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WORLD INTELLECnJAL PROPERTY ORGANIZATION 
lotemadonal Bureau 




PCX 

INTERNATIONAL APPUCATION PUBUSHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) intonatioiial Patent ClassificatioD ^ : 

C12N 15/19, C07K 13/00, A61K 37/02 



Al 



(11) InteniatioDal Publication NmnW: WO 94/24285 

(43) Isteniatioiial Publication Date: 27 October 1994 (27.10.94) 



(21) Internationa] Appfication Number: PCr/GB94/00822 

(22) Internationa] FlUng Date: 19 April 1994 (19.04.94) 



(30) Priority Data: 

9308060.4 



19 April 1993 (19.04.93) 



GB 



(71) Appficant {for aU designated Ssates except US): CANCER 

RESEARCH CAMPAIGN TECHNOLOGY LCMITED 
[GB/GB]; Cambridge House;, 6-10 Cambridge Teirace, 
Regent's Park, London NWl 4JL (GB). 

(72) Inventors; and 

(75) Inventors/Applicants (for US onfy)i GRAHAM, Gerard 
[GB/GB]; Beatson Institute for Cancer Research, Cancer 
Research rampaign Beatson Laboratories, Garscube Estate, 
Bearsden, Glasgow G61 IBD (GB). PRAGNELL, Ian 
[GB/GB]; Beatson Institute for Cancer Research, Canco* 
Research Campaign Beatson Lat>oratories, Garscube £statB, 
Bearsden, Glasgow G61 IBD (GB). 

(74) Agoits: CRESSWELL, Thomas. Anthony et al.; JA. Kemp 
& Co., 14 South Square, Gray's Inn, London WCIR 5LX 
(GB). 



(81) Designated States: JP, US, European patent (AT, BE, CH, DE, 
DK, ES, FR, GB, GR. IE. FT. LU, MC. NL. PT, SE). 



Published 

With international search report. 



(54) Title: MA<3lOPHAGE INFLAMMATORY PROIEIN VARIANTS 



M1P-10C NH2- 



-CC- 



— c- 



„o_-r 



-COOH 



(1) "-CC i*-t-.-Z--_c— C— COOH 



(2) NH2 CC --C C----~- COOH 



(3) NH2- CC *i-t~-:i-C C-^ COOH 



(57) Abstract 

The present invention provides a Stem Cell Inhibitcr (SQ) protein which comprises at least one amino add alteration from its native 
form which protein does not significantly aggregate but which retains substantially unaltered stem cell inhibitory activity. The alteration 
is preferably a conservative substitution of a charged amino add residue. Such proteins may be used in treating stem cells in a patient 
undeigCHng chemotherapy. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to idendfy States pany to the PCT on tbe front pages of pamphlets pubfishing intematioDal 
appHcati(»s under die PCT. 



AT 


Annru 


GB 


United fOogdoo 


MR 


Mauritania 


AU 


AustnliA 


GE 


GeocgU 


MW 


MaUvi 


BB 


ButadM 


GN 


Guinea 


NE 


Niga 


BE 


Belgium 


GR 


Grococ 


NL 


Nediertanda 


BF 


BurfcinA Fmo 


BV 


Hungjry 


NO 


Norway 


EG 


Bulgsii 


IE 


IicUod 


NZ 


New Zealand 


BJ 


BciUD 


IT 


ItMiy 


PL 


Polanl 


BR 


Bnzil 


JP 


Japan 


FT 


Portugal 


BY 


Bclmn 


KE 


Kcoya 


RO 


Romania 


CA 




KG 


Kyrgyttan 


m 


Rnslan Fedoidon 


CF 


Ceotnl AfriciD ReptdtUc 


KP 


Democntic People' a Repidilic 


SD 


Sudan 


CG 


Congo 




of Korea 


SE 


Sweden 


CH 


Switzerland 


KR 


Rq>dbtic of Korea 


SI 


Slovenia 


a 


C&te d*Ivoire 


KZ 


KazaktHtan 


SK 


Slovakia 


CM 


Cifflcrooo 


U 




SN 


Senegal 


CN 


ChiDB 


LK 


SriLaidU 


TD 




GS 


CzechodovakU 


LV 


Lineisbourg 


TG 


Togo 


CZ 


Czecb Re{NA)Uc 


LV 


LaMa 


XJ 


Tajikistan 


D£ 




MC 


Monaco 


TT 


l^indad and Tobago 


DK 


Denoiaik 


MD 


Republic of Moldova 


UA 


Ukiaine 


ES 


Sptto 


MG 


Madagaacar 


US 


United Stalea of Amc 


n 




ML 


Mafi 


UZ 


Uzbekiflan 


FR 


IVuee 


MN 


MoQgoUa 


VN 


VielNam 


GA 


Gaboo 











wo 94/24285 



- 1 - 



PCT/GB94/00822 



MACROPHAGE INFLAMMATORY PROTEIN VARIANTS 

* 

The present invention relates to variants of stem cell 
inhibitors • 

The treatment of cancer with chemotherapeutic agents is designed 
to attack and destroy cells which are undergoing division within 
5 the body. A side effect of such treatment is thus the 
destruction of normal cells, particularly the stem cells of the 
haematopoietic system and the epithelial stem cells which line 
the scalp and gut. Radiation can also cause similar destruction 
of such cells. 

10 It has been proposed that in order to improve the treatment of 
cancers by chemotherapy it would be desirable to protect stem 
cells from cell cycle specific cytotoxic drugs. WO89/10133 
discloses a stem cell inhibitor and describes the use of the 
inhibitor in the treatment of cancers. The inhibitor may be 

15 administered to a patient in order to protect stem cells during 
chemotherapy. 

Stem Cell Inhibitor (SCI),. also known as MlPl-a is a peptide of 
about 8kD which forms large self aggregates, the molecular weight 
of which is dependant upon the concentration of SCI/MIPl-of 

20 monomers (Graham et al, 1990, Nature 341; 442, Wolpe & C^rami, ' 
1989, FASEB J, 1; 2656) . It has been found that SCl/MIPl-Of has 
a native, aggregated molecular weight of about lOOkD at O.lmg/ml 
in physiological buffers such as PBS. It has been foxind that 
diluting SCI/MIPl-a to about 20-100ng/ml or less will bring about 

25 disaggregation of this protein. 

Human SCI/MIPl-a has been cloned by us {Graham et al (1992) , 
Growth Factors 2;151-160) . The cDNA has also been cloned by 
Nakao et ai (1990, Mol. Cell, Biol., 10;3646-;S8) and called 
LD78i3. A variant of the cDNA LD78Qr was also found, which has a 
30 very similar sequence. It differs by only 4 amino acid residues. 
The human cDNA and protein sequence of the factor cloned by us is 
shown is Seq. ID No. 1- The first 27 amino acicis are a leader 
sequence. The mature protein starts at residue 28 (ala) . The 



wo 94/24285 PCT/GB94/00822 

- 2 - 

amino acid sequence of the variant found by Nakao et al is shovm 
as Seq. ID No. 3. The leader sequence of the protein . is • one 
amino acid shorter and thus the mature protein starts at residue 
27 (ala) . The sequence of the murine homologue, upon which we 
5 have conducted our work, is also known and is very similar. It 
can be found for example in Graham et al (1994, J. Biol. Chem. , 
26 9 ; 4974-78) . 

It has been reported (Mantel et al, 1993, PNAS 9fi;2232) that 
monomeric SCI/MIPl-a is more active than the aggregated form in 

10 inhibiting in vitro and in vivo stem cell proliferation. In 
using SCI/MIPl-a in the treatment of humans it would be desirable 
to administer monomeric protein, not just from an activity point 
of view but also in order to provide reliable and reproducible 
formulations. However, it is likely that the low concentrations 

15 of SCI/MIPl-o which must be made in order to provide monomeric 
protein will be too low for use in practice. 

We have now surprisingly found that it is possible to obtain 
SCI/MIPl-cx variants which retain substantially the activity of 
the native protein but which do not form the same large 
20 aggregates. These mutants are stable as monomers or as small 
conglomerates (eg dimers or tetramers) at concentrations many 
fold higher than native SCI/MIPl-o. Thus for those variants 
which have activity comparable to native SCI/MIPl-a. the variants 
may have higher activity in vivo on a unit weight basis. 

25 Accordingly, the present invention provides a Stem Cell Inhibitor 
protein which comprises at least one amino acid alteration from 
its native form which does not significantly aggregate but which 
retains substantially unaltered stem cell inhibitory activity. 
The protein may comprise either the full length stem cell 

30 inhibitor or the mature processed form lacking the leader 
sequence . 

The invention also provides pharmaceutical compositions 
comprising a stem cell inhibitor according to the invention in 
combination with a pharmaceutical ly acceptable carrier or 
35 diluent, and optionally other therapeutic ingredients. The 



wo 94/24285 PCT/GB94/00822 

- 3 - 

carrier (s) must be "acceptable" in the sense of being compatible 
with the other ingredients of the formulation and not deleterious 
to the recipients thereof. 

The formulations include those suitable for parenteral (including 
5 subcutaneous, intramuscular, intravenous, intraperitoneal, 
intradermal, intrathecal and epidural) administration. The 
formulations may conveniently be presented in unit dosage form 
and may be prepared by any of the methods well known in the art 
of pharmacy. Such methods include the step of bringing into 
10 association the active ingredient with the carrier which 
constitutes one or more accessory ingredients. In general the 
formulations are prepared by uniformly and intimately bringing 
into association the active ingredient with liquid carriers. 

Formulations suitable for parenteral administration include 
15 aqueous and non-aqueous sterile injection solutions which may 
contain ant i -oxidants, buffers, bacteriostats and solutes which 
render the formulation isotonic with the blood of the intended 
recipient; and aqueous and non-aqueous sterile suspensions which 
may include suspending agents and thickening agents, and 
20 liposomes or other microparticulate systems which are designed to 
target the compound to blood components or one or more organs. 
Suitable liquid carriers include phosphate buffered saline at a 
pH of between 7.0 and 8.0, for example 7.4. The formulations may 
be presented in unit-dose or multi-dose containers, for example 
25 sealed ampoules and vials, and may be stored in a freeze-dried 
(lyophilized) condition requiring only the addition of the 
sterile liquid carrier, for example water for injections, 
immediately prior to use. 

Preferred unit dosage formulations are those containing a daily 
30 dose or unit, daily sub-dose, or an appropriate fraction thereof, 
of an active ingredient. 

Formulations of the SCI/MIP-lof proteins of the present invention 
preferably contain from 0.05 to 5 mg/ml of protein, for example 
0,1 to 1.0 mg/ml. We have found that the solubility of the 
35 variants of the invention do vary although the maximum solubility 



wo 94/24285 PCT/GB94/00822 

- 4 . 

of any one particular variant may be determined by simple 
titration by those of skill in the art. 

The invention also provides such proteins and compositions for 
use in a method of treatment of the human or animal body. 

5 The invention further provides a method for treating a subject 
who is to be exposed to an agent capable of killing dividing or 
cycling stem cells by administering to the subject an effective 
amount of a protein or composition according to the invention. 

The subject may also be treated with a protein or composition 
10 according to the invention during or after chemotherapy. In the 
latter case, this will usually be for a period sufficient to 
allow clearance of the agent from the body. 

The method of treatment according to the invention may be used in 
the treatment of solid tumours or leukemias. In the case of 

15 treatment of leukemias, it is possible to treat a sample of the 
patients bone marrow which has been removed from the body while 
the patient is undergoing treatment. The bone marrow is purged 
of cancer cells in the presence of a protein of composition 
according to the invention, and the treated marrow reintroduced 

20 into the patient. 

Although the dose of the variant protein according to the 
invention will ultimately be at the discretion of the physician, 
taking into account the nature of the condition being treated and 
the state of the patient, effective doses may be in the range of 
25 from about 10 body weight to about 5 mg/kg of variant 

protein, for example from about 50 to about 1000 fig/kg, ^g about 
500 fig/kg^ 

We have also found that SCI/MIPl-a can act to enhance the 
expansion of primitive haemopoietic cells in ex vivo cytokine 
30 driven stem cell expansion experiments. Thus, variant proteins 
of the invention may also be used in methods to expand stem cell 
populations removed from a patient ex vivo wherein such stem 



wo 94/24285 PCT/GB94/00822 

- 5 - 

cells are brought into contact with growth factors and the 
variant proteins of the invention under conditions which allow 
the growth and expansion in numbers of the cells, prior to 
reintroduction into the same or another patient. Such a method 
5 could be used in bone marrow transplant proceedures whereby a 
limited number of starting cells obtained from a donor are 
expanded prior to transplantation, or in certain therapies where 
a sample of bone marrow is removed from a patient prior to 
treatment and reintroduced following treatment. Such therapies 
10 include the treatment of leukemias, or other tumours including 
solid tumours where damage to the bone marrow may occur. The 
concentration of the variant proteins required to produce 
suitable activity will be in the range of from about 1 to about 
100 ng/ml, for example from about 10 to about 50 ng/ml . 

15 A protein or composition according to the invention may also be 
used in the treatment of disorders caused by proliferation of 
stem cells, eg. psoriasis. 

A protein according to the invention is preferably a protein 
which contains at least one change from the native protein 
20 resulting in the loss of of one of more charges on the protein, 
eg. by replacement of one or more charged amino acids. 

The change may be as a result of a deletion or substitution or 
insertion. In the case of a deletion or insertion, single base 
deletions or insertions are generally preferred, in order to 
25 retain a structure similar to the native protein. However, 
deletions of insertions of more than this, eg or 2, 3, 4, 5 or 
more amino acids are possible. In the case of a substitution, it 
is preferably a conservative substitution, such as Asp to Asn or 
Glu to Gin. 



30 



In addition, fragments of native protein which retain their stem 
cell inhibitory activity but which exhibit the reduced tendency 
to aggregate are within the scope or the invention. 



Preferably, the change to the protein is in the C-terminal 
region, -eg within the last 20 or even last 10 amino acids. This 



wo 94/24285 PCT/GB94/00822 

- 6 - 

may include C-terminal deletions. 

More than one change to a native stem cell inhibitor protein may 
be made. For example, 2, 3, 4 or 5 changes may be made. 

Another preferred region of the MIPl protein which may be altered 
5 is the putative heparin binding region between amino acids 68 and 
71 of Seq. ID No. 1. We have determined by experimentation and 
by comparison of this sequence with known heparin binding regions 
that this portion of MIPl has heparin binding activity. Thus 
suitable amino acids which may be altered in accordance with the 
10 invention include one, two or three of 68(lys), 69{arg) and 
71 (arg) . Such alterations may be made, if desired with an 
alteration to the c- terminal region of the MIPl protein as 
described above. 

Preferred stem cell inhibitor proteins of the invention are those 
15 based upon the human protein of Seq. ID, 2 or that of Seq. ID 3. 
Also preferred are the mature forms of such proteins, ie. from 
residues 28 onwards. 

Particular amino acids which may be altered in the protein 
sequence of Seq. ID No. 2 or Seq. ID No. 3 include alterations at 

20 any positively charged residue, eg. lys or arg, and/or at any 
negatively charged residue, eg asp or glu. The residues of Seq. 
ID. No. 2 which may be altered thus include: 29 (asp) , 41 (arg) , 
50 (asp), 53 (glu), 60 (lys), 68 (lys) , 69 (arg), 71<arg), 76 (asp) , 
79 (glu), 80 (glu), 84 (lys), 87 (asp) or 90 (glu) . The changes made 

25 to these positions may be as described above. 

Combinations of changes which may be made include changing the 
final 2, 3, 4, 5 or 6 charged residues of the stem cell 
inhibitor. In the case of the human protein, this results in a 
protein which corresponds to the native protein ex<:ept for 
30 changes at position 90 and/or one or more of positions 76, 79, 
80, 84 or 88. Preferably, all the changes are single amino acid 
substitutions. Preferably, all such substitutions are 

conservative changes. 



wo 94/24285 



PCT/GB94/00822 



- 7 - 

Proteins according to the invention may be made by any means 
available in the art. In the examples which follow, we have made 
modified stem cell inhibitory proteins . by site directed 
mutagenesis using PGR primers of the murine SCI cDNA, followed by 
5 expression of the modified cDNA in a vector in a host cell to 
produce the protein. The protein may be recovered from the host 
cell using protein purification techniques known per se. 
Analogous methods may be used to make modified human or other 
primate SCI. The murine cDNA may be obtained for example by 

10 reference to the methods disclosed in WO89/10133 or by reference 
to the published literature. Human cDNA may also be obtained by 
reference to the published literature or cloned using probes 
based on all or part of the DNA sequence of Seq. ID No. 1 to 
identify SCI cDNA in a cDNA library made from cells expressing 

15 SCI RNA. 

Accordingly, the present invention also provides a method for 
making a protein according to the invention which comprises: 

(i) modifying a DNA sequence coding for SCI protein in order to 
introduce at least one change which causes a change in the amino 

20 acid sequence of the SCI protein; 

(ii) expressing said DNA, operably linked to a promoter, in a 
vector in a host cell compatible with said promoter; and 

(iii) recovering said protein. 

The DNA may be modified by site directed mutagenesis as mentioned 
25 above or described in the examples, to obtain insertions, 
deletions or subsitutions in the amino acid sequence. 

The vector may contain one or more selectable marker genes, for 
example an ampicillin resistance gene in the case of a bacterial 
plasmid or a neomycin resistance gene for a mammalian vector. 

30 A further embodiment of the invention provides host cells 
transformed or transfected with the vectors for the replication 
and expression of DNA produced as described above, including the 
DNA Seq. ID No. 1 modified as mentioned above. The cells will be 
chosen to be compatible with the vector and may for example be 

35 bacterial, yeast, insect or mammalian. 



wo 94/24285 PCT/GB94/00822 

- 8 - 

The invention also provides monoclonal or polyclonal antibodies 
to a peptide according to the invention which is directed to a 
epitope containing an alteration of the native SCI. The 
invention further provides a process for the production of such 
5 monoclonal or polyclonal antibodies. Monoclonal antibodies may 
be prepared by conventional hybridoma technology using the 
proteins or peptide fragments thereof, as an immunogen. 
Polyclonal antibodies may also be prepared by conventional means 
which comprise inoculating a host animal, for example a rat or a 
10 rabbit, with a peptide of the invention and recovering immune 
serum. 

In either case, antibodies which recognise altered epitopes may 
be identified by screening them with native SCI and the altered 
SCI to which the antibody was raised and identifying an antibody 
15 which recognises only the altered SCI. 

The following examples illustrate the invention. 



Example 1 

Figure 1 shows a schematic representation of murine SCI/MIPl-a 
indicating the position of charged amino acids. A series of 
20 altered proteins (1) - (3) were made using PCR primers on cDNA 
encoding the protein together with a wild type 5' primer. The 
altered proteins all contained conservative changes, ie. 
glutanmate to glutamine and/or aspartate to asparagine. The 
primers used are as follows: 

25 Variant 1: 

5' TC AGG AAT TCA <3GC ATT CAG TTG CAG GTC 3' (SEQ ID NO. 4) . 
This alters the C-terminal end of the murine MlPl-Of protein from: 
VQEYITDLELNA (SEQ ID NO. 5) to VQEYITDLQLNA (SEQ ID NO. 6). 

Variant 2 : 

30 5'TC AGG AAT TCA ^GGC ATT CAG TTG CAG GTT AGT GAT 3' (SEQ ID NO. 7) 
whinch alters Seq. ID No. 5 to VQEYITNLQLNA (SEQ ID NO. 8). 



Variant 3 : 



wo 94/24285 PCT/GB94/00822 

- 9 - 

5' TC AGG AAT TCA GGC ATT CAG TTG CAG GTT AGT GAT GTA TTG 
TTG GAC 3' (SEQ ID NO. 9) 

which alters Seq, ID No. 5 to VQQYITNLQLNA (SEQ ID NO. 10) 

The varied cDNA molecules were ligated into a fusion protein 
5 expression vector and the altered proteins were produced. The 
native protein together with the three altered proteins were 
analysed by chromatographic techniques and the molecular weights 
of each estimated. 

The estimates were as follows: 
10 Native protein 100-150 kD 

Protein (1) 35 kD 

Protein (2) 18 kD 

Protein (3) 8 kD 

Protein (1) thus appears to exist as a tetramer, protein (2) as 
15 a dimer and protein (3) as a monomer under conditions in which 
native MlPl-cy exists as an aggregated protein. 

The above proteins were assesed for bioactivity using standard 
techniques (Pragnell et al Blood, 1988, 72; 196 and Lorimore et 
al, 1990, Leukaemia Research 14; 481) and found to be bioactive. 

20 Example 2 

Two 3' (carboxy terminus) primers were synthesised with the 
following sequences: 

5' GTA CGT GGA TCC TCA GGC ACT CAG CTG CAC GTT GCT <SAC ATA TTG 
CTG GAC 3' (SEQ ID NO. 11) 
25 and 

5' GTA CGT GGA TCC TCA GGC ACT CAG CTG CAG GTT GCT GAC ATA TTG 
CTG GAC CCA CTG CTC ACT 3' (SEQ ID NO. 12) . 

A Bam HI recognition site is underlined. 

The primer of Seq. ID No. 11 encodes amino acids 82 to 93 of Seq. 
30 ID No. 1 but alters the lysine at position 84 (84 (lys) ) to 
glutamine {gin), 88 (asp) to asn, and 90(glu) to gin. 



wo 94/24285 PCT/GB94/00822 

- 10 - 

The primer of Seq. ID. No. 12 encodes to amino acids 78 to 93 of 
Seq. ID No. 1 but contains the three changes described above for 
Seq. ID No. 11 and also a futher change, 80(glu) to gin. 

To produce the human variants incorporating the above changes the 
5 above primers are each used with an amino terminal primer of Seq. 
ID No. 13: 

5' GAC G GC CAT GG C TGA CAC GCC GAG CGC CTG C 3' (SEQ ID NO. 13) 
which encodes amino acids 28-35 of Seq. ID No. 1. An Ncol 
recognition site is underlined. This corresponds to the start of 
10 the mature SCI/MIP-1 protein. 

The primers are used in a PGR to provide full length clones 
encoding variants incorporating the changes described above, and 
the variant clones introduced into an expression vector to 
provide dissagregated variant proteins of the invention. 

15 The variants are tested in a similar manner as described above 
for activity. 

Example 3 

A internal primer which encodes a central portion of the murine 
MlPl-a protein was designed, incorporating changes which cause 
20 point mutations in two of the three positively charged residues 
between the third and fourth cysteine residues shown in Figure 
1(a). The primer is of the sequence: 

5' CGT CTA GAC GGC CAA CGA CAA TCA GTC CTT 3' (SEQ ID NO. 14) 
which alters the murine sequence: 
25 FLTKRNRQIC (SEQ ID NO. 15) to FLTNSNRQIC (SEQ ID NO. 16) . 

The mutagenesis was done in two halves using this primer and the 
wild type amino termial primer and a complemetary primer wae used 
with the wild type carboxy terminal primer. The two reaction 
products were then mixed and the full length molecule produced 
30 using the wild type amino and carboxy terminal primers. The 
variant is also tested for activity. 



wo 94/24285 



- 11 - 



PCT/GB94/00822 



SEQUENCE LISTING 



(I) GENERAL INFORMATION: 

(i) APPLICANT: 

(A) NAME; Cancer Research Campaign Technology Limited 

(B) STREET: €-10 Cambridge House 

(C) CITY: London 

(E) COUNTRY: GB 

(F) POSTAL CODE (ZIP): NWl 4JL 

(i) APPLICANT: 

(A) NAME: Graham, Gerard 

(B) STREET: Beatson Laboratories, Garsciabe Estate 

(C) CITY: Glasgow 

(E) COUNTRY: GB 

(F) POSTAL CODE (ZIP) : G61 IBD 

(i) APPLICANT: 

(A) NAME: Pragnell, Ian 

(B) STREET: Beatson Laboratories , Gars cube Estate 

(C) CITY: Glasgow 

(E) COUOTRY: GB 

(F) POSTAL CODE (ZIP) : G61 IBD 

(ii) TITLE OF INVENTION: Stem Cell Inhibitor 
(iii) NUMBER OF SEQUENCES: 16 

(iv) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) CCMPUTER: IBM PC compatible 

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

(D) SOFTWARE: Patentin Release #1.0. Version #1.25 (EPO) 

(v) CURRENT APPLICATION DATA: 
APPLICATION NUMBER: 

(2) INFORMATION FOR SEQ ID N0:1: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 282 base pairs 
(B> TYPE: nucleic acid 

(C) STRANDEDNESS : double 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 



(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 1..282 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 

ATG CAG GTC TCC ACT ^CT GCC CTT GCC GTC CTC CTC TGC ACC ATG 6CT 48 
Met Gin Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala 
15 10 15 

CTC TGC AAC CAG GTC CTC TCT GCA CCA CTT <3CT G€T <3AC AOG CCG ACC 96 
Leu Cys Asn Gin Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr 
20 25 30 

GCC TGC TGC TTC AGC TAC ACC TCC CGA CAG ATT CCA CAG AAT TTC ATA 144 
Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gin lie Pro Gin Asn Phe lie 
35 40 45 



wo 94/24285 



- 12 - 



PCT/GB94/00822 



240 



282 



GCT GAC TAC TTT GAG ACG AGC AGC CAG TGC TCC AAG CCC AGT GTC ATC. 192 
Ala ASP Tyr Phe Glu Thr Ser Ser Gin Cys Ser Lys Pro Ser Val lie 
50 55 60 

TTC CTA ACC AAG AGA GGC CGG CAG GTC TGT GCT GAC CCC AGT GAG GAG 
Phe Leu Thr Lys Arg Gly Arg Gin Val Cys Ala Asp Pro Ser Glu Glu 
65 70 75 eo 

TGG GTC CAG AAA TAC GTC AGT GAC CTG GAG CTG AGT GCC TGA 
Trp Val Gin Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala ♦ 
85 90 

{2} INFORMATION FOR SEQ ID N0:2: 

<i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 93 amino acids 

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

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2: 

Met Gin Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala 
1 5 10 15 

Leu Cys Asn Gin Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr 
20 25 30 

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gin He Pro Gin Asn Phe He 
35 40 45 

Ala Asp Tyr Phe Glu Thr Ser Ser Gin Cys Ser Lys Pro Ser Val He 
so 55 €0 

Phe Leu Thr Lys Arg Gly Arg Gin Val Cys Ala Asp Pro Ser Glu Glu 
65 70 75 80 

Trp Val Gin Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala 
85 90 

(2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 92 amino acids 

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

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 

Met Gin Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala 
1 5 10 15 

Leu cys Asn Gin Phe Ser Ala Ser Leu Ala Ala Asp Thr Pro Thr Ala 
20 25 30 

CVS CVS Phe ser Tyr Thr Ser Arg Gin He Pro Gin Asn Phe He Ala 
35 40 45 

ASP Tvr Phe Glu Thr Ser Ser -Gin Cys Ser Lys Pro Gly Val He Phe 
^ 50 55 60 

Leu Thr Lys Arg Ser Arg Gin Val Cys Ala Asp Pro Ser Glu ^lu Trp 
€5 70 75 fiO 

Val Gin Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala 
85 90 



wo 94/24285 



" 13 - 



PCT/GB94/00822 



(2) INFORMATION FOR SEQ ID N0:4: 

(i) SEQXJENCE CHARACTERISTICS: 

(A) LENGTH: 29 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: CDNA 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4: 



TCAGGAATTC AGGCATTCAG TTGCAGGTC 



(2) INFORMATION FOR SEQ ID NO: 5: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 12 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 

Val Gin Glu Tyr lie Thr Asp Leu Glu Leu Asn Ala 
1 5 10 

(2) INFORMATION FOR SEQ ID NO: 6: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 12 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 

Val Gin Glu Tyr He Thr Asp Leu Gin Leu Asn Ala 
1 5 10 



(2) INFORMATION FOR SEQ ID NO: 7: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 35 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 



TCAGGAATTC AGGCATTCAG TTGCAGGTTA GTGAT 



(2) INFORMATION FOR SEQ ID NO: 8: 
(i) SEQUENCE CHARACTERISTI<:S : 



wo 94/24285 



- 14 - 



PCT/GB94/00822 



(A) LENGTH: 12 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS : single 

(D) TOPOliOGY: linear 

(ii) MOLECULE TYPE: peptide 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 

Val Gin Glu Tyr He Thr Asn Leu Gin Leu Asn Gin 
1 5 10 



(2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 47 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: CDNA 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 



TCAGGAATTC AGGCATTCAG TTGCAGGTTA GTGATGTATT GTTGGAC 



(2) INFORMATION FOR SEQ ID NO: 10: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 12 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 

Val Gin Gin Tyr He Thr Asn Leu Gin Leu Asn Ala 
1 5 10 



(2) INFORMATION FOR SEQ ID NO: 11: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 51 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11: 
GTACGTGGAT CCTCAGGCAC TCAGCTGCAG GTTGCTGACA TATTGCTGGA C 



(2) INFORMATION FOR SEQ ID NO: 12: 

(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 63 base pairs 
<B) TYPE: nucleic acid 
(C) STRANDEDNESS : single 
{D) TOPOLOGY: linear 



wo 94/24285 PCT/GB94/00822 

- 15 - 

(ii) MOLECULE TYPE: cDNA 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 



GTACGTGGAT CCTCAGGCAC TCAGCTGCAG GTTGCTGACA TATTGCTGGA CCCACTGCTC 
ACT 



(2) INFORMATION FOR SEQ ID NO: 13: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 31 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS : single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: CDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 
GACGGCCATG GCTGACACGC CGACCGCCTG C 



(2) INFORMATION FOR SEQ ID NO: 14: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 30 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 
CGTCTAGACG GCCAACGACA ATCAGTCCTT 
(2) INFORMATION FOR SEQ ID NO: 15: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: 

Phe Ijeu Thr Lys Arg Asn Arg Gin lie Cys 
1 5 10 



(2) INFORMATION FOR SEQ ID NO: 16: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 10 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ .ID NO: 16: 

Phe Leu Thr Asn Ser Asn Arg Gin lie Cys 
1 5 10 



wo 94/24285 



PCT/GB94/00822 



- 16 - 
CLAIMS 

1. A Stem Cell Inhibitor (SCI) protein which comprises at least 
one amino acid alteration from its native form which protein 
does not significantly aggregate but which retains 
substantially unaltered stem cell inhibitory activity. * 

2. A protein according to claim 1 which exists as a tetramer, 
dimer or monomer under conditions in which the native 
protein exists as an aggregate. 

3. A protein according to claim 1 or 2 wherein the alteration 
is an amino acid substitution. 

4. A protein according to claim 3 wherein the amino acid 
substitution results in the loss of a charged amino acid. 

5. A protein according to claim 3 or 4 wherein the substitution 
is a conservative substitution. 

6 . A protein according to claim 5 wherein the substitution is 
of Asp to Asn or Glu to Gin. 

7. A protein according to any one of claims 1 to 6 which is a 
mature stem cell inhibitor. 

8. A protein according to any one of claims 1 to 7 wherein the 
native fonn of protein is human stem cell inhibitor. 

9. A protein according to <:laim 8 wherein ti^ amino acid 
alteration is at one or more of 29 (asp), 41(arg), 50 (asp), 
53 (glu), 60(lys), €8(lys), 69(arg), 71<arg) , 7€{asp), 
79 (glu), 80 (glu), 84 (lys) , 87<asp) or 90<glu) . 

10. A protein according to any one of the preceding claims which 
contains 2 or 3 amino acid alterationis . 



11. A pharmaceutical composition comprising a protein according 
to any one of claims 1 to 10 in cotriDination with a carrier 



wo 94/24285 



- 17 - 



PCT/GB94/00822 



or diluent. 

12. A protein according to any one of claims 1 to 10 or a 
composition according to claim 11 for use in a method of 
treatment of the human or animal body. 

13. A method for treating a subject who is to be exposed to an 
agent capable of killing dividing or cycling stem cells by 
administering to the subject an effective amount of a 
protein as defined in any one of claims 1 to 10 or a 
composition according to claim 11. 

14. A method for making a protein as defined in any one of 
claims 1 to 10 which comprises: 

(i) modifying a DNA sequence coding for SCI protein in order 
to introduce at least one change which causes a change in 
the amino acid sequence of the SCI protein; 

(ii) expressing said DNA, operably linked to a promoter, in 
a vector in a host cell compatible with said promoter; and 

(iii) recovering said protein. 



wo 94/24285 



1/1 



PCT/GB94/00822 



X 

o 
o 
o 



o 



o 
o 



CM 



I 
o 
o 
o 



o 
o 



CM 

z 



I 
o 
o 
o 



o 



o 
o 



CVJ 
X 

z 



X 

o 
o 
o 



o 



o 



o 
o 



CM 

X 

z 



8 

I 

QL 



CM 



CO 



SUBSTITUTE SHEET tRULt 26) 



INTERNATIONAL SEARCH REPORT 



Inter. dmI Application No 

PCT/GB 94/00822 



A. CLASSmCATION OF SUBJECT MATTER 

IPC 5 C12N15/19 C07K13/00 A61K37/02 



Acoonting to international Patent Qasafication (IPQ or to both national clasnfication and IPC 



B. FIELDS SEARCHED 



Minifluun documentation searched (dassificanon system followed by classification symbols) 

IPC 5 C12N C07K 



Documentation searched other than minimum documentation to the extent that such documents are included in the Oelds searched 



Beciromc rf«t> base consulted dunng the international search (name of data base and, whoc practical, search tenns used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category * Qtation of riocumcntt with indication, where appropriate, of the relevant passages 



Relevant to datm No. 



P.x 



W0,A,93 13206 (BRITISH BIO-TECHNOLOGY 
LTD.; GB) 8 July 1993 
see the whole document 

GROWTH FACTORS 

vol. 7, no. 2 , 1992 

pages 151 - 160 

GRAHAM, G.J. ET AL.; 'Purification and 
biochemical characterization of human and 
murine stem cell inhibitors (SCI).' 
see page 158, column 1, line 28 - page 
159, column 1, line 2 

W0,A,91 04274 (GENETICS INSTITUTE, INC.; 

US) 4 April 1991 

see the whole document 

-/-- 



1-14 



1-3.5,7. 
8,10-13 



1.3,5,7, 

8,10-13 

2 



II 



PuTtber documents arc listed in the continuation of box C. 



Patent family membm are listed in annex. 



* Special categories of dted documents : 

'A* document defining the general state of the art which is not 

considered to be of particular relevance 
*E* earlier document but published on or after the international 

filing date 

*L' document which may throw doubts on prionty daiffl(s) or 
which is dted to »g>fWi*»> the publication date of another 
dtation or other special reason (as spedfied) 

'O* document refcning to an oral disdosure, use, adu*taitiaa or 
(^er means 

'P* document published ptior to the international filing date but 
later than the phonty date r'-" — ' 



"T later document puUidted after the international filing date 
or priority dau and not in conflict with the application but 
dted to understand the prindpie or theory uuleriying (he 
invention 

"X' document of particular Tdevaner, the daimed invention 
cannot be considocd novd or cannot be considered to 
involve an inventive step when the document is taken alone 

'Y' document of particular tdevanoe; the daimed invention 
cannot be considered to involve on inventive step when the 
document is combined with one or more other such dooK 
mests, such combination being obvious to a penon i 
in the arL 

*&' document mcEDbcr of the tame patent family 



Date of the actual completion of the international search 



11 July 1994 



Date of mailing of the international search report 



1 V. S"! 



Name and mailing address of the ISA 

European Patent Office, P.B. 581 8 Patentlaan 2 
NL - mo HV Rijswijk 
Td.(-t' 31-70) 340-2040, !>£. 31 «S1 eponl. 
Fax (-t^ 31-70) 340-3016 



Authonzed offica 



Nauche, S 



Fonn PCT/ISA/aiO (sKODtf Chwt) (July 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



Intt' jml Application No 

PCT/GB 94/00822 



C^Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT 



Category* 



X 
Y 

P.X 



Qtation of document, with indication, where appropnate, of the relevant passages 



PROCEEDINGS OF THE NATIONAL ACADEMY OF 
SCIENCES OF USA. 

vol . 90 , 15 March 1993 , WASHINGTON US 
pages 2232 - 2236 

MANTEL, C. ET AL.; 'Polymerization of 
murine macrophage inflammatory protein 1 
alpha inactivates its myelosuppressive 
effects in vitro : The active form is a 
monomer . ' 

see the whole document 

W0.A,92 05198 (CHIRON CORPORATION) 2 April 
1992 

see the whole document 

JOURNAL OF BIOLOGICAL CHEMISTRY, 
vol. 269, no. 7 , 18 February 1994 , 
BALTIMORE US 
pages 4974 - 4978 

GRAHAM 6J;MACKENZIE J:LOWE S;TSANG. 
ML:WEATHERBEE JA;ISSACSON A:MEDICHERLA 
J:FAN6 F;WILKINSON PC;PRAGNELL IB; 
'Aggregation of the chemokine MIP-1 alpha 
is a dynamic and reversible phenomenon. 
Biochemical and biological analyses.' 
see the whole document 



Relevant to clum No. 



1.3,5,7, 

8,10-13 

2 

1-14 



Form PCT/IS A/310 <amtlntaitton of 



li BhMi) (July tm) 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 



.mationaJ application No. 

PCT/GB94/ 00822 



Box I Observations where certain claims were Tound unsearchable (Continuation of item 1 of first sheet) 



This international search report has not been established in respect of certain claims under Article 17(2Xa) for the following reasons: 



1. [Xj Claims Nos.: 

because they relau to subject matter not required to be searched by this Authority, namely. 

Remark : Although claim 13 Is directed to a method of treatment of the 
human/animal body as well as diagnostic methods (Rule 39.1(1v)PCT) the 
search has been carried out and based on the alleged effects of the 
compound/composition. 



Claims Nos.: 

because they relate to parts of the internaiionai appHcaiion that do not comply with the prescribed requirements to such 
an extent that no meaningful international search can be carried out. specifically: 



□ 



Claims Nos.: 

because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a). 



Box 11 Observations where unity of invention is tacking (Continuation of item 2 of first sheet) 



This International Searching Authority found multiple inventions in this inurnational application, as follows: 



. [ I As all required additional search fees were timely paid by the applicant, this intcmaiional search report covers all 



searchable claims. 



2. [[^ As all searchable claims could be searches without effort justifying an additional fee, this Authority did not invite payment 
of any additional fee. 



3. As only some of the required additional search fees were timely paid by the applicant, this international search report 
covers only those claims for which fees were paid, specifically claims Nos^ 



4, r~j No required additional search fees were timely paid by the applicant. Consequently, this inurnational search report is 
restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 



Remark on Protest [ | The additional search fees were accompanied by the applicant's proiest- 

j I No protest accompanied the payment of additional search fees. 



Form PCT;ISA;2I0 (continuation of first sheet (1)) (July 1992) 



INTERNATIONAL SEARCH REPORT 

mfannation on patent Esmily memben 



Intt onal Application No 

PCT/GB 94/00822 



Paient document 
dted in search report 


Publication 
d&te 


Patent family 
membeT(s) 


Publication 
dale 


WO-A-9313206 


08-07-93 


AU-B- 


3260493 


28-07-93 


WO-A-9104274 


04-04-91 






26-03-91 




EP-A- 


0494268 


15-07-92 






JP-T- 


5502443 


28-04-93 


WO-A-9205198 


02-04-92 


CA-A- 
EP-A- 
JP-T- 


2091266 
0548214 
6503710 


15-03-92 
30-06-93 
28-04-94 



Form PCT/ISA/aiO (patent fteiUy wmn} (July I9M) 



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