Note: Descriptions are shown in the official language in which they were submitted.
f~ r3
INTERFER0~-ALPHA 6L
Description
Technical Field
The invention is in the field of biotech-
5 nology. More particularly it relates to a polypeptidehaving interferon (IF~) activity, DNA that codes for
the polypeptide, a recombinant vector t~at includes
the DNA, a host organism transformed with the recom-
binant vector that produces the polypeptide and phar-
10 maceutical compositions containing the polypeptide.
Bac~ground Art
IF~s are proteins with antiviral, immuno-
modulatory, and antiproliferative activities produced
by mammalian cells in response to a variety of indu-
15 cers (see Stewart, W.E., The Interferon System,Springer-Verlag, New York, 1979). The activity of IFN
is largely species specific (Colby, C., and Morgan, M.
J., Ann. Rev. Microbiol. 25:333-360 (1971 ) and thus
only human IFN can be used for human clinical studies.
20 Human IFNs are classified into three groups, a, ~, and
y, (~ature, 286:110, (1980))~ The human IFN-a genes
ccmpose a multigene family sharing 85~-95% sequence
homology (Goeddel, D. V., et al, ~ature 290:20-27
(1981) ~agata, S., et al, J. Intereron Research
1:333-336 (1981)). Several of the IFN-a genes have
been cloned and expressed in E.coli (Nagata, S., et
al, Nature 284:316-320 (1980); Goeddel, D. V., et a1,
Nature 287:411-415 (1980); Yelverton, E., et al,
Nucleic Acids Research, 9:731-741, (1981); Streuli,
30 M., et al, Proc Nat Acad Sci (USA), 78:2848-2852. The
-
resulting polypeptides have been purified and tested
or biological activities associated with partially
`:
purified native human IFNs and found to possess simi-
lar activities. Accordingly such polypeptides are
potentially useful as antiviral, immunomodulatory, or
antiproliferative agents.
A principal object of the present invention
is to provide a polypeptide having interferon activity
that is produced hy an organism transformed with a
newly isolated and newly characterized IFN-~ gene that
is not expressed naturall~. This polypeptide is some-
times referred to herein as "IFN-a6L". Other objects
of the invention are directed to providing the compo-
sitions and organisms that are used to produce this
polypeptide and to therapeutic compositions and
methods that use this polypeptide as an active
ingredient.
Disclosure of the Invention
_
One aspect of the invention is a recombinantly
produced polYpeptide having inter~eron activity and com-
prising the amino acid sequence:
~s~spLeuProGln ThrHisThrLeuArg AsnArgArgAlaLeu IleLeuLeuGlyGln
~etGlyArgIleSer ProPheSerCysLeu LysAspArgHisAsp PheArgIleProGln
GluGluPheAsp~ly AsnGlnPheGlnLys AlaGlnAlaIleSer ValLeuHisGl~et
IleGlnGlnThrPhe AsnLeuPheSerThr Glu~pSerSerAla AlaTrpGluG~nSer
LeuLeuGluLysPhe SerThrGluIleTyr Gln51nLeu~snAsp LeuGluAlaCysVal
IleGlnGluValGly ValGluGluThrPro LeuMetAsnGluAsp SerIleLeuAlaVal
ArgLysTyrPheGln ArgIleThrLeuTyr LeuIleGluArgLys TyrSerPro~sAla
TrpGluValValArg AlaGluIleMetArq SerLeuSerPheSer ThrAsnLeuGlnLys
ArgLeuArgArgLys Asp.
A second aspect of the invention is a DNA
unit or fragment comprising a nucleotide s~quence that
encodes the above described polypeptide.
A third aspect of the invention is a cloning
2~ vehicle or vector that includes the above described
DNA.
~ ., , ~
A fourth aspect of the invention is a host
organism that is transformed with the above described
cloning vehicle and that produces the above described
polypeptide.
A fifth aspect of the invention is a process
for producing the above described polypeptide compri-
sing cultivating said transformed host organism and
collecting the polypeptide from the resulting culture.
Another aspect of the invention is a pharma-
ceutical composition having interferon activity com-
prising an efective amount of ~he above described
polypeptide admixed with a pharmaceutically acceptable
carrier.
Brief Descri~ n of the Drawings
Figure 1 is a partial restriction map ~hich
shows the two XhoII restriction sites that produce a
homologous 260 base pair DNA fragment from the IFN-al
and IFN-a2 structural genes. This fragment is used as
a probe in identifying and isolating the IFN-a6L
gene. Data for this map are from Streuli, M., et al
Science, 209:1343-1347 (1980).
Figure 2 depicts the sequencing strategy
used to obtain the complete D~A sequence of the
IFN-a6L gene coding region. Bacteriophage mp7:a6L-l
D~A served as the template ~or sequen~es obtained with
primers A, H and F and bacteriophage mp7:a6L-2 D~A was
the template for sequences obtained with primers E and
G. The crosshatched area of the gene depicts the
region that encodes the 23 amino acid signal peptide
(preinterferon) and the open box depicts the region
that encodes the mature polypeptide~ The scale, in
base pairs, is numbered with 0 representing the ATG
start codon of preinterferon. The arrows indicate the
direction and extent of sequencing with each primer.
Figure 3 is the nucleotide sequence of the
structural gene coding for IFN-a6L including some of
the flanking 5' and 3'- noncoding regions of the
gene. The region coding for preinterferon and the
mature polypeptide begins with the ATG codon at posi-
tion 42 and terminates with the TGA codon at posi-
tion 609.
Figure 4 is a partial restriction map of the
coding region of the IF~-a6L gene. The crosshatching
represents the region that encodes the 23 amino acid
signal peptide and the open box represents the gene
coding sequence for the mat1lre polypeptide. The
scale, in base pairs, is numbered with 0 representing
the ATG start codon of prein~erferon.
Figure 5 shows the amino acid sequence of
the 23 amino acid signal peptide and the 166 amino
acid mature IFN-6L coded for by the gene depicted in
~0 Figure 3. The 189 amino acid sequence is displayed
above the corresponding nucleotide sequence. Amino
acid 24, cysteine, is the first amino acid of the
mature IFN-a6L protein.
Figure 6 is the D~A sequence of the E. coli
trp promoter and the gene of Figure 3 which was
inserted between the EcoRI and AccI sites of the
plasmid pBR322. The amino acid sequence of Figure 5
is written above the corresponding DNA sequence and
the location of the restriction sites used in the
construction of the expression plasmid are indicated.
Figure 7 is a diagram of the expressi~n
plasmid, pGW21, used to transform bacteria with the
IF~-a6L gene.
~f~'74~
--5--
Modes for Carrying Out the Invention
In general terms IFN-~6L was made by identi-
fying and isolating the IF~-6L gene by screening a
library of human genomic DNA with an appropriate IFN-a
DNA probe, constructing a vector containing the
IFN-a6L gene, transforming microorganisms with the
vector, cultivating transformants that produce IFN-6L
and collecting IFN-~6L from the culture. A preferred
embodiment of this procedure is described below.
D~A Probe Preparation
Total cytoplasmic RNA was extracted from
human lymphoblastoid cells, Namalwa, which had been
induced for IF~ production by pretreatment with
5-bromodeoxyuridine and Newcastle Disease Virus
l~DV). The poly(A) (polyadenylic acid)-containing
messenger RNA (mR~A) was isolated from total RNA by
chromatography on oligo(dT)-cellulose (type 3 from
Collaborative Research; Aviv, H., and Leder, P., Proc
~atl Acad Sci (USA), 69:1408-1412, ~1972)) and
enriched for IF~ mRNA by density gradient centrifuga-
tion on 5~-20% sucrose gradients. Fractions contain-
ing IEN mR~A were identified by translating the mR~A
by microinjecting aliquots of each fraction int~
Xenopus oocytes and determining the IFN activity of
the products of the translations according to a method
described by Colman, A., and Morser, J., Cell, 17:517-
526 (1979).
The ~amalwa cell IF~ enriched mRNA was used
to construct complementary DNA (cDNA) clones in
E. coli by the G/C tailing method using the PstI site
of the cloning vec or pBR322 (Bolivarr F., et al,
Gene, 2:95-113 (1977)). A population of transformants
containing approximately 50,000 individual cDNA clones
7 4 ~ ~
--6--
was grown in one liter o medium overnight and the
total plasmid DNA was isolated therefrom.
The sequences of two IFN-a clones (IFN-al
and IFN 2) have been published (Streuli, M., et al,
5 Science, 209:1343-1347 (1980)). Examination of the
DNA sequences of these two clones revealed that the
restriction enzyme XhoII would excise a 260 bp frag-
ment from either the IFN-al or the IFN-~2 gene ~see
Figure 1~. XhoII was prepared in accordance with the
10 process described by Gingeras, T.R., and Roberts,
R.J., J Mol Biol, 118:113-122 (1978).
One mg of the purified total plasmid DNA
preparation ~as digested with XholI and the resul~ing
DNA fragments were separated on a preparative 6% poly-
15 acrylamide gel. DNA from the region of the gel cor-
responding to 260 bp was recovered by electroelution
and recloned by ligation into the BamHI site of the
single strand bacteriophage M13:mp7. Thirty-six
clones were picked at random and single stranded DNA
20 was isolated therefrom, and sequenced. The DNA
sequences of four of these clones were homologous to
known IFN-~ DNA sequences. Clone mp7:a-260, with a
DNA sequence homologous to IF~-al DNA (Streuli, M. et
al, Science, 209:1343-1347 ~1980)) was chosen as a
25 highly specific hybridization probe for identifying
additional IFN-a DNA sequences. This clone is herein-
after referred to as the "260 probe."
Screening of Genomic DNA Librar~
In order to isolate other IFN-a gene
30 sequences, a 32P-labelled 260 probe was u~ed to screen
a library of human genomic DNA by in situ hybridiza-
tion. The human gene bank, prepared by Lawn, R.M., et
al, Cell, 15:1157-1174 (1978), was generated by par-
tial cleavage of fetal human ~NA with HaeIII and AluIand cloned into bacteriophage ~ Charon 4A with syn-
thetic EcoRI linkers. Approximately 800,000 clones
were screened, of which about 160 hybridizea with the
260 probe. Each of the 160 clones was further charac-
terized by restriction enzyme mapping and comparison
with the published restriction maps of 10 chromosomal
IFN genes (Nagata, S., et al, J Interferon Research,
1:333-336 ~1981)). One of the clones, hybrid phage
~4A:a6L containing a 13.9 kb insert, was characterized
as follows. A DNA preparation of ~4A:5L was cleaved
with HindIII, BglII, and EcoRI respectively, the frag-
ments separated on an agarose gel, transferred to a
nitrocellulose filter, and hybridized with 32p_
labelled 260 probe. This procedure localized the
IF~-6L gene to a 2.0 kb EcoRI restriction fragment
which was then isolated and recloned, in both orienta-
tions, by ligation of the fragment into EcoRI cleaved
M13:mp7. The two subclones are designated mp7:a6L-l
and mp7:6L-2. The -1 designation indicates that the
single-stranded bacteriophage contains insert D~A
ccmplementary to the mRNA (the minus strand) and the
-2 designation indicates that the insert DNA is the
same sequence as the mR~A (the plus strand).
Sequencing of the IF~-a6L Gene
The Sanger dideoxy-technique was used to
determine the DNA sequence of the IFN-a6L gene. The
strategy employed is diagrammed in Figure 2, the D~A
sequence thus obtained is given in Figure 3, and a
30 partial restriction map of the IFN-~6L gene i8 illus-
trated in Figure 4. Unlike many genes from euXaryotic
organisms, but analogous to other IFN chromosomal
genes which have been characterised, the D~A sequence
~ -8-
of this gene demonstrates that it lacks introns.
Homology to protein sequences of the publi~hed IFN-a
genes made it possible to determine the correct trans-
lational reading frame and thus allowed the entire 166
amino acid sequence of IFN-a6L to be predicted from
the D~A sequence as well as a precursor segment, or
signal polypeptide, of 23 amino acids ~Figure 5).
The DNA sequence of the IFN-a6L gene (Fig 3)
and the amino acid sequence predicted therefrom (Fig
5) differ from the other known IF~-a DNA and IF~-a
amino acid sequences. Translation of the DNA sequence
of the IFN-~6L gene reveals that the gene is a pseudo-
gene that cannot be expressed naturally because of a
stop codon in the leader polypeptide sequence. Other-
15 wise the coding region of the gene is intact and it
can be expressed in transformed microorganisms as a
mature polypeptide. The IF~-6L is, therefore, a
truly novel polypeptide in that it has never been
produced by or isolated from human cells.
Goeddel, D.V., et al, Nature, 290:20-27
(1981) describes isolating an IFN-a gene, IF~ C, that
differs from the IF~-~6L gene by six nucleotides that
result in the stop codon in the leader sequence and
three amino acid changes in the rnature polypeptide.
25 The nucleotide change that causes the stop codon
occurs at position 101 and is a change from T to A.
The three substitutions that caus2 the amino acid
changes are: (1) a change from G to C at nucleotide
133 resulting in a change at amino acid 8 from Ser to
30 Thr, (2) a change from G to C at position 138 resul-
ting in a change at amino acid 10 from Gly to Arg, and
(3) a change from C to A at position 375 resulting in
a change at amino acid 89 from Leu to Ile. As regards
the other two changes one is a neutral change from C
to G at position 137 and the other is a change from C
to T at position 711 well outside the coding regionO
Plasmid Preparation and Host Transformation
Assembly of the plasmid for direct expres-
sion of the IFN-a6L gene involved replacing the DNA
fragment encoding the 23 amino acid signal peptide
with a 120 bp EcoRI/Sau3A promoter fragment E.coli trp
promoter, operator, and trp leader ribosome binding
site preceding an ATG initiation codon and using the
naturally occurring AccI site, 153 bp 3'- of the TGA
translational stop codon, to insert the gene into a
vector derived from the plasmid pBR322. The complete
DNA sequence of the promoter and gene fragments
inserted between the EcoRI and AccI sites of pBR322 is
shown in Figure 6 which also shows the exact location
of relevant cloning sites. Details of the construc-
tion are described below.
The IFN-a6L gene has a Sau3A restriction
site following the codon for the initial cysteine of
the mature protein, a second Sau3A site in the coding
region, and a third Sau3A site in the 3'- flanking
region. It also contains an AccI site on the 3'
flanking region (at nucleotide 760 in Fig 3) and a
second A I site approximately 240 nucleotides 5'- of
the sequence sho~ in Fig 3. The mp7:a6L clone was
digested with AccI and the ~one kb AccI fragment was
isolated on polyacrylamide gel. The AccI fragment was
then subject to partial digestion with Sau3A. Several
partial digestions of fragment were carried out using
a digestion mixture of 10 parts DNA, 12.5 parte buf-
fer, 1.25 parts Sau3A and 100 parts water (parts are
by volume). The digestions were made at 30~C for
varying times. The digests were resolved on 5% poly
~ l;3
--10-- `
acrylamide gel. The resulting 646 bp fragmentæ in the
digests were eluted from the gel, precipitated with
ethanol, and combinsd. The precipitates were spun
down, resuspended in Tris-EDTA containing 0.1 M NaCl,
5 filtered, reprecipitated and spun down, washed with
70% ethanol and resuspended in water.
The 646 bp Sau3A-AccI fragment was ligated
in a three-fragment, sticky end ligation with the pre-
viously described 120 bp promoter fragment and a
2116 bp _coRI-AccI vector fragment derived by diges-
ting p~R322 with EcoRI and AccI. The ligation was
carried out at 4C. The ligation mixture was used to
transform E.coli MM 294. The correct transformants
were identified by restriction enzyme mapping of
colonies that hybridized to a 32p labelled IFN-a
geno~ic fragment and by cytopathic effect activity on
human cells. Four out of 18 clones screened contained
the correct construction. Fig 7 is a diagram of the
correct expression construct, designated pGW21. Other
prokarytic hosts such as bacteria other than E.coli
may, of course, be transformed with this construct or
other suitable constructs either to replicate the
IFN-a6L gene and/or to produce IFN-a6L.
A sample of one of the correct transformants
was deposited in the American Type Culture Collection,
12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A.
on August 12, 1983. The sample was assigned ATCC
~o. 39409
Cultivation of Transformants
Bacteria transformed with the IFN-a6L gene
may be cultivated in an appropriate growth medium,
such as a minimum essential medium, that satisfies the
nutritional and other requirements needed to permit
the bacteria to grow and produce IFN-a6L. If the
bacteria are such that the protein is contained in
their cytoplasm, the IFN-~6L may be extracted from the
cells by lysing the cells such as by sonication and/or
treatment with a strong anionic solubilizing agent
such as sodium dodecyl sulfate. Further purification
of the extract may be achieved by affinity chromatog-
raphy, electrophoresis, or other protein purification
techniques.
Expression of the IFN-a6L gene by bacterial
hosts such as E.coli that utilize N-formyl-methionine
and/or methionine to initiate translation produces
IFN-a6L molecules that are preceded by an N-formyl-
methionine or a methionine group. Some of the N-
formyl-methionine or methionine groups could be
removed by natural in vivo bac~erial claavage mech-
anisms. This would result in a mixture of molecules,
some of which would include an initial N-formyl-
methionine or methionine and others that would not.
All such IFN-a6L molecules, those containing an
initial ~-formyl-methionine or methionine, khose not
containing an N-formyl-methionine or methionine and
any mixture thereof, are encompassed by the present
in~ention. Accordingly, the invention contemplate~
producing IFN-a~L-containing compositions having IFN
activity that is attributable solely to IFN-a6L and/or
said terminal N-formyl-mekhionine or methionine
derivative khereof.
Biological Testing of IFN-6L
IFN-6L-containing cell sonicates were
tested _ vitro and found to have the following
activities: (1) inhibition of viral replication of
vesicular stomatitis virus (VSV) and herpes simplex
C!~
-12-
virus-l (HSV-l); t2) inhibition of human tumor cell
growth, ~3) inhibition of colony formation by tumor
cells in soft agar; (4) activation of natural killer
(NK) cells; (5) enhancement of the level of 2',S'-
oligoadenylate synthetase (2',5'-A); and (6) enhance-
ment of the double-stranded RNA-dependent protein
kinase. IF~ a6L was active in inhibiting viral
replication in both human and other mammalian cell~,
such as hamster, monkey, bovine, and rabbit cells.
The tests show that IFN-a6L exhibits anti-
viral activity against D~A and R~A viruses, cell
growth regulating activity, and an ability to regulate
the production of intracellular enzymes and other
cell-produced substances. Accordingly, it is expected
IFN-a6L may be used to treat viral infections with a
potential for interferon therapy such as chronic hepa-
titis B inEection, ocular, local, or systemic herpes
virus infections, influenza and other respiratory
tract virus infections, rabies and other ~iral
zoonoses, arbovirus infections, and slow virus
diseases such as Kuru and sclerosing panencepha-
litis. It may also be useful for treating viral
infections in immunocompromised patients such as
herpes zoster and varicella, cytomegalovirus, Epstein-
25 Barr virus infection, herpes simplex infections,rubella, and progressive multifocal leukoencephalo-
pathy. Its cell growth regulating activity makes it
potentially useful for treating tumors and cancers
such as osteogenic sarcoma, multiple myeloma,
30 HodgXin's disease, nodular, poorly differentiated
lymphoma, acute lymphocytic leukemia, breast carci-
noma, melanoma, and nasopharyngeal carcinoma. The
fact that IF~-a6L increases protein kinase and 2',5'-
oligoadenylate sythetase indicates it may also
J
-13--
increase synthesis of other enzymes or cell-produced
substances commonly affected by IFNs such as hista-
mine, hyaluronic acid, prostaglandin E, tRNA methyl-
ase, and aryl hydrocarbon hydrolase. Similarly, it
may be useful to inhibit enzymes commonly inhibited by
IFNs such as tyrosine amino transferase, glycerol-3-
phosp~ate dehydrogenase glutamine synthetase, orni-
thine decarboxylase, S-adenosyl-l-methionine
decarboxylase, and UDP-N-acetylglucosamine-dolichol
monophosphate transerase. The ability of the IF~-a6L
to stimulate NK cell activity is indicative that it
may also possess other interferon activities such as
the abilities to induce macrophage activity and anti-
body production and to effect cell surface alterations
such as changes in plasma membrane density or cell
~urface charge, altered capacity to bind substances
such as cholera toxin, concanavalin A and thyroid-
stimulating hormone, and change in the exposur~ of
surface gangliosides.
Pharmaceutical compositions that contain
IF~-a6L as an active ingredient will normally be for-
mulated with an appropriate solid or liquid carrier
depending upon the particular mode of administration
being used. For instance, parenteral formulations are
usually injectable fluids that use pharmaceutically
and physiologically acceptable fluids such as physio-
logical saline, balanced salt solutions, or the like
as a vehicle. IFN-~6L will usually be formulated as a
unit dosage form that contains in the range of 104 to
107 in~ernational units, more usually 106 to 107
international units per dose.
IFN-6L may be administered to humans or
other mammals in various manners such as orally,
intravenously, intramuscularly, intraperitoneally,
-14-
intranasally, intradermally, ancl subcutaneously. The
particular mode of administration and dosage regimen
will be selected by the attending physician taking
into account the particulars o~ the patient, the
disease and the disease state involved. For instance,
viral infections are usually treated by daily or twice
daily doses over a few days ~o a few weeks, whereas
tumor or cancer treatment involves daily or multidaily
doses over months or years. IF~-~6L therapy may be
combined with other treatmentsO In this regard it may
be combined with or used in association with other
chemotherapeutic or chemopreventive agents for
providing therapy against viral diseases, cancer and
other conditions against which it i5 effective. For
instance, in the case of herpes virus keratitis treat-
ment therapy with native IF~ has been supplemented by
thermocautery, debridement and trifluorothymidine
therapy.
