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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCI) 



(51) International Patent Classification 5 : 

O07K 13/00, A61K 35/28 
C12N 15/19, A61K 37/02 



Al 



(11) International Publication Number: 
(43) International Publication Date: 



WO 91/04274 

4 April 1991 (04.04.91) 



(21) Internationa] Application Number: PCT/US90/0545 1 

(22) International Filing Date: 25 September 1990 (25.09.90) 



(30) Priority data: 

412,303 
581,713 



25 September 1989 (25.09.89) US 
13 September 1990 (13.09.90) US 



(71) Applicants: GENETICS INSTITUTE, INC. [US/US]; 87 

CambridgePark Drive, Cambridge, MA 02140 (US). 
CRC TECHNOLOGY LTD. [GB/GB]; 2 Carlton 
House Terrace, London SW1Y 5AR (GB). 

(72) Inventors: PRAGNELL, Ian, B. ; 14 Belmont Crescent, 

Glasgow G12 (GB). DONALDSON, Debra, D. ; 40 1/2 
Roberts Road, Cambridge, MA 02138 (US). GRAHAM, 
Gerald, J. ; 31 Edgemont Street, Glasgow G41 3 EH 
(GB). WONG, Gordon, G. ; 40 Jamaica Way, Apt. 10, 
Jamaica Plain, MA 02130 (US). 



(74) Agents: KAPINOS, Ellen, J. et al.; Genetics Institute, Inc., 
87 Cambridge Park Drive, Cambridge, MA 02140 (US). 



(81) Designated States: AT (European patent), BE (European 
patent), CA, CH (European patent), DE (European pa- 
tent)*, DK (European patent), ES (European patent), 
FR (European patent), GB (European patent), IT (Euro- 
pean patent), JP, LU (European patent), NL (European 
patent), SE (European patent). 



Published 

With international search report. 

Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 



(54) Title: METHOD FOR INHIBITING GROWTH OF STEM CELLS 



(57) Abstract 

The present invention discloses a method for protecting cycling or dividing stem cells from the cytotoxic effects of chem- 
otherapeutic agents and radiation by administering prior to exposure to these agents, an effective amount of Stem Cell Inhibitory 
Factor as well as a composition useful therefor. 



* See back of page 



DESIGNATIONS OF "DE" 



Until further notice, any designation of "DE* in any international application 
whose international filing date is prior to October 3, 1990, shall have effect in the 
territory of the Federal Republic of Germany with the exception of the territory of the 
former German Democratic Republic. 



FOR THE PURPOSES OF INFORMATION ONLY 

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international 
applications under the PCT. 



AT 


Austria 


ES 


Spain 


MC 


Monaco 


AU 


Australia 


Fl 


Finland 


MG 


Madagascar 


BB 


Barbados 


FR 


France 


ML 


Mali 


BE 


Belgium 


GA 


Gabon 


MR 


Mauritania 


BP 


Burkina Fasso 


GB 


United Kingdom 


MW 


Malawi 


BC 


Bulgaria 


GR 


Greece 


NL 


Netherlands 


BJ 


Benin 


HU 


Hungary 


NO 


Norway 


BR 


Brazil 


IT 


Italy 


PL 


Poland 


CA 


Canada 


JP 


Japan 


RO 


Romania 


CF 


Central African Republic 


KP 


Democratic People's Republic 


SD 


Sudan 


CC 


Congo 




of Korea 


SE 


Sweden 


CH 


Switzerland 


KR 


Republic of Korea 


SN 


Senegal 


CM 


(Cameroon 


LI 


Liechtenstein 


SU 


Soviet Union 


DE 


Germany 


LK 


Sri Lanka 


TO 


Chad 


DK 


Denmark 


LU 


taxembourg 


TG 


Togo 



US United Stales of America 



WO 91/04274 



PCT/US90/0S451 



] 

METHOD FOR INHIBITING GROWTH OF STEM CELLS 

The present invention relates generally to the 
treatment of human or animal subjects anticipating 
5 exposure to chemotherapeutic agents, radiation exposure, 
or other agents which irreparably damage cycling stem 
cells and a stem cell inhibitory composition useful 
therefore. 

Cross-Reference to Related Application 
10 This application is a continuation-in-part of 

U. S. Patent Application, Serial No. 07/412,303, filed 
September 25, 1989. 

Background of the Invention 

In the treatment of subjects with cancer, 
15 conventional chemotherapy involves the application of one 
or more cell cycle specific cytotoxic agents, which have 
the adverse effect of killing, or irreparably damaging, 



25 



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PCT/US90/054S1 



2 

both normal and cancer cells undergoing division. The 
goal of such Chemother apeutic agents, e.g., cytosine 
arabinoside (ara-C) , is to destroy or disable the 
dividing cancer cells, preventing further cancer cell 
5 growth. A presently unavoidable effect of chemotherapy, 
however, is the destruction of other normal dividing 
cells, particularly the stem cells of the hematopoietic 
system and the epithelial stem cells which line the scalp 
and gut. Stem cell damage caused by the administration 

10 of chemotherapy, result in the usual side effects 
thereof, such as hair loss, stomach and intestinal 
damage, skin damage, myelosuppression, anemia, reduced 
immune function or response and the resulting increased 
sensitivity to infection. 

15 A characteristic property of stem cells is 

their quiescent nature within the cell cycle. When there 
is 'insult 1 to the bone marrow, such as by drug 
treatment, radiation, severe blood loss, inflammatory 
reaction, or infection, the stem cell responds by a 

20 feedback mechanism to begin cycling to replenish the more 

mature progenitor cells, which in turn, differentiate 
into the required mature cells of the hematopoietic, 
immune or epithelial systems. 

Since the hematopoietic stem cells are 

25 necessary for the development of all of the mature cells 
of the hematopoietic and immune systems, their survival 
is essential in order to reestablish in the subject 



WO 91/04274 



PCT/US90/054S1 



treated with chemotherapy, a fully functional host 
defense system. Similarly, survival of epithelial stem 
cells is necessary for repair of the epithelial linings 
of organs, including the skin. Because high doses of 
5 cycle specific chemotherapeutic drugs, such as ara-C, 

effectively kill hematopoietic stem cells and stem cells 
of epithelial tissues, patients exposed to such agents 
suffer from serious side effects and are frequently at 
risk of serious infections, 

10 Similar destruction of stem cells occurs upon 

exposure to a variety of dosages of radiation, whether 
the radiation is used for therapeutic purposes or is the 
result of accidental or unavoidable exposure to 
radiation, e.g., incident to a clean-up of, or presence 

15 at the location of, a nuclear accident at a power plant, 
or on a nuclear submarine. Subjects exposed to radiation 
also experience the destruction of their hematopoietic 
stem cells, and consequent failure or serious damage to 
the hematopoietic and immune systems. Radiation also 

20 kills dividing epithelial cells with consequent damage to 

many epithelial tissues. 

While agents, such as the colony stimulating 
factors, e.g., M-CSF, CSF-1, GM-CSF, and others, are 
presently being employed to stimulate the development of 

25 certain hematopoietic cell lineages in subjects exposed 
to chemotherapy or radiation, such agents are not 
believed to be capable of restoring the hematopoietic 



WO 91/04274 



PCT/US90/05451 



system if an insufficient quantity of stem cells is 
present in the subject after such exposure. 

Thus, there remains a need in the art for other 
therapeutic agents capable of protecting stem cells from 
5 the damaging effects of chemotherapy or radiation. 

Summary of the Invention 

The present invention addresses this need in 
the art by providing a method of treating a subject 
anticipating exposure to an agent capable of killing 

10 dividing or cycling stem cells by administering to that 
subject an effective amount of a stem cell inhibitory 
composition. The stem cells protected by this method may 
be hematopoietic stem cells ordinarily present and 
dividing in the bone marrow. Alternatively, stem cells 

15 may be epithelial, located e.g., in the intestines or 
scalp or other areas of the body. The method of this 
invention may be desirably employed on humans, although 
animal treatment is also encompassed by this method. 

The stem cell inhibitory composition useful in 

20 this method comprises an effective amount of a 

polypeptide or fragment of a protein referred to herein 
as Stem Cell Inhibitor Factor (SCIF) and described with 
specificity below. 



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5 

In another aspect, the invention provides a 
method for protecting and restoring the hematopoietic, 
myeloid and immune systems of a patient undergoing 
chemotherapy, which includes administering to the patient 
5 an effective amount of SCIF. SCIF may be administered 

prior to the chemotherapy. Alternatively the SCIF may be 
administered during chemotherapy. Another alternative is 
administering the SCIF for a period after the 
chemotherapeutic regimen. Following chemotherapy, the 
10 patient may be treated with therapeutic factors, such as 
colony stimulating factors or other lymphokines that 
stimulate the stem cells to divide and further stimulate 
the production of more mature cells of the hematopoietic 
lineages. 

15 in still a further aspect, the present 

invention involves a method for adjunctively treating any 
cancer, including those characterized by solid tumors, by 
administering to a patient having cancer an effective 
amount of SCIF to protect the hematopoietic stem cells of 

20 the bone marrow, thereby allowing greater dosages of 
chemotherapeutics or radiation to be employed for 
treatment of the cancer. 



Yet another aspect of the present invention 
involves the treatment of leukemia comprising treating 
bone marrow cells having proliferating leukemia cells 
therein with an effective amount of SCIF to inhibit 
proliferation of normal stem cells, and treating the bone 
marrow with a cytotoxic agent to destroy leukemia cells. 
This method may be enhanced by the follow-up treatment of 
the bone marrow with other agents that stimulate its 
proliferation, e.g., lymphokines. This method may be 
performed in vivo . Alternatively, this method may be 
performed ex vivo and the resulting marrow purged of 
leukemia cells by the chemotherapeutic agent. The marrow 
may then be reinjected into the patient. 

In still a further aspect, the method involves 
treating a subject having any disorder caused by 
proliferating stem cells. Such a disorder, such as 
psoriasis, may be treated by administering to the subject 
an effective amount of SCIF to partially or wholly 
inhibit proliferation of the stem cell in question. 

Other aspects and advantages of the present 
invention will be apparent upon consideration of the 
following detailed description of preferred embodiments 
thereof. 



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PCT/US90/05451 



Detailed Description of the Invention 

The present invention provides a method for 
reversibly protecting stem cells from damage from a 
cytotoxic agent capable of killing dividing stem cells, 
5 The method involves administering to a subject 

anticipating exposure to such an agent an effective 
amount of a Stem Cell Inhibitory Factor (SCIF) . The 
method also may involve extending the; SCIF treatment 
throughout the cytotoxic treatment to enhance the stem 
10 cell protective effect. 

1 

SCIF can reversibly inhibit division of a 
variety of stem cells in the human body. Specifically 
SCIF is effective in temporarily inhibiting cell divisi- 
of hematopoietic stem cells. Additionally, SCIF also 

15 acts to inhibit cycling or dividing epithelial stem 
cells, located throughout the body. Other stem cell 
populations on which SCIF may exercise a reversibly 
inhibiting activity include male and female germinal 
cells. SCIF may then be employed to protect a male or 

2 0 female patient from post-chemotherapeutic germinal 

aplasia. Additionally, because chemotherapeutic agents 
commonly induce alopecia and mucositis, SCIF may be use* 
to reversibly protect hair follicles and oro- 
gastrointestinal epithelial stem cells from adverse sid« 

25 effects of chemotherapy or radiation. 



WO 91/04274 



PCT/US90/05451 



8 

Thus the method of this invention may be 
employed in alleviating the undesirable side effects of 
chemotherapy on the patient's hematopoietic, myeloid and 
immune systems by protecting stem cells from damage from 
5 a chemotherapeutic agent, such as a cytotoxic drug or 

radiation dosage normally used to destroy cancer cells. 
Such an application of SCIF may also serve to protect 
epithelial stem cells during chemotherapy. SCIF may be 
administered to the patient in a dosage sufficient to 

10 inhibit stem cell division for a time sufficient to allow 
the action of the chemotherapeutic agent. After the 
chemotherapeutic agent has performed its function, the 
stem cells inhibited by SCIF will without further 
treatment, revert to dividing cells. If it is desired to 

15 enhance the reversion of hematopoietic stem cells, the 

patient can receive doses of hematopoietic growth factors 
or cytokines used to stimulate the growth and development 
of hematopoietic cells. 

The majority of chemotherapeutic agents used 

20 for cancer chemotherapy have a relatively short in vivo 

half-life, usually less than 24 hours. This inhibitory 
effect of SCIF according to the present invention is 
maintained for at least the major proportion of the 
effective time during which the chemotherapeutic agent is 

25 active in vivo . For those cytotoxic agents which have 



WO 91/04274 



PCT/US90/05451 



prolonged half-lives (e.g., greater than 24 hours), it is 
expected that more prolonged treatment with SCIF would be 
required. The normal physiological mechanisms within the 
subject would limit the effective duration of activity of 
5 SCIF in relation to cycling stem cells. 

Additionally, the method has utility in 
providing a subject protection against other exposure to 
radiation, which damages the bone marrow cells of a 
subject. An individual may be administered SCIF where 

10 unintentional or accidental exposure to dangerous levels 

of radiation are anticipated. For example, such 
individuals anticipating entering sites of nuclear fall- 
out, persons responsible for examining and cleaning 
nuclear power plants after a leak of dangerous levels of 

15 radiation or nuclear submarines and the like may be 

treated by this method to inhibit replicating stem cell 
division in case of short term radiation exposure. SCIF 
administration during, as well as before radiation 
exposure may provide enhanced protection. 

20 Additionally, SCIF may be employed in this method as an 
adjunctive treatment with chemotherapy to treat any 
cancers. Because the bone marrow is the limiting organ 
in determining the amount of radiation or dosage of 
cytotoxic drug .that can be applied to a patient, SCIF may 

25 be used to protect the bone marrow hematopoietic cells 



10 

from radiation or chemotherapy, thereby allowing greater 
amounts of radiation or drugs to be applied to treat any 
cancer, normally amenable to either radiation or 
chemotherapeutic treatment- Since the myelotoxicity of 
the cytotoxic drugs is also limiting on their dosage 
during chemotherapy, the administration of SCIF to the 
patient is likely to enable increased drug dosages to be 
given to the patient without the serious side effects 
that would normally accompany such increased dosages . 

The method may also be employed to treat solid 
tumors by inhibiting during chemotherapeutic treatment, 
the division of epithelial stem cells. 

SCIF may also be employed in a method for 
preparing autologous bone marrow for transplantation. 
The marrow can be treated ex vivo with an effective 
amount of SCIF to inhibit stem cell division and then 
purged of cancerous cells by administering an effective 
amount of a chemotherapeutic agent or radiation. Marrow 
thus treated may be reinjected into the autologous donor. 
Optionally the patient may then be treated with an agent 
known to stimulate hematopoiesis . 

SCIF may also be employed in the method of this 
invention as an adjunctive therapy in the treatment of 
leukemia. For example, where the leukemic cells do not 
respond to SCIF, the leukemic bone marrow cells may be 



WO 91/04274 



PCT/US90/05451 



11 

treated ex vivo , with SCIF. The proliferation of normal 
stem cells is prevented by administration of SCIF. Thus, 
during the time that the proliferating leukemic cells are 
treated with a cytotoxic agent, a quantity of normal stem 
5 cells are protected from damage. Additionally, a 

stimulatory cytokine, such as IL-3 or GM-CSF, may be 
administered to induce cycling in the leukemic cells 
during drug or radiation treatment while the normal stem 
cells are protected with SCIF. 

10 For ex vivo use, the bone marrow may be treated 

with SCIF to inhibit stem cell division, thereby 
protecting stem cells from destruction by the subsequent 
application to the marrow or to the source of any other 
cancer, of chemotherapy designed to destroy cycling 

15 cells, e.g. leukemic cells. The resulting purged marrow 
may be reinjected into the patient, wherein the stem 
cells will begin to divide normally. To enhance cell 
division of the hematopoietic stem cells, the lymphokines 
and cytokines above-identif ied may also be administered. 

20 The same process may be accomplished by administering 
SCIF to the patient in vivo * 

The method of this invention may also be 
employed to treat disorders related to hyperprol iterative 
stem cells. For example, psoriasis is a disorder caused 

25 by hyperproliferating epithelial cells of the skin and is 



WO 91/04274 



PCIYUS90/0S451 



12 

sometimes treated with cytotoxic drugs. Similarly, a 
condition of in situ dysplasia of cervical epithelium is 
recognized which will proceed to cervical cancer if left 
untreated. Both of these transitional states may be 
treated prophylactically with SCIF. Other pre-neoplastic 
lesions in which stem cell proliferation is involved may 
also be amenable to effective amounts of SCIF employed to 
inhibit wholly or partially the proliferation of the stem 
cells. For these uses, topical or transdermal 
compositions containing SCIF may be employed, as well as 
other parenteral means of administering SCIF. 

The SCIF polypeptides may also be used in 
another method of this invention. Antibodies, monoclonal 
or polyclonal, may be developed by standard techniques to 
the SCIF polypeptides. These antibodies or SCIF 
polypeptides may be labelled with detectable labels of 
which many types are known in the art. The labeled SCIF 
or anti-SCIF antibodies may then be employed as stem cell 
markers to identify and isolate stem cells by 
administering to a patient directly for diagnostic 
purposes. Alternatively these labeled polypeptides or 
antibodies may be employed ex vivo to identify stem cells 
in a bone marrow preparation, to enable their removal 
prior to purging techniques. In the same manner such 



WO 91/04274 



PCT/US90/0S451 



13 

labeled polypeptides or antibodies may be employed to 
isolate and identify epithelial, or other stem cells. 

The inventors herein have discovered that SCIF, 
employed in the following methods and examples, is the 
5 murine homologue of protein factors previously 

identified. K. Obaru et al, J. Biochem. . 99:885-894 
(1986) identified the amino acid and DNA sequence of a 
gene found to be inducible by tumor promoters such as 
phorbol ester in human tonsillar lymphocytes. The 

10 authors predicted that this gene produced a protein which 

may signal proliferation of T cells. More recently, P. 
F. Zipfel et al, J - Immunol . . 142 ; 1582-1590 (1989) 
reported that the same protein was produced by mitogenic 
activation of human T cells and suggested that it may 

15 function as a lymphokine or cytokine. 

The present method thus desirably employs the 
human SCIF factor in human therapy. This SCIF factor may 
be the human polypeptide encoded by these prior art 
sequences. The Obaru/Zipfel SCIF sequence is reported 

20 below in Table I. Alternatively, this factor may be 
obtained as described in Example 1 below. The human 
analog of SCIF was obtained from a human T cell line 
using oligonucleotides based on the published LD 78 



14 

sequence of Obaru et al, cited above. Human SCIF 
demonstrates human activity identical to the murine 
protein in the SCIF assays described below. 

The human SCIF clone of Example 1 has been 
substantially sequenced. While exhibiting the required 
activity, this sequence has been found to differ in both 
nucleotide and amino acid sequence from the published 
sequence. The alternative human SCIF DNA and amino acid 
sequence of Example 1 is reported below in Table II. The 
sequence within the brackets has not yet been determined. 
The sequence that does appear within the brackets on 
Table II has been derived from the published LD 78 
sequence of Obaru et al and may or may not be identical 
with the human SCIF clone. The differences in the 
nucleotide sequence between the cloned SCIF sequence of 
Example 1 and the published Obaru sequence are indicated 
in Table II by asterisks above the changed or added amino 
acids and below the changed nucleotides. Dashes indicate 
non-coding sequences not found in the sequence of Example 
1. There are 18 nucleotide differences and 4 amino acid 
differences between the two SCIF sequences. Additionally 
the human clone includes 3 more nucleotides and an 
additional amino acid in contrast to the published 
sequence . 



WO 91/04274 



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15 

However, also useful in the present invention 
as a SCIF molecule are other proteins encoded by other 
DNA sequences, not identical to that of the prior art 
references. Such DNA sequences are characterized as 
5 capable of hybridizing under stringent hybridization 

conditions [see, T. Maniatis et al, Molecular Cloning (A 
Laboratory Manual) . Cold Spring Harbor Laboratory (1982) , 
pages 387 to 389] to the human SCIF (or Obaru et al) DNA 
sequences and coding on expression for polypeptides or 

10 proteins capable of demonstrating SCIF activity as 
described herein/ An example of one such stringent 
hybridization condition is hybridization at 4XSSC at 
65 °C, followed by a washing in 0.1XSSC at 65 °C for an 
hour. Alternatively an exemplary stringent 

15 hybridization condition is in 50% formamide, 4XSSC at 
42°C. 

Other human SCIF proteins or polypeptides may 
be encoded by other DNA sequences which hybridize to the 
sequences for human SCIF (Obaru et al sequence and the 

20 sequence obtained from the clone of Example 1) under 
relaxed hybridization conditions and which code on 
expression for peptides having SCIF biological 
properties. Examples of such non-stringent hybridization 
conditions are 4XSSC at 50 °C or hybridization with 30-40% 

25 formamide at 42 *C. For example, the molecule identified 
previously as human MlP-beta, may be useful in the method 



WO 91/04274 



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16 

of this invention, if capable of displaying stem cell 
inhibitory activity as defined hereinbelow by assay. 

In a like manner other SCIF polypeptides may be 
characterized by amino acid sequences which differ from 
5 the Obaru et al sequence or the sequence obtained from 
the clone of Example 1 due to allelic variations 
(naturally-occurring base changes in the species 
population which may or may not result in an amino acid 
change). SCIF polypeptides encoded by DNA sequences 

10 which differ in codon sequence due to the degeneracies of 

the genetic code or allelic variations are also expected 
to be useful in the method of this invention. Variations 
in the DNA sequence of SCIF which are caused by point 
mutations or by induced modifications to enhance the 

15 activity, half -life or production of the polypeptides 

encoded thereby are also encompassed in the invention and 
expected to be useful in the disclosed method. 

The method of the present invention may also 
employ the murine factor (also known as macrophage 

20 inflammatory protein I-alpha, MIP-1 alpha) . Murine MIP- 
1 beta does not have stem cell inhibitory activity. The 
sequence of the molecule here identified as murine SCIF 
is reported in Davatelis et al, J. Exp. Med. . 167:1939- 
1944 (1988) incorporated herein by reference. The stem 

25 cell inhibitory function of this molecule was not 



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17 

recognized in that reference. The murine factor may also 
be employed in the methods of this invention, provided 
that it provokes no antibody generation from the human 
immune system. 

5 Obviously other species analogs of SCIF may be 

employed in various veterinary uses of the above 
identified methods. 



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18 
TABLE I 

AAGGACACGG GCAGCAGACA GTGGTCATGC CTTTCTTGGC 40 
TCTGCTGACA CTCGAGCCCA CATTCCGTCA CCTGCTCAGA ATC 83 



ATG CAG GTC TCC ACT GCT GCC CTT GCT GTC CTC CTC TGC 122 
Met Gin Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys 
1 5 10 

ACC ATG GCT CTC TGC AAC CAG TTC TCT GCA TCA CTT GCT 161 
Thr Met Ala Leu Cys Asn Gin Phe Ser Ala Ser Leu Ala 
15 20 25 

GCT GAC ACG CCG ACC GCC TGC TGC TTC AGC TAC ACC TCC 200 
Ala Asp Thr Pro Thr Ala Cys Cys Phe Ser Tyr Thr Ser 
30 35 

CGG CAG ATT CCA CAG AAT TTC ATA GCT GAC TAC TTT GAG 239 
Arg Gin lie Pro Gin Asn Phe lie Ala Asp Tyr Phe Glu 
40 45 50 

ACG AGC AGC CAG TGC TCC AAG CCG GGT GTC ATC TTC CTA 278 
Thr Ser Ser Gin Cys Ser Lys Pro Gly Val lie Phe Leu 
55 60 65 

ACC AAG CGA AGC CGG CAG GTC TGT GCT GAC CCC AGT GAG 317 
Thr Lys Arg Ser Arg Gin Val Cys Ala Asp Pro Ser Glu 
70 75 

GAG TGG GTC CAG AAA TAT GTC AGC GAC CTA GAG CTG AGT 356 
Glu Trp Val Gin Lys Tyr Val Ser Asp Leu Glu Leu Ser 
80 85 90 

GCC TGA GGGGTCCAGA AGCTTCGAGG CCCAGCGACC 392 
Ala End 



TCGGTGGGCC AGTGGGGAGG AGCAGGAGCC TGAGCCTTGG GAAACATGCG 442 
TGTGACCTCC ACAGCTACCT CTTCTATGGA CTGGTTGTTG CCAAACAGCC 492 



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19 

ACACTGRGGG ACTCTTCTTA ACTTAAATTT TAATTTATTT ATTTATACTA 542 
TTTAGTTTTT GTAATTTATT TTCGATTTCA CAGTGTGTTT GTGATTGTTT 592 
GCTCTGAGAG TTCCCCTGTC CCCTCCCCCT TCCCTCACAC CGCGTCTGGT 642 
GACAACCGAG TGGCTGTCAT CAGCCTGTGT AGGCAGTCAT GGCACCAAAG 692 
CCACCAGACT GACAAATGTG TATCGGATGC TTTTGTTCAG GGCTGTGATC 742 
GGCCTGGGGA AATAATAAAG ACGCTCTTTT AAAAGGTAAA AAAAAAAAAA 792 
AAAAAA 798 



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20 

TABLE II 



* ** 

CTCGAGCCCA CATTCCATCA CCTGCTCCCA ATC ATG CAG GTC 42 

Met Gin Val 
1 



TCC 
Ser 


ACT 
Thr 
5 


GCT 
Ala 


GCC 
Ala 


CTT 
Leu 


GCC 
Ala 

*** 


GTC 
Val 
10 


CTC 
Leu 


CTC 
Leu 

* 


TGC 
Cys 


ACC 
Thr 


ATG 
Met 
15 


GCT 
Ala 


81 


CTC 
Leu 


TGC 
Cys 


AAC 
Asn 


CAG 
Gin 

. 20 


GTC 
Val 
* 


CTC 
Leu 

* 


TCT 
Ser 


GCA 
Ala 


CCA 
Pro 
*25 


CTT 
Leu 


GCT 
Ala 


GCT 
Ala 


GAC 
Asp 


12 0 


ACG 
Thr 
30 


CCG 
Pro 


ACC 
Thr 


GCC 
Ala 


TGC 
Cys 


TGC 
Cys 
35 


TTC 
Phe 


AGC 
Ser 


TAC 
Tyr 


ACC 
Thr 


TCC 
Ser 
40 


* 

CGA 
Arg 


CAG 
Gin 


159 


ATT 
He 


CCA 
Pro 


CAG 
Gin 
45 


AAT 
Asn 


TTC 
Phe 


ATA 
He 


GCT 
Ala 


GAC 
Asp 
50 


TAC 
Tyr 


TTT 
Phe 


GAG 
Glu 


ACG 
Thr 


AGC 
Ser 
55 


198 


AGC 
Ser 


CAG 
Gin 


TGC 

,Cys 


TCC 
Ser 


AAG 
Lys 
60 


CCC 
Pro 


* 

AGT 
Ser 
* 


GTC 
Val 


ATC 
He 


TTC 
Phe 
65 


CTA 
Leu 


ACC 
Thr 


AAG 
Lys 


237 




* 


























AGA 
Arg 


GGC CGG 
Gly Arg 
*70 


CAG 
Gin 


GTC 
Val 


TGT 
Cys 


GCT 
Ala 
75 


GAC 
Asp 


CCC 
Pro 


AGT 
Ser 


GAG 
Glu 


<GAG 
Glu 
80 


TGG 
Trp 


276 








* 




* 




* 














GTC 
Val 


CAG 
Gin 


AAA 
Lys 


TAC 
Tyr 


GTC 
Val 


AGT 
Ser 


GAC 
Asp 


CTG 
Leu 


GAG 
Glu 


CTG 
Leu 


AGT 
Ser 


GCC 
Ala 


TGA 
End 


315 



85 90 

[GGGGTCCAGA AGCTTCGAGG CCCAGCGACC TCGGTGGGCC AGTGGGGAGG 365 

AGCAGGAGCC TGAGCCTTGG GAAACATGCG TGTGACCTCC ACAGCTACCT 415 

CTTCTATGGA CTGGTTGTTG CCAAACAGCC ACACTGRGGG ACTCTTCTTA 465 



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21 



ACTTAAATTT 


TAATTTATTT 


ATTTATACTA 


TTTAGTTTTT 


GTAATTTATT 


515 


TTCGATTTCA 


CAGTGTGTTT 


GTGATTGTTT 


GCTCTGAGAG 


TTCCCCTGTC 


565 


CCCTCCCCCT 


TCCCTCACAC 


CGCGTCTGGT GACAACC]GA GTGGCTGTCA 


615 


TCGGCCTGTG 
* 


TAGGCAGTCA 


TGGCACCAAA 


GCCACCAGAC 


TGACAAATGT 


665 


GTATCAGATG 
* 


CTTTTGTTCA 


GGGCTGTGAT 


CGGCCTGGGG 


AAATAATAAA 


715 


GATGTTCTTT 


TAAACGGTAA 


AAA 






738 



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22 

This stem cell inhibitory function of LD78 or 
murine MIP alpha identified in the references described 
above, has heretofore never been identified as a function 
of this protein despite the extensive work of researchers 
5 on this factor. See, e.g., Obaru et al and Zip f el et al, 
cited above. 

The stem cell inhibitory factor acts on cycling 
stem cells by reversibly placing them in an undividing 
"resting" state. When the stem cells are initially 

10 treated in this way, the subsequent application of 

chemotherapy or radiation does not kill the stem cells 
because the cells are not dividing. Thus, after exposure 
to the chemotherapy or radiation, the stem cells may be 
"reactivated" to generate dividing progenitor cells, by 

15 discontinuing the administration of SCIF. 

An additional means of stimulating the resting 
stem cells into division may also be the administration 
to the subject of other colony stimulating factors, e.g., 
M-CSF, CSF-1, GM-CSF, G-CSF, Meg-CSF or other cytokines, 

20 such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, 

IL-11 and erythropoietin following the chemotherapy or 
radiation exposure. 



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23 

SCIF polypeptides or active fragments having 
stem cell inhibitory activity, may be produced by known 
conventional chemical synthesis or recombinant techniques 
employing the amino acid and DNA sequences of Table I or 
5 Table II, above. For example, SCIF polypeptides may be 

produced by culturing a suitable cell or cell line, which 
has been transformed with a DNA sequence coding on 
expression for a SCIF polypeptide or active fragment 
thereof under the control of known regulatory sequences. 

10 The resulting protein may be isolated from the cells, 
cell lysate, or medium by conventional techniques. 
Suitable techniques for such production of recombinant 
SCIF are described in, e.g., T. Maniatis et al, Molecular 
Cloning-A Laboratory Manual, Cold Spring Harbor 

15 Laboratory, Cold Spring Harbor, NY (1982). 

Methods for constructing SCIF polypeptides 
useful in the method of the present invention by chemical 
synthetic means are also known to those of skill in the 
art. See, e.g., Merrifield in J.A.C.S . 85 ; 2149-2154 

20 (1963) or Peptide Synthesis" by Bodanszky, et al, second 

edition, John Wiley and Sons, 1976. 

The recombinant or synthetically-constructed 
SCIF polypeptide sequences, by virtue of sharing primary, 
secondary, or tertiary structural and conformational 

25 characteristics with SCIF polypeptides may possess the 



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24 

same biological characteristic of inhibiting stem cell 
division in common with the human factor identified above 
in Table I or Table II. 

Modifications in the peptides or DNA sequences 
5 encoding SCIF or active fragments thereof may also be 

made and are believed to be useful also in the method of 
this invention, where the modified SCIF peptide or 
fragment thereof shares the desired stem cell inhibitory 
biological activity* Modifications of interest in the 

10 SCIF sequences may include the replacement, insertion or 

deletion of a selected amino acid residue in the coding 
sequences. Mutagenic techniques for such replacement, 
insertion or deletion are well known to one skilled in 
the art. [See, e.g., United States Patent No. 

15 4,518,584.] 

Specific mutations of the sequences of the SCIF 
polypeptide may involve the insertion of an asparagine- 
linked glycosylation site, either Asp-X-Thr or Asp-X-Ser, 
where X can be any amino acid, into the sequence or a 

20 sequence modification at any site of the molecule that is 
modified by addition of 0-1 inked carbohydrate. 

Other analogs and derivatives of the sequence 
of SCIF which would be expected to retain SCIF activity 
in whole or in part may also be easily made by one of 

25 skill in the art and may be useful in the methods of this 
invention. 



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25 

Recombinant production of SCIF is presently 
preferred. The human cDNA may be isolated and inserted 
into suitable cells or cell lines under the control of 
appropriate regulatory sequences. Suitable host cells 
5 for production of the protein may be mammalian cells, 

such as Chinese hamster ovary cells (CHO) or 3T3 cells. 
The selection of suitable mammalian host cells and 
methods for transformation, culture, amplification, 
screening and product production and purification are 

10 known in the art. See, e.g., Gething and Sambrook, 

Nature , 293 :620-625 (1981), or alternatively, Kaufman et 
al, Mol. Cell. Biol. . 5(7) : 1750-1759 (1985) or Howley et 
al, U. S. Patent No. 4,419,446. Other suitable mammalian 
cell lines are the monkey COS-1 cell line, and the CV-1 

15 cell line. 

Similarly useful as host cells suitable for the 
production of SCIF for use in the present invention are 
bacterial cells. For example, the various strains of JL_ 
coli (e.g., HB101, MC1061 and strains used in the 
20 following examples) are well-known as host cells in the 

field of biotechnology. Various strains of subtilis. 
Pseudgmonas, other bacilli and the like may also be 
employed in this method. 



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26 

Many strains of yeast cells known to those 
skilled in the art are also available as host cells for 
expression of the polypeptides of the present invention. 
Additionally, where desired, insect cells may be utilized 
5 as host cells in the method of the present invention. 

See, e.g., Miller et al, Genetic Engineering r 8:277-298 
(Plenum Press 1986) and references cited therein. 

It may also be possible to employ one or more 
peptide fragments of SCIF, which retain the stem cell 

10 inhibitory activity of intact SCIF molecule, for use in 

the present methods for protecting a subject's stem cells 
from destruction by exposure to an agent capable of 
killing dividing stem cells. 

For use in the method for protecting stem cells 

15 according to this invention a therapeutically effective 

amount of the SCIF protein or a therapeutically effective 
fragment thereof may be employed in admixture with a 
pharmaceutical^ acceptable carrier. Where desirable, 
this SCIF composition can be systemically administered 

20 parenterally. In clinical applications, it may be 

desirable to target the SCIF to the blood-forming tissue, 
e.g., bone marrow. This targeting can be achieved by 
injecting the SCIF, normally by infusion or bolus 
intravenous administration. Alternatively, the SCIF can 

25 be targeted by varying the pharmaceutical formulation of 
the drug, e.g. by linking it to agents which have been 



WO 91/04274 



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27 

shown to target to the bone marrow. This formulation can 
be administered intravenously. If desirable, the 
composition may be administered subcutaneously. 

When systematically administered, the 
5 therapeutic composition for use in this invention is in 
the form of a pyrogen-free, parenterally acceptable 
aqueous solution. The preparation of such a 
pharmaceutically acceptable protein solution, having due 
regard to pH, isotonicity, stability and the like, is 

10 within the skill of the art. For administration in the 
method for treating hyperproliferating stem cells, the 
composition containing SCIF may be administered topically 
or through a transdermal patch to localize its effect on 
the area of hyperprol iteration . 

15 The dosage regimen involved in a method for 

treating the subject anticipating exposure to such 
cytotoxic agents or for treatment of hyperproliferating 
stem cells will be determined by the attending physician 
considering various factors which modify the action of 

20 drugs, e.g. the condition, body weight, sex and diet of 
the patient, the severity of any infection, time of 
administration and other clinical factors. Generally, 
the daily regimen should be in the range of 1-1000 
micrograms of SCIF protein or fragment thereof per 

25 kilogram of body weight. 



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28 

Following the subject" s exposure to the 
cytotoxic agent or radiation, the therapeutic method of 
the present invention may also employ administering to 
the subject one or more lymphokines, colony stimulating 
5 factors or other cytokines, hematopoietins , interleukins, 
growth factors to generally stimulate the growth and 
division of the stem cells inhibited by the prior 
treatment with SCIF. Such therapeutic agents which 
encourage hematopoiesis include IL-1, IL-2, IL-3, IL-4, 

10 IL-5, IL-6, IL-7, Meg-CSF, M-CSF, CSF-1, GM-CSF, G-CSF or 

erythropoietin. The dosages of these agents may be 
employed in the same ranges as the dosages for SCIF 
recited above. In a like manner, these dosages would be 
adjusted to compensate for variations in the physical 

15 condition of the patient, and the amount and type of 

chemotherapeutic agent or radiation to which the subject 
was exposed. Progress of the reversal of the inhibition 
of the stem cells caused by administration of SCIF in the 
treated patient can be monitored by conventional methods. 

20 In the treatment of leukemia, it may prove 

beneficial to administer both SCIF to inhibit normal stem 
cell cycling and a stimulator of leukemic cell growth, 
such as IL-3 or GM-CSF, simultaneously with the cytotoxic 
drug treatment or during radiation. By this protocol, it 

25 should be possible to achieve the greatest differences 



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between the cycling statuses of normal and leukemic 
cells. 

The following examples illustratively describe 
the use of murine SCIF in the in vitro stem cell assay. 
5 These examples are for illustration and do not limit the 
scope of the present invention. 

Example 1 - Obtaining Human SCIF cDNA 

Polyadenylated RNA isolated from the human T 
cell line C10-MJ2 was used as a template for synthesis of 
10 single stranded complementary DNA by standard procedures 
[see, e.g., Maniatis et al., cited above]. The presence 
of cDNA's encoding human SCIF were demonstrated by the 
polymerase chain reaction (PCR) employing stringent 
conditions [R. K. Saiki et al., Science , 130:1350 (1985)] 
15 using the following oligonucleotides based on the 
sequence of Table I: 

5' AGCTCGAGAT CATGCAGGTC TCCACTG 3' 
5 1 GCGAATTCCC TCAGGCACTC AGCTCCA 3 1 . 

Double-stranded SCIF DNA obtained as the 
20 product of the PCR and labeled with (alpha 3Z P) dCTP by 
random priming was used to identify SCIF cDNAs in a 
previously constructed C10-MJ2 cDNA expression library 
[J. F. Moreau et al, Nature , 336:690 (1988)]. 



Conditioned media obtained from COS cells 
transfected with full-length SCIF cDNAs in the proper 
orientation were analyzed according to the assay 
described in Example 3. Sequencing of selected clones, 
named pXMT2.Al, pXMT2.A3 through pXMT2.A9, is ongoing. 

The human SCIF sequence of Table II differs 
substantially from the Obaru et al LD 78 sequence of 
Table I, from which the oligonucleotides were derived. 
There are 18 base changes, 4 amino acid changes as well 
as three additional bases and an additional amino acid. 

Example 2 - In Vitro Stem Cell Assay 

For the detection of stem cells In vitro 10 4 
murine bone marrow cells from the murine macrophage cell 
line, J774.2 [P. Ralph et al, J, Immunol. , 114:898-905 
(1975)] in 4 ml supplemented alpha-modified minimal 
essential medium (MEM) containing 25% fetal calf or horse 
serum and 0.3% agar were seeded on top of an underlayer 
of the same medium containing 0.6% agar, 10% L929 cell 
conditioned medium (L929 CM, a source of the growth 
factor CSF-1) and 10% AF1-19T cell conditioned medium 
(AF1-19T CM) (a source of the growth factor GM-CSF and 
other uncharacterized stem cell growth factors) in a 6 cm 
petri dish. Cultures were incubated at 37° C in a fully 
humidified atmosphere of 10% C0 2 , 5%, 0 2 , 85% N 2 for 11 
days. Colonies can be stained with INT 2- (p-iodophenyl) - 



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31 

3-(p-nitrophenyl)-5-phenyl-tetrazolium chloride hydrate 
overnight. 

While some colonies are present in the CFU-A 
assay having a diameter less than 2 mm, this value was 
5 selected as a useful cut-off point after preliminary 

experiments were performed using cytosine arabinoside. 
Within individual dishes, colonies with a diameter <2 mm 
were mainly derived from cells in cycle, whereas colonies 
>2 mm were found to be derived from minimally 

10 proliferating cells- Only colonies with diameters >2 mm 
were scored in these assays. 

The assay, also described in I. B. Pragnell et 
al, Blood , 72:196-201 (1988), demonstrates that more 
mature progenitor cells are unaffected by treatment with 

15 SCIF, while cycling stem cells, after treatment with 

SCIF, become resistant to the action of cytosine 
arabinoside. If the treated stem cells are then washed 
with buffered saline to remove the cytotoxic drug and 
SCIF the surviving stem cells proliferate in culture 

20 normally as illustrated below. 

Example 3 - Demonstration of the Effect of SCIF on CFU-A 

Bone marrow cells were incubated in paired 
tubes containing 5 x 10 6 cells in 1 ml Fischer" s medium 
supplemented with 20% horse serum. SCIF or alpha-MEM was 
25 added to each tube and Fischer f s medium was added to 



WO 91/04274 PCI7US90/0S451 

32 

control tubes. The mixtures were incubated at 37 °C for 5 
hours (inhibition assays) . For the last 60 minutes of 
the incubation 10" 3 *! cytosine arabinoside was added to one 
tube and an equal volume of medium to the other tube. 
5 Cells were then washed twice before being assayed in the 
CFU-A assay as described above. SCIF was found to reduce 
the number of stem cells in cycle from an average of 
greater than 30% to an average of less than 10%. The 
untreated cells were killed by the cytotoxic drug 
10 treatment in contrast to the inhibitor treated stem 
cells. 

Pure preparations of SCIF also reversibly 
trigger multipotential stem cells out of cycle when 
assayed in vivo using ara-C in the CFU-S assay described 

15 in Pragnell et al, cited above. 

The purified and sequenced SCIF of this 
invention is a cytokine which has the ability to 
specifically reduce the proportion of haemopoietic stem 
cells in DNA synthesis, thereby protecting them from 

20 cytosine arabinoside, a cell-cycle specific cytotoxic 

drug. In contrast, SCIF does not affect the 
proliferation of more mature progenitors and so appears 
to be a specific regulator of the stem cell compartment. 
SCIF is active in the 200-500 pM range, as measured in 

25 the CFU-A direct addition assays. 



WO 91/04274 . PCT/US90/05451 

33 

Example 4 - Expression of Recombinant Human SCIF 

To produce SCIF, the cDNA encoding it is 
transferred into an appropriate expression vector of 
which numerous types are known in the art for mammalian, 
5 insect, yeast, fungal and bacterial expression by 

standard molecular biology techniques. See, e.g., Y. C. 
Yang et al, Cell , 47:3-10 (1986). One such vector is the 
COS cell expression vector, pXM, containing the SV40 
enhancer, major adenovirus late promoter, DHFR coding 

10 sequence, SV40 late message poly A addition site and Val 
gene. This vector may be linearized with the 
endonuclease enzyme Xhol and ligated to equimolar amounts 
of SCIF cDNA which has been previously modified by the 
addition of synthetic oligonucleotides that generate 

15 complementary Xhol cohesive ends. Such oligonucleotides 

are commercially available [Collaborative Research, 
Lexington, MA]. The vector is then introduced into 
appropriate host cells by conventional genetic 
engineering techniques. 

20 a. Mammalian Cell Expression 

To obtain expression of the SCIF 
polypeptide for use in the assays described below, the 
pXM vector containing the SCIF DNA sequence is 
transfected onto COS cells, for example. The conditioned 

25 medium for the transfected COS cells contains SCIF 



WO 91/04274 



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34 

biological activity as measured in the assay described in 
Example 2. 

The mammalian cell expression vectors 
described herein may be synthesized by techniques well 
5 known to those skilled in this art. The components of 

the vectors , e.g. repl icons, selection genes, enhancers, 
promoters, and the like, may be obtained from natural 
sources or synthesized by known procedures. See, Kaufman 
et al, J. Mol. Biol. . 159:511-521 (1982); and Kaufman, 

10 Proc. Natl. Acad. Sci. . USA . 82:689-693 (1985). 

Exemplary mammalian host cells include particularly 
primate cell lines and rodent cell lines, including 
transformed cell lines. Normal diploid cells, cell 
strains derived from in vitro culture of primary tissue, 

15 as well as primary explants, are also suitable. 

Candidate cells need not be genotypically deficient in 
the selection gene so long as the selection gene is 
dominantly acting. For stable integration of the vector 
DNA, and for subsequent amplification of the integrated 

20 vector DNA, both by conventional methods, CHO cells may 
be employed. Alternatively, the vector DNA may include 
all or part of the bovine papilloma virus genome [Lusky 
et al, Cell . 36:391-401 (1984)] and be carried in cell 
lines such as C127 mouse cells as a stable episomal 

25 element. Other suitable mammalian cell lines include but 
are not limited to, HeLa, COS-1 monkey cells, mouse L-929 



WO 91/04274 PCI7US90/0S451 

35 

cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, 
BHK or HaK hamster cell lines. 

Stable trans formants are then screened for 
expression of the product by standard immunological or 
5 enzymatic assays. The presence of the DNA encoding the 

SCIF polypeptides may be detected by standard procedures 
such as Southern blotting. Transient expression of the 
DNA encoding the polypeptides during the several days 
after introduction of the expression vector DNA into 

10 suitable host cells, such as COS -1 monkey cells, is 

measured without selection by activity or immunologic 
assay of the proteins in the culture medium. 

One skilled in the art can also construct 
other mammalian expression vectors comparable to the 

15 pXM/SCIF vector by, e.g., inserting the DNA sequence of 

SCIF from the respective plasmids with Xhol and employing 
well-known recombinant genetic engineering techniques and 
other known vectors, such as pJL3 and pJL4 [Gough et al., 
EMBO J. , 4:645-653 (1985)] and pMT2 (starting with pMT2- 

20 VWF, ATCC #67122; see PCT application PCT/US87/00033) . 

The transformation of these vectors into appropriate host 
cells can result in expression of the SCIF polypeptides, 
b. Bacterial Expression Systems 

Similarly, one skilled in the art could 

25 manipulate the sequence of SCIF by eliminating any 
mammalian regulatory sequences flanking the coding 



WO 91/04274 PCT/US90/0S451 

36 

sequences and inserting bacterial sequences to create 
bacterial vectors for intracellular or extracellular 
expression of the SCIF polypeptides of the invention by 
bacterial cells. The DNA encoding the factor may be 
5 further modified to contain different codons for 

bacterial expression as is known in the art. Preferably 
the sequence is operatively linked in- frame to a 
nucleotide sequence encoding a secretory leader 
polypeptide permitting bacterial expression, secretion 

10 and processing of the mature variant protein, also as is 

known in the art. The compounds expressed in bacterial 
host cells may then be recovered, purified, and/or 
characterized with respect to physiochemical, biochemical 
and/ or clinical parameters, all by known methods. 

15 c. Insect or Yeast Cell Expression 

Similar manipulations can be performed for 
the construction of an insect vector [See, e.g., 
procedures described in published European patent 
application 155,476] for expression in insect cells. A 

20 yeast vector could also be constructed employing yeast 
regulatory sequences for intracellular or extracellular 
expression of the proteins of the present invention by 
yeast cells. [See, e.g., procedures described in 
published PCT application WO 86/00639 and European patent 

25 application EP 123,289.] 



37 

Example 5 - Construction of CHO C^ H Lines Expressing 
High Levels of SCIF 

One method for producing high levels of the 
SCIF polypeptides of the invention from mammalian cells 
involves the construction of cells containing multiple 
copies of the heterologous SCIF gene. The heterologous 
gene can be linked to an amplifiable marker, e.g., the 
dihydrofolate reductase (DHFR) gene for which cells 
containing increased gene copies can be selected for 
propagation in increasing concentrations of methotrexate 
(MTX) according to the procedures of Kaufman & Sharp, 
Mol. Biol. . (1982) supra . This approach can be employed 
with a number of different cell types. 

For example, the pXM vector containing a SCIF 
gene in operative association with other plasmid 
sequences enabling expression thereof and the DHFR 
expression plasmid pAdD26SV(A)3 (Kaufman & Sharp, Mol. 
Cell Biol. . 3(9) :1598-1608 (1983) can be co-introduced 
into DHFR-deficient CHO cells, DUKX-BII, by calcium 
phosphate coprecipitation and transf ection. 
Alternatively, the SCIF gene may be introduced into pMT2 
as previously mentioned and the resultant vector used in 
place of pXM/SCIF and pAdD26SV(A) 3 . DHFR expressing 
transformants are selected for growth in alpha media with 
dialyzed fetal calf serum, and subsequently selected for 
amplification by growth in increasing concentrations of 



WO 91/04274 



PCT/US90/05451 



38 

MTX (sequential steps in 0.02, 0.2, 1.0 and 5uM MTX) as 
described in Kaufman et al., Mol. Cell Biol, . 5:1750 
(1983). Transformants are cloned, and biologically 
active SCIF polypeptide expression is monitored by the 
5 assay of Example 2. SCIF polypeptide expression is 
expected to increase with increasing levels of MTX 
resistance. 

Numerous modifications and variations in 
practice of this invention are expected to occur to those 
10 skilled in the art. 



WO 91/04274 



PCT/US90/05451 



39 
CLAIMS 

1. A stem cell inhibitory protein substantially free 
from other human proteinaceous materials and comprising 
the amino acid sequence of Table II. 

2. A pharmaceutical composition useful for inhibiting 
stem cell division in a human subject exposed to or 
anticipating exposure to an agent capable of damaging or 
destroying stem cells undergoing division comprising an 
effective amount of a human stem cell inhibitory factor 
in admixture with a suitable carrier. 

3. The composition according to claim 2 wherein said 
stem cell inhibitor is all or a biologically active 
fragment of the human protein having the same or 
substantially the same amino acid sequence of Table I, an 
allelic variant thereof, a polypeptide having a DNA 
sequence capable of hybridizing to the DNA sequence of 
Table I and having stem cell inhibiting activity, or a 
biologically active fragment thereof. 

4. The composition according to claim 2 wherein said 
stem cell inhibitor is all or a biologically active 
fragment of the protein having the same or substantially 
the same amino acid sequence of Table II, an allelic 
variant thereof, a polypeptide having a DNA sequence 



WO 91/04274 



PCI7US90/05451 



40 

capable of hybridizing to the DNA sequence of Table II 
and having stem cell inhibiting activity, or a 
biologically active fragment thereof. 

5. A method for preparing an autologous bone marrow 
sample for transplantation comprising treating said 
marrow ex vivo with an effective amount of a human stem 
cell inhibitory factor substantially free from other 
human proteinaceous materials and comprising the amino 
acid sequence of Table II to inhibit stem cell division; 
and purging said marrow of cancerous cells by 
administering an effective amount of a chemotherapeutic 
agent or radiation. 

6. A DNA sequence consisting essentially of a DNA coding 
on expression for a stem cell inhibitory protein 
substantially capable of inhibiting growth of stem cells 
in humans, said DNA sequence encoding a peptide sequence 
encompassing the mature form of the polypeptide of Table 
II and allelic variations thereof also capable of 
inhibiting the growth of stem cells in humans. 

7. A vector comprising a DNA sequence consisting 
essentially of a DNA coding on expression for a stem cell 
inhibitory protein substantially capable of inhibiting 
growth of stem cells in humans, said DNA sequence 
encoding a peptide sequence encompassing the mature form 



WO 91/04274 



PCT/US90/0S451 



41 

of the polypeptide of Table II and allelic variations 
thereof in association with regulatory control sequences 
capable of directing the replication and expression 
thereof in host cells. 

8. A method for producing a stem cell inhibitory protein 
substantially free from other human proteinaceous 
materials and comprising the amino acid sequence of Table 
II comprising culturing a cell transfected with the DNA 
sequence encoding said protein, said DNA under the 
control of regulatory control sequences capable of 
directing the replication and expression thereof in said 
cell. 

9. The use of a stem cell inhibitory protein 
substantially free from other human proteinaceous 
materials and comprising the amino acid sequence of Table 
II in a pharmaceutical composition for the treatment of 
subjects anticipating exposure to agents which damage 
dividing stem cells. 



INTERNATIONAL SEARCH REPORT 

International Application No 



PCT/US 90/05451 



1. CLASSIFICATION OF SUBJECT MATTER (it »vtfH classification tymbolt apply, indicate •»> * 



According to International Patant Classification (IPC) or to both National Claseifieation and IPC 



IPC 



5. c 07 K 13/00, A 61 K 35/28, C 12 N 15/19, A 61 K 37/02 



II. FIELDS SEARCHED 



Minimum Documentation Searched ' 



Classification System 



Classtfication Symbols 



IRT 



C 07 K f A 61 K, C 12 N 



Documentation Searched other than Minimum Documentation 
to the Extent that such Documents are Included In the Fields Searched 



III. DOCUMENTS CONSIDERED TO »1 RELEVANT* 



Category ' 



r;,..,„» m foment " with Indication, where appropriate, of the relevant passsgea » 



The Journal of Immunology, vol. 142, no. 5, 
1 March 1989, The American Association 
of Iramunologists, (US), 

P.F. Zipfel et al.: "Mitogenic activation 
of human T cells induces two closely 
related genes which share structural 
similarities with a new family of 
secreted factors", pages 1582-1590 
see page 1582, column 2, lines 4-23, 
36-42; page 1583, column 1, lines 6-11; 
page 1585, figure 3B; page 1586, 
column 1, lines 8-18; discussion 
cited in the application 



EP, A, 0317141 (BECTON DICKINSON AND CO.) 
24 May 1989 
see claims 1,2,5 



1-4,6-9 



■ /. 



• Special categories of cited documents: « 

-A" document defining the general state of the art which la not 

considered to be of particular relevance 
"E* earlier documant but publiahed on or after the International 

filing data 

-L- document which may throw doubts on priority c ' ,im <"L2r" 
which ia cited to eatabliah the publication date of another 
citation or other apecial reaaon (aa specified) 

"O* document referring to an oral disclosure, use, aihlblUon or 
other means 

•P" document publiahed prior to the internetlonal filing data but 
later than the priority date claimed 



T" later document publiahed after the International filing date 
o^r priorSy date and not in conflict with the apphcat on but 
cited to understand the principle or theory underlying the 
invention 

-X- document of particular relevance; the £l™!fj^ tl0 | !! 

cannot be considered novel or cannot be conaWered to 

involve en inventive step 
»Y" document of particular relevance;' ^the claim** { ^lTSH 

cannot be considered to Involve an mveirtive 

document is combined with one or more j rthei jw^ jocu. 

ments, such combination being obvious to e person skilled 

tn tha art 

•t" document member of the same patent family 



IV. CIWTIFICATiOH __ 

Data of the Actual Completion of the International Search 

31st January 1991 



Date of Mailing of this International Search Report 

2 2.02 91 



International Searching Authority 

EUROPEAN PATENT OFFICE 



Signature of Authortied Officer 

miss T. MORTENSEiy /W/ 




Form PCT/ISAftlO (second sheet) (January 1916) 



lntarnattonal Application No PCT/US 90/05451 



III. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) 


Category 4 


Citation of Document. " with indication, whn approDflata, of th« ralavant paaaagas 


Rttavant to Claim No. 


X 


J. Biochem., vol. 99 f no. 3, 1986, 
( Tokyo , JP ) , 

K. Obaru et al. : "A cDNA clone used to 
study mRNA inducible in human tonsillar 
lymphocytes by a tumor promoter", 
pages 885-894 

see introduction; page 889, figure 3; 
discussion 
cited in the application 


1-4,6-9 


P,X 


Nucleic Acids Research, vol. 18 r no. 11, 
11 June 1990, 

S.G. Irving et al.: "Two inflammatory 
mediator cytokine genes are closely 
linked and variably amplified on 
chromosome 17q", pages 3261-3270 
see the whole article 


1-4,6-9 



Form PCT/ISA 210 (extra sheet) (January 1985) 



ANNEX TO THE INTERNATIONAL SEARCH REPORT 

ON INTERNATIONAL PATENT APPLICATION NO. US 9005451 

SA 41492 

This 8 nn« lists the patent famDy members rating to the _p.tem documents cited to the above^nenooned international search report. 
The members are as contained to the European Patent Office EDP file on 15/02/91 u,r„ TO ,ri B „ 
Se Eur^Sn PatfnJ : Offceis to no way liable for these particulars which are merely live* for the purpose of information. 



Patent document 
cited in search report 



Publication 
date 



Patent family 
members) 



Publication 
date 



EP-A 



- 0317141 24-05-89 JP-A- 2002345 



08-01-90 



S For more details about this annex 



: see Official Journal of the European Patent Office, No. 12/82 



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Defective images within this document are accurate representations of the original 
documents submitted by the applicant. 

Defects in the images include but are not limited to the items checked: 

□ BLACK BORDERS 

□ IMAGE CUT OFF AT TOP, BOTTOM OR SIDES 
□TfADED TEXT OR DRAWING 
[^BLURRED OR ILLEGIBLE TEXT OR DRAWING 

□ SKEWED/SLANTED IMAGES 

□ COLOR OR BLACK AND WHITE PHOTOGRAPHS 

□ GRAY SCALE DOCUMENTS 

□ LINES OR MARKS ON ORIGINAL DOCUMENT « 

□ REFERENCE(S) OR EXHIBIT(S) SUBMITTED ARE POOR QUALITY 

□ OTHER: . 

IMAGES ARE BEST AVAILABLE COPY. 
As rescanning these documents will not correct the image 
problems checked, please do not report these problems to 
the IFW Image Problem Mailbox.