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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 4 

A61K 37/02, C07K 15/00 
C12N 15/00, C07K 3/02 



Al 



(II) International Publication Number: WO 89/10133 

(43) International Publication Date : 2 November 1989 (02. 1 1.89) 



(21) International Application Number: PCT/GB89/00396 

(22) International Filing Date : 2 1 April 1 989 (2 1 .04.89) 



(30) Priority data: 
8809419.8 



21 April 1988(21.04.88) GB 



(71)(72) Applicant and Inventor: PRAGNELL, Ian, Bernard 
[GB/GB]; Beatson Institute for Cancer Research, Gar- 
scube Estate, Bearsden, Glasgow G61 1BD (GB). 

(74) Agents: GOLDIN, Douglas, Michael et al.; J.A. Kemp & 
Co., 14 South Square, Gray's Inn, London WC1R 5EU 
(GB). 



(81) Designated States: AT (European patent), BE (European 
patent), CH (European patent), DE (European patent), 
FR (European patent), GB (European patent), IT (Euro- 
pean patent), JP, LU (European patent), NL (European 
patent), SE (European patent), 



US. 



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: STEM CELL INHIBITORS 



(57) Abstract 



A polypeptide, capable of interacting with haemopoietic stem cells so as to protect them against the cell cycle specific cyto- 
toxic drugs used in cancer chemotherapy, is isolated from macrophage cells of bone marrow origin. 



FOR THE PURPOSES OF INFORMATION ONLY 



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



AT 


Austria 


FR 


France 


ML 


Mali 


AU 


Australia 


GA 


Gabon 


MR 


Mauritania 


BB 


Barbados 


GB 


United Kingdom 


MW 


Malawi 


BE 


Belgium 


HU 


Hungary 


NL 


Netherlands 


BG 


Bulgaria 


IT 


Italy 


NO 


Norway 


BJ 


Benin 


JP 


Japan 


RO 


Romania 


BR 


Brazil 


KP 


Democratic People's Republic 


SD 


Sudan 


CF 


Central African Republic 




of Korea 


SE 


Sweden 


CG 


Congo 


KR 


Republic of Korea 


SN 


Senegal 


CH 


Switzerland 


LI 


Liechtenstein 


su 


Soviet Union 


CM 


Cameroon 


LK 


Sri Lanka 


TD 


Chad 


DE 


Germany, Federal Republic of 


LU 


Luxembourg 


TG 


Togo 


DK 


Denmark 


MC 


Monaco 


US 


United States of America 


n 


Finland 


MG 


Madagascar 







WO 89/10133 



PCT/GB89/00396 



TITLE: STEM CELL INHIBITORS 

THIS INVENTION relates to stem cell inhibitors 
and is particularly concerned with improvements in the 
management of cancer chemotherapy. 

It is well-known that medullary aplasia 
5 represents a limiting factor for the clinical use of 

cytotoxic drugs which are active in cycling cells during 
chemotherapy of cancer. It has been recognised for many 
years that existing methods of chemotherapy could be 
improved if it were possible to protect the haemopoietic 

10 stem cells during treatment with the cytotoxic drug but, 
in spite of extensive research in this area, no suitably 
specific inhibitory agent from a readily amenable source 
has previously been discovered. One of the reasons for 
this lack of progress is believed to be the difficulty in 

15 developing a suitable in vitro assay to monitor stem cell 
regulation. 

We have now been able to isolate and identify a 
proteinaceous substance, obtainable from certain 
macrophage cell lines, that has the ability to interact 
20 with haemopoietic stem cells so as to protect them against 
the cell cycle specific cytotoxic drugs used in cancer 
chemotherapy. 

Accordingly, the present invention provides a 
haemopoietic stem cell inhibitor characterised by the 
25 following properties: 



WO 89/10133 



PCT/GB89/00396 



- 2 - 

1. The inhibitor activity is sensitive to 
degradation with trypsin and the inhibitor is therefore 
proteinaceous. 

2. The inhibitor when partially purified by 

5 treatment on an anion-exchanger shows a molecular weight 
range 45-60 Kd on a molecular weight analysis resin. 

3. The inhibitor, when purified to a single band on 
polyacrylamide gel electrophoresis, under reducing 

10 conditions, shows a molecular weight range of 8-10 Kd and 
a slightly higher molecular weight range under 
non-reducing conditions. 

4. The inhibitor is heat-stable as follows: 

(i) Inhibitory activity is retained after heating 
15 for 1 hour at 37°C, 55°C and 75°C. 

(ii) Inhibitory activity is retained after heating 
for 10 minutes at 100°C. 

5. The inhibitor binds to anion-exchangers and can 
be eluted with a salt gradient at between 0.26 and 0.28 

20 molar NaCl. 



WO 89/10133 



PCT/GB89/00396 



- 3 - 

6. The inhibitor binds to a Heparin Sepharose 
affinity chromatography resin and can be eluted from the 
resin with one molar sodium chloride buffer. 



7. The inhibitor binds avidly to Blue Sepharose 

5 affinity chromatography resin and can be eluted from the 
resin with 5M magnesium chloride. 

8. Cycling stem cells, when treated with the 
partially purified inhibitor, become resistant to the 
action of cytosine arabinoside. If the treated stem 

10 cells are then washed with buffered saline to remove the 
cytotoxic drug and inhibitor, the surviving stem cells 
proliferate in culture normally. 

The isolation and identification of the 
inhibitor has been facilitated by the development, for the 

15 first time, of an In vitro assay to monitor stem cell 

regulators. Cycling progenitor cells (more mature cells, 
not stem cells) are unaffected by the inhibitor. Direct 
addition of the purified inhibitor to this assay results 
in inhibition of macroscopic colony development but not on 

20 other colonies in the assay. 



WO 89/10133 



PCT/GB89/00396 



- 4 - 

The in vitro stem cell (CFU-A) assay 

For the detection of stem cells in vitro 10 4 
bone marrow cells in 4 ml supplemented alpha-modified 
minimal essential medium (MEM) containing 25% foetal calf 
5 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 

10 and other uncharacter ised 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 were stained INT 2- (p-iodophenyl)- 
3- (p-nitrophenyl)-5-phenyl-tetrazolium chloride hydrate 

15 overnight. Whilst there are colonies in the CFU-A assay 
with a diameter less than 2 mm, we have chosen this value 
as a useful cut-off point after preliminary experiments 
were performed using cytosine arabinoside. We found that 

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



WO 89/10133 



PCT/GB89/00396 



The inhibitor of the present invention is 
obtainable from several macrophage cell lines. Interest 
centres initially upon the various known macrophage cell 
lines of bone marrow origin, typically murine bone marrow 
5 as this represents a major source of such macrophage cell 
lines. Our screening of a population of such cell lines 
has identified at least two known cell lines capable of 
producing our inhibitor. In the Examples illustrating 
this invention, we describe the isolation from one such 
10 known macrophage cell line. 

As an alternative to the use of known macrophage 
cell lines, it is also possible to produce suitable 
macrophage cell lines by the transformation of bone marrow 
cells with a retrovirus transforming agent. 
!5 The inhibitor of the invention can be produced 

quite simply from the inhibitor-producing macrophage cell 
line by cultivating the cell line in an appropriate growth 
medium under conventional conditions, e.g. 37°C, the use 
of aerobic conditions with 5% v/v carbon dioxide in air 
20 provides an ideal growth environment, until the cell 
concentration is approximately 10 6 per ml. At this 
stage, the still growing cells are separated from the 
growth medium, e.g. by centr if ugation or preferably 
membrane filtration and the inhibitor can then be 
25 recovered from the supernatant. In order to demonstrate 



WO 89/10133 



PCI7GB89/00396 



inhibitor activity, it is desirable that the supernatant 
first be concentrated, e.g. using membrane dialysis to 
give a concentration of about 20^fold. The inhibitor can 
then be isolated from the concentrated supernatant by 
5 chromatographic procedures. 

A first step of purification may include passing 
the concentrated supernatant over an anion-exchange resin 
and eluting a fraction using a 0.25-0.30M NaCl solution. 
A second step of purification may include 

10 passing the product from the first step over a 

Sepharose-Heparin column and eluting a fraction using NaCl 
solution of molarity at least 1M. 

A third step of purification may include passing 
the product from the second step over Sepharose-Blue and 

15 eluting a fragment using MgCl 2 solution of molarity at 
least 3M. This elution gives a product that shows a 
single band on polyacrylamide gel electrophoresis under 
reducing conditions. 

While the main application of the inhibitor of 

20 the invention is in clinical practice, as will be 
described in more detail below, the inhibitor or 
imraunogenically active fragment thereof can also be 
used as an immunogen to raise antibodies that will 
recognise part or all of the inhibitor, by immunising a 

25 host animal and recovering from the host animal antibodies 



WO 89/10133 PCT/GB89/00396 

7 - 

or antibody producing cells. Such antibodies can be 
prepared e.g. in rabbits where polyclonal antibodies can 
be recovered from the serum of the rabbit or can be used 
as immunogens in mice for the production of monoclonal 
5 antibodies by conventional techniques. 

The present invention also extends to DNA 
sequences encoding the inhibitor of the invention. Such 
DNA is of interest in the production of the inhibitor by 
recombinant DNA techniques. Such techniques can involve 
10 two different approaches. Both approaches require, as a 
first step, the production of a gene library from the 
messenger RNA of the macrophage cell line that naturally 
produces the inhibitor. This involves the production of a 
complementary DNA expressible in a bacterial or other host 
15 cells. 

Having produced the cDNA library, a first 
approach involves the production and use of DNA probes. 
Limited sequence analysis of the inhibitor will permit the 
synthesis of oligonucleotides that can be used to probe 

20 the DNA library to identify those cDNA's in the library 
that hybridise with the probe and so permits the 
identification of the messenger RNA encoding the whole 
inhibitor. As an alternative to probing a gene library 
derived from the macrophage cell, a gene library of human 

25 origin can be probed to identify and isolate a DNA 



WO 89/10133 



PCT/GB89/00396 



- 8 - 

encoding an inhibitor of human origin which is 
expressable, using techniques now well-known, in a host 
cell system. 

Our preliminary investigation of the biologieal 
5 properties of the inhibitor indicate that its inhibitory 
effect upon cycling stem cells is reversible. The 
majority of chemotherapeutic agents used for cancer 
chemotherapy have a relatively short in vivo half-life, 
usually less than 24 hours so that the inhibitory effect 
0 of the inhibitor of the invention is maintained for at 
least the major proportion of the effective time during 
which the chemotherapeutic agent is active in vivo. Our 
expectation is that the normal physiological mechanisms 
within the body would limit the effective duration of 
5 activity of the inhibitor in relation to the cycling stem 
cells. 

In clinical application, it is desirable to 
target the inhibitor of the invention to the blood-forming 
tissue. This targetting can be achieved by injecting the 
3 inhibitor, normally by infusion or bolus intravenous 
administration and the present invention extends to 
pharmaceutical compositions containing the inhibitor and 
appropriate diluents or carriers suitable for such 
parenteral administration. 



WO 89/10133 



PCT/GB89/00396 



- 9 - 

The interest in the inhibitor of this invention 
is not restricted to stem cells specific to the 
haemopoietic system but extends to other stem cells e.g. 
epithelial stem cells, making the inhibitor of interest 
5 not only in relation to alleviating the side effects of 
cytotoxic drug therapy on bone marrow cells but also in 
the treatment of solid tumours. The inhibitor is also of 
interest in the treatment of leukaemia where leukaemic 
bone marrow cells are treated in vitro or iji vivo, with 
10 inhibitor so that proliferation of normal stem cells is 
prevented and the proliferating leukaemic cells can then 
be treated with a cytotoxic agent. 

The invention will now be further illustrated in 
the following Examples. 

15 EXAMPLE 

a) Cell line selected for inhibitor isolation 
and purification - . 

The widely available J774.2 was used. This is 
as described in J. Immunol. (1975) 114 r 898 

Cancer Res. (1977) 37^ 546 

20 Selection for serum-free growth in spinner culture 



J774.2 cells were originally growing in a 



WO 89/10133 



- 10 - 



PCT/GB89/00396 



modified Eagles medium supplemented with 10% foetal calf 
serum. The cells were subcultured into the medium: 



Dulbeccos x 10 50 ml 

SF12 x 10 50 ml 

5 Glutamine 200mM 10 ml 

Sodium pyruvate lOOmM 5 ml 

distilled water 840 ml 

Nutridoma SP 10 ml 

Sodium bicarbonate 7.5% 41 ml 



10 Nutridoma SP from Boehringer, Lewes , Sussex, 

Cat. No. 1011375 : other reagents from Flow Laboratories 
UK and Gibco-Biocult UK. 

The 774.2 cells were subcultured into the above 
medium with added foetal calf serum (5%) for one week, 

15 with a further subculture at three days in the same 

medium. Cells were further subcultured into medium plus 
2.5% foetal calf serum for a week, then into 1% foetal 
calf serum for a further week until finally all serum was 
removed. At this stage the cells were subcultured every 

20 two days, allowed to grow to a concentration of 8 x 10 5 /ml 
and diluted to 2 x 10 5 /ml at subculture. Cells were then 
transferred to spinner culture and acclimatised to growth 
in suspension, subcultering every two days as described 
above. This cell line designated J774.2(S) and capable of 



WO 89/10133 



PCT/GB89/00396 



-in- 
growth in serum free suspension culture was then used for 
inhibitor production. 



b) Growth of cells in large scale culture and processing 
of conditioned medium for purification of inhibitor. 

500 ml (8 x 10 5 /ml) J774.2(S) were seeded into 
5 large fermentors (Techne f 7L) containing 1500 ml of 
medium. Stirred fermentors were gassed with 5% COj in 
air i stirred at 20 rpm at 37°C for 2 days, then 
supplemented with 6L medium and allowed to continue to 
grow for 3 days. 

g 

10 Undiluted medium from 8 x 10 cells is separated 

from the cells using a Millipore Pellicon-Casette system 
with a 0.45u microporous membrane. The medium minus cells 
is then concentrated inthe same apparatus using a 10K 
cut-off membrane to a final volume of about 400 ml. This 

15 concentrate is then processed as described below for 
biochemical purification. 



Biochemical purification 

Stage 1 . The 400 ml concentrate is desalted on a G-25 
HR16/50 (Pharmacia) in 0.02M Tris-HCl pH 7.6, applied to 
20 the anion-exchange Mono Q HR10/10 column (Pharmacia, FPLC 
system) and finally eluted with a salt gradient (0 to 1M 



WO 89/10133 



PCT/GB89/00396 



- 12 - 

NaCl) in the same buffer. The active fractions of 
inhibitor elute between 0.26 and 0.28M NaCl. 

Stage 2. The impure inhibitor from Stage 1 is applied 
directly to a Sepharose-Heparin (Pharmacia) column, 
5 HR10/10. The inhibitor binds to the resin and the 

majority of proteins (more than 90%) can be washed off the 
resin using 0.1M NaCl-0.02M Tris HCl f pH 7.6. The 
inhibitor is thn eluted from the resin in 1M NaCl-0.02M 
Tris HC1, pH 7.6. 

10 Stage 3. The partially purified inhibitor from Stage 2 is 
applied directly to a Blue Sepharose (Pharmacia) column 
HR5/5 without desalting. The column is then washed with 
the buffer used to elute the inhibitor in Stage 2, to 
remove other protein. The purified inhibitor, which binds 

15 strongly, is eluted with 5M MgCl 2 -0.02M Tris-HCl pH 7.6 

Other biochemical characteristics of the 
inhibitor include: 

a) Heat stability. 

b) It is stable at 37°C , 55°C, 75°C for 1 hour. 



WO 89/10133 



PCT/GB89/00396 



- 13 - 

Molecular size 

The purified inhibitor can be seen as a single 
band of molecular weight 8-10Kd on polyacrylamide gels 
under reducing conditions. When the inhibitor is applied 
5 to polyacrylamide gel electrophoresis under non-reducing 
conditions, it can be seen to be of slightly higher 
molecular weight. These observations suggest that the 
inhibitor is a single chain polypeptide with internal 
disulphide bonds affecting electrophoretic mobility. 

10 Biological characteristics of the inhibitor 

Reversibly triggers multipotential stem cells 
(CFU-A, as mentioned in the 'in vitro assay description) 
out of cell cycle. 

Bone marrow cells were incubated in paired tubes 

15 containing 5 x 10 6 cells in 1 ml Fischer's medium 

supplemented with 20% horse serum. The inhibitor or 
alpha-MEM was added to each tube and Fischer's medium was 
added to control tubes. The mixtures were incubated at 
37°C for 5 hours (inhibition assays). For the last 60 

20 minutes of the incubation 10 M cytosine arabinoside was 
added to one tube and an equal volume of medium to the 
other tube. Cells were then washed twice before being 



WO 89/10133 



PCT/GB89/00396 



- 14 - 

assayed in the CFU-A assay as described above. The 
inhibitor was found to reduce the number of stem cells in 
cycle from an average of 30% to an average of about 3%. 
The untreated cells were killed by the cytotoxic drug 
5 treatment in contrast to the inhibitor treated stem cells. 
Impure preparations of the inhibitor also reversibly 
trigger multipotential stem cells out of cycle when 
assayed in vivo using the CPU-S assay described in the 
literature. 



WO 89/10133 



- 15 - 



PCT/GB89/00396 



CLAIMS 

1. A haemopoietic stem cell inhibitor 
characterised by the following properties: 

1. The inhibitor activity is sensitive to 
degradation with trypsin and the inhibitor is therefore 

5 proteinaceous . 

2. The inhibitor when partially purified by 

5 treatment on an anion-exchanger shows a molecular weight 
range 45-60 Kd on a molecular weight analysis resin. 

3. The inhibitor, when purified to a single band on 
polyacrylamide gel electrophoresis, under reducing 

15 conditions, shows a molecular weight range of 8-10 Kd and 
a slightly higher molecular weight range under 
non- reducing conditions. 

4. The inhibitor is heat-stable as follows: 

(i) Inhibitory activity is retained after heating 
20 for 1 hour at 37°C, 55°C and 75°C. 

(ii) Inhibitory activity is retained after heating 
for 10 minutes at 100°C. 



WO 89/10133 



PCT/GB89/00396 



- 16 - 

5. The inhibitor binds to anion-exchangers and can 
be eluted with a salt gradient at between 0.26 and 0,28 
molar NaCl. 

6. The inhibitor binds to a Heparin Sepharose 

5 affinity chromatography resin and can be eluted from the 
resin with one molar sodium chloride buffer. 

7. The inhibitor binds avidly to Blue Sepharose 
affinity chromatography resin and can be eluted from the 
resin with 5M magnesium chloride. 

8. Cycling stem cells, when treated with the 
partially purified inhibitor, 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 inhibitor, -the surviving stem cells 
proliferate in culture normally. 

2. An inhibitor according to claim 1 obtained 
from macrophage J774. 2 cells. 

3. A pharmaceutical composition comprising 
an inhibitor according to claim 1 or 2 together with a 

20 pharmaceutically acceptable carrier or diluent. 



10 



15 



WO 89/10133 



PCT/GB89/00396 



- 17 - 

4. A composition according to claim 3 for 
parenteral administration, 

5, An antibody to an inhibitor according to 
claim 1 or 2. 

5 6. A method of raising an antibody which 

comprises immunising a host animal with an inhibitor 
according to claim 1 or 2 or with an immunogenically 
active fragment of the inhibitor and recovering antibody 
or antibody producing cells from the host animal, 

10 7. Synthetic DNA including DNA encoding the 

inhibitor according to claim 1 or 2 expressable in a host 
cell. 

8. A method of isolating and purifying an 
inhibitor as defined in claim 1 which includes the steps 
15 of: 

1. growing an inhibitor producing macrophage cell 
line of bone marrow origin in a growth medium to produce a 
supernatant containing inhibitor; 

2. contacting the supernatant with an 

20 anion-exchanger and eluting the anion-exchanger with 



WO89/10133 



PCT/GB89/00396 



- 18 - 

0.25-0.30M NaCl solution to give a first eluant containing 
the inhibitor. 

3. contacting the first eluant with Sepharose- 
Heparin and eluting the Sepharose-Heparin with NaCl 

5 solution of molarity at least 1M to give a second eluant 
containing the inhibitor. 

4. contacting the second eluant with Sepharose-Blue 
and eluting the Sepharose-Blue with MgCl 2 solution of 
molarity at least 3M to give a third eluant containing the 

10 inhibitor. 

9. An inhibitor according to claim 1 or 2 for 
use in a method of treatment of the human or animal body 
by therapy. 

10. A method of treating a patient receiving a 
15 cell cycle specific cytotoxic drug which includes 

administration of an inhibitor according to claim 1 or 2 
so as to protect the patient's haemopoietic stem cells 
against damage by the cytotoxic drug. 



( 



\ 



INTERNATIONAL SEARCH REPORT 

International Application No PCT/GB 89/00396 



1. CLASSIFICATION OF SUBJECT MATTER (if cl.„,ficat,on .ymoo.. ,„»,«.. ..!> • 


Accordino 10 International Patent Cla.i.neat.on (IPC) or to Both National Cla.a.f.eation .no IPC 

IPC 4. A 61 K 37/02, C 07 K 15/00, C 12 N 15/00, C 


07 K 3/02 


II. FIELDS SEARCHED — — — — — — — . 


Minimum Documentation Searched 1 


Classification System 


■ 


Classification Symbols 




4 

IPC 


C 12 P, A 61 K 


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




III. DOCUMENTS CONSIDERED TO BE RELEVANT* 


Category • 


1 Citation of Document, 11 with indication, where appropriate, of the relevant passages ■» 


| Relevant to Claim No. " 


x,p 


Chemical Abstracts, vol. 109, 1988, 
( Columbus , Ohio , US ) , 
I.B. Pragnell et al.: "The effect 
of stem cell proliferation regulators 
demonstrated with an in vitro assay", 
see page 344, abstract 89048m, 
& Blood 1988, 72(1), 196-201 


1,8 


X,P 


Biological Abstracts, Reviews-Reports- 
Meetings, vol. 35, 1988, no. 
106933, 

E.G. Wright et al.: "The effect 
of stem cell proliferation regulators 
demonstrated with an in-vitro 
assay" , 

& Experimental Hematology, 1988, 
vol. 16, no. 6, page 473 
see title and terms 

./. 


1,8 


* Special categories of cited documents: to 

"A" document defining the general state ol the art which Is not 
considered to be of particular relevance 

"E" earlier document but published on or after the international 
filing date 

*T** document which may throw doubts on priority c)aim(e) or 
which is ctted to establish the publication cate of another 
citation or other special reason (as specified) 

**O m document referring to an oral disclosure, use. eahibitfon or 
other means 

"P" document published prior to the International filing date but 
later than the priority date claimed 


*T" later document published after the International filing date 
or priority date and not in conflict with the application but 
cited to understand the principle or theory underlying the 
invention 

"X" document of particular relevance; the claimed Invention 
cannot be considered novel or cannot be considered to 
involve an inventive step 

N Y" document of particular relevance;' the claimed Invention 
cannot be considered to involve an Inventive step when the 
document is comomed- with one or more other such docu- 
ments, such combination being obvious to a person skilled 
In the art. 

"A" document member of the same patent family 


IV. CERTIFICATION 


Dale of the Actual Completion ol the International Search 

10th August 1989 


Date of Mailing of this International Search Report 

1 4. flo 89 


International Searching Authority 

EUROPEAN PATENT OFFICE 


Signature of Authorised Officer _. 



- 2 - 



International Aoollcation No. PCT/GB 89 /00396 



III. DOCUMENTS CONSIDERED TO 31 RELgVANT (CONTINUED tno* TH E SECOND SHEET) 
Cat.gory * , Cation of Oocumoni, wth indication, why, appropnaf . of tr» rHvim paasages 



Ratavant to Claim No 



A 



I 



Biological Abstracts, Re views -Report s- 
Meetings,. vol. 33, 1987, no, 
61402, 

A. Janowska-Wieczorek et al.: 
"Protection of human hematopoietic 
stem cells from tubercidin toxicity 
by inhibitors of nucleoside 
transport", 

& Proc. Am. Assoc. Cancer Res. 
Annu. Meet. 1987, vol. 28, no. 0, 
page 409 

see title and terms 

Chemical Abstracts, vol. 96, 1982, 
( Columbus , Ohio , US ) , 
M. Guigon et al. : "Protection of 
mice against lethal doses of 
lbeta-D-arabinofuranosylcytosine 
by pluripotent stem cell inhibitors", 
see page 36, abstract no. 
U5633h, 

St Cancer Res. 1982, 42(2), 638-41 



Form PCT IS A. -210 (aura ahaat) (January IMS) 



International Application No. / GB 89 /O0396 



FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET 



V.(3§ OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE « ' 



This International search report hat not been established In respect of certain claims under Article 17(2) (a) for the following reasons: 
1.f%} Claim numbers ID., because they relate to subject matter not required to be searched by this Authority, namely: 

See PCT-Rule 3 9.1 (IV); methods for treatment of the human 

or animal body by surgery or therapy, 
as well as diagnostic methods . 



2.r~| Claim numbers because they relate to pans of the International application that do not comply with the prescribed require* 

ments to such en extent that no meaningful international search can be carried out. specifically : 



i f"! Claim numbers..... 
PCT Rule 6.4<a). 



. because they ire dependent claims end ere not drafted m accordance wtth the second and third sentences of 



VI.Q OBSERVATIONS WHERE UNITY OF INVENTION 18 LACKING * 



This International Searching Authority found multiple Inventions In this international application as follows; 



*Q A> a " required additional search fees were timely paid by the applicant, this Inttrnationsl search report covers all searchable claims 
of the international application. 

2-Q As only some of the required additional search fees were timely paid by the applicant, this international search report covers only 
those claims of the international application for which fees were paid, specifically claims: 

*0 No "Wired additional search fees were timely paid by the applicant Consequently, this Internatlonsl search report la restricted to 
the invention first mentioned In the claims; It la covered by claim numbers; 



4 *0 At all eearchable claims could be eearched without effort Justifying an additional fee. the International Searching Authority did not 
Invite payment of any additional tee. 



Remark on Protest 
I""! The additional search fe«s were accompanied by applicant's protest 
PI No protest accompanied the payment of additional eearch fees. 



Form PCT /IS A /210 (aupptemental sheet <2» (January 1985) 



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