CA2107463C - Totipotent hematopoietic stem cell receptors and their ligands - Google Patents

Abstract

Isolated mammalian nucleic acid molecules encoding receptor protein tyrosine kinases expressed in primitive hematopoie-tic cells and not expressed in mature hematopoietic cells are provided. Also included are the receptors encoded by such nucleic acid molecules; the nucleic acid molecules encoding receptor protein tyrosine kinases having the sequences shown in Figure 1 (flk-2) and Figure 2(flk-1); the receptor protein tyrosine kinases having the amino acid sequences shown in Figure 1 (flk-2) and Figure 2 (flk-1); ligands for the receptors; nucleic acid sequences that encode the ligands; and methods of stimulating the prolif-eration and/or differentiation of primitive mammalian hematopoietic stem cells comprising contacting the stem cells with a li-gand that binds to a receptor protein tyrosine kinase expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells.

Description

TOTIPOTENT HEMATOPOIETIC STEM CELL
RECEPTORS AND THEIR LIOANDS

The invention described in this application was made with U.S. government support from Grant Numbers R01-CA45339 and R01-DK42989 awarded by the National Institutes of Health.
The government has certain rights in this invention.

FIELD OF THE INVENI'ION
The present invention relates to hematopoietic stem cell receptors, ligands for such receptors, and nucleic acid molecules encoding such receptors and ligands.

BACKGROUND OF THE INVENTION

The mammalian hematopoietic system comprises red and white blood cells. These cells are the mature cells that result from more primitive lineage-restricted cells. The cells of the hematopoietic system have been reviewed by Dexter and Spooncer in the Annual Review of Cell Biology 3, 423-441 (1987).

The red blood cells, or erythrocytes, result from primitive cells referred to by Dexter and Spooncer as erythroid burst-forming units (BFU-E). The immediate progeny of the erythroid burst-forming units are called erythroid colony-forming units (CFU-E).

The white blood cells contain the mature cells of the lymphoid and myeloid systems. The lymphoid cells include B
lymphocytes and T lymphocytes. The B and T lymphocytes result from earlier progenitor cells referred to by Dexter and Spooncer as preT and preB cells.
The myeloid system comprises a number of cells including granulocytes, platelets, monocytes, macrophages, and SUBS"Ti"T'UTE SHEET

7 4. 63) CA 02107463 1995-08-17 WO 92/17486 PC1'/US92/02750 megakaryocytes. The granulocytes are further divided into neutrophils, eosinophils, basophils and mast cells.

Each of the mature hematopoietic cells are specialized 5 for specific functions. For example, erythrocytes are responsible for oxygen and carbon dioxide transport. T and B
lymphocytes are responsible for cell-and antibody-mediated immune responses, respectively. Platelets are involved in blood clotting. Granulocytes and macrophages act generally 10 as scavengers and accessory cells in the immune response against invading organisms and their by-products.

At the center of the hematopoietic system lie one or more totipotent hematopoietic stem cells, which undergo a series of differentiation steps leading to increasingly lineage-restricted progenitor cells. The more mature progenitor cells are restricted to producing one or two lineages. Some examples of lineage-restricted progenitor cells mentioned by Dexter and Spooncer include granulocyte/macrophage colony-forming cells (GM-CFC), megakaryocyte colony-forming cells (Meg-CFC), eosinophil colony-forming cells (Eos-CFC), and basophil colony-forming cells (Bas-CFC). Other examples of progenitor cells are discussed above.
The hematopoietic system functions by means of a precisely controlled production of the various mature lineages. The totipotent stem cell possesses the ability both to self renew and to differentiate into committed progenitors for all hematopoietic lineages. These most primitive of hematopoietic cells are both necessary and sufficient for the complete and permanent hematopoietic reconstitution of a radiation-ablated hematopoietic system in mammals. The ability of stem cells to reconstitute the entire hematopoietic system is the basis of bone marrow transplant therapy.

It is known that growth factors play an important role in the development and operation of the mammalian Sld BST{'T'!9"T~ SHEET

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hematopoietic system. The role of growth factors is complex, however, and not well understood at the present time. One reason for the uncertainty is that much of what is known about hematopoietic growth factors results from in vitro experiments. Such experiments do not necessarily reflect in vivo realities.

In addition, in vitro hematopoiesis can be established in the absence of added growth factors, provided that marrow stromal cells are added to the medium. The relationship between stromal cells and hematopoietic growth factors in vivo is not understood. Nevertheless, hematopoietic growth factors have been shown to be highly active in vivo.

From what is known about them, hematopoietic growth factors appear to exhibit a spectrum of activities. At one end of the spectrum are growth factors such as erythropoietin, which is believed to promote proliferation, only of mature erythroid progenitor cells. In the middle of the spectrum are growth factors such as IL-3, which is believed to facilitate the growth and development of early stem cells as well as of numerous progenitor cells. Some examples of progenitor cells induced by IL-3 include those restricted to the granulocyte/macrophage, eosinophil, megakaryocyte, erythroid and mast cell lineages.

At the other end of the spectrum is the hematopoietic growth factor that, along with the corresponding receptor, was discussed in a series of articles in the October 5, 1990 edition of Cell. The receptor is the product of the W locus, c-kit, which is a member of'the class of receptor protein tyrosine kinases. The ligand for c-kit, which is referred to by various names such as stem cell factor (SCF) and mast cell growth factor (MGF), is believed to be essential for the development of early hematopoietic stem cells and cells restricted to the erythroid and mast cell lineages in mice;
see, for example, Copeland et al., Cell 63, 175-183 (1990).

it appears, therefore, that there are growth factors Sl.&BSTITU T'E SHEET

+) CA 02107463 1995-08-17 17 /~_ t tl( WO 92C17486 PC'I /L7S92/02750 that exclusively affect mature cells. There also appear to be.growth factors that affect both mature cells and stem cells. The growth factors that affect both types of cells may affect a small number or a large number of mature cells.
There further appears to be an inverse relationship between the ability of a growth factor to affect mature cells and the ability of the growth factor to affect stem cells.
For example, the c-kit ligand, which stixnulates a small number of mature cells, is believed to be more important in the renewal and development of stem cells then is IL-3, which is reported to stimulate proliferation of many mature cells (see above).

Prior to the present specification, there have been no reports of growth factors that exclusively stimulate stem cells in the absence of an effect on mature cells. The discovery of such growth factors would be of particular significance.
As mentioned above, c-kit is a protein tyrosine kinase (pTK). It is becoming increasingly apparent that the protein tyrosine kinases play an important role as cellular receptors for hematopoietic growth factors. Other receptor pTKs include the receptors of colony stimulating factor 1 (CSF-1) and PDGF.

The pTK family can be recognized by the presence of several conserved amino acid regions in the catalytic domain.
These conserved regions are summarized by Hanks et al. in Science 241, 42-52 (1988), see Figure 1 starting on page 46 and by Wilks in Proc. Natl. Acad. Sci. USA 86, 1603-1607 (1989), see Figure 2 on page 1605.

Additional protein tyrosine kinases that represent hematopoietic growth factor receptors are needed in order more effectively to stimulate the self-renewal of the totipotent hematopoietic stem cell and to stimulate the development of all cells of the hematopoietic system both in SUBSTITUTE SHEET

~7 16 3 vitro and in vivo. Novel hematopoietic growth factor receptors that are present only on primitive stem cells, but are not present on mature progenitor cells, are particularly desired. Ligands for the novel receptors are also desirable 5 to act as hematopoietic growth factors. Nucleic acid sequences encoding the receptors and ligands are needed to produce recombinant receptors and ligands.

SUMAiARY OF THE INVENTION
These and other objectives as will be apparent to those with ordinary skill in the art have been met by providing isolated mammalian nucleic acid molecules encoding receptor protein tyrosine kinases expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells. Also included are the receptors encoded by such nucleic acid molecules; the nucleic acid molecules encoding receptor protein tyrosine kinases having the sequences shown in Figure 1(flk-2) and Figure 2(flk-i); the receptor protein tyrosine kinases having the amino acid sequences shown in Figure 1 (flk-2) and Figure 2(flk-1); ligands for the receptors;
nucleic acid sequences that encode the ligands; and methods of stimulating the proliferation of primitive mammalian hematopoietic stem cells comprising contacting the stem cells with a ligand that binds to a receptor protein tyrosine kinase expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells.

DESCRIPTION OF THE FIGURES
Figure 1a.1-1a.3 shows the cDNA and amino acid sequences of murine flk-2. The amino acid residues occur directly below the nucleotides in the open readir~g frame. Amino acids 1-27 constitute the hydrophobic leader sequence. Amino acids 28-544 constitute the extracellular receptor domain. Amino acids 545-564 constitute the transmembrane region. The remainder of the amino acids constitute the intracellular .catalytic domain. The following amino acid residues in the intracellular domain are catalytic sub-domains identified by SUBSTITUTE SHEET

dVO 92/17486 CA 02107463 1995-08-17 PCr/U.7"92/027Jo Hanks (see above): 545-564, 618-623, 811-819, 832-834, 857-862, 872-878. The sequence at residues 709-785 is a signature sequence characteristic of flk-2. The protein tyrosine kinases generally have a signature sequence in this region.

Figure lb shows the cDNA and amino acid sequences of a portion of human flk-2 from the extracellular domain. Amino acids 1-110 of the human flk-2 correspond to amino acids 43-152 of murine flk-2.

Figure lc shows the cDNA and amino acid sequences of a portion of human flk-2 from the intracellular (kinase) domain. Amino acids 1-94 of the human flk-2 correspond to amino acids 751-849 of murine flk -2.

Figure 2-2.3 shows the cDNA and amino acid sequences of flk-1. Amino acid residue 763-784 constitute the transmembrane region of flk-i.
Figure 3 shows the time response of binding between a murine stromal cell line (2018) and APtag-flk-2 as well as APtag-flk-1. APtag without receptor (SEAP) is used as a control. See Example 8.
Figure 4 shows the dose response of binding between stromal cells (2018) and APtag-flk-2 as well as APtag-flk-1.
APtag without receptor (SEAP) is used as a control. See Example 8.
DETAILED DESCRIPTION OF THE INVENTION
Receptors In one embodiment, the invention relates to an isolated mammalian nucleic acid molecule encoding a receptor protein tyrosine kinase expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells.

SUBSTITUTE SHEET

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The nucleic acid molecule may be a DNA, cDNA, or RNA
molecule. The mammal in which the nucleic acid molecule exists may be any mammal, such as a mouse, rat, rabbit, or human.
The nucleic acid molecule encodes a protein tyrosine kinase (pTK). Members of the pTK family can be recognized by the conserved amino acid regions in the catalytic domains.
Examples of pTK consensus sequences have been provided by Hanks et al. in Science 241, 42-52 (1988); see especially Figure 1 starting on page 46 and by Wilks in Proc. Natl.
Acad. Sci. USA 86, 1603-1607 (1989); see especially Figure 2 on page 1605. A methionine residue at position 205 in the conserved sequence WMAPES is characteristic of pTK's that are receptors.

The Hanks et al article identifies eleven catalytic sub-domains containing pTK consensus residues and sequences. The pTKs of the present invention will have most or all of these consensus residues and sequences.

Some particularly strongly conserved residues and sequences are shown in Table 1.

Conserved Residues and Sequences in pTKs' Residue or Catalytic Posiic-nZ Seguence Domain 1. See Hanks et al., Science 241, 42-52 (1988) 2. Adjusted in accordance with Hanks et al., Id.

SUBSTITUTE SHEET

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A pTK of the invention may contain all thirteen of these highly conserved residues and sequences. As a result of natural or synthetic mutations, the pTKs of the invention may contain fewer than all thirteen strongly conserved residues and sequences, such as 11, 9, or 7 such sequences.

The receptors of the invention generally belong to the same class of pTK sequences.that c-kit belongs to. It has surprisingly been discovered, however, t:hat a new functional class of receptor pTKs exists. The new functional class of receptor pTKs is expressed in primitive hematopoietic cells, but not expressed in mature hematopoietic cells.

For the purpose of this specification, a primitive hematopoietic cell is totipotent, i.e. capable of reconstituting all hematopoietic blood cells in vivo. A
mature hematopoietic cell is non-self-renewing, and has limited proliferative capacity - i.e., a limited ability to give rise to multiple lineages. Mature hematopoietic cells, for the purposes of this specification, are generally capable of giving rise to only one or two lineages in vitro or in vivo.

It should be understood that the hematopoietic system is complex, and contains many intermediate cells between the primitive totipotent hematopoietic stem cell and the totally committed mature hematopoietic cells defined above. As the stem cell develops into increasingly mature, lineage-restricted cells, it gradually loses its capacity for self-renewal.

The receptors of the present invention may and may not be expressed in these intermediate cells. The necessary and sufficient condition that defines members of the new class of receptors is that they are present in the primitive, totipotent stem cell or cells, and not in mature cells restricted only to one or, at most, two lineages.

An example of a member of the new class of receptor pTKs SUBSTITUTE SHEET

is called fetal liver kinase 2 (flk-2) after the organ in which it was found. There is approximately 1 totipotent stem cell per 10 cells in mid-gestation (day 14) fetal liver in mice. In addition to fetal liver, flk-2 is also expressed in fetal spleen, fetal thymus, adult brain, and adult marrow.
For example, flk-2 is expressed in individual multipotential CFU-Blast colonies capable of generating numerous multilineage colonies upon replating. It is likely, therefore, that flk-2 is expressed in the entire primitive (i.e. self-renewing) portion of the hematopoietic hierarchy.
This discovery is consistent with flk-2 being important in transducing putative self-renewal signals from the environment.
It is particularly relevant that the expression of flk-2 mRNA occurs in the most primitive thymocyte subset. Even in two closely linked immature subsets that differ in expression of the IL-2 receptor, flk-2 expression segregates to the more primitive subset lacking an IL-2 receptor. The earliest thymocyte subset is believed to be uncommitted. Therefore, the thymocytes expressing flk-2 may be multipotential. flk-2 is the first receptor tyrosine kinase known to be expressed in the T-lymphoid lineage.
The fetal liver mRNA migrates relative to 285 and 185 ribosomal bands on formaldehyde agarose gels at approximately 3.5 kb while the brain message is considerably larger. In adult tissues, flk-2 m-RNAfrom both brain and bone marrow migrated at approximately 3.5 kb.

A second pTK receptor is also included in the present invention. This second receptor, which is called fetal liver kinase 1(flk-1), is not a member of the same class of receptors as flk-2, since flk-l may be found in some more mature hematopoietic cells. The amino acid sequence of flk-1 is given in Figure 2.

Thepresent invention includesthe flk-1 receptor as SUBSTITUTE SHEET

WO 9b/17aW CA 02107463 1995-08-17 PCT/U542/02750 =~' .-well as DNA, cDNA.and RNA encoding flk-1. The DNA sequence of.flk-1 is also given in Figure 2. P'lk-1 may be found in the same organs as flk-2, as well as in fetal brain, stomach, kidney, lung, heart and intestine; and in adult kidney, 5 heart, spleen, lung, muscle, and lymph nodes.

The receptor protein tyrosine kinases of the invention are known to be divided into easily found domains. The DNA
sequence corresponding to the pTKs encode, starting at their 10 5'-ends, a hydrophobic leader sequence followed by a hydrophilic extracellular domain, which binds to, and is activated by, a specific ligand. Immediately downstream from the extracellular receptor domain, is a hydrophobic transmembrane region. The transmembrane region is immediately followed by a basic catalytic domain, which may easily be identified by reference to the Hanks et al. and Wilks articles discussed above.

The present invention includes the extracellular receptor domain lacking the transrnembrane region and catalytic domain. Preferably, the hydrophobic leader sequence is also removed from the extracellular domain. In the case of flk-2, the hydrophobic leader sequence includes amino acids 1-27.
These regions and domains may easily be visually identified by those having ordinary skill in the art by reviewing the amino acid sequence in a suspected pTK and comparing it to known pTKs. For example, referring to Figure la, the transmembrane region of flk-2, which separates the extracellular receptor domain from the catalytic domain, is encoded by nucleotides 1663 (T) to 1722 (C). These nucleotides correspond to amino acid residues 545 (Phe) to 564 (Cys). The amino acid sequence between the transmembrane region and the catalytic sub-domain (amino acids 618-623) i.dentified by Hanks et al. as sub-domain I (i.e., GXGXXG) is :haracteristic of receptor protein tyrosine kinases.

The extracellular domain may al.so be identified through SUBS1"t t'lJ tE SHEET

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commonly recognized criteria of extracellular amino acid sequences. The determination of appropriate criteria is known to those skilled in the art, and has been described, for example, by Hopp et al, Proc. Nat'l Acad. Sci. USA 78, 3824-3828 (1981); Kyte et al, J. Mol. Biol. 157, 105-132 (1982)y Emini, J. Virol. 55, 836-839 (1985); Jameson et al, CA BIOS 4, 181-186 (1988); and Karplus et al, Naturwissenschaften 72, 212-213 (1985). Amino acid domains predicted by these criteria to be surface exposed characteristic of extracellular domains.

As will be discussed in more detail below, the nucleic acid molecules that encode the receptors of the invention may be inserted into known vectors for use in standard recombinant DNA techniques. Standard recombinant DNA
techniques are those such as are described in Sambrook et al., "Molecular Cloning," Second Edition, Cold Spring Harbor Laboratory Press (1987) and by Ausubel et al., Eds, "Current Protocols in Molecular Biology," Green Publishing Associates and Wiley-Interscience, New York (1987). The vectors may be circular (i.e. plasmids) or non-circular. Standard vectors are available for cloning and expression in a host. The host may be prokaryotic or eucaryotic. Prokaryotic hosts are preferably E. coli. Preferred eucaryotic hosts include yeast, insect and mammalian cells. Preferred mammalian cells include, for example, CHO, COS and human cells.

Ligands The invention also includes ligands that bind to the receptor pTKs of the invention. In addition to binding, the ligands stimulate the proliferation of additional primitive stem cells, differentiation into more mature progenitor cells, or both.
The ligand may be a growth factor that occurs naturally in a mammal, preferably the same mammal that produces the corresponding receptor. The growth factor may be isolated and purified, or be present on the surface of an isolated SUBSTITUTE SHEET

41+~!!IIj3 WO 92/17486 PCI'/U592/02750 population of cells, such as stromal cells.

The ligand may also be a molecule that does not occur naturally in a mammal. For example, antibodies, preferably monoclonal, raised against the receptors of the invention or against anti-ligand antibodies mimic the shape of, and act as, ligands if they constitute the negative image of the receptor or anti-ligand antibody binding site. The ligand may also be a non-protein molecule that acts as a ligand when it binds to, or otherwise comes into contact with, the receptor.

In another embodiment, nucleic acid molecules encoding the ligands of the invention are provided. The nucleic acid molecule may be RNA, DNA or cDNA.

Stimulating Proliferation of Stem Cells The invention also includes a method of stimulating the proliferation and/or differentiation of primitive mammalian hematopoietic stem cells as defined above. The method comprises contacting the stem cells with a ligand in accordance with the present invention. The stimulation of proliferation and/or differentiation may occur in vitro or in vivo.

The ability of a ligand according to the invention to stimulate proliferation of stem cells in vitro and in vivo has important therapeutic applications. Such applications include treating mammals, including humans, whose primitive stem cells do not sufficiently undergo self-renewal. Example of such medical problems include those that occur when defects in hematopoietic stem cells or their related growth factors depress the number of white blood cells. Examples of such medical problems include anemia, such as macrocytic and aplastic anemia. Bone marrow damage resulting from cancer chemotherapy and radiation is another example of a medical problem that would be helped by the stem cell factors of the invention.

SUBST1 rlalTE SHEET

Functional Eguivalents The invention includes functional equivalents of the pTK
receptors, receptor domains, and ligands described above as well as of the nucleic acid sequences encoding them. A
protein is considered a functional equivalent of another protein for a specific function if the equivalent protein is immunologically cross-reactive with, and has the same function as, the receptors and ligands of the invention. The equivalent may, for example, be a fragment of the protein, or a substitution, addition or deletion mutant of the protein.
For example, it is possible to substitute amino acids in a sequence with equivalent amino acids. Groups of amino acids known normally to be equivalent are:

(a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);
(b) Asn(N) Asp(D) Glu(E) Gln(Q);
(c) His(H) Arg(R) Lys(K);
(d) Met(IM) Leu(L) Ile(I) Val(V); and (e) Phe(F) Tyr(Y) Trp(W).

Substitutions, additions and/or deletions in the receptors and ligands may be made as long as the resulting equivalent receptors and ligands are immunologically cross reactive with, and have the same function as, the native receptors and ligands.

The equivalent receptors and ligands will normally have substantially the same amino acid sequence as the native receptors and ligands. An amino acid sequence that is substantially the same as another sequence, but that differs from the other sequence by means of one or more substitutions, additions and/or deletions is considered to be an equivalent sequence. Preferably, less than 25%, more preferably less than 10%, and most preferably less than 5% of the number of amino acid residues in the amino acid sequence of the native receptors and ligands are substituted for, added to, or deleted from.

SUBSTITUTE SHEET

WO 92/17486 PCT/U +92/02750 Equivalent nucleic acid molecules include nucleic acid s'equences that encode equivalent receptors and ligands as defined above. Equivalent nucleic acid molecules also include nucleic acid sequences that differ from native nucleic acid sequences in ways that do riot affect the corresponding amino acid sequences.

ISOLATION OF NUCLEIC ACID MOLECULES AND PROTEINS
Isolation of Nucleic Acid Molecules Encoding Receptors In order to produce nucleic acid molecules encoding mammalian stem cell receptors, a source of stem cells is provided. Suitable sources include fetal liver, spleen, or thymus cells or adult marrow or brain cells.

For example, suitable mouse fetal liver cells may be obtained at day 14 of gestation. Mouse fetal thymus cells.
may be obtained at day 14-18, preferably day 15, of gestation. Suitable fetal cells of other mammals are obtained at gestation times corresponding to those of mouse.
Total RNA is prepared by standard procedures from stem cell receptor-containing tissue. The total RNA is used to direct cDNA synthesis. Standard methods for isolating RNA
and synthesizing cDNA are provided in standard manuals of molecular biology such as, for example, in Sambrook e't al., "Molecular Cloning," Second Edition, Cold Spring Harbor Laboratory Press (1987) and in Ausubel et al., (Eds), "Current Protocols in Molecular Biology," Greene Associates/Wiley Interscience, New York (1990).

The cDNA of the receptors is amplified by known methods.
For example, the cDNA may be used as a template for .35 amplification by polymerase chain reaction (PCR); see Saiki et al., Science, 239, 487 (1988) or Mullis et al,, U.S.
patent 4,683,195. The sequences of the oligonucleotide primers for the PCR amplification are derived from the sequences of known receptors, such as from the sequences SUBSTITUTE SHEET

71 p~
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WO 92/17486 PC'T/US92/02750 ~: ~..:=_ given in Figures 1 and 2 for flk-2 and flk-1, respectively, =preferably from flk-2. The oligonucleotides are synthesized by methods known in the art. Suitable me'thods include those described by Caruthers in Science 230, 281-285 (1985).

In order to isolate the entire protein-coding regions for the receptors of the invention, the upstream oligonucleotide is complementary to the sequence at the 5' end, preferably encompassing the ATG start codon and at least 10 5-10 nucleotides upstream of the start codon. The downstream oligonucleotide is complementary to the sequence at the 3' end, optionally encompassing the stop codon. A mixture of upstream and downstream oligonucleotides are used in the PCR
amplification. The conditions are optimized for each 15 particular primer pair according to standard procedures.
The PCR product is analyzed by electrophoresis for the correct size cDNA corresponding to the sequence between the primers.

Alternatively, the coding region may be amplified in two or more overlapping fragments. The overlapping fragments are designed to include a restriction site permitting the assembly of the intact cDNA from the fragments.

The amplified DNA encoding the receptors of the invention may be replicated in a wide variety of cloning vectors in a wide variety of host cells. The host cell may be prokaryotic or eukaryotic. The DNA may be obtained from natural sources and, optionally, modified, or may be synthesized in whole or in part.

The vector into which the DNA is spliced may comprise segments of chromosomal, non-chromosomal and synthetic DNA
sequences. Some suitable prokaryotic cloning vectors include plasmids from E. coli, such as colEl, DCRl, RBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA such as M13 and other filamentous single-stranded DNA phages.

SUBSTITUTE SHEET

WO 92/17486 2107 46 j PCI'/US92/02750 Isolation of Receptors DNA encoding the receptors of the invention are inserted into a suitable vector and expressed in a suitable prokaryotic or eucaryotic host. Vector:> for expressing proteins in bacteria, especially E.coli, are known. Such vectors include the PATH vectors described by Dieckmann and Tzagoloff in J. Biol. Chem. 260, 1513-1520 (1985). These vectors contain DNA sequences that encode anthranilate synthetase (TrpE) followed by a polylinker at the carboxy terminus. Other expression vector systems are based on beta-' galactosidase (pEX); lambda PL; maltose binding protein (pMAL); and glutathione S-transferase (pGST) - see Gene 67, 31 (1988) and Peptide Research 3, 167 (1990).
Vectors useful in yeast are available. A suitable example is the 2 plasmid.

Suitable vectors for use in mammalian cells are also known. Such vectors include well-known derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences and vectors derived from combination of plasmids and phage DNA.

Further eukaryotic expression vectors are known in the art (e.g., P.J. Southern and P. Berg, J. Mol. Appl. Genet. 1, 327-341 (1982); S. Subramani et al, Mol. Cell. Biol. 1, 854--864 (1981); R.J. Kaufmann and P.A. Sharp, tAmplification And Expression Of Sequences Cotransfected with A Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol.
Biol. 159, 601-621 (1982); R.J. Kaufmann and P.A. Sharp, Mol.
Cell. Biol. 159, 601-664 (1982); S.I. Scahill et al, "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells,"
Proc. Natl. Acad. Sci. USA 80, 4654-4659 (1983); G. Urlaub and L.A. Chasin, Proc. Natl. Acad. Sci. USA 77, 4216-4220, (1980).

The expression vectors useful in the present invention contain at least one expression control sequence that is SUBSTITUTE SHEET

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operatively linked to the DNA sequence or fragment to be expressed. The control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of fd coat protein, the glycolytic promoters of yeast, e.g., -the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, e.g., the early and late promoters or sV40, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations 'thereof.

Vectors containing the receptor-encoding DNA and control signals are inserted into a host cell for expression of the receptor. Some useful expression host cells include well-known prokaryotic and eukaryotic cells. Some suitable prokaryotic hosts include, for example, E. coli, such as E.
coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E.
coli X2282, E. coli DHI, and E. coli MRC1, Pseudomonas, Bacillus, such as Bacillus subtilis, and Strebtomyces.
Suitable eukaryotic cells include yeast and other fungi, insect, animal cells, such as COS cells and CHO cells, human cells and plant cells in tissue culture.

The human homologs of the mouse receptors described above are isolated.by a similar strategy. RNA encoding the receptors are obtained from a source of human cells enriched for -rimitive stem cells. Suitable human cells include fetal spl-en, thymus and liver cells, and umbilical cord blood as well as adult brain and bone marrow cells. The human fetal cells are preferably obtained on the day of gestation corresponding to mid-gestation in mice. The amino acid sequences of the human flk receptors as well as of the nucleic acid sequences encoding them are homologous to the amino acid and nucleotide sequences of the mouse receptors.

SUBST"1TUTE SHE

wo 9z/ ~ 7ass PCT/US92/02750 ~M
-2~ 18 In the present specification, the sequence of a first protein, such as a receptor or a ligand, or of a nucleic acid molecule that encodes the protein, is considered homologous to a second protein or nucleic acid molecule if the amino acid or nucleotide sequence of the first protein or nucleic acid molecule is at least abqut 30% homologous, preferably at least about 50% homologous, and more preferably at least about 65% homologous to the respective sequences of the second protein or nucleic acid molecule. In the case of proteins having high homology, the amino acid or nucleotide sequence of the first protein or nucleic acid molecule is at least about 75% homologous, preferably at least about 85%
hoinologous, and more preferably at least about 95% homologous to the amino acid or nucleotide sequence of the second protein or nucleic acid molecule.

Combinations of mouse oligonucleotide pairs are used as PCR primers to amplify the human homologs from the cells to account for sequence divergence. The remainder of the procedure for obtaining the human f'lk homologs are similar to those described above for obtaining mouse flk receptors. The less than perfect homology between the human flk homologs and the mouse oligonucleotides is taken into account in determining the stringency of the hybridization conditions.
Assay for expression of Receptors on StemCells In order to demonstrate the expression of flk receptors on the surface of primitive hematopoietic stem cells, antibodies that recognize the receptor are raised. The receptor may be the entire protein as it exists in nature, or an antigenic fragment of the whole protein.
Preferably, the fragment comprises the predicted extra-cellular portion of the molecule.
Antigenic fragments may be identified by methods known in the art. Fragments containing antigenic sequences may be selected on the basis of generally accepted criteria of potential antigenicity and/or exposure. Such criteria StJ BSTi'i't11'E SHEET

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include the hydrophilicity and relative antigenic index, as determined by surface exposure analysis of proteins. The determination of appropriate criteria is known to those skilled in the art, and has been described, for example, by Hopp et al, Proc. Nat'l Acad. Sci. USA 78, 3824-3828 (1981);
Kyte et al, J. Mol. Biol. 157, 105-132 (1982); Emini, J.
Virol. 55, 836-839 (1985); Jameson et al, CA BIOS 4, 181-186 (1988); and Karplus et al, Naturwissenschaften 72, 212-213 (1985). Amino acid domains predicted by these criteria to be surface exposed are selected preferentially over domains predicted to be more hydrophobic or hidden.

The proteins and fragments of the receptors to be used as antigens may be prepared by methods known in the art.
Such methods include isolating or synthesizing DNA encoding the proteins and fragments, and using the DNA to produce recombinant proteins, as described above.

Fragments of proteins and DNA encoding the fragments may be chemically synthesized by methods known in the art from individual amino acids and nucleotides. Suitable methods for synthesizing protein fragments are described by Stuart and Young in "Solid Phase Peptide Synthesis," Second Edition, Pierce Chemical Company (1984). Suitable methods for synthesizing DNA fragments are described by Caruthers in Science 230, 281-285 (1985).

If the receptor fragment defines the epitope, but is too short to be antigenic, it may be conjugated to a carrier molecule in order to produce antibodies. Some suitable carrier molecules include keyholelimpet hemocyanin, Ig sequences, TrpE, and human or bovine serum albumen.
Conjugation may be carried out by methods known in the art.
One such method is to combine a cysteine residue of the fragment with a cysteine residue on the carrier molecule.
The antibodies are preferably monoclonal. Monoclonal antibodies may be produced by methods known in the art.
These methods include the immunological method described by SUBSTITUTE SHEET

WO 92/174Q I d PCIf/U592/02750 Kohler and Milstein in Nature 256, 495-497 (1975) and Campbell in "Monoclonal Antibody Technology, The Production and Characterization of Rodent and Human Hybridomas01 in Burdon et al., Eds, Laboratory Techniques in Biochemistry and 5 Molecular Biology, Volume 13, Elsevier Science Publishers, Amsterdam (1985); as well as by the recombinant DNA method described by Huse et al in Science 246, 1275-1281 (1989).

Polyclonal or monoclonal antisera shown to be reactive 10 with receptor-encoded native proteins, such as with flk-1 and flk-2 encoded proteins, expressed on the surface of viable cells are used to isolate antibody-positive cells. One method for isolating such cells is flow cytometry; see, for example, Loken et al., European patent application 317,156.
15 The cells obtained are assayed for stem cells by engraftment into radiation-ablated hosts by methods known in the art;
see, for example, Jordan et al., Cell 61, 953-963 (1990).
Criteria for Novel Stem Cell Receptor Tyrosixse Kinases 20 Expressed in Stem Cells Additional novel receptor tyrosine kinase cDNAs are obtained by amplifying cDNAs from stem cell populations using oligonucleotides as PCR primers; see above. Examples of suitable oligonucleotides are PTK1 and PTK2, which were described by Wilks et al. in Proc. Nat1. Acad. Sci. USA 86, 1603-1607 (1989). Novel cDNA is selected on the basis of differential hybridization screening with probes representing known kinases. The cDNA clones hybridizing only at low stringency are selected and sequenced. The presence of the amino acid triplet DFG confirms that the sequence represents a kinase. The diagnostic methionine residue in the WMAPES
motif is indicative of a receptor-like kinase, as described above. Potentially novel sequences obtained are compared to available sequences using databases such as Genbank in order to confirm uniqueness. Gene-specific oligonucleotides are prepared as described above based on the sequence obtained.
The oligonucleotides are used to analyze stem cell enriched and depleted populations for expression. Such cell populations in mice are described, for example, by Jordan et SUBSTITUTE SHEET

WO 92/17486 Fcrius92i02750 al. in Cell 61, 953-956 (1990); Ikuta et al. in Cell 62, 863-864. (1990); Spangrude et al. in Science 241, 58-62 (1988);
and Szilvassy et al. in Blood 74, 930-939 (1989). Examples of such human cell populations are described as CD33'CD34+ by Andrews et al. in the Journal of Experimental Medicine 169, 1721-1731 (1989). Other human stem cell populations are described, for example, in Civin et al., European Patent Application 395,355 and in Loken et al., European Paten't Application 317,156.
isolating Ligands and Nucleic Acid Molecules Encoding Ligands Cells that may be used for obtaining ligands include stromal cells, for example stromal cells from fetal liver, fetal spleen, fetal thymus and fetal or adult bone marrow.
Cell lines expressing ligands are established and screened.
For example, cells such as stromal (non-hematopoietic) cells from fetal liver are immortalized by known methods.
Examples of known methods of immortalizing cells include transduction with a temperature sensitive SV40 T-antigen expressed in a retroviral vector. Infection of fetal liver cells with this virus permits the rapid and efficient establishment of multiple independent cell lines. These lines are screened for ligand activity by methods known in the art, such as those outlined below.

Ligands for the receptors of the invention, such as flk-1 and flk-2, may be obtained from the cells in several ways.
For example, a bioassay system for ligand activity employs chimeric tagged receptors; see, for example, Flanagan et al., Cell 63, 185-194 (1990). One strategy measures ligand binding directly via a histochemical assay. Fusion proteins comprising the extracellular receptor domains and secretable alkaline phosphatase (SEAP) are constructed and transfected into suitable cells such as NIH/3T3 or COS cells. Flanagan et al. refer to such DNA or amino acid constructs as APtag followed by the name of the receptor - i.e. APtag-c-Xit. The fusion proteins bind with high affinity to cells expressing S11BSTI'fU'TE SHEET

WO 92/174'q CA 02107463 1995-08-17 PL~'j'/US92/02750 p.,.. ..

surface-bound ligand. Binding is detectable by the enzymatic activity of the alkaline phosphatase secreted into the medium. The bound cells, which are often stromal cells, are isolated from the APtag-receptor complex.
For example, some stromal cells that bind APtag-flkl and APtag-flk2 fusion proteins include mouse fetal liver cells (see example 1); human fetal spleen cells (see example 3);
and human fetal liver (example 3). Some stromal fetal thymus cells contain flk-1 ligand (example 3).

To clone the cDNA that encodes the ligand, a cDNA
library is constructed from the isolated stromal cells in a suitable expression vector, preferably a phage such as CDMB, pSV Sport (BRL Gibco) or piH3, (Seed et al., Proc. Natl.
Acad. Sci. USA 84, 3365-3369 (1987)). The library is transfected into suitable host cells, such as COS cells.
Cells containing ligands on their surface are detected by known methods, see above.
in one such method, transfected COS cells are distributed into single cell suspensions and incubated with the secreted alkaline phosphatase-f1k receptor fusion protein, which is present in the medium from NIHf3T3 or COS
cells prepared by the method described by Flanagan et al., see above. Alkaline phosphatase-receptor fusion proteins that are not bound to the cells are removed by centrifugation, and the cells are panned on plates coated with antibodies to alkaline phosphatase. Bound cells are isolated following several washes with a suitable wash reagent, such as 5% fetal bovine serum in PBS, and the DNA is extracted from the cells. Additional details of the panning method described above may be found in an article by Seed et al., Proc. Natl. Acad. Sci. USA 84, 3365-3369 (1987).
In a second strategy, the putative extracellular ligand binding domains of the receptors are fused to the transmembrane and kinase domains.of the human c-fms tyrosine kinase and introduced into 3T3 fibroblasts. The human c-fms SUBSTITUTE SHEET

PL C=/US92/0275U
~
2~~~

kinase is necessary and sufficient to transduce proliferative signals in these cells after appropriate activation i.e. with the flk-1 or flk-2 ligand. The 3T3 cells expressing the chimeras are used to screen putative sources of ligand in a cell proliferation assay.

An alternate approach for isolating ligands using the fusion receptor-expressing 3T3 cells and insertional activation is also possible. A retrovirus is introduced into random chroanosomal positions in a large population of these cells. In a small fx=action, the retrovirus is inserted in the vicinity of the ligand-encoding gene, thereby activating it. These cells proliferate due to autocrine stimulation of the receptor. The ligand gene is "tagged" by the retrovirus, thus facilitating its isolation.
Examples Example 1. Cells containinrf mouse flk-1 and flk-2 liqands.
Murine stromal cell line 2018.

In order to establish stromal cell lines, fetal liver cells are disaggregated with collagen and grown in a mixture of Dulbecco's Modified Eagle's Medium (DMEM) and 10% heat-inactivated fetal calf serum at 37 C. The cells are immortalized by standard methods. A suitable method involves introducing DNA encoding a growth regulating- or oncogene-encoding sequence into the target host cell. The DNA may be introduced by means of transduction in a recombinant viral particle or transfection in a plasmid. See, for example, Hammerschmidt et al., Nature 340, 393-397 (1989) and Abcouwer et al, Biotechnology 7, 939-946 (1989). Retroviruses are the ,35 preferred viral vectors, although SV40 and Epstein-Barr virus can also serve as donors of the growth-enhancing sequences.
A suitable retrovirus is the ecotropic retrovirus containing a temperature sensitive SV40 T-antigen (tsA58) and a G418 resistance gene described by McKay in Ce11 66, 713-729 (1991). After several days at 37 C, the temperature of the medium is lowered to 32 C. Cells are selected.with G418 (0.5 SUBSTITUTE SHEET

4. CA 02107463 2 ~ 95-08-17 mg/ml).176The selected cells are expanded and maintained.

A mouse stromal cell line produced by this procedure is called 2018 and was deposited on October 30, 1991 in the American Type Culture Collection, Rockville, Maryland, USA
(ATCC); accession number CRL 10907.

Example 2. Cells containing human fik-1. and f1k-2 liaands.
Human fetal liver (18, 20, and 33 weeks after abortion), spleen (18 weeks after abortion), or thymus (20 weeks after abortion) is removed at the time of abortion and stored on ice in a balanced salt solution. After mincing into 1 mm fragments and forcing through a wire mesh, the tissue is washed one time in Hanks Balanced Salt Solution (HBSS).
The disrupted tissue is centrifuged at 200 xg for 15 minutes at room temperature. The resulting pelle=t is resuspended in 10-20 ml of a tissue culture grade trypsin-EDTA solution (Flow Laboratories). The resuspended tissue is transferred to a sterile flask and stirred with a stirring bar at room temperature for 10 minutes. One ml of heat-inactivated fetal bovine calf serum (Hyclone) is added to a final concentration of 10% in order to inhibit trypsin activity. Collagenase type IV (Sigma) is added from a stock solution (10 mg/ml in HBSS) to a final concentration of 100 g/ml in order to disrupt the stromal cells. The tissue is stirred at room temperature for an additional 2.5 hours;
collected by centrifugation (400xg, 15 minutes); and resuspended in "stromal medium," which contains Iscove's modification of DMEM supplemented with 10%
heat-inactivated fetal calf serum, 5% heat-inactivated human serum (Sigma), 4 mM L-glutamine, ix sodium pyruvate, (stock of 100x Sigma), lx non-essential amino acids (stock of l00x, Flow), and a mixture of antibiotics kanomycin, neomycin, penicillin, streptomycin. Prior to resuspending the pellet in the stromal medium, the pellet is washed one time with HBSS.- It is convenient to suspend the cells in 60 ml of medium. The number of cultures depends on the amount of St1BST1 rL11 ~ SHEET

tissue.

Example 3. Isolating Stromal cells Resuspended Cells (example 2) that are incubated at 37 C
with 5% carbon dioxide begin to adhere to the plastic plate within 10-48 hours. Confluent monolayers may be observed within 7-10 days, depending upon the number of cells plated 10 in the initial innoculum. Non-adherent and highly refractile cells adhering to the stromal cell layer as colonies are separately removed by pipetting and frozen. Non-adherent cells are likely sources of populations of self-renewing stem cells containing flk-2. The adherent stromal cell layers are 15 frozen in aliquots for future studies or expanded for growth in culture.

An unexpectedly high level of APtag-flk-2 fusion protein binding to the fetal spleeri cells is observed. Two fetal 20 spleen lines are grown in "stromal medium," which is described in example 2.

Non-adherent fetal stem cells attach to the stromal cells and form colonies (colony forming unit - CFU). Stromal 25 cells and CFU are isolated by means of sterile glass cylinders and expanded in culture. A clone, called Fsp 62891, contains the flk-2 ligand. Fsp 62891 was deposited in the American Type Culture Collection, Rockville, Maryland, U.S.A on November 21, 1991, accession number CRL 10935.
Fetal liver and fetal thymus cells are prepared in a similar way. Both of these cell types produce ligands of flk-1 and, in the case of liver, some flk-2. One such fetal thymus cell line, called F.thy 62891, and one such fetal liver cell line, called FL 62891, were deposited in the American Type Culture Collection, Rockville, Maryland, U.S.A
on November 21, 1991 and April 2, 1992, respectively, accession numbers CRL 10936 and CRL 11005, respectively.

Stable human cell lines are prepared from fetal cells SUBSTITUTE SHEET

r)76~ z 6 Pcrius92ro7750 with the same temperature sensitive immortalizing virus used to.prepare the murine cell line described in example 1.
Example 4. Isolation of human stromal cell clone Highly refractile cells overgrow patches of stromal cells, presumably because the stromal cells produce factors that allow the formation of the CFU. To isolate stromal cell clones, sterile glass cylinders coated with vacuum grease are positioned over the CFU. A trypsin-EDTA solution (100 ml) is added in order to detach the cells. The cells are added to 5-m1 of stromal medium and each (clone) plated in a single well of 6-well plate.

Exa=le S. Plasmid (AP-tag) for expressing secretable alkaline phosphatase (SEAP) Plasmids that express secretable alkaline phosphatase.
are described by Flanagan and Leder in Cell 63, 185-194 (1990). The plasmids contain a promoter, such as the LTR
promoter; a polylinker, including Hindill and BglII; DNA
encoding SEAP; a poly-A signal; and ampicillin resistance gene; and replication site.
Exa.mple 6. Plasmid for ex~ressinq APtag-flk-2 and A"Ptag-flk-1 fusion proteins Plasnrnids that express fusion proteins of SEAP and the extracellular portion of either flk-1 or flk-2 are prepared in accordance with the protocols of Flanagan and Leder in Cell 63, 185-194 (1990) and Berger et al., Gene 66, 1-10 (1988). Briefly, a Hindlil-Bam HI fragment containing the extracellular portion of flk-1 or flk-2 is prepared and inserted into the HindIII-Bg1II site of the plasmid described in example 5.

Example 7. Production Of APtapt-flk-1 or -flk-2 P'usion Protein SUBSTITUTE SHEET

WO 92/17486 2 10 7 PCf'/US92/02750 The plasmids from Example 6 are transfected into Cos-7 cells by DEAE-dextran (as described in Current Protocols in Molecular Biology, Unit 16.13, "Transient Expression of Proteins Using Cos Cells," 1991); and cotransfected with a selectable marker, such as pSV7neo, irito NIH/3T3 cells by calcium precipitation. The NIH/3T3 cells are selected with 600pg/ml G418 in 100 mm plates. Over 300 clones are screened for secretion of placexital alkaline phosphatase activity.
The assay is performed by heating a portion of the supernatant at 65 C for 10 minutes to inactivate background phosphatase activity, and measuring the OD405 after incubating with 1M diethanolamine (pH 9.8), 0.5 mM MgCIZ1 10 mM L-homoarginine (a phosphatase inhibitor), 0.5 mg/ml BSA, and 12 mM p-nitrophenyl phosphate. Human placental alkaline phosphatase is used to perform a standard curve. The APtaq-flk-1 clones (F-1AP21-4) produce up to 10 g alkaline phosphatase activity/m1 and the APtaq-flk-2 clones (F-2AP26-0) produce up to 0.5 Ag alkaline phosphatase activity/ml.
Example B. Assay For APtaq-flk-I Or APtaq-flk-2 Pindincx To Cells The binding of APtaq-flk-1 or APtag-flk-2 to cells containing the appropriate ligand is assayed by standard methods. See, for example, Flanagan and Leder, Cell 63:185-194, 1990). Cells (i.e., mouse stromal cells, human fetal liver, spleen or thymus, or various control cells) are grown to confluency in six-well plates and washed with HBHA (Hank's balanced salt solution with 0.5 mg/ml BSA, 0.02% NaN3, 20 mM
HEPES, pH 7.0). Supernatants from transfected COS or NIH/3T3 cells containing either APtaq-flk-1 fusion protein, APtag-flk-2 fusion protein, or APtag without a receptor (as a control) are added to the ce11 monolayers and incubated for two hours at room temperature on a rotating platform. The concentration of the APtaq-flk-1 fusion protein, APtag-flk-2 fusion protein, or APtag without a receptor is 60 ng/ml of alkaline phosphatase as determined by the standard alkaline SUBSTITUTE SHEET

phosphatase curve (see above). The cells are then rinsed seven times with HBHA and lysed in 350 l of 1m Triton X-100, mM Tris-HC1 (pH 8.0). The lysates are transferred to a microfuge tube, along with a further 150 l rinse with the 5 same solution. After vortexing vigorously, the samples are centrifuged for five minutes in"a microfuge, heated at 65 C
for 12 minutes to inactivate.cellular phosphatases, and assayed for phosphatase activity as described previously.
Results of experiments designed to show the time and dose 10 responses of binding between stromal cells containing the ligands to flk-2 and flk-1 (2018) and APtag-flk-2, APtag-flk-1 and APtag without receptor (as a control) are shown in Figures 3 and 4, respectively.

Example 8A. Plasmids for exoressina flkl/fms and flk2/fms fusion proteins Plasmids that express fusion proteins of the extracellular portion of either flk-1 or flk-2 and the intracellular portion of c-fms (also known as colony-stimulating factor-1 receptor) are prepared in a manner similar to that described under Example 6 (Plasmid for expressing APtag-flk-2 and APtag-flk-i fusion proteins).
Briefly, a Hind III - Bam HI fragment containing the extracellular portion of flkl or flk2 is prepared and inserted into the Hind III - Bgl II site of a pLH expression vector containing the intracellular portion of c-fms.

8B. Expression of flkl/fms or flk2/fms in 3T3 cells The plasmids from Example 11 are transfected into NIH/3T3 cells by calcium. The intracellular portion of c-fms is detected by Western blotting.

ExaanDle 9. Cloning and ExDression of oDNA Coding For Mouse Ligand To f1k-1 and flk-2 Recs tors cDNA expressing mouse ligand for flk-1 and flk-2 is SUBSTITUTE SHEET

WO 92/17486 CA 02107463 1995-08-17 PC j"/U6,''92/02750 ~:. . ..

prepared by known methods. See, for example, Seed, B., and .Piruffo, A. PNAS 84:3365-3369, 1987; Simmons, D. and Seed, B.
J. Immunol. 141:2797-2800; and D'Andrea, A.D., Lodish, H.F.
and Wong, G.G. Cell 57:277-285, 1989).
The protocols are listed below in sequence: (a) RNA
isolation; (b) poly A RNA preparation; (c) cDNA synthesis;
(d) cDNA size fractionation; (e) propagation of plasmids (vector); (f) isolation of plasmid DNA; (g) preparation of vector pSV Sport (BRL Gibco) for cloning; (h) compilation of buffers for the above steps; (i) Transfection of cDNA
encoding Ligands in Cos 7 Cells; (j) panning procedure; (k) Expression cloning of flk-1 or flk-2 ligand by establishment of an autocrine loop.
9a. Guanidinium thiocyanate/LiCl Protocol for RNA Isolation For each ml of mix desired, 0.5 g guanidine thiocyanate (GuSCN) is dissolved in 0.55 ml of 25% LiCl (stock filtered through 0.45 micron filter). 20 l of mercaptoethanol is added. (The resulting solution is not good for more than about a week at room temperature.) The 2018 stromal cells are centrifuged, and 1 ml of the solution described above is added to up to 5 x 10' cells.
The cells are sheared by means of a polytron until the mixture is non-viscous. For small scale preparations (<108 cells), the sheared mixture is layered on 1.5 ml of 5.7M CsCl (RNase free; 1.26 g CsCl added to every ml 10 mM EDTA pH8), and overlaid with RNase-free water if needed. The mixture is spun in an SW55 rotor at 50 krpm for 2 hours. For large scale preparations, 25 ml of the mixture is layered on 12 ml CsCl in an SW28 tube, overlaid as above, and spun at 24 krpm for 8 hours. The contents of the tube are aspirated carefully with a sterile pasteur pipet connected to a vacuum flask. Once past the CsCl interface, a band around the tube is scratched with the pipet tip to prevent creeping of the layer on the wall down the tube. The remaining CsCl solution is aspirated. The resulting pellet is taken up in SUBSTITUTE SHEET

~1 ~= ~ t' " '~ ~ CA 02107463 1995-08-17 WO 92/17486 PC1'/US92/02750 water, but not redissolved. 1/10 volume of sodium acetate and three volumes of ethanol are added to the mixture, and spun. The pellet is resuspended in water at 70 C, if necessary. The concentration of the RNA is adjusted to 1 mg/ml and frozen.

It should be noted that small RNA molecules (e.g., 5S) do not come down. For small amounts of cells, the volumes are scaled down, and the mixture is overlaid with GuSCN in RNase-free water on a gradient (precipitation is inefficient when RNA is dilute).

9b. Poly A' RNA preparation (All buffers men=tioned are compiled separately below) A disposable polypropylene column is prepared by washing with 5M NaOH and then rinsing with RNase-free water. For each milligram of total RNA, approximately 0.3 ml (final packed bed) of oligo dT cellulose is added. The oligo dT
cellulose is prepared by resuspending approximately 0.5 ml of dry powder in 1 ml of 0.1M NaOH and transferring it into the column, or by percolating 0.1M NaOH through a previously used column. The column is washed with several column volumes of RNase-free water until the pH is neutral, and rinsed with 2-3 ml of loading buffer. The column bed is transferred to a sterile 15 ml tube using 4-6 ml of loading buffer.

Total RNA from the 2018 cell line is heated to 70 C for 2-3 minutes. LiCl from RNase-free stock is added to the mixture to a final concentration of 0.5M. The mixture is combined with oligo dT cellulose in the 15 ml tube, which is vortexed or agitated for 10 minutes. The mixture is poured into the column, and washed with 3 ml loading buffer, and then with 3 ml of middle wash buffer. The mRNA is eluted directly into an SW55 tube with 1.5 ml of 2 mM EDTA and 0.1%
aDS, discarding the first two or three drops.

The eluted mRNA is precipitated by adding 1/10 volume of SUBSTITUTE SHEET

2~.07 A
WO 92/17486 PCr/US92/02750 3M sodium acetate and filling the tube with ethanol. The contents of the tube are mixed, chilled for 30 minutes at -20 C, and spun at 50 krpm at 5 C for 30 minutes. After the ethanol is decanted, and the tube air dried, the mRNA pellet is resuspended in 50-100 l of RNase-free water. 5 l of the resuspended mRNA is heated to 70 C in MOPS/EDTA/formaldehyde, and examined on an RNase-free 1% agarose gel.

9c. cDNA Synthesis The protocol used is a variation of the method described by Gubler and Hoffman in Gene 25, 263-270 (1983).

1. First Strand. 4 g of mRNA is added to a microfuge tube, hea=ted to approximately 100 C for 30 seconds, quenched on ice. The volume is adjusted to 70g1 with RNAse-free water. 20 i of RT1 buffer, 2 l of RNAse inhibitor (Boehringer 36 u/Ai), 1Al of 5 g/ l of oligo dT
(Collaborative Research), 2.5 Al of 20 mM dXTP's (ultrapure -US Biochemicals), I l of iM DTT and 4 l of RT-XL (Life Sciences, 24 u/ l) are added. The mixture is incubated at 42 C for 40 minutes, and inactivated by heating at 70 C for 10 minutes.
2. Second Strand. 320 l of RNAse-free water, 80 Ml of RT2 buffer, 5 l of DNA Polymerase I (Boehringer, 5 tJ/ul), 2 l RNAse H (BRL 2 u/g1) are added to the solution containing the first strand. The solution is incubated at 15 C for one hour and at 22 C for an additional hour. After adding 20 M1 of 0.5M EDTA, pH 8.0, the solution is extracted with phenol and precipitated by adding NaCl to 0.5M linear polyacrylamide (carrier) to 20 pg/ml, and filling the tube with EtOH. The tube is spun for 2-3 minutes in a microfuge, vortexed to dislodge precipitated material from the wall of the tube, and respun for one minute.

3. Adaptors. Adaptors provide specific restriction sites to facilitate cloning, and are available from BRL

StlBST1TlJTE SHEET

+~~ ~j ~ c ~~ ( /.!j ,~ CA 02107463 1995-08-17 WO 92/17486 PCT/bJS92/02750 .=~., Gibco, New England Biolabs, etc. Crude adaptors are r'esuspended at a concentration of 1Mg/ l. MgSO4 is added to a final concentration of 10 mM, followed by five volumes of EtOH. The resulting precipitate is rinsed with 70% EtOH and resuspended in TE at a concentration of 1gg/ l. To kinase, 25 Ml of resuspended adaptors is added to 3 l of lOX
kinasing buffer and 20 units of kinase. The mixture is incubated at 37 C overnight. The precipitated cDNA is resuspended in 240 l of TE (10/1). After adding 30 l of lOX low salt buffer, 30 l of 10X ligation buffer with 0.1mM
ATP, 3 l (2.4 g) of ]cinased 12-mer adaptor sequence, 2 l (1.6 g) of kinased 8-mer adaptor sequence, and 1 l of T4 DNA ligase (BioLabs, 400 u/ l, or Boehringer, 1 Weiss unit ml), the mixture is incubated at 15 C overnight. The cDNA is extracted with phenol and precipitated as above, except that the extra carrier is omitted, and resuspended in 100 l of TE.

9d. cDNA Size Fractionation.
A 20% KOAc, 2 mM EDTA, 1 gg/ml ethidium bromide solution and a 5% KOAc, 2 mM EDTA, 1;ug/ml ethidium bromide solution are prepared. 2.6 ml of the 20% KOAc solution is added to the back chamber of a small gradient maker. Air bubbles are removed from the tube connecting the two chambers by allowing the 20% solution to flow in'to the front chamber and forcing the solution to return to the back chamber by tilting the gradient maker. The passage between the chambers is closed, and 2.5 ml of 5% solution is added to the front chamber. Any liquid in the tubing from a previous run is removed by allowing the 5% solution to flow to the end of the tubing, and then to return to its chamber. The apparatus is placed on a stirplate,.and, with rapid stirring, the topcock connecting the two chambers and the front stopcock are opened. A polyallomer 5W55 tube is filled from the bottom with the KOAc solution. The gradient is overlaid with 100 l of cDNA solution, and spun for three hours at 50k rpm at 22 C. To collect fractions from the gradient, the SW55 tube is pierced close to the bottom of the tube with a butterfly SUBSTITUTE SHEET

infusion set (with the luer hub clipped off). Three 0.5 ml .fractions and then six 0.25 ml fractions are collected in microfuge tubes (approximately 22 and 11 drops, respectively). The fractions are precipitated by adding linear polyacrylamide to 20 g/ml and filling the tube to the top with ethanol. The tubes are cooled, spun in a microfuge tube for three minutes, vortexed, and respun for one minute.
The resulting pellets are rinsed with 70% ethanol and respun, taking care not to permit the pellets to dry to completion.
Each 0.25 ml fraction is resuspended in 10 l of TE, and 1 l is run on a 1% agarose minigel. The first three fractions, and the last six which contain no material smaller than 1 kb are pooled.

9e. Propagation of Plasmids SupF plasmids are selected in nonsuppressing bacterial hosts containing a second plasmid, p3, which contains amber mutated ampicillin and tetracycline drug resistance elements.
See Seed, Nucleic Acids Res., 11, 2427-2445 (1983). The p3 plasmid is derived from RP1, is 57 kb in length, and is a stably maintained, single copy episome. The ampicillin resistance of this plasmid reverts at a high rate so that amp' plasmids usually cannot be used in p3-containing strains. Selection for tetracycline resistance alone is almost as good as selection for ampicillin-tetracycline resistance. However, spontaneous appearance of chromosomal suppressor tRNA mutations presents an unavoidable background (frequency about 10-9) in this system. Colonies arising from spontaneous suppressor mutations are usually larger than colonies arising from plasmid transformation. Suppressor plasmids are selected in Luria broth (LB) medium containing ampicillin at 12.5 g/ml and tetracycline at 7.5 g/ml. For scaled-up plasmid preparations, M9 Casamino acids medium containing glycerol (0.8%) is employed as a carbon source.
The bacteria are grown to saturation.

Alternatively, pSV Sport (BRL, Gaithersberg, Maryland) may be employed to provide SV40 derived sequences for SUBSTITUTE SHEET

3 '%) CA 02107463 1995-08-17 34 replication, transcription initiation and termination in Cos 7-c.ells, as well as those sequences necessary for replication and ampicillin resistance in E. coli.

9f. Isolation of Vector DNA/Plasmid One liter of saturated bacterial cells are spun down in J6 bottles at 4.2k rpm for 25 minutes. The cells are resuspended in 40 ml 10 mM EDTA, pH 8. 80 ml 0.2M NaOH and 1% SDS are added, and the mixture is swirled until it is clear and viscous. 40 ml 5M KOAc, pH 4.7 (2.5M KOAc, 2.5IrY
HOAc) is added, and the mixture is shaken semi-vigorously until the lumps are approximately 2-3 mm in size. The bottle is spun at 4.2k rpm for 5 minutes. The supernatant is poured through cheesecloth into a 250 ml bottle, which is then filled with isopropyl alcohol and centrifuged at 4.2k rpm for 5 minutes. The bottle is gently drained and rinsed with 70%
ethanol, taking care not to fragment the pellet. After inverting the bottle and removing traces of ethanol, the mixture is resuspended in 3.5 ml Tris base/EDTA (20 mM/10 mM). 3.75 ml of resuspended pellet and 0.75 ml 10 mg/ml ethidium bromide are added to 4.5 g CsCl. VTi8O tubes are filled with solution, and centrifuged for at least 2.5 hours at 80k rpm. Bands are extracted by visible light with 1 ml syringe and 20 gauge or lower needle. The top of the tube is cut off with scissors, and the needle is inserted upwards into the tube at an angle of about 30 degrees with respect to the tube at a position about 3 mm beneath the band, with the bevel of the needle up. After the band is removed, the contents of the tube are poured into bleach. The extracted band is deposited in a 13 ml Sarstedt tube, which is then filled to the top with n-butanol saturated with 1M NaCl extract. If the amount of DNA is large, the extraction procedure may be repeated. After aspirating the butanol into a trap containing 5M NaOH to destroy ethidium, an approximately equal volume of 1M ammonium acetate and approximately two volumesof 95% ethanol are added to the DNA, which is then spun at 10k rpm for 5 minutes. The pellet is rinsed carefully with 70% ethanol, and dried with a swab SUBSTITUTE SHEET

{
WO 92/17486 2 _.~..~ 7 ~ ~ !1 Cl, 3 PCT/US92/02750 or lyophilizer.

9g. Preparation of Vector for Clonina 5 20 ug of vector is cut in a 200 gl reaction with 100 units of BstXI (New York Biolabs) at 50 C overnight in a well thermostated, circulating water bath. Potassium acetate solutions (5 and 20%) are prepared in 5W55 tubes as described above. 100 l of the digested vector is added to each tube 10 and spun for three hours, 50k rpm at 22 C. Under 300 nm UV
light, the desired band is observed to migrate 2/3 of the length of the tube. Forward trailing of the band indicates that the gradient is overloaded. The band is removed with a 1 ml syringe fitted with a 20 gauge needle. After adding 15 linear polyacrylamide and precipitating the plasmid by adding three volumes of ethanol, the plasmid is resuspended in 50 l of TE. Trial ligations are carried out with a constant amount of vector and increasing amounts of cDNA. Large scale ligation are carried out on the basis of these trial 20 ligations. Usually the entire cDNA prep requires 1-2 g of cut vector.

9h. Buffers 25 Loading Buffer: .5M LiCI, 10 mM Tris pH 7.5, 1 mM
EDTA .1% SDS.
Middle Wash Buffer: .15M LiCl, 10 mM Tris pH 7.5, 1 mM
EDTA .1% SDS.
RT1 Buffer: .25M Tris pH 8.8 (8.2,at 42'), .25M
30 KC1, 30 mM MgC12.
RT2 Buffer: .1M Tris pH 7.5, 25 mM MgC12, .5M
KC1, .25 mg/ml BSA, 50 mM
dithiothreitol (DTT).
lOX Low Salt: 60 mM Tris pH 7.5, 60 mM MgC12, 50 mM
35 NaCl, 2.5 mg/ml BSA 70 mM DME
lOX Ligation Additions: 1 mM ATP, 20 mM DTT, 1 mg/ml BSA 10 mM spermidine.
lOX Kinasing Buffer: .5M Tris pH 7.5, 10 mM ATP, 20 mM
DTT, 10 mM spermidine, 1 mg/ml BSA

SUBSTITUTE SHEET

WO 92/ ~ 74g5 CA 02107463 1995-08-17 pCY/US92/02750 ?U0"~ '4:03 36 loo mM MgCl2 9i. Transfection of cDNA encoding Ligands in Cos 7 Cells Cos 7 cells are split 1:5 into 100 mm plates in Dulbecco's modified Eagles medium (DME)/10% fetal calf serum (FCS), and allowed to grow overnight. 3 ml Tris/DME (0.039M
Tris, pH 7.4 in DME) containing 400 pg/ml DEAE-dextran (Sigma, D-9885) is prepared for each 100 mm plate of Cos 7 cells to be transfected. 10 pg of plasmid DNA preparation per plate is added. The medium is removed from the Cos-7 cells and the DNA/DEAE-dextran mixture is added. The cells are incubated for 4.5 hours. The medium is removed from the cells, and replaced with 3 ml of DME containing 2% fetal calf serum (FCS) and 0.1 mM chloroquine. The cells are incubated for one hour. After removing the chloroquine and replacing with 1.5 ml 20% glycerol in PBS, the cells are allowed to stand at room temperature for one minute. 3 ml Tris/DME is added, and the mixture is aspirated and washed two times with Tris/DME. 10 ml DME/10% FCS is added and the mixture is incubated overnight. The transfected Cos 7 cells are split 1:2 into fresh 100 mm plates with (DME)/10% FCS and allowed to grow.

9'i . Panning Procedure for Cos 7 cells Expressinct Ligand 1) Antibody-coated plates:

Bacteriological 100 mm plates are coated for 1.5 hours with rabbit anti-human placental alkaline phosphatase (Dako, California) diluted 1:500 in 10 ml of 50 mM Tris.HC1, pH 9.5.
The plates are washed three times with 0.15M NaCl, and incubated with 3 mg BSA/ml PBS overnight. The blocking solution is aspirated, and the plates are utilized immediately or frozen for later use.
2) Pannincs cel ls The medium from transfected Cos.7 cells is aspirated, SUBSTITUTE SHEET

WO 92/17486 ~ ~ ! ~ 4 I Pi '7 4 ~ 3 and 3 ml PBS/0.5 mM EDTA/0.02% sodium azide is added. The plates are incubated at 37 C for thirty minutes in order to detach the cells. The cells are triturated vigorously with a pasteur pipet and collected in a 15 ml centrifuge tube. The plate is washed with a further 2 ml PBS/EDTA/azide solution, which is then added to the centrifuge tube. After centrifuging at 200 xg for five minutes, the cells are resuspended in 3 ml of APtaq-flk-1 (F- 1AP21-4) or flk-2 (F-2AP26-0) supernatant from transfected NIH/3T3 cells (see Example 7.), and incubated for 1.5 hours on ice. The cells are centrifuged again at 200 xg for five minutes. The supernatant is aspirated, and the cells are resuspended in 3 ml PBS/EDTA/azide solution. The cell suspension is layered carefully on 3 ml PBS/EDTA/azide/2% Fico11, and centrifuged at 200 xg for four minutes. The supernatant is aspirated, and the cells are resuspended in 0.5 ml PBS/EDTA/azide solution. The cells are added to the antibody-coated plates containing 4 ml PBS/EDTA/azicle/5o FBS, and allowed to stand at room temperature one to three hours. Non-adhering cells are removed by washing gently two or three times with 3 ml PBS/5% FBS.

3) Hirt Supernatant:

0.4 ml 0.6% SDS and 10 mM EDTA are added to the panned plates, which are allowed to stand 20 minutes. The viscuous mixture is added by means of a pipet into a microfuge tube.
0.1 ml 5M NaCl is added to the tube, mixed, and chilled on ice for at least five hours. The tube is spun for four minutes, and the supernatant is removed carefully. The contents of the tube are extracted with phenol once, or, if the first interface is not clean, twice. Ten micrograms of linear polyacrylamide (or other carrier) is added, and 'the tube is filled to the top with ethanol. The resulting precipitate is resuspended in 0.1 ml water or TE. After adding 3 volumes of EtQH/NaOAc, the cells are reprecipitated and resuspended in 0.1 ml water or TE. The cDNA obtained is transfected into any suitable E. coli host by electroporation. Suitable hosts aredescribed in various SUBSTITUTE SHEET

e) 38 catalogs, and include MC1061/p3 or Electromax DH10B Cells of BRL Gibco. The cDNA is extracted by conventional methods.

The above panning procedure is repeated until a pure E.
coli clone bearing the cDNA as a unique plasmid recombinant capable of 'transfecting mammalian cells and yielding a positive panning assay is isolated. Normally, three repetitions are sufficient.

9k. Expression cloning of flkl or flk2 ligand by establishment of an autocrine loop Cells expressing flkl/fms or flk2/fms (Example 10) are transfected with 20-30 ug of a cDNA library from either flkl ligand or flk2 ligand expressing stromal cells, respectively.
The cDNA library is prepared as described above (a-h). The cells are co-transfected with 1 g pLTR neo cDNA. Following transfection the cells are passaged 1:2 and cultured in 800 g/m1 of G418 in Dulbecco s medium (DME) supplemented with 10% CS. Approximately 12 days later the colonies of cells are passaged and plated onto dishes coated with poly -D-lysine (1 mg/ml) and human fibronectin (15 g/ml). The culture medium is defined serum-free medium which is a mixture (3:1) of DME and Ham's F12 medium. The medium supplements are 8 mM NaHCO3r 15 mM HEPES pH 7.4, 3 mM
histidine, 4 M MnC1Z1 10 AM ethanolamine, 0.1 M selenous acid, 2AM hydrocortisone, 5 g/m1 transferrin, 500 Ag/ml bovine serum albumin/linoleic acid complex, and 20 Ag/ml insulin (Ref. Zhan, X, et al. Oncogene 1: 369-376,1987). The cultures are refed the next day and every 3 days until =the only cells capable of growing under the defined medium condition remain. The remaining colonies of cells are expanded and tested for the presence of the ligand by assaying for binding of APtag - flkl or APtag - flk2 to the cells (as described in Example 8). The DNA would be rescued from cells demonstrating the presence of the flkl or flk2 ligand and the sequence.

StJBS CITtJ TE SHE

WO 92/17486 PCr/US92/02750 .~:~:gr3 Example 10. Expression of Ligand cDPIA

The cDNA is sequenced, and expressed in a suitable host cell, such as a mammalian cell, preferably COS, CHO or NIH/3T3 cells. The presence of the ligand is confirmed by demonstrating binding of the ligand to APtag-flk2 fusion protein (see above).

Example 11. Chemical Cross Linking of Receptor and Ligand Cross linking experiments are performed on intact cells using a modification of the procedure described by Blume-Jensen et al et al., EMBO J., 10, 4121-4128 (1991). Cells are cultured in 100mm tissue culture plates to subconfluence and washed once with PBS-0.1% BSA.

To examine chemical cross linking of soluble receptor to membrane-bound ligand, stromal cells from the 2018 stroma], cell line are incubated with conditioned media (CM) from transfected 3T3 cells expressing the soluble receptor Flk2-APtag. Cross linking studies of soluble ligand to membrane bound receptor are performed by incubating conditioned media from 2018 cells with transfected 3T3 cells expressing a Flk2-fms fusion construct.
Binding is carried out for 2 hours either at room temperature with CM containing 0.02% sodium azide to prevent receptor internalization or at 4 C with-CM (and buffers) supplemented with sodium vanadate to prevent receptor dephosphorylation. Cells are washed twice with PBS-0.1a BSA
and four times with PBS.

Cross linking is performed in PBS containing 250 mM
disuccinimidyl suberate (DSS; Pierce) for 30 minutes at room temperature. The reaction is quenched with Tris-HCL pH7.4 to a final concentration of 50 mM.

.Ce1ls are solubiliied in solubilization buffer: 0.5%
Triton - X100, 0.5% deoxycholic acid, 20 mM Tris pH 7.4, 150 SlJBS'TITLI'fE SHEET

WO 92/171~ g~ ~ CA 02107463 1995-08-17 pC7'/U592/02750 mM NaCl, 10mM EDTA, 1mM PMFS, 50 mg/ml aprotinin, 2 mg/ml bestatin, 2 mg/ml pepstatin and 10mg/ml leupeptin. Lysed cells are immediately transferred to 1.5 ml Nalgene tubes and solubilized by rolling end to end for 45 minutes at 4 C.
5 Lysates are then centrifuged in a microfuge at 14,000g for 10 minutes. Solubilized cross linked receptor complexes are then retrieved from lysates by incubating supernatants with 10% (v/v) wheat germ lectin-Sepharose 6MB beads (Pharmacia) at 4 C for 2 hours or overnight.
Beads are washed once with Tris-buffered saline (TBS) and resuspended in 2X SDS-polyacrylamide nonreducing sample buffer. Bound complexes are eluted from the beads by heating at 95 C for 5 minutes. Samples are analyzed on 4-12%
gradient gels (NOVEX) under nonreducing and reducing conditions (0.35 M 2-mercaptoethanol) and then transferred to PVDF membranes for 2 hours using a Novex blotting apparatus.
Blots are blocked in TBS-3% BSA for 1 hour at room temperature followed by incubation with appropriate antibody.

Cross linked F1k2-APtag and f'lk2-fms receptors are detected using rabbit polyclonal antibodies raised against human alkaline phosphatase and fms protein, respectively.
The remainder of the procedure is carried out according to the instructions provided in'the ABC Kit (Pierce). The kit is based on the use of a biotinylated secondary antibody and avidin-biotinylated horseradish peroxidase complex for detection.
SUPPLEMENTAL ENABLEMENT

The invention as claimed is enabled in accordance with the above specification and readily available references and starting materials. Nevertheless, Applicants have deposited with the American Type Culture Collection, Rockville, Md., USA (ATCC) the cell lines listed below:

2018, ATCC accession no. CRL 10907, deposited SUBSTITUTE SHEET

October 30, 1991.

Fsp 62891, ATCC accession no. CRL 10935, deposited November 21, 1991.
F.thy 62891, ATCC accession no. CRL 10936, deposited November 21, 1991.

FL 62891, ATCC accession no. CRL 11005, deposited April 2, 1992.

These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the regulations thereunder (Budapest Treaty).
This assures maintenance of a viable culture for 30 years from date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Applicants and ATCC which assures unrestricted availability upon issuance of the pertinent U.S.
patent. Availability of the deposited strains is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

SUBSTITUTE SHEET

f,'~''1 ; ,I=

SEQUENCE LISTING
(1) GENERAL INFORMATION:

(i) APPLICANT: TRUSTEES OF PRINCETON UNIVERSITY

(ii) TITLE OF INVENTION: Totipotent Hematopoietic Stern Cell Receptors And Their Ligands (iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: IMCLONE SYSTEMS INCORPORATED
(B) STREET: 180 VARICK STREET
(C) CITY: NEW YORK
(D) STATE: NEW YORK
(E) COUNTRY: US
(F) ZIP: 10014 (v) 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 #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 02-APR-1992 (C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: FEIT, IRVING N.

(B) REGISTRATION NUMBER: 28,601 (C) REFERENCE/DOCKET NUMBER: LEM-3-PPPPT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 212-645-1405 (B) TELEFAX: 212-645-2054 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3453 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 31..3009 (ix) FEATURE:
(A) NAME/KEY: mat_peptide SUBSTITUTE SHEET

WO 92/17486 . ?WA-63 PCT/US92/02750 (B) LOCATION: 31..3006 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GCGGCCTGGC TACCGCGCGC TCCGGAGGCC ATG CGG GCG TTG GCG CAG CGC AGC
Met Arg Ala Leu Ala Gln Arg Ser GAC CGG CGG CTG CTG CTG CTT GTT GTT TTG TCA GTA ATG ATT CTT GAG
Asp Arg Arg Leu Leu Leu Leu Val Val Leu Ser Val Met Ile Leu Glu ACC GTT ACA AAC CAA GAC CTG CCT GTG ATC AAG TGT GTT TTA ATC AGT
Thr Val Thr Asn Gln Asp Leu Pro Val Ile Lys Cys Val Leu Ile Ser CAT GAG AAC AAT GGC TCA TCA GCG GGA AAG CCA TCA TCG TAC CGA ATG
His Glu Asn Asn Gly Ser Ser Ala Gly Lys Pro Ser Ser Tyr Arg Met GTG CGA GGA TCC CCA GAA GAC CTC CAG TGT ACC CCG AGG CGC CAG AGT
Val Arg Gly Ser Pro Glu Asp Leu Gln Cys Thr Pro Arg Arg Gin Ser GAA GGG ACG GTA TAT GAA GCG GCC ACC GTG GAG GTG GCC GAG TCT GGG
Glu Gly Thr Val Tyr Glu Ala Ala Thr Val Glu Val Ala Glu Ser Gly TCC ATC ACC CTG CAA GTG CAG CTC GCC ACC CCA GGG GAC CTT TCC TGC
Ser T1e Thr Leu Gln Val Gln Leu Ala Thr Pro Gly Asp Leu Ser Cys CTC TGG GTC TTT AAG CAC AGC TCC CTG GGC TGC CAG CCG CAC TTT GAT
Leu Trp Val Phe Lys His Ser Ser Leu Gly Cys Gln Pro His Phe Asp TTA CAA AAC AGA GGA ATC GTT TCC ATG GCC ATC TTG AAC GTG ACA GAG
Leu Gln Asn Arg Gly Ile Val Ser Met Ala Ile Leu Asn Val Thr Glu ACC CAG GCA GGA GAA TAC CTA CTC CAT ATT CAG AGC GAA CGC GCC AAC
Thr Gln Ala Gly Glu Tyr Leu Leu His :C1e Gln Ser Glu Arg Ala Asn TAC ACA GTA CTG TTC ACA GTG AAT GTA AGA GAT ACA CAG CTG TAT GTG
Tyr Thr Val Leu Phe Thr Val Asn Val Arg Asp Thr Gln Leu Tyr Val .155 160 165 CTA AGG AGA CCT TAC TTT AGG AAG ATG GAA AAC CAG GAT GCA CTG CTC
Leu Arg Arg Pro Tyr Phe Arg Lys Met Glu Asn Gln Asp Ala Leu Leu TGC ATC TCC GAG GGT GTT CCG GAG CCC ACT GTG GAG TGG GTG CTC TGC
Cys Ile Ser Glu Gly Val Pro Glu Pro Thr Val Glu Trp Val Leu Cys S11~STI"ClJ TE SHEET

AGC TCC CAC AGG GAA AGC TGT AAA GAA GAA GGC CCT GCT GTT GTC AGA
Ser Ser His Arg Glu Ser Cys Lys Glu Glu Gly Pro Ala Val. Val Arg AAG GAG GAA AAG GTA CTT CAT GAG TTG TTC GGA ACA GAC ATC AGA TGC
Lys Glu Glu Lys Val Leu His Glu Leu Phe Gly Thr Asp Ile Arg Cys TGT GCT AGA AAT GCA CTG GGC CGC GAA TGC ACC AAG CTG TTC ACC ATA
Cys Ala Arg Asn Ala Leu Gly Arg Glu Cys Thr Lys Leu Phe Thr Ile GAT CTA AAC CAG GCT CCT CAG AGC ACA CTG CCC CAG TTA TTC CTG AAA
Asp Leu Asn Gln Ala Pro Gln Ser Thr Leu Pro Gln Leu Phe Leu Lys GTG GGG GAA CCC TTG TGG ATC AGG TGT AAG GCC ATC CAT GTG AAC CAT
Val Gly Glu Pro Leu Trp Ile Arg Cys Lys Ala Ile His Val Asn His GGA TTC GGG CTC ACC TGG GAG CTG GAA GAC AAA GCC CTG GAG GAG GGC
Gly Phe Gly Leu Thr Trp Glu Leu Glu Asp Lys Ala Leu Glu Glu Gly AGC TAC TTT GAG ATG AGT ACC TAC TCC ACA AAC AGG ACC ATG ATT CGG
Ser Tyr Phe Glu Me't Ser Thr Tyr Ser Thr Asn Arg Thr Met Ile Arg ATT CTC TTG GCC TTT GTG TCT TCC GTG GGA AGG AAC GAC ACC GGA TAT
Ile Leu Leu Ala Phe Val Ser Ser Val Gly Arg Asn Asp Thr Gly Tyr TAC ACC TGC TCT TCC TCA AAG CAC CCC AGC CAG TCA GCG TTG GTG ACC
Tyr Thr Cys Ser Ser Ser Lys His Pro Ser Gin Ser Ala Leu Val Thr ATC CTA GAA AAA GGG TTT ATA AAC GCT ACC AGC TCG CAA GAA GAG TAT
Ile Leu Glu Lys Gly Phe Ile Asn Ala Thr Ser Ser Gln Glu Glu Tyr GAA ATT GAC CCG TAC GAA AAG TTC TGC TTC TCA GTC AGG TTT AAA GCG
Glu Ile Asp Pro-Tyr Glu Lys=Phe Cys Phe Ser Val Arg Phe Lys Ala TAC CCA CGA ATC CGA TGC ACG TGG ATC TTC TCT CAA GCC TCA TTT CCT
Tyr Pro Arg Ile Arg Cys Thr Trp Ile Phe Ser Gln Ala Ser Phe Pro TGT GAA CAG AGA GGC CTG GAG GAT GGG TAC AGC ATA TCT AAA TTT TGC
Cys Glu G.ln Arg Gly Leu Glu Asp Gly Tyr Ser Ile Ser Lys Phe Cys GAT CAT AAG AAC AAG CCA GGA GAG TAC ATA TTC TAT GCA GAA AAT GAT
Asp His Lys Asn Lys Pro Gly Glu Tyr Ile Phe Tyr Ala Glu Asn Asp SUBS Ti"9'UZ'E SHEET

WO 92i17486 Pcrius92i02750 GAC GCC CAG TTC ACC AAA ATG TTC ACG CTG AAT ATA AGA AAG AAA CCT
Asp Ala Gln Phe Thr Lys Met Phe Thr Leu Asn Ile Arg Lys Lys Pro CAA GTG CTA GCA AAT GCC TCA GCC AGC CAG GCG TCC TGT TCC TCT GAT
Gln Val Leu Ala Asn Ala Ser Ala Ser Gln Ala Ser Cys Ser Ser Asp GGC TAC CCG CTA CCC TCT TGG ACC TGG AAG AAG TGT TCG GAC AAA TCT
Gly Tyr Pro Leu Pro Ser Trp Thr Trp Lys Lys Cys Ser Asp Lys Ser CCC AAT TGC ACG GAG GAA ATC CCA GAA GGA GTT TGG AAT AAA AAG GCT
Pro Asn Cys Thr Glu Glu Ile Pro Glu Gly Val Trp Asn Lys Lys Ala AAC AGA AAA GTG TTT GGC CAG TGG GTG TCG AGC AGT ACT CTA AAT ATG
Asn Arg Lys Val Phe Gly Gln Trp Val Ser Ser Ser Thr Leu Asn Met AGT GAG GCC GGG AAA GGG CTT CTG GTC AAA TGC TGT GCG TAC AAT TCT
Ser Glu Ala Gly Lys Gly Leu Leu Val Lys Cys Cys Ala Tyr Asn Ser ATG GGC ACG TCT TGC GAA ACC ATC TTT TTA AAC TCA CCA GGC CCC TTC
Met Gly Thr Ser Cys Glu Thr Ile Phe Leu Asn Ser Pro Gly Pro Phe CCT TTC ATC CAA GAC AAC ATC TCC TTC TAT GCG ACC ATT GGG CTC TGT
Pro Phe Ile Gln Asp Asn I1e Ser Phe Tyr Ala Thr Ile Gly Leu Cys CTC.CCC TTC ATT GTT GTT CTC ATT GTG TTG ATC TGC CAC AAA TAC AAA
Leu Pro Phe Ile Val Val Leu Ile Val Leu Ile Cys His Lys Tyr Lys AAG CAA TTT AGG TAC GAG AGT CAG CTG CAG ATG ATC CAG GTG ACT GGC
Lys Gln Phe Arg Tyr Glu Ser Gln Leu Gln Met Ile Gln Val Thr Gly CCC CTG GAT AAC GAG TAC TTC TAC GTT GAC TTC AGG GAC TAT GAA TAT
Pro Leu Asp Asn Glu Tyr Phe Tyr Val Asp Phe Arg Asp Tyr Glu Tyr GAC CTT AAG TGG GAG TTC CCG AGA GAG AAC TTA GAG TTT GGG AAG GTC
Asp Leu Lys Trp Glu Phe Pro Arg Glu Asi'i Leu Glu Phe Gly Lys Val CTG GGG TCT GGC GCT TTC GGG AGG GTG ATG AAC GCC ACG GCC TAT GGC
Leu Gly Ser Gly Ala Phe Gly Arg Val Met Asn Ala Thr Ala Tyr Gly ATT AGT AAA ACG GGA GTC TCA ATT CAG GTG GCG GTG AAG ATG CTA AAA
Ile Ser Lys Thr Gly Val Ser Ile Gln Val Ala Val Lys Met Leu Lys SUBSTITUTE SHEET

w6 92/17486 .PCT/US92/02750 CA 02107463 1995-08-17 .?3'S
/F.. 46 GAG AAA GCT GAC AGC TGT GAA AAA GAA GCT CTC ATG TCG GAG CTC AAA
Glu Lys Ala Asp Ser Cys Glu Lys Glu Ala Leu Met Ser Glu Leu Lys ATG ATG ACC CAC CTG GGA CAC CAT GAC AAC ATC GTG AAT CTG CTG GGG
Met Met Thr His Leu Gly His His Asp Asn Ile Val Asn Leu Leu Gly GCA TGC ACA CTG TCA GGG CCA GTG TAC TTG ATT TTT GAA TAT TGT TGC
Ala Cys Thr Leu Ser Gly Pro Val Tyr Leu Ile Phe Glu Tyr Cys Cys TAT GGT GAC CTC CTC AAC TAC CTA AGA AGT AAA AGA GAG AAG TTT CAC
Tyr Gly Asp Leu Leu Asn Tyr Leu Arg Ser Lys Arg Glu Lys Phe His AGG ACA TGG ACA GAG ATT TTT AAG GAA CAT AAT TTC AGT TCT TAC CCT
Arg Thr Trp Thr Glu Ile Phe Lys Glu His Asn Phe Ser Ser Tyr Pro ACT TTC CAG GCA CAT TCA AAT TCC AGC ATG CCT GGT TCA CGA GAA GTT
Thr Phe Gln Ala His Ser Asn Ser Ser Met Pro Gly Ser Arg Glu Val CAG TTA CAC CCG CCC TTG GAT CAG CTC TCA GGG TTC AAT GGG AAT TCA
G1n Leu His Pro Pro Leu Asp G1n Leu Ser Gly Phe Asn Gly Asn Ser ATT CAT TCT GAA GAT GAG ATT GAA TAT GAA AAC CAG AAG AGG CTG GCA
Ile His Ser Glu Asp Glu Ile Glu Tyr Glu Asn Gln Lys Arg Leu Ala GAA GAA GAG GAG GAA GAT TTG AAC GTG CTG ACG TTT GAA GAC CTC CTT
Glu Glu Glu Glu Glu Asp Leu Asn Val Leu Thr Phe Glu Asp Leu Leu TGC TTT GCG TAC CAA GTG GCC AAA GGC ATG GAA TTC CTG GAG TTC AAG
Cys Phe Ala Tyr Gin Val Ala Lys Gly Met Glu Phe Leu Glu Phe Lys TCG TGT GTC CAC AGA GAC CTG GCA GCC AGG AAT GTG TTG GTC ACC CAC
Ser Cys Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Thr His GGG AAG GTG GTG AAG ATC TGT GAC TTT GGA CTG GCC CGA GAC ATC CTG
Gly Lys Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Leu AGC GAC TCC AGC TAC GTC GTC AGG GGC AAC GCA CGG CTG CCG GTG AAG
Ser Asp Ser Ser Tyr Val Val Arg Gly Asn Ala Arg Leu Pro Val Lys TGG ATG GCA CCC GAG AGC TTA TTT GAA GGG ATC TAC ACA ATC AAG AGT
Trp Met Ala Pro Glu Ser Leu Phe Glu Gly Ile Tyr Thr Ile Lys Ser SUBSTITUTE SHEET

WO 92/17486 ~ ~ ~ ~ rE ~ '~ PCT/US92/02750 GAC GTC TGG TCC TAC GGC ATC CTT CTC TGG GAG ATA TTT TCA CTG GGT
Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile Phe Ser Leu Gly GTG AAC CCT TAC CCT GGC ATT CCT GTC GAC GCT AAC TTC TAT AAA CTG
Val Asn Pro Tyr Pro Gly Ile Pro Val Asp Ala Asn Phe Tyr Lys Leu ATT CAG AGT GGA TTT AAA ATG GAG CAG CCA TTC TAT GCC ACA GAA GGG
Ile G1n Ser Gly Phe Lys Met Glu Gln Pro Phe Tyr Ala Thr Glu Gly ATA TAC TTT GTA ATG CAA TCC TGC TGG GCT TTT GAC TCA AGG AAG CGG
I1e Tyr Phe Val Met Gln Ser Cys Trp Ala Phe Asp Ser Arg Lys Arg CCA TCC TTC CCC AAC CTG ACT TCA TTT TTA GGA TGT CAG CTG GCA GAG
Pro Ser Phe Pro Asn Leu Thr Ser Phe Leu Gly Cys Gln Leu Ala Glu GCA GAA GAA GCA TGT ATC AGA ACA TCC ATC CAT CTA CCA AAA CAG GCG
Ala G1u Glu Ala Cys Ile Arg Thr Ser Ile His Leu Pro Lys Gln Ala GCC CCT CAG CAG AGA GGC GGG CTC AGA GCC CAG TCG CCA CAG CGC CAG
Ala Pro G1n G1n Arg Gly Gly Leu Arg Ala Gln Ser Pro Gln Arg Gln GTG AAG ATT CAC AGA GAA AGA AGT TAGCGAGGAG GCCTTGGACC CCGCCACCCT
Val Lys Ile His Arg Glu Arg Ser AGCAGGCTGT AGACCGCAGA GCCAAGATTA GCCTCGCCTC TGAGGAAGCG CCCTACAGCG
CGTTGCTTCG CTGGACTTTT CTCTAGATGC TGTCTGCCAT TACTCCAAAG TGACTTCTAT
AAAATCAAAC CTCTCCTCGC ACAGGCGGGA GAGCCAATAA TGAGACTTGT TGGTGAGCCC
GCCTACCCTG GGGGCCTTTC CACGAGCTTG AGGGGAAAGC CATGTATCTG AAATATAGTA
TATTCTTGTA AATACGTGAA ACAAACCAAA CCCGTTTTTT GCTAAGGGAA AGCTAAATAT
GATTTTTAAA AATCTATGTT TTAAAATACT ATGTAACTTT TTCATCTATT TAGTGATATA
TTTTATGGAT GGAAATAAAC TTTCTACTGT Ai9.AAAAA
(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 992 amino acids SUBS i ITUTE SHEET

WO 92/17486 CA 02107463 1995-08-17 PCr/US92/02750 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Arg Ala Leu Ala Gln Arg Ser Asp Arg Arg :Leu Leu Leu Leu Val Val Leu Ser Val Met Ile Leu Glu Thr Val Thr Asn Gln Asp Leu Pro Val Ile Lys Cys Val Leu Ile Ser His Glu Asn Asn Gly Ser Ser Ala Gly Lys Pro Ser Ser Tyr Arg Met Val Arg Gly Ser Pro Glu Asp Leu Gln Cys Thr Pro Arg Arg Gln Ser Glu Gly Thr Val Tyr Glu Ala Ala Thr Val Glu Val Ala Glu Ser Gly Ser Ile Thr Leu Gln Val Gln Leu Ala Thr Pro Gly Asp Leu Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu Gly Cys Gln Pro His Phe Asp Leu Gln Asn Arg Gly Ile Val Ser Met Ala Ile Leu Asn Val Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu His Ile Gln Ser Glu Arg Ala Asn Tyr Thr Val Leu Phe Thr Val Asn Va1 Arg Asp Thr G1n Leu Tyr Val Leu Arg Arg Pro Tyr Phe Arg Lys Met Glu Asn Gln Asp Ala Leu Leu Cys Ile Ser G1u Gly Val Pro Glu Pro Thr Val Glu Trp Va1 Leu Cys Ser Ser His Arg Glu Ser Cys Lys Glu Glu Gly Pro Ala Val Val Arg Lys G1u Glu Lys Val Leu His Glu Leu Phe Gly Thr Asp Ile Arg Cys Cys Ala Arg Asn Ala Leu Gly Arg Glu Cys Thr Lys Leu Phe Thr I1e Asp Leu Asn Gln Ala Pro Gin Ser Thr Leu Pro Gln Leu Phe Leu Lys Val Gly Glu Pro Leu Trp Ile Arg a11BSTllCUli'E SHEET

P~') Cys Lys Ala Ile His Val Asn His Gly Phe Gly Leu Thr Trp Glu Leu Glu Asp Lys Ala Leu Glu Glu Gly Ser Tyr Phe Glu Met Ser Thr Tyr Ser Thr Asn Arg Thr Met I1e Arg Ile Leu Leu Ala Phe Val Ser Ser 30 : 310 315 320 Val Gly Arg Asn Asp Thr Gly Tyr Tyr Thr Cys Ser Ser Ser Lys His Pro Ser Gln Ser Ala Leu Val Thr Ile Leu Glu Lys Gly Phe 21e Asn Ala Thr Ser Ser Gln Glu Glu Tyr Glu Ile Asp Pro Tyr Glu Lys Phe Cys Phe Ser Val Arg Phe Lys Ala Tyr Pro Arg Ile Arg Cys Thr Trp I1e Phe Ser Gln Ala Ser Phe Pro Cys Glu Gln Arg Gly Leu Glu Asp Gly Tyr Ser 21e Ser Lys Phe Cys Asp His Lys Asn Lys Pro Gly Glu Tyr Ile Phe Tyr Ala Glu Asn Asp Asp Ala Gln Phe Thr Lys Met Phe Thr Leu Asn Ile Arg Lys Lys Pro Gin Val Leu Ala Asn Ala Ser Ala Ser G1n Ala Ser Cys Ser Ser Asp Gly Tyr Pro Leu Pro Ser Trp Thr Trp Lys Lys Cys Ser Asp Lys Ser Pro Asn Cys Thr Glu Glu Ile Pro Glu Gly Val Trp Asn Lys Lys Ala Asn Arg Lys Val Phe Gly Gln Trp Val Ser Ser Ser Thr Leu Asn Met Ser Glu Ala Gly Lys Gly Leu Leu Val Lys Cys Cys Ala Tyr Asn Ser Met Gly Thr Ser Cys Glu Thr I1e Phe Leu Asn Ser Pro Gly Pro Phe Pro Phe Ile G1n Asp Asn Ile Ser Phe Tyr Ala Thr Ile Gly Leu Cys Leu Pro Phe Ile Val Val Leu Ile Val Leu Ile Cys His Lys Tyr Lys Lys G1n Phe Arg Tyr Glu Ser Gln SUBSTITUTE SHEET

~-~-iy7 WO 92/17486 CA 02107463 1995-08-17 P~ d/ VS92/02750 Leu Gln Met Ile Gln Val Thr Gly Pro Leu Asp Asn Glu Tyr Phe Tyr Val Asp Phe Arg Asp Tyr Glu Tyr Asp Leu Lys Trp Glu Phe Pro Arg Glu Asn Leu Glu Phe Gly Lys Val Leu Gly Ser Gly Ala Phe Gly Arg Val Met Asn Ala Thr Ala Tyr Gly Ile Ser Lys Thr Gly Val Ser I1e Gln Val Ala Val Lys Met Leu Lys Glu Lys Ala Asp Ser Cys G1u Lys Glu Ala Leu Met Ser Glu Leu Lys Met Met Thr His Leu Gly His His Asp Asn Ile Val Asn Leu I,eu Gly Ala Cys Thr Leu Ser Gly Pro Val Tyr Leu Ile Phe Glu Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Tyr Leu Arg Ser Lys Arg Glu Lys Phe His Arg Thr Trp Thr Glu I1e Phe Lys Glu His Asn Phe Ser Ser Tyr Pro Thr Phe G1n Ala His Ser Asn Ser Ser Met Pro Gly Ser Arg Glu Val Gln Leu His Pro Pro Leu Asp Gln Leu Ser Gly Phe Asn Gly Asn Ser Ile His Ser Glu Asp Glu Ile Glu Tyr Glu Asn Gin Lys Arg Leu Ala Glu Glu Glu Glu Glu Asp Leu Asn Va1 Leu Thr Phe Glu Asp Leu Leu Cys Phe Ala Tyr G1n Val Ala Lys Gly Met Glu Phe Leu Glu Phe Lys Ser Cys Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Thr His Gly Lys Val Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Leu Ser Asp Ser Ser Tyr Val Val Arg Gly Asn Ala Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ser Leu Phe Glu Gly I1e Tyr Thr Ile Lys Ser Asp Val Trp Ser Tyr Gly Ile Leu SUBSTITUTE SHEET

PCI'/US92/02750 Leu Trp Glu Ile Phe Ser Leu Gly Val Asn Pro Tyr Pro Gly Ile Pro Va1 Asp Ala Asn Phe Tyr Lys Leu Ile Gln Ser Gly Phe Lys Met Glu G1n Pro Phe Tyr Ala Thr Glu Gly Ile Tyr Phe Val Met Gln Ser Cys Trp Ala Phe Asp Ser Arg Lys Arg Pro Ser Phe Pro Asn Leu Thr Ser Phe Leu Gly Cys Gln Leu Ala Glu Ala Glu Glu Ala Cys Ile Arg Thr Ser Ile His Leu Pro Lys Gln Ala Ala Pro Gln Gln Arg Gly Gly Leu Arg Ala Gln Ser Pro Gln Arg G1n Val Lys Ile His Arg Glu Arg Ser (2) INFOR'','ATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 332 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..332 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

AAC AAT GAT TCA TCA GTG GGG AAG TCA TCA TCA TAT CCC ATG GTA TCA
Asn Asn Asp Ser Ser Val Gly Lys Ser Ser Ser Tyr Pro Met Val Ser GAA TCC CCG GAA GAC CTC GGG TGT GCG TTG AGA CCC CAG AGC TCA GGG
Glu Ser Pro Glu Asp Leu Gly Cys Ala Leu Arg Pro G1n Ser Ser Gly ACA GTG TAC GAA GCT GCC GCT GTG GAA GTG GAT GTA TCT GCT TCC ATC
Thr Val Tyr Glu Ala Ala Ala Val Glu Val Asp Val Ser Ala Ser Ile ACA CTG CAA GTG CTG GTC GAT GCC CCA GGG AAC ATT TCC TGT CTC TGG
Thr Leu Gln Val Leu Val Asp Ala Pro Gly Asn Ile Ser Cys Leu Trp SUBSTITUTE SHEET

o CA 02107463 1995-08-17 Val Phe Lys His Ser Ser Leu Asn Cys Gln Pro His Phe Asp Leu Gln AAC AGA GGA GTT GTT TCC ATG GTC ATT TTG AAA ATG ACA GAA ACC CAA
Asn Arg Gly Val Val Ser Met Val Ile Leu Lys Met Thr Glu Thr Gln GCT GGA GAA TAC CTA CTT TTT ATT CAG AGT GAA GCT ACC AAT TA
Ala Gly Glu Tyr Leu Leu Phe Ile Gln Ser Glu Ala Thr Asn (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 110 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Asn Asn Asp Ser Ser Val Gly Lys Ser Ser Ser Tyr Pro Met Val Ser Glu Ser Pro Glu Asp Leu Gly Cys Ala Leu Arg Pro Gln Ser Ser Gly Thr Val Tyr Glu Ala Ala Ala Val G1u Val Asp Val Ser Ala Ser Ile Thr Leu Gln Val Leu Val Asp Ala Pro Gly Asn Ile Ser Cys Leu Trp Val Phe Lys His Ser Ser Leu Asn Cys Gin Pro His Phe Asp Leu G1n Asn Arg Gly Val Val Ser Met Val Ile Leu Lys Met Thr Glu Thr Gln Ala Gly Glu Tyr Leu Leu Phe Ile Gln Ser Glu Ala Thr Asn (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 284 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS

S1JBS1'ITl1TE SHEET

'~J n WO 92/17486 : ~3 ~-<,,. =

(B) LOCATION: 1..282 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GAT CAA ATC TCA GGC TTC ATG GAA TTC ATT CAC TCT GAA GAT GAA ATT
Asp Gln Ile Ser Gly Phe Met Glu Phe Ile His Ser Glu Asp Glu Ile GAA TAT GAA AAC CAA AAA AAG AGG CTG GAA GAA GAG GAG GAC TTG AAT
Glu Tyr Glu Asn Gln Lys Lys Arg Leu Glu Glu Glu Glu Asp Leu Asn GTG CTT ACA TTT GAA GAT CTT CTT TGC TTT GCA TAT CAA GTT GCC AAA
Val Leu Thr Phe G1u Asp Leu Leu Cys Phe Ala Tyr Gln Val Ala Lys GGA ATG GAA TTT AAG TCG TGT GTT CAC AGA GAC CTG GCC GCC AGG AAC
Gly Met Glu Phe Lys Ser Cys Val His Arg Asp Leu Ala Ala Arg Asn GTG CTT GTC ACC CAC GGG AAA GTG GTG AAG ATA TGT GAC TTT GGA TTG
Val Leu Val Thr His Gly Lys Val Val Lys I].e Cys Asp Phe Gly Leu GCT CGA GAT ATC ATG AGT GAT TCC GGC TAT GTT GTC AGG CAA
Ala Arg Asp Ile Met Ser Asp Ser Giy Tyr Val Val Arg Gln (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 94 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Asp Gln Ile Ser Gly Phe Met Glu Phe Ile His Ser Glu Asp Glu Ile Glu Tyr Glu Asn Gln Lys Lys Arg Leu Glu Glu Glu Glu Asp Leu Asn Val Leu Thr Phe Glu Asp Leu Leu Cys Phe Ala Tyr Gin Val Ala Lys G1y Met Glu Phe Lys Ser Cys Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Va1 Thr His Gly Lys Val Val Lys Ile Cys Asp Phe Gly Leu SUBSTITUTE SHEET

(.~ J. U 4, i>i 1") J.

WO 92/17486 PCf/US92/02750 Ala Arg Asp Ile Met Ser Asp Ser Gly Tyr Val Val Arg Gln (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5406 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 208..4311 (ix) FEATURE:
(A) NAME/KEY: mat_peptide (B) LOCATION: 208..4308 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

CTGTGTCCCG CAGCCGGATA ACCTGGCTGA CCCGATTCCG CGGACACCCG TGCAGCCGCG
GCTGGAGCCA GGGCGCCGGT GCCCGCGCTC TCCCCGGTCT TGCGCTGCGG GGGCCGATAC
CGCCTCTGTG ACTTCTTTGC GGGCCAGGGA CGGAGAAGGA GTCTGTGCCT GAGAAACTGG
GCTCTGTGCC CAGGCGCGAG GTGCAGG ATG GAG AGC AAG GGC CTG CTA GCT
Met Glu Ser Lys Gly Leu Leu Ala GTC GCT CTG TGG TTC TGC GTG GAG ACC CGA GCC GCC TCT GTG GGT TTG
Val Ala Leu Trp Phe Cys Val Glu Thr Arg Ala Ala Ser Val Gly Leu CCT GGC GAT TTT CTC CAT CCC CCC AAG CTC AGC ACA CAG AAA GAC ATA
Pro Gly Asp Phe Leu His Pro Pro Lys Leu Ser Thr Gln Lys Asp Ile CTG ACA ATT TTG GCA AAT ACA ACC CTT CAG ATT ACT TGC AGG GGA CAG
Leu Thr Ile Leu Ala Asn Thr Thr Leu Gln Ile Thr Cys Arg Gly Gln CGG GAC CTG GAC TGG CTT TGG CCC AAT GCT CAG CGT GAT TCT GAG GAA
Arg Asp Leu Asp Trp Leu Trp Pro Asn Ala Gln Arg Asp Ser Glu Glu AGG GTA TTG GTG ACT GAA TGC GGC GGT GGT GAC AGT ATC TTC TGC AAA
Arg Val Leu Val Thr Glu Cys Gly Gly Gly Asp Ser Ile Phe Cys Lys SUBSTITUTE SHEET

"1,~.~~

wo 92/17486 PCT/uS92/02750 ACA CTC ACC ATT CCC AGG GTG GTT GGA AAT GAT ACT GGA GCC TAC AAG
Thr Leu Thr Ile Pro Arg Val Val Gly Asn Asp Thr Gly Ala Tyr Lys TGC TCG TAC CGG GAC GTC GAC ATA GCC TCC ACT GTT TAT GTC TAT GTT
Cys Ser Tyr Arg Asp Val Asp I1e Ala Ser Thr 'Val Tyr Val Tyr Val CGA GAT TAC AGA TCA CCA TTC ATC GCC TCT GTC AGT GAC CAG CAT GGC
Arg Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp Gln His Gly ATC GTG TAC ATC ACC GAG AAC AAG AAC AAA ACT GTG GTG ATC CCC TGC
I1e Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr 'Val Val I1e Pro Cys CGA GGG TCG ATT TCA AAC CTC AAT GTG TCT CTT TGC GCT AGG TAT CCA
Arg Gly Ser Ile Ser Asn Leu Asn Va1 Ser Leu Cys Ala Arg Tyr Pro GAA AAG AGA TTT GTT CCG GAT GGA AAC AGA ATT TCC TGG GAC AGC GAG
Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Glu ATA GGC TTT ACT CTC CCC AGT TAC ATG ATC AGC TAT GCC GGC ATG GTC
Ile Gly Phe Thr Leu Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Va1 TTC TGT GAG GCA AAG ATC AAT GAT GAA ACC TAT CAG TCT ATC ATG TAC
Phe Cys Glu Ala Lys Ile Asn Asp Glu Thr Tyr Gln Ser I1e Met Tyr ATA GTT GTG GTT GTA GGA TAT AGG ATT TAT GAT GTG ATT CTG AGC CCC
Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Ile Leu Ser Pro CCG CAT GAA ATT GAG CTA TCT GCC GGA GAA AAA CTT GTC TTA AAT TGT
Pro His Glu Ile Giu Leu Ser Ala Gly Glu Lys Leu Vai Leu Asn Cys ACA GCG AGA ACA GAG CTC AAT GTG GGG CTT GAT TTC ACC TGG CAC TCT
Thr Ala Arg Thr G1u Leu Asn Val Gly Leu Asp Phe Thr Trp His Ser CCA CCT TCA AAG TCT CAT CAT AAG AAG ATT GTA AAC CGG GAT GTG AAA
Pro Pro Ser Lys Ser His His Lys Lys Ile Va1 Asn Arg Asp Val Lys CCC TTT CCT GGG ACT GTG GCG AAG ATG TTT TTG AGC ACC TTG ACA ATA
Pro Phe Pro Gly Thr Val Ala Lys Met Phe Leu Ser Thr Leu Thr Ile GAA AGT GTG ACC AAG AGT GAC CAA GGG GAA TAC ACC TGT GTA GCG TCC
Glu Ser Va1 Thr Lys Ser Asp Gin Gly Glu Tyr Thr Cys Val Ala Ser SUBSTITUTE SHEET

'{.r ~. t~ ~ ~~= ~ 3 CA 02107463 1995-08-17 ;~:=-;, AGT GGA CGG ATG ATC AAG AGA AAT AGA ACA TTT GTC CGA GTT CAC ACA
Ser Gly Arg Met Ile Lys Arg Asn Arg Thr Phe Val Arg Val His Thr AAG CCT TTT ATT GCT TTC GGT AGT GGG ATG AAA TCT TTG GTG GAA GCC
Lys Pro Phe Ile Ala Phe Gly Ser Gly Met Lys Ser Leu Val Glu Ala ACA GTG GGC AGT CAA GTC CGA ATC CCT GTG AAG TAT CTC AGT TAC CCA
Thr Val Gly Ser Gln Val Arg I1e Pro Val Lys Tyr Leu Ser Tyr Pro GCT CCT GAT ATC AAA TGG TAC AGA AAT GGA AGG CCC ATT GAG TCC AAC
Ala Pro Asp Ile Lys Trp Tyr Arg Asn Gly Arg Pro Ile Glu Ser Asn TAC ACA ATG ATT GTT GGC GAT GAA CTC ACC ATC ATG GAA GTG ACT GAA
Tyr Thr Met Ile Val Gly Asp Glu Leu Thr Ile Met Glu Val Thr Glu AGA GAT GCA GGA AAC TAC ACG GTC ATC CTC ACC AAC CCC ATT TCA ATG
Arg Asp Ala Gly Asn Tyr Thr Val xle Leu Thr Asn Pro Ile Ser Met GAG AAA CAG AGC CAC ATG GTC TCT CTG GTT GTG AAT GTC CCA CCC CAG
Glu Lys Gln Ser His Met Val Ser Leu Val Val Asn Val Pro Pro Gln ATC GGT GAG AAA GCC TTG ATC TCG CCT ATG GAT TCC TAC CAG TAT GGG
Ile Gly Glu Lys Ala Leu Ile Ser Pro Met Asp Ser Tyr Gln Tyr Gly ACC ATG CAG ACA TTG ACA TGC ACA GTC TAC GCC AAC CCT CCC CTG CAC
Thr Met G1n Thr Leu Thr Cys Thr Val Tyr Ala Asn Pro Pro Leu His CAC ATC CAG TGG TAC TGG CAG CTA GAA GAA GCC TGC TCC TAC AGA CCC
His Ile Gln Trp Tyr Trp G1n Leu Glu Glu Ala Cys Ser Tyr Arg Pro GGC CAA ACA AGC CCG TAT GCT TGT AAA GAA TGG AGA CAC GTG GAG GAT
Gly Gln Thr Ser Pro Tyr Ala Cys Lys Glu Trp Arg His Val Glu Asp TTC CAG GGG GGA AAC AAG ATC GAA GTC ACC AAA AAC CAA TAT GCC CTG
Phe Gln Gly Gly Asn Lys Ile Glu Val Thr Lys Asn Gln Tyr Ala Leu ATT GAA GGA AAA AAC AAA ACT GTA AGT ACG CTG GTC ATC CAA GCT GCC
Ile Glu Gly Lys Asn Lys Thr Val Ser Thr Leu Va1 Ile G1n Ala Ala AAC GTG TCA GCG TTG TAC AAA TGT GAA GCC ATC AAC AAA GCG GGA CGA
Asn Val Ser Ala Leu Tyr Lys Cys Glu Ala Ile Asn Lys Ala Gly Arg SUBSTITUTE SHEET

WO 92/17486 (: 1. PCT/US92/02750 GGA GAG AGG GTC ATC TCC TTC CAT GTG ATC AGG GGT CCT GAA ATT ACT
Gly Glu Arg Val Ile Ser Phe His Val Ile Arg Gly Pro Glu Ile Thr GTG CAA CCT GCT GCC CAG CCA ACT GAG CAG GAG AGT GTG TCC CTG TTG
Val Gln Pro Ala Ala Gln Pro Thr Glu Gln Glu Ser Val Ser Leu Leu TGC ACT GCA GAC AGA AAT ACG TTT GAG AAC CTC ACG TGG TAC AAG CTT
Cys Thr Ala Asp Arg Asn Thr Phe G1u Asn Leu Thr Trp Tyr Lys Leu GGC TCA CAG GCA ACA TCG GTC CAC ATG GGC GAA TCA CTC ACA CCA GTT
Gly Ser Gln Ala Thr Ser Val His Met Gly Glu Ser Leu Thr Pro Va1 TGC AAG AAC TTG GAT GCT CTT TGG AAA CTG AAT GGC ACC ATG TTT TCT
Cys Lys Asn Leu Asp Ala Leu Trp Lys Leu Asn G1y Thr Met Phe Ser AAC AGC ACA AAT GAC ATC TTG ATT GTG GCA TTT CAG AAT GCC TCT CTG
Asn Ser Thr Asn Asp Ile Leu Ile Val Ala Phe Gln Asn Ala Ser Leu CAG GAC CAA GGC GAC TAT GTT TGC TCT GCT CAA GAT AAG AAG ACC AAG
Gln Asp Gln Gly Asp Tyr Val Cys Ser Ala Gln Asp Lys Lys Thr Lys AAA AGA CAT TGC CTG GTC AAA CAG CTC ATC ATC CTA GAG CGC ATG GCA
Lys Arg His Cys Leu Val Lys Gin Leu Ile Ile Leu Glu Arg Met Ala CCC ATG ATC ACC GGA AAT CTG GAG AAT CAG ACA ACA ACC ATT GGC GAG
Pro Met Ile Thr Gly Asn Leu Glu Asn G1n Thr Thr Thr Ile Gly Glu ACC ATT GAA GTG ACT TGC CCA GCA TCT GGA AAT CCT ACC CCA CAC ATT
Thr Ile Glu Val Thr Cys Pro Ala Ser Gly Asn Pro Thr Pro His I1e ACA TGG TTC AAA GAC AAC GAG ACC CTG GTA GAA GAT TCA GGC ATT GTA
Thr Trp Phe Lys Asp Asn Glu Thr Leu Val Glu Asp Ser Gly Ile Val CTG AGA GAT GGG AAC CGG AAC CTG ACT ATC CGC AGG GTG AGG AAG GAG
Leu Arg Asp Gly Asn Arg Asn Leu Thr Ile Arg Arg Val Arg Lys Glu GAT GGA GGC CTC TAC ACC TGC CAG GCC TGC AAT GTC CTT GGC TGT GCA
Asp Gly Gly Leu Tyr Thr Cys G1n Ala Cys Asn Val Leu Gly Cys Ala AGA GCG GAG ACG CTC TTC ATA ATA GAA GGT GCC CAG GAA AAG ACC AAC
Arg Ala Glu Thr Leu Phe Ile Ile Glu Gly Ala Gln G1u Lys Thr Asn SUBSTITUTE SHEET

912 CA 02107463 1995-08-17 Pi.~-T/US(~2/OZ750 VVII / /~ A .~~

TTG GAA GTC ATT ATC CTC GTC GGC ACT GCA GTG ATT GCC ATG TTC TTC
Leu Glu Val Ile Ile Leu Val Gly Thr Ala Val Ile Ala Met Phe Phe TGG CTC CTT CTT GTC ATT CTC GTA CGG ACC GTT AAG CGG GCC AAT GAA
Trp Leu Leu Leu Va1 I1e Leu Va1 Arg Thr Val Lys Arg Ala Asn Glu GGG GAA CTG AAG ACA GGC TAC TTG TCT ATT GTC ATG GAT CCA GAT GAA
Gly Glu Leu Lys Thr Gly Tyr Leu Ser Ile Val Met Asp Pro Asp Glu TTG CCC TTG GAT GAG CGC TGT GAA CGC TTG CCT TAT GAT GCC AGC AAG
Leu Pro Leu Asp Glu Arg Cys G1u Arg Leu Pro Tyr Asp Ala Ser 'Lys TGG GAA TTC CCC AGG GAC CGG CTG AAA CTA GGA AAA CCT CTT GGC CGC
Trp Glu Phe Pro Arg Asp Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg GGT GCC TTC GGC CAA GTG ATT GAG GCA GAC GCT TTT GGA ATT GAC AAG
Gly Ala Phe Gly Gln Val Ile Glu Ala Asp Ala Phe Gly I1e Asp Lys ACA GCG ACT TGC AAA ACA GTA GCC GTC AAG ATG TTG AAA GAA GGA GCA
Thr Ala Thr Cys Lys Thr Val Ala Val Lys Met Leu Lys Glu Gly Ala ACA CAC AGC GAG CAT CGA GCC CTC ATG TCT GAA CTC AAG ATC CTC ATC
Thr His Ser Glu His Arg Ala Leu Met Ser Glu Leu Lys Ile Leu Ile CAC ATT GGT CAC CAT CTC AAT GTG GTG AAC CTC CTA GGC GCC TGC ACC
His Ile G1y His His Leu Asn Val Val Asn Leu Leu Gly Ala Cys Thr AAG CCG GGA GGG CCT CTC ATG GTG ATT GTG GAA TTC TCG AAG TTT GGA
Lys Pro Gly Gly Pro Leu Met Val Ile Val Glu Phe Ser Lys Phe Gly AAC CTA TCA ACT TAC TTA CGG GGC AAG AGA AAT GAA TTT GTT CCC TAT
Asn Leu Ser Thr Tyr Leu Arg Gly Lys Arg Asn Glu Phe Val Pro Tyr AAG AGC AAA GGG GCA CGC TTC CGC CAG GGC AAG GAC TAC GTT GGG GAG
Lys Ser Lys Gly Ala Arg Phe Arg G1n Gly Lys Asp Tyr Val Gly Glu CTC TCC GTG GAT CTG AAA AGA CGC TTG GAC AGC ATC ACC AGC AGC CAG
Leu Ser Val Asp Leu Lys Arg Arg Leu Asp Ser Ile Thr Ser Ser G1n AGC TCT GCC AGC TCA GGC TTT GTT GAG GAG AAA TCG CTC AGT GAT GTA
Ser Ser Ala Ser Ser Gly Phe Val Glu Glu Lys Ser Leu Ser Asp Val SUBSTITUTE SHEET

VI'O 92/17486 2 1. o 7 ~4 . ~ PCT/~US92/(32750 ~r.

GAG GAA GAA GAA GCT TCT GAA GAA CTG TAC AAG GAC TTC CTG ACC TTG
Glu Glu Glu Glu Ala Ser Glu Glu Leu Tyr Lys Asp Phe Leu Thr Leu GAG CAT CTC ATC TGT TAC AGC TTC CAA GTG GCT AAG GGC ATG GAG TTC
Glu His Leu Ile Cys Tyr Ser Phe Gln Val Ala Lys Gly Met Glu Phe TTG GCA TCA AGG AAG TGT ATC CAC AGG GAC CTG GCA GCA CGA AAC ATT
Leu Ala Ser Arg Lys Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile CTC CTA TCG GAG AAG AAT GTG GTT AAG ATC TGT GAC TTC GGC TTG GCC
Leu Leu Ser Glu Lys Asn Val Val Lys Ile Cys Asp Phe Gly Leu Ala CGG GAC ATT TAT AAA GAC CCG GAT TAT GTC AGA AAA GGA GAT GCC CGA
Arg Asp Ile Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg CTC CCT TTG AAG TGG ATG GCC CCG GAA ACC ATT TTT GAC AGA GTA TAC
Leu Pro Leu Lys Trp Met Ala Pro Glu Thr Ile Phe Asp Arg Val Tyr ACA ATT CAG AGC GAT GTG TGG TCT TTC GGT GTG TTG CTC TGG GAA ATA
Thr Ile Gin Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile TTT TCC TTA GGT GCC TCC CCA TAC CCT GGG GTC AAG ATT GAT GAA GAA
Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Lys Ile Asp G1u Glu TTT TGT AGG AGA TTG AAA GAA GGA ACT AGA ATG CGG GCT CCT GAC TAC
Phe Cys Arg Arg Leu Lys Glu Gly Thr Arg Met Arg Ala Pro Asp Tyr ACT ACC CCA GAA ATG TAC CAG ACC ATG CTG GAC TGC TGG CAT GAG GAC
Thr Thr Pro Glu Met Tyr Gln Thr Met Leu Asp Cys Trp His Glu Asp CCC AAC CAG AGA CCC TCG TTT TCA GAG TTG GTG GAG CAT TTG GGA AAC
Pro Asn Gin Arg Pro Ser Phe Ser Giu Leu Val G1u His Leu Gly Asn CTC CTG CAA GCA AAT GCG CAG CAG GAT GGC AAA GAC TAT ATT GTT CTT
Leu Leu Gln Ala Asn Ala Gln Gin Asp Gly Lys Asp Tyr I1e Val Leu CCA ATG TCA GAG ACA CTG AGC ATG GAA GAG GAT TCT GGA CTC TCC CTG
Pro Met Ser G1u Thr Leu Ser Met Glu G1u Asp Ser Gly Leu Ser Leu CCT ACC TCA CCT GTT TCC TGT ATG GAG GAA GAG GAA GTG TGC GAC CCC
Pro Thr Ser Pro Val Ser Cys Met Glu Glu Glu Glu Val Cys Asp Pro SUBS I I fU1~ E SHEET

AAA TTC CAT TAT GAC AAC ACA GCA GGA ATC AGT CAT TAT CTC CAG AAC
Lys Phe His Tyr Asp Asn Thr Ala Gly Ile Ser His Tyr Leu Gln Asn 1210 1215' 1220 AGT AAG CGA AAG AGC CGG CCA GTG AGT GTA AAA ACA TTT GAA GAT ATC
Ser Lys Arg Lys Ser Arg Pro Val Ser Val Lys Thr Phe Glu Asp Ile CCA TTG GAG GAA CCA GAA GTA AAA GTG ATC CCA GAT GAC AGC CAG ACA
Pro Leu Glu Glu Pro Glu Val Lys Val Ile Pro Asp Asp Ser Gln Thr GAC AGT GGG ATG GTC CTT GCA TCA GAA GAG CTG AAA ACT CTG GAA GAC
Asp Ser Gly Met Val Leu Ala Ser Glu Glu Leu Lys Thr Leu G:1u Asp AGG AAC AAA TTA TCT CCA TCT TTT GGT GGA ATG ATG CCC AGT AAA AGC
Arg Asn Lys Leu Ser Pro Ser Phe Gly Gly Met Met Pro Ser Lys Ser AGG GAG TCT GTG GCC TCG GAA GGC TCC AAC CAG ACC AGT GGC TAC CAG
Arg Glu Ser Val Ala Ser Glu Gly Ser Asn Gln Thr Ser Gly Tyr Gln TCT GGG TAT CAC TCA GAT GAC ACA GAC ACC ACC GTG TAC TCC AGC GAC
Ser Gly Tyr His Ser Asp Asp Thr Asp Thr Thr Val Tyr Ser Ser Asp GAG GCA GGA CTT TTA AAG ATG GTG GAT GCT GCA GTT CAC GCT GAC TCA
Glu Ala Gly Leu Leu Lys Met Val Asp Ala Ala Val His Ala Asp Ser GGG ACC ACA CTG CAG CTC ACC TCC TGT TTA AAT GGA AGT GGT CCT GTC
Gly Thr Thr Leu Gln Leu Thr Ser Cys Leu Asn Gly Ser Gly Pro Val CCG GCT CCG CCC CCA ACT CCT GGA AAT CAC GAG AGA GGT GCT GCT
Pro Ala Pro Pro Pro Thr Pro Gly Asn His Glu Arg Gly Ala Ala TAGATTTTCA AGTGTTGTTC TTTCCACCAC CCGGAAGTAG CCACATTTGA TTTTCATTTT
TGGAGGAGGG ACCTCAGACT GCAAGGAGCT TGTCCTCAGG GCATTTCCAG AGAAGATGCC
CATGACCCAA GAATGTGTTG ACTCTACTCT CTTTTCCATT CATTTAAAAG TCCTATATAA
TGTGGTCTCA CTACCAGTTA AAGCAAAAGA CTTTCAAACA CGTGGACTCT GTCCTCCAAG
TGTGCCCTGC AAGTGGCAAC GGCACCTCTG TGAAACTGGA TCGAATGGGC AATGCTTTGT
GTGTTGAGGA TGGGTGAGAT GTCCCAGGGC CGAGTCTGTC TACCTTGGAG GCTTTGTGGA
GGATGCGGCT ATGAGCCAAG TGTTAAGTGT GGGATGTGGA CTGGGAGGAA GGAAGGCGCA
AGAGCGGTTG GAGCCTGCAG ATGCATTGTG CTGGCTCTGG TGGAGGTGGG CTTGTGGCCT
SUBSTITUTE SHEET

7 4. ~ rs WO 92/17436 9'C'I'/U592/02750 GTCAGGAAAC GCAAAGGCGG CCGGCAGGGT TTGGTTTTGG AAGGTTTGCG TGCTCTTCAC
AGTCGGGTTA CAGGCGAGTT CCCTGTGGCG TTTCCTACTC CTAATGAGAG TTCCTTCCGG
ACTCTTACGT GTCTCCTGGC CTGGCCCCAG GAAGGAAATG ATGCAGCTTG CTCCTTCCTC
ATCTCTCAGG CTGTGCCTTA ATTCAGAACA CCAAFIAGAGA GGAACGTCGG CAGAGGCTCC
TGACGGGGCC GAAGAATTGT GAGAACAGAA CAGAAACTCA GGGTTTCTGC TGGGTGGAGA
CCCACGTGGC GCCCTGGTGG CAGGTCTGAG GGTTCTCTGT CAAGTGGCGG TAAAGGCTCA
GGCTGGTGTT CTTCCTCTAT CTCCACTCCT GTCAGGCCCC CAAGTCCTCA GTATTTTAGC
TTTGTGGCTT CCTGATGGCA GAAAAATCTT AATTGGTTGG TTTGCTCTCC AGATAATCAC
TAGCCAGATT TCGAAATTAC TTTTTAGCCG AGGTTATGAT AACATCTACT GTATCCTTTA
GAATTTTAAC CTATAAAACT ATGTCTACTG GTTTCTGCCT GTGTGCTTAT GTTAAAAAAA

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1367 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Met Glu Ser Lys Gly Leu Leu Ala Val Ala Leu Trp Phe Cys Val Glu Thr Arg Ala Ala Ser Val Gly Leu Pro Gly Asp Phe Leu His Pro Pro Lys Leu Ser Thr Gln Lys Asp Ile Leu Thr Ile Leu Ala Asn Thr Thr Leu Gln Ile Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro Asn Ala Gln Arg Asp Ser Glu Glu Arg Val Leu Val Thr Glu Cys Gly Gly Gly Asp Ser Ile Phe Cys Lys Thr Leu Thr Ile Pro Arg Val Val Gly Asn Asp Thr Gly Ala Tyr Lys Cys Ser Tyr Arg Asp Val Asp Ile Ala Ser Thr Val Tyr Val Tyr Va1.Arg Asp Tyr Arg Ser Pro Phe Ile SUBS T!TU'f E SHEET

s (3r 7 L4 ~~ CA 02107463 1995-08-17 WO(((~~~92...333/17486 PCT/US92/02750 62 Ala Ser Val Ser Asp G1n His Gly Ile Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val Ile Pro Cys Arg Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser Trp Asp Ser Glu Ile Gly Phe Thr Leu Pro Ser Tyr Met Ile Ser Tyr Ala Gly Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Thr Tyr Gln Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Ile Leu Ser Pro Pro His Glu Ile Glu Leu Ser Ala Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Leu Asp Phe Thr Trp His Ser Pro Pro Ser Lys Ser His His Lys Lys Ile Val Asn Arg Asp Val Lys Pro Phe Pro Gly Thr Val Ala Lys Met Phe Leu Ser Thr Leu Thr Ile Glu Ser Val Thr Lys Ser Asp Gin Gly Glu Tyr Thr Cys Val Ala Ser Ser Gly Arg Met Ile Lys Arg Asn Arg Thr Phe Val Arg Val His Thr Lys Pro Phe Ile Ala Phe Gly Ser Gly Met Lys Ser Leu Val Glu Ala Thr Val Gly Ser G1n Val Arg Ile Pro Val Lys Tyr Leu Ser Tyr Pro Ala Pro Asp Ile Lys Trp Tyr Arg Asn Gly Arg Pro Ile Glu Ser Asn Tyr Thr Met 21e Val Gly Asp Glu Leu Thr Ile Met Glu Val Thr Glu Arg Asp Ala Gly Asn Tyr Thr Val Ile Leu Thr Asn Pro Ile Ser Met Glu Lys G1n Ser His Met Val Ser Leu Va1 Val Asn Val Pro Pro Gin Ile Gly Glu Lys Ala Leu Ile Ser SUBSTITUTE SHEET

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Pro Met Asp Ser Tyr Gln Tyr Gly Thr Met Gln Thr Leu Thr Cys Thr Val Tyr Ala Asn Pro Pro Leu His His I1e Gln Trp Tyr Trp G1n Leu Glu Glu Ala Cys Ser Tyr Arg Pro Gly Gln Thr Ser Pro Tyr Ala Cys Lys Glu Trp Arg His Val Glu Asp Phe Gln Gly Gly Asn Lys Ile Glu Va1 Thr Lys Asn Gln Tyr Ala Leu Ile Glu Gly Lys Asn Lys Thr Val Ser Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr Lys Cys Glu Ala I1e Asn Lys Ala Gly Arg Gly Glu Arg Val Ile Ser Phe His Val Ile Arg Gly Pro Glu Ile Thr Val Gln Pro Ala Ala Gln Pro Thr Glu Glri Glu Ser Val Ser Leu Leu Cys Thr Ala Asp Arg Asn Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Ser Gln Ala Thr Ser Val His Met Gly Glu Ser Leu Thr Pro Val Cys Lys Asn Leu Asp Ala Leu Trp Lys Leu Asn Gly Thr Met Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile Val Ala Phe Gln Asn Ala Ser Leu Gin Asp Gln Gly Asp Tyr Val Cys Ser Ala Gln Asp Lys Lys Thr Lys Lys Arg His Cys Leu Val Lys Gln Leu Ile Ile Leu Glu Arg Met Ala Pro Met Ile Thr Gly Asn Leu Glu Asn Gln Thr Thr Thr Ile Gly Glu Thr Ile Glu Val Thr Cys Pro Ala Ser Gly Asn Pro Thr Pro His Ile Thr Trp Phe Lys Asp Asn Glu Thr Leu Va1 Glu Asp Ser Gly Ile Val Leu Arg Asp Gly Asn Arg Asn Leu Thr Ile Arg Arg Val Arg Lys Glu Asp Gly Gly Leu Tyr Thr Cys Gln SUBSTITUTE SHEET

a(~. ~~/17~t86 3 PCT/LJS92/02750 Ala Cys Asn Val Leu Gly Cys Ala Arg Ala Glu Thr Leu Phe Ile Ile Glu Gly Ala G1n G1u Lys Thr Asn Leu Glu Val Ile I1e Leu Val Gly Thr Ala Val Ile Ala Met Phe Phe Trp Leu Leu Leu Val Ile Leu Val Arg Thr Val Lys Arg Ala Asn Glu Gly'Glu Leu Lys Thr Gly Tyr Leu Ser Ile Val Met Asp Pro Asp Glu Leu Pro Leu Asp Glu Arg Cys Glu Arg Leu Pro Tyr Asp Ala Ser Lys Trp Glu Phe Pro Arg Asp Arg Leu Lys Leu Gly Lys Pro Leu Gly Arg Gly Ala Phe Gly Gln Val Ile Glu Ala Asp Ala Phe Gly Ile Asp Lys Thr Ala Thr Cys Lys Thr Val Ala Val Lys Met Leu Lys Glu Gly Ala Thr His Ser Glu His Arg Ala Leu Met Ser Glu Leu Lys Ile Leu Ile His Ile Gly His His Leu Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys Pro Gly Gly Pro Leu Met Val Ile Val Glu Phe Ser Lys Phe Gly Asn Leu Ser Thr Tyr Leu Arg Gly 915 . 920 925 Lys Arg Asn Glu Phe Val Pro Tyr Lys Ser Lys Gly Ala Arg Phe Arg Gln Gly Lys Asp Tyr Val Gly Glu Leu Ser Val Asp Leu Lys Arg Arg Leu Asp Ser I1e Thr Ser Ser Gln Ser Ser Ala Ser Ser Gly Phe Val Glu Glu Lys Ser Leu Ser Asp Val Glu Glu Glu Glu Ala Ser Glu Glu Leu Tyr Lys Asp Phe Leu Thr Leu Glu His Leu Ile Cys Tyr Ser Phe G1n Val Ala Lys Gly Met Glu Phe Leu Ala Ser Arg Lys Cys I1e His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu Ser Glu Lys Asn Val Val SdlBS'fITLD'ffi'E SHEET

WO 92/17486 ~ ~ ~ a/ ~. ~ =:-i?f Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asp Pro Asp Tyr Val Arg Lys Gly Asp Ala Arg Leu Pro Leu Lys Trp Met Ala Pro Glu Thr Ile Phe Asp Arg Val Tyr Thr Ile G1n Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Ala Ser Pro Tyr Pro Gly Val Lys Ile Asp Glu Glu Phe Cys Arg Arg Leu Lys Glu Gly Thr Arg Met Arg Ala Pro Asp Tyr Thr Thr Pro Glu Met Tyr Gln Thr Met Leu Asp Cys Trp His Glu Asp Pro Asn Gln Arg Pro Ser Phe Ser Glu Leu Val Glu His Leu Gly Asn Leu Leu Gln Ala Asn Ala G1n Gln Asp Gly Lys Asp Tyr I1e Val Leu Pro Met Ser Glu Thr Leu Ser Met Glu Glu Asp Ser Gly Leu Ser Leu Pro Thr Ser Pro Val Ser Cys Met Glu Glu Glu Glu Val Cys Asp Pro Lys Phe His Tyr Asp Asn Thr Ala Gly Ile Ser His Tyr Leu Gln Asn Ser Lys Arg Lys Ser Arg Pro Val Ser Val Lys Thr Phe Glu Asp Ile Pro Leu Glu Glu Pro Glu Val Lys Val I1e Pro Asp Asp Ser Gln Thr Asp Ser Gly Met Val Leu Ala Ser Glu Glu Leu Lys Thr Leu Glu Asp Arg Asn Lys Leu Ser Pro Ser Phe G1y Gly Met Met Pro Ser Lys Ser Arg Glu Ser Val Ala Ser Glu G1y Ser Asn Gln Thr Ser Gly Tyr Gln Ser Gly Tyr His Ser Asp Asp Thr Asp Thr Thr Val Tyr Ser Ser Asp Glu Ala Gly Leu Leu Lys Met Val Asp Ala Ala Val His Ala Asp Ser Gly Thr Thr Leu Gln Leu Thr Ser SUBSTITUTE SHEET

~~~.~"1 ~ ~:.~-'~92/oZ750 92~17~1 b CA 02107463 1995-08-17 YCT/UC~

Cys Leu Asn Gly Ser Gly Pro Val Pro Ala Pro Pro Pro Thr Pro Gly Asn His Glu Arg Gly Ala Ala SUBS fi'f'l!TE SHEET

Claims (37)

What I claim is:

1. An isolated mammalian nucleic acid molecule encoding either (a) a receptor protein tyrosine kinase expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells that is flk-2 consisting essentially of the sequence shown in Figure 1a or (b) a receptor tyrosine kinase fragment expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells that is a flk-2 fragment consisting essentially of the sequence shown in Figure 1b, or 1c.

2. The nucleic acid molecule according to claim 1 wherein the nucleic acid molecule is DNA.

3 The nucleic acid molecule according to claim 1 wherein the nucleic acid molecule is cDNA.

4. The nucleic acid molecule according to claim 1 wherein the nucleic acid molecule is RNA.

5. The nucleic acid molecule according to claim 1 that is a mouse nucleic acid molecule.

6. The nucleic acid molecule according to claim 1 that is a human nucleic acid molecule.

7. The nucleic acid molecule according to claim 6 that is DNA.

8. An isolated nucleic acid molecule that is either (a) flk-2 consisting essentially of the sequence shown in Figure 1a or (b) a flk-2 fragment consisting essentially of the sequence shown in Figure 1b, or 1c.

9. The nucleic acid molecule according to claim 8 consisting essentially of the sequence shown in Figure 1b, or 1c.

10. The nucleic acid molecule according to claim 8 wherein the nucleic acid molecule is DNA.

11. The nucleic acid molecule according to claim 8 wherein the nucleic acid molecule is RNA.

12. An isolated nucleic acid molecule that is flk-1 having the sequence shown in Figure 2.

13. The nucleic acid molecule according to claim 12 wherein the nucleic acid molecule is DNA.

14. The nucleic acid molecule according to claim 12 wherein the nucleic acid molecule is cDNA.

15. The nucleic acid molecule according to claim 12 wherein the nucleic acid molecule is RNA.

16. A vector comprising a mammalian nucleic acids molecule encoding a receptor protein tyrosine kinase expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells that is flk-1 having the nucleic acid sequence of Figure 2.

17. A vector comprising a mammalian nucleic acid molecule encoding either (a) a receptor protein tyrosine kinase expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells that is flk-2 consisting essentially of the sequence shown in Figure 1a or (b) a receptor tyrosine kinase fragment expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells that is a flk-2 fragment consisting essentially of the sequence shown in Figure 1b, or 1c.

18. The vector according to claim 16 wherein the vector is capable of being cloned in a host.

19. The vector according to claim 17 wherein the vector is capable of being cloned in a host.

20. The vector according to claim 18 wherein the host is a prokaryotic host.

21. The vector according to claim 19 wherein the host is a prokaryotic host.

22. The vector according to claim 16 that is capable of expressing flk-1 in a host.

23. The vector according to claim 17 that is capable of expressing flk-2 in a host.

24. The vector according to claim 22 wherein the host is a prokaryotic host.

25. The vector according to claim 23 wherein the host is a prokaryotic host.

26. The vector according to claim 22 wherein the host is a eucaryotic host.

27. The vector according to claim 23 wherein the host is a eucaryotic host.

28. An isolated protein tyrosine kinase expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells that is either (a) flk-2 consisting essentially of the amino acid sequence shown in Figure 1a or (b) a flk-2 fragment consisting essentially of the amino acid sequence shown in Figure 1b, or 1c.

29. The protein tyrosine kinase according to claim 28 that is human flk-2.

30. The protein tyrosine kinase according to claim 29 that is flk-2 consists of the sequence shown in Figure 1b or Figure 1c.

31. An isolated protein tyrosine kinase that is flk-1 consists of the sequence shown in Figure 2.

32. Isolated antibody that binds specifically to either (a) the extracellular portion of a protein tyrosine kinase that is flk-2 consisting essentially of the amino acid sequence 28-544 encoded by the nucleic acid molecule consisting of the sequence shown in Figure 1a or (b) a flk-2 fragment encoded by the nucleic acid molecule consisting essentially of the sequence shown in Figure 1b.

33. The antibody according to claim 32 wherein flk-2 is the human flk-2.

34. The antibody according to claim 32 wherein flk-2 is the murine flk-2.

35. Isolated antibody that binds specifically to the extracellular portion of a protein tyrosine kinase that is flk-1, encoded by a nucleic acid molecule consisting of the sequence shown in Figure 2.

36. The antibody according to claim 36 wherein flk-1 is the murine flk-1.

37. Isolated antibody that binds to a protein having the amino acid sequence shown in Figure 2.