WO1996022693A9 - Self-renewing pluripotent hematopoietic stem cell compositions, methods of use, and culture systems therefor - Google Patents

Abstract

Disclosed are compositions comprising undifferentiated pluripotent hematopoietic stem cells, and methods for long-term maintenance of pluripotent hematopoietic stem cell lines. Novel compositions for controlling the differentiation of these cells into particular cell types are also provided. These compositions (including antibodies against F factor and hematopoietic stem cell surface antigens) and methods have application in bone marrow transplantation, cancer therapy and diagnosis, somatic gene therapy, and treatment of leukemias and other immunohematocompromising disorders. Also disclosed are novel nucleic acid segments and proteins comprising the mammalian A3 nuclear-envelope-associated protein, and antibodies against the mammalian transmembrane glycoprotein CD98, a protein which plays an important role in the development of hematopoietic cells.

Description

DESCRIPTION

SELF-RENEWING PLURIPOTENT HEMATOPOIETIC STEM CELL COMPOSITIONS, METHODS OF USE, AND CULTURE SYSTEMS THEREFOR

BACKGROUND OF THE INVENTION

The present application is a continuation-in-part of U.S. Serial No. 08/378, 144, filed January 24, 1995, which is a continuation-in- part of U.S. Serial No. 08/462, 108, filed June 5, 1 995. The entire text and figures of which disclosures are specifically incorporated herein by reference without disclaimer.

A. Field of the Invention

The present invention relates generally to the field of molecular biology, and in particular, maintaining undifferentiated hematopoietic precursor cells in culture. More particularly, it concerns compositions comprising particular cytokine combinations for use in the culturing and maintenance of undifferentiated, pluripotent hematopoietic stem cells. These compositions (including antibodies against F factor and hematopoietic stem cell surface antigens) and methods have application in bone marrow transplantation, cancer therapy and diagnosis, somatic gene therapy, and treatment of leukemias and other immunohematocompromising disorders. Nucleic acid segments and proteins comprising the mammalian A3 nuclear-envelope-associated protein, and antibodies recognizing the mammalian transmembrane glycoprotein CD98, which plays an important role in the development of hematopoietic cells, are also disclosed.

B. Description of the Related Art

Because all mature blood cells have a limited life span, they must therefore be continuously generated by less-differentiated precursor cells with various degrees of proliferative capability throughout life. This process is called hematopoiesis (Metcalf and Moore, 1971 ).

1. Hematopoietic Cells

Hematopoietic cells can be arbitrarily divided in three main compartments: The first is constituted by pluripotent stem cells that can give rise to new stem cells (self-renew) and generate progenitor cells for all blood cell types. The second compartment consists of progenitor cells that exhibit less self-renewal capability and more- restricted precursor potential, i.e. they are oligo-, bi- or mono-potent precursors. The third compartment is constituted by mature blood cells, most of which no longer possess self-renewal capabilities and which exert specialized functions.

The existence of hematopoietic stem cells in the bone marrow of adult mice and in the fetal liver has been demonstrated by several groups (Wu et al. , 1967; Abramson et al. , 1977; Spangrude et al. , 1988; Keller and Snodgrass, 1990; Jordan et al. , 1990), but procedures designed to enrich stem cell populations have only recently been developed (see e.g. , Spangrude, 1 989; Visser and von Bekkum, 1990). 2. Limited Availability of Pluripotent Hematopoietic Stem Cells

The fact that pluripotent hematopoietic stem cells (PHSC) give rise to both new stem cells (self-renewal) and all blood cell types makes them very unique. Unfortunately, the extremely low number of these cells in primary hematopoietic organs and the lack of culture systems that support proliferation of undifferentiated PHSC have both precluded the study of the biology of these cells and their exploitation in clinical applications.

Moreover, determination of the molecular characteristics of PHSC and how the self-renewal vs. differentiation choice has not been possible. This shortcoming of the prior art has also prevented the development of effective and safe gene-transfer protocols for somatic gene therapy involving PHSC.

Although PHSC can be enriched, the numbers obtained are very low (reviewed in Spangrude and Scollay, 1 990; Visser and van Bekkum, 1 990; Jordan et al. , 1990; Uchida et al. , 1 993) .

Additionally, there currently are no specific surface markers and no single in vitro assay for PHSC. The single most-limiting cause for this is the fact that no culture conditions presently exist which permit the large-scale culture of these cells without their differentiation into other cell types.

Purified PHSC are still heterogeneous in terms of size, cell cycle status and Rh 1 23 staining (Li and Johnson, 1 992; Harrison and Zhong, 1 992). The only way to generate homogeneous PHSC in large number is to establish culture conditions which would support proliferation without differentiation, but unfortunately no such culture conditions currently exist.

Despite the fact that several research groups have tested cytokines in various combinations for their capacity to support proliferation of PHSC from mice and man (e.g. , Migliaccio et al. , 1991 ; Musashi et al. , 1991 ; Lowry et al. , 1991 ; Leary et al. , 1992; Fletcher et al. , 1991 ; Lyman et al. , 1993), in all cases proliferation was associated with differentiation usually along the myeloid/erythroid lineages. Thus, the continuous proliferation of undifferentiated PHSC in short or long-term culture has not been achieved.

If the division of a true stem cell always gives rise to different daughter cells, growth and differentiation cannot be segregated.

However, the fact that even less-differentiated totipotent cells such as the embryonic stem cells derived from the inner cell mass have been successfully grown in long-term culture with limited or no differentiation with appropriate growth factors (Martin, 1981 ; Robertson, 1 987) supports the view that proliferation without differentiation of PHSC could be achieved provided the appropriate culture conditions are found.

3. The Lack of Continuously-Proliferating Non-Transformed PHSC Cell Lines Limits Somatic Gene Therapy

Although continuously-proliferating non-transformed cell lines have been isolated from progenitor cells (the second compartment of hematopoietic cells) including oligopotent myeloid-restricted progenitor lines (Greenberger et al. , 1983; Spooncer et al. , 1 986), progenitor clones able to generate B-lymphocytes (Palacios et al. , 1987; Palacios et al. , 1 989; Kathoh et al. , 1990), monopotent Pro-B lymphocyte progenitor clones (Palacios and Steinmetz, 1985; McKearn et al. , 1986; Palacios and Samaridis, 1992) and monopotent Pro-T lymphocyte progenitor clones (Palacios and Pelkonen, 1 988), no continuously-proliferating non-transformed PHSC cell lines have ever been reported.

Several fundamental issues in hematopoiesis, particularly those concerning early stages of blood cell formation, remain unknown. For instance, nothing is known about the processes by which stem cells are formed, self-renew, or differentiate. It is also not known whether stem cells differentiate into lymphoid or myeloid progenitors directly or if they do it in a progressive manner, nor whether this is achieved in a stochastic or instructive mechanism, or a combination of both.

Neither is it understood how a given stem cell, or its putative immediate multipotent progeny, develop into particular cell lineages but not others (i.e. , how the cells become committed to develop along a given pathway) . The isolation of large quantities of PHSC has been technically very difficult because very few stem cells and their immediate progeny are present in bone marrow or fetal liver cells. This limitation has also hampered the molecular characterization and isolation of specific PHSC surface markers.

Currently, PHSC and their immediate progeny can only be properly identified by functional tests. No single assay exists for these cells, making a number of functional tests required for unambiguous classification of cells at such early stages of hematopoietic development.

4. Improvements Needed Over the Prior Art IL 3, LIF, FLT3-lig., and Steel Factor have previously been reported to have direct or indirect effects on PHSC (Lowry et al. , 1991 ; Fletcher et al. , 1991 ; Musashi et al. , 1991 ; Migliaccio et al. , 1991 ; Lyman et al. , 1993). Attempts to use known cytokines such as IL 1 to IL 12, TNF, TGF, FLT3-ligand, GM-CSF, M-CSF, G-CSF, activin, fibroblast growth factor to prevent cell differentiation have been unsuccessful.

The establishment of culture conditions that would allow the long-term proliferation, expansion and cloning of undifferentiated PHSC would constitute a breakthrough in the field and substantially improve the state of the art in fields such as hematology, immunology, cancer therapy, cell biology and clinical research. Indeed, it would revolutionize the fields of bone marrow transplantation, therapy of malignant and non-cancerous blood cell diseases and somatic gene therapy. It also would offer a unique opportunity to specifically stimulate or inhibit PHSC differentiation, and permit the identification of compounds that affect growth or differentiation of PHSC.

The identification of genes involved in self-renewal and differentiation of PHSC would facilitate marked improvements in diagnosis and therapy of leukemic or otherwise immunohematocompromised patients. SUMMARY OF THE INVENTION

1. Aspects of the Present Invention

The present invention overcomes one or more limitation of the prior art by providing for the first time undifferentiated PHSC hematopoietic cell lines and clones.

Also disclosed are methods for the culture of PHSC cell lines and clones which permit the long-term growth of such cells in undifferentiated state. Such methods have been demonstrated in murine cell lines, and are particularly important for permitting long- term growth of PHSC cell lines and clones from human and other animals.

Another important aspect of the present invention is the discovery of a soluble factor, termed F factor, which is produced by the FLS4.1 stromal cell line. This F factor is demonstrated herein to maintain PHSC in their undifferentiated state, and together with other cytokines, to support proliferation of undifferentiated PHSC. F activity (proliferation without differentiation of PHSC lines) was assayed following gel exclusion chromatography. The results indicate an apparent molecular weight of 1 5-45 kDa for F factor.

While FLS4.1 stromal cells were used to obtain F factor, other fetal stromal cell lines, or other similar cell lines would be expected to produce one or more factors of similar activity. Likewise, non- stromal cell lines may also produce active F factor. As used herein, "F factor" is understood to include one or more components which may contribute to the activity herein described. This composition may be obtained from intact cells, cell- free lysates, or culture medium.

While gel filtration was used to obtain an active fraction comprising F factor, other separation methods known to those of skill in the art may be used to isolate such active fractions. The determination of the molecular weight of F factor may vary in accordance with the particular method used for the determination of molecular weight.

Methods of the present invention also employ a specific combination of cytokines and F factor to permit proliferation (but not differentiation) of PHSC. By way of example, the method may be used to expand PHSC obtained from any tissue that contains these cells in limited numbers, such as fetal liver and bone marrow.

Methods for the isolation of murine and human cDNAs encoding F factor employing either a) direct cloning and expression systems or b) by constructing oligonucleotide probes from the N- terminal amino acid sequence of F factor and their use as probes for screening a cDNA library made from FLS4.1 stromal cells also constitute an important aspect of this invention.

A further aspect of the invention is the ability of undifferentiated PHSC (maintained in the presence of F factor) to reconstitute the hematopoietic system of hematocompromised animals. Successful restoration of the hematopoietic system of X- irradiated mice has been demonstrated using the methods disclosed herein. Continuously-proliferating PHSC cell lines and clones maintained according to the present invention have been preserved in their undifferentiated state, and have been able to regenerate and to provide a continuing stem cell population in the animal.

Because the invention achieves long-term growth of undifferentiated PHSC, it is now possible to identify surface antigens on such cells. This permits for the first time generation of antibodies against specific surface markers for PHSC.

It is also now possible to discover new genes expressed in PHSC as a result of the present invention. Genes involved in the growth and differentiation of PHSC as well as genes involved in the malignant transformation (Protooncogenes) or the suppression of tumor development (tumor suppressor genes); genes coding for new growth factor receptors, adhesion molecules, or their ligands expressed in PHSC can now be identified and molecularly cloned. Aside from the impact in basic research of PHSC physiology, early identification of aberrant genes by this method are contemplated to be useful in not only gene therapy, but in early detection and diagnosis of genetic disorders.

Using the compositions of the present invention, several PHSC cell lines and clones have been isolated. These cell lines and clones differentiate in vivo and in vitro onlv if induced.

Also provided are methods for generating antibodies specific for F factor. Such antibodies find utility in screening for F factor and crossreactive species. The present invention relates, in one aspect, to the molecular cloning and expression of a new gene called A3. A3 is related to the NIP1 gene product (Gu et al. , 1992) which is an essential protein required for nuclear transport in yeast. The sequence of A3 predicts a 53,598 Daltons polypeptide with one membrane-spanning region and two potential N-linked glycosylation sites, suggesting that A3 is a new mammalian integral membrane glycoprotein presumably involved in nuclear import of macromolecules.

Other aspects of the present invention concern isolated DNA segments and recombinant vectors, and the creation and use of recombinant host cells through the application of DNA technology, that express the gene products disclosed herein. As such, the invention concerns DNA segment comprising an isolated gene that encodes a protein or peptide that includes an amino acid sequence essentially as set forth by a contiguous sequence from SEQ ID NO:4. These DNA segments are represented by those that include a nucleic acid sequence essentially as set forth by a contiguous sequence from SEQ ID NO:3. Compositions that include a purified protein that has an amino acid sequence essentially as set forth by the amino acid sequence of SEQ ID NO:4 are also encompassed by the invention.

Regarding the novel protein A3, the present invention concerns DNA segments, that can be isolated from virtually any bacterial source, that are free from total genomic DNA and that encode proteins having A3-like activity. DNA segments encoding A3-like species may prove to encode proteins, polypeptides, subunits, functional domains, and the like. As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding A3 refers to a DNA segment that contains A3 coding sequences yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the term "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified A3 gene refers to a DNA segment including A3 coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, extra-genomic and plasmid- encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides. Such segments may be naturally isolated, or modified synthetically by the hand of man.

"Isolated substantially away from other coding sequences" means that the gene of interest, in this case, a gene encoding A3, forms the significant part of the coding region of the DNA segment,' and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that encode a species that includes within its amino acid sequence an amino acid sequence essentially as set forth in SEQ ID NO:4. In other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences that include within their sequence a nucleotide sequence essentially as set forth in SEQ ID NO:3.

The term "a sequence essentially as set forth in SEQ ID NO:4" means that the sequence substantially corresponds to a portion of SEQ ID NO:4 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:4. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and about 80%; or more preferably, between about 81 % and about 90%; or even more preferably, between about 91 % and about 99%; of amino acids that are identical or functionally equivalent to the amino acids of SEQ ID NO:4 will be sequences that are "essentially as set forth in SEQ ID NO:4" .

In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:3. The term "essentially as set forth in SEQ ID NO:3" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:3 and has relatively few codons that are not identical, or functionally equivalent, to the codons of SEQ ID NO:3. Again, DNA segments that encode proteins exhibiting A3-like activity will be most preferred.

It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various upstream or downstream regulatory or structural genes.

Naturally, the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:3. Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:3, under relatively stringent conditions such as those described herein. The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared that include a short contiguous stretch identical to or complementary to SEQ ID NO:3, such as about 14 nucleotides, and that are up to about 10,000 or about 5,000 base pairs in length, with segments of about 3,000 being preferred in certain cases. DNA segments with total lengths of about 2,000, about 1 ,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths) are also contemplated to be useful.

It will be readily understood that "intermediate lengths" , in these contexts, means any length between the quoted ranges, such as 14, 1 5, 16, 1 7, 1 8, 19, 20, etc. ; 21 , 22, 23, etc ; 30, 31 , 32, etc. ; 50, 51 , 52, 53, etc ; 100, 101 , 102, 103, etc ; 1 50, 1 51 , 1 52, 1 53, etc. ; including all integers through the 200-500; 500- 1 ,000; 1 ,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000 ranges, up to and including sequences of about 1 2,001 , 12,002, 1 3,001 , 13,002 and the like.

It will also be understood that this invention is not limited to the particular nucleic acid and amino acid sequences as disclosed in SEQ ID NO:3 and SEQ ID NO:4, respectively. Recombinant vectors and isolated DNA segments may therefore variously include the A3 coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include A3 coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompass biologically functional equivalent A3 proteins and peptides, in particular those A3 proteins isolated from prokaryotic sources, and particularly bacteria. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g. , to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g. , where the A3 coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g. , proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively) . Recombinant vectors form further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter. The promoter may be in the form of the promoter that is naturally associated with a A3 gene, as may be obtained by isolating the 5 ' non-coding sequences located upstream of the coding segment, for example, using recombinant cloning and/or PCR™ technology, in connection with the compositions disclosed herein.

In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a A3 gene in its natural environment. Such promoters may include A3 promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, organism, or even animal, chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al. , 1 989. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. Prokaryotic expression of nucleic acid segments of the present invention may be performed using methods known to those of skill in the art, and will likely comprise expression vectors and promotor sequences such as those provided by tac, trp, lac, lacilVδ or T7. When expression of the recombinant A3 proteins is desired in eukaryotic cells, a number of expression systems are available and known to those of skill in the art. An exemplary eukaryotic promoter system contemplated for use in high-level expression is the Pichia expression vector system (Pharmacia LKB Biotechnology).

In connection with expression embodiments to prepare recombinant A3 and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire A3 or functional domains, epitopes, ligand binding domains, subunits, etc. being most preferred. However, it will be appreciated that the use of shorter DNA segments to direct the expression of A3 peptides or epitopic core regions, such as may be used to generate anti-A3 antibodies, also falls within the scope of the invention. DNA segments that encode peptide antigens from about 1 5 to about 100 amino acids in length, or more preferably, from about 1 5 to about 50 amino acids in length are contemplated to be particularly useful.

In addition to their use in directing the expression of A3, the nucleic acid sequences disclosed herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 14 nucleotide long contiguous sequence of SEQ ID NO:3 will find particular utility. Longer contiguous identical or complementary sequences, e.g. , those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to homologous sequences will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so, identical or complementary to SEQ ID NO:3, are particularly contemplated as hybridization probes for use in, e.g. , Southern and Northern blotting. This would allow structural or regulatory genes to be analyzed, both in diverse cell types and also in various bacterial cells. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 14 and about 100 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect. The use of a hybridization probe of about 14-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 1 5 to 25 contiguous nucleotides, or even longer where desired.

Hybridization probes may be selected from any portion of any of the sequences disclosed herein. All that is required is to review the sequence set forth in SEQ ID NO:3 and to select any continuous portion of the sequence, from about 14-25 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a probe or primer. The choice of probe and primer sequences may be governed by various factors, such as, by way of example only, one may wish to employ primers from towards the termini of the total sequence.

The process of selecting and preparing a nucleic acid segment that includes a contiguous sequence from within SEQ ID NO:3 may alternatively be described as preparing a nucleic acid fragment. Of course, fragments may also be obtained by other techniques such as, e.g. , by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR™ technology of U.S. Patent 4,683,202 (incorporated herein by reference), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of the entire gene or gene fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g. , one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.1 5 M NaCl at temperatures of 50°C to 70°C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as about 0.1 5 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55 °C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G + C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc. ). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.

2. Cytokines

Although several groups have reported combinations of cytokines that apparently support proliferation of PHSC, in all cases this was accompanied by differentiation (specially along the myeloid cell lineages). Actually, it was not possible to rule out that the proliferating cells were the differentiated progeny of PHSC rather than the PHSC themselves.

The identification of combinations of soluble cytokines that will support growth but not differentiation of PHSC requires that both the number of cells, and any signs of differentiation of the proliferating cells be monitored. The first is determined by visual observation using an inverted microscope and by counting the number of viable cells. The latter is monitored by FACS analysis using monoclonal antibodies specific for erythroid (TER 1 1 9), myeloid (8C5, Mac-1 ,F4/80), T-lymphoid (JORO 75, JORO 30-8) and B-lymphoid (B-220) surface markers. Combinations of soluble cytokines (most of them in recombinant form) are tested for their capacity to support growth of cell sorter purified single PHSC in microplate wells and the positive cultures allowed to expand for 10- 20 days (this will require re-feeding of the cultures) to have enough cells to detect evidence for or against differentiation. Combinations of growth factors which support growth but not differentiation in a manner similar to F factor are then used to establish continuously-proliferating PHSC clones. In another embodiment of the cell culture system the characterized stromal cell lines previously developed will be included with F factor. In this case, stromal cells which have been exposed to irradiation or mitomycin C (to prevent DNA synthesis) are tested in the presence and the absence of exogenous growth factors.

The use of PHSC from mutant mice that lack the p53 gene the product of which is involved in the control of the cell cycle and of programmed cell death (apoptosis) (Kuerbitz et al. , 1992, Yonish- Rouach et al. , 1991 ) would present an important advantage. PHSC from p53 deficient mice should survive longer in culture and would probably have a lower threshold to enter into mitosis. P53 controls the G 1 to S phase transition of the cell cycle without affecting hematopoiesis (Kuerbitz et al. , 1 992, Donehower et al. , 1 992).

The culture of PHSC in medium containing Steel factor (c-Kit ligand), LIF, rIL 3 and F factor (supernatant from two-day confluent cultures of the FLS4.1 fetal liver stromal line supported their growth without differentiation. Cultures that received Steel factor and IL 3 show proliferation but most cells differentiate into erythroid/myeloid cells. Only cultures that received F factor or were carried out on monolayers of the FLS4.1 stromal cells have reproducibly supported proliferation of Thy 1 ⁺ PgP -1 ⁺ c-Kit ⁺B-220 JORO 75^" Mac-1 TER 1 1 9 cells without differentiation during a period of up to 6 months' observation. Moreover, several lines and clones have been established in culture using the combination of factors indicated above, and these cell lines have retained their phenotypic and functional properties of PHSC > 6 months. cDNAs encoding these factors are cloned in the pcDNA3 mammalian expression vector followed by transfection into X63 Ag8 myeloma cells by electroporation, and selection of transfectant producing stably high levels of the cytokines. These factors are tested both in short- and long-term culture of the cells to determine whether or not they have adverse effects on the functional potential of the cell lines. The efficiency of cultures initiated with soluble cytokines only is compared to those using monolayers of FLS4.1 stromal cells in terms of both short- and long-term cultures. By using the culture conditions that support best growth of undifferentiated PHSC, it is possible to establish additional PHSC cell lines by passing the cells into freshly prepared culture medium every 3-5 days.

3. Pluripotent Hematopoietic Stem Cells

Various criteria have been used to define a hematopoietic cell as a stem cell. As used in the description of the present invention, this term is used to define a cell that a) gives rise to all blood cell types and b) possesses extensive self-renewal which is manifested in the long-term ( > 6 months) repopulation of the hematopoietic system. Bone marrow cells and fetal liver cells with such properties were recently found to express Thy1 and Ly6A (previously called Sca-1 ) surface markers and to lack most lineage-restricted surface markers (e.g. , B-220 for B lymphocyte lineage cells; Mac-1 for myeloid lineage cells) (Spangrude et a/. , 1988; Jordan et al. , 1990).

4. Gel Filtration Chromatography of F Factor

The F factor of the present invention is particularly characterized as comprising a polypeptide exhibiting an apparent molecular weight of about 1 5- to about 45-kDa as determined by gel filtration column chromatography. However, it is, of course, generally understood by those of skill in the art that both the migration of a polypeptide using SDS/PAGE, and the mobility of a polypeptide using different sizing columns can vary with different experimental conditions. It will therefore be appreciated that under differing electrophoretic and chromatographic conditions, the molecular weight assignments quoted above may vary.

It will be further understood that certain of the polypeptides may be present in quantities below the detection limits of the

Coomassie brilliant blue staining procedure usually employed in the analysis of SDS/PAGE gels, or that their presence may be masked by an inactive polypeptide of similar M_r. Although not necessary to the routine practice of the present invention, it is contemplated that other detection techniques may be employed advantageously in the visualization of each of the polypeptides present within the growth factor. Immunologically-based techniques such as Western blotting using enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies are considered to be of particular use in this regard.

5. Production of Recombinant F Factor

For the expression of the gene encoding F factor, once suitable (full-length if desired) clone(s) are obtained, whether they be cDNA based or genomic, one may prepare an expression system for the recombinant preparation of F factor. The engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may be employed in the expression of F factor. F factor may be successfully expressed in eukaryotic expression systems with the production of active protein, however, it is envisioned that bacterial yeast or baculovirus expression systems may ultimately be preferred for the preparation of F factor for all purposes. The cDNA for F factor may be separately expressed in bacterial systems, with the encoded proteins being expressed as fusions with β-galactosidase, ubiquitin, Schistosoma japonicum glutathione S-transferase, and the like. It is believed that bacterial expression will ultimately have numerous advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained thereby.

It is proposed that transformation of host cells with DNA segments encoding F factor will provide a convenient means for obtaining biologically active protein. However, separate expression followed by reconstitution is also certainly within the scope of the invention. Both cDNA and genomic sequences are suitable for eukaryotic expression, as the host cell will, of course, process the genomic transcripts to yield functional mRNA for translation into protein.

It is similarly believed that almost any eukaryotic expression system may be utilized for the expression of F factor (e.g., baculovirus-based, glutamine synthase-based or dihydrofolate reductase-based systems) could be employed. For example, plasmid vectors incorporating an origin of replication and an efficient eukaryotic promoter, as exemplified by the eukaryotic vectors of the pCMV series, such as pCMV5, will be of most use. For expression in this manner, one would position the coding sequences adjacent to and under the control of the promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame of the protein between about 1 and about 50 nucleotides "downstream" of (i.e. 3' of) the chosen promoter.

Where eukaryotic expression is contemplated, one will also typically desire to incorporate into the transcriptional unit which includes the enzyme, an appropriate polyadenylation site (e.g., 5'-AATAAA-3') if one was not contained within the original cloned segment. Typically, the poly-A site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.

It is contemplated that virtually any of the commonly employed host cells can be used in connection with the expression of F factor in accordance herewith. Examples include cell lines typically employed for eukaryotic expression such as FLS4.1 , 239, X63Ag8, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK, COS-7, 558L, RIN and MDCK cell lines.

It is contemplated that the F factor polypeptide of the invention may be "overexpressed," i.e. , expressed in increased levels relative to its natural expression in FLS4.1 cells, or even relative to the expression of other proteins in the recombinant host cell. Such overexpression may be assessed by a variety of methods, including radiolabeling and/or protein purification. However, direct methods are preferred, for example, those involving SDS/PAGE and protein staining or Western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein or peptide in comparison to the level in natural FLS4.1 cells is indicative of overexpression, as is a relative abundance of the specific protein in relation to the other proteins produced by the host cell as determined by methods such as gel electrophoresis.

As used herein, the term "engineered" or "recombinant" cell is intended to refer to a cell into which an exogenous DNA segment or gene, such as a cDNA or gene encoding an F factor has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced exogenous DNA segment or gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a cDNA gene (i.e., they will not contain introns), a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.

Generally speaking, it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. However, the inventor does not exclude the possibility of employing a genomic version of a particular gene where desired. 6. Recombinant Host Cells and Vectors

Particular aspects of the invention concern the use of plasmid vectors for the cloning and expression of recombinant peptides, and particular peptide epitopes comprising either native, or site- specifically mutated epitopes. The generation of recombinant vectors, transformation of host cells, and expression of recombinant proteins is well-known to those of skill in the art. Prokaryotic hosts are preferred for expression of the peptide compositions of the present invention. An example of a preferred prokaryotic host is E. coli, and in particular, E. co/i strains JM101 , XL1 -Blue™, RR1 , LE392, B, X1776 (ATCC31 537), and W31 10 (F-, lambda-, prototrophic, ATCC273325). Alternatively, other Enterobacteriaceae species such as Salmonella typhimurium and Serratia marcescens, or even other Gram-negative hosts including various Pseudomonas species may be used in the recombinant expression of the genetic constructs disclosed herein.

In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli may be typically transformed using vectors such as pBR322, or any of its derivatives (Bolivar et al., 1 977) . pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins. In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as ΛGEM™-1 1 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.

Those promoters most commonly used in recombinant DNA construction include the Mactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979) or the tryptophan (trp) promoter system (Goeddel et al., 1 980). The use of recombinant and native microbial promoters is well-known to those of skill in the art, and details concerning their nucleotide sequences and specific methodologies are in the public domain, enabling a skilled worker to construct particular recombinant vectors and expression systems for the purpose of producing compositions of the present invention.

In addition to the preferred embodiment expression in prokaryotes, eukaryotic microbes, such as yeast cultures may also be used in conjunction with the methods disclosed herein. Saccharomyces cerevisiae, or common bakers^' yeast is the most commonly used among eukaryotic microorganisms, although a number of other species may also be employed for such eukaryotic expression systems. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1 979; Tschemper et al., 1 980) . This plasmid already contains the trpL gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977) . The presence of the trpL lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., 1 980) or other glycolytic enzymes (Hess et al., 1968; Holland et al., 1 978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose- 6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, an origin of replication, and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from muiticellular organisms may also be used as hosts in the routine practice of the disclosed methods. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years. Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al. , 1978) . Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the HindlU site toward the Bgl\ site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

The origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g. , Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

It will be further understood that certain of the polypeptides may be present in quantities below the detection limits of the

Coomassie brilliant blue staining procedure usually employed in the analysis of SDS/PAGE gels, or that their presence may be masked by an inactive polypeptide of similar M_r. Although not necessary to the routine practice of the present invention, it is contemplated that other detection techniques may be employed advantageously in the visualization of particular polypeptides of interest. Immunologically- based techniques such as Western blotting using enzymatically-, radiolabel-, or fluorescently-tagged antibodies described herein are considered to be of particular use in this regard. Alternatively, the peptides of the present invention may be detected by using antibodies of the present invention in combination with secondary antibodies having affinity for such primary antibodies. This secondary antibody may be enzymatically- or radiolabeled, or alternatively, fluorescently-, or colloidal gold-tagged. Means for the labeling and detection of such two-step secondary antibody techniques are well-known to those of skill in the art.

Prokaryotic hosts are preferred for expression of the F factor protein. An example of a prokaryotic host which is particularly useful is E. coli strain RR1 . Other strains of E. coli which are also useful include LE392, B, X1 776 (ATCC31 537), and W31 10 (F , λ , prototrophic, ATCC273325). Enterobacteriaceae species such as Salmonella typhimurium and Serratia marcescens, various Pseudomonas species, or Gram-positive bacilli such as Bacillus subtifis may also be used. In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322 (Bolivar et al. , 1977), or one of its many derivatives. pBR322 contains genes which express ampicillin and tetracycline resistance in Gram-negative hosts and thus provides a convenient means for identifying transformed cells. pBR322, its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbe for expression of endogenous proteins.

In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as ΛGEM-1 1™ may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.

Those promoters most commonly used in recombinant DNA construction include the Mactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1 979) or the tryptophan (trp) promoter system (Goeddel et al., 1 980; EPO Appl. Publ. No. 0036776). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (EPO Appl. Publ. No. 0036776). In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may also be used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1 979; Tschemper et al., 1980). This plasmid already contains the trpL gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC44076 or PEP4-1 (Jones, 1977). The presence of the trpL lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolytic enzymes (Hess et al., 1 968; Holland et al., 1 978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose- 6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, an origin of replication, and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years. Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et a/. , 1 978) . Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the /-///.dill site toward the Bgl\ site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

The origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g. , Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

7. Purification of F factor

Further aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of F factor. The phrase "purified F factor" as used herein, is intended to refer to a polypeptide composition, present in FLS4.1 supernatant, wherein the F factor is purified to any degree relative to its naturally-obtainable state, i.e., in this case, relative to its purity within the supernatant of FLS4.1 cell cultures. A purified F factor, therefore, also refers to isolated F factor, free from the environment in which it may naturally occur.

Generally, "purified" will refer to an F factor composition which has been subjected to fractionation to remove various non- polypeptide components, and which composition substantially retains its ability to support growth without differentiation of PHSC. Where the term "substantially purified" is used, this will refer to a composition in which F factor forms the major component of the composition, such as constituting from about 50% to about 60% of the protein in the composition or more. Various methods for quantifying the degree of purification of the F factor will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of an F factor fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial F factor source (e.g. , FLS4.1 supernatant, and to thus calculate the degree of purity, herein assessed by a "-fold purification number."

The actual units used to represent the amount of inhibitory activity will, of course, be dependent upon the particular assay technique chosen to follow the purification. As discussed above, the present inventor prefers to use an assay based upon the inhibition of hematopoietic stem cell differentiation.

As is generally known in the art, to determine the specific activity, one would calculate the number of units of activity per milligram of total protein. In the purification procedure, the specific activity of the starting material, i.e. , of the FLS4.1 culture supernatant containing F factor, would represent the specific activity of the F factor in its natural state. At each step, one would generally expect the specific activity of the F factor to increase above this value, as it is purified relative to its natural state. In preferred embodiments, it is contemplated that one would assess the degree of purity of a given F factor fraction by comparing its specific activity to the specific activity of the starting material, and representing this as X-fold purification. The use of "-fold purification" is advantageous as the purity of an inhibitory fraction can thus be compared to another despite any differences which may exist in the actual units of activity or specific activity.

It is contemplated that the F factor of the present invention be purified to between about between about 90-fold and about

100-fold, and even more preferably, to about 100-fold, relative to its natural state.

To prepare a substantially purified F factor in accordance with the present invention one would concentrate by ultrafiltration in the FLS4.1 supernatants. Followed by DEAE-cellulose-anion-exchange chromatography, ultra gel ACA54 gel filtration chromatography, wheat-germ agglutinin agarose chromatography, and reverse phase chromatography. Analysis of purified F factor is then done for biological activity (preventing PHSC cell lines from differentiation) and by SDS-PAGE analysis of silver stained gels.

Generally, "purified" will refer to a composition comprising an F factor which has been subjected to fractionation to remove various non-polypeptide components such as other cell components. Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. A specific example presented herein is the purification of F factor using gel filtration chromatography. The preferred purification method disclosed herein below contains several steps. This preferred mode of F factor purification involves the execution of certain purification steps in the order described herein below. However, as is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified F factor.

As mentioned above, although preferred for use in certain embodiments, there is no general requirement that the F factor always be provided in its most-purified state. Indeed, it is contemplated that less substantially purified F factor, which is nonetheless enriched in F factor activity relative to the natural state, will have utility in certain embodiments. These include, for example, the inhibition of cell differentiation of PHSC cells. Partially purified F factor fractions for use in such embodiments may be obtained by subjecting FLS4.1 cell culture supernatant to one or a combination of the steps described above.

8. Therapeutic and Diagnostic Kits Comprising F Factor

Therapeutic/diagnostic kits comprising F factor form another aspect of the invention. Such kits will generally contain, in suitable container means, a pharmaceutically-acceptable formulation of F factor. The kit may have a single container means that contains F factor alone or a combination of F factor and other cytokines such as IL-3, LIF, Steel Factor, FLT3/FLK2-ligand, IL7, IL6, BMP4, follistatin, or it may have distinct container means for each compound. When the components of the kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The F factor polypeptide may also be formulated into a syringeable composition. In which case, the container means may itself be a syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, inoculated into cell culture medium, or even applied to and mixed with the other components of the kit.

However, the components of the kit may be provided as dried powder(s). When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the F factor may be placed, preferably, suitably allocated. Where a second cytokine is provided, the kit will also generally contain a second vial or other container into which this cytokine may be placed. The kits may also comprise a second/third container means for containing a sterile, pharmaceutically acceptable buffer or other diluent.

The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained. Irrespective of the number or type of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate F factor composition within the body of an animal or within a cell culture. Such an instrument may be a syringe, pipette, forceps, measuring spoon, eye dropper or any such medically-approved and/or suitable delivery vehicle.

Also disclosed in a method of generating an immune response in an animal. The method generally involves administering to an animal a pharmaceutical composition comprising an immunologically effective amount of a peptide composition disclosed herein. Preferred peptide compositions include the peptide disclosed in SEQ ID NO:4.

The invention also encompasses A3 and A3-derived peptide antigen compositions together with pharmaceutically-acceptable excipients, carriers, diluents, adjuvants, and other components, such as additional peptides, antigens, or outer membrane preparations, as may be employed in the formulation of particular vaccines. The nucleic acid sequences of the present invention encode A3 and are useful to generate pure recombinant A3 for administration to a host.

Antibodies may be of several types including those raised in heterologous donor animals or human volunteers immunized with the peptides of the present invention, monoclonal antibodies (mAbs) resulting from hybridomas derived from fusions of B cells from immunized animals or humans with compatible myeloma cell lines, so-called "humanized" mAbs resulting from expression of gene fusions of combinatorial determining regions of mAb-encoding genes from heterologous species with genes encoding human antibodies, or antibody-containing fractions of plasma from human donors.

It is contemplated that any of the techniques described above might be used for the vaccination of subjects for the purpose of antibody production. Using the peptide antigens described herein, the present invention also provides methods of generating an immune response, which methods generally comprise administering to an animal, a pharmaceutically-acceptable composition comprising an immunologically effective amount of a peptide composition.

Preferred animals include mammals, and particularly humans. Other preferred animals include murines, bovines, equines, porcines, canines, and felines. The composition may include partially or significantly purified peptide epitopes, obtained from natural or recombinant sources, which proteins or peptides may be obtainable naturally or either chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing DNA segments encoding such epitopes. Smaller peptides that include reactive epitopes, such as those between about 10 and about 50, or even between about 50 and about 100 amino acids in length will often be preferred. The antigenic proteins or peptides may also be combined with other agents, such as other peptide or nucleic acid compositions, if desired.

By "immunologically effective amount" is meant an amount of a peptide composition that is capable of generating an immune response in the recipient animal. This includes both the generation of an antibody response (B cell response), and/or the stimulation of a cytotoxic immune response (T cell response). The generation of such an immune response will have utility in both the production of useful bioreagents, e.g., CTLs and, more particularly, reactive antibodies, for use in diagnostic embodiments, and will also have utility in various prophylactic or therapeutic embodiments.

Further means contemplated by the inventors for generating an immune response in an animal includes administering to the animal, or human subject, a pharmaceutically-acceptable composition comprising an immunologically effective amount of a nucleic acid composition encoding an epitope as disclosed herein, or an immunologically effective amount of an attenuated live organism that includes and expresses such a nucleic acid composition. The "immunologically effective amounts" are those amounts capable of stimulating a B cell and/or T cell response.

Immunoformulations of this invention, whether intended for vaccination, treatment, or for the generation of antibodies. As such, antigenic functional equivalents of the proteins and peptides described herein also fall within the scope of the present invention. An "antigenically functional equivalent" protein or peptide is one that incorporates an epitope that is immunologically cross-reactive with one or more epitopes derived from any of the particular proteins disclosed. Antigenically functional equivalents, or epitopic sequences, may be first designed or predicted and then tested, or may simply be directly tested for cross-reactivity.

The identification or design of suitable epitopes, and/or their functional equivalents, suitable for use in immunoformulations, vaccines, or simply as antigens (e.g., for use in detection protocols), is a relatively straightforward matter. For example, one may employ the methods of Hopp, as enabled in U.S. Patent 4,554,101 , incorporated herein by reference, that teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences, for example, Chou and Fasman (1974a,b; 1978a,b; 1979); Jameson and Wolf (1 988); Wolf et al. ( 1988); and Kyte and Doolittle (1982) address this subject. The amino acid sequence of these "epitopic core sequences" may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.

It is proposed that the use of shorter antigenic peptides, e.g., about 25 to about 50, or even about 15 to 25 amino acids in length, that incorporate particular epitopes will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.

In still further embodiments, the present invention concerns immunodetection methods and associated kits. It is contemplated that the proteins or peptides of the invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect peptides.

In general, the preferred immunodetection methods will include first obtaining a sample suspected of containing a reactive antibody, such as a biological sample from a patient, and contacting the sample with a first peptide under conditions effective to allow the formation of an immunocomplex (primary immune complex). One then detects the presence of any primary immunocomplexes that are formed.

Contacting the chosen sample with the protein or peptide under conditions effective to allow the formation of (primary) immune complexes is generally a matter of simply adding the protein or peptide composition to the sample. One then incubates the mixture for a period of time sufficient to allow the added antigens to form immune complexes with, i.e., to bind to, any antibodies present within the sample. After this time, the sample composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antigen species, allowing only those specifically bound species within the immune complexes to be detected.

The detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches known to the skilled artisan and described in various publications, such as, e.g., Nakamura et al. (1987), incorporated herein by reference. Detection of primary immune complexes is generally based upon the detection of a label or marker, such as a radioactive, fluorescent, biological or enzymatic label, with enzyme tags such as alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase being suitable. The particular antigen employed may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of bound antigen present in the composition to be determined. Alternatively, the primary immune complexes may be detected by means of a second binding ligand that is linked to a detectable label and that has binding affinity for the first protein or peptide. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labelled secondary antibodies and the remaining bound label is then detected.

For diagnostic purposes, it is proposed that virtually any sample suspected of containing the antibodies of interest may be employed. Exemplary samples include clinical samples obtained from a patient such as blood or serum samples, bronchoalveolar fluid, ear swabs, sputum samples, middle ear fluid or even perhaps urine samples may be employed. Furthermore, it is contemplated that such embodiments may have application to non-clinical samples, such as in the titering of antibody samples, in the selection of hybridomas, and the like. Alternatively, the clinical samples may be from veterinary sources and may include such domestic animals as cattle, sheep, and goats. Samples from feline, canine, and equine sources may also be used in accordance with the methods described herein.

In related embodiments, the present invention contemplates the preparation of kits that may be employed to detect the presence of specific antibodies in a sample. Generally speaking, kits in accordance with the present invention will include a suitable protein or peptide together with an immunodetection reagent, and a means for containing the protein or peptide and reagent.

The immunodetection reagent will typically comprise a label associated with a protein or peptide, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first protein or peptide or antibody, or a biotin or avidin (or streptavidin) ligand having an associated label. Detectable labels linked to antibodies that have binding affinity for a human antibody are also contemplated, e.g. , for protocols where the first reagent is a protein or peptide that is used to bind to a reactive antibody from a human sample. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention. The kits may contain antigen or antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.

The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antigen may be placed, and preferably suitably allocated. Where a second binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed. The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow- molded plastic containers into which the desired vials are retained. It is expected that to achieve an "immunologically effective formulation" it may be desirable to administer polypeptides or protein antigens to the human or animal subject in a pharmaceutically acceptable composition comprising an immunologically effective amount of the composition mixed with other excipients, carriers, or diluents which may improve or otherwise alter stimulation of B cell and/or T cell responses, or immunologically inert salts, organic acids and bases, carbohydrates, and the like, which promote stability of such mixtures.

Immunostimulatory excipients, often referred to as adjuvants, may include salts of aluminum (often referred to as Alums), simple or complex fatty acids and sterol compounds, physiologically acceptable oils, polymeric carbohydrates, chemically or genetically modified protein toxins, and various particulate or emulsified combinations thereof. Peptides within these mixtures, or each variant if more than one are present, would be expected to comprise about 0.0001 to 1 .0 milligrams, or more preferably about 0.001 to 0.1 milligrams, or even more preferably less than 0.1 milligrams per dose.

It is also contemplated that attenuated organisms may be engineered to express recombinant gene products and themselves be delivery vehicles for the invention. Alternatively, pox-, polio-, adeno-, or other viruses, and bacteria such as Salmonella, Shigella, Listeria, Streptococcus species may also be used in conjunction with the methods and compositions disclosed herein.

The naked DNA technology, often referred to as genetic immunization, has been shown to be suitable for protection against infectious organisms. Such DNA segments could be used in a variety of forms including naked DNA and plasmid DNA, and may administered to the subject in a variety of ways including parenteral, mucosal, and so-called microprojectile-based "gene-gun" inoculations. The use of nucleic acid compositions of the present invention in such immunization techniques is thus proposed to be useful as a vaccination strategy against Lyme disease.

It is recognized by those skilled in the art that an optimal dosing schedule of a vaccination regimen may include as many as five to six, but preferably three to five, or even more preferably one to three administrations of the immunizing entity given at intervals of as few as two to four weeks, to as long as five to ten years, or occasionally at even longer intervals.

9. Immunoassays

As noted, it is proposed that F factor polypeptides of the invention will find utility as immunogens, e.g. , in connection with vaccine development, or as antigens in immunoassays for the detection of anti-F factor antigen-reactive antibodies. Turning first to immunoassays, in their most simple and direct sense, preferred immunoassays of the invention include the various types of enzyme linked immunosorbent assays (ELISAs) known to the art. However, it will be readily appreciated that the utility of F factor peptides is not limited to such assays, and that other useful embodiments include RIAs and other non-enzyme linked antibody binding assays or procedures.

In one such ELISA, peptides incorporating the F factor antigen sequences of invention may be first immobilized onto a selected surface, e.g., a well of a surface exhibiting a protein affinity, such as a well in a polystyrene microtiter plate. In such an ELISA, generally, labelled anti-F factor antibodies would then be added to the wells, allowed to bind, and detected by means of their label. The amount of F factor in an unknown sample would be determined by mixing the sample with the labeled anti-F factor antibodies before or during incubation in an appropriate container means.

In another form of ELISA, an antibody capable of binding a F factor protein or peptide of the invention may be immobilized onto the solid surface, or well, and used directly in conjunction with labeled F factor compositions. In these ELISAs, generally, labeled F factor is added to the wells, allowed to bind, and detected by means of the label. The amount of F factor in an unknown sample is here determined by mixing the sample with the labeled F factor before or during incubation with the anti-F factor antibody in the wells. The presence of F factor in the sample again acts to reduce the amount of labeled F factor available for binding to the well and thus reduces the ultimate signal.

In coating a plate with either antigen or antibody, one will generally wash the wells of the plate to remove incompletely adsorbed material and then bind or "coat" a nonspecific protein onto the wells of the plate. Nonspecific proteins are those that are known to be antigenically neutral with regard to the test antisera, and include bovine serum albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface. Where an antibody capable of binding an F factor polypeptide is immobilized onto an ELISA plate, it is more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the control F factor and/or clinical or biological sample to be tested in a manner conducive to immune complex (antigen/antibody) formation. Detection of the F factor then requires a labeled secondary antibody, or a secondary antibody and a labeled tertiary antibody. The labeled secondary antibody is, of course, an anti-F factor antibody that is conjugated to a detectable label. When using a tertiary approach, the secondary antibody is an unlabeled anti-F factor antibody and the tertiary antibody is a labeled antibody that is specific for the species, or isotype, of the secondary antibody employed.

A "manner conducive to immune complex (antigen/antibody) formation" means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween™. These added agents also tend to assist in the reduction of nonspecific background.

Incubation steps are typically from about 1 to 2 to 4 hours, at temperatures preferably on the order of 25 °C to 27 °C, or may be overnight at about 4°C or so. Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non- immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween™, or borate buffer.

Following the formation of specific immunocomplexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immunocomplexes may be determined. As mentioned above, this may be achieved by subjecting the first immunocomplex to a second antibody having specificity for the first, or even a third antibody having specificity for the second. Where a second antibody alone is used, given that the control and test F factor samples will typically be of human origin, the second antibody will preferably be an antibody having specificity in general for human or mouse F factor. Where a third antibody is also used, the second antibody will still preferably be an antibody having specificity for human or mouse F factor, and the third antibody will then be an antibody having specificity in general for the second antibody. A second rabbit antibody and a third anti-rabbit Ig antibody is a particular example.

To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the first or second immunocomplex with a urease, glucose oxidase or peroxidase- conjugated antibody for a period of time and under conditions that favor the development of further immunocomplex formation (e.g. , incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween™). After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline- 6-sulfonic acid [ABTS] and H₂0₂, in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g. , using a visible spectra spectrophotometer.

10. Cloning of Human and Murine F Factors

The present inventor contemplates cloning the gene encoding F factor identified in Example 1 . A technique often employed by those skilled in the art of protein production today is to obtain a so- called "recombinant" version of the protein, to express it in a suitable cell and to obtain the protein from such cells. These techniques are based upon the "cloning" of a DNA molecule encoding the protein from a DNA library, i.e., on obtaining a specific DNA molecule distinct from other portions of DNA. This can be achieved by, for example, cloning a cDNA molecule, or cloning genomic DNA. Techniques such as these would also, of course, be appropriate for the production of F factor in accordance with the present invention.

The first step in such cloning procedures is the screening of an appropriate DNA library, such as, in the present case, a murine FLS4.1 cell line-derived library. The screening procedure may be an expression screening protocol employing antibodies directed against the protein, or activity assays. For example, one may employ methods as described in Young et al. (1983), specifically incorporated herein by reference. Alternatively, screening may be based on the hybridization of oligonucleotide probes, designed from a consideration of portions of the amino acid sequence of the protein, or from the DNA sequences of genes encoding related proteins. The operation of such screening protocols are well known to those of skill in the art and are described in detail in the scientific literature, for example, in Sambrook et al. (1 989), specifically incorporated herein by reference. Moreover, as the present invention encompasses the cloning of genomic segments as well as cDNA molecules, it is contemplated that other suitable methods known to those in the art, such as, e.g., those described by Spoerel et al. (1 987), may also be used in connection with cloning a gene encoding F factor polypeptide.

After identifying an appropriate DNA molecule, it may be inserted into any one of the many vectors currently known in the art and transferred to a prokaryotic or eukaryotic host cell where it will direct the expression and production of the so-called recombinant version of the protein. This is also, of course, routinely practiced in the art and described in various publications, such as, e.g., in Green et al. ( 1988) and Sambrook et al. ( 1 989).

It will be understood that recombinant F factor may differ from naturally-produced F factor in certain ways. In particular, the degree of post-translational modifications, such as, for example, glycosylation and phosphorylation may be different between the recombinant F factor and the F factor purified from a natural source, such as the FLS4.1 cell line. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A. The presence of rearrangements of TCRd in the progeny of induced BMp53 A1 1 (lane 3) and FLp53^'B4 (lane 4) cells to differentiate into TCR⁺ cells, uninduced BMp53 A1 1 (lane 1 ), uninduced FLp53^'B4 (lane 2) and positive control (16-day fetal thymocytes or adult thymocytes) (lane 5) was determined by DNA- based PCR™ assays. Amplification products were fractionated on agarose gels, blotted to nitrocellulose filters and hybridized with ³²P- labeled J<51 -, yλ - or J ?2-specific probes.

FIG. 1 B. The presence of rearrangements of TCRj in the progeny of induced BMp53 Al 1 (lane 3) and FLp53 B4 (lane 4) cells to differentiate into TCR⁺ cells, uninduced BMp53^'A1 1 (lane 1 ), uninduced FLp53 B4 (lane 2) and positive control ( 1 6-day fetal thymocytes or adult thymocytes) (lane 5) was determined by DNA- based PCR™ assays. Amplification products were fractionated on agarose gels, blotted to nitrocellulose filters and hybridized with ³²P- labeled Jό1 -, Jj 1 - or J ?2-specific probes.

FIG. 1 C. The presence of rearrangements of TCR ? in the progeny of induced BMp53 A1 1 (lane 3) and FLp53^"B4 (lane 4) cells to differentiate into TCR⁺ cells, uninduced BMp53 A1 1 (lane 1 ), uninduced FLp53 B4 (lane 2) and positive control (16-day fetal thymocytes or adult thymocytes) (lane 5) was determined by DNA- based PCR™ assays. Amplification products were fractionated on agarose gels, blotted to nitrocellulose filters and hybridized with ³²P- labeled J<51 -, Jj 1 - or J ?2-specific probes.

FIG. 1 D. The PCR™ amplification of the actin gene from the samples in FIG. 1 A, FIG. 1 B, and FIG. 1 C.

FIG. 2A. In vitro differentiation of the BMp53^" A1 1 and FLp53 B4 clones along the B-lymphocyte pathway. The presence of rearrangement of the Ig heavy gene in the progeny of induced BMp53^'A1 1 (lane 3) and FLp53 B4 (lane 4) cells, uninduced BMp53 A1 1 (lane 1 ), uninduced FLp53^'B4 (lane 2) and positive control (bone marrow cells) (lane 5) was determined by DNA-based PCR™ assays.

FIG. 2B. In vitro differentiation of the BMp53 A1 1 and FLp53 B4 clones along the B-lymphocyte pathway. The presence of rearrangement of the kappa light gene in the progeny of induced BMp53 A1 1 (lane 3) and FLp53 B4 (lane 4) cells, uninduced BMp53 A1 1 (lane 1 ), uninduced FLp53 B4 (lane 2) and positive control (bone marrow cells) (lane 5) was determined by DNA-based PCR™ assays.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. A3 NUCLEIC ACID SEGMENTS

As used herein, the term "A3 gene" is used to refer to a gene or DNA coding region that encodes a protein, polypeptide or peptide that is related to the NIP1 gene of yeast and that encodes a nuclear- envelope associated protein.

The definition of an "A3 gene", as used herein, is a gene that hybridizes, under relatively stringent hybridization conditions (see, e.g. , Maniatis et al. , 1982), to DNA sequences presently known to include A3 gene sequences. It will, of course, be understood that one or more than one genes encoding an A3 protein or peptide may be used in the methods and compositions of the invention. The nucleic acid compositions and methods disclosed herein may entail the administration of one, two, three, or more, genes or gene segments. The maximum number of genes that may be used is limited only by practical considerations, such as the effort involved in simultaneously preparing a large number of gene constructs or even the possibility of eliciting a significant adverse cytotoxic effect.

In using multiple genes, they may be combined on a single genetic construct under control of one or more promoters, or they may be prepared as separate constructs of the same of different types. Thus, an almost endless combination of different genes and genetic constructs may be employed. Certain gene combinations may be designed to, or their use may otherwise result in, achieving synergistic effects on formation of an immune response, or the development of antibodies to gene products encoded by such nucleic acid segments, or in the production of diagnostic and therapeutic assays. Any and all such combinations are intended to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature, so that one of ordinary skill in the art would readily be able to identify likely synergistic gene combinations, or even gene-protein combinations.

It will also be understood that, if desired, the nucleic segment or gene could be administered in combination with further agents, such as, e.g. , proteins or polypeptides or various pharmaceutically active agents. So long as genetic material forms part of the composition, there is virtually no limit to other components which may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or tissues.

B. THERAPEUTIC AND DIAGNOSTIC KITS

Therapeutic kits comprising, in suitable container means, a composition of the present invention in a pharmaceutically acceptable formulation represent another aspect of the invention. The composition may be nucleic acids, native or truncated peptides, site-specifically mutated, or peptide epitopes, or alternatively antibodies which bind native peptides disclosed herein or to peptide epitopes. Such nucleic acid segments may be DNA or RNA, and may be either native, recombinant, or mutagenized nucleic acid segments.

The kits may comprise a single container means that contains the composition. The container means may, if desired, contain a pharmaceutically acceptable sterile excipient, having associated with it, a composition as described herein, and, optionally, a detectable label or imaging agent. However, the single container means may contain a dry, or lyophilized, mixture of a composition, which may or may not require pre-wetting before use. Alternatively, the kits of the invention may comprise distinct container means for each component. In such cases, one container would contain the preferred composition, either as a sterile DNA solution or in a lyophilized form, and the other container would include the matrix, which may or may not itself be pre-wetted with a sterile solution, or be in a gelatinous, liquid or other syringeable form.

The kits may also comprise a second or third container means for containing a sterile, pharmaceutically acceptable buffer, diluent or solvent. Such a solution may be required to formulate the component into a more suitable form for application to the body, e.g. , as a topical preparation, or alternatively, in oral, parenteral, or intravenous forms. It should be noted, however, that all components of a kit could be supplied in a dry form (lyophilized), which would allow for "wetting" upon contact with body fluids. Thus, the presence of any type of pharmaceutically acceptable buffer or solvent is not a requirement for the kits of the invention. The kits may also comprise a second or third container means for containing a pharmaceutically acceptable detectable imaging agent or composition.

The container means will generally be a container such as a vial, test tube, flask, bottle, syringe or other container means, into which the components of the kit may placed. The matrix and gene components may also be aliquoted into smaller containers, should this be desired. The kits of the present invention may also include a means for containing the individual containers in close confinement for commercial sale, such as, e.g. , injection or blow-molded plastic containers into which the desired vials or syringes are retained. Irrespective of the number of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the placement of the ultimate composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.

C. AFFINITY CHROMATOGRAPHY

Affinity chromatography is generally based on the recognition of a protein by a substance such as a ligand or an antibody. The column material may be synthesized by covalently coupling a binding molecule, such as an activated dye, for example to an insoluble matrix. The column material is then allowed to adsorb the desired substance from solution. Next, the conditions are changed to those under which binding does not occur and the substrate is eluted. The requirements for successful affinity chromatography are:

1 ) that the matrix must specifically-adsorb the molecules of interest;

2) that other contaminants remain unadsorbed; 3) that the ligand must be coupled without altering its binding activity;

4) that the ligand must bind sufficiently tight to the matrix; and

5) that it must be possible to elute the molecules of interest without destroying them.

A preferred embodiment of the present invention is an affinity chromatography method for purification of antibodies from solution wherein the matrix contains protein or peptide epitopes derived from the compositions disclosed herein. This matrix binds the antibodies of the present invention directly and allows their separation by elution with an appropriate gradient such as salt, GuHCI, pH, or urea. Another preferred embodiment of the present invention is an affinity chromatography method for the purification of proteins and peptide epitopes from solution. The matrix binds the amino acid compositions of the present invention directly, and allows their separation by elution with a suitable buffer as described above.

D. METHODS OF NUCLEIC ACID DELIVERY AND DNA TRANSFECTION

In certain embodiments, it is contemplated that the nucleic acid segments disclosed herein will be used to transfect appropriate host cells. Technology for introduction of DNA into cells is well- known to those of skill in the art. Four general methods for delivering a nucleic segment into cells have been described:

( 1 ) chemical methods (Graham and VanDerEb, 1 973) ;

(2) physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al. , 1985) and the gene gun (Yang et al. , 1990);

(3) viral vectors (Clapp, 1993; Eglitis and Anderson, 1988); and

(4) receptor-mediated mechanisms (Curiel et al. , 1 991 ; Wagner et a/. , 1992).

E. LIPOSOMES AND NANOCAPSULES

In certain embodiments, the inventors contemplate the use of liposomes and/or nanocapsules for the introduction of particular peptides or nucleic acid segments into host cells. Such formulations may be preferred for the introduction of pharmaceutically-acceptable formulations of the nucleic acids, peptides, and/or antibodies disclosed herein. The formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et a/. , 911 which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half- times (Gabizon and Papahadjopoulos, 1 988; Allen and Choun, 1987).

Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al. , 1 987) . To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur et al., 1977; 1988) .

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs) . MLVs generally have diameters of from 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.

In addition to the teachings of Couvreur et al. (1988), the following information may be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.

F. METHODS FOR PREPARING ANTIBODY COMPOSITIONS In another aspect, the present invention contemplates an antibody that is immunoreactive with a polypeptide of the invention. As stated above, one of the uses for the proteins and epitopic peptides according to the present invention is to generate antibodies. Reference to antibodies throughout the specification includes whole polyclonal and monoclonal antibodies (mAbs), and parts thereof, either alone or conjugated with other moieties. Antibody parts include Fab and F(ab)₂ fragments and single chain antibodies. The antibodies may be made in vivo in suitable laboratory animals or in vitro using recombinant DNA techniques. In a preferred embodiment, an antibody is a monoclonal antibody. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Harlow and Lane, 1 988).

Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood. The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen, as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal) . The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs (below).

One of the important features provided by the present invention is a polyclonal sera that is relatively homogenous with respect to the specificity of the antibodies therein. Typically, polyclonal antisera is derived from a variety of different "clones, " i. e. , B-cells of different lineage. mAbs, by contrast, are defined as coming from antibody-producing cells with a common B-cell ancestor, hence their "mono" clonality.

When peptides are used as antigens to raise polyclonal sera, one would expect considerably less variation in the clonal nature of the sera than if a whole antigen were employed. Unfortunately, if incomplete fragments of an epitope are presented, the peptide may very well assume multiple (and probably non-native) conformations. As a result, even short peptides can produce polyclonal antisera with relatively plural specificities and, unfortunately, an antisera that does not react or reacts poorly with the native molecule. Polyclonal antisera according to present invention is produced against peptides that are predicted to comprise whole, intact epitopes. It is believed that these epitopes are, therefore, more stable in an immunologic sense and thus express a more consistent immunologic target for the immune system. Under this model, the number of potential B-cell clones that will respond to this peptide is considerably smaller and, hence, the homogeneity of the resulting sera will be higher. In various embodiments, the present invention provides for polyclonal antisera where the clonality, i.e. , the percentage of clone reacting with the same molecular determinant, is at least 80% . Even higher clonality - 90%, 95% or greater - is contemplated.

To obtain mAbs, one would also initially immunize an experimental animal, often preferably a mouse, with a protein or peptide composition. One would then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal. The spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the desired peptide.

Following immunization, spleen cells are removed and fused, using a standard fusion protocol with plasmacytoma cells to produce hybridomas secreting mAbs against the particular peptides of interest. Hybridomas which produce mAbs to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods. Hybridoma clones can then be cultured in liquid media and the culture supernatants purified to provide the specific mAbs.

It is proposed that the mAbs of the present invention will also find useful application in immunochemical procedures, such as

ELISA and Western blot methods, as well as other procedures such as immunoprecipitation, immunocytological methods, etc. which may utilize specific antibodies. In particular, antibodies may be used in immunoabsorbent protocols to purify native or recombinant proteins or epitopic-derived peptide species or synthetic or natural variants thereof.

The antibodies disclosed herein may be employed in antibody cloning protocols to obtain cDNAs or genes encoding proteins such as that encoded by the A3 gene from other species or organisms, or to identify proteins having significant homology to A3. They may also be used in inhibition studies to analyze the effects of A3 in cells, tissues, or whole animals. Anti-A3 antibodies will also be useful in immunolocalization studies to analyze the distribution of A3 peptide in vivo, and its cellular localization, for example, to determine the cellular or tissue-specific distribution of the A3 gene product under different physiological conditions. A particularly useful application of such antibodies is in purifying native or recombinant A3s, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure. G. RECOMBINANT EXPRESSION OF A3

Recombinant clones expressing the A3 nucleic acid segments may be used to prepare purified recombinant A3 (rA3), purified rA3- derived peptide antigens as well as mutant or variant recombinant protein species in significant quantities.

Additionally, by application of techniques such as DNA mutagenesis, the present invention allows the ready preparation of so-called "second generation" molecules having modified or simplified protein structures. Second generation proteins will typically share one or more properties in common with the full- length antigen, such as a particular antigenic/immunogenic epitopic core sequence. Epitopic sequences can be provided on relatively short molecules prepared from knowledge of the peptide, or encoding DNA sequence information. Such variant molecules may not only be derived from selected immunogenic/ antigenic regions of the protein structure, but may additionally, or alternatively, include one or more functionally equivalent amino acids selected on the basis of similarities or even differences with respect to the natural sequence.

H. ANTIBODY COMPOSITIONS AND FORMULATIONS THEREOF

Means for preparing and characterizing antibodies are well known in the art (See, e.g. , Harlow and Lane ( 1 988); incorporated herein by reference). The methods for generating mAbs generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) . Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, 77-maleimidobenzoyl-/V- hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4, 196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g. , a purified or partially purified protein, polypeptide or peptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions. Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody- producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately about 5 x 10⁷ to about 2 x 10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1 986; Campbell, 1 984) . For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1 /1 .Ag 4 1 , Sp210-Ag14, FO, NSO/U, MPC-1 1 , MPC1 1 -X45-GTG 1 .7 and S1 94/5XX0 Bui; for rats, one may use R210.RCY3, Y3-Ag 1 .2.3, IR983F and 4B210; and U-266, GM 1 500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1 -Ag4-1 ), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 ratio, though the ratio may vary from about 20: 1 to about 1 : 1 , respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1 976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. ( 1977) . The use of electrically induced fusion methods is also appropriate (Goding, 1986) .

Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10⁶ to about 1 x 10⁸. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium) . Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g. , hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific mAb produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

I. IMMUNOASSAYS As noted, it is proposed that native and synthetically-derived peptides and peptide epitopes of the invention will find utility as immunogens, e.g. , in connection with vaccine development, or as antigens in immunoassays for the detection of reactive antibodies. Turning first to immunoassays, in their most simple and direct sense, preferred immunoassays of the invention include the various types of enzyme linked immunosorbent assays (ELISAs), as are known to those of skill in the art. However, it will be readily appreciated that the utility of proteins and peptides is not limited to such assays, and that other useful embodiments include RIAs and other non-enzyme linked antibody binding assays and procedures.

In preferred ELISA assays, proteins or peptides incorporating protein antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity, such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, one would then generally desire to bind or coat a nonspecific protein that is known to be antigenically neutral with regard to the test antisera, such as bovine serum albumin (BSA) or casein, onto the well. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween™. These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for, e.g., from 2 to 4 hours, at temperatures preferably on the order of about 25 ° to about 27°C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween™, or borate buffer.

Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and the amount of immunocomplex formation may be determined by subjecting the complex to a second antibody having specificity for the first. Of course, in that the test sample will typically be of human origin, the second antibody will preferably be an antibody having specificity for human antibodies. To provide a detecting means, the second antibody will preferably have an associated detectable label, such as an enzyme label, that will generate a signal, such as color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions that favor the development of immunocomplex formation (e.g. , incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween™).

After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H₂0₂, in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g. , using a visible spectrum spectrophotometer.

ELISAs may be used in conjunction with the invention. In one such ELISA assay, proteins or peptides incorporating antigenic sequences of the present invention are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of powdered milk. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by . nonspecific binding of antisera onto the surface.

J. IMMUNOPRECIPITATION The antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation. Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein.

In an alternative embodiment the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g. , enzyme-substrate pairs.

K. WESTERN BLOTS

The compositions of the present invention will find great use in immunoblot or western blot analysis. The antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof. In conjunction with immunoprecipitation, followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background. This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal. Immunologically-based detection methods in conjunction with Western blotting (including enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety) are considered to be of particular use in this regard.

L. VACCINES

The present invention contemplates vaccines for use in both active and passive immunization embodiments. Immunogenic compositions proposed to be suitable for use as a vaccine may be prepared most readily directly from the novel immunogenic proteins and/or peptide epitopes described herein. Preferably the antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle.

The preparation of vaccines that contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251 ; 4,601 ,903; 4,599,231 ; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines. Vaccines may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations that are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1 -2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70% .

The proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines may be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g. , the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered will be readily determinable by the skilled practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.

Various methods of achieving adjuvant effect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol^®) used as 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 70° and about 101 °C for 30 second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated F(ab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram- negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide monooleate (Aracel-A™) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA™) used as a block substitute may also be employed.

In many instances, it will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations. The vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1 -5 years, usually three years, will be desirable to maintain protective levels of the antibodies. The course of the immunization may be followed by assays for antibodies for the supernatant antigens. The assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescers, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791 ,932; 4, 1 74,384 and 3,949,064, as illustrative of these types of assays.

Of course, in light of the new technology on DNA vaccination, it will be understood that virtually all such vaccination regimens will be appropriate for use with DNA vectors and constructs, as described by Ulmer et al. (1 993), Tang et al. ( 1 992), Cox et al.

( 1993), Fynan et al. (1993), Wang et al. ( 1 993) and Whitton et al. (1993), each incorporated herein by reference. In addition to parenteral routes of DNA inoculation, including intramuscular and intravenous injections, mucosal vaccination is also contemplated, as may be achieved by administering drops of DNA compositions to the nares or trachea. It is particularly contemplated that a gene-gun could be used to deliver an effectively immunizing amount of DNA to the epidermis (Fynan et al. , 1993).

The present invention contemplates vaccines for use in both active and passive immunization embodiments. Immunogenic compositions, proposed to be suitable for use as a vaccine, may be prepared most readily directly from immunogenic peptides prepared in a manner disclosed herein. Preferably the antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle. The preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251 ; 4,601 ,903; 4,599,231 ; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables. Either as liquid solutions or suspensions: solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines. M. PHARMACEUTICAL COMPOSITIONS

The pharmaceutical compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1 % of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The 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. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

For oral prophylaxis the polypeptide may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution) . Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.

The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

The composition can be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 1 5th Edition, pages 1035-1038 and 1 570-1 580) . Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards. N. EPITOPIC CORE SEQUENCES

The present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which incorporates an epitope that is immunologically cross-reactive with one or more of the antibodies of the present invention.

As used herein, the term "incorporating an epitope(s) that is immunologically cross-reactive with one or more anti-antibodies" is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a particular polypeptide. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.

The identification of epitopes, gene products and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter. For example, one may employ the methods of Hopp, as taught in U.S. Patent 4,554, 101 , incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, for example, Jameson and Wolf, 1988; Wolf et al. , 1 988; U.S. Patent Number 4,554, 101 ) . The amino acid sequence of these "epitopic core sequences" may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention will generally be on the order of about 5 to about 25 amino acids in length, and more preferably about 8 to about 20 amino acids in length. It is proposed that shorter antigenic peptide sequences will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.

It is proposed that particular advantages of the present invention may be realized through the preparation of synthetic peptides which include modified and/or extended epitopic/immunogenic core sequences which result in a "universal" epitopic peptide sequences. It is proposed that these regions represent those which are most likely to promote T-cell or B-cell stimulation in an animal, and, hence, elicit specific antibody production in such an animal.

An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is "complementary" to, and therefore will bind, antigen binding sites on epitope-specific antibodies. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term "complementary" refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.

In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences. The smallest useful core sequence expected by the present disclosure would generally be on the order of about 5 amino acids in length, with sequences on the order of 8 or 25 being more preferred. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.

The identification of epitopic core sequences is known to those of skill in the art, for example, as described in U.S. Patent 4,554, 101 , incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. Moreover, numerous computer programs are available for use in predicting antigenic portions of proteins (see e.g. , Jameson and Wolf, 1988; Wolf et al. , 1988) .

Computerized peptide sequence analysis programs (e.g. , DNAStar^® software, DNAStar, Inc., Madison, WI) may also be useful in designing synthetic peptides and peptide analogs in accordance with the present disclosure. In this regard, particular advantages may be realized through the preparation of synthetic peptides that include epitopic/immunogenic core sequences. These epitopic core sequences may be identified as hydrophilic and/or mobile regions of the polypeptides or those that include a T cell motif. It is known in the art that such regions represent those that are most likely to promote B cell or T cell stimulation, and, hence, elicit specific antibody production.

To confirm that a protein or peptide is immunologically cross- reactive with, or a biological functional equivalent of, one or more epitopes of the disclosed peptides is also a straightforward matter. This can be readily determined using specific assays, e.g., of a single proposed epitopic sequence, or using more general screens, e.g., of a pool of randomly generated synthetic peptides or protein fragments. The screening assays may be employed to identify either equivalent antigens or cross-reactive antibodies. In any event, the principle is the same, i.e., based upon competition for binding sites between antibodies and antigens.

Suitable competition assays that may be employed include protocols based upon immunohistochemical assays, ELISAs, RIAs, Western or dot blotting and the like. In any of the competitive assays, one of the binding components, generally the known element, such as the peptide disclosed herein, or a known antibody, will be labeled with a detectable label and the test components, that generally remain unlabeled, will be tested for their ability to reduce the amount of label that is bound to the corresponding reactive antibody or antigen. In addition to the peptidyl compounds described herein, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions of the peptide structure. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and hence are also functional equivalents. The generation of a structural functional equivalent may be achieved by the techniques of modelling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention.

Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence, are readily achieved using conventional synthetic techniques such as the solid phase method (e.g. , through the use of a commercially-available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer) . Peptide antigens synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g. , up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at 4°C, or more preferably, frozen. Of course, where the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g. , in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.

O. SITE-SPECIFIC MUTAGENESIS

Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique, well-known to those of skill in the art, further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 14 to about 25 nucleotides in length is preferred, with about 5 to about 10 residues on both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M 1 3 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art. Double-stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected peptide- encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants. Specific details regarding these methods and protocols are found in the teachings of Maloy et al. , 1 994; Segal, 1976; Prokop and Bajpai, 1 991 ; Kuby, 1994; and Maniatis et al. , 1982, each incorporated herein by reference, for that purpose.

The PCR™-based strand overlap extension (SOE) (Ho et al. , 1 989) for site-directed mutagenesis is particularly preferred for site- directed mutagenesis of the nucleic acid compositions of the present invention. The techniques of PCR™ are well-known to those of skill in the art, as described hereinabove. The SOE procedure involves a two-step PCR™ protocol, in which a complementary pair of internal primers (B and C) are used to introduce the appropriate nucleotide changes into the wild-type sequence. In two separate reactions, flanking PCR™ primer A (restriction site incorporated into the oligo) and primer D (restriction site incorporated into the oligo) are used in conjunction with primers B and C, respectively to generate PCR™ products AB and CD. The PCR™ products are purified by agarose gel electrophoresis and the two overlapping PCR™ fragments AB and CD are combined with flanking primers A and D and used in a second PCR™ reaction. The amplified PCR™ product is agarose gel purified, digested with the appropriate enzymes, ligated into an expression vector, and transformed into E. coli JM101 , XL1 -Blue™ (Stratagene, LaJolla, CA), JM 105, or TG1 (Carter et al., 1985) cells. Clones are isolated and the mutations are confirmed by sequencing of the isolated plasmids. Beginning with the native gene sequence, suitable clones and subclones may be made in BG26:pB/2.5(5), from which site-specific mutagenesis may be performed. Alternatively, the use of pET vectors (Novagen, Inc., Madison, WI; U.S. Patent 4, 952,496, disclosed herein by reference) is contemplated in the recombinant production of polypeptides. P. BIOLOGICAL FUNCTIONAL EQUIVALENTS

Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table: TABLE 1

For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1 982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1 982), these are: isoleucine ( + 4.5); valine ( + 4.2); leucine ( + 3.8); phenylalanine ( + 2.8); cysteine/cystine ( + 2.5); methionine ( + 1 .9); alanine ( + 1 .8); glycine (-0.4); threonine (-0.7); serine (- 0.8); tryptophan (-0,9); tyrosine (-1 .3); proline (-1 .6); histidine (- 3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. , still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those which are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554, 101 , incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U.S. Patent 4,554, 101 , the following hydrophilicity values have been assigned to amino acid residues: arginine ( + 3.0); lysine ( + 3.0); aspartate ( + 3.0 ± 1 ); glutamate ( + 3.0 ± 1 ); serine ( + 0.3); asparagine ( +0.2); glutamine ( + 0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1 ); alanine (-0.5); histidine (-0.5); cysteine (-1 .0); methionine (-1 .3); valine (-1 .5); leucine (-1 .8); isoleucine (-1 .8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4) . It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within + 2 is preferred, those which are within ± 1 are particularly preferred, and those within ± 0.5 are even more particularly preferred. As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side- chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

Q. NUCLEIC CYTOPLASMIC TRAFFICKING

Several gene products are thought to participate in the nucleocytoplasmic trafficking of macromolecules. Nuclear import has been divided in two stages: 1 ) Initial NLS-mediated targeting to the nuclear envelope/nuclear pore complex and 2) ATP-dependent translocation across the nuclear envelope (Nigg et al. , 1 991 ; Silver, 1 991 ; Yamasaki and Lanford, 1 992; Dingwall and Laskey, 1 992; Newmeyer, 1 993) . Following synthesis in the cytoplasm, proteins enter the nucleus through nuclear pore complexes. Certain proteins are transported to the nucleus if they contain an active nuclear localization sequence (NLS) . Proteins that recognize NLSs- containing proteins may first bind in the cytoplasm and deliver proteins to the nuclear pore complex, while other NLS-binding proteins may be nuclear enveloped associated (Nigg et al. , 1 991 ; Silver, 1 991 ; Newmeyer, 1993; Hinshaw et al. , 1 992). A number of laboratories, using cell-free or permeabilized-cell transport assays have identified cytosolic transport factors (reviewed in Newmeyer, 1 993) . Recently, the NIP1 gene which encodes an essential protein required for nuclear transport was identified and cloned by using a genetic selection with the yeast Saccharomyces Cerevisiae for the isolation of mutants that are defective in the nuclear import of proteins (Gu et al. , 1 992).

Heat shock proteins have been shown to play a role in many intracellular protein trafficking systems. It has been recently demonstrated that heat shock protein 70 can bind to NLS peptides and to participate in nuclear import of the NLS peptide-conjugates (Shi and Thomas, 1992; Imamoto, ef al. , 1992).

R. NUCLEAR PORE COMPLEX

The nuclear pore complex (NPC) has been considered as an organelle composed of a unique set of proteins necessary for transporting macromolecules across the nuclear envelope. Because the nuclear pore complex associates with both the nuclear membrane and the underlying lamina it has been difficult to isolate the nuclear pore complex in pure form and consequently, only some of the nuclear pore complex-associated proteins have been identified (Silver, 1 991 ; Newmeyer, 1993; Hinshaw et al. , 1992). One set of pore complex proteins called nucleoporins are O-glycosylated and have been found to play a specific role in protein translocation and the assembly of functional nuclear pore complexes (Hart et al. , 1989; Feldherr, et al. , 1993; Wimmer et al. , 1992) . cDNA clones for two vertebrate nucleoporins, p62 and Nup1 53 have been isolated (Starr er al. , 1 990; Cordes et a/. , 1991 ; Sukegawa and Blobel, 1993) . Several nucleoporin genes have been cloned in yeast, usually by screening expression libraries with antibodies to mammalian nucleoporins (Wimmer et a/. , 1 992; Wente et al. , 1 992; Nehrbass et al. , 1990; David and Fink, 1990). Often these genes are essential for viability. Recently, the nup1 53 gene encoding a rat liver nucleoporin was isolated (Sukegawa and Blobel 1 993). Interestingly, Nup1 53 was shown to actually bind to DNA in a zinc- dependent fashion. This observation suggests that Nup1 53 may play a role in getting genes to the nuclear pore complex, facilitating export of transcribed in mRNA (Blobel, 1985).

S. GENE EXPRESSION IN LYMPHOHEMATOPOIETIC PRECURSORS

Lymphohematopoietic precursors must express sets of genes whose products acting in the nucleus, cytoplasm or in the cell membrane, participate in the decisions of self-renewal versus differentiation and/or cell lineage determination. Critical transcription regulators have been identified through the study of nuclear factors binding cis-regulatory elements involved in lineage- specific gene expression and by pursuit of genes aberrantly activated in leukemia (reviewed in Kehrl et al. , 1 995) . Support for the view that these factors play a role in at least some steps during lineage determination comes from the findings that the knockout of a lineage-restricted (and in some cases of a broadly expressed regulator gene, e.g. E2A) transcription factor has often lead to a selective loss of the relevant hematopoietic lineage in the animal. For instance, loss of embryonic erythropoiesis in the absence of Gata-q, and Tal-1 (Pevni ef a/. , 1 991 ; Shivdasani et al. , 1995) and of lymphoid precursors and mature lymphocytes in Ikaros-null mice (Georgopoulos et al. , 1994) illustrate the importance of these proteins in establishing lineage-commitment decisions.

T. LONG-TERM CULTURE OF PHSCs

To find culture conditions that support long-term growth of undifferentiated PHSC the inventors thought that it would be advantageous to use PHSC from mutant mice that lack the p53 gene (Donehower et al. , 1992; Kuerbitz et al. , 1992; Yonish- Rouacch et al. , 1991 ) . PHSC from p53-deficient mice might survive longer in culture and might have a lower threshold to enter into mitosis. P53 controls the G1 to S phase transition of the cell cycle without affecting hematopoiesis (Donehower et al. , 1 992; Kuerbitz et al. , 1 992; Yonish-Rouacch et al. , 1991 ) . Enriched populations of PHSC from day 1 2-13 fetal liver cells or bone marrow of young adult p53 deficient mice were obtained by a combination of negative selection using magnetic beads and positive selection by FACS cell sorter (Palacios et a/. , 1 993; Palacios et al. , 1995) . Two cytokine combinations out of several tested were found to support proliferation with no or little differentiation, namely, LIF, Steel Factor, F (supernatants from the FLS4.1 fetal liver stromal cell line) and either IL3 of FLT3-ligand. These mixtures will be referred to as 3LSF or FLT3-ligLSF, respectively. If F was removed from the cytokine mixture the cells will continue to proliferate but will also differentiate usually into, F4/80, 8C5 and TER1 1 9-positive myeloid/erythroid cells. Both 3LSF and FLT3-Lig. LSF conditioned medium supported the initiation and long-term ( > 5 months) maintenance of PHSC lines. In cultures that received FLT3-lig. LSF- medium, the frequency of positive wells (1 2 to 19%) and the time required for the cultures to reach confluency (3-5 weeks) was lower and longer, respectively, when compared to the frequency of growing wells (up to 26%) and the time required to reach confluency (2-3 weeks) observed if the same Lin fetal liver cells were cultured in 3LSF-medium. Periodic monitoring by FACS for signs of differentiation revealed few myeloid or erythroid-lineage cells in some cell lines mostly in cultures containing 3LSF. The differentiated cells were depleted by magnetic bead selection and 1 2 out of 49 lines were discarded because of persistent and significant (10-20% F4/80, 8C5, TER1 1 9 positive cells) differentiation. A total of six p53 cell lines with stable Lin phenotype, three from fetal liver (called FLp53 A, B, C . . .) and three from bone marrow (called BM p53 A, B, C . . .), were cloned first by micromanipulation and selected clones were recloned twice by limiting dilution. The same protocol was then used to establish Lin cell lines from day 1 2-1 3 fetal liver of normal C57BL/5 mice. Two out of several cell lines with stable Lin phenotype established from C57BL/6 normal mice were cloned as indicated above. The following describes the details of the characterization of two clones derived from fetal liver of normal C57BL/6 mice (called FLSC 8, FLSC 14) and two clones, one derived from BM (BMp53 A1 1 ) and one from FL (Flp53 B4), of p53- deficient mice. All studies were performed 3 to 5 months after the establishment of the cell lines in culture. The p53 clones and the clones from normal mice divided approximately every 10-12 and 1 6- 20 hours, respectively. The clones from wild type mice die within 36-48 hours while the clones from p53 mice can survive up to 5-6 days in the absence of their exogenous growth factors.

All four clones are approximately 5-8 μm in diameter, round in shape, and exhibit a scant cytoplasm and a prominent dense nucleus, as determined by Giemsa staining. Scanning electron microscopy shows that they have a homogeneous morphology and display microvillae uniformly on their cell surface.

The phenotypic characteristics of all clones as determined by FACS analysis using a panel of antibodies against several hematopoietic surface markers, can be summarized as follows. First, the clones are negative for several hematopoietic lineage restricted surface markers normally present on myeloid cells (8C5, F4/80), erythroid cells (TER1 19), immature and mature B- lymphocyte lineage cells (B-220 and Ig) and immature and mature T- lymphocyte lineage cells (Joro 75, CD4, CD8, CD3). Intriguingly, the fetal liver-derived, but not the marrow-derived, clones expressed low levels of Mac-1 antigen. Second, the cells are positive for Sca- 1 , PgP-1 , c-kit receptor, and express no or very low levels of Thy 1 surface antigen. Third, they stained with the Joro 1 77, Joro 1 84 and Joro 96 monoclonal antibodies indicating that besides reacting with early lymphoid progenitors and myeloid precursors, these surface proteins are also expressed by PHSC (Palacios et a/. , 1 990). Fourth, the cells are positive for the VLA-5, VLA-6, lcam-1 , lcam-2, heat stable antigen (HSA), PgP-1 and very weakly for VLA-4 adhesion molecules and are negative for VLA-2, and VCAM-1 . Expression was somewhat variable for c-Kit, Thy 1 , Joro 1 84, Joro 96, Mac-1 , VLA 4 and 6, lcam 1 and lcam 2.

Northern blot analysis revealed the absence of RNA transcripts for genes expressed at very early stages of lymphocyte development (CD3 gamma, CD3 zeta, Rag-1 , Rag-2, TCF-2, Gata 3, LEF-1 , MB-1 , vPRE-B, lambda 5, μ Ig heavy chain, T-cell receptor delta), and a Gata-1 mRNA expressed at early stages of erythroid/myeloid differentiation. All clones synthesized mRNA for the A3 nuclear transport gene and for the A52 gene encoding ribosomal protein L1 3. Reverse-transcribed polymerase chain reaction (RT-PCR) assays revealed that all four clones synthesize RNA for CD34 and for Tal-1 , Gata-2, E1 2 and Id- 1 transcriptional regulator genes and were negative for CD1 9, B-cell lineage specific gene. These results indicate that the Lin- p53 and the Lin- p53⁺ clones are at an earlier stage of differentiation than lymphoid, erythroid and myeloid committed progenitors. In addition, the phenotypic and genotypic characteristics readily distinguish these clones from previously described multipotent, Pro-B cell, Pro-T cell, B-cell/Myelocytic and myeloid progenitor cell lines (Palacios et a/. , 1 993; Palacios et al. , 1992; Cross et al. , 1994) .

The clones were tested for their capacity to provide long-term reconstitution of the hematopoietic system of X-irradiated mice. It has been shown that sublethally irradiated Scid mice are well suited to assess the differentiation of not only lymphocyte precursors, but also of lymphohematopoietic stems cells (Palacios et al. , 1995;

Phillips et al. , 1 991 ). The Scid mutation renders cells more sensitive to X-irradiation and hampers their hematopoietic recovery after non = lethal doses of irradiation (Phillips et al. , 1991 ) . This provides with a competitive assay between host PHSC and the PHSC population under test without the need to expose the recipients to lethal doses of irradiation, which may cause severe damage of the microenvironments required for survival and differentiation of PHSC.

Two markers were used to distinguish the hematopoietic progeny of the donor cells (Lin clones) from C3H Scid recipient cells, namely, H-2b assessed using an MHC class 1 H-2b specific antibody and FACS analysis (Palacios et al. , 1993; Palacios et al. , 1 995) and the Neo^r gene [present in the genome of the p53- deficient mice (Donehower et al. , 1992)] assessed by DNA-based PCR (Palacios et al. , 1 993; Palacios et al. , 1995). It has been shown that the H-2b MHC class 1 antibody used here does not react with cells bearing H-2d or H-2k (C3H Scid mice) MHC antigens (Palacios et al. , 1 995). Thus, both markers are expressed by the donor cells but not by the recipient mice. Five to six months after transfer of the Lin-clones the bone marrow and/or spleens of C3H Scid mice contained donor cells which included granulocytes/macrophage (H^"2b⁺ Mac-1 /F4/80/8C5 ⁺ ), erythroid-cells (H-2b⁺ TER1 19⁺), B-cells (H-2b⁺ B-220⁺, lgM ⁺ ) and T-cells (H-2b⁺ CD4/CD8/TCR/CD3 ⁺ ) (Table 2). No H-2b⁺ cells were found in C3H Scid mice which received no cells. The Neo^r' gene was found in thymocytes, splenic lymphocytes and myeloid cells and bone marrow lymphoid, myeloid, and erythroid cells of Scid mice reconstituted with the Lin clones from p53 Neo^r+ mice. No Neo^r- positive cells were detected in cells from tissues of the control Scid mice which received fetal liver mononuclear cells from p53⁺ Neo^r- normal C57BL/6 mice. These findings indicate that the Lin clones have the capacity to provide long-term reconstitution of the lymphohematopoietic system.

In another set of studies, it was determined whether the Lin clones could rescue mice from a lethal dose of irradiation. Thus, the p53 Lin and the Lin p53⁺ clones from normal mice were injected I.V. either alone or together with bone marrow cells from Rag-2 deficient mice into lethally irradiated Rag-2 deficient mice. Control groups included irradiated mice that received either no cells or marrow cells from Rag-2 deficient mice only. Survival of the mice was monitored daily during the first 20 days and twice weekly afterwards. The presence of donor derived mature T and B lymphocytes in the mice that survived was monitored by FACS analysis of peripheral blood for the presence of TCR⁺ mature T- lymphocytes and lgM ⁺ mature B-lymphocytes. Rag-2 deficient mice cannot rearrange their antigen-receptor encoding genes and thereby do not have mature T and B lymphocytes (Shinkai et al. , 1992). Thus, the presence of mature T and/or B lymphocytes in the Rag-2 deficient mice that received the p53 Lin clones or the p53⁺ Lin clones alone all died between 5 to 14 days after exposure to a lethal dose of irradiation, like the control group of mice that received no cells did. The Lin clones failed by themselves to rescue lethally irradiated mice at all cell concentrations tested (10⁶,5 10⁶, 10⁷ cells per mouse). The latter also precluded to determine their potential to give rise to CFU-S, a property of less immature progenitor cells than PHSC (Uchida et al. , 1993; Jones et al. , 1 990). In contrast, all lethally irradiated Rag-2 deficient mice that received the p53 Lin clones or the p53⁺ Lin clones together with few marrow cells from Rag-2 deficient mice survived during the period of observation (6 months). These mice had mature T and B lymphocytes (range of percentages of TCRab⁺ plus lgM⁺ mature lymphocytes detected was 37 to 63 six months after transfer of the Lin clones) progeny of the Lin lymphohematopoietic precursor clones injected. The latter results ruled out the possibility that the Lin clones were unable to survive, engraft and differentiate in these lethally irradiated mice. Eight out of ten lethally irradiated Rag-2 deficient mice that only received marrow cells from Rag-2 deficient mice survived, but none of these mice had detectable mature T or B lymphocytes. The finding that the p53 Lin and p53⁺ Lin lymphohematopoietic precursor clones could not by themselves rescue mice from a lethal does or irradiation agree with some (Jones et al. , 1990; Kiefer et al. , 1 991 ) but not with others (Uchida et al. , 1993; Spangrude et al. , 1 995) previous studies in which the ability of freshly isolated enriched PHSC populations to rescue mice from lethal irradiation was also investigated. There are several potential explanations for these contrasting results. For instance, the PHSC population might be heterogenous in terms not only of cell cycle status, Rh 1 23 staining and size (Uchida et al., 1993; Harrison et al. , 1992; Li et al. , 1992), but also in their ability to rescue mice from a lethal dose of irradiation.

The clones described here would represent the PHSC subset that lacks this property. Another explanation is that perhaps a few more differentiated precursor cells contained in a given preparation of freshly isolated enriched PHSC cells could account for survival of lethally irradiated mice. The latter implies that PHSC would not possess the capacity of rescuing mice from a lethal dose of irradiation (Jones ef al. , 1990; Kiefer ef a/. , 1991 ) and would be consistent with the results obtained here with the Lin clones.

* * * * * * * * * *

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 ISOLATION AND CHARACTERIZATION OF F FACTOR

The present example provides a method that was used to obtain and characterize the active component, termed "F factor", identified by the present inventor. When F factor was present in stem cell cultures, differentiation into other blood cell types was prevented. When F factor was not added to the cultures, evidence of differentiated cell types, including B-cells, T-cells, platelets, and other cell types of myeloid/erythroid lineage was present.

Active F factor was characterized in the cell supernatant of a culture of the FLS4.1 cell line. This mouse fetal liver stromal cell line is described in Palacios et al. , ( 1 992) which is specifically incorporated herein by reference, for this purpose. Although, F factor was isolated from a murine cell line, the factor is also expected to be produced by other cell lines as well, including human cell lines. The soluble F factor produced spontaneously in cultures of FLS4.1 maintained PHSC in their undifferentiated state. Together with three other cytokines, F factor supports growth without differentiation of PHSC.

F factor was found to have a molecular weight of about 1 5- to 45-kDa as determined by gel filtration chromatography. Supernatant from FLS4.1 stromal cells were concentrated in Amicon ultrafiltration membranes ( 10PM 10, 62 mm) and layered on a Sephadex G-100 (2.5 x 60 cm) column which was equilibrated with phosphate-buffered saline and standardized with dextran blue, bovine albumin, ovalbumin, chymotrypsinogen and ribonuclease A (Pharmacia) . The column was run at a flow rate of 6 drops/minute, and 3.7- ml fractions were collected and sterilized by filtration through 0.45 μm Acrodisc filters. Alternate fractions were assayed for their ability to support proliferation without differentiation of Lin hematopoietic cells from fetal liver in the presence of IL3, LIF, Steel Factor.

F activity (proliferation without differentiation of PHSC lines) was assessed following Sephadex G-100 gel exclusion chromatography of concentrated supernatant from the FLS4.1 stromal cells. Alternate fractions were assayed for their ability to promote proliferation without differentiation of the BMp53 A1 1 PHSC cell line. Supernatants from FLS4.1 stromal cells were concentrated in AMICON ultrafiltration membranes (10PM10,62 mm) and layered on a Sephadex G-100 (2.5 x 60 cm) column (Pharmacia) which was equilibrated with PBS and standardized with dextran blue, bovine albumin, ovalbumin, chymotrypsinogen and ribonuclease A size markers. The column was run at 4°C at a flow rate of 6 drops/min and 3.7 ml fractions were collected, filtered and assayed for biological activity as described previously. F activity was found in between fractions 34 and 46(between the ovalbumin and ribonuclease A size markers). The results indicate an apparent molecular weight between about 1 5 and 45 about kDa as determined by gel exclusion chromatography.

EXAMPLE 2

LONG-TERM CULTURE OF LYMPHOHEMATOPOIETIC STEM CELLS A. MATERIALS AND METHODS

1. Animals

C57BL/6 normal mice and C3H Scid mice were bred and maintained in an animal barrier facility. Female 8- to 1 2-week-old Scid mice which had no detectable serum Ig were used in studies described herein. P53-deficient mice (Donehower et al., 1992) were obtained from GenPharm International (Mountain View, CA). Homozygous p53-deficient mutant embryos and young adult mice were screened by using an exon 5 deletion-specific probe and liver or tail DNA digested with BamHl, in Southern hybridization analysis as described (Donehower et al., 1 992). The day of detection of vaginal plug was taken as day 0 of gestation.

2. Cytokines Recombinant IL1 beta was obtained from F. Hoffmann-

LaRoche, (Basel, Switzerland) . Supernatants from X63Ag8 or J558/L myeloma cells transfected with cDNAs coding for mlL2, mlL3, mlL4, mlL5, HIL6, mlL7 (Karasuyama and Melchers 1 988, Samaridis et al., 1 991 ), or hIL 10 (as pHIL-10-550p), was obtained from P. Dellabona, H. S. Raffaele, Milan); Cos-1 cells transfected with cDNA coding for Steel Factor (Palacios and Samaridis, 1992), cDNA coding for hLIF (pC10-6R was obtained from A. Smith Centre For Animal Genome Research, Edinburgh); cDNA coding for Fibroblast Growth Factor (as pbFGF), was obtained from G. Neufeld, Israel Institute of Technology, Haifa, Israel). mlL9 was obtained from J. Van Snicke (University of Brussels, Brussels, Belgium) ; B9-transfected cells producing IL1 1 were obtained from R. G. Hawley (University of Toronto, Toronto, Ontario, Canada). Erythropoietin, M-CSF, G-CSF, TNF α, TGF β, IL1 2 were purchased from B&D Systems (Minneapolis, MN) and mGM-CSF was obtained from BIOGEN SA (Geneva, Switzerland). F factor (cell free supernatants collected and filtered from three day-confluent cultures of the FLS4.1 stromal cell line) were obtained as described in Example 1 .

3. Antibodies

Biotin, phycoerythrin (PE) or fluorescein isothiocyanate (FITC)- conjugated antibodies against Thy1 , LyT2 (CD8), L3T4 (CD5), CD3 (hybridoma 145-2c1 1 ), B-220 (hybridoma 6B2), MHC Class I of the H-2 b haplotype (hybridoma AF6-88.5), MHC class I of the H-2k haplotype (hybridoma AF3-1 2.1 ), Mac-1 (hybridoma m1 /70), TER1 19, 8C5, T-cell receptor (TCR) aβ (hybridoma H57-597), TCR yδ (hybridoma GL3), VLA-4, VLA-5, lcam-1 , lcam-2, Heat Stable antigen, V-cam 1 , and Sca-1 were purchased from Pharmingen (San Diego, CA), FITC- and PE-conjugated F4/80 antibody was obtained from SEROTEC (Kidlington, Oxford, England). Purified EA1 antibody against VLA-6 was obtained from B. A. Imhof, Basel Institute For Immunology, Basel, Switzerland. FITC-conjugated anti-mouse IgM, kappa, lambda and IgG and PE-streptavidin were from Southern Biotechnology Associates (Birmingham, AL); FITC-streptavidin was from Vector Laboratories, (Eugene, Oregon), FITC-conjugated anti- rat IgG, mouse Ig and Rat Ig were from Jackson Immunoresearch Labs (West Grove, PA). FITC-anti-rat IgM was from the Binding Site (Birmingham, UK) . Antibodies against the following cell surface markers were prepared as described herein: Joro 37-5, 75, 3, 96, 1 84, 177; PgP-1 (I/45), c-Kit (ACK2), LFA-1 (FD441 .8), heat stable antigen (J 1 1 D). 4. Cell Lines

The development, characterization and culture conditions for FLS4.1 fetal liver, RP.O.10 BM stromal cell lines (Palacios and Samaridis, 1 992), EH6 and ET thymic epithelial cell lines (Palacios et al., 1 988; Golunski and Palacios, 1994) have been described.

5. Isolation of Lin^' Mononuclear Cells

Bone marrow mononuclear cells from 3- to 6-week-old mice and day 12-13 fetal liver mononuclear cells free of erythrocytes from p53 deficient homozygous or normal mice were prepared as described (Palacios and Samaridis, 1 992). BM and fetal liver cells were first depleted of CD4 ⁺ , CD8\ CD3 ⁺ , lg ⁺ , Joro 75 ⁺ - B-220 ⁺ , F4/80⁺ , Mac-1 ⁺, 8C5⁺ and TER 1 1 9⁺ cells using magnetic beads coupled with sheep anti-mouse Ig (Dynabeads Dynal, Oslo, Norway) by incubating the cells with saturating concentrations of the antibodies at 4°C for 40 min. The cells were washed and incubated with beads (beads/cell ratio: 30 to 1 ) at 4°C for 20 min.

The magnetic particle-bound cells were removed by applying a magnetic force, and the magnetic particle-free cells were collected and re-exposed to the magnetic force for 5 min to further remove the particle-bound cells. The magnetic-particle-free cells were collected, spun, counted and incubated with appropriate dilutions of PE-or Biotin-conjugated antibodies against F4/80, B-220, Mac-1 , TER 1 1 9, 8C5, Joro 75 and CD4 at 4°C for 30 min. The cells were washed and exposed to PE-streptavidin at 4°C for 20 min. The cells were then washed twice, suspended in cell sorter buffer (PBS + 5% FCS + gentamycin), and viable cells in the lymphoid gate (determined by forward and side scatters) that were negative for all these markers were purified by cell sorter using an ELITE sorter (Coulter, Miami, FL) essentially as described (Palacios and Samaridis, 1993). A proportion of the cell sorter selected cells were used for re-analysis and showed that > 99.5 of the cells were negative for all the surface markers indicated above. The cells are referred to as Lin cells.

The fetal liver or BM Lin^" cells were washed and resuspended in Iscove's Modified Dulbecco culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS) (Hyclone Laboratories, (Miami, FL), 0.05 mM 2-mercaptoethanol, 2 mM L-glutamine, and 50 μg/ml gentamycin.

6. Screening for Proliferation Without Differentiation of Lin^" Cells This was performed with p53 Lin cells from fetal liver or bone marrow in 24-well Linbro tissue culture plates. 5-10 x 10³ cell sorter-purified Lin cells per well were cultured in the presence of several cytokine mixtures (cytokines were used at final concentrations of 10, 100, 500 and 1000 units/ml; F factor was

used at a final concentration of 10%) in a final volume of 1 ml per well. Among the cytokine mixtures tested were:

Cell growth was monitored visually using an inverted microscope during one to three weeks. Cytokine mixtures which supported growth of the cells were selected for the next round of screening: support of proliferation without differentiation of Lin cells. To this end, similar cultures to those indicated above were set up but this time 3-5 wells per each group to obtain enough cells to screen by FACS analysis for signs of differentiation. The cells from the different cultures were stained with FITC- or biotin-conjugated antibodies (Palacios et al. , 1990) to lineage-restricted surface markers (B-220, Joro 75, Mac-1 , 8C5, TER1 19 and F4/80) followed by FITC-or PE-streptavidin and the presence of positive cells was determined by flow cytometry in a Coulter Profile Instrument (Coulter, Miami, FL).

7. Establishment of Lin^' Fetal Liver, Bone Marrow Cell Lines Of all cytokine mixtures tested, the mixture of IL 3, LIF, Steel

Factor and F factor (3LSF) was found to best support proliferation with no or little differentiation. This mixture was then used to try to establish Lin cell lines. To this end, Lin cells (1 to 10 cells) were placed in round-bottomed microculture wells containing 100 μl of either 3LSF-medium (culture medium supplemented with 50-100 units of IL-3, 500 to 10³ units of hLIF, 100 to 300 units of Steel factor, and F factor to a final concentration 10% vol/vol).

Alternatively, FLT3-ligLSF-medium was employed (culture medium supplemented with 100 to 500 units of FLT3-Lig. , and LIF, Steel Factor and F at the concentrations indicated for 3LSF-medium above) . The cultures were incubated at 37°C in a 7.5% C0₂ air atmosphere. Every 5-7 days the cultures were supplemented with 50-100 μl of freshly prepared 3LSF-medium (prior removal of an approximately equal volume of old medium).

Similar bulk cultures at higher cell densities (3 x 10⁴ Lin cells per 0.5 ml of 3LSF-medium) were also performed in 24-well Linbro tissue culture plates. Between 20 and 36% of the microculture wells were positive for cell growth (assessed by visual inspection with an inverted microscope) after two-three weeks' culture in three separate studies. A total of sixty cultures were transferred to 24-well Linbro plates containing a final volume of 0.5 ml of 3LSF- medium.

When cultures reached confluence, each well was split into three new Linbro wells in 0.5-1 ml of 3LSF-medium. Forty nine of the original sixty cell lines showed continued growth in culture, could be transferred to tissue culture flasks, and were thus considered established cell lines. They were propagated in 3LSF- medium at a density of 2-4 x 10⁵ cells per ml by transferring them into fresh medium every 3-4 days. Aliquots of each line were then frozen (DMSO 14%, FCS 14% in IMDM medium) by standard procedures. (Palacios and Samaridis, 1993) All cell lines, termed FLp53 or BMp53^" according to tissue of origin, were phenotyped by FACS analysis using antibodies to several surface markers on different hematopoietic cell lineages. Twelve out of forty-nine cell lines studied comprised some ( < 25%) Mac-1 , 8C5, or TER 1 1 9- positive cells and these cell lines were therefore discarded. A total of six FLp53 or BMp53 cell lines that exhibited a stable Lin phenotype were first cloned by micromanipulation (Palacios and Steinmetz, 1 985) and were re-cloned by limiting dilution (0.1 cells/well) in 3LSF-medium. The expanded clones were phenotyped and aliquots were frozen by standard procedures.

Essentially the same protocol was used to establish Lin cell lines from fetal liver of C57BL/6 normal mice, which were termed SC. The frequency and time required to establish Lin cell lines from normal mice was lower and took longer, respectively, than that observed with Lin cells from p53 deficient homozygous mice. 8. Isolation and Analysis of Nucleic Acids

DNA and total RNA preparation, restriction enzyme digestion, agarose gel electrophoresis, DNA, RNA blotting, probe preparations, hybridization procedures and autoradiography were performed as described (Palacios and Samaridis, 1992, 1 993; Samaridis et al., 1991 ; Pelkonen et al., 1988).

9. DNA Probes

The following DNA probes were used: a) PB10AT3 y cDNA for mouse CD3 y (0.9 kb Hind\\\-EcoH\ fragment) (Krissassen et al., 1 987); b) pGEM-3Z^® cDNA for CD3 ζ (1 .0-kb EcoRI fragment); c) the C ό cDNA for constant region of TCR 6 (0.9-kb EcoRI fragment) (obtained from K. Karjalainen, Basel Institute For Immunology, Basel); d) M6-BSK cDNA for mouse RAG-1 ( 1 .4-kb EcoRI-EcoRI fragment); e) MR2-1 cDNA for mouse Rag-2 (1 .0-kb PstΛ-PstΛ fragment) (Oettinger et al., 1 990); f) the PuCN374 cDNA for cμ Ig chain ( 1 .3-kb _?a/r.HI-βa/7,HI fragment) (Marcu et al., 1 980); g) the pZ183-1 a cDNA for mouse lambda 5 (0.7-kb Hinc\\-Xho\ fragment, (Sakaguchi and Melchers 1 986); h) pZ121 cDNA for mouse vPRE B1 (0.9-kb EcoRI fragment (Kudo and Melchers, 1987); i) A3 cDNA (0.6-kb Bam\- \-Hind\\\ fragment) (Xie and Palacios); j) A52 cDNA (0.8-kb BamH\-Xho\ fragment) (Xie and Palacios); k) B-actin probe ( 1 .1 -kb Pst\-Pst\ fragment, obtained from S. Carsson, MRC, London, England); I) 0.94-kb EcoR\-Bgl\\ fragment of the MB-1 cDNA; m) Neo probe (the 1 .4-kb V/πdlll-Smal fragment of the PSV-2 Neo plasmid); n) Gata-1 ( 1 .3-kb Xba\ fragment); o) TCF-1 (0.8-kb Nsi\-Xho\ fragment); p) LEF-1 (0.6-kb Sal\ fragment); and q) Gata-3 (0.8-kb Hinά\\\ fragment), obtained from H. Clevers, Rijksuniversiteit te Utrecht, Utrecht, Holland. 10. PCR™ Assays

DNA-based PCR™ was carried out with cell lysates obtained as described (Palacios and Samaridis, 1993). The following primers were used: Vd1 , Vd4, Vd6, Jd1 , Vg1 , Vg5, Vg6, Vg7, Jg1 , 5 'Db2, 5'Db1 , 3'Jb2, DHL, VQ52, VH7183, VH558, JH3, Vk, Jk2, 5'actin, 3'actin, 5'Neo: 5'-TTCGGCTATGACTGGGCACAAC-3' (SEQ ID N0: 1 ) and 3'Neo: 5 '-TCAGTGACAACGTCGAGCACAG-3' (SEQ ID N0:2); cycles were performed in a Perkin-Elmer Model 9600 Instrument (La Jolla, CA) as described (Palacios and Samaridis, 1993). The PCR™ products were fractionated on agarose gels, blotted to nitrocellulose filters, and hybridized with ³²P-labeled probes, followed by autoradiography (Palacios and Samaridis, 1 993) . Exposure times were usually between 0.5 to 4 hr.

1 1. In Vivo Functional Assays

For repopulation of sublethally-irradiated (300 rads of gamma rays) C3H-Scid-Scid mice, the p53 Lin clones, freshly isolated fetal liver mononuclear cells from C57BL/6 embryos ( 10⁶ cells/0.4 ml of PBS) or PBS alone were injected IV into the recipient animal 2-4 hours after irradiation. All mice were housed in sterile isolators with sterile food in a laminar flow hood. Hematopoietic reconstitution in bone marrow and spleen of Scid mice was assessed by single- and two-color FACS analysis five- to six-months later. For some studies, thymuses, spleens and bone marrow of three Scid mice injected with the same p53 clone were pooled and used to isolate thymocyte subsets, splenic T-and B-lymphocytes, bone marrow B- cell precursors and lg⁺ B-lymphocytes, myeloid cells and erythroid cells by using magnetic beads-coupled with sheep anti-mouse or rat IgG or by cell sorter, as required, using appropriate antibodies as indicated above. DNA was isolated from these various populations to detect the presence of the Neo^r gene by PCR™ assay.

12. In Vitro T-Cell Differentiation Assay Lin cells ( 10⁵ cells/well) were cultured on monolayers of the

EH6 subcapsular thymic epithelial clone on six well plates (Costar, Inc., Cambridge, MA) in the presence of recombinant IL 7 (500 units/ml), Steel Factor (100 units/ml) and FLT3-ligand (200 units/ml) in a final volume of 2 ml of culture medium per well at 37°C for 7 days. Cells were harvested, washed and cultured (10⁵ cells/well) on monolayers of the ET cortical thymic epithelial clone in the presence of IL 7, FLT3-ligand and Steel Factor in a final volume of 2 ml of culture medium per well at 37°C for 5-7 days. The cells were harvested, washed and were used for FACS analysis, DNA-based PCR™ analysis for TCR rearrangements and scanning electron microscopy (Palacios and Samaridis, 1 993; Palacios and Imhof, 1993) .

13. In Vitro B-Cell Differentiation Assay Lin cells (10⁵ cells/ml) were cultured on monolayers of irradiated (2000 rads) FLS4.1 stromal cells on six well plates (Costar) in the presence of IL 7 (500 units/ml), Steel Factor ( 100 units/ml) and IL 1 1 (100 units/ml) in a final volume of 2 ml of culture medium at 37°C for 7-8 days. The cells were harvested, washed and a portion of the cells were used for scanning electron microscopy. The rest of the cells were cultured ( 10^B cells/well) on irradiated (3500 rads) RP010 BM stromal cells in the presence of IL 7 and lipopolysaccharide (50 g/ml) in a final volume of 2 ml of culture medium per well at 37 °C for 5-7 days. The cells were harvested and washed and used for FACS analysis and for DNA- based PCR™ analysis for Ig heavy and kappa light chain rearrangements (Palacios and Samaridis, 1 993, Palacios and Imhof, 1993) .

14. Myeloid/Erythroid/Megakaryocyte Cell Differentiation

Lin^' cells ( 10⁵ cells /well) were cultured on six well plates (Costar) containing GM-CSF (200 units/ml), Steel Factor ( 100 units/ml), Erythropoietin (2 units/ml), IL 3 ( 10 Units/ml), IL 1 1 (100 units/ml) in a final volume of 2 ml of culture medium per well at 37°C for 8-10 days. The cells were harvested, washed and a portion of them were used for cytospin preparations followed by Giemsa or Benzidine staining and the rest were used for FACS analysis (Palacios and Samaridis, 1 993; Palacios and Imhof, 1993).

15. FACS Analysis

FACS analysis was performed as described previously (Palacios and Samaridis, 1 992; Palacios et al., 1 990) . All staining was performed with cell samples which were preincubated with heat inactivated hamster serum (10-1 5%) and purified rat IgG (250 μg/ml) to prevent nonspecific Fc-receptor binding of labeled antibodies. Single- and two-color FACS analysis were performed using Coulter Profile and ELITE V instruments. BM, spleen and thymocytes from normal mice were used as positive controls as required and to set up electronically green and red compensations. Fluorescence emitted by single viable cells was measured with logarithmic amplification. Dead cells were excluded from analysis by forward and side scatter gating. Data collected from 10⁴ cells were analyzed and displayed in the form of fluorescence histograms (single color) or dot plots (two color). 16. Scanning Electron Microscopy

Electron microscopy was performed as previously described (Fabra et al., 1 992) . Briefly, the uninduced or induced cells were plated on glass coverslips and fixed (3% glutaraldehyde, 2% paraformaldehyde in cacodylate buffer, pH 7.2) for 1 hr at room temperature. The samples were then incubated in the same buffer containing 1 % osmium tetroxide for 1 hr. Samples were then placed in 1 % aqueous thiocarbohydrazide for 10 min before fixation under similar conditions. The samples were dehydrated in a graded series of ethanol followed by three changes of absolute ethanol and then transferred into 1 , 1 ,3,3,3-hexamethyldisilane (Eastman Kodak, Rochester, NY) for 5 min, air dried for 2 hr, and sputter coated with Pt/Pd for 2 to 4 min in a Med 010 Evaporator (Balzer, Inc., Hudson, NH) . The samples were examined in an Amray 1000A scanning electron microscope (Burlington, MA) at an accelerating voltage of 5 kV.

B. RESULTS

1 . Phenotypic Characterization of PHSC The phenotypic characteristics of the proliferating cells in the long-term cultures is assessed by FACS analysis using a panel of antibodies specific for:

1 ) Cell surface markers on stem cells (Thy 1 , Sca-1 , PgP-1 , c-Kit), erythroid (TER 1 19), myeloid (8C5, F4/80, Mac-1 ), B- lymphoid (B-220, IgM), T-lymphoid (JORO 75 and 30-8, CD4, CD8, TCR/CD3);

2) Adhesion molecules (σ 4, α 5, α 6, β 1 integrins; LFA- 1 ,V cam 1 , lcam 1 and 2, L-selectin) to gain information on the adhesion molecules that PHSC may use to interact with other cells in microenvironments where these cells differentiate and;

3) Cytokine receptors (c-kit, FLT3, IL3, IL4, IL5, IL6, IL7,

IL1 1 , LIF, GM-CSF and erythropoietin) to determine to what extent PHSC express a wide range of receptors for cytokines that influence their differentiation into erythroid, myeloid or lymphoid-lineage cells or if most such receptors are induced by interacting with cells of specialized microenvironments where PHSC generate the distinct blood cell lineages.

2. Genotypic Characterization of PHSC

The genotypic characterization is performed using Northern and RT-PCR™ assays with DNA probes for genes expressed very early during the formation of the different blood cell lineages. The following genes are tested: Rag-1 and 2, GATA 1 , 2 and 3; MB-1 , lambda 5, vPre-B and Ig; TCF-1 , LEF-1 , CD3 y,TCR ό, y and β; globin, myeloperoxidase and Id. These are genes which are expressed from very early stages of one or more lymphoid, myeloid or erythroid cell lineages.

In vivo tests are conducted by injecting the presumptive PHSC into lethally or sublethally irradiated CB1 7 or C3H Scid and normal mice and assessing for short (1 -6 months) and long ( > 6 months) term reconstitution of the erythroid, myeloid and lymphoid cell lineages in these mice. This is accomplished using multi-parameter FACS analysis with cell-lineage specific surface markers of cells obtained from bone marrow, thymus, spleen, lymph nodes and blood of these animals. Donor-derived cells may be unambiguously identified by using MHC Class l-specific monoclonal antibodies as described previously (Palacios et al. , 1 990; Palacios and Samaridis, 1993; Palacios and Nishikawa, 1 992; Palacios and Imhof, 1 993) .

3. T-Cell Differentiation

Cells may be cultured on monolayers of the EH6 subcapsular thymic epithelial clone (Palacios and Samaridis, 1993) on six-well plates (Costar, Inc.) in the presence of rlL7 and Steel factor in a final volume of 1 .5 ml per well of culture medium (Iscove's modified Dulbecco's medium + 2 mM L-glutamine + 50 μM 2- mercaptoethanol + gentamycin at 50 μg/ml and 7.5% FCS) at 37°C for 8-10 days. The cells are harvested, washed and a portion of the cells are used for FACS analysis and DNA-based PCR™ analysis. The remaining cells are cultured on monolayers of the ET cortical thymic epithelial clone (Palacios and Samaridis, 1993) in six- well plates (Costar, Inc.) in a final volume of 1 .5 ml of culture medium per well at 37°C for 6-8 days. These cells are subsequently harvested, washed and used for both FACS (Palacios and Samaridis, 1993; Palacios and Imhof, 1993) and DNA-based PCR™ analysis (Palacios and Samaridis, 1993; Palacios and Imhof, 1 993) to determine the presence of T-lineage cells (JORO 75 ⁺ , CD4 ⁺ , CD8 ⁺ , TCR/CD3 ⁺ ) and rearrangement of the T cell receptor genes δ, y, and β using methods the described elsewhere (Palacios and Samaridis, 1 993; Palacios and Imhof, 1993) . These culture conditions are known to support differentiation of primitive hematopoietic stem cells (Palacios and Imhof, 1993). 4. B-Lymphocyte Differentiation

Cells are cultured on monolayers of FLS4.1 stromal cells in six-well plates (Costar, Inc.) in the presence of rlL1 1 , rlL7 and Steel Factor, in a final volume of 1 .5 ml of culture medium at 37°C for 8- 10 days. The cells are harvested, washed and cultured on monolayers of the RP010 bone marrow stromal cell line in the presence of IL7 and LPS in a final volume of 2 ml of culture medium per well at 37°C for 6-8 days. The cells are subsequently harvested, washed and used for FACS (Palacios and Samaridis, 1 993; Palacios and Imhof, 1993) and DNA-based PCR™ (Palacios and Samaridis, 1 993; Palacios and Imhof, 1 993) analysis to determine the presence of B-220⁺ lgM ⁺ B lymphocytes and rearrangements of the heavy and light-chain Ig encoding genes as described (Palacios and Samaridis, 1 993; Palacios and Imhof, 1993). These culture conditions were found to support efficiently differentiation of yolk sac stem cells into B-lymphocytes (Palacios and Imhof, 1 993) .

5. Long-Term Culture and Characterization of PHSC

PHSCs are unique in that they give rise both to new stem cells (self-renewal, manifested in the repopulation of the hematopoietic system for long time [ > 5 months]) and to all blood cell types (Till et al. , 1961 ; Fleming et a/. , 1993). Mouse bone marrow (BM) and fetal liver (FL) cells with properties of PHSC were found to express Sca-1 surface marker and C-Kit receptor and to lack most lineage- restricted hematopoietic surface markers (Spangrude er /. , 1988; Jordan et a/. , 1 990) . IN man, putative PHSC were found to be contained in the CD34 + 33-38-DR- or DR + marrow populations (Andrews et a/. , 1 990; Baum et a/. , 1 992). The extremely low number of these cells in primary hematopoietic organs and the lack of culture systems that support proliferation of undifferentiated PHSC have both precluded the study of the biology of these cells and their clinical application. PHSC can be enriched (Reviewed in Spangrude et a/. , 1990; Visser et a/. , 1990) but the numbers obtained are very low. Purified PHSC appears still to be heterogeneous in terms of size, cell cycle status and Rh 123 staining (Uchida et al. , 1993; Harrison et al. , 1 992; Li et al. , 1 992) . The only way to generate homogeneous PHSC in large number is to establish culture conditions which support proliferation without differentiation. Clones were established from hematopoietic cells from the fetal liver or bone marrow of normal and p53-deficient mice using a combination of four growth factors. The clones share with the freshly-isolated populations enriched for PHSC, phenotypic and the two functions characteristics of PHSC, namely, the ability to provide long-term repopulation of the hematopoietic system and the capacity of giving rise to lymphoid, myeloid, and erythroid-cell lineages. Pluripotent hematopoietic stem cells (PHSC) were isolated from 1 2- to 1 3-day liver of embryos and 3-week-old bone marrow of CBA/J or C57BL/6 mice. To this end, mononuclear cells are first depleted of myeloid, erythroid, T- and B-lymphocyte mature and precursor cells by using a mixture of monoclonal antibodies against surface markers on such cell populations followed by magnetic-bead separation. The negatively-selected cells were stained with FITC- Thy 1 -, PE-c-Kit- and APC-Sca-1 -specific antibodies. Cells coexpressing two or all three surface markers were positively selected by cell sorter analysis. Single-positive cells were deposited in microtiter plates using an Epics Elite cell sorter (Coulter, Miami, FL) equipped with single cell deposition system.

Several cell lines and clones were established using purified Lin-mononuclear cells from the fetal liver or bone marrow of normal and p53-deficient mutant mice. Most cell lines were Sca-1 ⁺, c-Kit ⁺ , PgP-1 ⁺ , HSA ⁺ , LIN (B-220, Joro 75, Joro 37-5, Mac-1 , 8C5, F4/80, CD4, CD8, CD3, IgM, TER1 1 9 negative) and expressed three new surface markers Joro 1 77, Joro 1 84 and Joro 3. They did not express RNA transcripts for several genes expressed at early stages of lymphocyte and myeloid/erythroid cell development.

6. Isolation and Characterization of Pro-T Cell and PHSC- Reactive Antibodies A panel of rat monoclonal antibodies (mAb) against the Pro-T cell clones have been isolated. Three of them, named JORO 37-5, JORO 30-8 and JORO 75, have been characterized in detail (Palacios et al. , 1990). Marrow cells that bind JORO 30-8 generate in vivo both T- and B-lymphocytes, whereas marrow cells that bind JORO 37-5 or JORO 75 give rise to T lymphocytes only, after their transfer into sublethally irradiated Scid mice. Since JORO 30-8 does not bind to any of the Pro-B lymphocyte clones tested, this antibody must recognize not only Pro-T lymphocytes but also putative common lymphoid progenitors and even less-differentiated multipotent precursor cells (Palacios et al. , 1 990). To study this directly continuously-proliferating JORO 30-8⁺ clones have been established from the bone marrow of young (3-week-old) CBA/J mice. Six such clones have been studied and they represent a transitional stage of development between stem cells and lineage restricted progenitors. Indeed, these multipotent progenitor clones are able to give rise, both in vitro and in vivo, to T-lymphocytes, B- lymphocytes, granulocytes and macrophages, but not to cells of the erythroid lineage (Palacios and Samardis, 1 993) . Cytokines and specialized microenvironments have been shown to direct the fate of these multipotent progenitor cells (Palacios and Samaridis, 1993).

EXAMPLE 3 ESTABLISHMENT OF LIN CLONES

Enriched populations of PHSC from day 12-13 fetal liver cells from p53 deficient mice were obtained by a combination of negative selection using magnetic beads and positive selection by FACS cell sorter. Briefly, the fetal liver mononuclear cells were depleted of TER1 19⁺ erythroid-lineage cells, 8C5⁺ myeloid-lineage cells, F4/80 ⁺ macrophage-lineage cells, B-220⁺ B-lymphocyte-lineage cells and

Joro 75 ⁺ T-lymphocyte precursor cells using appropriate antibodies and magnetic-beads coupled with anti-rat Ig antibody. Following two rounds of depletion of the positive cells, the negative cells were collected and stained with a mixture of labeled-antibodies against hematopoietic-lineage restricted surface markers (B-cell precursors, granulocytes, erythroid, myeloid, macrophages, T-cell progenitors, mature T- and B-lymphocytes) . The viable cells included in the "lymphoid" gate (determined by forward and side scatters) which were negative for all these antigens (Lin ) were isolated by cell sorter and were placed in microculture wells in culture medium supplemented with various combinations of cytokines. Further positive selection was not used for the Sca-1 , c-Kit, Joro 1 77, Thy 1 or PgP-1 surface markers known to be expressed by PHSC to avoid any potential biological effect that the antibodies might exert on the cells.

Two cytokine combinations were found to support proliferation with no or little differentiation, namely, LIF, Steel Factor, F (supernatants from the FLS4.1 fetal liver stromal cell line) and either IL 3 or FLT3-ligand. These mixtures will be referred to as 3LSF or FLT3-ligLSF, respectively. If F was removed from the cytokine mixture the cells continued to proliferate and also differentiated (usually into Mac-1 , F4/80, 8C5 and TER1 1 9-positive myeloid/erythroid cells). Both 3LSF and FLT3-Lig. LSF-conditioned medium supported the initiation and long-term ( > 6 months) maintenance of PHSC lines.

In cultures that received FLT3-lig. LSF-medium, the frequency of positive wells ( 1 2 to 19%) and the time required for the cultures to reach confluency (2-4 weeks) was lower and longer, respectively, when compared to the frequency of growing wells (up to 36%) and the time required to reach confluency (2-3 weeks) observed if the same Lin fetal liver cells were cultured in 3LSF-medium. The frequency of cultures containing myeloid/erythroid-differentiated cells was lower in Lin cell cultures initiated and maintained with the FLT3-lig. LSF- cytokine mixture.

Weekly monitoring by FACS for signs of differentiation revealed few myeloid or erythroid-lineage cells in some cell lines mostly in cultures containing 3LSF. The differentiated cells were depleted by magnetic bead selection and 1 2 out of 49 lines were discarded because of persistent and significant (10-20% Mac-1 , 8C5, TER1 1 9 positive cells) differentiation.

p53 cell lines with stable Lin phenotype from fetal liver (called FLp53 A, B, C, etc. ) and from bone marrow (called BMp53 A, B, C, etc.) were cloned first by micromanipulation and recloned twice by limiting dilution. The clones were designated by number, e.g. , BMp53 A3 is a clone 3 of the line A obtained from bone marrow of p53-deficient young mice.

EXAMPLE 4 PHENOTYPIC AND GENOTYPIC CHARACTERISTICS OF LIN P53 CLONES

The FLp53 or BMp53 clones were approximately 5-8 microns of diameter, round in shape, and exhibited scanty cytoplasm and a prominent dense nucleus, as determined by Giemsa staining. Scanning electron microscopy show that they have a homogeneous morphology and display microvilli uniformly on their cell surface.

FACS analysis of the BMp53 A1 1 clone using a panel of antibodies against several hematopoietic surface markers provided the following results: a) Cells were negative for several hematopoietic lineage restricted surface markers normally present on myeloid cells (8C5, Mac-1 , F4/80), erythroid cells (TER1 19), immature and mature B-lymphocyte lineage cells (B-220 and Ig) and immature and mature T- lymphocyte lineage cells (Joro 75, CD4, CD8, CD3).

b) Cells were positive for Sca-1 , PgP-1 , c-kit receptor, and express no or very low levels of Thy 1 surface antigen.

c) Cells stained brightly with the Joro 177 and moderately with the Joro 184 and Joro 3 monoclonal antibodies indicating that, besides reacting with early lymphoid progenitors and myeloid precursors, these surface proteins were also expressed by PHSC (Palacios et a/., 1 990) .

d) Cells were positive for the VLA-5, VLA-6, lcam-1 , lcam-2, heat stable antigen (HSA), and PgP-1 adhesion molecules, and were negative for VLA-2, VLA-4, and VCAM-1 . The other clones and cell lines tested showed a similar pattern to the BMp53^"A1 1 clone. Expression was somewhat variable for c-Kit, Thy 1 , Joro 1 84, Joro 3, VLA 6, lcam 1 and lcam 2.

Northern blot analysis revealed the absence of RNA transcripts for genes expressed at very early stages of lymphocyte development (CD3 gamma, CD3 zeta, Rag-1 , Rag-2, TCF-1 , Gata 3, LEF-1 , MB-1 , vPRE-B, lambda 5, μ Ig heavy chain, T-cell receptor delta), and of Gata-1 mRNA expressed at early stages of erythroid/myeloid differentiation. All clones expressed a newly identified homeobox gene A3 and the leucine-zipper transcriptional activator gene A52. These results indicate that the Lin p53 clones were at an earlier stage of differentiation than lymphoid-, erythroid- and myeloid-committed progenitors.

1 . Hematopoietic Reconstitution In Vivo

For repopulation of sublethally irradiated (300 rads of gamma rays) C3H-Scid-Scid mice (female 8-1 2 weeks old mice which had no detectable serum Ig), the Lin clones from normal mice (FLSC 8, FLSC 14) or from p53-deficient mice (Bmp53 A1 1 , FLp53 B4), freshly isolated fetal liver mononuclear cells from C57BL/6 embryos (10⁶ cells/0.4 ml of PBS) or PBS alone were injected IV into the recipient animal 2-4 hours after irradiation. All mice were housed in sterile isolators with sterile food in a laminar flood hood. Hematopoietic reconstitution in bone marrow and spleen of Scid mice was assessed by single - and two-color FACS analysis five to six months later.

2. Radioprotection Assay

The Lin⁺ clones from p53 deficient mice (BMp53A1 1 , FLp53B4) or from normal mice (FLSC 8, FLSC 14) alone ( 10⁶,5x10⁶, or 10⁷ cells/0.4 ml PBS) or the Lin clones ( 10⁶ cells) together with freshly isolated bone marrow cells from Rag-2 deficient mice ( 10⁵ cells) were injected I.V. into lethally irradiated (1000 rads of gamma rays provided in two equal exposures given 4 hr apart) Rag-2 deficient mice (Shinkal et a/. , 1992, 6-8 weeks old male and female). Control groups included lethally irradiated Rag-2 deficient mice that received no cells and mice that received 1 .1 x 10⁵ freshly isolated marrow cells from syngeneic Rag-2 deficient mice. All mice were housed in sterile isolators with sterile food and antibiotics in the drinking water. Radioprotection was assessed by following the survival of recipient mice. The presence of donor derived mature T- cells (TCRab ⁺ ) and mature B-lymphocytes (lgM ⁺ ) in peripheral blood of reconstituted Rag-2 deficient mice was assessed by FACS analysis with TCRab and mouse IgM-specific antibodies at the time indicated in the text.

EXAMPLE 5

CONTROL OF DIFFERENTIATION

Cell-sorter-purified PHSC from day 1 2 fetal liver cultured on monolayers of mitomycin-C treated FLS4.1 stromal cell line (Palacios and Samaridis, 1 992) and exogenous Steel Factor and LIF proliferate without differentiating. The addition of rlL3 to the cultures increased the rate of proliferation but also promoted some differentiation along the erythroid-myeloid lineages (5-1 5% of the cells become Mac-1 ⁺ , TER1 1 9 ⁺ , F480 ⁺ ) . Interestingly, cell free supernatants from confluent cultures of FLS4.1 stromal cells together with Steel Factor, LIF and rlL3 also supported growth with little or no differentiation of PHSC. The F factor-containing FLS4.1 - supernatant prevented differentiation of PHSC. Recombinant Steel Factor + rlL3 support proliferation but also clear differentiation of PHSC. Only cultures that received F factor or were carried out on monolayers of FLS4.1 stromal cells have supported proliferation of Thy1 ⁺ PgP-1 ⁺ c-Kit⁺ B-220 JORO 75 Mac-1 F4/80- TER1 1 9 cells without differentiation. Furthermore, it has been shown that continuously-proliferating PHSC can repopulate the lymphoid, myeloid and erythroid-lineages after transfer into irradiated Scid mice recipients. Indeed, these cells gave rise to B-cell precursors, T-cell precursors, myeloid and erythroid-cells in the bone marrow and to mature T- and

B-lymphocytes in the spleen of Scid mice recipients as assessed by two color FACS analysis using MHC-Class l-specific antibody which unambiguously identified the presence of donor cells and distinguished them from cells of host origin (Table 2). The lymphohematopoietic precursor potential of these long-term cultured presumptive PHSC was also documented in vitro by inducing these cells in appropriate assays systems which are described herein.

Similar results have been obtained in three separate studies each of which started with cell-sorter-purified PHSC from embryos of different pregnant mice. Also, several ( > 26) cell lines with phenotypic and functional properties of PHSC have been propagated in long-term culture (6 months). These cell lines have retained both phenotypic (Table 3) and functional (Table 4) properties of PHSC cultured for shorter time in the culture system outlined herein. No evidence was found that these cell lines are transformed malignant cells as their growth has remained dependent on exogenous growth factors and they did not give rise to malignant tumors in immunocompromised mice (Nude, Scid, RAG-2 deficient) . Using these cell lines, an assay for F factor from FLS4.1 supernatants has been developed which monitors cell proliferation (³H-thymidine uptake) and cell differentiation (expression of myeloid/erythroid surface markers assessed by FACS analysis. EXAMPLE 6

LIN P53 CLONES CAN DIFFERENTIATE

IN VITRO INTO ALL HEMATOPOIETIC LINEAGES

A. T-CELL PRECURSOR POTENTIAL

The capacity of Lin p53 clones to give rise to T-lymphocyte lineage cells was tested in a two step induction assay (Palacios and Imhof, 1 993) . The clones were cultured in medium containing IL 7, FLT3-Lig. and Steel Factor on monolayers of the subcapsular thymic epithelial clone EH6 for 7-8 days and on monolayers of the cortical thymic epithelial clone ET for 5-7 days. In the first stage of the culture with EH6 cells the Lin clone BMp53^"A1 1 showed morphological changes as determined by scanning electron microscopy. FACS analysis of cells harvested from the second- stage cultures showed the presence of Joro 75 ⁺ T-cell progenitors, CD4⁺TCR/CD3 cells, CD4⁺TCR/CD3⁺ cells (mostly TCR aβ⁺) and of CD4TCR/CD3 ⁺ cells (mostly TCR <T). The uninduced BMp53 A1 1 cells were negative for all these markers. PCR™ analysis of DNA from induced cells showed the presence of rearrangements of the TCRtJ, y and β genes involving different V-(D)-J gene elements (FIG . 1 A, FIG. 1 B, FIG. 1 C, and FIG. 1 D). Several other Lin p53 clones could also be induced to differentiate into T-cells.

B. B-CELL PRECURSOR POTENTIAL

The potential of Lin p53 clones to give rise to B-lymphocytes was also assessed in a two-step culture assay (Palacios and Imhof, 1993) . The cells were cultured first on monolayers of irradiated FLS4.1 fetal liver stromal cells in the presence of IL 7, IL 1 1 and Steel Factor for 7-8 days and then on monolayers of irradiated RP 010 BM stromal cells in the presence of IL 7 and Lipopolysaccharide for 5-7 days. It was shown that the BMp53^"A1 1 cells underwent morphological changes in the first-culture stage as determined by scanning electron microscopy. These changes were distinctly different from those seen in the T-cell induction cultures.

FACS analysis of induced cells showed the presence of B- 220 ⁺ lgM and B-220 ⁺ IgM ⁺ immature and mature B cells whereas uninduced cells were negative for these surface markers. DNA- based PCR™ analysis confirmed the appearance of B-cells in the induced cultures. V-D-J rearrangements of the Ig heavy chain genes and V-J kappa rearrangements of the Ig light chain were seen in the induced but not in the uninduced BMp53 A1 1 cells (FIG. 2A, FIG. 2B) . FIG. 2A and FIG. 2B (lane 5) show the results of the positive control DNA from freshly isolated bone marrow cells, for comparison. Thus, BMp53 A1 1 cells possess B-lymphocyte precursor activity. Several other Lin p53 FL and BM clones could also be induced to differentiate into B-cells.

C. MYELOID/ERYTHROID/MEGAKARYOCYTE PRECURSOR POTENTIAL

To test the capacity to differentiate into myeloid/erythroid/megakaryocyte-cell lineages Lin p53 clones were cultured in the presence of GM/CSF, Steel Factor, IL 1 1 , IL 3 and erythropoietin for 10 days. The cells were examined by Giemsa and Benzidine staining of cytospin preparations and by FACS analysis. Induced but not uninduced BMp53 A1 1 cells included Mac-1 ⁺ , F4/80 ⁺ , 8C5 ⁺ granulocyte/macrophage-lineage cells and TER 1 1 9 ⁺ erythroid-lineage cells. Giemsa staining showed the presence of myelomonocytic cells, mast cells, and megakaryocytes. Benzidine staining of induced cells confirmed the presence of erythroid-lineage cells. Similar results were obtained with several other p53 Lin FL and BM clones.

EXAMPLE 7 LI CLONES RECONSTITUTE THE HEMATOPOIETIC SYSTEM IN VIVO

Lin^' p53 clones were tested for their capacity to provide long- term reconstitution of the hematopoietic system of X-irradiated mice. Sublethally-irradiated Scid mice are well-suited to assess the differentiation of not only lymphocyte precursors, but also of hematopoietic stem cells. The Scid mutation renders cells more sensitive to X-irradiation and hampers their hematopoietic recovery after non-lethal doses of irradiation (Phillips and Spaner, 1 991 ) . This provides a competitive assay between host PHSC and the PHSC population under test without the need to expose the recipients to lethal doses of irradiation, which may cause severe damage of the microenvironment required for survival and differentiation of PHSC.

Two markers were used to distinguish the hematopoietic progeny of the donor cells (Lin p53 clones) from C3H Scid recipient cells, namely, H-2b assessed using an MHC class I H-2b specific antibody and FACS analysis (Palacios and Samaridis, 1 993) and the Neo^r gene assessed by DNA-based PCR™. Both markers were expressed by the donor cells but not by the recipient mice (Table 2) . EXAMPLE 8

F FACTOR MAINTAINS A PLURIPOTENT POPULATION

OF STEM CELLS

The present example demonstrates the utility of the claimed invention for maintaining pluripotent hematopoietic stem cells, and the utility of the method in techniques for reconstituting the hematopoietic system by stem cell transplant over an extended period of time.

Five to six months after transfer of the p53 Lin clones the bone marrow and/or spleens of C3H Scid mice contained donor cells which included granulocytes/macrophages (H-2b ⁺ Mac-1 /F4/80/8C5 ⁺ ), erythroid-cells (H-2b⁺ TER1 19 ⁺ ), B-cells (H-2b⁺ B-220\ lgM ⁺ ), and T-cells (H-2b⁺ CD4/CD8/TCR/CD3 ⁺ ) (Table 2). No H-2b⁺ cells were found in C3H Scid mice which received no cells. The Neo^r gene was found in thymocytes, splenic lymphocytes, splenic myeloid cells, and bone marrow lymphoid, myeloid, and erythroid cells of Scid mice reconstituted with the Lin clones from p53 Neo^{r +} mice. No Neo'-positive cells were detected in cells from tissues of the control Scid mice which received fetal liver mononuclear cells from p53 ⁺ Neo^r normal C57BL/6 mice. These findings indicate that the p53 Lin clones have the capacity to provide long-term reconstitution of the hematopoietic system. Moreover, they confirm and extend the results obtained in the in vitro assays demonstrating that they are pluripotent. TABLE 2

Clone Scid Time of Percentage positive donor-derived cells (FACS Analysis) injected Mouse analysis Bone Marrow Spleen recipient months

H-2b⁺ H-2b⁺ H-2b⁺ H-2b⁺ H-2b⁺ H- 2b⁺ B-220⁺ 8C5⁺ Mac-1⁺ TER119⁺ TCR/CD3⁺ IgM⁺

o

I

TABLE 2 (continued)

The clones or control buffer without cells were injected into sublethally irradiated C3H Scid mice (H-2K). Five to six months later, the presence of donor derived (H-2b ⁺ ) cells in the bone marrow and spleen was determined by two color - FACS analysis. ιT_ O

EXAMPLE 9 LI CLONES ESTABLISHED FROM P53⁺ NORMAL MICE

To determine whether PHSC lines could also be established from normal P53⁺ mice, the same procedures to Lin cells from 1 2- 1 3 day fetal liver of normal C57BL/6 mice. After three to four weeks' culture in 3LSF- medium, growing cell cultures were found in 23.6, 30.3 and 20.5% of the wells, respectively, in three independent studies. Approximately 7-1 1 % of the wells contained stromal-type cells in addition to the hematopoietic cells and these cultures were not followed further. A total of twenty positive cultures were chosen for further expansion to establish cell lines, and these cell lines were termed SC. FACS analysis using a panel of surface markers against different hematopoietic cell lineages showed that 14 lines had a phenotype similar to the p53^" Lin clones and cell lines (i.e. , Sca-1 ⁺ , Joro 1 77 ⁺ , PgP-1 ⁺ , Lin [(B-220, Joro 75, Mac-1 , F4/80, 8C5, TER 1 19, negative)]) . The other six lines contained between 4 and 1 3% erythroid myeloid cells (TER1 1 9 ⁺ , Mac-1 ⁺ , 8C5 ⁺ ) (Table 3) . Depletion of the erythroid/myeloid cells from two of the latter cell lines by magnetic-beads and appropriate antibodies for three consecutive times, resulted in the propagation of virtually pure ( = 100%) Lin cells.

TABLE 3 EXPRESSION OF SURFACE MARKERS IN SC CELL LINES

SURFACE MARKERS EXPRESSED B-220 8C5 Mac-1 F4/80 TER1 1 9

+ + +

+ + to

+ + + + + + +

TABLE 3 (continued)

SURFACE MARKERS EXPRESSED B-220 8C5 Mac-1 F4/80 TER1 1 9

+ +

I

The phenotype of the SC cell lines was determined by single color FACS analysis. A means the presence of 4 to 1 3% positive cells for the surface marker indicated.

All ten SC Lin cell lines were found to have the potential to generate T-cells, B-cells, and myeloid/erythroid-lineage cells in vitro (Table 4). Taken together the results indicate that the SC Lin cell lines obtained from day 1 2-13 fetal liver of normal C57BL/6 mouse embryos, like the Lin clones obtained from fetal liver and bone marrow of p53 deficient mutant mice, possess properties of pluripotent lymphohematopoietic stem cells.

EXAMPLE 10 ISOLATION OF cDNA-ENCODING F FACTOR

The F factor produced in the FLS4.1 cell line may be used to isolate cDNA encoding F factor. This is accomplished using a direct cloning-expression system in which cDNAs from a FLS4.1 library are transiently transfected into Cos-1 cells (which do not produce F factor) with the supernatants being tested for the biological effects on PHSC. Purification of the FLS4.1 derived F factor facilitates the partial amino acid sequencing of F factor to produce oligonucleotides probes for screening a cDNA library made from FLS4.1 stromal cells. Re-screening of cDNA libraries and utilization of anchored PCR™ assays permits the isolation of full-length cDNAs.

The first one utilizes a direct cloning-expression strategy. Double-stranded cDNA is synthesized using Poly (A) ⁺ RNA from FLS4.1 cells, ligated and cloned into the Pst\ site of the mammalian expression vector pcDNAI (Invitrogen, San Diego, CA) and transfected into E. coli P3. Plasmid DNA from pools of 500-100 individual transformants are isolated and transfected into subconfluent COS-1 cells by Lipofection (Promega, Palo Alto, CA) . After 2-3 days, supernatants from the transfectant COS cells and control untransfected COS cells are screened for their ability to replace the biological activity of F factor in the assay described above using the PHSC cell lines and assaying for signs of differentiation by FACS analysis 3-5 days later. The positive pool(s) are subcloned and assayed until a single clone encoding the biological activity of F factor is obtained. The cDNA inserts from the selected clones are isolated and sequenced using the Sequenase^® 2.0 kit and T7 and Sp6 primers. Subsequently, 20mer oligonucleotides are prepared in a 394 DNA synthesizer (ABI, La

Jolla, CA) for use as primers to generate complete DNA sequences of both strands (Sambrook et al. , 1989).

A restriction map of the cDNA inserts permits characterization of the gene and facilitates assembling DNA segments into contigs (Sambrook et al. , 1989) . The advantage of this method is that it allows one to isolate cDNA containing the entire coding region of the gene encoding F factor. This method has also been used to identify genes encoding the JORO surface markers cloned by the inventor.

A second method for obtaining the gene for encoding F factor involves obtaining the partial amino acid sequence of F factor polypeptide, preparation of oligonucleotide probe families based on this amino acid sequence and screening of the cDNA library from FLS4.1 cells to isolate the corresponding cDNA. To this end, F factor is produced in large quantity (50 to 100 liters) by culturing FLS4.1 cells in a 20-liter perfusion culture system (Biolafitte, Boston, MA). 10

The SC cell lines had been cultured in the presence of F were induced to differentiate along either the T-lymphocyte, the B-lymphocyte or the myeloid/erythroid cell lineages. The induction procedure employed is described below. At the end of the cultures, the cells were harvested and the presence of the cell-lineage characteristic surface markers indicated was 5 assessed by single or two color FACS analysis.

Cells coexpressing Mac-1 and 8C5 surface markers were often detected, this explains why the total percentage of positive cells sometimes exceeded 100%.

The supernatants are collected, concentrated by ultrafiltration

10 and diafiltered at 4°C against 50 mM Tris-HCI (pH 7.8). The protein fraction is then applied to a DEAE-cellulose anion exchange column

(Whatman DE-52, Maidstone, England) equilibrated in 50 mM Tris-

I HCI (pH 7.8). The column is eluted with a 0- to 300-mM NaCl gradient in Tris buffer (10 column volumes). Fractions are tested for

1 5 their ability to replace F factor in the biological assay described herein. Positive fractions are pooled, concentrated and applied to an

Ultra gel AcA54 gel filtration column (LKB Pharmacia, Uppsala, Sweden) equilibrated in 50 mM Tris-HCI (pH 7.4) containing, 50 mM NaCl. Positive fractions in the biological assay are pooled and

20 applied to a wheat-germ agglutinin-agarose column (Pharmacia,

Uppsala, Sweden) equilibrated with 20 mM Tris-HCI (pH 7.4)

containing 500 mM NaCl. After washing with column buffer, bound material is eluted by applying a gradient of 0-750 mM NaCl (in column buffer). Pooled fractions from the Sepharose^® column are then applied to a reverse-phase column equilibrated with 60 mM ammonium acetate (pH 6.0) in isopropanol. After washing the column, a linear gradient made by two buffers based on ammonium acetate and isopropanol, is applied and fractions are collected.

Fractions are assayed for F factor activity and analyzed by SDS-PAGE followed by silver staining of the gels to document the purity of the protein. PHSC Line cell lines (e.g. , BMp53-lin-A1 1 ) are cultured in the presence of IL3, LIF, SF and preparations to be assayed for F factor activity. Only those preparations containing F factor activity will prevent differentiation of the PHSC Lin cells into myeloid/erythroid cells (this is determined by FACS analysis using Mac-1 , 8L5, TER 1 19, Fy 180 antibodies) and by cytospin preparations and Geimsa staining.

Non-full length cDNAs sequenced in this manner may be used as probes to screen a genomic library made of liver from 1 29Sv/Olac mice (Stratagene, La Jolla, CA) with standard procedures (Sambrook et al. , 1989) . Alternatively, anchored-PCR™ (Sambrook et al. , 1 989) using a T7-specific primer (the cDNA library will be constructed in a vector containing T7 and Sp6 promoters) and a downstream primer specific for the sequence already determined from the incomplete cDNAs may be used to obtain the full-length cDNA sequence. The PCR™ products are cloned into the modified pBlueScript^® vector using a commercially-available PCR™ cloning kit (Stratagene). cDNAs encoding F factor from human and murine cell lines are used to determine expression of F factor by various hematopoietic and nonhematopoietic cells/tissues in both embryo and adult mice by Northern blot analysis or RT-PCR™ assays. The possibility that the gene could belong to a given gene family will be tested by using mouse chromosomal DNA restricted with various enzymes, blotted to membrane filters and hybridized to labelled fragments of the cDNA coding for F.

EXAMPLE 1 1

PRODUCTION OF F-FACTOR-SPECIFIC ANTIBODIES

One hundred micrograms of purified F factor emulsified in complete Freund's adjuvant are injected in the hind foot of rat or hamster. Three days later a similar amount of purified protein diluted in phosphate buffered saline (without Freund's adjuvant) is injected against in the same anatomical site. Three days later this procedure is repeated once more. One day after the third immunization, the regional lymph node mononuclear cells are recovered and used to fuse with the HAT -resistant myeloma

X63Ag8 or J558L cells lines. The cells are selected for hybridomas by culturing in HAT-selection medium and the presence of antibodies against F factor in individual culture wells is determined by ELISA. The anti-F antibody secreting hybridoma cells identified are then expanded in culture medium and the F factor-specific antibody is purified by Protein G-column chromatography. The purified antibody is then tested for its ability to neutralize F factor biological activities as well as to immunoprecipitate (Western blot or SDS-PAGE analysis) F factor or quantitate F factor in different samples by ELISA.

EXAMPLE 12 LABELED RECOMBINANT F FACTOR

PROTEINS TO STUDY CELL SURFACE RECEPTORS

F Factor produced in recombinant form and purified as described in Example 1 1 can be labeled by biotinylation or iodination with ¹²⁵l or ³H-Leucine. The labeled F-factor is incubated with cells known to be sensitive or insensitive to F factor at different temperatures (4°C, 37 °C), ratios of labeled-factor to number of cells. The binding of F factor to the cells is then determined by measuring the fluorescence intensity (in case of biotin-labeled F Factor, using FITC-conjugated Streptavidin which binds to biotin) or the radioactivity contained in the cell pellet (measures bound F factor to cells) and in the supernatant free of cells (measures unbound labeled F factor) . Scatchard plots are then performed to determine the presence and the number of binding sites with high, intermediate and low affinities for the F factor. Similar studies can be used to determine the dissociation rate of bound labeled-F factor. These studies reveal the presence of receptors for the F factor and identifies cell lines that express the highest number of receptors for F. Such cell lines can then be used to generate monoclonal antibodies against the receptor for the F factor as described in

Example 13. In this case, antibodies are screened for their ability to block the binding of labeled-F factor to receptor-positive cells. Once monoclonal antibodies reactive against the F factor are obtained, immunoprecipitations of cell lysates from the receptor-expressing cells can be carried out using the anti-F factor receptor antibody to determine the biochemical characteristics and the components of the F factor receptor. Also, the gene(s) encoding the component(s) of the F factor receptor may be cloned by cloning expression systems described in Example 1 1 .

The availability of cDNAs encoding and monoclonal antibodies reactive with F factor receptor will allow the development of ELISA assays to detect and quantitate the presence of this receptor in serum, urine or other fluids from patients with abnormalities of the hematopoietic system; to assess by FACS analysis, immunohistochemistry, Southern Blot or Northern blot assays abnormalities in the expression of the F factor receptor in blood cell disorders including immunodeficiencies, AIDS, leukemias, lymphomas, aplastic anemia, graft versus host diseases, septicemia and blood cell deficiencies secondary to chemotherapy and radiotherapy. Finally, together with the availability of F factor in recombinant form, it will offer the possibility of immunohematotherapy in diseases in which the F factor or its receptor is deficient. Also, the availability of the gene encoding the F factor or its receptor will offer the possibility of somatic gene therapy in cases where these genes are found to be defective or to produce a biologically inactive protein. EXAMPLE 13

DEVELOPMENT OF A TRANSGENIC ANIMAL MODEL

EXPRESSING F FACTOR

The cDNA encoding F factor protein is subcloned in the expression vector pCDNA 2 (contains CMV promoter) or the PHT4- YK-CEH expression vector (contains a heavy chain core enhancer and promoter and a kappa light chain promoter elements) . The fragments containing the regulatory elements (enhancers, promoters), cDNA for F factor, polyadenylation and splicing signals, is released from the vector and purified by agarose gel electrophoresis. The purified fragment is then injected into the pronuclei of mouse fertilized eggs from (C57BL/6 x SJDF1 or (C57BL/6 x DBA/2)F1 mice and subsequently placed in the uterus of foster pseudopregnant mice. The pups are then screened for expression of the transgenic mice by using DNA obtained from their tails which is subjected to enzyme restriction digestion followed by Southern blot hybridization and hybridization with radioactive- labelled DNA probes specific for the transgene. Transgenic mice are bred by crossing brother sisters or progeny to parents. The presence of circulating F factor in the serum of transgenic mice can be detected by ELISA assays with F factor specific antibodies. The cells and tissues producing the transgenic F factor will depend on the regulatory elements used to control the expression of the transgene F factor. In the examples above, the transgene using the CMV promoter will be expressed in most if not all cell types while the transgene under the control of the Ig heavy and kappa chain enhancer/promoters will be expressed in B-lymphocytes only. The consequences of overexpression of F factor on the hematopoietic system and on the development of mouse embryos can be determined by comparing wild type animals with the transgenic mice by using FACS analysis to study the proportion of cells in a given tissue or organ, immunohistochemistry to study the morphogenesis of tissues and organs and functional tests including transfer of hematopoietic precursor cells into immunohematocompromised animals to determine the frequency and function of hematopoietic precursors comprising stem cells. An example of developing transgenic mice for a cytokine is illustrated in a previous publication (Samaridis et al. 1 991 ) .

EXAMPLE 14 USING UNDIFFERENTIATED PHSC CLONES ESTABLISHED IN CULTURE IN F-FACTOR CONTAINING CYTOKINE MIXTURES

To generate antibodies against surface proteins expressed on pluripotent hematopoietic stem cells (PHSC), pluripotent hematopoietic stem cell lines such as those established by the inventor using F factor in the cytokine-conditioned culture medium (e.g. , BMp53LinA1 1 ) are critical/essential. One successful protocol (there are of course other variations to it) to produce monoclonal antibodies reactive with PHSC consists in injecting 30 million PHSC cells emulsified in complete Freund's adjuvant in the hind foot of a rat or a hamster. Three days later, the animals are injected with 30 million of PHSC cells diluted in phosphate buffered saline (without Freund's adjuvant) in the same anatomical site. This injection is repeated a third time three days later. One day after the last immunization, the regional lymph node mononuclear cells are obtained and fused with a HAT-resistant myeloma cell (e.g. , X63Ag8, J558L, SP2/0) and the cells are selected for hybridomas by culturing in medium containing HAT selection drugs. Supernatants from cultures containing hybridomas (growing cells) are tested for antibodies binding to the PHSC cells used in the immunization. This can be conveniently done by FACS analysis.

The hybridomas secreting antibodies reactive against the PHSC cells are then expanded to obtain large quantities of the antibody. Aliquots of the hybridoma cells are frozen. The monoclonal antibody can be purified by using Protein-G chromatography. The purified antibody is now used in immunoprecipitations of cell lysates of the PHSC cells to determine the biochemical characteristics of the protein recognized by the antibody. The purified antibody also can be used to determine whether the antigen recognized by the antibody is expressed only PHSC cells or in other type of cells. This is usually done by FACS analysis or standard immunohistochemistry assays (see Palacios et al. , 1990). The antibodies can then be used to identify, quantitate, and isolate stem cells from different tissues and at different stages of mouse development. Also, the selected antibodies can be used to clone the gene encoding the protein recognized by them using cloning expression systems such as that described in Example 1 1 . The selected antibodies also can be used to test for their ability to interfere with proliferation or differentiation of stem cells by adding the antibodies to appropriate cultures in which the stem cells will proliferate only or will differentiate into lymphoid, myeloid and erythroid cell lineages as described in Examples 7, 8 and 9. EXAMPLE 15

PRODUCTION OF MOUSE MUTANTS DEFICIENT IN F FACTOR

A targeting vector is constructed to perform disruption of one allele of the gene coding for F factor in Embryonic Stem cells following standard protocols (Ramirez-Soliz, et al. , 1992). Briefly, between 1 .5 and 3 kb of genomic DNA containing the F factor gene is subcloned upstream and downstream of an expression vector containing Neo^r gene and TK genes to confer sensitivity of FIAU. The linearized vector is then injected into ES cells (e.g. , ESD3, ESE 14.1 ) and ES cells have underdone homologous recombination are selected by culturing the transfected cells in the selective drugs FIAU and G418. DNA is then isolated from the Neo^r cells and the presence of a disrupted allele of the F factor gene is determined by Southern blot analysis with appropriate radiolabeled specific probes. ES cells which are found to carry one disrupted allele are then injected into the blastocyst of normal mice and blastocysts are then implanted in the uterus of pseudopregnant mice. Pups born are then screened for those than carry one disrupted F factor gene. Brother and sisters or son with mother that score positive in the screening are mated to generate mice which the F factor gene disrupted in BOTH alleles (i.e. , mice homozygous for the null mutation). Homozygous mice are then studied to determine the lack of F factor in the development of hematopoietic stem cells and other blood cell types as well as for any other abnormalities that might occur in other tissues or organs. The latter will reveal other functions of the F factor in other cells than hematopoietic stem cells. Thus, by developing mice lacking the F factor it is possible to directly document the physiological functions of this cytokine in both the development of the embryo and in the physiology of the entire adult mouse.

EXAMPLE 16 NUCLEIC ACID SEGMENTS COMPRISING A NOVEL MAMMALIAN

GENE ISOLATED FROM A LYMPHOHEMATOPOIETIC PRECURSOR

Another aspect of the present invention concerns the use of lymphohematopoietic precusor cells in the isolation of novel nucleic acid segments comprising nuclear-envelope associated protein- encoding genes. In this example, the A3 gene has been isolated and cloned from a cDNA library constructed with poly(A) RNA from the mouse lymphohematopoietic progenitor clone PR-23. The A3 gene sequence predicts a polypeptide of 53,598 Daltons with one potential membrane-spanning region and two potential N- glycosylation sites. Northern blot analysis shows that the A3 gene is expressed in hematopoietic cells (from hematopoietic stem cells onwards), nonhematopoietic cells (from hematopoietic stem cells onwards), nonhematopoietic cells (thymic epithelial cells, fetal liver and bone marrow stromal cells) and in several tissues (thymus, bone marrow, spleen, kidney, brain, heart) . The A3 deduced protein was found to be related to the NIP1 gene that codes for an essential protein required for nuclear transport in yeast. Together with its broad pattern of expression the A3 protein could be a nuclear envelope-associated protein perhaps forming part of the nuclear pore complex and functioning in the binding of nuclear localization signal (NLS) containing proteins. A. MATERIALS AND METHODS

1. Cell lines

The reports describing the origin and characterization of the cell lines BMP53A1 1 , PR-23, PR-5, PR-8, LyD9, Ba/C1 , FLB32, FLB41 , 32Dcl, FTH5, FTg1 2, Scid27F/ET, EA2, ET, RPO10, FLS4.1 , 97.2, J558L, P388D1 are listed elsewhere (Palacios and Samaridis, 1993; Palacios and Nishikawa, 1992; Palacios and Samaridis, 1992).

2. Cloning and analysis of the A3 cDNA

The A3 clone was isolated from the cDNA library constructed with poly (A) ⁺ RNA from the lymphohematopoietic clone PR-23

(Palacios and Samaridis, 1 993) in the mammalian expression vector pcDNAI (InVitrogen) as described (Xie and Palacios, 1 994).

The A3 clone contained an ~ 1 .7 kb insert, and it was initially sequenced with an Applied Biosystems 370A automated sequencer (Applied Biosystems, Foster City, CA) using T7 and SP6 promoter- specific primers. Comparison using the BLAST program of the nucleotide sequence obtained with the genes deposited in GenBank and SwissProt databases suggested that A3 could be a new gene. Sequenase^® kit (US Biochemical, Cleveland, OH) by the dideoxynucleotide chain-termination method (Sambrook et al. , 1 989) with specific oligonucleotide primers. Searches of the GenBank, EMBL and SwissProt databases were performed with the BLAST program. Analysis of the A3 sequence was performed with the GCG software package.

3. Isolation of Nucleic Acids

The Tri-Reagent protocol was followed to isolate total RNA from the different hematopoietic and nonhematopoietic cells lines and from tissues (liver, brain, heart, kidney, thymus, spleen, bone marrow) of 6-8 weeks old C57BL/6 and CBA/J mice.

4. Northern Blot analysis

After electrophoresis of 10-1 5 μg of total RNA and transfer to Zeta probe nylon membranes, the filters were hybridized with ³²P- labeled ~ 1 .2 kb BamH\-XHO\ (probe B) or 0.4 kb Bam \-Bam \ (probe A) isolated fragments of the A3 cDNA overnight at 65 °C. Following one wash in 0.3X SSC + 0.1 % SDS at room temp for 5 min and one wash in 3. OX SSC + 0.1 % SDS at 65 °C for 30 min, the filters were processed for autoradiography and the films were exposed at -70°C for 1 to 5 days. The probe was stripped and the filters were hybridized with a beta actin specific probe (Palacios and Samaridis, 1993; Xie and Palacios, 1994), processed for autoradiography and the films were exposed for 6-24 hrs.

B. RESULTS AND DISCUSSION

The A3 cDNA contained an ~ 1 .7kb insert and Northern blot analysis showed the presence of a single — 1 .6 kb RNA transcript synthesized by PR-23 cells and other hematopoietic precursor clones. This suggested that the A3 clone represented a full-length cDNA. The A3 clone comprised 1 721 nucleotides and included a poly(A) ⁺ tract (SEQ ID NO:3) . The largest open reading frame found in this clone comprised 458 codons that extended from a methionine initiation codon at nucleotides 61 through 63 to a stop codon at 1437 through 1439 (SEQ ID NO:4). The initiation codon at nucleotides 61 through 63 was embedded in a context favorable for translation (Kozak, 1 981 ) and it was preceded by upstream stop codons at nucleotides 3-5, 30-33 and 48-51 . The start of the poly(A) additional signal was 1 8 nucleotides from the poly(A) tall and had the sequence AUUAAA, which is less common than AAUAAA, but is found in other mammalian genes (Xie and Palacios, 1994). The protein deduced from the A3 nucleotide sequenced had a predicted M_r 53,598 and a predicted isoelectric point of 6.67. Analysis of the A3 deduced amino acid sequence with Tmpred^®, a program for the prediction of transmembrane regions, showed the existence of one potential transmembrane helix-spanning residues 269 to 287. Consistent with this analysis the hydropathy plot (Kyte and Doolittle, 1 982) showed a hydrophobic region spanning residues 271 to 287. Two potential N-glycosylation sites at residues 1 98- 201 and 219-222 were found. In addition, seven potential protein kinase C phosphorylation sites (motif [ST]a[RK]) were found at amino acid residues 8-10, 25-27, 1 1 7-1 19, 149-1 51 , 225-227, 320-322, 375-377; nine potential Casein kinase II phosphorylation sites (motif [ST]-aa[DE]) were found at amino acid residues 6-9, 34- 37, 83-86, 1 1 2-1 1 5, 1 1 8-1 21 , 1 20-1 23, 320-323, 385-388 and 394-397. These results are consistent with the view that the A3 gene codes for a type I transmembrane glycoprotein with potential phosphorylation sites for protein kinase C and Casein kinase II . Searches in the gene databases with the BLAST program revealed that the A3 deduced protein has about 30.6% overall amino acid identity to the portion comprising amino acid residues 304 to 800 of the yeast NIP1 protein. More precisely, the A3 deduced protein displayed three regions with significant identity to the yeast NIP1 gene which encodes a nuclear transport protein (Gu et al. , 1 992). Thus, the A3 region spanning residues 27 to 101 had about 34.5% amino acid identity and 59% amino acid similarity to the NIP1 region spanning the amino acids 364 to 443, a second A3 segment comprising residues 1 1 7 to 132 shows 37% amino acid identity and 62% amino acid similarity to resides 32 to 47 of the NIP1 protein and a third segment of the predicted A3 protein comprising residues 143 to 448 has 23% amino acid identity and 39% amino acid similarity to the region spanning residues 489 to 794 of the NIP1 yeast predicted protein. Also, a short region of the deduced A3 protein (residues 361 to 443) showed 26% amino acid identity and 51 % amino acid similarity to a region comprising residues 362 to 444 of the YMJ5 CAEEL cosmid clone from c. elegans of unknown function. This analysis shows that the mouse A3 protein is related to the yeast NIP1 protein, an essential polypeptide required for nuclear transport in yeast (Gu et al. , 1992).

Northern blot analysis indicate that the A3 gene is expressed in hematopoietic and nonhematopoietic cells. Within the hematopoietic lineage A3 was expressed in the hematopoietic stem cell clones BMP53A1 1 and PSB6/8, the multipotent progenitors PR- 23, PR-8 and PR-5, the B-lymphocyte/myelocytic progenitor lines LyD9 and Ba/C1 , the pre-B cells FLB32 and FLB41 , the myeloma J558L cells, the Pro-T cell clones FTH5 and FTg1 2, the pre-T cells Sci27F/ET, the myeloid progenitor 32Dci, and the macrophage lines 97.2 and P338D1 . A3-mRNA was also synthesized by the thymic epithelial lines EA2, the bone marrow RPO10 and the fetal liver FLS4.1 stromal lines. Among tissues, A3 was expressed in thymus, bone marrow, spleen, kidney and very weakly in heart and brain. The same results were obtained using the probe A and the probe B which covers the NH₂-terminal and the mid and COOH-terminal encoding regions, respectively, of the A3 cDNA. These observations show that the A3 gene is broadly expressed in cells of both hematopoietic and nonhematopoietic origin.

The nuclear pore complex has been considered as an organelle composed of a unique set of proteins necessary for transporting macromolecules across the nuclear envelope. Only some of the nuclear pore complex-associated proteins have so far been identified (Silver, 1 991 ; Yamasaki and Lanford, 1 992; Newmeyer, 1 993; Hinshaw et al. , 1 992) . One set of pore complex proteins, called nucleoporins, are O-glycosylated and have been postulated to provide a docking site for NLS-binding protein complexes (Wimmer et al. , 1 992; Wente et al. , 1 992) . A3 seems unrelated to the rat P62, the yeast NSP1 and NUP1 (Nehrbass et al. , 1 990; Davis and Fink, 1990), and the rat NUP1 53 (Sukegawa and Blobel, 1 993) nucleoporins or the Heat shock protein 70 recently shown to participate in nuclear import. A3 also is unrelated to the 70 kDa NLS-binding phosphoprotein required for nuclear import in permeabilized Drosphi/ia cells (Stochaj and Silver, 1 992) and to the human 70 kDa cytoplasmic NLS-binding protein described by Li et a/. , (1 992) . The A3 deduced protein was found to be related to the NIP1 gene encoding an essential protein required for nuclear transport in yeast (Gu et a/. , 1992) . Together with its broad pattern of expression it seems likely that the A3 protein may participate in nuclear transport of proteins in mouse cells. Like the yeast NIP1 and NRS1 nuclear transport proteins (Gu et al. , 1992), the predicted NH2-terminal region of A3 is rich in serine and glutamic acid and has several potential Casein kinase II phosphorylation sites, features which were interpreted to suggest that these proteins might function in the transport of NLS-containing proteins via their serine- rich acidic NH2-terminal (Gu et al. , 1 992) . Unlike the NIP1 amino acid sequence which reveals no obvious membrane-spanning region, the gene sequences of A3 predicts a 53,598 Daltons polypeptide with one potential membrane-spanning region and two potential N- linked glycosylation sites suggesting that A3 is a transmembrane protein, indicating A3 may belong to the group of nuclear-envelope associated NLS-binding proteins.

EXAMPLE 17 ANTIBODY COMPOSITIONS RECOGNIZING THE CD98

TRANSMEMBRANE PROTEIN

In the search for cell surface markers expressed on hematopoietic stem cells and/or very early progenitor cells, it was found that the Joro 177 monoclonal antibody (mAb) bound to most hematopoietic cells in day 8/8.5 yolk sac, day 1 2 fetal liver, and day 1 3 fetal thymocytes; it stained hematopoietic stem cells and less immature lymphoid, myeloid, and erythroid-lineage cells, but not most thymocytes and splenic lymphocytes in adult mice. Joro 177 mAb stimulated tyrosine phosphorylation of a -1 25 kDa protein and induced homotypic aggregation of lymphoid progenitor cells. Importantly, Joro 1 77 mAb inhibited cell survival/growth and consequently the generation of lymphoid, myeloid and erythroid lineage cells in vitro from early Lin hematopoietic precursors. Joro 1 77 mAb induced apoptosis of hematopoietic progenitor cells. Molecular cloning and expression indicated that Joro 1 77 mAb recognizes a type II transmembrane protein which is the mouse homologue of the human CD98 heavy chain gene. CD98 appears to function as a cell membrane receptor involved in the control of cell survival/death of hematopoietic cells.

During mouse development, primitive hematolymphopoiesis occurs in district tissues/organs as the embryo develops. These include the yolk sac which appears at day 7 of gestation, the liver primordium which appears at day 8-9, the thymic anlage which appears at day 9-10, and the spleen and bone marrow which appear at day 1 5-1 6. The heart starts beating at day 9, at which point blood circulation commences in the embryo. Definitive hematolymphopoiesis occurs mainly in the bone marrow throughout the life time of the mouse (Metcalf and Moores, 1 981 ). Hematolymphopoiesis is a dynamic process marked by the stepwise loss of both self-renewal and multifunctional potential as the PHSC gives rise to progenitors for the different mature blood cell types. Mouse bone marrow (BM) and fetal liver (FL) cells with properties of PHSC express Sca-1 surface marker and c-Kit receptor and lack most lineage-restricted surface markers (Lin) (e.g. , B-220 for B- lymphocyte, Joro 75 for T-lymphocyte, Mac-1 for myeloid, Ter1 1 9 for erythroid, 8C5 for granulocyte-blood cell lineages) (Dexter et al. 1 976; Jordan et al. , 1990; Keller and Snodgrass, 1990; Uchida et al., 1 993). In man, putative PHSC are included in the CD34 + 33- DR- and CD34 + 33-DR + marrow populations (Andrews et al. , 1992; Verfaille, CM., 1 992; Baum, et al. , 1992).

The cell surface molecules on PHSC that participate in interactions with other cells from particular microenvironments, the growth factors that support cell division without differentiation of these cells and the cellular and molecular events responsible for the choice between self-renewal and differentiation are still poorly understood. PHSC must express sets of genes whose products, acting in the nucleus, cytoplasm, or the cell membrane, participate in these processes. The molecules expressed on the cell membrane of PHSC are likely to play key roles, interacting with other cells (e.g. , stromal cells) and transmitting signals to and receiving them from a given microenvironment. Actually, these most probably dictate whether a PHSC will self-renew, differentiate, or even die.

A. MATERIALS AND METHODS

1 . Animals

Male or female normal C57BL/6 (3-10 weeks old) and female C3H Scid mice (8-12 weeks old) that had no detectable Ig in their serum were used. C57BL/6 mouse embryos were obtained from timed matings; the day of detection of a vaginal plug was designated day 0. 2. Cell lines

The FTH5 Pro-T cell line (Pelkonen et a/. , 1988), the fetal liver stromal line FLS4.1 (Palacious and Samaridis, 1 992), the ET cortical thymic epithelial cell line (Palacious, et a/. , 1 988), and Cos-1 cells (provided by Beat A. Imhof, Basel Institute for Immunology, Basel, Switzerland), were used. The development, characterization, and conditions used to propagate these cells in culture have been described before (Palacious and Samaridis, 1 992; Palacious, et a/. , 1 988) .

3. Cytokines

Recombinant mGM-CSF (gift from Biogen S.A., Geneva, Switzerland), supernatants from X63Ag8 or J558/L myeloma cells transfected with cDNAs coding for mlL2, mlL3, mlL4, hlL6, mlL7 (Karasuyama and Melchers, 1 988; Samaridis, et a/. , 1 991 ), Cos-1 cells transfected with cDNA coding for Steel Factor (Palacious and Samaridis, 1992); B8 transfected cells producing IL1 1 were provided by R.G. Hawley (University of Toronto, Toronto) .

Erythropoietin was purchased from B&D Systems (Minneapolis, MN) . J558/L/FLT3-Ligand/2 transfectant cells producing recombinant mouse FLT3-ligand were developed by subcloning FLT3-ligand-mouse cDNA into the EcoRI-Xhol site of the pCDNA3 mammalian expression vector. 4. Antibodies

Biotin, phycoerythrin (PE) or fluorescein isothiocyanate (FITO- conjugated antibodies against Thy1 , LyT2 (CD8), L3T4 (CD5), CD3 (Hybridoma 145-2c1 1 ), B-220 (hybridoma 6B2), MHC class I of the H-2b haplotype (hybridoma AF6-B8.5) . MHC class I of the H-2K haplotype (hybridoma AF3-1 2.1 ), Mac-1 (hybridoma H57-597) , TER1 19, 8C5, T-cell receptor (TCR) aβ (hybridoma H57-H97), TCR yδ (hybridoma GL3), alpha-4 integrin (PS/2), alpha-5 integrin (5H10), lcam-1 (YN 1 /1 .7), lcam-2 (MIC2), and Sca-1 were purchased from Pharmingen (San Diego, CA) . FITC- and PE-conjugated F4/80 antibody was from SEROTEC (Kidlington, Oxford). FITC-conjugated anti-mouse IgM, Kappa light chain, and IgG and PE-streptavidin were from Southern Biotechnology Associates (Birmingham, AL) . FITC- streptavidin was from Vector Laboratories. Unconjugated and FITC- conjugated anti-rate IgG, purified mouse Ig and Rate Ig were from Jackson Immunoresearch Labs (West Grove, PA) . Joro 75 and Joro 1 77 (Palacious, et a/. , 1992) were prepared as described earlier. Antibody against CD44 (KM703) was obtained from P.W. Kincade (Oklahoma Medical Research Foundation, Oklahoma) and LFA-1 (H 1 55-78) was obtained from M. Pierres (Centre d'lmmunologie, Marseille, France). Antibodies against B1 (9EG7) and B7 (FIB30.1 ) integrins were provided by D. Vestweber (MPI, Freiburg, Germany) and by E. Butcher (Stanford University, Stanford, CA), respectively. Antiserum against FAK and anti-phosphotyrosine antibody (4G10) were from Upstate Biotechnology Incorporated (Lake Placid, NY) . mAbs were purified using the Monotrap II^® system from Pharmacia (Uppsala, Sweden) and conjugated to biotin as described before (Palacious, et a/. , 1988). 5. Preparation of mononuclear cells

Mononuclear cells free of erythrocytes were prepared from spleen, thymus, bone marrow of adult mice, and fetal liver and thymus as described before (Palacious, et a/. , 1 990). Yolk sac mononuclear cells from day 8/8.5 embryos were obtained as described (Palacious and Imhof, 1993) . The cells were washed and resuspended in the required buffers or in culture medium (Iscove's modified Dulbecco medium supplemented with 5-7.5% heat- inactivated fetal calf serum, L-glutamine [2 mM], 2-mercaptoethanol [5 x 10⁵ M], and gentamycin [50 μg/ml]).

6. isolation of Lin and Joro 177 Lin mononuclear cells

Bone marrow mononuclear cells from 3-6-week old mice and day 1 2-1 3 fetal liver mononuclear cells were first depleted of lg ⁺ , Joro 75 ⁺ , B-220 ⁺ , F4/80 ⁺ , 8C5 ⁺ , CD3 ⁺ (in BM samples), and TER1 19⁺ cells using magnetic beads coupled with sheep anti-rat Ig (Dynabeads, Dynal, N.Y.) by incubating the cells with saturating concentrations of the antibodies at 4°C for 20 min. The magnetic- particle bound cells were removed by applying a magnetic force; the magnetic-particle-free cells were collected and re-exposed to the magnetic force for 5-10 min. to further remove the particle-bound cells. The magnetic-particle-free cells were collected, spun, counted, and resuspended in culture medium; they are referred to as Lin hematopoietic cells. Joro 1 77 ⁺ Sca-1 ⁺ Lin cells from day 1 2 fetal liver were purified by further incubating fetal liver Lin cells obtained as described above, with appropriate dilutions of Pl-or biotin-conjugated antibodies against F4/80, B-220, Mac-1 , TER1 1 9, 8C5, Joro 75, CD4, and FITC- Sca-1 at 4°C for 30 min. The cells were washed and exposed to PE-streptavidin at 4°C for 20 min. After this, the cells were washed two times and resuspended in cell sorter buffer (PBS + 5%FCS + gentamycin), and the viable cells that were Sca-1 ⁺ and negative for all the other markers (Lin) were purified by cell sorter using an ELITE sorter instrument (Coulter, Florida) essentially as described before (Palacious and Samaridis, 1 993). A proportion of the cell sorter purified cells were used for staining with biotin-conjugated Joro 1 77 and APC-streptavidin followed by FACS analysis. This analysis showed that > 99% of the cells were Sca-1 ⁺ Lin and 100% Joro 1 77 ⁺ ; they are referred to as Joro 177 ⁺ Lin cells.

T-Cell Differentiation Assay

Lin cells (10⁵ cells/well) were cultured on monolayers of irradiated (2-3 Gy) ET cortical thymic epithelial cells on six well Costar plates in the presence of IL7 (500 units/ml) in a final volume of 2 ml of culture medium per well at 37°C for 8-10 days. Cultures were performed in the presence or absence of purified (from 1 to 20 μg/ml) Joro 1 77 or Joro 75 mAbs. The cells were harvested, washed, and used for FACS analysis (Palacious and Imhof, 1 993; Palacious and Samaridis, 1 993) .

8. B-Cell Differentiation Assay

Lin cells ( 10⁵ cells/ml) were cultured on monolayers of irradiated (2-3 Gy) FLS4.1 stromal cells on six well Costar plates in the presence of IL7 (500 units/ml), Steel Factor ( 100 units/ml) , and IL1 1 ( 100 units/ml) in a final volume of 2 ml of culture medium at 37°C for 8-10 days. Cultures were performed in the presence or absence of purified (from 1 to 20 μg/ml) Joro 1 77 or Joro 75 mAbs. The cells were harvested and washed and used for FACS analysis (Palacious and Imhof, 1 993; Palacious and Samaridis, 1 993).

9. Myeloid/Erythroid/Megakaryocyte-Cell Differentiation Assay

Lin cells ( 10⁵ cells/well) were cultured in six-well Costar plates containing GM-CSF (200 units/ml), Steel Factor (100 units/ml), Erythropoietin (2 units/ml), IL3 (2 units/ml), and IL1 1 ( 100 units/ml) in a final volume of 2 ml of culture medium per well at 37 °C for 8-10 days. Cultures were performed in the presence or absence of purified (from 1 to 20 μg/ml) Joro 1 77 or Joro 75 mAbs. The cells were harvested, washed, and used for FACS analysis (Palacious and Imhof, 1 993; Palacious and Samaridis, 1 993) .

10. In Vivo Hematopoietic Reconstruction Assay

For repopulation of sublethally irradiated (300 rads of gamma rays) C3H-Scid-Scid mice, the cell sorter purified Joro 1 77 ⁺ Lin fetal liver mononuclear cells from day 1 2 C57BL/6 embryos (5 x 10³ cells/0.4 ml of PBS) or PBS alone were injected IV into the recipient animal 2-4 hr after irradiation. All mice were housed in sterile isolators with sterile food in a laminar flood hood. Hematopoietic reconstitution in bone marrow and spleen of Scid mice was assessed by single- and two-color FACS analysis 5 to 6 months later. 1 1. Homotypic Cell Aggregation Assay

FTH5 pro-T cells (1 .5-2 x 10⁶) suspended in 100 ul of culture medium supplemented with IL2 (final concentration 10 units/ml) were placed in 96-well flat-bottomed microplates in the presence or absence of Joro 1 77 mAb, PS/2 alpha 4-integrin-specific mAb, or CD44-specific mAb KM703 (final dilution 1 .3 of hybridoma culture supernatants) at 37 °C for 2-3 hr. Cell aggregation was determined by direct visualization of the cultures with an invested microscope. In the studies in which the various drugs or the cell-adhesion- specific mAbs indicated in the results section were tested for their inhibitory effect on cell aggregation, the FTH5 cells were preincubated with the drugs (at 37°C for 1 hr) or with the blocking antibodies (at 4°C for 30 min) before use in the cell aggregation assay.

12. Western Blot Analysis for Detection of Tyrosine Phosphorylated Proteins

FTH5 cells were harvested, and washed twice in PBS, and

5 x 10³ cells were preincubated at 37 °C for 30 min before addition of Joro 1 77 mAb or MK/2 control antibody. At various intervals, the cells were pelleted by centrifugation at 2000 x g for 20 sec. and lysed in 1 ml of lysis buffer ( 1 % NP-40™, 50 mM Tris-HCI pH 7.4, 1 50 mM NaCl, 2 mM PMSF, 1 mM Na₂V0₄, 2 mM NaF and 5 mM EDTA) at 4°C for 60 min. Insoluble material was pelleted by centrifugation at 14000 x g at 4°C for 15 min. The supernatant was transferred to a fresh tube containing 5 μg phosphotyrosine- specific antibody 4G 10 and Protein A-Sepharose and precipitation was allowed to proceed at 4°C overnight. The Sepharose beads were washed three times in lysis buffer, and boiled in Laemmli sample buffer, and proteins were separated under reducing conditions by 7.5% SDS polyacrylamide gel electrophoresis. Following transfer into nitrocellulose filters, the proteins were western blotted with antibody 4G 10 (0.25 μg/ml) . Detection was with horseradish peroxidase-coupled secondary antibodies and the enhanced chemiluminescence system (Amersham).

13. Analysis of DNA Fragmentation

Apoptosis was assessed essentially as described before (Fernandez et a/. , 1 995). Lin cells (0.5-1 x 10⁶ cultured in medium containing GM-CSF, IL 1 1 , Steel Factor) were treated with Joro 1 77 mAb or Joro 75 mAb (final concentration 10 μg/ml) in the presence or absence of F(ab)₂-anti-rat IgG (final concentration 5 μg/ml) at 37°C for 24 hr. The cells were harvested, and washed, and DNA was isolated and fractionated by electrophoresis on 1 .5% agarose gels DNA was visualized by staining with ethidium bromide and photographed under UV light using a transilluminator.

14. cDNA Library Construction and Expression Cloning

Poly(A) RNA isolated from the FTH5 cell line was used to generate a cDNA library constructed in the pCDNAI mammalian expression vector (InVitrogen). To isolate cDNA encoding the protein recognized by Joro 1 77 mAb, the cloning expression system developed by Seed and Aruffo (Seed and Aruffo, 1 987) was used with some modifications. Briefly, Cos-1 cells were transfected with 25 μg plasmid DNA from the FTH5 cDNA library by electroporation (0.25 kV 960 μF, in a 0.4-ml volume). Three days later, Cos-1 cells expressing Joro 1 77 were isolated by FACS. Plasmid DNA was recovered from the selected Cos-1 transfectants by the Hirt procedure, expanded in E. coli. P3, and used for transfection into Cos-1 cells. Following three rounds of transfection and enrichment for cDNAs encoding the Joro 1 77 antigen, individual clones were screened by DEAE-Dextran/chloroquine-mediated transfection into Cos-1 cells and FACS analysis. One clone, called Joro 1 77-87, out of 80 screened conferred expression of the Joro 1 77 antigen in Cos- 1 cells and contained an = 1 .8 kb insert. DNA sequencing of both strands was carried out by primer walking in an ABI automatic DNA sequencer. DNA and protein sequence analysis and comparisons were made using the GCG and MacDNASIS^® 2 software packages. Searches in the GeneBank, SwissproT, and OWL databases were made using the BLAST programs.

15. FACS Analysis

FACS was carried out as described in detail previously

(Palacious, et al. , 1990; Palacious and Samaridis, 1992; Palacious and Imhof, 1993). All stainings were performed with cell samples which were preincubated with heat-inactivated hamster serum (10- 1 5%) and purified rate IgG (250 μg/ml) to prevent nonspecific Fc- receptor binding of labeled antibodies. Single- and two-color FACS analysis were performed using Coulter Profile and ELITE V instruments. Bone marrow (BM), spleen, and thymocytes from normal mice were used as positive controls as required and to set up electronically green and red compensations. Cells exposed to second-step reagents were used as negative controls. Fluorescence emitted by single viable cells was measured with logarithmic amplification. Dead cells were excluded from analysis by forward and side scatter gating. Date collected from 10⁴ cells were analyzed and displayed in the form of fluorescence histograms (single color) or dot plots (two color).

B. RESULTS

The Joro 1 77 mAb is a rat lgG2a. Immunofluorescence staining of cell lines representing different hematopoietic cell lineages and FACS analysis showed that Pro-T cell clones (C4-77, FTH5), the thymic lymphoma BW5147, activated IL2-dependent mature cytolytic CFL1 T- cells, B-cell/myelocytic progenitors (LyD9), the Pro-B cell line CB/Bm7, the Pre-B cell line 18.81 , the Immature B-cell lymphoma WEHI 279, the myeloid progenitor cell line Mye5, and the macrophage cell line P388D 1 were all positive for the Joro 1 77 surface antigen (Palacious, et al. , 1990).

1 . Identification of Joro 177 cells in hematopoietic tissues of adult mice and embryos

The inventors found that in the bone marrow 70-80% of mononuclear cells included in the "lymphoid" gate (defined by forward and side scatters) and 42-45% mononuclear cells included in the "myeloid" gate were Joro 1 77 ⁺ . In the adult thymus, only less than 2% of the small thymocytes (which comprise - 95 % of the thymocytes), but 85-100% of the large thymocytes (which usually represent < 5% of the thymocytes and includes most of the CD4 CDB CD3 thymocyte precursors) bound Joro 1 77. The spleen contained 5-12% Joro 177 ⁺ cells, but following activation with the polyclonal T-cell mitogen Concanavalin A or with the polyclonal B- cell mitogen Lipopolysaccharide (LPS) the percentage of Joro 1 77 ⁺ increased up to 45-73% (Table 5).

TABLE 31

Tissue Distribution of Hematopoietic Cells

Reactive with Joro 177 Antibody

Mononuclear cells isolated from the tissues indicated of C57BL/6 mice were stained with Joro 177 antibody and the percentage of positive cells was determined by FACS analysis. Small/large cells and lymphoid/myeloid gates were defined by forward and side scatters. The numbers represent the range of percentage of positive cells in three separate studies. Two-color FACS analysis using lineage-characteristic surface markers and Joro 1 77 mAb were carried out to further determine the expression of Joro 177 among the different blood cell lineages. About 30-39% of the B-220⁺ B-lymphocyte lineage cells but less than 1 % of the lgM ⁺ mature B-lymphocytes in the bone marrow were Joro 177 ⁺ . Between 19 and 24% of the marrow granulocytes identified with the 8C5 surface marker and 1 5 to 1 7.6% of the myeloid cells identified with the Mac-1 surface marker in the bone marrow were also Joro 177 ⁺ . About 4.6-6.4% of marrow cells co- expressed the erythroid-lineage marker TER1 1 9 and Joro 1 77, and less than 1 % marrow cells stained with Joro 1 77 and either Thy 1 expressed on PHSC and T-lymphocytes or the Joro 75 surface marker for T-cell progenitors (Table 6).

TABLE 32

Joro 177 Positive Cells Co-Expressing

Lineage— Restricted Hematopoietic Cell Surface Markers

FACS analysis of mononuclear cells included in the "lymphoid" or the "myeloid" gates (determined by forward and side scatters) was carried out to determine the percentage of Joro 1 77 ⁺ cells co— expressing the lineage — restricted surface markers indicated. The numbers are the range of double positive cells detected in three separate studies.

Similar FACS analysis showed that 73-89% of hematopoietic cells in the yolk sac at day 8/8.5 of gestation (before blood circulation has started in the embryo) and essentially 100% of mononuclear cells from fetal liver at day 1 2 of gestation were strongly positive for Joro 1 77 (Table 5). Two-color FACS analysis using lineage-characteristic surface markers and Joro 1 77 mAb of mononuclear cells from fetal liver at day 1 3-14 of gestation revealed that 14.4-1 5.7% B-220⁺ B-cell precursors were Joro 1 77 ⁺ ; as expected no detectable mature lgM ⁺ B-lymphocytes were found at this stage of development. Most if not all detectable Thy 1 ⁺ immature cells and Joro 75 ⁺ T-cell progenitors were Joro 1 77 ⁺ . Between 4 and 5% of the mononuclear cells stained with 8C5 granulocytic marker and between 2.8 and 4.1 % of the cells that bound the Mac-1 myeloid marker co-expressed Joro 177, and 1 9.1 to 23.4% of the mononuclear cells bound both the TER1 19 erythroid-lineage marker and Joro 177. Finally, most thymocytes ( > 90%) at day 1 3, 14, and 1 5 of gestation were Joro 1 77 ⁺ (Table 6) .

Most adult thymocytes and splenic mature T-lymphocytes and B-lymphocytes carry very low or not detectable Joro 1 77 antigen on the cell membrane. Interestingly, a significant proportion of the splenic mature T- and B-lymphocytes become Joro 1 77⁺ after their activation in vitro by polyclonal mitogens.

The latter results, together with the finding that most large thymocytes (known to contain actively dividing cells) and most proliferating cell lines including transformed lymphoma cells representing mature B-lymphocytes and T-lymphocytes, brightly stained with the Joro antibody (Palacious, et al., 1990), suggest that proliferating lymphoid cells express higher levels of Joro 1 77 than quiescent lymphocytes. Taken together, these results show that Joro 1 77 is expressed on hematopoietic stem cells, T-and B-cell precursors, and a subset of myeloid and erythroid-lineage cells. Most mature peripheral T- and B-lymphocytes are Joro 1 77 negative but become positive following their activation by mitogens.

2. Joro 177 is expressed on Lin fetal liver PHSCs

To test directly whether Joro 177 was expressed on PHSC, Joro 1 77 ⁺ Seal ⁺ Lin (B-220, F4/80, 8C5, Joro 75, TER1 99 negative) cells were purified from fetal livers of day 1 2 C57BL/6 mouse embryos using FACS. The purified Joro 177⁺ Lin fetal liver mononuclear cells were then tested for their ability to give rise to lymphoid, myeloid, and erγthroid-hematopoietic cell lineages and to reconstitute the hematopoietic system for long time ( > 5 months), the two characteristic properties of PHSC. To this end, purified Joro 1 77 ⁺ Lin fetal liver hematopoietic precursor cells of C57BL/6 origin H-2b) was injected I.V. into sublethally irradiated immunodeficient C3H Scid (H-2k) mice. The progeny of donor origin (C57BL/6, H-2b) were distinguished from cells of host origin (C3H, H-2k) by a H-2b-specific monoclonal antibody known to react with cells expressing class 1 MHC of the H-2b but not with cells bearing class 1 MHC of the H-2k haplotypes (Palacious and Samaridis, 1993; Palacious, et al. , 1 995), along with appropriate hematopoietic lineage-specific surface markers and two-color FACS analysis.

The results are summarized in Table 7. Scid mice that received Joro 1 77 Lin fetal liver cells 5 months before analysis, but not the control mice (which received no cells), contained significant numbers of donor-derived B-lymphocyte precursors (H-2b B-220 ⁺ ), T-lymphocyte precursors (H-2b⁺ Joro 75 ⁺ ), myeloid-lineage cells (H- 2b⁺ Mac-1 ⁺) and erythroid-lineage cells (H-2b + TER1 1 9 + ) in the bone marrow and mature T-lymphocytes (H-2⁺ CD3 ⁺ ) and mature B lymphocytes (H-2b⁺ lgM ⁺ ) in the spleen. These results strongly argue that Joro 1 77 is expressed by fetal liver hematopoietic cells with properties of PHSC.

TABLE 33

Cell Sorter Purified Fetal Liver Joro 177 ⁺ Lin (B220, Mac- 1 , Joro 75^~, TER1 19, 8C5 )

Cells Give Rise to Lymphoid, Myeloid and Erythroid Lineage Cells In Vivo

10

Cell sorter purified Joro 1 77 ⁺ Lin cells obtained from day 1 2 C57BL/6 (H — 2b) fetal livers were

1 5 injected (5 x 10³ cells/mouse) i.v. into sublethally irradiated C3H Scid (H — 2k) mice. Two colour FACS analysis using a H — 2b specific antibody (to identify donor derived cells) was carried out

with bone marrow cells and spleen from C3H Scid mice that received Joro 1 77 ⁺ Lin^" fetal liver cells or no cells twenty weeks before analysis.

3. Joro 177 induces homotypic aggregation of lymphocyte progenitor clones

It was found that Joro 1 77 mAb induced homotypic aggregation of nontransformed, growth-factor-dependent lymphoid progenitor lines such as the Pro-T cell clone FTH5 (Pelkonen, et al. , 1 988). FTH5 cells grow as a single-cell suspension in culture medium supplemented with their required exogenous growth factors IL 2 or IL 3. The addition of Joro 1 77 mAb to these cultures increased the adhesiveness of FTH5 cells so that they aggregated. Aggregation was at least as extensive as that induced by an σ 4 integrin-specific antibody. The latter integrin was previously found to participate in aggregation of peripheral blood mononuclear cells (Bednarczyk and Mclntyre, 1 990). Maximal homotypic aggregation of FTH5 cells stimulated by Joro 1 77 was apparent after 2-4 hr of treatment and persisted for an additional 1 6-20 hr. Several pieces of evidence ruled out the possibility that the clumping of FTH5 cells triggered by the Joro 1 77 mAb was either an Fc-mediated phenomenon or a nonspecific in vitro artifact. Firstly, the isotype- matched CD44-specific antibody KM703 (which is expressed at high levels by FTH5 cells) did not cause homotypic aggregation of FTH5 cells. Secondly, as Table 8 shows homotypic aggregation was not observed. If the treatment was performed at 4°C, and it was abolished by metabolic energy blockers such as sodium azide, by chelation of divalent cations with EDTA, and by disruption of microfilaments with cytochalasin B. Colchicine, which disrupts microtubule assembly, failed to inhibit the Joro 177- induced aggregation of FTH5 cells. TABLE 34

Characteristics of Homotypic Aggregation of

FTH5 Pro— T Cells Induced by Joro 177 Antibody

Agent Homotypic Aggregation Induced by Joro 177

None +

37 °C

4°C cytochalasin B 1 .25 μM colchicine 10 μM + sodium azide 10 mM

EDTA 2.5 mM genistein 1 .25 μg/ml herbimycin A 500 nM

ZnCI₂ 0.5 mM

Na₂ V0₄ 5 mM

NaF 5 mM

H7 1 2.5 μM staurosporine 2 μM +

H8 12.5 μM bepridil 25 μM

The effect of the different temperatures or drugs indicated on the Joro 1 77-induced homotypic aggregation of FTH5 cells was studied by exposing the cell to the agent 60 min before addition of the Joro 1 77 Mab. + = presence of homotypic aggregation; — = absence of homotypic aggregation.

Two unique features to the homotypic aggregation elicited by Joro 177 mAb were observed. Joro 177-induced aggregation of FTH5 cells required IL 2 or IL 3, but IL 4, IL 7, IL 1 1 or Steel Factor were ineffective in this regard (Table 9). The other feature is that unlike previously described homotypic aggregations of hematopoietic cells induced by several means, the aggregation induced by Joro 1 77 mAb required protein synthesis (Table 8) . None of the antibodies tested against CD44 (KM703), alpha 4 integrin (PS/2), alpha 5 integrin (5H 10), alpha 6 integrin (EA-1 ), LFA-1 (H1 55-78), lcam-1 (YN1 /1 .7), and lcam-2 (MIC2) blocked the Joro 1 77 mAb-induced homotypic aggregation of FTH5 cells.

TABLE 35

IL2 or IL3 is Required for Joro 177 — Induced Homotypic Aggregation of FTH5 Cells

Cytokine Induction of Homotypic Aggregation

Joro 1 77 KM703

None — IL2 +

IL3 +

IL4 -

IL7 - IL1 1 Steel Factor -

FTH5 cells were cultured in the presence and the absence of the cytokines indicated and exposed to either Joro 1 77 Mab or KM703 (CD44) control Mab. The presence of homotypic aggregation was assessed by direct visualization of the cultures with an inverted microscope.

+ = presence and — = absence of homotypic aggregation of FTH5 cells.

Subsequent experiments (also compiled in Table 8) showed that inhibitors of protein tyrosine kinases (genistein, herbimycin B) or of protein tyrosine phosphatases (ZnCI₂, sodium o-vanadate) inhibited Joro 177 mAb-induced homotypic aggregation, suggesting the involvement of these enzymes in this process. In contrast, staurosporine, a specific inhibitor of PKC, did not affect homotypic aggregation. An inhibitor of PKC, PKG, and PKA (H7), a specific inhibitor of PKA (H8), and an inhibitor of Na ⁺ /CA^{2 +} exchange (bepridil) significantly decreased homotypic aggregation of FTH5 cells elicited by Joro 177 mAb.

4. Joro 177 mAb stimulates tyrosine phosphorylation of a

125 kDa protein in FTH5 Pro-T cells

Since protein tyrosine kinase- and phosphatase-specific inhibitors abolished Joro 1 77 mAb-induced homotypic aggregation of FTH5 cells, tyrosine phosphorylation of proteins in these cells was studied after exposure to Joro 177 mAb. These studies were carried out in the absence of cytokines to exclude the possibility that any tyrosine-phosphorylated protein detected could be due to the cytokine rather than to Joro 1 77 mAb stimulation. Western blot analysis of lysates from FTH5 cells treated with Joro 1 77 mAb or a control MK/2 antibody, using a phosphotyrosine-specific antibody, showed increased tyrosine phosphorylation of a 1 24 kDa protein in FTH5 cells exposed to Joro 1 77 mAb but not in lysates of cells treated with the control antibody. Interestingly, the increased phosphorylation was apparent 2-5 min after Joro 1 77 mAb stimulation; phosphorylation levels returned to basal levels 20-60 min later. These results and those demonstrating the indication of homotypic aggregation upon Joro 1 77 mAb binding to FTH5 cells lead to the conclusion that the cell membrane protein recognized by the Joro 1 77 mAb can transduce signals into the cell.

5. Joro 177 mAb inhibits cell survival/growth and differentiation of Lin^' hematopoietic precursors along with the lymphoid, myeloid and erythroid cell pathways

To determine whether the Joro 1 77 molecule participates during development of very early Lin hematopoietic precursors into the lymphoid, myeloid, or erythroid cell lineages, Joro 1 77 mAb or Joro 75 mAb (used as an isotype-matched control antibody) were added to cultures in which purified Lin hematopoietic precursor cells isolated from either day 1 3-14 fetal liver or the bone marrow of young adult mice were induced to differentiate along the T- lymphocyte, the B-lymphocyte, or the myeloid/erythroid cell pathways using appropriate in vitro induction assay systems described in detail before (Palacious and Imhof, 1 993; Palacious and Samaridis, 1 993) . Table 10 illustrates the results obtained in these studies. Joro 1 77 mAb significantly reduced the number of viable cells recovered at the end of all three different types of culture. The absolute number of CD4⁺ TCR precursors and CD4⁺ TCR⁺ T-cells and CD4 TCR⁺ produced in the cultures supporting T-cell differentiation, B-220 ⁺ lgM-B-cell precursors and B-220⁺ lgM ⁺ mature-B-cells generated in the cultures promoting B-lymphocyte differentiation, and Mac-1 \ 8C5 ⁺ myeloid cells and TER1 1 9 ⁺ erythroid cells produced in the assay cultures supporting differentiation along these hematopoietic lineages, was significantly decreased in the cultures exposed to the Joro 1 77 mAb. 10

TABLE 36 (continued)

Absolute Number of Positive Cells ( x 10⁴) Cultures Treated With

Induction Assays None Joro 1 77 (μg/ml) Joro 75 (μg/ml)

10 20 10 20

Myeloid/Erythroid Differentiation

8C5 ⁺ 1 1 2.4

Mac - 1 ⁺ 1 53.2 I

H

TER1 19⁺ 36.2 00

^

5 Lin^" fetal liver cells were induced to differentiate along the distinct lineages indicated in appropriate induction assays (see experimental procedures for details). The antibodies indicated were added at the beginning of the cultures. The number of viable cells at the end of the cultures was determined by trypan blue dye exclusion. Input cells per assay group were 3 x 10⁵ cells. FACS analysis was carried out to determine the presence and to calculate the 10 number of positive cells developed in the cultures. The numbers represent the absolute number ( x 10⁴) of cells positive for the surface markers indicated.

ln contrast, the isotype-matched control antibody Joro 75 did not affect the number of cells recovered at the end of the cultures nor the absolute number of T-lymphoid, B-lymphoid, and myeloid and erythroid lineage cells generated by Lin fetal liver precursor cells (Table 10) . Similar results were obtained using Lin bone marrow precursor cells purified from young adult mice. Also, the same phenomenon was found in similar cultures in which we used long-term cultured multipotent precursor lines instead of freshly isolated Lin fetal liver or bone marrow cells. In some experiments, Joro 177 mAb blocked completely the generation of mature CD4⁺ TCR⁺ T-cells or lgM ⁺ B-cells. In the appropriate induction assay cultures, but in most of the experiments a few mature T- or B-cells were detected. Thus, the results suggest that the Joro 1 77 cell surface molecule may play an important role in cell survival/growth of Lin⁺ hematopoietic precursors induced to differentiate along the lymphoid, myeloid and erythroid-cell lineages.

6. Joro 177 mAb can induced apoptosis

The results summarized above showed that Joro 1 77 mAb affected cell survival/growth of developing hematopoietic precursors and that the molecule recognized by Joro 177 mAb can transduce signals into hematopoietic precursor cells. The results suggested that Joro 1 77 mAb could initiate programmed cell death in hematopoietic precursor cells. Treatment of Lin hematopoietic precursor cells with Joro 1 77 mAb induced DNA fragmentation; this sign of apoptosis (McConkey et a/. , 1994) was more apparent. If cross linking of Joro 177 mAb bound to Lin cells was promoted by F(ab)₂ anti-rat IgG specific antibody. In contrast, Lin cells exposed to control Joro 75 mAb and F(ab)₂ anti-rat IgG antibody showed no signs of DNA fragmentation. Thus, Joro 1 77 mAb can initiate programmed cell death in hematopoietic progenitors.

7. Molecular cloning and expression of cDNA encoding the protein recognized by Joro 177 mAb

To characterize further the cell membrane molecule recognized by Joro 1 77 mAb, cDNA encoding this protein was isolated using a transient expression assay in Cos-1 cells (Seed and Aruffo, 1 987) . Following three consecutive rounds of selection of transfectant Cos- 1 cells, one clone, Joro 1 77- B7, was obtained that, upon transfection into Cos cells, conferred expression of detectable Joro 1 77 antigen on the cell membrane. The Joro 1 77 87 cDNA was ^" 1 .8 kb in length and included a poly (A) ⁺ tract. This clone contained a single open reading frame of 533 codons that extended from a Met initiation codon at nucleotides 1 10 thorough 1 1 2 to a stop codon at 1 706 through 1 708. The Met initiation codon at nucleotides 1 10-1 1 2 was preceded by an inframe UAG stop codon at nucleotides 56-58 and it was embedded in a context favorable for translation (Kozak, 1 981 ). The start of the poly (A) addition signal was 1 7 nucleotides from the poly (A) tall and had the sequence AAUAAA.

The protein deduced from the clone Joro 1 77- 87 nucleotide sequence had a predicted M_r = 5881 7 and a predicted isoelectric point of 5.55. The deduced amino acid sequence of Joro 1 77-87 cDNA contains: seven potential N-glycosylation (Asn-Xaa-Ser/Thr) sites mostly located in the COOH-terminal half of the protein (aa residues at positions 1 72-1 74, 265-267, 269-271 , 307-309, 391 - 393, 405-407, and 51 5-51 7), two potential cAMP/cGMP-kinase phosphorylation (R/K-Xaa-S/T) sits (aa residues at positions 197-200 and 423-426), eight potential protein kinase C phosphorylation (S/T- Xa-R/K) sites (aa residues at positions 1 52-1 55, 1 95-198, 407-409, 471 -473, 474-476, 491 -493, 500-502, and 510-512), and eight potential Casein Kinase II phosphorylation (S/T-Xaa-D/E) sites (aa residues at positions 5-8, 64-67, 145-148, 185-188, 294-297, 367- 370, 445-448, and 504-508). Neither potential tyrosine kinase phosphorylation nor potential tyrosine kinase catalytic motifs could be found in the Joro 177-87 deduced amino acid sequence as determined by the SOPM program (Geourjon and Delcage, 1994). Two cysteine residues, at positions 109 and 331 may serve for covalent heterodimer formation between this and other proteins. Kyte and Doolittle analysis of the deduced amino acid sequence for the presence of membrane-associated segments showed the presence of a typical transmembrane-spanning region between amino acids 76 and 98; as found in other membrane proteins, this region is preceded and followed by basic residues. The other additional hydrophobic regions do not fit the requirements of typical transmembrane segments since they are interrupted by charged residues and lack the basic amino acids in the flanking sequences. The observations that the protein exhibits a strongly hydrophilic NH₂-terminal region and lacks the signal peptide found on most transmembrane protein precursors, together with the presence of potential N-glycosylation sites COOH-terminal to the putative membrane spanning region, indicate that the Joro 177-87 cDNA codes for a type II transmembrane glycoprotein. Searches in the Genbank, Swissprot and OWL databases using the BLASTn, BLASTx, and GAP programs showed that the Joro 1 77-87 cDNA has 77.5% nucleotide sequence identity and 76.4% and 86.4% amino acid sequence identity and similarity, respectively, to the heavy chain of human CD98 (Teixelra et al. , 1987; Lumadue et a/. , 1987; Quackenbush et a/. , 1987) and 100% nucleotide sequence identify to a cDNA coding for the mouse CD98 heaving chain gene which was isolated by screening cDNA libraries using human CD98-specific probes (Parmacek et a/. , 1 989) .

The Joro 1 77 mAb proved to be unsatisfactory for immunoprecipitation of the protein, and could only weakly immunoprecipitate a protein of approximately 1 20- 1 30 kDa from cell lysates of both FTH5 cells and Cos cell transfectants. The Joro r177 mAb apparently recognizes the heavy chain of the mouse CD98 type 1 1 transmembrane glycoprotein.

Molecular cloning and expression revealed that the Joro 1 77 mAb recognizes the heavy chain of the mouse homologue of human CD98 and that this molecule is a type II transmembrane glycoprotein with several potential phosphorylation sites for PKC, PKA and Casein Kinase II, but not tyrosine kinase, and without apparent protein tyrosine kinase or phosphatase catalytic motifs.

The human and mouse CD98 is a - 1 25 kDa heterodimer with a glycosylated heavy chain of 85 kDa covalently associated by a disuifide bridge to a nonglycosylated 40 kDa light chain (Haynes et a/. , 1 981 ; Hemler and Strominger, 1982). While specific mAbs and cDNAs coding for the human (Teixelra et a/. , 1 987; Lumadue er a/. , 1 987; Quackenbush et al. , 1 987) and for the mouse (Parmacek et al. , 1989) heavy chain have been isolated, neither antibodies nor cDNAs coding for the 40 kDa light chain have been reported. The nature of the light-chain component of CD98 remains unknown.

Results indicated that the Joro 177 antigen may be among the first cell surface molecules expressed by the developing blood cells at sites of primitive hematopoiesis (8/8.5 day yolk sac and 1 2 day fetal liver) . Furthermore, studies show that Joro 1 77 mAb is expressed on pluripotent hematopoietic stem cells, T-and B- lymphocyte precursors, a subset of myeloid and erythroid-lineage cells, most day 1 3-14 fetal thymocytes and the subset of large double negative cells in the adult thymus. Joro 177 mAb does not, however, bind to most double-positive (CD4⁺8 ) and single-positive (CD4⁺8 or CD4 8 ^{+ l} thymocytes nor to most mature T-lymphocytes and B-lymphocytes in the spleen of adult mice. Mature lymphocytes and B-lymphocytes in the spleen of adult mice. Mature lymphocytes could be induced to express Joro 1 77 antigen on the cell surface following their activation in vitro; this finding points out that Joro 1 77 may also serve as a marker for activated peripheral lymphocytes. Northern blot studies have shown that CD98 heavy chain RNA transcripts are synthesized in the brain, kidney, lung, and testis (Parmacek et al. , 1989), suggesting that CD98 may also be expressed by some non-hematopoietic cells.

To uncover functions of the molecule recognized by the Joro 1 77 mAb in the hematopoietic system, evidence was obtained which demonstrated that CD98 can transduce signals into the cell which was manifested in both the tyrosine phosphorylation of a - 1 25 kDa protein and the induction of homotypic aggregation in a Pro-T cell progenitor line. As the CD98 heavy chain protein lacks protein tyrosine kinase catalytic sites, it is possible that Joro 1 77 mAb binding to CD98 on FHT5 progenitor cells stimulates a tyrosine kinase that in turn phosphorylates the - 1 25 kDa target protein, the nature of this phosphotyrosine protein remains to be elucidated. The tyrosine phosphorylation levels of the - 1 25 kDa protein returned to basal levels 20-60 min. after Joro 1 77 mAb stimulation, implying that this target protein might be subject to regulation by a tyrosine phosphatase.

The homotypic aggregation triggered by Joro 1 77 mAb binding to CD98 required divalent cations, protein synthesis, and microfilament assembly and was dependent on metabolic energy and temperature. Results using available blocking antibodies against mouse adhesion molecules suggest that alpha 4B1 , alpha 5B1 , alpha 6B1 , LFA-1 /lcam -1 /lcam-2 integrins indicate that either these molecules do not participate in the homotypic aggregation elicited by the Joro 1 77 mAb or that the antibodies used do not interfere with epitopes critical for homotypic aggregation in this system.

Other cell adhesion molecules (e.g. , cadherins) could be involved in this process. It seems likely that the stimulation of tyrosine phosphorylation of the 1 25 kDa protein and the induction of homotypic aggregation (which was abolished by both tyrosine kinase and tyrosine phosphatase-specific inhibitors) following Joro 1 77 mAb binding to CD98 on FTH5 Pro-T cells are related.

Very interestingly, it was also found that Joro 177 mAb specifically inhibited growth/cell survival of Lin early hematopoietic precursor cells induced to develop along the lymphocyte, myeloid, and erythrocyte pathways. Joro 177 mAb induced apoptosis of the developing hematopoietic cells in the cultures. Several potential mechanisms could account for these results.

One possibility is that CD98 controls the uptake or exchange of essential nutrients (amino acids, divalent cations) necessary for cell survival/growth of cells. Joro 1 77 mAb would block this function by binding to CD98. Support for this mechanism comes from previous studies reporting that the human CD98 heavy chain stimulated the uptake of neutral and dibasic amino acids in Xenopus oocytes (Wells et al. , 1 992; Bertran et al. , 1992), and that human CD98-specific antibodies inhibited sodium-dependent calcium exchange in sarcolemmal vesicles (Michalak et al. , 1 985) . However, two findings argue strongly against this possibility. mRNA for the CD98 heavy chain gene is expressed at very low levels in heart and skeletal muscle (Parmacek et al. , 1 989), two tissues very active in sodium-calcium exchange, and, in contrast to other previously described ion channels that contain multiple membrane-spanning domains (Kopito and Lodish, 1985), the CD98 heavy chain specific protein contains a single transmembrane region. Also, the putative role of CD98 as controller of ion channels or as an actual ion channel exchanger cannot satisfactorily explain the finding that the human CD98-heavy chain-specific antibody 4F2 inhibits lectin-induced proliferation of peripheral blood lymphocytes but has no effect on other ion-dependent cell responses such as antibody-dependent cellular cytotoxicity or killing by alloantigen- specific cytotoxic T-cells (Haynes et a/. , 1 981 ) . Furthermore, while this mechanism could explain the blockage of hematopoietic growth/differentiation of Lin^' precursor cells caused by Joro 1 77 mAb shown here. It does not readily explain the induction of both tyrosine phosphorylation and homotypic aggregation elicited by binding of Joro 1 77 mAb to CD98 in hematopoietic progenitor cells.

A more likely mechanism postulates that CD98 and its putative ligand constitute a receptor/ligand pair normally involved in controlling cell survival/apoptosis. Thus, the CD98 receptor/ligand pair would control cell numbers and/or the extent of cell expansion of cells that are:

a) resting CD98 which become CD98⁺ following activation by antigens, cytokines (e.g. , resting T-lymphocytes become highly CD98⁺ after their activation by mitogens or antigens); and

b) already CD98⁺ cells present in different tissues in the developing embryo and tissues with continued cell renewal in the adult (e.g. , developing hematopoietic progenitors).

The postulated role of CD98 in controlling cell survival/death is directly supported by the results showing that Joro 177 mAb can induce programmed cell death in hematopoietic progenitors and by the data showing that this antibody significantly decreased the number of lymphoid, myeloid, and erythroid cells generated in culture by developing Lin hematopoietic precursor cells. Moreover, also consistent with this view is the finding that ligation of CD98 on the cell membrane by Joro 1 77 mAb elicited signal transduction events manifested by tyrosine phosphorylation of proteins and increased adhesiveness, resulting in homotypic aggregation of cells. This suggested function for CD98 also readily explains results showing that CD98-specific antibodies inhibited the growth of the human tumor cell lines (Yagita et a/. , 1 986), the proliferation of normal human lymphocytes following their activation by mitogens (Haynes er a/. , 1981 ) and the reported existence of a subset of human natural killer cells that specifically recognizes and kills target cells bearing CD98 (Moingeon et al. 1 985).

* * * * * * * *

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. REFERENCES

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SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: BOARD OF REGENTS, THE UNIVERSITY OF

TEXAS

(B) STREET: 201 West 7th Street

(C) CITY: Austin

(D) STATE: Texas (E) COUNTRY: United States of America

(F) POSTAL CODE (ZIP) : 78701

(G) TELEPHONE: (512) 499-4462 (H) TELEFAX: (512) 499-4523

(ii) TITLE OF INVENTION: SELF-RENEWING PLURIPOTENT

HEMATOPOIETIC STEM CELL COMPOSITIONS, METHODS OF USE, AND CULTURE SYSTEMS THEREFOR

;iii) NUMBER OF SEQUENCES: 4

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version

#1.30 (EPO)

(v) CURRENT APPLICATION DATA:

APPLICATION NUMBER: US UNKNOWN

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US SN 08/462,108 (B) FILING DATE: 05-JUN-1995 (vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US SN 08/378,144

(B) FILING DATE: 24-JAN-1995

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TTCGGCTATG ACTGGGCACA AC 22

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TCAGTGACAA CGTCGAGCAC AG 22

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1721 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION:63..1436 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CATGAAGCCA GAGATGTGGA AGATGTGTCT AGACTGCATC AATGAACTGA TGGATACGTT 60

5 GG ATG CAC ATT CCA ACA TCT CTG TCG GAG AGA ACA TTT TGG CAG AGA 107 Met His lie Pro Thr Ser Leu Ser Glu Arg Thr Phe Trp Gin Arg 1 5 10 15

GTG AGA ACT TAC ACA ACT TTG ATC AGT TCA CTC CGT GTA CGA CGC TGC 155 10 Val Arg Thr Tyr Thr Thr Leu lie Ser Ser Leu Arg Val Arg Arg Cys

20 25 30 _t_

M .1-

ATC CTA ACT TTG GTG GAG CGA ATG GAT GAA GAA TTT ACC AAA ATA ATG 203 lie Leu Thr Leu Val Glu Arg Met Asp Glu Glu Phe Thr Lys lie Met 15 35 40 45

CAA AAT ACT GAT CCT CAC TCC CAA GAG TAT GTG GAG CAC CTG AAG GAT 251 Gin Asn Thr Asp Pro His Ser Gin Glu Tyr Val Glu His Leu Lys Asp

50 55 60

20

GAG GCA CAA GTG TGT GCC ATC ATT GAG CGA GTG CAG CGC TAC CTG GAG 299

Glu Ala Gin Val Cys Ala lie He Glu Arg Val Gin Arg Tyr Leu Glu

65 70 75

GAG AAA GGT ACC ACT GAG GAG ATC TGC CAG ATC TAC TTA AGG CGC ATC 347

Glu Lys Gly Thr Thr Glu Glu He Cys Gin He Tyr Leu Arg Arg He

80 85 90 95

5 CTG CAC ACG TAC TAC AAG TTT GAC TAC AAG GCC CAT CAG CGG GAG CTT 395 Leu His Thr Tyr Tyr Lys Phe Asp Tyr Lys Ala His Gin Arg Glu Leu

100 105 110

ACT CCT CCT GAA GGA TCC TCA AAG TCT GAG CAA GAC CAG GCA GAA AAT 443 10 Thr Pro Pro Glu Gly Ser Ser Lys Ser Glu Gin Asp Gin Ala Glu Asn

115 120 125 ,_ r-> U.

I

GAG GGT GAG GAC TCA GCT GTG CTA ATG GAA AGA CTG TGC AAG TAC ATC 491 Glu Gly Glu Asp Ser Ala Val Leu Met Glu Arg Leu Cys Lys Tyr He 15 130 135 140

TAT GCC AAG GAC CGT ACA GAC CGG ATC CGT ACC TGT GCC ATC CTC TGC 539 Tyr Ala Lys Asp Arg Thr Asp Arg He Arg Thr Cys Ala He Leu Cys

145 150 155

20

CAT ATC TAC CAT CAT GCG CTC CAC TCC CGC TGG TAT CAG GCC CGT GAC 587

His He Tyr His His Ala Leu His Ser Arg Trp Tyr Gin Ala Arg Asp

160 165 170 175

CTC ATG CTC ATG AGC CAC CTA CAG GAC AAC ATT CAG CAC GCA GAC CCG 635

Leu Met Leu Met Ser His Leu Gin Asp Asn He Gin His Ala Asp Pro

180 185 190

5 CCG GTG CAG ATC CTG TAT AAC CGT ACT ATG GTG CAA CTG GGC ATC TGT 683 Pro Val Gin He Leu Tyr Asn Arg Thr Met Val Gin Leu Gly He Cys 195 200 205

GCT TTC CGC CAA GGC CTG ACA AAG GAT GCA CAC AAT GGC ACT TCT GGA 731 10 Ala Phe Arg Gin Gly Leu Thr Lys Asp Ala His Asn Gly Thr Ser Gly 210 215 220 NJ

TAT TCA GTC AAG TGG TCG AGC CAA GGA GCT TCT AGG TCA GGG TCT GCT 779 Tyr Ser Val Lys Trp Ser Ser Gin Gly Ala Ser Arg Ser Gly Ser Ala 15 225 230 235

GCT GCG CGC TTG CAG GAG CGA AAT CAG GAA CAG GAA AAG GTA GAG CGA 827 Ala Ala Arg Leu Gin Glu Arg Asn Gin Glu Gin Glu Lys Val Glu Arg

240 245 250 255

20

CGC CGG CAG GTG CCC TTT CAC CTG CAC ATC AAC CTG GAG CTG CTG GAG 875 Arg Arg Gin Val Pro Phe His Leu His He Asn Leu Glu Leu Leu Glu

260 265 270

TGT GTC TAT CTG GTG TCA GCT ATG CTC CTG GAG ATC CCC TAC ATG GCT 923

Cys Val Tyr Leu Val Ser Ala Met Leu Leu Glu He Pro Tyr Met Ala 275 280 285

5 GCC CAT GAG AGC GAT GCC CGC CGA CGC ATC ATC AGC AAG CAG TTC CAC 971 Ala His Glu Ser Asp Ala Arg Arg Arg He He Ser Lys Gin Phe His 290 295 300

CAC CAA CTG CGG GTG GGC GAG CGG CAC GCC CTG CTA GGT CCT CCC GAG 1019 10 His Gin Leu Arg Val Gly Glu Arg His Ala Leu Leu Gly Pro Pro Glu 305 310 315 I

M M

I

TCA ATG AGG GAG CAT GTG GTC GCT GCC TCC AAG GCC ATG AAG ATG GGC 1067 Ser Met Arg Glu His Val Val Ala Ala Ser Lys Ala Met Lys Met Gly 15 320 325 330 335

GAC TGG AAG ACC TGC CAC AGT TTC ATC ATT AAT GAA AAG ATG AAT GGG 1115 Asp Trp Lys Thr Cys His Ser Phe He He Asn Glu Lys Met Asn Gly

340 345 350

20

AAA GTG TGG GAC CTT TTC CCT GAG GCT GAC AAA GTT CGC ACC ATG CTA 1163

Lys Val Trp Asp Leu Phe Pro Glu Ala Asp Lys Val Arg Thr Met Leu

355 360 365

GTT CGG AAG ATC CAG GAA GAG TCT CTG AGG ACC TAC CTT TTT ACC TAC 1211

Val Arg Lys He Gin Glu Glu Ser Leu Arg Thr Tyr Leu Phe Thr Tyr

370 375 380

5 AGC AGT GTC TAT GAC TCA ATC AGT ATG GAG ACA CTA TCA GAT ATG TTT 1259 Ser Ser Val Tyr Asp Ser He Ser Met Glu Thr Leu Ser Asp Met Phe 385 390 395

GAG CTG GAT CTA CCC ACT GTT CAC TCC ATC ATC AGC AAG ATG ATC ATT 1307 10 Glu Leu Asp Leu Pro Thr Val His Ser He He Ser Lys Met He He 400 405 410 415 I

CD

I

AAC GAA GAA TTG ATG GCT TCC CTG GAC CAG CCG ACA CAG ACT GTG GTG 1355 Asn Glu Glu Leu Met Ala Ser Leu Asp Gin Pro Thr Gin Thr Val Val 15 420 425 430

ATG CAC CGT ACT GAG CCC TCT GCC CAG CAA GAA ACT TGG CTC TGC AAG 1403 Met His Arg Thr Glu Pro Ser Ala Gin Gin Glu Thr Trp Leu Cys Lys

435 440 445

20

CTG GCT GAG AAA ACT TGG CAC CCT AGT GGA GAA TAATGGACGG GTGTTTGACC 1456

Leu Ala Glu Lys Thr Trp His Pro Ser Gly Glu

450 455

AAAAACAGGG AACCTATGGT GGCTATTTCC GAGACCCCAA GGGTGGCTAC CGGAAAAATG 1516

GAGGCTAAAT GCCCCGTGGT GGCTACCCCC AGCAACAGTC TCAGACAACC TCCTGAGTTC 1576

5 CCCACTTCAG TCACCCTGTG GACAGACCAT CTAACCTTTT TCTCCTAACT CACCCCAATC 1636

ATTAAAGATC TTTTGAGGAA TTAAAAAAAA AGAAAGAAAA AAAAAAAAAA AAAAAAAAAA 1696

AAAAAAAAAA AAAAAAAAAA AAAAA 1721

10

I

( 2 ) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS: 15 (A) LENGTH: 458 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met His He Pro Thr Ser Leu Ser Glu Arg Thr Phe Trp Gin Arg Val

10 15

Arg Thr Tyr Thr Thr Leu He Ser Ser Leu Arg Val Arg Arg Cys He 20 25 30

Leu Thr Leu Val Glu Arg Met Asp Glu Glu Phe Thr Lys He Met Gin 5 35 40 45

Asn Thr Asp Pro His Ser Gin Glu Tyr Val Glu His Leu Lys Asp Glu 50 55 60

10 Ala Gin Val Cys Ala He He Glu Arg Val Gin Arg Tyr Leu Glu Glu 65 70 75 80 to

NJ O

I

Lys Gly Thr Thr Glu Glu He Cys Gin He Tyr Leu Arg Arg He Leu

85 90 95

15

His Thr Tyr Tyr Lys Phe Asp Tyr Lys Ala His Gin Arg Glu Leu Thr 100 105 110

Pro Pro Glu Gly Ser Ser Lys Ser Glu Gin Asp Gin Ala Glu Asn Glu

20 115 120 125

Gly Glu Asp Ser Ala Val Leu Met Glu Arg Leu Cys Lys Tyr He Tyr 130 135 140

Ala Lys Asp Arg Thr Asp Arg He Arg Thr Cys Ala He Leu Cys His

145 150 155 160

He Tyr His His Ala Leu His Ser Arg Trp Tyr Gin Ala Arg Asp Leu

165 170 175

Met Leu Met Ser His Leu Gin Asp Asn He Gin His Ala Asp Pro Pro 180 185 190

10 Val Gin He Leu Tyr Asn Arg Thr Met Val Gin Leu Gly He Cys Ala 195 200 205 I

N) NJ

Phe Arg Gin Gly Leu Thr Lys Asp Ala His Asn Gly Thr Ser Gly Tyr 210 215 220

15

Ser Val Lys Trp Ser Ser Gin Gly Ala Ser Arg Ser Gly Ser Ala Ala 225 230 235 240

Ala Arg Leu Gin Glu Arg Asn Gin Glu Gin Glu Lys Val Glu Arg Arg

Arg Gin Val Pro Phe His Leu His He Asn Leu Glu Leu Leu Glu Cys 260 265 270

Val Tyr Leu Val Ser Ala Met Leu Leu Glu He Pro Tyr Met Ala Ala 275 280 285

His Glu Ser Asp Ala Arg Arg Arg He He Ser Lys Gin Phe His His 5 290 295 300

Gin Leu Arg Val Gly Glu Arg His Ala Leu Leu Gly Pro Pro Glu Ser 305 310 315 320

10 Met Arg Glu His Val Val Ala Ala Ser Lys Ala Met Lys Met Gly Asp

325 330 335 NJ NJ N)

Trp Lys Thr Cys His Ser Phe He He Asn Glu Lys Met Asn Gly Lys 340 345 350

15

Val Trp Asp Leu Phe Pro Glu Ala Asp Lys Val Arg Thr Met Leu Val 355 360 365

Arg Lys He Gin Glu Glu Ser Leu Arg Thr Tyr Leu Phe Thr Tyr Ser

Ser Val Tyr Asp Ser He Ser Met Glu Thr Leu Ser Asp Met Phe Glu 385 390 395 400

Leu Asp Leu Pro Thr Val His Ser He He Ser Lys Met He He Asn

405 410 415

Glu Glu Leu Met Ala Ser Leu Asp Gin Pro Thr Gin Thr Val Val Met 420 425 430

His Arg Thr Glu Pro Ser Ala Gin Gin Glu Thr Trp Leu Cys Lys Leu 435 440 445

10 Ala Glu Lys Thr Trp His Pro Ser Gly Glu 450 455 NJ NJ >

Claims

CLAIMS:

1 . A composition comprising a factor, purified relative to its naturally-occurring state, said factor having a molecular weight of about 1 5- to about 45-kDa as determined by gel filtration chromatography, wherein said factor inhibits differentiation of pluripotent hematopoietic stem cells.

2. The composition of claim 1 , wherein said factor is obtainable from a fetal stromal cell line such as FLS4.1 .

3. A method of preparing a pluripotent hematopoietic stem cell differentiation inhibiting factor, said method comprising the steps of:

a) culturing a fetal stromal cell line such as FLS4.1 ; and

b) collecting the culture supernatant from said cell line.

The method of claim 3, wherein said culture supernatant is fractionated on the basis of size to obtain an active fraction.

The method of claim 4, wherein said active fraction is F factor.

6. The method of claim 3, wherein said factor inhibits differentiation of an erythroid, myeloid, or lymphoid cell.

A pluripotent hematopoietic stem cell differentiation inhibitory factor prepared by culturing a fetal stromal cell line such as FLS4.1 and collecting the stem cell differentiation inhibitory factor from the cell culture supernatant.

8. A method of inhibiting the differentiation of a pluripotent hematopoietic stem cell comprising contacting said cell with the pluripotent hematopoietic stem cell differentiation inhibitory factor of claim 7.

9. The method of claim 8, wherein said stem cell is located within an animal.

10. The method of claim 8, wherein said factor further comprises Steel factor, LIF, and IL-3 or FLT3-ligand.

1 1 . A method for preparing an undifferentiated pluripotent hematopoietic stem cell comprising admixing a pluripotent hematopoietic stem cell with the pluripotent hematopoietic stem cell differentiation inhibitory factor of claim 7.

1 2. The method of claim 1 1 , wherein said pluripotent hematopoietic stem cell differentiation inhibitory factor has a molecular weight of about 1 5- to about 45-kDa as determined by gel filtration chromatography.

13. The method of claim 12, wherein said factor is obtainable from a fetal stromal cell line such as FLS4.1 .

14. The method of claim 1 2, wherein said composition further comprises Steel factor, LIF, and IL-3 or FLT3-ligand.

1 5. A DNA segment comprising an isolated coding region that encodes a pluripotent hematopoietic stem cell differentiation- inhibiting factor having a molecular weight of about 1 5- to about 45-kDa as determined by gel filtration chromatography.

16. An antibody that specifically binds to the pluripotent hematopoietic stem cell marker Joro 1 77, Joro 1 84, or Joro 3.

17. The antibody of claim 1 6 that specifically binds to the pluripotent hematopoietic stem cell marker Joro 1 77.

1 8. The antibody of claim 1 6, wherein said antibody induces apoptosis of hematopoietic progenitor cells.

1 9. The antibody of claim 16, wherein said antibody binds to a type II transmembrane protein.

20. The antibody of claim 19, wherein said type II transmembrane protein is CD98.

21 . The antibody of claim 1 6, wherein said antibody binds to a type II transmembrane protein, and induces apoptosis of hematopoietic progenitor cells.

22. The antibody of claim 1 6, wherein said antibody is linked to a detectable label.

23. The antibody of claim 1 6, wherein the antibody is a monoclonal antibody.

24. A method for detecting an undifferentiated pluripotent hematopoietic stem cell comprising: a) contacting a suspected undifferentiated pluripotent hematopoietic stem cell, such as the lymphohematopoietic progenitor cell PR-23, with a composition according to claim 7; and

b) identifying the presence of an A3 gene in the nucleic acids of said cell line, wherein an undifferentiated pluripotent hematopoietic stem cell is indicated by the presence of said gene.

25. A method for detecting a pluripotent hematopoietic stem cell in a biological sample, comprising the steps of:

a) obtaining a biological sample suspected of containing a pluripotent hematopoietic stem cell;

b) contacting said sample with a first antibody that binds to a pluripotent hematopoietic stem cell, under conditions effective to allow the formation of complexes; and

c) detecting the complexes so formed.

26. The method of claim 25, further comprising identifying cell surface markers that bind said antibody.

27. A method for providing an undifferentiated pluripotent hematopoietic stem cell to an animal, comprising preparing an undifferentiated pluripotent hematopoietic stem cell according to claim 1 1 , and administering said cell to said animal.

28. The method of claim 27, wherein said cell is obtainable from a bone marrow sample.