Skip to main content

Full text of "USPTO Patents Application 09804625"

See other formats


PCT 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 4 : 

C12N 15/00, C12P 21/02 
A61K 37/02 



Al 



(11) International Publication Number: WO 86/ 00639 

(43) International Publication Date: 30 January 1986 (30.01.86) 



(21) International Application Number: PCT/EP85/00326 

(22) International Filing Date: 4 July 1985 (04.07.85) 



(31) Priority Application Numbers: 



628,342 
652,447 
652,742 



(32) Priority Dates: 



(33) Priority Country: 



6 July 1984(06.07.84) 
19 September 1984(19.09.84) 
19 September 1984(19.09.84) 

US 



(71) Applicant (for all designated States except US): SAN- 

DOZ AG [CH/CH]; Lichtstrasse 35, CH-4002 Basel 
(CH). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only) : CLARK, Steven, C. 
[US/US]; 122 Johnson Road, Winchester, MA 01890 
(US). KAUFMAN, Randal, J. [US/US]; 1 1 1 Marlboro 
Street, Boston, MA 02116 (US). WONG, Gordon, G. 
[CA/US]; 1137 Massachusetts Avenue, Cambridge, 
MA 02138 (US). WANG, Elizabeth, A. [US/US]; 



136 Wolf Rock Road, Carlisle, MA 017419 (US). 

(74) Common Representative: SAND02 AG; Lichtstrasse 
35, CH-4002 Basel (CH). 



(81) Designated States: AT (European patent), AU, BE (Eu- 
ropean patent), CH (European patent), DE (Euro- 
pean patent), DK, FI, FR (European patent), GB 
(European patent), HU, IT (European patent), JP, 
KR, LU (European patent), NL (European patent), 
NO, SE (European patent), SU, US. 



Published 

With international search report 



(54) Tide: LYMPHOKINE PRODUCTION AND PURIFICATION 



(57) Abstract 

A method for preparing and isolating a transfor- 
mation vector containing CSF/cDNA. The method 
comprises: preparing RNA from a cell that produces 
CSF; preparing polyadenylated messenger RNA from 
said RNA; preparing single stranded cDNA from said 
messenger RNA; converting the single stranded 
cDNA to double stranded cDNA; inserting the double 
stranded cDNA into transformation vectors and trans- 
forming bacteria with said vector to form colonies; 
picking pools of 200 to 500 colonies each and isolating 
plasmid DNA from each pool; transfecting the plas- 
mid DNA into suitable host cells for expressing CSF 
protein; culturing the transfected cells and assaying 
the supernatant for CSF activity; and selecting CSF 
positive pools and screening the colonies used to make 
the pool to identify a colony having CSF activity. Also 
described are a cDNA coding for a protein having 
CSF activity (i.e. CSF/cDNA), a microorganism or ceil 
line transformed with a recombinant vector containing 
such CSF/cDNA, and a method for producing CSF 
protein by expressing said CSF/cDNA by culturing a 
microorganism or cell line. The invention also pro- 
vides a method of purifying the CSF proteins and the 
purified proteins so produced. 



kt turn ati rw cn cm mc cti en <rc rrt see kt irt tee 

ACT Tr» Im tin ir In In In In lly TV v«i *ii 
It 

^ i* CSF-fi *M CSF-fl 

jHK»rerCTKacceieeeieTtf.cesMeecetft«aeMCceT 



cat 

it* 



AAT ICC iTC CM IM ICC CM tff CTC CTI AM CTt MY Ml UC ACT KT 



lit CSF-6 



Vat C5F-G 

ICT IM ATI Mf IM If* IT* |M fit ATC TCI IM iff TTT MG 

*u fit « *n iii n» v«i n« nt tu i» n« mt n* «•» 



CCJ MS TK CTA CM MS CM CTt IM Of riC AM CM MC CT1 
fp# rv Cm In flt» TV art lw ll» Im fy> tu «■ lit In 



CTC CM 
U» It* 



I 

CM MC 
art «1 



ere tee an crc am Me etc rn mc an an nt mc eM tm im cm 

La IP Hi l« 10 fl| N l« 1k» NT RT Mi *■» lit Ta> ly» 11* 



TK CCT CCI ACC CO IM aCT 
Z\ff m Prt TV tr* TV 



nt Mi IM AM CTt AM 6M 

F*t l*» «• *M lt% Irt Aa 

<Sff Ml 

ll« CSf-3 

cca ore cm cm tea uccccccm 
f»l ll» lit . 

127 



lit CSF* lit 
T 

Tte tit cca ate en act ate ace rrr cAt 

St? Cm. Ala Tap Itn TV Ut Th* At lit 

100 

TV cst-g 
e 

rrTencnncArcccCTTTueTtCTci 
u» i«« m in n n« Hi cyt Tr» 



aroaiMTci -ccamccom iamtktct crcirtAAM 



311 



321 



SI 



wutcrnt aacrcAiear ccrcircrri 

T 3M in Ml 

C-WACCTte CCTMfCCM JCIIACCCTt 

-M HI 4/1 

lUaarcMT mtatttat* Titmrarr 

ii nt 7*t 

CATAfTTATT CAAUTITTT TACCfTAATA 



9 311 3S* JM 371 

CACHACCAt MCirillCC ACATeCATM TtMAfTMC 

111 A2I ill AM 

ATACAMCAT CtCAUAUI. rQttttTiTT TTATACTtM 

_ *m in in in 

TTTAAMTAT TTAITTirTT AmanTAA CTTCITITTt 

m 7M 771 7ft 

ATTATTATTA AAAATATRT TCTJU 



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 


GA 


Gabon 


MR 


Mauritania 


All Australia 


GB 


United Kingdom 


MW 


Malawi 


BB Barbados 


HI! 


Hungary 


NL 


"Netherlands 


BE Belgium 


rr 


Italy 


NO 


Norway 


BG Bulgaria 


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 


; u 


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 


FI Finland 


MG 


Madagascar 






FR France 


ML 


Mali 







WO 86/00639 



PCT/EP85/00326 



LYMFHOKINB PRODUCTION AND PURIFICATION 

Field of the Invention 

This invention relates to the production of a protein 
having the ability to stimulate the growth and differentiation 
of primate hematopoietic progenitor cells* in particular 
colony stimulating factor (CSF). The invention in one aspect 
provides a method for producing CSF protein by recombinant 
DNA techniques, to vectors containing the gene for expressing 
said protein, to microorganisms and cell, lines transformed 
with said vectors and to CSF protein thus produced. In a 
second aspect > the invention provides a method for isolating 
and purifying CSF protein , from either natural or recombinant 
sources, and thus purified CSF protein having a degree of 
purity and level of activity well above any that has been 
previously reported. 

Background of the Invention 

The many different cell types found in blood are all derived 
from pluripotent hematopoietic stem cells. Stem cells perform 
two functions: (1) they reproduce themselves, thereby 
maintaining a stem cell population in the body -and (2) they 
provide progeny cells committed to differentiate into any of the 
mature blood cell types. The cell which is committed to 
differentiate along a particular hematopoietic pathway is termed 
a progenitor cell. Progenitor cells for T lymphocytes r 3 



ERSATZBLATT 



WO 86/00639 PCT/EP85/00326 

lymphocytes, granulocytes, red blood cells, platelets, and 
eosinophils, as well as earlier progenitors which can 
individually give rise to several of the mature cell types, have 
been studied experimentally both in vivo and in vitro (Dexter, 
•f .M. 1983 J. Fathology 111 415-433) . It has been determined in 
vitro that proliferation and/or differentiation of each 
progenitor cell type depends upon specific "factors" which have 1 
Seen derived from various sources. For example, the later 
progenitors of red blood cells require a factor called 
erythropoietin. The factors required for survival, proliferation 
and differentiation of the myeloid progenitors committed to form 
mature neutrophilic granulocytes, monocytes and mature 
macrophages are called colony stimulating factors (CSFs) . 

CSF activity has been studied extensively in the mouse. 
Most adult mouse organs produce CSF activity. However, - 
compositions containing CSF activity that have been obtained from 
various tissues and by various methods appear to differ in their 
biochemical characteristics. Thus, the structural relationships 
between the different factors remain unknown. Furthermore, CSF 
activity appears to act at more than one step of granulocyte and 
macrophage development, and again it has been uncertain whether a 
single factor is responsible for all of the observed activities 
or whether a different factor acts at each step. (Burgess, A. 
and Metcalf , D. 1980 Blood 5£. 947-957.). 

Human CSF active has been obtained from placenta, certain 
fetal tissues, macrophages, and stimulated T cells. A line of T 

-2- 

ERSATZSLATT 



WO 86/00639 



PCT/EP85/00326 



cells (Mo) that produces one or more potent CSF activities was 
established from a patient with a T cell variant of hairy cell 
leukaemia (leukaemic reticuloendotheliosis) (Golde et al 1978 
Blood £2. 1068-1072). 

The ability of CS activity to stimulate granulocyte and 
macrophage production indicated that pharmaceutical compositions 
having CSP activity are clinically useful in situations where 
increased production of these (myeloid) cell types is required. 
Indeed, several patients with extremely high levels of apparently 
normal circulating granulocytes have been shown to have tumors 
which over-produce CSF* In one case, upon surgical removal of 
the tumor, the granulocyte count rapidly declined towards a 
normal level, strongly suggesting that CSP may be useful in 
regulating the numbers of circulating granulocytes. (Hocking, 
W., Goodman, J., and Golde, D. Blflfid £1 600 (1983)). In 
particular, CSF compositions are useful clinically for the 
treatment of myelo-suppression caused by chemotherapeutical or 
irradiation treatment of cancer. In addition, CSP compositions 
are useful in treating severe infections because CSF can increase 
and/or activate the number of granulocytes and/or monocytes. 

There are various different types of known CSF activities, 
including granulocyte. CSF (G-CSF), macrophage-CSF (M-GSF) , 
granulocyte-macrophage CSF (GM-CSF) and multi-CSF. The present 
invention is particularly concerned with GM-CSF . CSF proteins 
are known from various animal sources. However, the present 
invention is particularly concerned with primate OSF, more 
particularly human CSF and ape CSF. 



-3- 



ERSATZBLATT 



WO 86/00639 



PCT/EP85/00326 



Biological and biochemical characterization of compositions 
having CSP activity, and study of these compositions in the 
clinical setting have been hampered to date by the scarcity and 
impurity of human and/or other primate CSP compositions. It can , 
be appreciated that it would be desirable to identify the protein 
or proteins responsible for CSF activity. Furthermore, it would . 
be desirable to have a primate, preferably human source of such 
CSF that could readily supply these proteins in quantities and 
purity sufficient for biological and biochemical characterization 

and for use as therapeutic agents* 

i^iif W<^"t^*ii^ V«BKM« cloninq make it 
possible to clone . nucleotide sequence which encodes a protein 
and to produce that protein in quantity usinq « suitable 
host-vector system Maniatis, T. HOl-Hllnr rlflritOT - » Moratory 
Banaal Cold Sprin, Harbor Laboratory. Cold Sprlnq Harbor, N.Y. 
1982) . The protein can then be recovered by known separation and 
purification techniques. Cloninq methods which have been used to 
aate can be orouped into three q.neral cat.qori.s, (» methods 
• based upon knowledge of the protein structure, for example, its 
amino acid sequence, (2) methods based upon identification of 
the protein expressed by the cloned qen. usin, an antibody 
specific for that protein, and (3) methods based upon 
identification of an RHA species which can be translated to yield 
the protein or activity encoded by the qen. of interest. 



ERSATZBLATT 
-4- 



WO 86/00639 



PCT/EP85/00326 



Each of these classes of methods becomes difficult to 
apply when the protein of interest, such as CSF protein, 
is available in very low amount. Thus, if it is difficult 
to obtain an adequate quantity of purified protein, then 
it is difficult to determine the amino acid sequence or even 
partial sequences of the protein. Similarly, identification 
of an expressed protein by antibody binding is preferentially 
carried out using a high-titer monospecific polyclonal anti- 
serum. Such an antiserum cannot be obtained in the absence 
of quantities* of the pure protein (antigen). A monoclonal 
antibody offers an alternative approach, but the required 
antibody can also be difficult to obtain in the absence- 
of suitable antigen, and such monoclonal antibody may not react 
with the protein in the form in which the protein is expressed by 
available recombinant host-vector systems. Finally , translation 
of an RNA species to yield an identifiable protein or activity 
requires that the RNA in question be present in the aHA source in 
sufficient abundance to give a reliable protein or activity 
signal. The relative abundance of an RNA encoding a pacsicular 
protein generally parallels the abundance of the .protein, so that 
a rare protein is usually encoded by a rare mRSA. 

The Mo cell line has been used both as a starting material 
for purifying human CSFs and for identifying the corresponding 
messenger RNAs. However, -even with this relatively good source 
of CSF activity, it has proved to be extremely difficult to 
isolate enough of the protein for structural studies. 

In order to overcome the problems inherent in -cloning the 
nucleotide sequence encoding a rare protein such as CSF by the 
methods descciced above, a novel wethed was developed- This 
method requires only that the gene product or .its activity can be 



ERSATZSLATT 
-s- 



WO 86/00639 PCT/EP85/00326 

reliably measured. Suitable methods of CSF assay are described 
in Example 2 hereinafter. In a. second aspect, a purification 
process has been developed which enables the CSF protein 
to be isolated and purified from either recombinant or natural 
sources in a level of purity and activity much higher than 
was previously possible. 

Summary of the recombinan t DNA process of the Invention 

In its first aspect the present invention overcomes 
the problems of the prior art and provides a ready source 
of protein having CSF activity using recombinant DNA technol- 
ogy, in accord with the present invention, a novel cloning 
technique that requires only an assay for CSF acitivity is 
utilized to clone cDNA coding for a protein having CSF 
activity. Thus, the present invention provides a cDNA 
coding for a protein having CSF activity (i.e. CSF/cDNA) , a 
microorganism or cell line transformed with a recombinant vector 
containing such CSF/cDNA, and a method for producing CSF protein 
by expressing said CSF/cDNA by culturing a microorganism or cell 
line. Because the CSF protein is produced from a clone in accord 
with the present invention, we can be sure that it is a protein 
that has CSF activity. The invention further comprises a method 
for preparing and isolating a transformation vector containing 
CSF/cDNA, said method comprising: 

preparing RNA from a cell that produces CSF; 

preparing polyadenylated messenger RNA from said RNA,* 

preparing single stranded cDNA from said messenger RNA; 

converting the single stranded cDNA to double stranded 

cDNA; 



ERSATZBLATT 

-6- 



WO 86/00639 



PCT/EP8S/00326 



inserting the double stranded cDNA into transformation 
vectors and transforming bacteria with said vector to form 
colonies; 

picking pools of 200 to 500 colonies each and isolating 

plasmid DNA from each pool? 

transfecting the plasmid DNA into suitable host cells 

for expressing CSF protein; 

culturing the transfected cells and assaying the 

m 

supernatant for CSP activity; and 

selecting CSF positive pools and screening the colonies 
used to make the pool to identify a colony having CSP activity. 

The CSP proteins of this invention are growth and 
differentiation hormones for the cells of the myeloid system. 
They are for example indicated for use clinically for the 
treatment of myelo-suppression especially ( sympotomatic) 
granulocytopenia following chemotherapeucical or irradiation 
treatment of cancer. 
Brief Description of the Orawings 

Fig. 1 illustrates DNA sequences that code for a CSP 
protein in accord with the present invention. The DNA sequence set 
out in full codes for one variation of human CSF, referred to as 
CSF-Thr. Another allele codes for' an identical product except 
that Thr at position numbered 1 ioo is replaced by lie (CSF-Ile). 
The changes illustrated above for the human sequence are for 
differences in the DNA sequence coding for gibbon CSF (CSF 
of the Gibbon ape)(CSF-G). Deduced amino aci<i sequences are 

also illustrated. 

Fig.. 2 is a schematic illustrating the preparation of 

plasmid pTPL from plasmid pAdD26SVpA(3) . 

Fig. 3 is a schematic continuing from Fig. 2 and 
illustrating the preparation of plasmid p9i023 from plasmid 
pTPL. 

Fig. 4 is a schematic continuing from Fig. 3 and 
illustrating plasmid ?9L023(8). 

ERSATZ8LATT 

-7- 



WO 86/00639 



PCT/EP85/00326 



Fig. 6 is a schematic representation of vector pTALC-L8SR. 
Fig. 7 is a schematic representation of vector AJ-14. 

Detailed Description of the Process 

The following definitions are supplied in order to 
faciliate the understanding of this case. To the extent that * 
the definitions vary from meaning circulating within the art, the 

definitions below are to control. 

Amplification means the process by which cells produce gene 
repeats within their chromosomal DNA. 

CSF is a biological activity defined by the assays as 

described herein* 

CSF protein is a protein fren a primate source that exhibits 
CSF activity. For purposes of the present invention the teen CSF 
protein includes modified CSP protein, alleleic variations of CSF 
protein, and CSF protein preceded by a MET residue. 

Downstream means the direction going towards the 3» end of & 

nucleotide sequence. 

An enhancer is a nucleotide sequence that can potentiate the 
transcription of a gene independent of the position of the 
enhancer in relation to the gene or the orientation of the 
sequence. 

A gene is a deoxy ribonucleotide sequence coding for a given , 
protein. For the purposes herein, a gene shall not include 
untranslated flanking regions such as RNA transcription 
initiation signals, polyadenylation addition sites, promoters or 
enhancers. 

Ligation is the process of forming a phosphodiester bond 

between the 5' and 3' ends of two DNA strands. This may be 

ERSATZBLATT 

-8- 



WO 86/00639 PCT/EP8S/00326 

accomplished by several well known enzymatic techniques , 

including blunt end ligation by T4 ligase. 

Orientation refers to the order of nucleotides in a DNA 

sequence. An inverted orientation of a DNA sequence is one in 

which the 5' to 3' order of the sequence in relation to another 

sequence is reversed when compared to a point of reference in the 

DNA from which the sequence was obtained* Such points of 

reference can include the direction of transcription of other 

specified DNA sequences in the source DNA or the origin of 

replication of replicable vectors containing the sequence. 
Transcription means the synthesis of RNA from a DNA 

template* 

Transformation means changing a cell's genotype by the 
cellular uptake of exogenous DNA, Transformation may be detected 
in some cases by an alteration in cell phenotype. Transformed 
cells are called transf ormants, Pre-transf ormation cells are 
referred to as parental cells. 

Translation means the synthesis of a polypeptide from 
messenger RNA* 

Colony-stimulating factor activity (CSF) can be derived from 
a number of cellular sources including conditioned medium from 
peripheral blood mononuclear cells , lung and placental tissue, 
and bone marrow, urine from anemic patients, serum, and normal 
and neoplastic cells of T-lymphocyte and mononuclear phagocyte 
lineage. One cell line that produces CSF is the «o cell line 
deposited with and available from ATCC under the code number 
CRL8066. The CSF produced by this cell line is known as granulocyte 
macrophage CSF (or GM-CSF) and it is of course a human CSF. 
One source of Gibbon CSF is the T-cell line designated 4JCD MLA- 
144 and deposited with and available from the ATCC under code 
number HB 9370 deposited September 29, 1983. 

ERSATZBLATT 

-9- 



WO 86/00639 



PCT/EP85/00326 



In order to isolate a CSF clone in accord with the present 
invention, a novel procedure was used that requires only an assay 
technique for CSF activity. First, a cell that produces CSF 
activity such as T-lyraphocyte cells (or other sources such as set 
forth above) is identified. The mRNA of the cell is then 
harvested. Preferably, T-lymphocyte cells are used. In such 
case the membrane bound mRNA, which contains the mRNA for 
lymphokines , is separated from free mRNA in the cells. This 
separation is believed to enrich the collected mRNA 5-10 times 
for lymphokine sequences and thus reduces the effort involved in 
identifying the desired CSP clone. Polyadenylated messenger RNA 
is then prepared by chromatography on oligo dT cellulose. 

A cONA library is prepared from the mRNA using a vector 
suitable for transfection into a host to express the desired 
protein having CSF activity. First, sttand. cDNA is prepared using 
standard methods using the mRNA prepared above. The RNA/cDNA 
hybrid is. then converted to double-stranded cDNA form. The cdna 
can then be inserted into a suitable vector. 

The preferred host-vector system for the isolation of a CSF 
clone is based on expression of the CSF cDNA in a suitable 
transformation vector. A suitable transformation vector can rely 
on the transient introduction of DNA into mammalian cells 
(Mellon, P., V. Parker, Y. Gluzman, T. Maniatis 1981 Cell 21 
279-288) . In order to isolate the desired CSF transf ormants, it 
is not required that all cells of the population stably contain 
exogenous genes that express the desired CSF product. It is 
possible to transiently introduce exogenous genes into a 
subpopulation of cells such that the subpopulation will express 
the desired product over a period of several days. Because a 

ERSATZBLATT 
-in- 



WO 86/00639 



PCT/EP85/00326 



100-6415 

selectable marker is not required in the transformation vector 
for the DNA transfection and expression system in accord with the 
present invention, the exogenous DNA can be lost upon growth of 
the cells over a 1-2 week period. However, 2-3 days after 
transfection of suitable mammalian cells, the desired products 
are found to be synthesized and can be detected. 

The host-vector system of choice is based on the development 
of CV-1 monkey cell lines transformed with a 

replication-origin-defective SV40 DNA molecule (Gluzman, Y., Cell 
21 175-182, 1981). The transformed monkey CV-1 cells containing 
defective SV40 DNA, designated COS (CV-1, origin defective, 
SV40) , do not contain a. complete copy of the SV40 genome, but 
produce high levels of large T antigen and are permissive for 
SV40 DMA replication. They also efficiently support the 
replication of SV40 containing deletions in the early region and 
of bacterial plasaids which contain the SV40 origin of 
replication (Myers, R.M. & Tjian, R. 1980 PNAS 21 6491-6495) . 
Thus, this system provides a means of amplifying transfected 
exogenous DNA via SV4Q mediated DNA replication in order to 
increase the level off raRNA and protein expressed from the 
exogenous DNA. However, other similar systems are also useful. 

Vectors used for CSP expression typically contain various 
elements such as enhancers, promoters, introns, poiyadenylation 
sites, 3' noncoding regions and translational activators as will 
be described below. 

The vectors herein may include enhancers. Enhancers are 



ERSA7ZBLATT 
-11- 



WO 86/00639 



PCT/EP85/00326 



functionally distinct from promoters, but appear to operate in 
concert with promoters. Their function on the cellular level is 
not well understood, but their unique characteristic is the 
ability to activate or potentiate transcription without being 
position or orientation dependent. Promoters need to be upstream 
of the gene, while enhancers may be present upstream or 5* from 
the promoter, within the gene as an intron, or downstream from 
the gene between the gene and a polyadenylation site or 3» from 
the polyadenylation site. Inverted promoters are not. functional , 
but inverted enhancers are. Enhancers are cis-acting, i.e., they 
have an effect on promoters only if they are present on the same 
DNA strand. For a general discussion of enhancers see Khoury et 
al., Cell 21:313-314 (1983). 

preferred enhancers for use with mammalian cells are 
obtained from animal viruses such as simian virus 40, polyoma 
virus, bovine papilloma virus, retrovirus or adenovirus. 
Ideally, the enhancer should be from a virus for which the host 
cell is permissive, i.e. which normally infects cells of the host 
type. Viral enhancers may be obtained readily from publically 
available viruses. The enhancer regions for several viruses, 
e.g., Rous sarcoma virus and simian virus 40, are well known. 
See Luciw et al., Cell 21:705-716 (1983). It would be a matter 
of routine molecular biology to excise these regions on the basis 
of published restriction maps for the virus in question and, if 
necessary, modify the sites to enable splicing the enhancer into 
the vector as desired. For example, see Kaufman et al, J. Hoi. 



ERSAT2SLATT 
-12- 



WO 86/00639 



PCT/EP85/00326 



Biol., 151:601-621 (1982) and Mol. Cell Biol. 2<11> * 1304-1319 
(1982). Alternatively, the enhancer may be synthesized from 
sequence data? the sizes of viral enhancers (generally less than 
about 150 bp) are sufficiently small that this could be 
accomplished practically. 

Another element which should be present in the vector 
assembly is a polyadenylation splicing (or addition) site. This 
is a DNA sequence located downstream from the translated regions 
of a gene, shortly downstream from which in turn transcription 
stops and adenine ribonucleotides are added to form a polyadenine 
nucleotide tail at the 3 ' end of the messenger RNA. 
Polyadenylation is important in stabilizing the messenger RNA 
against degradation in the cell, an event that reduces the level 
of messenger RNA and hence the level of product protein. 

Eucaryotic polyadenylation sites are well known. A 
concensus sequence exists among eucaryotic genes: the 
hexanucleotide 5'-AAUAAA-3' is found 11-30 nucleotides from the 
point at which polyadenylation starts. DNA sequences containing 
polyadenylation sites may be obtained from viruses in accord with 
published reports. Exemplary polyadenylation sequences can be 
obtained from mouse beta-globin, and simian virus 40 late or 
early region genes, but viral polyadenylation sites are 
preferred. Since these sequences are known, they may be 
synthesized in yitro and ligated to the vectors in conventional 
fashion. 

The sequence which separates the polyadenyl&tion site from 

ERSATZBLATT 
-13- 



WO 86/00639 



PCT/EP85/00326 



the translational stop codon is preferably an untranslated DNA 
sequencs such as an unpromoted eucaryotic gene. Since such 
sequences and genes are not endowed with a promoter they will not * 
be expressed. The sequence should extend for a considerable 
distance, on the order of up to about 1,000 bases, from the stop 
codon to the polyadenylation site. This 3' untranslated sequence 
generally results in an increase in product yields. The vector 
may terminate from about 30 bp downstream from the concensus 
polyadenylation sequence, but it is preferable to retain the - V . 
sequences found downstream from the polyadenylation site in its 
wild-type environment. These sequences typically extend about 
from 200 to 600 base pairs downstream from the polyadenylation 
site. 

The presence of introns in the untranslated transcribed 
portion of the vector may increase product yields. Such introns 
nay be obtained from other sources than either the host cells or 
the gene sources. For example, a hybrid inton comprising a 5» 
splice site from the second intron of the adenovirus tripartite 
leader and a 3' splice site from an immunoglobulin gene inserted 
downstream from transcription start site in the adenovirus major 
late promoter results in increased product yield. 

In the preferred embodiment of the CSP cloning and 
expression vector there is a translational activator gene. 
Translational activators are genes which encode either protein or 
RNA products which affect translation of a desired mRNA. The 
best example is the adenovirus virus-associated (VA) gene (VAI) 

ERSATZBLATT 
-14- 



WO 86/00639 



PCT/EP85/00326 



which is transcribed into a short UNA specie3 that interacts with 
sequences in the 5' untranslated region of the adenovirus major 
late mRNAs (ThimraaFpaya et al., 1982 Cell 3 S43). The necessary 
sequences for translational activation by VA RNA lie within the 
adenovirus late mRNA tripartite leader. The adenovirus 
tripartite leader is spliced together from noncontiguous regions 
of the adenovirus genome and is present on the 5* end of the 
adenovirus major late transcripts. VA RNA can interact to 
activate translation of mRNAs which contain the tripartite leader 
sequence. Thus, the preferred cONA cloning- and expression vector 
contains the spliced form of the tripartite leader, and the 
adenovirus VA genes. 

These vectors can be synthesized by techniques well known to 
those skilled in this art. The components of the vectors such as 
enhancers, promoters, and the like may be obtained from natural 
sources or synthesized as described above. Basically, if the 
components are found in DNA available in large quantity, e.g. 
components such as viral functions, or if they may be 
synthesized, e.g. polyadenylation sites, then with appropriate 
use of restriction enzymes large quantities of vector may be 
obtained by simply culturing the source organism, digesting its 
DNA with an appropriate endonuclease, separating the DNA 
fragments, identifying the DNA containing the element of interest 
and recovering same. Ordinarily, a transformation vector will be 
assembled in small quantity and then ligated to a suitable 
autonomously replicating synthesis vector such as a procaryotic 

ERSATZBLATT 



WO 86/00639 



PCT/EP85/00326 



plasmid or phage. The pBR322 plasmid may be used in most cases. 
See Kaufman et al., QS.. cj£. 

The synthesis vectors are used to clone the ligated 
transformation vectors in conventional fashion, e.g. by 
transfection of a permissive procaryotic organism, replication of 
the synthesis vector to high copy number and recovery of the 
synthesis vector by cell lysis and separation of the synthesis 
vector from cell debris. 

The vectors containing cDNA prepared from a cell that 
produces CSP aactivity are then transfected into E. col i and 
plated out on petri dishes at approximately 2000 colonies per 
dish. The colonies are lifted off onto a nitrocellulose filter 
and the filter is transferred to a new plate which is kept as a 
master. "After growing these colonies, replicas are made and 
aligned with the original by careful marking so that sections of 
the replica filters can be identified with the corresponding 
portion of the master plate. 

Each replica filter is cut into sections containing a 
predetermined number of colonies per section, preferably* about 
200-500 colonies per section. The colonies from each section are 
scraped into mediuim such as L-Broth, the bacteria collected by 
centrifugation and the plasmid DNA separated. The plasmid DNA 
from each section is transfected into a suitable host for 
expression of protein. The preferred synthesis vector herein is 
a mutant of the e. coli plasmid pBR322 in which *equences have 
been deleted that are deleterious to eucaryotic cells. See 

ERSATZBLATT 



WO 86/00639 



PCT/EP85/00326 



Kaufman et al., ££• £i£. Use of this mutant obviates any need to 
delete the plasmid residue prior to transf ection . After growing 
the transf ected cells , the medium is assayed for CSF activity. 
A positive assay indicates that a < colony containing CSF/coNA is 
on a particular section of a filter* 

To determine which of the clones on the section of the 
original master filter contains CSF/cDNA, each clone on the 
filter section is picked and grown. The cultures are then placed 
in a matrix. Pools are prepared from each horizontal row and 
vertical column of the matrix. DNA samples are prepared from 
each pooled culture and transf ected into the host cells for 
expression. Supernatants from these pools are assayed for CSF 
activity. One vertical column pool and horizontal row pool 
should produce CSF activity. The clone common to these pools 
will contain CSF/cDNA. If the matrix contains more than one 
positive clone , more than one column and row will be positive. 
In such case, further screening of a small number of clones may 
be necessary. 

The CSF/cDNA is excised from the clones by restriction 
enzymes and can be sequenced by known techniques. It can be 
readily appreciated that the procedure described herein can be 
used to obtain CSF/cDNA from any source. The complete DNA 
sequence of a CSF/cDNA in accord with the invention is 
illustrated in Fig. 1 along with the predicted amino acid 
sequence of the translated CSF protein product. 

The DNA sequence coding for a protein exhibiting CSF 

m 

ERSATZ BLATT 



WO 86/00639 



PCT/EP85/00326 



activity in accord' with the present invention, such as 
illustrated in Pig. 1, can be modified by conventional techniques 
to produce variations in the final CSF protein which still have 
CSP activity in the assay tests described herein. Thus, for 
example, one, two, three, four or five amino. acids can be 
replaced by other amino acids. Belgian Patent No. 898,016, which 
is incorporated herein by reference, describes one such typical 

technique for replacing cysteine by, e.g., serine. 

CSF/cDNA in accord with this invention includes the nature 
CSF/cDNA gene preceded by an ATG codon and CSF/cDNA coding for 
allelic variations of CSF protein. One allele is illustrated in 
Fig. 1. Another allele that we discovered has a thymidine 
residue at position 365 instead of the cytosine residue 
illustrated in Fig. 1. The CSF protein of this invention 
includes the 1-methionine derivative of CSF protein (Met-CSF) and 
allelic variations of CSF protein. The mature CSF protein 
illustrated by the sequence in Fig. 1 begins with the sequence 
Ala' Pro- Ala* Arg— the beginning of which is depicted- by an arrow 
after nucleotide number 59 in Fig. 1. The Met-CSF would begin 
with the sequence MefAla«Pro»Ala»Arg — The allele variation 
illustrated in Fig. 1 has a Thr at amino acid residue number 
100 (beginning at Ala after the arrow) and can be referred 
to as CSF(Thr). Another variation has an lie residue at 
position 100 and can be referred to as CSF (lie). Purified 
CSF protein of the present invention exhibits a specific 
activity of at least 10 7 units/mg of protein and preferably 
at least 4 x 10 7 units/mg when assayed with human bone marrow 
cells . 



ERSATZSLATT 

-L8- 



WO 86/00639 



PCT/EP85/00326 



Host-vector systems for the expression of CSF may be 
procaryotic or eucaryotic, but the complexity of CSF may make the 
preferred expression system a mammalian one. Expression is 
easily accomplished by transforming procaryotic or eucaryotic 
cells with a suitable CSF vector. The DNA sequence obtained by 
the above described procedure can be expressed directly in 
mammalian cells under the control of suitable promoter. 
Heterologous promoters well-known by those skilled in the art can 
be used. In order to express CSF in procaryotic or in yeast 
cells, the leader sequence (or secretory sequence) must be 
removed. The position of the codon for the N- terminus of the 
mature CSF protein is illustrated in Fig. 1. This can be done 
using standard techniques known by those skilled in the art. 
Once the desired CSF/cDNA clone is obtained, known and 
appropriate means are utilized to express the CSF protein, e.g. 
insertion into an appropriate vector, and transfection of the 
vector into an appropriate host cell, selection of transformed 
cells, and culture of these transf ormants to express CSF 
activity. Suitable host cells include bacteria, e.g. E. coli, yeast, 
mammalian e.g. CHO, and insect cells. The CSF protein thus produced 
may have a methionine group at the N-terminus of the protein 
(herein called Met-CSF) . The mature protein produced by procaryotic 
and eucaryotic cells will be otherwise identical in amino acid 
sequence, but the eucaryotic product may be glycosylated to the 
same or a different extent as in the natural product. 
Various methods of obtaining CSF protein in accordance with 
the convention are illustrated in the Examples hereinafter. 
Other methods or materials, e.g. vectors, will be readily 
apparent to those skilled in the art on the basis of the 
Examples and the foregoing description. 

CSF protein expressed in appropriate procaryotic or 
eucaryotic cells can be recovered by purification and separa- 



ERSATZBLATT 

-19- 



WO 86/00639 PGT/EP85/00326 
tion techniques known to those skilled in the art. However, 
as indicated the present invention also provides a purification 
process which enables CSF protein from both recombinant and 
natural sources to be obtained in high purity and activity. 

Summary off the Purification process of the Invention 

The present invention overcomes the problems off the 
prior art and provides a method for purifying protein having 
CSF activity. CSF protein in accord with the present invention, 
has specific activity off at least about 1 x 10 units per 
mg off protein, preferably at least 2 x 10 7 units per mg off 
protein and more preferably at least about 4 x 10 units 
per mg off protein when assayed the human bone marrow assay. 

In accord with the present invention, a method for 
purifying CSF protein comprises? precipitating the protein 
with ammonium- sulfate- at, 80%. saturation to form a pellet 
containing the CSF protein? resuspending the pellet in a 
buffered solution at a pK in the range off about 6 to about 
8; applying the buffered solution containing CSF to a chromato- 
graphic column, eluting with the buffered solution containing 
sodiunt chloride- and collecting the fractions having CSF 
activity; pooling the active fractions, applying them to- 
a C4 reverse phase column and eluting with a 0 to 90% aceton- 
itrile gradient to collect the active fraction. 

Brief Description of the Drawings relating to the purif ication 
process - 

Fig. 5 illustrates SDS-PAGE analysis off the purified 
CSF protein. 

Detailed Description of the purification process of the 
Invention 

The CSF protein to be purified in accordance with the 
process of the invention can be derived from any of the natural 
sources described above as starting sources for the recombinant 

ERSATZBLATT 
-zo- 



WO 86/00639 



PCT/EP85/00326 



DMA process, for example the Mo cell line or the UCO MLA-144 
Gibbon cell line. 

Alternatively, the CSF protein may be produced using 
the recombinant DNA techniques of the invention. 

CSFs from any source can be purified by the process 
o£ the present invention. The conditioned medium from any 
source of CSF protein is preferably concentrated by ultra- 
filtration to a protein concentration of at least about 0.1 
mg protein per ml. The protein is then precipitated by adding 
ammonium sulfate to 80* of saturation. The resulting pellet 
is resuspended in an aqueous solution buffered at a pH in 
the range of about 6 to about 8. Examples of suitable buffers 
include Tris-HCl, HEPES, sodium citrate, and the like. 

The buffered solution is fractionated by column chromato- 
graphy. Suitable materials for use in the chromatography 
column are octylsepharose, DEAE-ultrogel, AcA44-ultrogel, 
AcA-54 ultogel, and the like. One or more of these materials 
can be used in sequence to obtain higher purity. 

Fractions form each column are collected and assayed 
for CSF activity. The active fractions are pooled and diluted 
with trifluoroacetic acid (TFA), heptaf luorobutyric acid 
(HFBA), or the like, and applied to a C4 reverse phase column 
The CSF activity is then eluted using a 0-90% acetonitrile 
gradient in TFA or HFBA, preferably at a concentration of 
0.101 or 0.15% (vol/vol) respectively, depending upon which 
acid was used to apply the pooled fractions to the column. 

The fractions having CSF activity are analyzed by SOS 
polyacrylamide gel electrophoresis (13.5% gel as described 
by Lammli, U. Nature 227 , 680 (1970)). Additional treatments 
using the above mentioned chromatographic column materials 
can further purify the CSF protein to homogeneity. 

Purified CSF protein fractionated by SOS-PAGE revealed 
a heterogeneous CSF protein having an apparent molecular 



ERSATZBLATT 

-21- 



WO 86/00639 



PCT/EP85/00326 



weight in the range of about 15,000 to about 26,000 daltons. 
This apparent size heterogeneity is due to the extensive 
glycosylation of the protein and is a common feature of gly- 
coproteins. Fractionation of less purified samples from Mo 
cell conditioned medium by SDS-PAGE (under non-reducing 
conditions) and assaying protein eluted from the gel revealed 
the presence of a second protein having CSP acitivity having 
an apparent molecular weight of about 28,000 to 30,000. 

CSF activity binds and elutes from octylsepharose, DEAE 
ultrogel and the C4 reverse phase column. Roughly 60% of 
the CSF activity binds a Con-A sepharose (40% flow through) 
and can be eluted with alpha methylmannoside. 

Molecular weight analysis of recombinant CSF by gel 
filtration in low salt revealed that about 30% of the activity 
eluted with an. estimated molecular weight of about 19,000 
but 70% of the material behaved as dimers, eluting at a 
position corresponding to a molecular weight of about 38,000. 
If 1M NaCL is included in this column, all of the activity 
elutes in a broad peak at about 19,000 daltons. 

The purified CSF is stable for at least 16 hours when 
incuba?«d at 4 8 C (pH 7.4) in 4M guanidine hydrochloride; 
in lOmM EDTA; lOmM 2-mercaptoethanol: and in 30% (v/v) ethanol. 
The CSF activity also is stable in 0.1% trif luroacetic acid 
(TFA) (pH 2.0) and 0.1% TFA plus 25% (v/v) acetonitrile. 

As aforesaid, the CSF protein in accord with the present 
invention is indicated for use in- the treatment of myelo- 
suppression such as (symptomatic) granulocytopenia, for example 
caused by chemotherapeutical or radiation treatment of cancer . 
In addition, CSF proteins of the invention are indicated 
for use in the treatment of severe infection. For such use, 
an indicated dosage of about 200 to 1-000 ug per patient is 
typically indicated. The CSF protein is preferably injected 
into the patient intravenously in a suitable pharmacological 
carrier. Exa.?.= las of such carriers include pharmacological 
saline and human serum albumin in saline. 



ERSATZBLATT 

-12- 



WO 86/00639 



PCT/EP85/00326 



In addition, the CSP proteins of the invention have 
other activities and uses. For instance, it has been shown 
that murine CSFs activate neutrophils. Thus, it would be 
expected that the primate CSFs of the present invention will 
also activate neutrophils. Therefore, physiological functions 
of CSF may be server al fold. In the bone marrow, this lymphokine 
can stimulate proliferation and differentiation of effector 
cells for host defense while, in the periphery, new and 
existing cells can be activated. In a localized immunological 
response CSF can retain circulating neutrophils in or away* 
from areas of inflammation. Inappropriate localization and/or 
activation of neutrophils can be involved in the patho- 
physiology of a variety of immune -mediated disorders such 

as rheumataoid arthritis. 

The invention will be further understood with reference to 

the following illustrative embodiments, which are purely 

exemplary, and should not be taken as limitive of the true scope 

of the present invention, as described in the claims. 

in the examples, unless otherwise specified, temperatures 

are in *C. 

Restriction endonucleases are utilized under the conditions 
and in the manner recommended by their commercial suppliers. 
Ligation reactions are carried out as described by Maniatis et 
al., auora at 245-*, the disclosure of which is incorporated 
' herein by ref 3ranc*, using the buffer described at page 246 
thereof and using a DNA concentration of 1-100 ag/ml, at a 
temperature of 23"C for blunt ended DNA and 16'C for "sticky 
ended- DNA. Electrophoresis is dene in 0.5-1 .5% Agarose eels 
containing 90 mM Tris-boca:e, 10 mM E0TA. All radiolabeled DNA 
is labeled with 32 P , whatever labeling technique was used. 

By "rapid ocep- is meant * rapid, small scale production of 
bacteriophage cr plasmid DMA, e.g., as described ty flaniatis et 
al., auajca, at p. 3SS-371. ERSATZBLATT 

-23- 



WO 86/00639 



PCT/EP8S/00326 



EXAMPLE A 
Step 1. ma rpM Line Cultures 

Mo cells (ATCC CRL 8066) were grown routinely in Alpha (6% 
C02> or Iscove's (10% CO2) medium containing 20% Petal Calf Serum^ 
(PCS) , 2mM glutamine, 100 O/ml streptomycin and 100 ug/ml 
penicillin. The cells should be subcultured every 4-5 days. * 
Cells are counted and seeded into Palcon T-175 flasks in 100-150 
*■ ml medium at density of 3-4 x 105 cells/ml. Cells will double in 
20% FCS every 4-7 days. Growth rate is not constant and cells 
may sometimes appear to stop growing then go through bursts of 
growth. Mo cells can be grown in serum-free medium. Survival is 
much better when cells are not washed when transferred from FCS 
to serum-free medium. Optimal density in Serum-Free medium (SF) 
is 5 x 105 cells/ml. Cells will grow slightly (or at least 
maintain constant number) for 3 days in serum-free medium, and 
then should be fed 20% FCS for at least 4 days. This growth 
schedule (3 days SP, 4 days 20% PCS) can be repeated weekly if SP 
medium is required, with no apparent harm to the cells for 
several months. 

Step. 2 Assays for rsP Activity 
A. Bone Marrow Assay 

Obtain fresh bone marrow. Break apart spicules by drawing 
through 20, 22, then 25 gauge needle. Dilute 1:1 with sterile ^ 
phosphate-buffered saline (PBS) (room temperature) and layer over 
Picoll-Paque (about 30 ml BM-PBS over 6 ml Picoll) . Spin at 1500" 



EflSATZBLATT 

-24- 



WQ 86/00639 



PCT/EP85/00326 



rp« for 40 minutes at room temperature Remove fat and PBS layer 
and discard. Pipette off the light density layer. Wash 2x with 
PBS and count. Plate cells in RPMI (purchased from GIBCO as RPMI 
1640) plus 10% HIFCS (heat inactivated PCS) for 3 hours to remove 

adherent cells. 

Plating medium (make fresh): 
20% PCS 

0.3% agar dissolved in H2O cooled to 40*C 
2x Iscoves (1:1 v/v with Agar) 

1% P/S final concentration of lOOU/ml streptomycin, 
100 ug/ml penicillin 

10-4M alpha thioglycerol in 2x iscoves from 10~2m 
stock 

' Cool agar to about 40*. Mix with other ingredients. 
Cool in H 2 0 bath to 37-38* and hold at that 
temperature. 



After 3 hours, pipette off the non-adherent cells. Spin and 
count. Add 2 x 10 5 cells/ml of plating medium and keep in 
controlled temperature water bath at 37-38°. Add samples (e.g., 
medium from transfected cells; usually 10 /il sample) to the first 
row of wells of a microtiter plate in duplicate. Add 100 /il cell 
suspension to each well. Add additional SO ,ul of cell suspension 
to each well in the first row. Mix thoroughly and transfer 50 /il 
of solution from the first row into the next row, .etc. and 
continue 1:3 dilutions across plate. Wrap the plate in paraf ilm. 



EflSATZSLATT 
-25- 



WO 86/00639 PCT/EP85/00326 

incubate 10-14 days at 10% C0 2 , 37"C in fully humidified 
atmosphere and score colonies. 

To scoce the colonies, the total number of colonies that 
grow in each well is counted. In each assay, several wells are 
plated without including a sample (blank) to obtain a background 
colony count. The average number of colonies that grew in the 
blank wells is subtracted from the number of colonies found in 
each of the wells containing samples. One unit of CSP is the 
amount that will stimulate the formation of one colony above the 
background level per 10 s human bone marrow cells (plated at 10* 
cells per ml) when the CSP concentration is sub-saturating. The 
sub-saturating concentration is determined by dilution and 
comparing the number of colonies at various dilutions to find the 
concentration just below the saturation level. 

For this assay., the colonies containing granulocytes , 
nonocytes oc both types cf cells are counted. The types of cells 
in the colonies are determined oy picking colonies and staining 
individual ceils. 

B. KG-1 Cell Assay 

KG-1 cells f Blood , vol. 56, No. 3 (1980)) are yrown in 
Iscovea medium + 10% FCS passed 2x per week and saeded for each 
passage at 2x10* cells/ml. The calls are used for assay only 
between passage 30-35. The assay is the same as for bone narrow 
as described above, except the KG-1 cells are plated in agar 
mixture at 4xl0 3 cells/ml. 



ERSATZ8LATT 

-26- 



WO 86/00639 PCT/EP85/00326 

The number of colonies growing in each well is determined 
and the background count is subtracted as in the Bone Marrow 
assay described above. One KG-1 CSF unit/ml is that 
concentration off CSF that will stimulate half off the maximum 
number (saturation) of KG-1 colonies to grow. The maximum number 
is obtained by including a saturating level of CSF in several 
wells. 

Step 3. C onstruefcion Qf Vector 091023(B) 

The transformation vector was pAdD26SVpA(3) described by 
(Kaufman at al., Hoi. Cell Biol. 2(11) : 1304-1319 (19821. It has 
the structure illustrated in Fig. 2. Briefly this plasm id 
contains a mouse dihydrof olate reductase (DHFR) cDNA gene that is 
under transcriptional control of the adenovirus 2 (Ad2) major 
late promoter. A 5' splice site is included in the adenovirus 
DNA and a 3* splice site* derived from an immunoglobulin gene, is 
present between the Ad2 major late promoter and the DHFR coding 
sequence. The SV40 early pcly&denylation 3ite is present 
downstream from the DHFR coding sequence. The 
procaryotic-darived section of pAd026SVpA(3) is from pSVOd 
(Mellon, p., Parker, V. # Gluzaan, ?. and Maniatis, T. 1981, Cell 
22:279-288) and does not contain the pBR322 sequences known to 
inhibit replication in mammalian cells (Lucky, M. , and Botohar., 
M. 1S31, Hacure (London) 221:79-81. 

pAdE26SV?A(3) is converted into plasmid pCVSVL2 as 
illustrated in Pig. 2. pAdD2'iSVpA(3) is converted into plasmid 



ERSATZBLATT 

-27- 



WO 86/00639 PCT/EP85/00326 

pAdD26SVpA(3) <d) by deletion of one of the two Pstl sites in 
pAdD26SVpA<3) . This is accomplished by a partial digestion with 
pstl (using a deficiency of enssyme activity so that a 
subpopulation of linearized plasraids can be obtained in which 
only one Pstl site is cleaved) , then treatment with Klenow, 
ligation to recircularize the pla3mid, transformation of Ei CQll 

« 

and screening for deletion of the Pstl site located 3* of the 
SV40 polyadenylation sequence* 

The adenovirus tripartite leader and virus associated genes 
(VA genes) were inserted into pAdD26SVpA<3) (d> as illustrated in 
Pig. 2. First, pAdD26SVpA(3) (d) was cleaved with PvuII to make a 
linear molecule opened within the 3* portion of the first off the 
three elements comprising the tripartite leader. Then, ?JAW 43 
(zain et al. 1979, Cell ifi 8S1) was digested with Xho 1, treated 
with Klenow, digested with PvuII, and the 140 base pair fragment 
containing the second and part of the third leaders was isolated 
by electrophoresis on an acrylaoide gel (6% in Tris berate 
buffer; Maniatis et al. 119821 supra). The 140 bo fragment was 
then ligated to the PvuII digested pAd026SVpA<3) (d) . The 
ligation product was used to transform S. S?li to tetracycline 
resistance and colonies were screened using the Grunstein-Hogness 
procedure using a 32 P labelled probe hybridising to the 140 base 
pair fragment. DMA was prepared from positively hybridizing 
colonies to test whether the PvuII site reconstructed was S'or 3 8i 
of the inserted 140 base pair DNA specific to the 2nd *nd 3rd 
adenovirus late leaders. In the correct orientation of the PvuII 



ERSATZBLATT * 
-2a- 



WO 86/00639 PCT/EP85/00326 

site is on the 5 1 side of the 140 base pair insert. This plasmid 
is designated pTPL in Fig. 2. 

The Ava II 0 fragment of SV4Q containing the SV40 enhancer 
sequence was obtained by digesting SV40 DMA with Ava II, blunting 
the ends with Klenow fraagment of Pol I, ligatlng Xho 1 linkers 
to the fragments, digesting with xho 1 to open the Xho 1 site, 
and isolating the fourth largest (D) fragment by gel 
electrophoresis. This fragment was then Ugated to Xho 1 cut " 
pTPL yielding the plasmid pCVSVL2-TPL. The orientation of the 
SV40 D Fragment in pCVSVL2-TPL was such that the SV40 late 
promoter is in the same orientation as the adenovirus major late 
promoter. 

To introduce the adenovirus virus associated (VA) genes into 
the pCV3VL2-TPL, first a plasmid p3R322 is constructed that 
contains the adenovirus type 2 Eind III B fragment. Adenovirus 
type 2 DNA is digested with Hind III and the 3 fragment is 
isolated after gel electrophoresis. This fragment is then 
inserted into pBR322 which has previously been digested with Hind 
III. After transformation of E. coli to ampicillin resistance, 
recombinants are screened for insertion of the Hind III B 
fragment and the inserted orientation is determined by 
restriction enzyme digestion. pBB322 - Ad Hind III B contains 
the adenovirus type 2 Hind III B fragment in the orientation 

depicted in Pig. J* 

As illustrated in Pig. 3, the va genes are conveniently 
obtained from plasmid p3R322-Ad Hind III 3 by diaesting with Hpa 



ERSAT2BLATT 

-29- 



WO 86/00639 PCT/EP85/00326 

I, adding EcoRl linkers and digesting with EcoRl, and recovering 
the 1.4kb fragment. The fragment having EcoRl sticky ends is 
then ligated into the EcoRl site of pTPL (which had previously 
been digested with ScoRl) . After transformation of S. co li HB1Q1 
and selection foe tetracycline resistance, colonies are screened 
by filter hybridization to a DNA probe specific to the VA genes. 
DNA is prepared from positively hybridizing clones and 
characterized by restriction endonuclease digestion. The product 
plasmid is designated p91Q23. 

The 2 EcoRl sites in p91Q23 are removed. p91023 is cut to 
completion with EcoRl, generating two DNA fragments, one about 
7Kb and the other about a 1.3 Kb fragment containing the VA 
genes. The ands of both fragmsnts are filled in using the Xienow 
fragment of Poll, and then both fragments i.e. 1.3 Kb, 7Kb, are 
religated together. A plasmid p91G23<A> containing the VA genes 
and similar to p91023 but deleted for the 2 EcoRl sites is 
idewtif ied by Grunscein-Hcgress screening with the VA gene 
fragment, and by conventional restriction site analysis. 

Then the single Pstl site in *91023 (A) is removed and 
replaced with an EcoRl site. p91023<A> is cut to completion with 
Pstl, and then treated with Klsnow fragment of Poll to generate 
flush ends. EcoRl linkers are ligated to the blunted Psel site 
of p91Q23(A). The linear p91023'.A> , with EcoRl linkers attached 
at the blunted ?stl site is separated frcm unligated linkers and 
digested to completion with EcoRl, and then religated, k pl&smid 
F 91023(S) is recovered and identified to have a structure similar 



ERSATZBLATT 

-30- 



WO 86/00639 PCT/EP85/00326 

to p91023(A), but with an EccRl site situated at the previous 
pstl site. 



Step 4. Preparatio n of cDHA Library 

Mo cells were induced for 16-20 hrs. with PHA and PHA to 
enhance their lymphokine production. Cells were plated at 5 x 
105 cells/ml in Iscove's medium with 20% PCS, 0.3% (v/v) PKA and 
5 ng/ml TP A. The cells were collected by centrifugation. The 
pelleted cells were resuspended in 20 ml of ice cold hypotonic 
lysis buffer (RSB bufferi 0.01H Tris-HCl, .PH 7.4, 0.01M KC1, 
0.GG1SM MgCl2, 1 ug/ml-cycloheximide, 50 units/ml RNAsin and 5nM 
dithiothreitol) . The cells were allowed to swell on ice for five 
minutes then were ruptured mechanically with 10 strokes of a 
tight fitting dounce glass homogenixer. Th« homogenate was 
centrifuged at low speed (2000 RPM in a Beckman J* centrifuge) to 
remove nuclei and unlysed cells. The supernatant was held on ice 
while the nuclear pellet was resuspended in 10 ml of RSB and 
re-cent cifuged at low speed. This second supernatant was pooled 
with the first and the combined supernatants were centrifuged at 
low speed to remove residual contamination with nuclei and 
unlysed cells. The supernatant from this spin was brought no 
0.15H KC1 by addition of 2M KC1 then centrifuged at high speed 
(25,000 RPM, Beckman Sw 28 rotor for 30 minutes) to pellet the 
nembranes. The membrane pellet was carefully washed with cold 
\SB then resuspended in 12 ml of RSB containing 2 M sucrose and 
J.15M XC1. Two discontinuous gradients were prepared in Becknan 



ERSATZBLATT 

-31- 



WO 86/00639 PCT/EP85/00326 

SM41 centrifuge tubes by layering 6 ml of the membrane solution 
in 2 M sucrose over 2 ml of RSB with 2.5 « sucrose and 0.15M KC1. 
The tubes were filled to the top by overlaying with 2.5 ml of RSB 
containing 1.3M sucrose and 0.15H RCl. These gradients were spun 
for 4 hours at 27,000 RPM (Beckman, SW41 rotor) at 4«C. The 
membrane layer (at the interface between the 2.0M and 1.3M 
sucrose) was carefully removed from the side using an 18 gauge . 
needle and syringe. The membrane fractions from the two 
gradients were pooled and diluted with 1 volume of distilled H 2 0 
then brought to 0.5% Triton X-l 00 and 0.5% sodium deoxycholate 
then extracted with an equal volume of phenol. The aqueous layer 
was re-extracted with a 1:1 mixture of phenol and chloroform and 
finally an equal volume of chloroform. Finally, the membrane 
bound RNA was precipitated by addition of Nad to 0.2SM and 2.5 
volumes of cold ethanol and incubated overnight at -2Q*C. The 
precipitated RNA was collected by centrifugation (4000 RPH for 10 
min. in the Beckman J-« centrifuge) and was resuspended in 1 ml 
of distilled water. From 2x10* cells, approximately 1 ag of RNA 
was obtained. The messenger RNA CmRNA) was isolated from the 
total RNA by chromotography on a 0.5 ml oligo dT-cellulose 
column. Briefly the RNA was heated to 70-C for 5 min., quick 
chilled on ice, then diluted 5 fold with room temperature binding 
buffer (0.5M LiCl, 0.01* Tris-BCl, ?H 7.4, 0.002 H EDTA, and O.U 
SDS) . The RNA in binding buffer was passed over the oligo 
dT-cellulose column equilibrated with binding buffer at room 
temperature. The column was washed with 5 ml of binding buffer 



ERSATZBLATT 
-32- 



WO 86/00639 PCT/EP85/00326 

then with 5 ui of 0.15M LiCl, 0.01M Tri3-HCl pH7.4, 0.C02M EDTA, 
and 0.1% SOS. Finally, mRUA was eluted with 2 al of 0.01M 
TtiS-HCl PH7.4, 0.002M EDTA, and 0.1% SDS. The mRNA was 
precipitated by addition of NaCl to 0-2S (4 and 2.S volumes of 
ethanol and incubation overnight at -20*C. The precipitated aRNA 
was collected by centrifugation (30,000 RPK foe 30 minutes in a 
Becknan SW55 rotor). The tube was carefully drained and the mRNA 
pellet was reauapended in SO ml of. H 2 0. The resuspended mRNA was 
brought to 0.25M Nad then extracted 1 time with a 1»1 mix of 
phenol and chloroform then 3 times with chloroform. The mRMA was 
precipitated by the addition of 2.5 volumes of ethanol. The 
mixture waa f cosen and thawed several times in a dry ice/ethanol 
bath then cantrifugad IS min. in an Sppendocfi centrifuge. The 
tube was carefully drained and the mRNA pellet was resuspended in 
20 ul cf distilled H 2 0. The final yield was approxiawtaiy 30 uc 
of mRMA. 

First strand c3SA was prepared using standard methods. 
Briefly, 10 ug of membcaca mRMA was diluted into a 100 ul cDSA 
synthesis reaction mixture containing 300 aN Tris pH 3.4. 140 art 
KC1, 10 on MgCl 2 , 10 n»M 3-marcaptoethanol , 500 uM each of dATF, 
dGTP, dCTP and dTTP, 5 ug. of oligo-dT (phosphorylated and 
average size of 12-19) as primer, 150 uCi of 32? dCTP (400 
Ci/mmole) and 20 units of the ribonuclease inhibitor RNAain. Tha. 
reaction was initiated by addition of 1C0 units of reverse 
transcriptase and incubated for 3C minutes at 42*C. The reaction 
was stopped by addition of EDTA to 40 bM and the RNA was degraded 



ERSATZSLATT 

-33- 



WO 86/00639 PCT/EP8S/00326 

by incubation Jot 20 mln. at «S'C in 0.2M HaOH. The has. was 
attained by addition of 20 aX 2M Tris, pH 7.4. Th. reaction 
„ix wa. then extracted with phenol/chloroform, oacx extracted 
with 50 ol 10 mH trie pB 7.5, 1 * EDTA CTE> and th. aou.ou. 
phases were pooled. Th. first strand cONA was converted to 
double stranded cDNA by Incubation for 12 hours at ICC with 40 
units of th. U.no« f r.caent of DNA polymeras. X in a 100 ul 
ruction containing 50 oM potassium phosphate, pB 7.4, 2.3 mM 
DTI, 2-..ccaptoethanol, 10»H NoClj. ISO uMolar each of th. 4 
d.oxynucleotid. triphoaphate. and 25 «Ci of »F dCTP. Th. 
reaction was stopped by extraction with phenol/ehlorofor- and th. 
unincorporated triphosphate, w.r. raaoved by passin, th. aqueous 
phas. over a 1 »1 seph.de. S-50 column. Thi excluded fraction, 
were pooled and ethanoi precipitated. 

'he CDMA pellet was washed with cold .thanol rhen 
resuspended in 200 ul of 20«M Tri. PB S.0. 1 =* EDTA, OOuMoler 
S-.d.no.yX-M«thioni»., and 300 units of ScoSi -ethyl... for SO 
oinutes at 37«C. Th. reaction wa. stopped by extraction with 
Phenol/chlorofom and th. «.thylat.d cONA wa. collect.d by 

ethanoi precipitation. 

Th. CDHA pallet wa. rinsed with 70% ethanoi then re.usp.nded 
i« 200 ul SI buffer (Maniati. et .1) and incubated with 200 units 
of 31-nucl.as. at 30'C for 30 minut... Th. reaction was stopp.d 
by .xtraction with phenol/chloroform and th. cONA collected by 
ethanoi precipitation. 

Th. double stranded am was blunted by incubation in 100 ul 



ERSATZSLATT 
-34- 



WO 86/00639 PCT/EP85/00326 

of 20mM Tris, pa 7.4» 50mM NaCl# lOmM 2 mercaptoethanol and 5C0 
uMolar o£ all four deoxynucleotide triphosphates with 25 units of 
Klenow at room temperature for 30 minutes. The reaction was 
stopped by extraction with phenol/chlocoforn and the cONA 
collected by ethanol precipitation. 

The CDMA was ligated in SO ul of T4 ligase buffer (aaniatis 
et al) with 500 pMoles of Rl linkers purchased from New England 
Biolaba (sequence: pCGGAATTCCG) using 2000 units of T4 ligase 
overnight at 16 *C. The reaction was stopped by incubation at 70" 
for 20 minutes then diluted to 300 ul such that the final salt 
concentration was 0.1 M NaCl, 10 aM, MgCl 2 , 50 m Tris-Cl, pH 
7.4. The cONA was then digested for 2 minutes at 37* with 700 
units of EcoRl. The reaction was stopped by extraction with 
phenol/chloroform and the cONA collected by ethanol 
precipitation. The pellet was resuspended in 50 ul of Tfi and 
passed over a 5ml C1-4B column. The excluded fractions were 
pooled and ethanol precipitated. The precipitated cONA was 
electrophoresed through a 1% agarose gel in Tris acetate buffer 
in the presence of 1 ug/ml ethidium bromide. cDNA in the size 
range 500-4000 base pairs was isolated from the gel using the 
standard glass powder procedure. The eluted cDNA was extracted 
with phenol/chloroform, ethanol precipitated and the pellet 
(after an ethanol rinse) was resuspended in 30ul of TB. The 
final yield was 100-500 ng of cDNA. 

•The preparation of the expression vector p91023<5) is 
described above. The ScoRl digested and phosphatase treated 



ERSATZBLATT - 
-35- 



WO 86/00639 PCT/EP85/00326 

vector (SOOng) was ligated with 100 ng of cONA in a lOOul 
reaction (standard T4 ligase reaction) overnight at 16 a C. The 
reaction was stopped by extracting with phencl/chlorofocra then 
the ligated cDNA was collected by ethanol precipitation after 
adding 5 ug of tRNA as carrier. 

The ethanol precipitated DNA was rinsed with 70% ethanol 
then resaspended in WO ul of TE. This DNA was used in 4 ul 
aliquots to transform fi, coll MC1061 (4 ul in a 100 ul 
transformation). Each of the 25 transformations was spread onto 
a ISO nan petri dish with 1% agar, L-broth and 10 ug/ml 
tetracycline (Tet plate) and incubated overnight at 37*. 
Approximately 2G00 colonies grew on each plate, resulting a total 
of about 30,000 colonies. After reaching approximately 0.5 mm in 
diameter, the colonies were transferred to nitrocellulose disks 
{137 mm) by carefully placing a dry filter on the surface of the 
plate then smoothly peeiing off the f ilcer. All of the colonies 
on the plate transferred to the filter which was then placed 
(colony side up) on a fresh Tet plate. After allowing the 
colonies to grow several hours, one replica was prepared fcou 
each of the filters by placing a fresh wetted filter exactly ever 
the original filter, pressing them together, peeling chea apart 
then returning each filter to a fresh Tet plate and incubating 
the plates overnight at 37". Each replica was carefully marked 
such that it would be realigned with the original filter. 



ERSA7Z8LATT 

-36- 



WO 86/00639 



PCT/EP85/00326 



Step 5. Plaamid QMA Preparation 

Each o£ the 25 replica filters was carefully sectioned into 
eighths using a scalpel and noting the orientation of each eighth 
relative to the original master filter. The colonies were 
scraped f rem each section into 10 ml of L-Broth. The bacteria 
were collected by centrifugation (3000 RPM, 10 min., aeckman J-6. 
centrifuge) resuspended in 0.6 ml of 25% sucrose, SO M Tris-HCl 
pa 8.0 and converted to protoplasts by addition of 0.12 ml of 5 
ng/ml lysezyme and incubation on ice for 5-10 min. The 
protoplasts were next incubated at room temperature foe 10 min. 
following the addition of 0.125 ml of 0.5M EDTA then lysed by 
addition of 0.12ml of 10% SDS in 50 mM Tris-HCl, P H 8.0. The 
lysate was mixed gently, incubated at coom temperature for 15 
sin. then protein and chromosomal DNA precipitated by the 
addition of 0.3 ml of 5M Nad. After incubation on ice for 15 
min., the lysate was centrifuged in an Eppendorf centrifuge for 
20 min. in the cold. The supernatant was carefully removed 
leaving behind the viscous DNA/protein pellet and was diluced by 
the addition of 2.5 ml H 2 0. The mixture was extracted with 1 nl 
of phenol, the layers separated by centrifugation (10K for 10 
min. in the Sorvall SS-34 rotor) and the aqueous layer remcved to 
a fresh tube. DNA was precipitated by adding 0.5 ml of SX Nad 
and 7.5 ml of cold ethanol and freezing the mixture several iimes 
in a dry ica ethanol bath. The precipitate was collected by 
centrifugation UOK, 15 min. in the Sorvall 3S-24) , resuspended 



ERSATZSLATT 

-37- 



WO 86/00639 



PCT/EP85/00326 



in 0.3 ml of 0.3M Sodium acetate and te-precipitated (in an 
Eppendorf tube) by the addition of 1 ml of ethanol. After ifl-is 
min. in a dty ice ethanol bath, the precipitated DNA was 
collected by centrifugation (5 min. in the Eppendorf) and the 
final pellet was resuspended in 100 ul of sterile TE (10 mM Tris 
pH8. lmH EDTA) . From a typical preparation, 5-10 ug of plasmid 
DNA was obtained. Each preparation contained the DNA from 
2C0-50O colonies on the original filter. A total of 200 DNA . 
samples were prepared from. the 25 filters. 
Step 6. Isolating CSF Clone 

Each of the DNA samples from Step 5 were separately 
transfected into M6 COS monkey cells as described below. 

The M6 cells are grown routinely in Dulbecco's modified 
Eagle's Medium (DME available from Gibco) containing 10% 
heatinactivated fetal calf serum (HIFCS), split twice a week 
at 1:6 dilution. Twenty-four hours after splitting 1:6 the 
M6 cells are ready for transfection. Twenty-four hours prior 
to transfection, 1.2 x 10 8 M6 cells (split 1:6) are seeded 
into a Cell Factory (available from Nunc) in 1.5 liters of 
DME + 10% HIFCS. Immediately before transfection, -plates 
are aspirated and washed twice with 7 ml of serum-free (SF> 
DME. The DNA is dissolved in 0.1 M Tris (pB 7.3) and added 
to DME medium containing 2mM glutamine, 100 ug/ml streptomycin, 
100 U/ml penicillin and 0.25 mg/ml DEAE Dextran totalling 
4 ml with the Tris -DNA solution. The 4 ml of medium containing 
dissolved DNA is added to the plate containing M6 COS cells 
and incubated for 12 hours. 

After incubation, the cells are rinsed once or twice 
with 7 ml SF DME. Then, 5 ml of DME with 10% HIFCS , 100 U/ml 
penicillin, LOO ug/ml streptomycin, 2mM glutamine, and 0.1 
mM chloroquin was added and tne cells were incubated for 
2 1/2 hours. 

m 

EfSSATZBLATT 



WO 86/00639 



PCT/EP85/00326 



After 2 1/2 hour 3, rinse once with ST DHE and add 10 al ome 
♦ 10% BIFCS/plate. After 30 hours aspirate media and feed 4 
ml/plate DHE + 10% HIFCS. Harvest by removing the conditioned 
medium after 24-26 houcs further incubation. 

The conditioned medium from each transection was assayed 
for CSP activity using the KG-1 assay. Tor each sample, positive 
for CSP activity, the clone on the original master filter 
responsible for the CSP activity had to be identified.* For 
example, for one tcansfection positive for CSP activity, all of 
the colonies of the section of the original master filter from 
where the transfection DHA sample was derived, were picked. Some 
320 of these colonies were picked into 3 ml of L-3roth plus 10 
ug/ml tetracycline. The cultures were grown overnight* The 320 
colonies were placed in an 18 x 18 matrix. Pools were prepared 
from each horizontal row and vertical column of the matrix <26 
total pools) (note: the last horizontal row had only 14 clones). 
DNA samples were prepared from eacn pooled culture then used to 
transfect CO 5 cells. The supernatants from these traaaf ec-tiona 
w«re assayed using the XG-i colony assay. Two positives were 
obtained from this set of transf actions: one in a vertical 
column, the other a horizontal row. The culture common to these 
pools contained the C£P clone. 

Twelve individual clones frcar this culture were isolated and 
miniprep DMA was prepared from 10 ml. cultures in L-Broth as 
described above. 10 ui samples of as* from these preparations 



CRSATZBLATT 

-39- 



WO 86/00639 PCT/EP85/00326 

were digested with EcoRl and the resulting ONA fragments analyzed 
by agarose gel electrophoresis. Nine of the twelve clones had a 
common approximately 750 base pair insert. The DMAs from four of 
these clones and the remaining three clones were introduced into 
146 COS cells as described above. The supernatants from these 
transfections were assayed using the KG-1 assay as well as the 
bone marrow assay for CSP. The four clones which each contained 
the 750 bone pair fragment all directed the expression by the H6 
COS cells of high levels of CSF activity as detected in either 
assay whle the other three clones did not. Thus, the coding 
region for CSP must be located within the 750 base pair insert. 

The SNA sequence coding for CSP was removed f rem the 
transformation vector in the positive clone' by digestion with 
EcoRl and sequenced using standard dideoxy sequencing methods 
after subcloning fragments into M13 vectors to obtain the 
sequence illustrated in Fig. 1. The plasmid, P 91023(B> - CSP. 
that was first shown to direct CSP expression in COS cells has 
been designated pCSF-1. This plasmid has been deposits* with -the 
American Type Culture collection in a strain of S^sali - «CIQ61 
undec the deposit number ATCC 39754 on July 2, 1984. 

step ?. am&aa.^n rfif "rotsin 

' H6 COS monk-y cells transformed with vector p91G23<B> 
containing CSF/=DHA as isolated in Step 6 are grown as descried 
in Step 6 to produce CSP protein in the culture medium. 

Namely, one ac of this SNA <pO:P-l) «as dissolved in 1 ml of 



ERSATZBLATT 

-40- 



WO 86/00639 



PCT/EP85/00326 



0.1 M Trls, pH 7.3 and added to 600 ml of OME containing 2 mM 
glutamine, 100 U/ml streptomycin, 100 ug/ml penicillin (P/S) and 
0.25 mg/ml DEAE Dextran (Molecular weight 500,000 from 
Pharmacia) . The 600 nd of DNA DEAE Dextran solution is added to 
the M6 COS cells in the cell factory and incubated at 37* for 12 
hours. After the incubation, the cells are rinsed once with ?00 
ml of SP DME then incubated for 2.5 hours with 600 ml of DM3 
containing 0.1 odl chloroquin, 10% HIFCS, 2 mtt glutamine and P/S. 
After aspirating the chloroquin containing medium, the cells are 
rinsed with SP DME and fed 1500 ml of DME with 10% HIFCS. After 
20 hours the cells are washed with SF DME, the medium is replaced 
with 800 ml of SF DME and the transf ected cells are allowed to 
condition the medium for 24 hours at 37«C. The conditioned 
medium is aspirated and replaced with another 800 ml of SP DME. 
The cells are allowed to condition this medium for 24 hours then 
the conditioned medium is collected. As soon as possible after 
harvesting, the conditioned media sample are concentrated 20 fold 
by pressurised ultrafiltration using the Amicon 2.5 liter chamber 
with the ra5 membrane (5,000 MW cutoff). 

ste p 3 . m1 fusion of Rrrnrchinant CSE. 

ivo hundred ml of concentrated conditioned medium (from 4 
liters of starting material - Step 7) was brought to 30% 
saturation of ammonium sulfate by addition of solid ammouiu* 
sulfate and the precipitated protein was. removed by 
centrifugation. The supernatant was brought to 80% saturation of 



ERSATZBLATT 

-41- 



WO 86/00639 PCT/EP85/00326 

ammonium sulfate by adding more solid ammonium sulfate and the 
ot-cipitated protein collected by centrifugation. The pellet was 
^suspended in 5 ml of 20 n* sodium citrate, P H 6.1, containing I 
» NaCl. The dissolved protein was applied to a 1.6 x 100 cm 
column of Ultrog*! AcA54 equilibrated in the sama buffer. The 
CSP activity eluted from the column with an apparent molecular 
weight of 19 * Daltons or after about 90 ml. It has been 
observed that if the gel filtration is performed at low ionic 
strength, CSP activity is elated from the column in two positions 
with apparent molecular weights of about 19 k Daltons and 38 * 
Daltons, suggesting that GH-CSF may readily for* dimers.) The 
active fractions were pooled and o,o«ght to 0.15% TPA (by 
addition of 10% TFA) and applied to a Vydacr C4 column (0.46 x 2S 
cm) equilibrated in 0.1% TPA. The coiuan was developed with a 
linear gradient off 0-90% aeatonitril* (1 "l/*in., 340 ml total) 
in 0.1% TPA. The CSP activity e'lutec between 39 and 43% 
acetcnitrile (Fractions 16-20) . A 20 ul sample of Fraction 19 
was analyzed by SDS polyacrylamide gei electrophoresis (13.5% gel 
a3 described by Lammii, 2^221, 630 (1970)). A .ingle broad 
protein band with an apparent h» of 18-26 * Daltons was observed. 
The rather broad size range for CSP is a common feature of 
glycoproteins and is thought to reflect extensive but variable 
addition of carbohydrate. Proteia from Fraction 19 was submitted 
to Edman Degradation using the Applied Biosystems gas phase 
microsecuenator. From approximately 20 ug of protein applied, 
the sequence of >-he Zizzt x6 amino acids vas obtained 



ERSATZBLATT 

-42- 



WO 86/00639 PCT/EP85/00326 

(A-P-A-R-S-P-S-P-S-T-Q-P-W-E-H) • Tlle hi 9 n yield of this single 
Dtotein sequence strongly suggested that the CSP protein in 

m 

Fraction 19 had been purified to homogeneity. Bioassay indicated 
that Fraction 19 had 3 x 10? units per A28O absorbance units. 
Since typical proteins in aqueous solution exhibit, a range of 
extinction coefficients of 0.8 to 1.2 A28O absorbance unit3 per 
milligram of protein, the specific activity of the purified CSF 
is between about 1 x 10 7 and about 4 x 10 7 units/rag when assayed 
using the human bone marrow cell assay. 



RXftHPLE B 
CLONING GIBBON CSP 
Step 1. Proratio n of mRWA from ftlhhnn T-Cfilla 

A sample of the gibbon T-Cell line designated UCD-MLA 144 
was cultured for several weeks in R?MI ic40 (purchased from 
Gibco) and 20% fetal calf serum (FCS) until there was obtained 1 
.x 10 9 total cells. The ceil- were induced to product high levels 
of CSF by activation for 24 hours in the preaenca of 10 nanograms 
per ml 12-0-tetrdecanoyl phosbol 13 -acetate CTPA) in RPHI 1540 
plus 1% FCS. The cells were harvested by centrifugation (1000 
rpm., 5 min.), washed once with phosphate buffered saline (P3S) 
and finally collected by centrifugation. 

Membrane bcund polysome (MBP) ntfiNA was prepared from these 
cells using the same procedure as described in Example A for 
preparation of Mo call RNA. 



1Ef?S A7ZB LATT 

-43- 



WO 86/00639 



PCT/EP85/00326 



step 2. ***** strand rrorc Reaction 

6 ug o£ HBP mRNA (from Step 1) was diluted into a SO ul cONA 
synthesis reaction mixture (see Example A - Step 4) and the 
reaction initiated by the addition of reverse transcriptase. 
After incubation for 30 minutes at 42«C, the reaction was stopped 
by addition of EDTA to 50 alt, and diluted with K 2 0 to 100 ul. 
The mixture was extracted with phenol/chloroform and further 
extracted with chloroform. The cONA/RNA hybrids were separated, 
from unincorporated triphosphates by chromatography on a 2 ml 
Sepharose CL-4B column. The excluded fractions were pooled and 
the hybrids collected by ethanol precipitations. The final yield 
was 570 ng. 

step 3 . TfrnM strand mm Reaction 

The first strand cDNA pellet (Step 2) was resuspended in 50 
ml off H 2 0, and second strand synthesis carried out in a standard 
reaction mixture with fi, coli Polymerase I, T i t «U ligase, and 
RNAse a. The reaction was incubated overnight at 16«C and then 
incubated for 1 hour at 37«C. The reaction was stopped by 
addition of EDTA and extracted with phenol/ chloroform. The cDNA 
was separated from unincorporated triphosphates by chromatography 
on a Sepharose CL-48 column, the excluded fractions pooled and 
the cONA collected by ethanol precipitation. 

step 4 . ftrffrh«nin.fe Preparation 

The cDNA pei.lec 'Step 3) was resuspended in 7S ul of H 2 o. 



ERSATZSLATT 

-44- 



WO 86/00639 PCT/EP85/00326 

Hoaopolyaeric C "tails" were added to the ends of the cONA by 
adding 10 ul of the cDNA solution to a 25 ul standard reaction 
mixture with terminal transferase, and incubating at 30°C for S 
minutes. The reaction was stopped by the addition of EDTA to 40 
mM and heat inactivation at 68"C for 10 minutes. 10 ng of this 
tailed cDNA was annealed with SO ng of G-tailed pBR322 (purchased 
from NEN) in 10 ul of 10 mM Tris, pH 7.5, 1 mM SD7A, and 100 mM 
NaCl. The annealing reaction was incubated for 10 minutes at 
68"C and then foe 2 hours at 57". 

Step S. ^a^arlal Transformation 

e. coll strain MC1061, was grown in L-broth, chilled on ice, 
harvested by centrif ugation, and treated wth CaClj to prepare 
them for transformation. 5 ul of the cDNA annealing reaction wa* 
then incubated with 200 ul of the CaCl 2* treated bacteria. 
Fifteen such transformations were performed, using all of the 
annealed cDNA, and spread on 15 cm, 1% agar L-broth plates 
containing 10 ug/ml tetracycline. Approximately 1000 colonies 
grew on each plate. 

Stap 6. flfP ,<ff » Ptafcina 

10,000 colonies from the transformation were each picked 
with a toothpick, transferred to fresh plates (500 per plate in a 
grid) , and grown overnight at 37 # C. The colonies were then 
lifted from each plane by pressing a dry nitrocellulose filter 
firmly over the surface of the plate. Two replica filters were 



EFiSATZBLATT 

-45- 



WO 86/00639 PCT/EP85/00326 

prepared from each of these master filters. The master filters 
were stored at 4*C, and the replica filters treated with base, 
and baked to prepare them for hybridization. 

step 7. P rfT? f» MQn af 32p r-abei'i™* Hvhridi rat ion Probea 

The cDNA insert from pCSF-I was isolated by digestion with 
the restriction enzyme ecori . and electrophoresis in an agarose 
gel with Tris acetate and ethidium bromide. The band containing 
the cDNA fragment was cut from the gel and purified by the glass 

powder technique. 

300 ng of the cDNA fragment was then added to 1 ul of 10 x 
T4 DNA Polymerase Buffer <0.33 M Tris Acetate, pB 7.9, 0.66 X 
potassium acetate, 0.1 M Magnesium acetate and 10 mM 
dithiothreitol) , and 3 units of T4 DNA Polymerase (New England 
Biolabs), and diluted with water to 10 ui. After incubation for 
5-10 minutes at 37»C, this fixture was combined with 1 ul 10 x T4 
DNA Polymerase Buffer; .1 ul of a 2 mM solution of each of dCTP, 
dTTP, dGTP? 10 ul of 32pdATP UQuCi/ul, 3,000 Ci/mmole> ; and 3 
units of Tr DNA polymerase. The reaction was incubated for 20 
minutes at 37 9 C. Then 1 ul of 2 mM dATP was added and the 
reaction incubated for an additional 10 minutes at 37°C. 

The unincorporated triphosphates were separated from the 
labelled cDNA by chromatography on a Sephadex G100 column. A 
second probe was prepared from a synthetic oligonucleotide having 

the sequence t 

ATC TGG CTG CAC AG 



■ERSA72SLATT 

-46- 



WO 86/00639 PCT/EP85/00326 

which is complimentary to the amino terminus of the CSP coding 
region. This oligonucleotide was lab-lied with 3 *p dATP at its 
5» end using a standard polynucleotide kinase reaction. 

step 8. iaaJaffffr flff car rnMA glcnes 

in a standard hybridization screening procedure, some 45 
clones hybridized with the T4 labelled pCSF-1 cDNA. Of thes-3, 
approximately 20 also hybridized to the labelled oligonucleotide 
probe. The coding region of one of these has been sequenced, and 
the sequence data revealed a number of base substitutions, some 
of which result in amino acid difference in the expressed 
protein. These differences are illustrated in Figure 1 above the 
DNA sequence for the human CSP gene cloned in Example A. 

CLONING CSP FROM PERIPHERAL 3L00D LYMPHOCYTE mRNA 

step i. nam r~r?*r»+* nn from peripheral ninoil T . Yirehosyte a 

Peripheral blood lymphocytes were prepared from four 
plasmapheresis by-products (purchased from the Red Cross) by 
fractionation on a Ficoll-Hypaque gradient. The light density in 
RPMI-1S40 in the presence of 5% fetal calf serum, 0.17% 
phytoheoaaglutinin, and lOng/ml phorbal myristate acetate (?MA) 
at a density of 2 x 10« cells/ml (a total of 6 x 10* cells were 
obtained). The cells were harvested by centrif ugation (1000 rmp, 
S min.) , washed once with phosphate buffered saline (?as) and 
finally collecteo oy centrifugation. Cytoplasmic RNA was 



ERSATZBLATT 

-47- 



WO 86/00639 



PCT/EP85/00326 



prepared by a gentle lysis procedure in which the cells were 
resuspended in SO ml cold Triton lysis buffer (140 rati NaCl, 1.5 
mM MgCl2, 10 mM Tris, pH 8.6, 0.5% Triton X-100) with 10 mM 
dithiothreitol (DTT) and 50 units/ml RNAsin (purchased from 
Biotec) • This lysate was divided into 2 equal parts and each 
part was layered over a 10 ml cushion of lysis buffer containing 
20% sucrose* The cell nuclei were removed by centrifugation in 
the cold (4*C, 400 rpm for 5 minutes). Th« upper layer 
(cytoplasmic extract) was carefully removed and sodium 
dodecylsulfate (SOS) was added to a final concentration of 1%. 
This solution was extracted twice with an equal volume of phenol 
chloroform (1:1 mixture) and the RNA was precipitated by adding 
2.5 volumes of cold ethanol. The precipitated RNA was collected 
by centrifugation (15 min. at 4000 rpm) and resuspended in 0.01 M 
Tris, pH 7.5, 1 mM EDTA, 0.25 H NaCl (TE buffer plus 0.25 M MaCl) 
and reprecipitated -by addition of 2.5 volumes of cold ethanol. 
Finally, the RNA was collected by centrifugation and resuspended 
in 5 ml of B2O. The final yield was 7.5 mg. 

Messenger RNA was isolated from tha total cytoplasmic RNA by 
selection on oligo dT cellulose. 2.5 mg of total RNA was heated 
to 65° for five minutes. NaCl was added to 0.5 M and the RNA was 
allowed to cool to room temperature. This RNA was passed over a 
one ml column of oligo dT cellulose equilibrated in TE + 0.5 m 
NaCl (binding buffer) . Unbound RNA was removed by washing the 
column extensively with binding buffer. Bound messenger RNA was 
eluted with 3 ml of H2O and precipitated by addition of C.2 ml 



ERSATZSLATT 
-48- 



WO 86/00639 



PCT/EP85/00326 



of 4 H Nad and 2.5 volumes of cold ethanol. The precipitated 
mRNA was collected by centrifugation (30 minutes at 23,000 rpm) . 
The final pallet (approximately 100 ug) was resuspended in 50 ul 
of H2O. 

step 2. rlrnt gtega n1 r nM * wction 

20 ug of PBL mRNA was diluted into a SO ul cDNA synthesis 
reaction containing 100 mM Tris, pH 8.4, 140 mM Kd, 10 mil Mgd 2 , 
10 mM 2-mercaptoethanol, 400 uM each of dATP, dGTP, dCTP, and 
dTTP, 5 ug of oligo-dT (average sixe 12-13) as primer, 25 uCi of 
32pdCTP (400 uCi/nmole) and 20 units of the cibonuclease 
inhibitor RNAsin. The reaction was initiated by addition of 60 
units of reverse transcriptase at 37-C and incubated for 30 
minutes at 42-C. The reaction was stopped by addition of EDTA to 
40 mM and extracted with an equal volume of H 2 0 saturated phenol. 
The phenol phase was back extracted with 50 ul of TB buffer. The 
aqueous phases were pooled. The cDNA/RNA hybrids were separated 
from unincorporated triphosphates by passing the pooled aqueous 
phase over a 5 ml Sepharose Ct-4B column (purchased from Sigma) . 
equilibrated with TB. The fractions that were excluded from the 
column were pooled, brought to 250 mM Nad and the nucleic acids 
precipitated by addition of 2.S volumes of cold ethanol. The 
hybrid, were collected by centrifugation for 30 minute, at 40,000 
rP». Th. final pellet (2.5 ug of cDNA) was resuspended in SO ul 
of H2O. 



EflSATZBLATT 
-49- 



WO 86/00639 PCT/EP85/00326 

stap 3 . fi* gQ nH strand- rnNA Reaction 

Second strand cDNA was synthesized by the combined action of 
the enzymes col* DNA Polymerase I, B. «li DNA u 9 aae and 
spjj, RHAse H. The reaction mixture (50 ul> contained 20 mM Tris, 
pH 8.0, 4 mM MgCl 2 , 1.2 mM EDTA, 25 uM NAD, 100 uM each of dATP, 
dGTP, dCTP, and dTTP; and 50 uCI 32 PdC TP (3,000 Ci/mmole) . The 
reaction was performed by adding 3 units DNA polymerase I, 0.5 
units DMA ligase, and 0.75 units of RNAse H and incubating at 16* 
for 18 hours, then at 37« for 1 hour, and then stopped by adding 
EDTA to 40 mM and extracted with an equal volume of phenol. The 
phenol phase was back extracted with 50 ul TB, the aqueous phases 
pooled, and the cDNA was separated fr-m the unincorporated 
triphosphates by chromatography on a Sepharose CL-4B column as 
described obove for the first strand. Based on incorporation of 
32?, the first strand cDNA was quantitatively converted to a 
double-stranded form. 

step 4. flffomMnant mna Proration 

Homopolymeric c "tails- were added to the ends of the cDNA 
by gently heating 400 ng of cDNA in a 50 ul reaction mixture 
containing 1 mM 2-m«rcapto«thanol, 1 mM CoCl 2 , and 9 units of 
terminal deoxynucleotidyl transferase at 30*C for five minutes. 
The reaction was stopped by the addition of EDTA to 40 mM and 
heating to 68«C for 10 minutes. 200 ng of this tailed cDNA was 
annealed with 500 ng of G-tailed ?AT1S3 'purchased from Amersham) 
in 100 ul of 10 Tris, pB 7.5, I mM EDTA, and 100 mM Nad. The 



ERSATZSLATT 
-50- 



WO 86/00639 PCT/EP85/00326 • 

annealing reaction was performed at 57* foe 2 hours after a 5 
ainute preincubation at 68*C. 

Step 5. Bacterial Transformation 

The cDNA annealing reaction product was used directly to 
transform the g. colt strain MC1061. A frash colony of bacteria, 
cells was used to inoculate 50 ml of L-broth and grown for 
r several hours until the optical density at 5S0 na was 0.25. The 
cells were chilled on ice and harvested by centrif ugation (2000 
rpm for 10 min.) • The pellet was resuspended in 10 ml cf cold 
0.1 a CaCl£ and allowed to sit on ice for 10 minutes. The cells 
were collected by centrif ugation <200Q rpa for 5 minutes) and 
resuspended in 2.S ml of 0.1 H Ca&2* 10 ul of the cONA 
annealing reaction was then incubated with 200 ul of 
CaCl2~ treated bacterial for 30 minutes on ice and then for 2 
minutes at 37*C, followed by addition of 0.8 ml of L-broth and 
final incubation for 30 minutes at 37*C. 

Twenty of these transformations were performed, utilizing 
all of the annealed cDNA. Each transformation mixture was spreac 
onto 1% Agar L-broth plates (IS cm diameter) containing 10 ug/ml 
tetracycline. Prom the twenty transformations a total of 20 suet 
plates were 3pread and incubated overnight ae 37"C. On the 
average approximately 1,500 bacterial colonies grew on each plate 
for a total of 30,000 clones . 



ERSATZBLATT 



WO 86/00639 



PCT/EP85/00326 



Step 6. Plating 

The original colonies growing on each plate were transferred 
to 137 mm nitrocellulose filters by pressing a dry filter on top 
of the colonies and lifting them off the plate. Two identical 
replicas were prepared from each original filter by standard 
replica plating methods, in which each original filter was 
carefully placing methods, in which each original filter was 
carefully placed colony side ap on a sterile aquare off filter 
paper <Whataan 3 MM) resting on a square piece of glass. A new 
pre-wetted nitrocellulose filter was carefully aligned on top of 
the master filter, covered with a second sterile square of filter 
paper and the complete sandwich then pressed together firmly with 
a second piece of glass. The sandwiched filters were numbered 
and 3 pinholes were punched through them asymmetrically so that 
they could be exactlu aligned agan in the future. The replica 
was then removed from the master and placed colony side up on a 
new tetracycline-containing L-broth agar plate. A second 
replica was immediately prepared in identical fashion, each 
master filter was returned to a plate and all of the plates were 
incubated at 37» for several hours until the bacterial colonies 
had reached approximately 1 ma in diameter. The original master 
filters were stored at 4«C and the replicas prepared for 
hybridization as described below. 

step 7 . ?r m r »rm*i*n Pilars f or. Hybridization 

Each replica filter (Step 5) was placed colony side up on 



ERSAT2SLATT 
-32- 



WO 86/00639 



PCT/EP85/00326 



filter paper (Whatman 3 MM) soaked in 0.5 M NaOH, 1.5 M Nad for 
seven minutes. The filters were transferred to neutralization 
filter papers, soaked in 1 m Tris, pH 7.5, 1.5 M Nad, for 2 
minutes and then transferred to a second set of neutralization 
filters for 5-10 minutes. Finally, the filters were placed on 
filters soaked in SSC buffer (0.015 M Sodium Citrate, 0.15 M 
NaCl, pH 7.4) for five minutes, air-dried and baked in vasafl at 
80*C for 1-2 hours. 

step a. T« rt1afetQn Qf r * p ffnwA ^lonea 

Duplicate filters were probed with the radioactively 
labelled pCSP-1 cDNA insert, prepared as described above in 
Example B. Some 20 colonies hybridized with the cDNA. Twelve 
of these were picked from the master filter and grown overnight, 
in L-broth for further analysis. Restriction enzyme digests (Est 
1) of DMA samples (rapid prep) from these clones indicated that 3 
were nearly full length. One of these has been sequenced. The 
sequence of the CSP coding region of this clone was identical to 
the corresponding sequence of pCSF-1 (i.e. having a T at position 
3S5-CS?(Ile)) . 

EXAMPLE 0 

Purification of CSF from Mo Cell Line 

Mo serum free conditioned medium (40 liters) was incubated 
at 55«C for 30 minutes to inactivate the HTLV-II virus associated 
with the cell line. This medium was concentrated by pressurized 



ERSATZBLATT 



WO 86/00639 



PCT/EP85/00326 



ultrafiltration using the Pellicon Casctte with membrane PTGC 
<1.5 square feet) which has a 10,000 molecular weight cut-off. 
The protein was further concentrated by ammonium sulfate 
precipitation (80% saturation). The final pcottin pellet (800 
mg) was resuspended in 100 ml of 20 mM 

tris (hydroxynethyl) aminomethane hydrochloride (Tris-HCl) , pH 7.4, 
and dialyzed against the same buffer (3 times with 4 liter 
changes each time) . The dialywd protein was applied to a 2.S x 
10 cm column of DEAB (diethylaminoethyl) -ultrogel equilibrated in 
the same buffer. The column was washed wit* 800 ml of 20 mM 
Tris-HCl, P H 7.4, then the CSP activity eluted with 800 ml of 20 
' mM Tris-HCl, PH 7.4, containing 0.12 M NaCl. 10 ml fractions 
were collected and assayed for CSP. The active fractions (3) 
were pooled, and concentrated 6 fold (to 5 ml) by pressurised 
ultrafiltration (Amicon ¥MS* membrane, S,000 molecular weight 
cut-off) . The concentrated sample from the DBAE column was 
applied to a 1.6 x 100 ca AcA44 ultrogel (an acrylamide agarose 
' ultrogel having 10 to 130 k Dalton fractionation) column 
equilibrated in 20 mM H-2-hydroxyethylpipera*ine-N-2-ethane 
sulfonic acid (HEPBS) , pH 7.4, 50 mM Had, and 0.01% polyethylene 
glycol (PEG-8000) . CSP activity eluted from the column with an 
apparent molecular weight of 30 k Oaltons. The active 
fractions were pooled and brought to 0.15% (v/v) tri- 
fluoroacetic ac.d (TFA) by addition of 10% TFA and applied *o ^ 
a vydac C 4 reverse phase column (1 * 25 cm) . The column was . 
developed with a linear gradient of 0-90% acetonitrile in 0.1% 
TFA (v/v) at 4 l/ln (1,000 ml total). The CSP activity eluted 



ERSATZBLATT 

-54- 



WO 86/00639 PCT/EP85/00326 

at approximately 47% (v/v) acetonitrile. The pooled active 
fractions were brought to 0.05% (v/v) heptafluorobutyric acid 
(HFBA) by addition of one half volume of 0.15% (v/v) HFBA and 
applied to a Vydac C4 column (0.46 x 25 cm) equilibrated in 0.15% 
(v/v) HFBA. The column was developed with a linear gradient of 
0-90% (v/v) acetonitrile in 0.15% (v/v) HFBA at 1 ml/rain. (340 ml 
total) . The CSF activity eluted at about 53% (v/v) acetonitrile. 
Fractions 37-44 (l.al each) were found to be active. 0.15 ml of 
fraction 40 was concentrated 4 fold (using the SAVANT Speed Vac 
Concentrator) and 40 ul of 2 x SDS gei sample buffer added (0.125 
M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol and 0.004% Bromophenol 
blue). These samples were boiled for 2 minutes and applied to a 
13.5% Laamli, O. Mataxft 221, 680 (1970) SOS gel (See Figure 2). 
Fraction (140) was determined to have 110,000 bone marrow CSF 
units/ml. This corresponds to about 3.0 x 10 7 units per A 2 90 
absorbance unit. Since typical proteins have extinction 
coefficients ranging between 0.8 and 1.2. A 2 ao P« r milligraa, 

the purified CSF had a specific activity in- the range of about 1 
x 10 7 to about 4 x 10 7 units per mg in the bone marrow assay. A ' 
1 ug sample of purified GM-CSF was submitted to Edman Degradation 
using the Applied Biosystems Gas Phase Microsequenator. The 
sequence of residues 3 through 5 was determined to be Ala Arg 
Ser. 



ERSA7ZBLATT 
-ss- 



WO 86/00639 PCT/EP85/00326 



EXAMPLE E 



Cotransformation and Amplification of CSF Sequence in 
CHO Cells Plasmid P 91023(B)-CSF was introduced into CHO DHFR 
deficient cells DUKX-Bll (Chasin 4 Orlaub PNAS 77:4216, 1980) 
by protoplast fusion as described (Sandri-Goldin et al. Mol. 
Cell. Bio. 1 743-752. 1981). The growth and maintenance of 
the CHO cells has been described (Kaufman * Sharp, J. Mol. 
Bio. 150 601-621 1981). For protoplast fusion, p91023(B)-CSF-l 
was introduced into E. Coli HB101 and bacteria grown in SO 
ml of m9 salts containing 0.5% casainino acids, 0.4% glucose, 
0.012% MgS04, 5ug/ral thiamine, and 10 ug/ml tetracycline 
to an absorbance of 0.6 at 600 nm. Chloramphenical was added 
to 250 ug/ml and the culture incubated at 37 °C for an 
additional 16 hours in order to amplify the plasmid copy 
number. The cells were centrifuged at 3,000 x g for 10 min. 
at 4°C and suspended in 2.5 ml of chilled 20% sucrose in 
50 mM Tris-Cl pH8.0. Lysozyme was added (0.5 ml of a 5mg/ml 
solution in 0.25M Tris-Cl P H8.0) and the mixture held on 
ice for 5 min. EDTA ( 1 ml of 0.25 M EDTA pH8.0) was added 
for an additional 5 min. on ice, and then 1.0ml of 0.05 M 
Tris-Cl pHS.O was added slowly. The suspension was incubated 
for 15 minutes at 37°C until the bacteria were converted 
to protoplasts. The suspension was then slowly diluted with 
20ml of prewarmed medium containing 10% sucrose and lflmm 

and held at 37°C for 15 min. The solution of protoplasts 



MgC1 2 

(approximately 10 9 /tul) was added to CHO, DHFR deficient DUKX- 
Bll cells in a 6-well plate (approximately 1X10 cells/well) 
at a ratio of approximately 1-2X10 4 protoplasts/cell and 
the protoplasts were pelleted onto the cells by centrifuging 
at 2000 RPM for 8 min. in a swinging microtiter dish rotor 
of an IEC Model K centrifuge. After centrifugation, the super- 



ERSATZBLATT 

-56- 



WO 86/00639 



PCT/EP85/00326 



natant was removed by aspiration. A 2 ml amount of polyethylene 
glycol solution 50 g of PEO - 1450, (Baker Chem. Co.) in 
50 ml of medium was added to each well of the 6-well plate). 
The cells were again centrifuged at 2000 RPM for 90 seconds , 
the polyethylene glycol solution removed, and the plates 
rinsed 3 times with 4ml of medium/well. Cells were then try* 
psinized, suspended in 10ml media containing 10% fetal calf 
serum/ and centrifuged in a conical tube at 500 RPM in a 
clinical centrifuge. Pelleted cells from 3 wells were pooled 
and plated into a 10 cm tissue culture dish. Fresh medium 
containing 100 ug/ml of kanamycin, thymidine, adenoxine, 
deoxyadenosine, penicillin and streptomycin and 10% dialyz^d 
fetal calf serum was added to each plate. The kanamycin was 
included to prevent the growth of any bacteria which had 
escaped conversion to protoplasts. - 

Two days later the cells were subcultered It 15 into 
alpha-media with 10% dialyzed fetal calf serum, penicillin 
and streptomycin, but lacking the nucleosides. Cells were 
then fed again with the same selective media (lacking nucleo- 
sides) after 4-5 days. 

Colonies appeared 10-12 days after subculturing into 
selective media. Two schemes for methotrexate (MTX) s-election 
and amplification have been followed. In the first scheme, 
single independent cloned transf ormants were isolated on 
the basis of DHFR expression and subsequently each clone 
was propagated under conditions to amplify the copy number 
of the foreign DNA i.e., growth in increasing concentrations 
of methotrexate. In the second scheme a pool of multiple 
independent transf ormants was isolated on the basis of DHFR 
expression and propagated together under conditions to 



ER5A7ZBLATT 

-57- 



WO 86/00639 PCT/EP8S/00326 



amplify the foreign DNA, i.e. growth in increasing concentra- 
tions of methotrexate. Then individual clones were isolated 
from the mass selected population and analyzed for GM-CSF 
expression. Those clones exhibiting highest levels of GM-CSF 
expression were grown again under conditions to further amplify 
the foreign DNA (i.e. growth in increasing concentration 
of methotrexate in the culture media) . 

In one experiment, seven DHFR* transformants were pooled 
into alpha medium lacking nucleosides. These cells were sub- 
sequently grown in stepwise increasing concentrations of 
MTX starting at 0.02 uM then steps to 0.1, 0.5 and 2.0 uM» 
MTX. When assayed for GM-CSF activity in the KG-1 cell assay, 
these cells produced from 3,000 to 12,000 units per ml. The 
selected population was cloned 0.5 uM-MTX and in 2.0 uM»MTX. 
Clones obtained in 0.5 uM-MTX (010, D2, and B6) were subse- 
quently selected for growth in 2.0 uM- MTX. When assayed for 
GM-CSF activity in the KG-1 cell assay, the cloned cell lines 
produced from 15,000 to 300,000 units per ml of GM-CSF 
activity. The GM-CSF produced according to this Example has 
the amino acid sequence given for CSF-Thr in Figure 1. 

EXAMPLE F 

EXPRESSION OF GM-CSF IN E. COLI 

GM-CSF was expressed in E. coli from vector pTALC-lS5R, 
a diagramatic description of which is provided in figure 
6. The GM-CSF encoding sequence begins with the synthetic 
sequence ATG- CCA- CCA- CCT* CCT' TCT- CCA- TCT- CCA-TCT- ACT, which 
determines the intitial 11 amino acid residues of mature 
GM-CSF. The remainder of the GM-CSF encoding sequence in 



ERSATZBLATT 
-5«- 



WO 86/00639 PCT/EP85/00326 



pTALC-185R is identical to that of pCSF-l, nucleotides 97-447, 
followed by the sequence TAR* TAR. TAG. Immediately following 
the triple terminator there is the pUC-18 polylinker. The 
tetracycline resistance gene from pBR322 has been inserted, 
in the opposite orientation to the CSF gene, 100 bases down- 
stroam from the pUC-18 polylinker. The tetracycline resistance - 
gene carries its own promoter. Continuing counterclockwise 
there is next the gene f or ft -lactamase followed by the pUC-18 
(CoLEl) origin of replication. 

The final structural festure of the plasmid before re- 
turning to CSP sequences is the PL promoter. This promoter 
is essentially as described by A. Skatzman and M. Rosenberg 
(in "Molecular cloning, a laboratory manual" (1982), Cold 
Spring Harbor Laboratory, page 419). CSP expression is driven 
by the PL promoter after thermal induction in a suitable 
E. coli host strain. 

The parental strain used for all the strain constructions 
was W3110 lacI°L8 (R. Brent and M. Ptashne PNAs 78 (1981) 
4204-4208. 

A fragment of^DNA (A- nucleotides 34499 to 38214) was 
integrated into the chromosome of W3110 lacI°L8 at the lacZ 
locus. The integration was performed using an integration 
vector composed of pBR32S sequences carrying the genes for 
chloramphenicol and ampicillin resistance as well as the 
PBR322 replication origin, (F. Bolivar Gene 4 (1978) 121-136). 
The A DNA fragment is inserted into the lacZ gene, which itself 
is present on the plasmid as a fragment extending from the 
BstEil site in LacI to a Tthilll site downstream of lacZ. 



ERSATZBLATT 

-59- 



WO 86/00639 



PCT/EP85/00326 



Integration of theA-DNA into the chromosomal copy of 
lacZ was achieved by homologous recombination and lac , 
ampicillin sensitive, chloramphenicol resistant colonies 
were found. A second recombinational event leading to the 
removal of all extra plasmid sequences but leaving the A DNA 
fragment integrated was screened for on lactose-MacConkey 
plates.. The initial lac + , amp S , cam R phenotype changed to 
a lac", amp s , cam 3 phenotype following the second recombi- 
national event. The resulting strain was called GL400 and 
wasA R at 30° andA S at 42°. This phenotype demonstrates^ 
the existence of a functional chromosomal copy of the CI 
allele. 

GL400 was rendered Ion* by PL transduction from a lysate 
grown on strain SG202S2 (lacAUl69, araAl39 rpsl lorulOO:: 
TnlO > . The TnlO was cured by screening for Tet on selective 
media (S. Maloy, W. Nunn J. Bacterid. 145 (1981) 1110-1112). 

The final host strain was called GI413 (lacl°L8, LacZA* 
(ACI, REX, N), lonAlOO). 

pTALC-185R was transformed into GI413. An overnight 
culture of this strain was grown at 30 °C in 5mls of induction 
medium containing Tugml" 1 tetracycline. Induction medium 
contains, per liter: 

20g Casainino Acids 
6g Na 2 HP0 4 7H 2 0 
3g KH 2 P0 4 
0.5g NaCI 
lg NH 4 CI 



ERSATZBLATT 

-60- 



WO 86/00639 



PCT/EP85/00326 



1% glycerol 
2mg vitamin Bl 
2mg CaCI 2 -2H 2 0 
0.2g MgCI 2 .6H 2 0 

This medium (25mls), containing Tugml" 1 tetracycline, 
was innoculated with 125ul of the overnight culture and shaken 
at 30°C in a water bath until the culture reached a density 
of A 0.5. It was then rapidly moved to a 40° water bath 
and shaken for a further 2 hours to allow synthesis of GM-CSP. 
Cells were harvested and checked for their content of CSF 
by SDS-polyacrylamide gel electrophoresis. Under these con- 
ditions GM-CSF accumulates to approximately 5% of the cellular 
protein. 

EXAMPLE G 

Expression of GM-CSF in Saccharomyce s Cerevisiae 
A. Vector Construction 

A plasmid was constructed which contained the gene for 
an enzyme in the uracil biosynthetic pathway (URA3) as a 
selection gene and the 2u origin of replication. This plasmid 
was derived from Ylp5 (Botstein et al.. Gene 8, pp. 17-24 
(1979)) with the addition of a fragment containing the origin 
of replication from the 2 micron plasmid of yeast. 

B. Isolation of the gene for Glycer aldehyde Phosphate 



ERSATZBLATT 

-61- 



WO 86/00639 



PCT/EP85/00326 



Dehydroqenese (GPDH) 

Two genes for GPDH have been isolated from yeast (Holland 
and Holland Journal of Biological Chemistry 255 pp' 2596-2605 
(1980)). An oligonucleotide probe synthesized from the pub- 
lished sequence was used to isolate a GPDH gene frota a plasmid ' 
library of yeast genomic DNA by standard methods. A plasmid 
containing the entire GAP491 gene has been deposited previously 
(ATCC No. 39777). 

c . Preparation of the glycer aldehvde phosphate dehydrogenase 
promoter for heterologous gen e expression 

A plasmid was constructed which allows for the natural 
spacing of the GPDH promoter from the start of the desired 
heterologous structural gene. This was accomplished by in- 
troducing a Kpnl site immediately adjacent to the initiator 
methionine codon of the GPDH structural gene. The promoter 
"cassette" was then inserted into the yeast expression vector 
YOpl. 

D. isolation of the ge ne f or <* factor 

A gene for the* factor mating pheromone has been isolated 
from yeast (Kurjan and Herskowitz Cell, Vol. 30, pp. 933-943 
(1982)). An oligonucleotide probe synthesized from this 
sequence was used to isolate the gene from a plasmid library 
of yeast genomic DNA by standard methods. 

E. Preparation of the CSF Express ion Plasmid 

Ef?S ATZS L ATT 

-62- 



WO 86/00639 



PCT/EP85/00326 



From the elements described above, and the human CSF 
gene # an expression vector (AJ14, Figure 7 ) was constructed 
by standard methods. In this vector, the natural leader 
sequence of CSF has been removed and the sequence coding 
for mature CSF has been inserted adjacent to the factor 
pre-pro sequence. The junctions between the GPDH promoter, 
dL factor pre-pro sequence, and mature CSF sequence are precised 
(below) and have been confirmed by dideoxynucleotide sequenc- 
ing. AAAT AAAC AAAATG . CGTTTTCCTTC A AAA AGA GAG GCG GAA 

GCT.GCA CCC GCC CGC TCG... 



F, Expression of GM-CSF 

The plasmid AJ14 was transformed into a strain of 
Saccharomyces Cerevisiae. Cells were cultured to produce 
CSF. 



ERSATZSLATT 

-€3- 



WO 86/00639 



PCT/EP85/00326 



In accordance with the above the following specific 
embodiments of the invention are i.a. envisaged and form 
part of the invention: 

1. A method foe preparing and isolating a transformation * 
vector containing CSF/cDNA, said method comprising: 

preparing RNA from a cell that produces CSF; 
preparing polyadenylated messenger RNA from said RNA; 
preparing single stranded cDNA from said messenger Re- 
converting the single stranded cDNA to double stranded 

cDNA; 

inserting the double stranded cDNA into transformation- 
vectors and transforming bacteria with said vector to form 
colonies; 

picking pools of 200 to 500 colonies each and isolating 

plasmid DNA from each pool; 

transf ecting the plasmid DNA into suitable host cells 

for expressing CSF protein; 

culturing the transfected cells and assaying the 
supernatant foe CSP activity; and 

selecting CSP positive pools and screening the colonies 
used to make the pool to identify a colony having CSP activity. 

2. The method of i wherein said cell that produces 

CSP is a T lymphocyte cell* * 

3. An eukaryotic cell that secretes protein, said cell 
being transformed by the expression vector prepared by the .-nethed 

of I- 

ERSATZBLATT 

-44- 



WO 86/00639 



PCT/EP85/00326 



4 # The cell of 5 wherein said cell is a mammalian 

cell. 

5. The method of 1, further comprising isolating the 

DNA coding for CSP protein f rom the colony having CSF activity, 
and insetting said DNA coding for CSF into an expression vector 
having a promoter heterologous to the CSF DMA. 

6. A cell transformed by said expression vector prepared by 
the method of 5. 

7. The cell of 6 wherein "said cell is prokaryotic. 

8. The cell of 6 wherein said cell is eukaryotic. 

9. cDMA that codes CSP. 

10. CDMA having the nucleotide sequence illustrated in Fig. 

1. 

.11. An expression vector comprising cDMA coding for CSF. 

12. An egression vector comprising a nucleotide sequence 
illustrated in Pig* 1* 



ERSATZBLATT 



WO 86/00639 



PCT/EP85/00326 



13. A transformed cell comprising the expression vector of 
11 or an allelic variation thereof. 

14. A transformed cell comprising the expression vector of 

12. 

15. The cell of 13 wherein said cell is prokaryotic. 

16. The cell of 14 wherein said cell is prokaryotic. 

17. The cell of 13 wherein said cell is eukaryotic. 

18. The cell of 14 wherein said cell is eukaryotic. 

19. The cell of 17 wherein said cell is a yeast cell. 

20. The cell of 18 wherein said cell is a yeast cell. 

21. The cell of 17 wherein said cell is a mammalian 

cell. 

22. The cell of 18 wherein said cell is a mammalian 

cell. 

23. The cell of 17 wherein said cell is an insect 

cell. 



ERSATZBLATT 

-66- 



WO 86/00639 PCT/EP85/00326 



24. The. ceil of 18 wherein said cell is an insect 
cell. 

23. CSP protein made by expressing cDMA coding for CSP in a 
transformed cell. 

26. The protein of 25 wherein said cell is a 
prokaryotic cell. 

27. The protein of 25 wherein said cell is a 
eukaryotic cell. 

28. The protein of 27 wherein said cell is a yeast 
cell. 

29. The protein of 27 wherein said cell is a mammalian 
roil. 

30 # The protein of 27 wherein said cell, is an insect 
cell. 

31. CSF protein substantially free of the protein of native 
origin* 



ERSATZBLATT 

-67- 



WO 86/00639 



PCT/EP85/00326 



32. The CSP protein of 31 substantially free of 
protein of human origin. 

33. CSF protein substantially free of glycosylation. 

34. CSP protein glycosylated by expression of cDNA coding 
foe CSP protein in a transformed eukaryotic cell. 

35. The CS? protein of 34 wherein, said eukaryotic cell 
is a mammalian cell or an insect cell. 

36. CSF protein made by expressing cONA having the 
nucleotide sequence illustrated in Pig. 1. 

37. The protein of 36 wherein said cell is a 
prokaryotic cell. 

33. The protein of 33 wherein said cell is a 
eukaryotic cell. 

39. The protein of 38 wherein said cell is a yeast 
cell. 

40. The procein of 38 wherein said cell is a mammalian 
cell. 

ERSATZBLATT 

-€8- 



WO 86/00639 



PCT/EP85/00326 



41. The pcotein o£ 38 wherein said cell is an insect 
cell. 

42. Hunan CSP protein having substantially the amino acid 
sequence illustrated in Pig. 1 or an allele variation thereof. . 

43. The CSP protein of 42 wherein said CS? is 
.CSF (The) » 

44. The CSP protein of 42 wherein said CSF is 
CSP (lie) . 

45. The CSF pcotein of 42 wherein said CSF is Met-CSF. 

46. The CSP pcotein of 42 wherein said CSF is a 
mixture of CSP and Ket-CSF. 

47. A therapeutic composition foe the treatment of 
myelo-suppression comprising a myelo-suppcession treatment amount 
of CSP protein in a pharmacological carrier. 

48. A method for the treatment of aanmals having 
myelo-suppression, said cetbod comprising treating said m«uiOil 
with CSP protein. 



ERSATZBLATT 

-69- 



WO 86/00639 



PCT/EP85/00326 



49. The method of 48 wherein said treating step 
comprises intravenously injecting said mammal with a therapeutic 
composition comprising CSF protein in a pharmacological carrier. 

50. A method of treating mammals to increase the number of - 
circulating granulocytes, said method comprising treating said 
mammal with an effective amount of CSP protein. 

51. Gibbon CSF having substantially the amino acid sequence 
illustrated in Fig. 1 or an allele variation thereof. 

52. The CSF protein of 51 wherein said CSF is 
CoF(Thr). 

53. The CSF protein of 51 wherein said CSF is 
CSF (lie). 

54. The CSP protein of 51 wherein said CSF is Met-CSF. 

55. The CSP protein of 51 wherein said CSF is. a 
mixture of CSF and ttetrCSP. 



ERSATZBLATT 

-70- 



WO 86/00639 



PCT/EP85/00326 



56. A method foe purifying CSP protein from a mixture of 
proteins suspended in aqueous medium, said method comprising: 

precipitating the protein with ammonium sulfate at 801 
saturation to form a pellet containing the CSP protein; 

resuspending the pellet in a buffered solution at a pH in 
the range of about 6 to about 8; 

applying the buffered solution containing CSP to a 
chromatographic column, eluting the CSP activity with the 
buffered solution containing sodium chloride and collecting the 
fractions having CSP activity; and 

pooling the active fractions, applying the pooled fractions 
to a C4 reverse phase column and eluting the CSP activity with a 
G to 30% acetonitrile gradient to collect the fractions 
containing CSP activity. 

57. The method of 56 wherein said buffer 'is selected 
from tris(hydroxymethyl)aminoiaethane hydrochloride, 
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid and sodium 

citrate. 

58. The method of 56 wherein the chromatographic column 
is loaded with octylsepharose, diethylamino-ethyl-ultrogel or 
acrylamide-agacose-ultrocel. 



E3SATZSLATT 

-71- 



WO 86/00639 



PCT/EP85/00326 



59. The method of 56 wherein prior to applying the 
pooled fraction* to the C4 column, the pooled fractions are 
treated with the acetonitrile gradient in a trifluoroacetic acid 
solution or a heptafluorobutyric acid solution, respectively. 

60. The method of. 59 wherein the concentration of 
trifluoroacetic acid or heptafluorobutyric acid in the eluting 
solution is 0.10% or 0.15% <v/v), respectively. 

61 The method of 56 wherein the aqueous medium 
contain!,,, CSr prot.in is first t«e»t.d with senium sulfat. « 
J0% saturation to pracipitat. protain and th. sup.rnat.ne toe th. 
remaining steps. 

62. The method of 56 wherein after the step of 
precipitating the protein with ammonium sulfate to form a 
pellet, the method comprises*. 

cesuspending the pellet in a solution of Tris-HCl and 
dialyzing the resulting solution; 

applying the dialyzed solution to a column of DEAE-ultragei; 
eluting the column with a solution of Tris-HCl containing at 
least 0.1 M Nad and collecting the fractions containing CSP . 
activity; 

pooling the active fractions and applying the pooled 
• fractions to a column containing AcA44-ultrogei equilibrated with 
! a solution of HEPES containing NaCl and polyethylene glycol; 

ERSATZBLATT 



WO 86/00639 PCT/EP85/00326 

eluting the column with a solution of HEPES with NaCl and 
polyethylene glycol and collecting the fractions containing CSP 
activity; 

pooling the fractions containing CSP activity and treating 
the pooled fractions with trif luoroacetic acid; 

applying the trifluoroacetic acid treated pool to a C4 
reverse phase column, eluting with a 0 to 90% acetonitrile 
gradient in a trifluoroacetic acid solution and collecting the 
fractions containing CSP activityj 

pooling the fractions containing CSP activity , treating the 
pooled fractions with heptaf luorobutyric acid and applying the 
treated solution to a second C4 reverse phase column* and 

eluting the second C4 reverse phase column with a 0 to 90% 
acetonitrile gradient in a heptaf luorobutyric acid solution to 
collect the fractions having CSP activity. 

63. The method of 56 wherein after the step of 
precipitating the protein with ammonium sulfate to form a 
pellet, the method comprises! 

resuspending the pellet in a sodiium citrate solution 
containing NaCl and applying the solution to a column containing 
acrylaraide-agarose-ultrogel equilibrated in the same buffer; 

eluting the column with a solution of sodium citrate and 
NaCl and collecting the fractions having CSP activityj 

pooling the fractions having CSP activity, treating with 
trifluoroacetic acid and applying the treated solution to a C4 



ERSATZBLATT 

-73- 



WO 86/00639 



PCT/EP85/00326 



reverse phase column; and 

eluting the column with a 0 to 90% acetonitrile gradient in 
a trif luoroacetic acid solution to collect the fractions having 
CSF activity. 

64. CSP protein having a specific activity of at least about 
1 x 10 7 units/mg in the bone marrow assay. 

• 65. The CSP protein of 64 having a molecular weight in 
the range of about 15 ,000 to about 26,000 Daltons. 

66. The CSF protein of 64 having a specific activity of 

at least about 4 x 10* units per ng of protein in the bone marrow 
assay. 

67. The method of 56 wherein said CSF is purified from 
a MO cell conditioned medium. 

68. The method of 56 wherein said CSF is purified from 
a medium obtaind by culturing cells transfected with a 
recombinant vector containing an expressible CSP gene. 

69. The method of 56 wherein said CSF is purified from 
a medium obtained by culturing COS cells transfected with 
p91023(B)-CSP. 



ERSATZSLATT 

-74- 



WO 86/00639 



PCT/EP8S/00326 



70. A method of producing a primate colony stimulating 
factor (CSF) protein, which comprises inserting a gene coding 
for such protein into an appropriate vector, transforming 

said vector containing said gene into eukaryotic or prokaryotic 
host cells and expressing and isolating said CSF protein. 

71. A method according to 70, in which the CSF protein 
is human granulocyte-macrophage CSF. 

72. A method according to 70, in which the CSF protein 
has the amino acid sequence shown for CSF-thr in Fig. 1. 

73. A method according to 70, in which the CSF protein 
has che amino acid sequence shown for CSF-ile in Fig. 1. 

74. A method according to 70, in which the CSF protein 
is Gibbon ape granulocyte-macrophage CSF. 

75. A method according to 70, in which the CSF protein- 
has the amino acid sequence shown for CSF-G in Fig. 1. 

76. A method according to 70, in which the CSF protein 
is a protein corresponding in amino acid sequence to a 
naturally occurring CSF, except that one or more amino acids 
has been added, substituted or removed without substantially 
affecting the biological activity of the natural CSF. 

77. A method according to 76, in which the CSF protein 
corresponds in amino acid sequence to a natural CSF protein 
except that one or more cysteine residues have been replaced 
by residues of other amino acids. 



ERSAT2SLATT 



WO 86/00639 



PCT/EP85/00326 



78. A method according to 77, in which the CSF protein has 
the amino acid sequence of a natural CSF except that it is 
proceeded by a methionine residue. 

79. CSF protein of 42, 43, 45, 46 in mature form. 

80. CSF protein of 42, 43, 45, 46 containing a signal 
poten.tia.ter start region. 

81. CSF protein having 127 amino acids. 

82. CSF protein obtainable by expressing E. coli MCt06l 
deposited in the ATCC under the number ATCC 39754 or the 
127 amino acid CSF coding nucleotide sequence of plasmid 
p91023(B)-CSF deposited therein. 



EKSATZBLATT 

-76- 



WO 86/00639 



PCT/EP85/00326 



WE CLAIM 

1. Recombinant CSF protein. 

2. CSF protein having a specific activity of 1 x 10 7 
units/mg in the bone marrow assay. 

3. CSF protein as claimed in claim 2 which is recombinant 
CSF protein. 

4. CSF protein as claimed in claim 1 which is human 
granulocyte-macrophage CSF. 

5. CSF protein as claimed in claim 1 which has the 
amino acid sequence shown for CSF-thr in Fig. 1, or CSF-ile 
in Fig. 1, or CSF-G in Fig. 1. 

6. A CSF protein as claimed in _ ■ claim I 
which contains the amino acid sequence as shown in 

Fig. 1 commencing with Ala* Pro ... or wherein the amino acid 
sequence commencing Ala •Pro ... is proceeded by a methionine 
residue. 

7. A CSF protein according to claim 1 which is a CSF 
protein corresponding in amino acid sequence to a naturally 
occurring CSF f except that one or more amino acids has been 
added, substituted or removed without substantially af.fectinq 
the biological activity of the natural CSF. 

8. A CSF protein according to claim which is a CSF 
protein having the amino acid sequence of a natural CSF except 
that it is proceeded by a methionine residue. 



ERSATZBLATT 

-77- 



WO 86/00639 PCT/EP85/00326 

9. A method of producing a CSF protein as claimed in 
claim 1 which comprises isolating said CSF protein as expressed 
from eukaryotic or prokaryotic host cells into which has 
been transformed a vector, said vector having inserted therein 
a gene coding for said CSF protein. 

10. A method as claimed in claim 9 wherein expression 
occurs from an E. coli, CHO or yeast . 



11. A method for preparing and isolating a transformation 
vector containing CSF/cDNA, said method comprising: 

preparing RNA from a cell that produces CSF? 
preparing polyadenylated messenger RNA from said RNA; 
preparing single stranded cDNA from said messenger RNA; 
converting the single stranded cDNA to double stranded 

cDNA; 

inserting the double stranded cDNA into transformation 
vectors and transforming bacteria with said vector to form 
colonies; 

picking pools of 200 to 500 colonies each and isolating 
plasmid DNA from each pool; 

transfecting the plasmid DNA into suitable host cells 
for expressing CSF protein; 

culturing the transfected cells and assaying the 
supernatant for CSF activity; and 

selecting CSF positive pools and screening the colonies 
used to make the pool to identify a colony having CSF activity. 



ERSATZBLATf 

-7-8- 



WO 86/00639 



PCT/EP85/00326 



12. A method for purifying CSF protein from a mixture of 
proteins suspended in aqueous medium, said method comprising: 

precipitating the protein with ammonium sulfate at 80% 
saturation to form a pellet containing the CSF protein; 

resuspending the pellet in a buffered solution at a pH in 
the range of about 6 to about 8; 

applying the buffered solution containing CSF to a 
chromatographic column, eluting the CSF activity with the 
buffered solution containing sodium chloride and collecting the 
fractions having CSF activity; and 

pooling the active fractions, applying the pooled fractions 
to a C4 reverse phase column and eluting the CSF activity with a 
0 to 90% acetonitrile gradient to collect the fractions 
containing CSF activity. 

13. cDNA, or an expression vector coding for CSF 
according to claim 1. 

14. A pharmaceutical composition comprising a CSF 
according to claim 1, or CSF according to claim 1 for use 
in therapy. 

15. A method of treating infection or granulocytopenia 
or activating neutrophils in animals which comprises 
administering CSF of claim 1 to an animal in need of much 
treatment, or use of CSF according to claim 1 for use in 

the manufacture of pharmaceutical compositions for such method. 



EK3A7ZBLA7T 

-79- 



WO 86/00639 



PCT/EP85/00326 



16. A pharmaceutical composition comprising a CSF pre- 
pared according to the process of claim 12. 

17. A method of treating infection or granulocytopenia 
or actiyatin neutrophils in animals which comprises 
administering CSF of claim 12 to an animal in need of much 
treatment, or use of CSF according to claim 12 for use in 

the manufacture of pharmaceutical compositions for such method. 

| 8 . CSF protein obtainable by expressing E. coli MCI061 
deposited in the ATCC under the number ATCC 39754 or the 
127 amino acid CSF coding nucleotide sequence of plasmid 
p91023(B)-CSF deposited therein. 



EfiSATZBLATT 

-80- 



WO 86/00639 PCT/EP85/00326 

20 

SCT G6A68 ATS T6S CT6 CM AGC CT6 CT6 CTC TTG 66C ACT 6TS 6CC T6C 

BET Trp Ltu Bin Str Ltu Liu Leu Leu Big The Val AU Cys 

80 

, Str CSF-6 CSF-G 

A6C ATC TCT 6CA CCC BCC CGC TC8 CCC AGC CCC AGC ACS CAO CCC TGG GAG CAT 
CSF-thr Str lit Str Ala Pro Alt Art Str Pro Str Pro Str Thr Gin Pro Trp Glu His 

140 

* 

6T8 AAT GCC ATC CAO 6A6 GCC C68 COT CTC CTS AAC CTO A6T A6A 6AC ACT 6CT 
Val Asn AU IU Gin Glu Alt Art *ro Ltu Ltu Asn Ltv Str Arf Asp Thr Ala 

200 

tit CSF-G Val CSF-G. 

A 0 
SCT 6A0 ATS AAT 6AA ACA 6TA 6AA STC ATC TCA 6AA ATS nT 6 AC CTC CAO 6A0 
Ala Glu ACT Asn Glu Thr Val 6lu Vat lit Str Slu SET Pht Asp Leu Gin Slu 

260 

0 T 

CCO ACC TOC CTA CAO ACC CGC CTO GAS CTS TAC AM CAO GGC CTO CAO 60C AGC 

Pro Thr Cys Ltii Gin Thr Aro Ltv Glu Ltu Tyr Lys Gin 6ly Ltu Arf Gig Str 

320 

CTC ACC AAO CTC AAG G6C CCC TTO ACC ATO ATS GCC AGC CAC TAC AAO CAS CAC 
Ltu. Thr Lys Ltu Lys Oly Pro Ltv Thr RET ACT Ala Str His Tyr Lys Sin His 

lit CSF-ile 

AS T 

TOC CCT CCA ACC CCS GAA ACT TCC TGT GCA ACC CAS ACT ATC ACC TTT GAA A6T 

Cyr Pro Pro Thr Pro Glu Thr Str Cys Ala Thr 6ln Thr lit Thr Pht Glu Str 

* 100 
... 380 

Thr CSF-G 
C 

TTC AAA 6A0 AAC CTO AAO GAC TTT CTO CTT 6TC ATC CCC TTT SAC T6C TGB GAS 

Pht Lys Glu Asn Ltu Lys Asp Pht Ltu Ltu Val lit Pro Pht Asp Cys Trp 6lu 

430 440 470 480 490 500 

Gly CSF-G 
0 

CCA GTC CAO GAS TGA GACCGGCCAS ATGAG6CT-GG CCAAGCCGGO GAGCT6CTCT CTCAT6AAAC 
Pro Val Bin Glu . 

127 

310 520 530 540 330 560 370 

A G * 8 

AAGAGCTGGA AACTCA6GAT GGTCATCTT6 CAGGGACCAA GGGGTGG6CC ACATCCAT68 T6G6AGT66C 

580 570 400 610 620 630 640 

T 

C660ACCT6C CCT66GCCAC ACT6ACCCT0 ATACAGGCAT G6CAGAAGAA TG66AATATT TTATACT6AC 

630 660 670 680 690 700 710 

A6AAATCA6T AATATTTATA TATTTATATT TTTAAAATAT TTATTTATTT ATTTATTTAA BTTCATATTC 

720 730 740 730 740 770 780 

CATATTTATT CAA6ATSTTT TACC6TAATA ATTATTATTA AAAATATSCT TCTAAAAAAA AAAAAAAAAA 



ERSATZSLATT 



WO 86/00639 



2/7 
FIG. 2 



PCT/EP85/00326 




ERSATZBLATT 



WO 86/00639 PCT/EP85/00326 

3/7 

FIG. 3 




ERSATZBLATT 



WO 86/00639 



4/7 

FIG. 4 



PCT/EP85/00326 



p 91023 

EcoRI DIGEST / KLENOW / LIGATE 

p 91023 (A) 

PstI / KLENOW /EcoRI LINKER 
EcoRI DIGEST / LIGATE 



r 




ERSATZBLATT 



WO 86/00639 



5/7 

FIG. 5 



PCT/EP85/00326 




SDS PAGE OF 
GM-CSF 



ERSATZBLATT 



6/7 

FIG. 6 



PCT/EP85/00326 



p TALC- 185 R 



Hind III 




^puc 18 



Bom HI P0lylinker 
Kpnl 

TER (UAA UAA UAG) 
Apa I 



CSF 



Nsi I 



Ndel 



ERSATZBLATT 



WO 86/00639 



7/7 

FIG. 7 



PCT/EP8S/00326 



CSF Expression Plasmid AJ-14 




i 



ER5ATZBLATT 



INTERNATIONAL SEARCH REPORT 

International Appliolien No PCT/EP 85/00326 



1. CLASSIFICATION OF SUBJECT MATTER 01 several classif.cal.of. symbols apoly. indicate all) « 


Accoidmo to International Patent Classification (IPC) or to both National Classification and IPC 


IPC : C 1 2 


N 15/00; C 12 P 21/02; A 61 K 37/02 


II. FIELDS SEARCHED 


Minimum Documentation Searched 7 


Classification System 


Classification Symbols 




C 12 N 


IPC 4 


C 12 P 




A 61 K 




Documentation Searched other than Minimum Documentation 




to the Eitent that such Documents are Included In the Fields Searched ' 



III. DOCUMENTS CONSIDERED TO BE RELEVANT 1 



Category * 


( Citation ot Document, 11 with indication, where appropriate, of the relevant passages " 


Relevant to Claim No. 11 


A 


Nature, volume 298, 1 July 1982, 

Aldons J. Lusis et al. : "Translation of 
mRNA for human granuJocyte - macrophage 
colony stimulating factor", 
pages 75-77, see the whole document 


1 


A 


Nature, volume 309, 28 June 1984, 

Nicholas M. Gough et al. : "Molecular 
cloning of cDNA encoding a murine 
haematopoietic growth regulator, 
granulocyte- macrophage colony stimula- 
ting factor", pages 763-737, see the 
whole document 


1 


A 


Nature, volume 307, 19 January 1984, 

M.C. Fung et al. : "Molecular cloning of 
cDNA for murine interleukin-3" , pages 
233-237, see the whole document 


1 


A 


Pfcbc. Natl. Acad. Sci USA, volume 81, Febru- 
ary 1984, 

Takashi yokota et al. : "Isolation and 
characterisation of a mouse cDNA -clone 
that expresses mast-cell errowth- factor 





* Special categories of cited 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 priority etaim(s) or 
which is cited to esiabdsn the publication date of another 
citation or other special reason {as specified) 

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

M P" document published prior to the international filing date but 



"T later document published after the international filing date 
or priority dale ano not in conflict with the aooiiceuon but 
cited to understand the principle or theory unaerljing the 
invention 

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

**Y" document of particular relevance:' the claimed invention 
cannot be considered to involve an inventive step wnen the 
document <t combined with one or more other such docu- 
ments, such combination being obvious 10 a person ehillea 
In the art 



IV. CERTIFICATION / g \ 




Date of the Actual Completion ot the International Search 

15th October 1985 


Date of Mailing of thii International jfeeorchflj^ 

1 4 NOV. 1985 T 1 


5ort 


International Searching Authority 

EUROPEAN PATENT OFFICE 


Signature of Authorized Officer 1 I k J / f IJJ 

G . T, .NTT fpuvdenfcfira J 



International Application No. pCJi /EP 85/00326 



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



Category • | 



Citation of Document, with indication, wnere appropriate, of the relevant passages 



Relevant to Claim No 



activity in monkey cells", pages 1070- 
1074, see the whole .document 

US, A, 4438032 (D.W. GOLDE) 20 March 1984, 
see claims; column 3-12 

GB, A, 2092159 (KABUSHIKI KAISHA HAYA-SHIBARA 
SEIBOTSU KAGAKU KENKYUJO) 11 August 1982, 
see claims; page 3, lines 31-42 

EP, A, 0089062 (AJINOMOTO) 9 September 1983, 
see claims; page 9, line 7 - page 11, 
line 10 



P , X • Chemical Abstracts, volume 103, nr. 7, 19 
August 1985, Columbus, Ohio, (US) 
Wong, Gordon G. et al.:"Human GM-CSF: 
molecular cloning of the complementary 
DNA and purification of the natural and 
recombinant proteins", see page 140, 
abstract number 49T05x 
Science (Washington, D.C. 1883-) 1985, 
228(4701), 810-15 (Eng) 



P,X, 



1/2,12 

1,2,12 
1,2,12 



1,4,9,11,13 



Chemical Abstracts, volume 103, nr. 3, 22 
July 1985, Columbus, Ohio, (US). 
Greenberger, Joel S. et al . : "Molecularly 
cloned 'and expressed murine T-cell gene 
product is biologically similar to inter- 
leukin-3" , see page 452, abstract number 
21037c, Exp. Hematol (N.Y. ) 1985, 13(4), 
249-60 (Eng) 

Proc. Natl. Acad Sci: USA, volume 82, July 
1985 

Frank Lee et al. : "Isolation of cDNA for 
a human granulocyte- macrophage colony- 
stimulating factor by functional express- 
ion in mammalion cells", pag^s 4360-4364,; 
see the whole document i 1,4,13 



J 



P,XiProc. Natl. Acad. Sci: USA, volume 82, January 
i 1985 : 
j Lindsay Sparrow et al. r "Purification and j 
| partial amino acid sequence of asialo i 
! ' murine granulocyte-macrophage stimulating 
■j factor", pages 292-296, see the whole 
document 



1,2,12 



Form PCT ISA/210 (eitra sheet) (January 1965) 



International Appiicat.on No. PCT/EP 85/00326 
FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET 



Vjg OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE 



Th* International search report ha. no, been established ,„ re.pec, of certain Calm, under Artel. 17(2) (a) for «h« follow!*, rea.on.- 
1-KJ Claim numbef.lS.l.yb.cau.e they relate to aubject m.tta r not required to bo ..arched by thl. Authority, namely: 
SEE RULE PCT 39.1. Civ)*. 

Methods for treatment of the human or animal body 

by surgery or therapy, as well as diagnostic methods. 



3Q P^R^Hi: "** 18 * Pe0 * fll eW,nS "* "* ^ m aCC0fainC8 third stance, of 

YlQ OBSERVATIONS WHERE UNITY OP INVENTION IS tACKIMc" ' " 

Thl. International Searching Authority found multiple Inv.ntlon. in thl. International application .. follow.: 



,,D E£2Z£Z? *"* W,,# ^ P " d by ' h ' — «•« co- - MM da... 

A. only iom. of the required addiUonal March fat. were timely paid by the aBolicant thl. im.„..h~..i - k 
tho.e turn, of th. International appHe.l.on for which f M . war. paw. .pe^'X^im* M " natUmt ' ,Weh "">« 



! covera only 



lD ^.^TiT^ ~ ■>»-— to- 



**LJ A, .? H ""'enable claim, could be searched without effort ju.tifyino an *rfri.t;»*.i ...... . * 

invite payment of any additional fee. juemymg an additional fee. the International Searching Authority did not 

Remark on Protest 
Q The additional search fees were accompanied by applicant's protect 
Q No protest accompanied the payment of additional search fees. 



Form PCT/ISA/J10 (supplemental sheet (2)) (January 1985) 



ANNEX TO THE INTERNATIONAL SEARCH REPORT ON 



INTERNATIONAL APPLICATION NO. PCT/EP 85/00326 (SA 10038) 



This Annex lists the patent family members relating to the 
patent documents cited in the above-mentioned international 
search report. The members are as contained in the European 
Patent Office EDP file on 08/11/85 

The European Patent Office is in no way liable for these * 

particulars which are merely given for the purpose of 

information. 



Patent document Publication Patent family Publication 

cited in search date member (s) date 
report 

US-A- 4438032 20/03/84 None 



GB-A- 2092159 11/08/82 FR-A,B 2497099 02/07/82 

JP-A- 57114525 16/07/82 
CH-B- 649786 14/06/85 



EP-A- 0089062 21/09/83 JP-A- 58157723 19/09/83 



Or 



For. more details about this annex : 

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



This Page is Inserted by IFW Indexing and Scanning 
Operations and is not part of the Official Record 

BEST AVAILABLE IMAGES 

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 
□f FADED 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.