, and can thus both be suitable for HSC maintenance.

In situ hybridization At 11.5 and at 12.5 dpc a hybridization signal was detected that localizes to the luminal (endothelial) and perivascular mesenchymal cells in some blood vessels throughout the embryonic body. Thus, results of the embryonic in-situ hybridization point to the possible involvement of the CTLA2alpha gene product in hematopoiesis in the liver, tissue remodeling associated with placenta formation and embryonic angiogenesis.

Adult tissues show little hybridization signal. This signal locates to the most apical cells of the surface epithelium of the stomach, tubular epithelium of the renal cortex and skeletal muscle. A weak signal is associates with lymphocytes in the spleen and thymic medulla.

In vitro and in vivo validation 1. CTLA2alpha-flag was cloned into the retroviral vector pLNCX, MS-5, FBMD-1 cells were infected and over-expressing cells selected to produce stably expressing lines. For control, MS-5 and FBMD-1 cells were infected with the empty retroviral vector (mock-infected).

2. MS-5 cells and FBMD-1 stably over-expressing the full length CTLA2alpha-flag protein (as verified by western blot analysis) were used in LTC-IC assays: Long term cultures were

grown with sorted sca-1+/lin~/c-kit+ cells in the presence of hydrocortisone on CTLA2alpha over-expressing and control (mock-infected) cells.

3. In vivo assays were performed by maintenance of the cells during one or two weeks on CTLA2alpha over-expressing MS-5 or FBMD-1 cells. HSC and stromal cells were then collected and injected into irradiated mice for a re-population assay. Control cells were maintained and assayed identically, with the exception of being grown on stromal cells that do not over-express CTLA2alpha.

Results In-vitro validation : Two independent LTC-IC experiments were performed on FBMD-1 stromal cells overexpressing CTLA-2alpha (harvested after 4 weeks): Numbers are expressed as percent of control (compared to MS-5 containing empty vector) BFU-E CFU-GM CFU-GEMM Total colonies CTLA-2alpha--101. 728 980. 392 181. 4433 -- 75. 2542 56.2092 71.82426 average--88. 49132 518.3007 126.6338 Standard deviation--18. 72006 653.4961 77.51237 Table 7: LTC-IC performed on FBMD-1 Three independent LTC-IC experiments were performed on MS-5 stromal cells overexpressing CTLA-2alpha (harvested after 5 weeks): Numbers are expressed as percent of control (compared to MS-5 containing empty vector) BFU-E CFU-GM CFU-GEMM Total colonies CTLA-2alpha 100 78. 0083 37. 5 76. 8 --243. 827 263.636 245.0867 300 0 276.5217 average--207. 2785 100.3788 199.4695 Standard deviation--115. 4207 142.6231 107.3913 Table 8: LTC-IC performed on MS-5 In three out of five experiments performed, the presence of CTLA-2alpha strongly improves the maintenance of HSC (1.8-2. 7 times)

Example 4 Gene Therapy By gene therapy as used herein refers to the transfer of genetic material (e. g DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype. The genetic material of interest encodes a product (e. g. a protein, polypeptide, peptide, functional RNA, antisense fragment, GIE) the production of which in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value. Alternatively, the genetic material of interest may encode a suicide gene. For a review see, in general, the text"Gene Therapy" (Advances in Pharmacology 40, Academic Press, 1997).

Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy. In ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vitro. Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc. ) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically reimplanted cells have been shown to express the transfected genetic material in situ.

In in vivo gene therapy, target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ, that is within the recipient. In an alternative embodiment, if the host gene is defective, the gene is repaired in situ (Culver, 1998. Site-Directed recombination for repair of mutations in the human ADA gene.

(Abstract) Antisense DNA & RNA based therapeutics, February, 1998, Coronado, CA). These genetically altered cells have been shown to express the transfected genetic material in situ.

The gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell. The expression vehicle can include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5'UTR and/or 3'UTR of the gene can be replaced by the 5'UTR and/or 3'UTR of the expression vehicle. Therefore as used herein the expression vehicle can, as needed, not include the 5'UTR and/or 3'UTR of the actual gene to be transferred and only include the specific amino acid coding region.

The expression vehicle can include a promotor for controlling transcription of the heterologous material and can be either a constitutive or inducible promotor to allow selective transcription.

Enhancers that can be required to obtain necessary transcription levels can optionally be included.

Enhancers are generally any non-translated DNA sequence which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. The expression vehicle can also include a selection gene as described herein below.

Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art. Such methods can be found generally described in Sambrook et al., Molecular Cloning : A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, MI (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, MI (1995), Vectors : A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see United States patent 4,866, 042 for vectors involving the central nervous system and also United States patents 5,464, 764 and 5,487, 992 for positive- negative selection methods.

Introduction of nucleic acids by infection offers several advantages over the other listed methods.

Higher efficiency can be obtained due to their infectious nature. Moreover, viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events.

A specific example of DNA viral vector for introducing and expressing recombinant sequences is the adenovirus derived vector Adenop53TK. This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences. This vector can be used to infect cells that have an adenovirus receptor which includes most cancers of epithelial origin as well as others. This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vitro or ex vivo culture of cells, a tissue or a human subject.

Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy. Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus. An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation can not occur.

Features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired cell type.

In addition, recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. The vector to be used in the methods of the invention depends on desired cell type to be targeted and is known to those skilled in the art. For example, if breast cancer is to be treated then a vector specific for such epithelial cells is used. Likewise, if diseases or pathological conditions of the hematopoietic system are to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, is used.

Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection. In the former case, the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles which are capable of undergoing further rounds of infection. The vector's genome is also engineered to encode and express the desired recombinant gene. In the case of non-infectious viral vectors, the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed do not contain a genome and therefore cannot proceed through subsequent rounds of infection. The specific type of vector depends upon the intended application.

The actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.

The recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a

quicker and more effective treatment, and administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neuro-degenerative diseases. Following injection, the viral vectors circulate until they recognize host cells with the appropriate target specificity for infection.

An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid. Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement required with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal. Such non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like. Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known by one skilled within the art.

Example 5 Pharmacolosv and drug delivery The medicament or pharmaceutical composition of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the disease to be treated, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.

The pharmaceutically"sufficient dose"for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.

The treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated. It is noted that humans are treated generally longer than the mice or other experimental animals exemplified herein.

The medicament or pharmaceutical composition of the present invention can be administered by any of the conventional routes of administration. It should be noted that it can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or as an

active ingredient in combination with pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles. The medicament or pharmaceutical composition can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques. Implants of the medicament or pharmaceutical composition are also useful. Liquid forms may be prepared for injection, the term including subcutaneous, transdermal, intravenous, intramuscular, intrathecal, and other parental routes of administration. The liquid compositions include aqueous solutions, with and without organic cosolvents, aqueous or oil suspensions, emulsions with edible oils, as well as similar pharmaceutical vehicles. In addition, under certain circumstances the compositions for use in the novel treatments of the present invention may be formed as aerosols, for intranasal and like administration. The patient being treated is a warm- blooded animal and, in particular, mammals including man. The pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention.

When administering the medicament or pharmaceutical combination of the present invention parenterally, it is generally formulated in a unit dosage injectable form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

Nonaqueous vehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, can also be used as solvent systems for compound compositions. Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it is desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.

A pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres. Examples of delivery systems useful in the present invention include U. S. Patent Nos. 5,225, 182; 5,169, 383; 5,167, 616; 4,959, 217; 4,925, 678; 4,487, 603; 4,486, 194; 4,447, 233; 4,447, 224; 4,439, 196; and 4,475, 196. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

A pharmacological formulation of the compound utilized in the present invention can be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable.

Known techniques which deliver it orally or intravenously and retain the biological activity are preferred. In one embodiment, the compound of the present invention can be administered initially by intravenous injection to bring blood levels to a suitable level. The patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition and as indicated above, can be used.

In general, the active dose for humans is in the range of from lng/kg to about 20-100 mg/kg body weight per day, preferably about 0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of one dose per day or twice or three or more times per day for a period of 1-2 weeks or longer, preferably for 24-to 48 hrs or by continuous infusion during a period of 1-2 weeks or longer.

It will be appreciated that the most appropriate administration of the pharmaceutical compositions of the present invention will depend on the type of injury or disease being treated. Thus, the treatment of an acute event will necessitate systemic administration of the active composition comparatively rapidly after induction of the injury. On the other hand, diminution of chronic degenerative damage may necessitate a sustained dosage regimen.

Example 6 Preparation of polypeptides Polypeptides may be produced via several methods, for example: 1) Synthetically;

Synthetic polypeptides can be made using a commercially available machine, using the sequence of the CTLA2alpha and/or CTLA2beta polypeptide, as described in Figure 1.

2) Recombinant Methods: A preferred method of making CTLA2alpha and/or CTLA2beta is to clone the cDNA or a fragment thereof of the gene, as described in Figure 1 or 2, into an expression vector and culture the cell harboring the vector so as to express the encoded polypeptide, and then purify the resulting polypeptide, all performed using methods known in the art (Bibl Haematol.

1965 ; 23 : 1165-74 Appl Microbiol. 1967 Ju1 ; 15 (4) : 851-6 ; Can JBiochem. 1968 May ; 46 (5) : 441-4 ; Biochemistry. 1968 Jul;7(7):2574-80; Arch Biochem Biophys. 1968 Sep 10;126(3):746-72 ; Biochem Biophys Res Commun. 1970 Feb 20 ; 33 (4) : 825-30).

The expression vector can include a promoter for controlling transcription of the heterologous material and can be either a constitutive or inducible promoter to allow selective transcription.

Enhancers that can be required to obtain necessary transcription levels can optionally be included.

The expression vehicle can also include a selection gene.

Vectors can be made and subsequently introduced into cells or tissues by any one of a variety of methods known within the art. Such methods can be found generally described in Sambrook et al., Molecular Cloning : A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989,1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Vega et al., Gene Targeting, CRC Press, Ann Arbor, MI (1995), Vectors. A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al. (1986).

3) Polypeptides (such as CTLA2alpha and/or CTLA2beta) can be purified from natural sources (such as tissues or cultured stromal cells) using many methods known to one of ordinary skill in the art, such as for example: immuno-precipitation, or matrix-bound affinity chromatography with any molecule known to bind the desired polypeptide.

Protein purification is practiced as is known in the art as described in, for example, Marshak et al., "Strategies for Protein Purification and Characterization. A laboratory course manual."CSHL Press (1996).

Example 7 Preparation of polvnucleotides The polynucleotides of the subject invention can be constructed by using a commercially available DNA synthesizing machine; overlapping pairs of chemically synthesized fragments of the desired gene can be ligated using methods well known in the art (e. g. , see U. S. Patent No.

6,121, 426) and, for example, the nucleotide sequence described in Figure 1 or 2.

Another means of isolating a polynucleotide is to obtain a natural or artificially designed DNA fragment based on that sequence. This DNA fragment is labeled by means of suitable labeling systems which are well known to those of skill in the art; see, e. g. , Davis et al. (1986). The fragment is then used as a probe to screen a lambda phage cDNA library or a plasmid cDNA library using methods well known in the art; see, generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Colonies can be identified which contain clones related to the cDNA probe and these clones can be purified by known methods. The ends of the newly purified clones are then sequenced to identify full-length sequences. Complete sequencing of full-length clones is performed by enzymatic digestion or primer walking.

A similar screening and clone selection approach can be applied to clones from a genomic DNA library.

Example 8 Preparation of antibodies Antibodies which bind to the CTLA2alpha and/or CTLA2beta polypeptide may be prepared using an intact polypeptide or fragments containing smaller polypeptides as the immunizing antigen. For example, it may be desirable to produce antibodies that specifically bind to the N-or C-terminal or any other suitable domains of the CTLA2alpha and/or CTLA2beta polypeptide.

The polypeptide used to immunize an animal can be derived from translated cDNA or chemical synthesis which can be conjugated to a carrier protein, if desired. Such commonly used carriers which are chemically coupled to the polypeptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA) and tetanus toxoid. The coupled polypeptide is then used to immunize the animal. Methods of immunization, including all necessary steps of preparing the immunogen in a suitable adjuvant, determining antibody binding, isolation of

antibodies, methods for obtaining monoclonal antibodies, and humanization of monoclonal antibodies are all known to the skilled artisan If desired, polyclonal or monoclonal antibodies can be further purified, for example by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound. Those skilled in the art know various techniques common in immunology for purification and/or concentration of polyclonal as well as monoclonal antibodies (see, for example, Coligan et al, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994).

Methods for making antibodies of all types, including fragments, are known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988) ). Methods of immunization, including all necessary steps of preparing the immunogen in a suitable adjuvant, determining antibody binding, isolation of antibodies, methods for obtaining monoclonal antibodies, and humanization of monoclonal antibodies are all known to the skilled artisan.

The antibodies may be humanized antibodies or human antibodies. Antibodies can be humanized using a variety of techniques known in the art including CDR-grafting (EP239, 400: PCT publication WO. 91/09967; U. S. patent Nos. 5,225, 539; 5,530, 101; and 5,585, 089, veneering or resurfacing (EP 592,106 ; EP 519,596 ; Padlan, Molecular Immunology 28 (4/5): 489-498 (1991); Studnicka et al. , Protein Engineering 7 (6): 805-814 (1994); Roguska et al. , PNAS 91: 969-973 (1994) ), and chain shuffling (U. S. Patent No. 5,565, 332).

The monoclonal antibodies as defined include antibodies derived from one species (such as murine, rabbit, goat, rat, human, etc. ) as well as antibodies derived from two (or more) species, such as chimeric and humanized antibodies.

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences.

See also U. S. Patent Nos. 4,444, 887 and 4,716, 111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, each of which is incorporated herein by reference in its entirety.

Additional information regarding all types of antibodies, including humanized antibodies, human antibodies and antibody fragments can be found in WO 01/05998, which is incorporated herein by reference in its entirety.

Example 9 Screening systems The CTLA2alpha and/or CTLA2beta gene or polypeptide may be used in a screening assay for identifying and isolating compounds which inhibit or stimulate stem cell proliferation. The compounds to be screened comprise inter alia substances such as small chemical molecules, antibodies, antisense oligonucleotides, antisense DNA or RNA molecules, siRNAs, ribozymes, polypeptides and dominant negatives, and expression vectors.

Many types of screening assays are known to those of ordinary skill in the art. The specific assay which is chosen depends to a great extent on the activity of the candidate gene or the polypeptide expressed thereby. Thus, if it is known that the expression product of a candidate gene has enzymatic activity, then an assay which is based on inhibition (or stimulation) of the enzymatic activity can be used. If the candidate polypeptide is known to bind to a ligand or other interactor, then the assay can be based on the inhibition of such binding or interaction. When the candidate gene is a known gene, such as CTLA2alpha and/or CTLA2beta, then many of its properties can also be known, and these can be used to determine the best screening assay. If the candidate gene is novel, then some analysis and/or experimentation is appropriate in order to determine the best assay to be used to find inhibitors of the activity of that candidate gene. The analysis can involve a sequence analysis to find domains in the sequence which shed light on its activity.

As is well known in the art, the screening assays can be cell-based or non-cell-based. A cell-based assay is performed using eukaryotic cells such as HeLa cells, or possibly stem cells or stromal cells, and such cell-based systems, if employed, offer a possibility of directly measuring the activity of candidate genes which are stem cell proliferation related genes, such as the CTLA2alpha and/or CTLA2beta gene. One way of running such a cell-based assay uses tetracycline-inducible (Tet- inducible) gene expression. Tet-inducible gene expression is well known in the art; see for example, Hofmann et al, 1996, Proc Natl Acad Sci 93 (11): 5185-5190.

Tet-inducible retroviruses have been designed incorporating the Self-inactivating (SIN) feature of a 3'Ltr enhancer/promoter retroviral deletion mutant. Expression of this vector in cells is virtually

undetectable in the presence of tetracycline or other active analogs. However, in the absence of Tet, expression is turned on to maximum within 48 hours after induction, with uniform increased expression of the whole population of cells that harbor the inducible retrovirus, thus indicating that expression is regulated uniformly within the infected cell population.

If the gene product of the candidate gene phosphorylates with a specific target protein, a specific reporter gene construct can be designed such that phosphorylation of this reporter gene product causes its activation, which can be followed by a color reaction. The candidate gene can be specifically induced, using the Tet-inducible system discussed above, and a comparison of induced versus non-induced genes provides a measure of reporter gene activation.

In a similar indirect assay, a reporter system can be designed that responds to changes in protein- protein interaction of the candidate protein. If the reporter responds to actual interaction with the candidate protein, a color reaction occurs.

One can also measure inhibition or stimulation of reporter gene activity by modulation of its expression levels via the specific candidate promoter or other regulatory elements. A specific promoter or regulatory element controlling the activity of a candidate gene is defined by methods well known in the art. A reporter gene is constructed which is controlled by the specific candidate gene promoter or regulatory elements. The DNA containing the specific promoter or regulatory agent is actually linked to the gene encoding the reporter. Reporter activity depends on specific activation of the promoter or regulatory element. Thus, inhibition or stimulation of the reporter is a direct assay of stimulation/inhibition of the reporter gene; see, for example, Komarov et al (1999), Science vol 285, 1733-7 and Storz et al (1999) Analytical Biochemistry, 276,97-104.

Various non-cell-based screening assays are also well within the skill of those of ordinary skill in the art. For example, if enzymatic activity is to be measured, such as if the candidate protein has a kinase activity, the target protein can be defined and specific phosphorylation of the target can be followed. The assay can involve either inhibition of target phosphorylation or stimulation of target phosphorylation, both types of assay being well known in the art; for example see Mohney et al (1998) J. Neuroscience 18,5285 and Tang et al (1997) J Clin. Invest. 100, 1180 for measurement of kinase activity. It is possible that CTLA2alpha and/or CTLA2beta interact with an enzyme and regulate its enzymatic activity through protein-protein interaction. In addition, it is possible that CTLA2alpha and/or CTLA2beta inhibit cysteine proteases (see above); this function can also be used to construct a non-cell based screening system relying on said inhibition as a measurable output.

One can also measure in vitro interaction of a candidate polypeptide with interactors. In this screen, the candidate polypeptide is immobilized on beads. An interactor, such as a receptor ligand, is radioactively labeled and added. When it binds to the candidate polypeptide on the bead, the amount of radioactivity carried on the beads (due to interaction with the candidate polypeptide) can be measured. The assay indicates inhibition of the interaction by measuring the amount of radioactivity on the bead.

Any of the screening assays, according to the present invention, may optionally include a step of identifying the chemical compound or other moiety (as described above) which tests positive in the assay and can also include the further step of producing as a medicament that which has been so identified. It is considered that medicaments comprising such compounds, or chemical analogs thereof, are part of the present invention. The use of any such compounds identified for inhibition or stimulation of stem cell proliferation, is also considered to be part of the present invention.