U.S. patent number 4,935,233 [Application Number 06/803,748] was granted by the patent office on 1990-06-19 for covalently linked polypeptide cell modulators.
This patent grant is currently assigned to G. D. Searle and Company. Invention is credited to Leslie D. Bell, Keith G. McCullagh, Alan G. Porter.
United States Patent |
4,935,233 |
Bell , et al. |
June 19, 1990 |
**Please see images for:
( Certificate of Correction ) ** |
Covalently linked polypeptide cell modulators
Abstract
Described is a new class of polypeptide cell modulators
characterized by being composed of two covalently linked cell
modulators in a linear polypeptide sequence. Such dual function
polypeptides have new and particularly useful activities when the
component polypeptide cell modulators are interferons, lymphokines
or cytotoxins which act through different and specific cell
receptors to initiate complementary biological activities.
Inventors: |
Bell; Leslie D. (Thame,
GB), McCullagh; Keith G. (Princes Risborough,
GB), Porter; Alan G. (High Wycombe, GB) |
Assignee: |
G. D. Searle and Company
(Chicago, IL)
|
Family
ID: |
25187335 |
Appl.
No.: |
06/803,748 |
Filed: |
December 2, 1985 |
Current U.S.
Class: |
424/85.5;
424/85.7; 530/351; 424/85.6; 435/69.51 |
Current CPC
Class: |
A61P
31/12 (20180101); C07K 14/57 (20130101); A61P
35/00 (20180101); C07K 14/565 (20130101); C07K
14/52 (20130101); A61P 37/00 (20180101); C12N
15/70 (20130101); C07K 14/475 (20130101); C12N
15/62 (20130101); C07K 2319/55 (20130101); Y10S
930/142 (20130101); A61K 38/00 (20130101); C07K
2319/00 (20130101); Y10S 930/143 (20130101); C07K
2319/75 (20130101) |
Current International
Class: |
C12N
15/62 (20060101); C12N 15/70 (20060101); C07K
14/435 (20060101); C07K 14/565 (20060101); C07K
14/57 (20060101); C07K 14/52 (20060101); C07K
14/475 (20060101); A61K 38/00 (20060101); A61K
045/02 (); C07K 015/26 (); C07K 013/00 (); C12P
021/00 () |
Field of
Search: |
;530/351
;424/85,85.5,85.6,85.7 ;435/68 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Shepard et al., Nature, vol. 294, pp. 563-565, 1981..
|
Primary Examiner: Hazel; Blondel
Attorney, Agent or Firm: Kanady; Mary Jo Matukaitis; Paul
D.
Claims
What is claimed is:
1. A composition represented by the formula
wherein R.sub.1 is gamma interferon or a biologically active
modified gamma interferon; R.sub.2 is beta interferon or a
biologically active modified beta interferon; and L is a peptide
linker segment of 1 to 500 amino acid residues.
2. A composition according to claim 1 wherein R.sub.1 is gamma
interferon and R.sub.2 is a modified beta interferon wherein amino
acids 36-48 of natural beta interferon have been replaced by amino
acids 34-46 of alpha interferon.
3. A composition according to claim 1 wherein R.sub.1 is gamma
interferon and R.sub.2 is a modified beta interferon wherein amino
acids of 36-48 of natural beta interferon have been replaced by
amino acids 34-46 of alpha interferon and the cysteine at position
17 of natural beta interferon is replaced by serine.
4. The protein identified in FIG. 8 and FIG. 9 as IFNX 601 and
having the amino acid sequence shown in FIG. 8 and FIG. 9.
5. The protein identified in FIG. 10 and FIG. 11 as IFNX 602 and
having the amino acid sequence shown in FIG. 10 and FIG. 11.
6. A pharmaceutical composition for use in the treatment of viral
infections, regulating cell growth or regulating the immune system
in an animal comprising a therapeutically effective amount of the
composition of claim 1 admixed with a pharmaceutically acceptable
carrier.
7. A pharmaceutical composition for use in the treatment of viral
infections, regulating cell growth or regulating the immune system
in an animal comprising a therapeutically effective amount of the
protein of claim 4 admixed with a pharmaceutically acceptable
carrier.
8. A pharmaceutical composition for use in the treatment of viral
infections, regulating cell growth or regulating the immune system
in an animal comprising a therapeutically effective amount of the
protein of claim 5 admixed with a pharmaceutically acceptable
carrier.
9. A method of treating viral infections in an animal in need of
such treatment comprising the administration of an effective
therapeutic amount of the protein of claim 4.
10. A method of regulating cell growth in an animal in need of such
treatment comprising the administration of an effective therapeutic
amount of the protein of claim 4.
11. A method of regulating the immune system in an animal in need
of such treatment comprising the administration of an effective
therapeutic amount of the protein of claim 4.
12. A method of treating viral infections in an animal in need of
such treatment comprising the administration of an effective
therapeutic amount of the protein of claim 5.
13. A method of regulating cell growth in an animal in need of such
treatment comprising the administration of an effective therapeutic
amount of the protein of claim 5.
14. A method of regulating the immune system in an animal in need
of such treatment comprising the administration of an effective
therapeutic amount of the protein of claim 5.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to covalently linked polypeptide cell
modulators, each of which acts through a different and specific
cell receptor to initiate complementary biological activities.
Polypeptide cell modulators include lymphokines, monokines,
interferons, polypeptide hormones or cytotoxins as well as
modifications and active segments of such peptides. Also described
are DNA sequences, plasmids and hosts capable of expressing the
linked polypeptide cell modulators.
2. Description of Prior Art
One class of polypeptide cell modulators can be defined whose
members exert an antiproliferative effect almost specifically on
tumour cells and possess immunomodulatory activity, but lack
antiviral activity. Among the members of this class are human
lymphotoxin and tumour necrosis factor (Gray, P. W. et al. Nature
312, 721, 1984; Pennica D. et al. Nature 312, 724, 1984).
Human lymphotoxin (hLT) is a cytotoxin induced in lymphocytes by a
specific antigen or by bacteria or parasites and has a cytotoxic or
cytostatic action on a variety of tumour cells in vivo or in vitro.
hLT has been implicated to play a role in cell-mediated immunity
and its potent anti-tumour effect suggests it may be of value
therapeutically (Ruddle, N. H. et al. Lymphokine Res. 2, 23,
1983).
Another class of lymphokine can be defined whose members induce an
antiviral state in responsive cells, and also have
antiproliferative and immunomodulating activity. Among the members
of this class are leukocyte interferon (IFN-alpha), fibroblast
interferon (IFN-beta) and immune interferon (IFN-gamma).
It has been reported that mixtures of type I interferons (IFN-beta
or IFN-alpha) and type II interferons (IFN-gamma) are highly
synergistic in exerting an antiviral or antiproliferative effect.
(Fleishmann, W. R. et al. Infect. Immun. 26, 248, 1979; Czarniecki,
C. W. et al. J. Virol. 49, 490, 1984).
In mixtures, much lower concentrations of type I and type II
interferons can achieve a particular level of response. Several
authors have also described IFN-gamma/hLT and IFN-alpha/hLT synergy
or related synergies (Lee, S. H. et al. J. Immunol. 133, 1083,
1984; Stone-wolff, D. S. et al. J. Exp. Med. 159, 828, 1984;
Williams, T. W. Lymphokine Res. 3, 113, 1984). European Patent
Application (EPO 107 498), (EPO 128009).
However, in these instances, there was no disclosure of covalent
linkage of the two classes of molecules that were synergistic.
Additional patent publications have described the primary amino
acid sequences of human IFN-gamma (GB 2 107 718 A), the IFN-gamma
(IFN X918) described herein (PCT 83/04053), IFN-alphas (U.S. Pat.
No. 4 414 150-08.11.83) and IFN-beta (e.g. GB 0689 70B; GB
2098996A). A modified IFN-beta (IFN X430) described herein is
identical to human fibroblast IFN-beta except that amino acids 36
to 48 inclusive are replaced with amino acids 34 to 46 inclusive
from human IFN-alpha 1 (European Patent Application 85105914.7 and
(Taniguchi, T. et al. Nature 285, 547, 1980).
BRIEF DESCRIPTION OF THE INVENTION
This invention encompasses mixed function proteins formed from
covalently linked polypeptide cell modulators, each of which acts
through a different and specific cell receptor to initiate
complementary biological activities. Novel compounds of this
invention are represented by the formula
where R.sub.1 is a polypeptide cell modulator with one activity,
R.sub.2 is a polypeptide cell modulator with a different but
complementary activity. By complementary activity is meant activity
which enhances or changes the response to another cell modulator.
The polypeptide cell modulators are either directly bonded to one
another or are each bound to a polypeptide linker segment. Thus L
represents a chemical bond or a polypeptide linker segment to which
both R.sub.1 and R.sub.2 are bound, most commonly L is a linear
peptide to which R.sub.1 and R.sub.2 are bound by amide bonds
linking the carboxy terminus of R.sub.1 to the amino terminus of L
and the carboxy terminus of L to the amino terminus of R.sub.2. The
linking group is generally a polypeptide of between 1 and 500 amino
acids in length.
The term polypeptide cell modulator encompasses a large variety of
peptides which elicit a biological response by binding to a
specific binding site on a cell. It is known that mixtures of
polypeptide cell modulators such as beta and gamma interferon
exhibit a synergistic effect. In this invention the polypeptide
cell modulators are bound together to produce the same synergistic
effect as a mixture of the polypeptide cell modulators or a further
enhanced effect or a different effect with the advantage of a
single dosage form.
Compounds of this invention are preferably made by genetic
engineering techniques. Thus genetic material (DNA) coding for one
polypeptide cell regulator, peptide linker segment and the other
polypeptide cell regulator is inserted into a suitable vector which
is used to transform bacteria, yeast or mammalian cells. The
transformed organism is grown and the protein isolated by standard
techniques. The resulting product is therefore a new protein which
has two complementary cell regulatory regions joined by a peptide
linker segment as shown in the formula R.sub.1 --L--R.sub.2,
wherein R.sub.1 and R.sub.2 represent polypeptide cell regulator
regions and L represents the peptide linker segment.
BRIEF DESCRIPTION OF THE CHARTS, TABLES, AND FIGURES
Table 1 shows the origin and identification of the plasmids used in
the construction of polypeptide cell modulators.
Table 2 shows expression and molecular weight data for IFN
X601.
Table 3 shows a comparison of the antiviral activity of IFN X601
with that of the parental IFNs.
Table 4 shows a comparison of the antiproliferative activity of IFN
X601 on Daudi lymphoblastoid cells and HEp-2 carcinoma cells with
that of the parental IFNs.
Table 5 demonstrates synergy between human IFN-gamma and IFN
X430.
Table 6 shows the antigenic properties of IFN X601 as judged by
enzyme-linked immunoadsorbent assay (ELISA).
Table 7 shows a comparison of the binding to Daudi cell IFN alpha 2
receptors of IFN X601 with that of the parental interferons, IFN
X918 and IFN X430.
Table 8 shows the antiviral, antiproliferative and HLA DR inducing
activity of IFN X601 eluted from monoclonal antibody affinity
columns.
Table 9 shows the antiviral, antiproliferative, HLA DR inducing and
ELISA activity of IFN X602 compared with IFN X601.
Table 10 shows the antiviral, antiproliferative, HLA DR inducing
and ELISA activity of IFN X603.
FIG. 3 depicting Chart 1A shows the path to construction of the
plasmid vector pGC269, which expresses IFN X601. FIG. 4 and FIG. 5
depicting Charts 1Aa and 1Ab show preparation of starting plasmid
pAP8.
FIG. 6 depicting Chart 1B shows the path to construction of the
plasmid vector pZZ102, which expresses IFN X603.
FIG. 7 depicts Chart 2; Chart 2A shows the ligated DNA duplex
coding for the spacer amino acids and used to prepare an
intermediate plasmid (pGC262) in the construction of pGC269.
Chart 2B shows the DNA duplex coding for (Ala-Gly-Ser).sub.7, an
alternative spacer for linking IFN X918 to IFN X430.
FIG. 8 and FIG. 9 depicting Chart 3 shows the complete nucleotide
and amino acid sequences of the IFN X601 gene and IFN X601,
respectively.
FIG. 10 and FIG. 11 depicting Chart 4 shows the complete nucleotide
and amino acid sequences of the IFN X602 gene and IFN X602,
respectively.
FIG. 12 and FIG. 13 depicting Chart 5 shows the complete nucleotide
and amino acid sequences of the IFN X603 gene and IFN X603,
respectively.
FIG. 14 and FIG. 15 depicting Chart 6 shows the complete nucleotide
and amino acid sequences of the IFN X604 gene and IFN X604,
respectively.
FIG. 16 and FIG. 17 depicting Chart 7 shows SDS-PAGE analysis of
immunoprecipitates of .sup.35 S-labelled E. coli extracts made with
anti IFN-.beta. anti IFN-.gamma. monoclonal antibodies.
FIG. 18 and FIG. 19 depicting Chart 8 shows Western blotting
confirmation of co-identity of IFN-.beta. immunoreactivity with IFN
X601 36 kd protein.
FIG. 1 shows the enhanced antiproliferative activity of IFN X601
and a mixture of IFN X918 and IFN X430 against HEp-2 carcinoma
cells.
FIG. 2 shows the activity of IFN X601 in inducing HLA DR expression
on human fibroblasts in comparison with the parental IFNs used
either individually or as a mixture.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptide cell modulators include soluble protein modulators
released by differentiated cells which have their principle effect
on other cell types and include lymphokines, monokines, peptide
hormones or peptide growth factors.
Among the polypeptide cell modulators are cytokines, that is, all
soluble protein modulators released by a differentiated cell that
have their principle effect on other cell types. Included within
this cytokine class are lymphokines, monokines, products of the
endocrine, paracrine or autocrine hormone systems and polypeptide
growth factors.
Specifically included within this cytokine class are the following
polypeptides: interleukins 1, 2 and 3, alpha interferons (all
types), beta interferon, gamma interferon, lymphotoxin, tumour
necrosis factor, epidermal growth factor or urogastrone, B-cell
growth factor, insulin like growth factors I & II, bone-derived
growth factor, chondrocyte growth factor, T-cell growth factors,
endothelial-derived growth factors, nerve growth factor,
macrophage-derived growth factors, platelet-derived growth factor,
neurotrophic growth factors, transforming growth factor (Type I or
II), transforming growth factors, T-cell replacing factor,
cartilage-derived growth factor, growth hormone, colony-stimulating
factors, insulin, endothelial-cell growth factors, placental
lactogen, erthropoietin, plasminogen activators, eye-derived growth
factor, prolactin, fibroblast-derived growth factor, relaxin,
fibroblast growth factors, thrombin, glial growth factor,
transferrin, osteosarcoma-derived growth factor, vasopressin,
thymosin, follicle stimulating hormone, luteinizing hormone,
thyroid stimulating hormone, calcitonin, adrenal corticotropin,
melanoctye stimulating hormone, parathyroid hormone, oxytocin,
glucagon, secretin, cholecystokinin, gastrin, angiotensin,
angiogenin and the polypeptide releasing factors from the
hypothalmus.
Those skilled in the biochemical arts will recognize that
modification of the polypeptide cell modulators such as changing
amino acid sequences and derived or synthetic portions or regions
of active cell modulators are equally useful as polypeptide cell
modulators and are included as polypeptide cell modulators.
These polypeptide cell modulators are either linked directly or
through a peptide linker segment. The peptide linker segment is
generally a polypeptide derived from 1 to 500 amino acids. Other
peptide linker segments such as dicarboxylic acids, diaminoalkyls
and the like are useful for chemically linking polypeptide cell
modulators. Peptide linker segments from the hinge region of heavy
chain immunoglobulins IgG, IgA, IgM, IgD or IgE provide an angular
relationship between the attached polypeptide cell modulators.
Especially useful are those hinge region sections where the
cysteines are replaced by serines.
Since the preferred methods for preparing these linked polypeptide
cell modulators are through genetic engineering, it is understood
that variations in the genetic code can produce polypeptide cell
modulators which have the general structure of
which is a peptide in which R.sub.1 and R.sub.2 are regions which
have sequences which have the above described polypeptide cell
modulator activity and L is a peptide linker segment. Large numbers
of variations will produce equivalent results. The invention also
encompasses glycosylated proteins which for example are produced as
a result of expression in yeast or mammalian cells. Also
encompassed are variations in the composition of oligasaccharide
chains attached to the protein through specific amino acid
glycosylation sites. Such variations can be introduced by
expression in cells or organisms of varying type of by modification
of amino acid glycosylation sites by genetic engineering
techniques.
DESCRIPTION OF PREFERRED EMBODIMENTS
Plasmids used in the construction of, or expression of linked
polypeptide cell modulator genes are listed in Table 1. One
preferred embodiment of the present invention is plasmid pGC269
which codes for IFN X601 (Chart 3) and was derived from plasmids
pGC262 (Chart 1A) and pJA39 (Chart 1A). Plasmid pGC262 was derived
from plasmid pCC203 (deposited at ATCC no. 39,494) via plasmid pJB9
(Chart 1A); pJA39, which codes for the IFN X430 gene, was derived
from plasmid pAP8.
Another preferred embodiment of the present invention is IFN X601
which is composed of sequentially from the N-terminus 1. IFN-gamma
in which the N-terminal cys-tyr-cys has been replaced by met
(designated IFN X918; Chart 3); (2) a 22 amino acid peptide linker
segment coded by synthetic DNA (Chart 2A), related to the mouse IgG
2b "hinge" region (Chart 3, amino acids 145 to 147; and Nature 283,
786, 1980), except that the four cysteines are replaces by serines
(Chart 3; serine residues 156, 159, 162 and 166); (3) IFN X430,
which is identical to human IFN-beta, except that amino acid
residues 36 to 48 inclusive are replaced by the equivalent residues
from human IFN-alpha 1 (Chart 3, residues 202 to 214).
The plasmid pGC269 of example 1 below (Chart 1A; Table 1) was used
in the expression of a polypeptide cell modulator (IFN X601) of
example 2 having the antiviral, antiproliferative and
immunomodulatory properties described in example 3.
IFN X918 is just one version of IFN-gamma which may be used (i.e.,
the N-terminal cys-tyr-cys may be present). IFN X430 is just one
example of a type I IFN which may be linked to IFN-gamma, or a
modified IFN-gamma, such as IFN X918. Other type I IFNs which may
be used include IFN-beta or any IFN-alpha (e.g., IFN-alpha 2;
Streuli, M. et al. Science 209, 1343, 1980).
Any suitable peptide linker segment may be used which correctly
aligns and separates the two polypeptides comprising the
polypeptide cell modulator, for example, the mouse IgG gamma 2b
"hinge" region (Nature 283, l786, 1980) with the four cysteines
converted to serines (e.g., Chart 3; residues 145 to 167); or a
seven times repeated unit coding for alanine-glycine-serine (Chart
2B; and Chart 4; residues 145 to 165) which separates IFN X918 and
IFN X430, giving rise to IFN X602 (Chart 4).
A further embodiment is expression plasmid pZZ102 of example 1
which codes for IFN X603 (Chart 5), which was derived from plasmids
pZZ101 and pLT101 (Chart 1B and Table 1). Plasmid pZZ101 was
derived from plasmid pJB9 by insertion of a 106 bp peptide linker
segment coding for the C-terminus of IFN X918 and the
amino-terminal 21 amino acids of hLT (Chart 5; resdidues 132 to
166); plasmid pLT 101 contains a synthetic human lymphotoxin gene
(i.e., amino acid residues 146 to 316; Chart 5) cloned between the
ClaI and BamHI sites of plasmid pAT153 (Twigg, A. J. Nature 283,
216, 1980). IFN X603 is composed of sequentially from the
N-terminus; 1) IFN X918; a single methionine; and (2) human
lymphotoxin (Chart 5).
Alternatively, any suitable peptide linker segment may be used
which results in significant potentiation of biological activity,
but preferably the mouse IgG gamma 2b "hinge" with the four
cysteines converted to serines. This modified hinge region may be
inserted between IFN X918 and hLT (Chart 6).
It must be appreciated that the DNA sequences coding for IFN X601,
IFN X602, IFN X603 and IFN X604 disclosed in charts 3 to 6, are
examples of many possible combinations given that alternative
triplet codons exist for all amino acids except methionine and
tryptophan. Other DNA sequences can code for the amino acid
sequences defined in the charts (e.g., Gln-2 in IFN X601 in Chart 3
may be coded by CAG or CAA, etc.).
Expression of polypeptide cell modulators, as in example 2, may be
in E. coli K12 HB 101, or other E. coli strain; from any strong
promoter and ribosome binding site combination of prokaryotic or
eukaryotic origin, but preferably the E. coli strain; from any
strong promoter and ribosome binding site combination of
prokaryotic or eukaryotic origin, but preferably the E. coli trp
promoter minus attenuator (Patent applications EP 130 564 and EP
130 564 A) linked to the following ribosome binding site sequence:
##STR1## where S.D. is the Shine Dalgarno region and I.C. is the
Initiation codon of IFNsX601, or X602, or X603 or X604.
The novel, polypeptide cell modulators of the present invention can
be formulated by methods well known for pharmaceutical
compositions, wherein the active chimaeron is combined in admixture
with a pharmaceutically acceptable carrier substance, the nature of
which depends on the particular mode of administration being used.
Remington's Pharmaceutical Sciences by E. W. Martin, hereby
incorporated by reference, describes compositions and formulations
suitable for delivery of the compounds of the present invention.
For instance, parenteral formulations are usually injectable fluids
that use phsiologically acceptable fluids such as saline, balanced
salt solutions, or the like as a vehicle.
The novel, polypeptide cell modulators of the invention may be
administered to humans or other animals on whose cells they are
effective in various ways such as orally, intravenously,
intramuscularly, intraperitoneally, intranasally, intradermally or
subcutaneously. Administration of the polypeptide cell modulators
is indicated for patients with malignancies or neoplasms, whether
or not immunosuppressed, or in patients requiring immunomodulation,
or antiviral treatment. Dosage and dose rates may parallel those
employed in conventional therapy with naturally occurring
interferons--approximately 10.sup.5 to 10.sup.8 antiviral units
daily. Dosages significantly above or below these levels may be
indicated in long term administration or during acute short term
treatment. A novel, polypeptide cell modulators may be combined
with other treatments or used in association with other
chemotherapeutic or chemopreventive agents for providing therapy
against the above mentioned diseases and conditions, or other
conditions against which it is effective.
EXAMPLE 1
Chemical Synthesis of Oligonucleotide Fragments; and Plasmid
Constructions
(a) Chemical Synthesis of Oligonucleotides
Oligodeoxyribonucleotides were synthesized by the phosphoramidite
method (M. H. Caruthers, in "Chemical and Enzymatic Synthesis of
Gene Fragments", ed. H. G. Gasen and A. Lang, Verlag chemie, 1982,
p. 71) on controlled pore glass (H. Koster et al., Tetrahedron,
1984, 40, 103). fully protected 2'-deoxyribonucleotide
3'-phosphoramidites were synthesized from the protected
deoxyribonucleotide and chloro-N,N-(diisopropylamino)
methoxyphosphine (L. J. McBride and M. H. Caruthers, Tetrahydron
Lett., 1983, 24, 245 and S. A. Adams et al., J. Amer. Chem. Soc.,
1983, 105, 661). Controlled pore glass supports were synthesized as
described (F. Chow et al., Nuc. Acids Res., 1981, 9, 2807) giving
30-50 umol deoxynucleoside per gram.
After completion of the synthesis, the protecting groups were
removed and the oligomer cleaved from the support by sequential
treatment with 3% (v/v) dichloroacetic acid/dichloromethane (120s),
thiophenol/triethylamine/dioxane 1/1/2 v/v) (1 hour) and
concentrated ammonia at 70.degree. C. (4 hour). The deprotected
oligonucleotides were purified either by HPLC on a Partisil.RTM. 10
SAX column using a gradient from 1M to 4M triethylammonium acetate
pH4.9 at 50.degree. C. or by electrophoresis on a denaturing 15%
polyacrylamide gel (pH8.3).
(b) Ligation of Oligonucleotide Blocks
500 pmole aliquots of the oligonucleotides were phosphorylated with
1 unit of T4 induced polynucleotide kinase in 20 ul of a solution
containing 1000 pmole [.sup.32 p]gamma-ATP (2.5 Ci/mMole), 100 uM
spermidine, 20 mM DTT, 10 mM MgCl.sub.2, 50 mM Tris-HCl (pH9.0) and
0.1 mM EDTA for 60 minutes at 37.degree. C. The mixtures were then
lyophilized and each oligonucleotide purified in a denaturing 15%
polyacrylamide gel (pH8.3). After elution from the gel, the
recovery was determined by counting the radiactivity.
Blocks (length 30-50 bases were assembled by combining 25 pmole of
each phosphorylated component with equimolar amounts of the
unphosphorylated oligomers from the complementary strand. The
mixtures were lyophilized and then taken up in 15 ul water and 2 ul
10.times.ligase buffer (500 mM Tris-HCl pH7.6, 100 mM mgCl.sub.2).
The blocks were annealed at 90.degree. C. for 2 minutes, then
slowly cooled to room temperature (20.degree. C.). 2 ul 200 mM DTT
and 0.5 ul 10 mM ATP were added to give final concentrations of 20
mM DTT and 250 uM ATP in 10 ul. 1.25 units of T4 DNA ligase were
also added. After 18 hours at 20.degree. C., the products were
purified in a 15% polyacrylamide gel under denaturing
conditions.
The final duplexes were then constructed from the single-stranded
pieces. 1.5 pmole of each piece was taken and the mixtures
lyophilized. Annealing was carried out in 15 ul water and 2 ul
10.times.ligase buffer at 100.degree. C. for 2 minutes, then slowly
cooled to 10.degree. C. 2 ul 200 mM DTT, 0.5 ul and 10 mM ATP and
1.25 units T4 DNA ligase were added. The reaction was left at
10.degree. C. for 18 hours. The final products were then purified
in a 10% native polyacrylamide gel.
(c) Plasmid Constructions
(i) Plasmid pGC269 (Table 1)
STEP 1
DNA corresponding to the amino-terminal cys-tyr-cys of human
IFN-gamma in the plasmid pCC203 (ATCC No. 39, 494) was deleted by
ClaI/BamH double restriction enzyme digestion as in Chart 1A
(Methods in Molecular Cloning, a Laboratory manual, eds. Maniatis
et al., Cold Spring Harbor Laboratory, 1982). The resultant
expression plasmid, pJ89, codes for IFN X918 which has the
cys-tyr-cys replaced by methionine (PCT No. 83/04053).
STEP 2
A 171 bp chemically synthesized duplex (Chart 2A) coding for the
C-terminal 13 amino acids of IFN X918, 22 amino acids of the mouse
immunoglobulin gamma 2b "hinge" region (cys-ser) and 20 N-terminal
amino acids of IFN X430, was ligated to the BglII to SalI large
vector fragment of pJB9 (Chart 1A). The resultant plasmid, pGC 262
(table 1) contains a HindIII site for insertion of the remainder of
the IFN X430 gene.
STEP 3
To create an IFN X416 gene (European Patent application No.
85105914.7) with a unique HindIII site, plasmid pAP8 was cut with
ClaI and XhoI (chart IA), and the 230 bp fragment replaced by an
identical chemically synthesized fragment except that codons 19 and
20 are AAGCTT (HindIII) instead of AAGCTC. The resultant plasmid
was designated pJA39 (Table 1).
STEP 4
Since IFN X416 and IFN X430 are identical except at amino acid
position 17, the HindIII to SalI 719 bp fragment from pJA39
(equivalent to amino acids 19 to 166 of IFN X430 or IFN X416) was
ligated to the large HindIII/SalI vector fragment of pGC262 to give
plasmid pGC269, which codes for the IFN X918-IFN X430 polypeptide
cell modulator, designated IFN X601 (Chart 3).
(ii) Plasmid pZZ102 (Table 1)
A similar strategy was used to construct pZZ102.
STEP 1
Plasmid pJ89 (Chart 1B) was cut with BglII and SalI and a 106 bp
chemically synthesized duplex, coding for the C-terminal 13 amino
acids of IFN X918 (as in Chart 2A); and a single methionine
followed by the 21 N-terminal amino acids of human lymphotoxin
(Chart 5; residues 132 to 166) was ligated to the BglII to SalI
large vector fragment of pJB9 (Chart 1B). The resultant plasmid,
pZZ101, contains an NsiI site at hLT codons 20 and 21 (Gray, P. W.
et al. Nature 312, 721, 1984) for insertion of the remainder of the
hLT gene, i.e. ##STR2##
STEP 2
Plasmid pZZ101 was cleaved with NsiI and SalI and the large vector
fragment isolated in preparation for insertion of the remainder of
the hLT gene, which was isolated from pLT101 (Table 1; chart
1B).
pLT101 contains a complete synthetic hLT gene modified from Gray,
P. W. et al. Nature 312, 721, 1984 (equivalent to amino acid
residues 145 to 316 in Chart 5). The hLT gene in pLT 101 was cloned
on a ClaI to BamHI fragment in the ClaI/BamHI sites of plasmid
pAT153. The nucleotide sequences of the ClaI and BamHI junctions
are, respectively: A T C G A T A A G C T A T G. and T A G A G G A T
C C (ATG=initiation codon, TAG=termination codon).
Plasmid pLT101 was cleaved with NsiI and SalI and the resultant 725
bp small fragment was ligated to the NsiI and SalI large vector
fragment of ppZZ101 (Chart 1B) to give plasmid pZZ102, which codes
for the IFN X918-lymphotoxin polypeptide cell modulator, designated
IFN X603 (Chart 5).
EXAMPLE 2
Expression and Isolation of Polypeptide Cell Modulators
(a) Expression of plasmids coding for IFN X601, X602, X603 and
X604
Overnight cultures (10 ml.) of transformed bacteria were grown in
M9/casamino acids medium (EP 131 816A) supplemented with tryptophan
(40 ug/ml) and ampicillin (100 .mu.g/ml). Inocula (0.5 ml.) were
added to 50 ml. M9/casamino acids medium containing 100 ug/ml.
ampicillin. Growth was continued at 37.degree. C. until the A 670
nm had reached 0.5, at which time the cultures were made 20 ug/ml.
with respect to beta-indole acrylic acid in order to induce the
synthesis of polypeptide cell modulators. Growth was at 37.degree.
C. with vigorous shaking, and samples for biological assay (as
described in example 3 below) and electrophoretic analysis were
removed at 4 hours after induction.
(b) SDS-polyacrylamide gel electrophoresis of total E. coli
proteins for estimation of expressed protein content
The volume of cells equivalent to 0.5 optical density units at 670
nm was removed from the culture immediately and at 4 hours after
adding IAA, and the bacteria recovered by centrifugation. The cells
were immediately resuspended in 50 ul of 60 mM tris-HCl pH 6.8,
0.05% bromophenol blue, 5% glycerol, 1% sodium dodecylsulphate,
0.5% 2-mercaptoethanol, heated at 100.degree. C. for 3 min. and
quick frozen on dry ice. The boiling-freezing cycles were repeated
2-3 times to reduce the viscosity of the sample before a final
boiling 5 minutes prior to loading 7.5 .mu.l on a 15%
SDS-polyacrylamide gel (Molecular Cloning, A Laboratory Manual,
ibid.). The gel was stained with coomassie brilliant blue and
dried. The dried gel was scanned with a Joyce-Loebl `chromascan 3`
gel scanner, which computes the percentage of total protein for
each polypeptide band.
Results
Table 2 shows that for IFN X601, a polypeptide of approximately the
size expected for an IFN X918/hinge/IFN X430 fusion is expressed in
the range 5.4 to 10% of total bacterial protein.
This polypeptide is absent from cultures of E. coli K12 HB 101
harbouring plasmid pJB9 expressing IFN X918 (.about.17K) or pIL201
expression IFN X430 (.about.19K).
(c) Preparation of bacterial extracts for biological assay
10 to 20 ml. of bacterial culture was removed at the optical
density (670 nm) of 1.5-2.0 (middle to late log phase of growth)
and centrifuged to recover the cells. After suspension in 25 mM
tris-HClpH 7.5, 50 mM NaCl (1 ml.) and 1 mM EDTA (1.4 ml.) at
0.degree. C., 28 ul lysozyme was added to a final concentration of
50 ug/ml and the suspension incubated at 0.degree. C. for 30 min.
The suspension was sonicated for 24 sec., the cell debris removed
by centrifugation and the supernatants assayed for biological
activity as described in Example 3 or gel analysis as described in
Example 2.
Alternatively, lysis without sonication was used as follows. 10 ml.
culture was centrifugated and the bacterial pellet resuspended in 2
ml. 30 mM NaCl, 50 mM tris-HCl pH 7.5, 0.05 to 1 mg/ml lysozyme.
Following incubation at 25.degree. C. for 10 min. and 0.degree. C.
for 15-30 min. three freeze-thaw cycles were performed (-70.degree.
C.). The supernatant from a 15,000 rpm, 15 min. centrifugation was
divided for gel analysis, protein estimation and assay.
EXAMPLE 3
Biological Activity of Polypeptide Cell Modulators in Crude
Bacterial Extracts
(a) Antiviral assay
The cellular extract prepared as in Example 2 (together with 1 log
dilutions to 10.sup.-6) was assayed for antiviral activity by
monitoring the protection conferred on Vero (African Green Monkey)
cells against the cytopathic effect of encephalomyocarditis (EMC)
virus infection in an in vitro microplate assay system; for
example, Dahl, H. and Degre, M. Acta. Path. Microbiol. Scan., 1380,
863, 1972.
Results
A comparison is made in Table 3 of the antiviral (AV) activity in
crude bacterial extracts of IFN X601 and the parental IFNs, derived
from equivalent numbers of bacterial cells. IFN X601 consistently
exhibited 2.5-3.0 fold higher AV activity than IFN X430 and a 4-6
fold higher AV activity than XFN X918, despite a .about.2-fold
lower level of protein expression (Table 2).
A 1:1 mixture of the separately expressed IFNs X918 and X430 also
exhibited a significantly enhanced AV activity, which was 4 fold
higher than the value expected if the AV activities of the
individual IFNs X918 and X430 were additive (Table 3). This is a
reflection of the known synergy between Type I and Type II IFNs
(Czarniecki, C. W. et al. J. Virol. 49, 490, 1985; and EP 0107
498).
In conclusion, the polypeptide cell modulator IFN X601 displayed a
significant enhancement of AV activity compared with the parental
IFNs, which was similar to that of equimolar mixtures of IFN X918
and IFN X430.
(b) Antiproliferative assays
(i) Daudi (lymphoblastoid) cells
Antiproliferative (AP) activity was assessed by the ability of the
polypeptide cell modulator to inhibit the replication of Daudi
(lymphoblastoid) cells (Horoszewicz et al. Science 206, 1091,
1979). Daudi cells in log phase were cultured for 6 days in 96 well
plates in the presence of various dilutions of chimaeron or IFN.
The phenol red in the medium changes from red to yellow (more acid)
with progressive cell growth. Liquid paraffin was added to prevent
pH change on exposure to the atmosphere, and the pH change in the
medium measured colorimetrically on a Dynatech plate reader.
Inhibition of cell growth is reflected by a corresponding reduction
in the colour change.
Results
A comparison is made in Table 4A of the Daudi lymphoblastoid cell
antiproliferative activity in crude bacterial extracts of IFN X601
and the parental IFNs derived from equivalent numbers of bacterial
cells. Daudi cells are known to be unresponsive to IFN-gamma and in
a similar fashion did not respond to the antiproliferative action
of IFN X918, being more than 100X less sensitive to IFN X918 than
to IFN X430 (Table 4A). By contrast, IFN X601 exhibited similar
activity to that of IFN X430. Mixtures of IFN X918 and IFN X430
gave a lower titre than with IFN X430 alone i.e., synergy was not
evident. These results are expected as the Daudi cell line is
capable of responding to the antiproliferative effect of only the
IFN X430 portion of the polypeptide cell modulator. These results
also indicate that the IFN X430 portion of the polypeptide cell
modulator is functionally active, contributing to its biological
activity (Tables 3 and 4B).
Consistent with these findings is the observation that there is a
similar level of binding of IFN X430 and IFN X601 to Daudi
receptors (Table 7), while the lack of AP activity of IFN X918
correlates with very low receptor binding.
(ii) HEp-2 (human laryngeal carcinoma) cells
Antiproliferative activity was also assessed in HEp-2 cells Growth
inhibition was measured by methylene blue staining of the cell
monolayer by a modification of the method of Ito. (Ito, M. J.
Interferon Res. 4, 603, 1984). Inhibitory concentration (IC.sub.50)
end point is the log dilution giving 50% reduction of methylene
blue staining.
Results
A comparison is made in Table 4B of the HEp-2 antiproliferative
activity in crude bacterial extracts of IFN X601 and the parental
IFNs, derived from equivalent numbers of bacterial cells. IFN X601
consistently displayed a 3 fold higher AP activity than IFN X430
and a 15 fold higher AP activity than IFN X918, despite a
.about.2-fold lower level of protein expression (Table 2).
Furthermore, when equivalent antiviral units of these interferons
were compared it was seen that IFN X601 had an enhanced
antiproliferative effect as shown in FIG. 1. For the individual
IFNs X430 and X918 there is a maximum achievable level of growth
inhibition which cannot be increased despite adding a hundredfold
excess of interferon. This is not seen with IFN X601 where a
markedly increased level of growth inhibition is seen.
These properties of IFN X601 are reminiscent of the
antiproliferative effect of mixtures of IFN X430 and IFN X918. For
example, Table 4B shows that equivalent concentrations of these two
IFNs mixed together gave 1.8-8.6 fold higher AP activity than
either alone. In this case, AP activity was almost 3 fold higher
than the value expected if the AP activities of the individual IFNs
X918 and X430 were additive (Table 4B). Further, like IFN X601,
equimolar mixtures of IFN X918 and IFN X430 have enhanced
antiproliferative activity against HEp-2 cells (FIG. 1).
Potentiation of AP activity by mixtures of IFN X918 and IFN X430 is
a reflection of the synergy which can be demonstrated between
IFN-gamma (equivalent to IFN X918) and IFN X430 and is illustrated
by the results presented in Table 5. Where the FIC index (as
defined in Table 5) is less than 0.5, synergy is evident. Maximum
synergy was observed at equivalent numbers of antiviral units of
IFN-gamma and IFN X430 (10 U/ml). Since the specific activities of
IFN-gamma and IFN X430 differ only by a factor of approximately
two, similar amounts of IFN protein are also present.
Taken together, these results indicate that (i) a covalent
combination of IFN X918 and IFN X430 via a peptide linker segment
potentiates cytotoxicity in a manner analogous to simple mixtures;
(ii) a covalent combination of IFN X918 and IFN X430 is a suitable
ratio to potentiate biological activity; (iii) the IC.sub.50 end
point on HEp-2 cells for IFN X601 was significantly higher than the
values for the parental IFNs. Potentiation was similar to that
observed with synergistic mixtures of IFN X918 and IFN X430.
(c) HLA-DR Antigen presentation on human fibroblasts
IFN-gamma, but not IFN-beta or IFN X430, induces the expression on
the surface of normally DR-negative human foetal lung fibroblasts
(17/1 strain). This is detected and measured by the binding of
monoclonal antibody against HLA-DR.
Fibroblasts are grown to confluence in DMEM/10%FCS (Dulbecco's
Modified Eagles Medium) in 96-well tissue culture plates. IFN-gamma
or modified IFN is serially diluted in DMEM/0.1% BSA and dilutions
are added to the medium on the fibroblasts. The fibroblasts are
incubated at 37.degree. C. for a further 3 days and then the medium
is removed and the cells are washed once with PBS. Admixtures in
Herpes-buffered DMEM of a monoclonal antibody directed against
HLA-DR and peroxidase conjugated antibody against mouse IgG, is
added to the cells and incubated at room temperature for 2 hours.
The cells are washed five times with PBS and then the amount of
anti-DR antibody bound to the cells is measured by assaying for
bound peroxidase using tetramethyl benzidine (TMB) as a chromogen.
The colour generated is measured with a Dynatech.TM. microelisa
reader.
Results
IFN X601 and IFN X918 clearly caused expression of HLA-DR antigens
on the surface of 17/1 fibroblasts while IFN X430 did not (table
9). The level of HLA DR induction by IFN X601 was markedly lower
than that induced by equivalent antiviral units of IFN X918. This
may be due to suppression by the IFN X430 domain because the HLA DR
induction by IFN X918 was seen to be reduced in a 1:1 mixture with
IFN X430. The HLA DR induction by IFN X601 can be increased more
than ten fold by blocking the activity of the IFN X430 domain with
anti IFN-.beta. monoclonal antibody. These results demonstrate that
IFN-gamma biological activity is present in the polypeptide cell
modulator IFN X601.
(d) Analysis of IFN X601 with Antibodies Against beta and
gamma-IFNs
(i) Enzyme linked immunoadsorbent assay (ELISA) for interferon
The ELISA for both beta and gamma interferons utilizes an indirect
two site sandwich technique. Dilutions of the interferon samples
(or standards) are allowed to bind to interferon antibodies
attached to the wells of a 96 well microplate. A second antibody to
interferon, but raised in a different species from that attached to
the plate, is included in the incubation mixture, which then binds
to a second epitope on the interferon molecule. After washing away
the unbound molecules, an enzyme labelled antispecies antibody is
added which binds to the second interferon antibody. The presence
of bound enzyme is detected by adding a substrate which changes
color in the presence of enzyme. The amount of color produced is
proportional to the amount of interferon, since the other reagents
are present in excess.
For the beta and gamma interferon ELISA's, two antibodies against
the corresponding interferon are used, while for a hybrid ELISA, an
antibody directed against beta interferon is bound to the plate,
while the second antibody used is one directed against gamma
interferon.
The general scheme of the assay is illustrated below:
MICROTITER PLATE
ANTIBODY TO INTERFERON
INTERFERON SAMPLE
SECOND ANTIBODY TO INTERFERON
ANTI SPECIES ANTIBODY
(ENZYME LABELLED)
BETA INTERFERON ELISA
96 well microplates (Nunc Immunoplate 1) are coated with a goat
anti human beta interferon antibody (Rega Institute). To each well
of a microplate, is added 100 microliter of a 5 microgram/ml
solution of immunoglobulin (obtained by a 40% ammonium sulphate
precipitation of the interferon antibody) in 0.05M sodium carbonate
buffer, pH 9.8, and incubated for two hours at room temperature.
After removal of the well contents, unoccupied binding situes are
blocked by incubation with 100 microliters of phosphate buffered
saline containing 0.5% casein (PBS/C), for 30 minutes at room
temperature. The plates are then washed six times with phosphate
buffered saline containing 0.05% Tween 20 (PBS/T), and stored at
+4.degree. C. in a covered moist box until required.
Serial dilutions of interferon samples are made in the plates, by
dilution in PBS/C containing a mouse monoclonal antibody to beta
interferon at a 1/100 dilution. Each plate also contains an
internal standard which has been calibrated against the
International Reference Standard. After incubation overnight at
+4.degree. C., the well contents are removed and the plates washed
six times with PBS/T.
100 microliters of peroxidase conjugated goat anti-mouse
immunoglobin (Sigma a7282, diluted 1/2000 in PBS/T), are added to
each well and incubated for thirty minutes at room temperature. The
well contents are removed and the plates are washed six times with
PBS/T. 100 microliters of TMB (Tetramethyl benzidine, Sigma, 50
mcg/ml in 0.1N acetate/citrate buffer pH 6.0, containing 0.0022%
hydrogen peroxide) are added and incubated for one hour at room
temperature. 25 microliters of 2.5M sulphuric acid is added to stop
the reaction and the optical density read at 450 nm in an automatic
plate reader (Titertek Multiscan MC). Data is fed into a computer
and the 50% end points determined by linear regression analysis of
the logic log transformed data. Corrections are then made to the
internal standard included on each plate.
GAMMA INTERFERON ELISA
This assay is carried out in the same way as the beta ELISA, with
the following changes: the plates are coated with a mouse
monoclonal antibody to gamma interferon (Meloy Laboratories) at
1/200 in carbonate buffer. Serial dilutions of the gamma interferon
samples are made in PBS/C containing a rabbit antiserum to human
gamma interferon (Immunomodulator laboratories, diluted to 1/5000).
A peroxidase conjugated goat anti rabbit immunoglobulin (Tago
Laboratories, diluted to 1/3000) is used as the indicator
molecule.
HYBRID BETA/GAMMA INTERFERON ELISA
The only difference from the beta ELISA is that the interferon
samples are diluted in PBS/C containing a mouse monoclonal to human
gamma interferon (Meloy Laboratories, at a dilution of 1/1000).
This assay will only detect interferon molecules containing both a
beta and a gamma epitope.
Results
The results of testing the polypeptide cell modulator IFN X601 and
the appropriate controls in the beta, gamma and hybrid ELISA's are
given in Table 6. In the beta ELISA, IFN X430 (equivalent to beta)
reacts, the gamma interferon shows no sign of cross reactivity,
while a 50/50 mixture of the two gives a titre reduced by 0.4 log
unit/ml, close to the expected 0.3 reduction. The IFN X601 also
reacts strongly, showing that the two beta interferon epitopes are
still available to bind antibodies.
In the gamma ELISA, the gamma interferon reacts, the IFN X430 shows
no cross reactivity, while a 50/50 mixture of the two gives a titre
reduced by the expected 0.3 log units/ml. IFN X601 also reacts,
though with a reduced titre compared to the other positive
reactions, which might indicate that one of the gamma epitopes is
slightly sterically affected by the presence of the beta hybrid
interferon.
In the hybrid ELISA, the only sample to react is IFN X601, which
conclusively demonstrates that the molecule contains both beta and
gamma epitopes covalently bonded to each other. Quantitatively the
results from this assay cannot be compared to the other two ELISA's
since there is no standard available and the 50% end points are
dependent on relative affinities and concentrations of the various
reagents used, which differ for the three assays used. However, the
results indicate that a substantial proportion of the polypeptide
cell modulators is present in the covalently linked state in sample
X601.
(ii) Immunoprecipitation
Interferons were labelled by including .sup.35 S-methionine in
bacterial growth medium and extracts were prepared by treatment by
lysozyme and sonication. .sup.35 S-labelled E. coli extracts were
immunoprecipitated with either monoclonal antibodies directed
againt IFN-.beta. or IFN-.gamma. and the immunoprecipitates were
analyzed by SDS-PAGE.
Results
The results in Chart 7 show that anti IFN-.beta. monoclonal
antibody precipitates IFN X430 but not IFN X918, anti IFN-.gamma.
monoclonal antibody precipitates IFN X918 but not IFN X430 while
both monoclonal antibodies precipitate a .about.36 kd protein in
the IFN X601 extract. The material precipitated from the IFN X601
extracts by both antibodies therefore has the predicted molecular
weight for the chimaeric protein and has both X430 and X918
antigenic activity.
(iii) Western Blot Analysis
Bacterial extracts containing IFNs were run out on SDS-PAGE and
analyzed by Western blotting with anti IFN-.beta. monoclonal
antibody.
Results
Chart 8 shows that anti-IFN-.beta. monoclonal antibody detects IFN
X430 in lanes A, does not recognize IFN X918 in lanes B and
recognizes a .about.36 kd band in the IFN X601 extract in lanes C.
This again demonstrates that a band in the IFN X601 extract which
is recognized by anti-IFN-.beta. monoclonal antibody has the
predicted MW for the chimaeric protein IFN X601.
(iv) Monoclonal antibody affinity column purification
Bacterial extracts containing IFN X601 were loaded on to monoclonal
antibody affinity columns consisting of either anti-IFN-.beta.
bound to CNBr sepharose or anti-IFN-.gamma. bound to CNBr sepharose
(Celltech MAb). The loaded columns were extensively washed, bound
material was eluted and fractions were assayed for
antiproliferative activity against Daudi and HEp-2 cells and for
HLA DR inducing activity on human lung fibroblasts.
Results
The results in Table 8 demonstrate that material from an E. coli
lysate containing IFN X601 can be bound to and eluted from both
anti-IFN-.beta. and anti-IFN-.gamma. affinity columns. The material
eluted from the anti-IFN-.beta. column must have IFN X430
antigenicity and has been shown to have IFN X430 biological
activity (Daudi antiproliferative assay) as well as IFN X918
activity in the HLA DR induction assay. The material eluted from
the anti-IFN-.gamma. column must have IFN X918 antigenicity and has
been shown to have IFN X918 biological activity (HLA DR induction
activity) as well as IFN X430 activity in the Daudi
antiproliferative assay. In addition, eluted material from both
columns showed enhanced antiproliferative activity against HEp-2
cells which is taken to indicate that both the IFN X430 and IFN
X918 domains are biologically active.
Biological Activity of IFN X602 (IFN X918 (AGS), IFN X430)
Table 9 shows X602 to have similar biological properties as
X601.
Biological Activity of IFN X603 (IFN X918-LT)
Table 10 shows that IFN X602 retains both lymphotoxin and
interferon-like activities. Antiproliferative activity against
mouse L cells is characteristic of LT activity, while AV, HLA DR
and ELISA give characteristic IFN-gamma activities. (HEp-2
antiproliferative activity could be due to IFN-gamma or
lymphotoxin/IFN-gamma combination but not to lymphotoxin
alone.)
EXAMPLE 4
Construction of the Plasmid pAP8 Expressing IFNX416
Charts 1Aa and 1Ab illustrate the path to constructing a high level
expression vector for IFN-.beta.[.beta.(36-48).fwdarw..alpha..sub.1
(34-46)][cys.sup.17 .fwdarw.ser.sup.17 ], also referred to as
IFNX416, in the host E. coli HB101 (European Patent No.
85105914.7). The starting vector was pl/24C (.about.4,440 bp) which
was identical to plasmid pl/24 U.K. Patent 8,102,051, except for
the underlined sequences which follows: ##STR3##
Step 1 (Chart 1Aa)
The subcloning of the natural human IFN-.beta. gene from plasmid
pl/24C (Taniguchi et al., Gene, 10, 11, 1980) in phage M13mp8
(Sanger, F. et al., J. Mol. Biol., 143, 161, 1981) was performed,
and the presence of the whole fragment was confirmed by restriction
endonuclease mapping of M13 plasmid mAP2.
Step 2 (Chart 1Aa)
The technique of "site-directed mutagenesis" (Zoller and Smith,
Nucl. Acids Res., 10, 6487, 1982) was employed to introduce two
base changes, one each in the IFN-.beta. codons 74 and 75 so as not
to change the encoded amino acid sequence. Supercoiled DNA
resulting from transcription/ligation was separated from
non-ligated DNA in a 1% agarose gel and used to transform E. coli
JM101. Total plasmid DNA was prepared.
Step 3 (Chart 1Aa)
Mutant DNA bearing a unique XhoI site was separated from non-mutant
DNA by XhoI restriction and electrophoresis in 1% agarose. The
linear DNA was electroeluted from the agarose (Molecular cloning, A
Laboratory Manual, eds. Maniatis et al., p.168, Cold Spring Harbor
Laboratories). Following self-ligation of the linear DNA and
transformation of E. coli JM101, M13 clones were obtained all of
which had a unique XhoI site, one of which was designated mAP3.
Step 4 (Chart 1Ab)
The complete IFN-.beta. gene with an XhoI site spanning codons
74-76 was recloned back in pAT153. This generated a vector (pAP4)
similar to pl/24C, except for the changed codons 74 and 75 and the
deletion of the .about.546 base pair BglII-BamHI fragment,
originally lying 3' to the IFN-.beta. coding sequence. The new
sequence of the Serine codons 74 and 75 is given in Chart 1Aa.
Step 5 (Chart 1Ab)
The .about.230 bp synthetic DNA fragment, assembled as described
above, was cloned in the ClaI-XhoI sites of plasmid pAP4 to give
pAP8 (Chart 1Ab), a plasmid expressing IFNX416 in the host E. coli
HB101.
Modifications of the above described mode for carrying out the
invention such as, without limitation, use of alternative vectors,
alternative expression control systems, and alternative host
micro-organisms and other therapeutic or related uses of the novel
polypeptide cell modulators that are obvious to those of ordinary
skill in the biotechnology, pharmaceutical medical and/or related
fields are intended to be within the scope of the following
claims.
TABLE 1 ______________________________________ Table of Plasmids
Plasmid Properties Source ______________________________________
pAP8 Expression vector coding for EP 85105914.7 IFN X416 gene U.K.
Patent 8,102,051, Chart 1Aa and 1Ab and example 4 pJA39 Expression
vector containing Amino acids 19/20 IFN X416 gene plus HindIII
coded by AAG.CTT site instead of AAG.CTC (pAp8) pGC262 Intermediate
vector in Chart 1A construction of pGC269 - codes for IFN-gamma +
22 amino acid mouse gamma 2b IgG "hinge" pCC203 Expression vector
containing Chart 1A and synthetic human IFN-gamma PCT 83/04053 gene
pJB9 Expression vecror containing Chart 1A and synthetic IFN-gamma
gene PCT 83/04053 with DNA coding for N- terminal Cys--Tyr--Cys
deleted and replaced by Met. (IFN X918) LT3/1 Expression vector
containing Charts 1A, 3 synthetic human lymphotoxin Nature 312,
721, gene 1984 pGC279 Intermediate vector in Chart 1B construction
of pZZ102; codes for IFN X918 plus 22 N- terminal amino acids of
lymphotoxin pGC282 Expression vector containing Charts 1B, 5 IFN
X603 gene (IFN X918 - metlymphotoxin polypeptide cell modulator).
pGC269 Expression vector containing Charts 1A, 3 IFN X601 gene.
______________________________________
TABLE 2 ______________________________________ Molecular Weight and
Expression in E. coli of IFN X601 Molecular weight Range of
expression (from polyacrylamide (% of total bacterial Interferon
gel) protein) ______________________________________ X918* 17,000
13.6-15.6 (N = 14.6) X430.sup.+ 19,000 12.3-17.0 (N = 14.65) X601
37,500 5.4-10.0 (N = 7.7) ______________________________________
*IFN-gamma with Nterminal cystyr-cys deleted and replaced by met
(Chart 3 .sup.+ IFNbeta with amino acids 36 to 48 inclusive
replaced by amino acid 34 to 46 inclusive from IFNalpha 1. N
mean.
TABLE 3 ______________________________________ Antiviral Activity
of IFN X601 Antiviral activity Increase compared I.U/ml at 10 A670
.times. with: Interferon 10 -6 IFN X430 IFN X918
______________________________________ .sup. X918.sup.1 0.59 (0.5X)
-- X430 1.1 -- 2.9X X601 2.87 2.6X 4.9X X918 + X430.sup.2 3.47 3.2X
5.9X ______________________________________ *IU/m110 A670 .times.
10.sup.-6. Mean of 3 determinations in 2 separate experiments: 1.
IFNgamma with Nterminal Cys--Tyr--Cys replaced by Met (chart 3). 2.
Approximately 1:1 mixture of each IFN (protein).
TABLE 4 ______________________________________ Increase compared
Antiproliferative with: Interferon Activity* IFN X430 IFN X918
______________________________________ A. Daudi lymphoblastoid
cells X918 0.004 -- -- X430 2.7 -- -- X601 3.3 1.2X -- X918 plus
X430.sup.1 1.9 (0.7X) -- B. HEp-2 carcinoma cells X918 0.57 (0.2X)
-- X430 2.8 -- 4.9X X601 9.0 3.2X 15.8X X918 plus X430.sup.1 4.9
1.8X 8.6X ______________________________________ *Units/ml .times.
10.sup.-4 = dilution of IFN at 50% cell growth inhibition. Mean of
2 determinations. Mixture 1:1 w/w
TABLE 5 ______________________________________ IFN X430/IFN-gamma
synergy on HEp-2 carcinoma cells A. IFN X430 B. IFN-gamma.sup.+
Antiviral FIC* Anitiviral FIC* FIC Index Units/ml "A" units/ml "B"
("A" + "B") ______________________________________ 168 1.000 0
0.000 1.000 56 0.334 0.3 0.003 0.337 40 0.230 1.0 0.009 0.239 32
0.188 3.1 0.029 0.217 10 0.059 10 0.094 0.153 3.1 0.018 27 0.252
0.270 2.2 0.013 32 0.298 0.311 1.0 0.006 81 0.767 0.773 0.8 0.004
100 0.940 0.944 0 0 106 1.000 1.000
______________________________________ *FIC. Fractional Inhibitory
Concentration Ratio: antiviral units at 50% cell growth inhibition
of a given IFN (e.g. `A`) in combination with another IFN 9 e.g.
`B`) to antiviral units to IFN`A` alone. Concentration of IFN alone
or in combination required to produce 50.degree. inhibition of HEp2
growth. Synergy is present when FIC index is equal to or less than
0.5
TABLE 6 ______________________________________ ACTIVITY (LOG
UNITS/ML) Beta Gamma Hybrid ELISA ELISA ELISA E F E F E F
______________________________________ A Gamma interferon ND ND
4.47 5.44 ND ND B IFN X430 (= beta) 3.95 5.84 ND ND ND ND C
Interferon X601 4.13 6.02 2.98 3.95 3.73 -- D Mixture of A and B
3.59 5.48 4.16 5.13 ND ND (1:1)
______________________________________ Notes 1. E represents the
50% end points 2. F represents teh corrected activities 3. ND is
not detectable activity
TABLE 7 ______________________________________ COMPETITION BY IFN
X601 FOR THE BINDING OF .sup.125 I-IFN alpha 2 TO DAUDI CELL
RECEPTORS IFN Activity Log U/ml.*
______________________________________ X430 7.0 X918 3.6 X601 6.6
______________________________________ *IFN .alpha.2 antiviral unit
equivalents. The activity in each sample was calculated by
interpolation from a standard dose curve of the compet:tion by IFN
.alpha.2 for the binding of .sup.125 IIFN.alpha.2.
TABLE 8 ______________________________________ MONOCLONAL ANTIBODY
AFFINITY PURIFICATION OF CRUDE LYSATES OF IFN X601 IFN Activity*
Fraction Daudi HEp-2** HLA DR
______________________________________ Anti IFN-Beta Column 3 3.00
Not done 2.3 4 3.25 2.89 2.3 5 4.25 3.79 2.47 6 4.20 3.85 2.65 7
3.82 3.25 Not done Anti IFN Gamma Column 3 3.24 2.72 2.3 4 3.72
4.31 2.4 5 3.70 4.15 2.3 6 3.28 3.95 2.3 7 3.22 3.67 Not done
______________________________________ *Log units/ml = dilution of
IFN at 50% assay end point. **Enhanced antiproliferative activity
seen.
TABLE 9
__________________________________________________________________________
BIOLOGICAL ACTIVITY OF IFN X602 COMPARED WITH IFN X601 Antiviral
Antiproliferative HLA DR Induction ELISA IFN EMC/Vero HEp-2 Daudi
Lung Fibroblasts Beta Gamma Mixed
__________________________________________________________________________
X601 6.49 4.74* 4.28 3.30 5.93 4.08 3.50 X602 6.46 3.89* 3.55 2.81
5.94 3.46 2.75
__________________________________________________________________________
Antiviral plus Beta and Gamma ELISA activities expressed as Log
IU/ml/10 A670. Antiproliferative, HLA DR and Mixed ELISA activities
expressed as Log dilution/ml/10 A670 at 50% end point. 1. Assayed
in presence of anti IFN beta monoclonal antibody to overcome
inhibitory activity of the X430 domain. *Enhanced growth inhibitory
activity typical of IFN gamma/IFN X430 mixtures.
TABLE 10
__________________________________________________________________________
BIOLOGICAL ACTIVITY OF IFN X603 Antiviral Antiproliferative HLA DR
Induction ELISA IFN EMC/Vero HEp-2 L Cell Lung Fibroblasts Gamma
__________________________________________________________________________
X603 4.47 3.19 4.02 2.80 4.31
__________________________________________________________________________
Antiviral and Gamma ELISA activities expressed as Log IU/ml/10
A670. Antiproliferative and HLA DR activities expressed as Log
dilution/ml/10 A670 at 50% end point.
* * * * *