Note: Descriptions are shown in the official language in which they were submitted.
WO93/1769X PCI/US93/01665 ~
~7 7 ','~1 i t ~
USE ~F INTERLEUKIN-10 TO SUPPRE~SS GRAFT-VS.~HOST
DISEASE
Field of the Inventio
The invention relates generally to a method for treating and
inhibiting graft-vs.-host disease or tissue rejection by administering to an
afflicted individual an effective amount of interleukin-10.
SUMIVIARY OF THE INVENTIQ~I
The invention r~lates to the use of interleukin-10 (IL-10) to suppress
graft-vs.-host disease or the rejection of transplanted tissues. The
invention also includes pharmaceutical compositions comprising
interleukin-10 or active variants thereof. Preferably, ~he interleukin-10 of
the invention is select~d from the group consisting of the mature
polypeptidss having the open reading frames that are defined by the
amino acid sequences given in SEQ. ID. NOS. 1 and 2 herein (all SEQ.
1~ IDs. are given immediately before the Claims~, wherein the standard three-
letter abbreviation is used to indicate L-amino acids, starting from the
N-terminus. These two forms of IL-10 are sometimes referred to as human
IL-10 (or human cytokine synthesis inhibitory factor) and viral IL-10 (ora
BCRF1), respectively: e.g. Moore et al., Science, Vol. 248, ~gs. 1230-1234
2~ (1990); Vieira et al., Proc. Natl. Acad. Sci., Vol. 88, pgs. 1 172-1 176 (1991 );
Fiorentino ~ al., J. Exp. Med, Vol. 170, pgs. 2081-2095 (1989~; Hsu et al.,
S~isn~e, Vol. 50, pgs. 830-832 (1990). More preferably, the mature IL-10
or varian~s thereof used in the methods of the invention are selected from
the group consisting of the mature polypeptides having the open reading
frames that are defined by the amino acid sequences given in SEQ. ID.
NOS. 3 and 4 herein.
B_ief Des~ription of the l:)rawin~s
Figure 1 is a diagram of the vector pcD(SRc~) used for expressing
IL-10 in mammalian cells.
Figure 2 is a diagram of the vector TRP-C11 used for expressing
IL-10 in bacteria.
WO 93/1769X PCI/US93/r-
'' Figure 3 shows plasmid pGSRG carrying the open reading ~rame
(ORF) of mouse IL-10, viral IL-10, or human IL-10 inserted into its XhoI
restriction site; it also shows the sequence of the RBS-ATG-polylinker
regions of the final construction (called TAC-RBS).
Figure 4 shows the effects of endogenous and exogenous IL-10 on
the proliferative responses in MLC. PBMC (1 x 105/well) and allogeneic
irradiated PBMC (1 x 1 05/well) (PBMC donor A x PBMC donor B (A)
PBMC donor B x PBMC donor A (B)) were cultured for 5 days in the
presence of increasing concentrations of IL-10 (open bars) and anti-lL-10
mAb (solid bars). MLC were carried out in the absence (solid bars) or in
the presence (hatched bars) of 100 U/ml IL-10 and increasing
concentrations of anti-lL-10 mAb (C). :
Figure 5 shows the effects of IL-10 on the proliferative responses of
purified T cells stimulated with various allogeneic cells. Purii?i~d T cells
1~ (1 x 10~/well) were cultured for 5 days with allogeneic irradiated elutriated
monocytes (2 x 104/well) (A), positively sorted C[:)14+ monocytes (2 x
104/well~ (B), purified B cells (3.3 x 104/well) (C), EBV-LCL (1 x 104/well)
(D) in the presence of increasing concentrations of IL-10. ~:
Figure 6 shows how $he kinetics of the IL-10 effec~s depend upon
the time that the IL-10 is added to the culture. PBMC ~1 x 1 05/wcll) and
allogeneic irradiated PBMC (1 x 1 05/well~ were cultured for 5 days. The
indicated concentrations of IL-10 were added at times indicated.
Figur~ 7 shows the effects of? iL-10 on IL-2-production-in MLC.
PBMC (1 x 105/well) and allogeneic irradiated P~MC (1 x 105/w~ll) were
cultured with increasing concentrations of IL-1 û and in the presence or in
the absence of 1011g/ml of the anti-lL-2 R antibody BB10. Three days later
the supematants were harvested and assayed for their IL-2 content by
cytokine-specific ELISA.
Figure 8 shows the effPct of exogenous IL-2 on the reduced
alloantigen-induced proliferative response of T cells induced by IL^10.
Purified T cells (105/well) stimulated with aliogeneic irradiated PBMC
~105/well) (A), or purified B cells (3.3 x 1 04/well) (B), were cuitured with
incraasing amounts of IL-2 in the absence (open symbols) or in the :
presence (closed symbols) of 1oo Ulml of IL-10.
WO 93/17698 , ~ ~ PCI/US93/01665
DETAILED DE~ÇRtPTlON OF THE INVENTIQN
The invention is directed to a method of using IL-10 or agonists
thereof to suppress graft-vs.-host disease or tissue rejection in individuals,
e.g., transplant patients. The invention also includes pharmaceutical
5 compositions comprising IL-10 for carrying out the method. IL-10 ~or use in
the invention is selected from the group of mature polypeptides encoded
by the open reading frames defined by the cDNA inserts of pH5~, pH15C,
and pBCRF1 ~SR), which are deposited with the American Type Culture
Collection (ATCC), Rockville, Maryland, under accession numbers 68191,
10 68192, and 68193, respectively, and active variants thereof, e.g., agonists.
Agonists include both muteins and post-translational variants of, e.g.,
processing, truncation, glycosylation.
L Assays for Interleukin-10
IL-1 0s exhibit several biological activities which could form me basis -
15 of assays and units. In particular, IL-1 0s have the property of inhibiting the
synthesis of at least one cytokine in the group consisting of IFN-y,
Iymphotoxin, IL-2, IL-3, and GM-CSF in a population of T helper cells ~`;
induced to synthesize one or more of these cytokines by exposure to
syngeneic antigen-presenting cells (APCs) and antigen. In this activity, the
20 APCs are treated so that they are incapable of replication, but that their~
antigen-processing machin~ry remains hJnctional. This is conveniently
accomplished by irradiating the APCs, e.g. with about 1 5Q0-3000 R
(gamma or X-radiation) before mixing with the T cells.
Alternatively, cytokine inhibition may be assayed in primary or,
25 preferably, secondary mixed Iymphocyte reactions (MLR), in which case
syngeneic APCs need not be used. MLRs are well known in the art, e.g.
Bradley, p~s. 162-166, in Mishell et al., eds. Sele~ted MethQds in Celiular
Immunolo~y (Freeman, San Francisco, 1980); and Battisto et al., Me~h. in
Enzymol., Vol. 150, pgs. 83-91 (1987). Briefly, two populations of
30 allogeneic Iymphoid cells are mixed, one of the populations having been
treated prior to mixing to prevent proliferation, e.g~ by irradiation.
Preferably, the cell populations are prepared at a concentration of about
2x 106 cells/ml in supplemented medium, e.g. RPMI 1640 with 10% fetal
calf serum. For both controls and test cultures, mix 0.1 ml of each
~/0 93/17698 ~ . PCI~/~JS93/1~ ~
~. ~-
population for the assay. For a secondary MLR, the cells remaining after 7
days in the primary MLR are re-stimulated by freshly prepared, irradiated
stimulator cells. The sample suspected of containing IL-10 may be added
to the test cultures at the time of mixing, and both controls and test cultures
may be assayed for cytokine production from 1 to 3 days after mixing.
Obtaining T cell populations and/or APC populations for IL-10
assays employs techniques well known in the art which are fully described
in DiSabato et al., eds., Meth. ~n Enzymol., Voi. 108 (1984). APCs forthe
preferred IL-10 a-csay are peripheral blood monocytes. These are
1 0 obtained ~Jsing standard techniques, e.g. as described by Boyum, Meth. in
Fnzymo/., Vol. 108, pgs. 88-102 (1984); Mage, Meth. in Enzymol., Vol.
108, pgs. 118-132 (1984); Litvin et al., Meth. in Enzymol., Vol. 108, pgs.
298-302 (1984); Stevenson, Meth. in Enzymol., Vol. 103, pgs. 242-249
(1989); and Romain et al., Meth. in Enzymol., Vol. 108, pgs. 148-153
1 ~ (1984); all of which references are incorporated her~ir by reference.
Preferably, helper T cells are used in the IL-10 assays, which are obtained
by first separating Iymphocytes from the peripheral blood and then
selecting, e.g. by panning or ~low cytometry, helper cells using a
commercially available anti-CD4 antibody, e.g. OKT4 described in U.S.
patent 4,381,295 and avaiiable from Ortho Pharmaceutical Corp. The
requisite techniques are fully disclosed by Boyum in Scand. J. Clin. Lab.
Invest., Vol. 21 (Suppl. 97), pg. 77 (1968), and in Meth. in EnzymoL, Vol.
108 (cited above), and by Bram et al. in Meth. in Enzymol.~ ol. 121, pgs.
737-748 (1986). Generally, PBLs are obtained from ~resh blood by Ficoll-
Hypaque density gradient centrifugation.
A variety of antigens can be employed in the assay, e.g. Keyhole
limpet hemocyanin (KLH), fowl ~-globulin, or the like. More preferably, in
place of aniigen, helper T cells are stimulated with anti-CD3 monoclcnal
antibody, e.g. OKT3 disclosed in U.S. patent 4,361,549, in the assay.
Cytokine concentrations in control and test samples are measured
by standard biological and/or immunochemical assays. Construction of
immunochemical assays for specific cytokines is well known in the art
when the purified cytokine is available: e. g. Campbell, Monoclonai
Antibody Technology (Elsevier, Amsterdam, 1984); Tijssen, Practice and
Theory of Enzyme Immunoassays (Elsevier, Amsterdarn, 1985); and U.S.
patent 4,486,530 are exemplary of the extensive literature on the subject.
~WO 93/17698 ~ PCl/lJS9i/0166~
... ,,. ,` , ~ :
ELISA kits for human IL-2, human IL-3, and human GM-CSF are
commerciatly available from Genzyme Corp. (Boston, MA); and an ELISA
kit for human IFN-~ is commercially available from Endogen, Inc. (Boston,
MA). Polyclonal antibodies specific for human Iymphotoxin are available
5 trom Genzyme Corp. which can be used in a radioimmunoassay for human
Iymphotoxin, e.g. Chard, An Introduction to Radioimmunoassay and
Related Techniques (Elsevier, Amsterdam, 1982).
Biological assays of the cytokines listed above can also be used to
determine IL-10 activity. A biological assay for human Iymphotoxin is
10 disclosed by Aggarwal, Meth. in Enzymo/., Vol. 1 ~6, pgs. 441-447 (1985),
and Matthews et al., pgs. 221-225, in Clemens et al., eds., Lymphokines
and Interferons: A Practical Approach (IRL Press, Washington, D.C., 1987).
Human IL-2 and GM-CSF can be assayed with factor dependent cell lines
CTLL-2 and KG-1, available from the ATCC under accession numbers TIB
214 and CCL 246, respectively. Human IL-3 can be assayed by R ability to -~
stimulate the formation of a wide range of hematopoietic cell colon~es in
soft agar cultures, e.g. as described by Metcalf, The Hemopoietic Colony
Stimulating Factors ~EIsevier, Amsterdam, 1984). IFN-y can be quantffled
~th anti-viral assays, e.g. Meager, pgs. 129-147, in Clemens et al., eds.
(cited above). See also, Roitt (1992) Encyclopedia of immunolQ~y,
Academic Press, New York, and Coligan (1992 and periodic supplements)
Current Protocols in Immunologv GreeneNViley, New York.
Cytokine production can also be determined by mRNA analysis.
Cytokine mRNAs can be measured by cytoplasmic dot hybridi~ation as
described by White et al., J. Biol. Chem.1 Vol. 257, pgs. 8~69-8~72 (19B2),
and Gillespie et al., U.S. patent 4,483,920. Accordingly, these references
are incorporated by re~erence. Other approaches include dot blotting
using purified RNA, e.g. chapter 6, in Hames et al., eds., Nucleic Acid-
Hybridization A Practical Approach (IRL Press, Washington, D.C., 1985).
Some samples to be tested for IL-10 activity may require
pretreatment to remove predetermined cytokines that might interfere with
the assay. For example, IL-2 increases the production of IFN-y in some
cells. Thus depending on the helper T cells used in the assay, IL-2 may
have to be removed from the sample being tested. Such removals are
conveniently accomplished by passing the sample over a standard anti-
cytokine affinity column.
.
~;' ..
~ Y,'~.~
WO93/1769X PCr/US93/~!- 6~ ~
J;~J~
h _ _
For convenience, units of IL-10 activity are defined in terms of
IL-10's ability to augment the IL-4-induced proliferation of MC/9 celis,
which are described in U.S. patent 4,559,310 and available from the ATCC
under accession number CRL 8306. 1 unit/ml is defined as the
5 concentration of IL-10 which gives 50% of maximum stimulation of MC/9
proliferaticn above the leve~ of IL-4 in the following assay. Prepare
duplicate or tripiicate dilutions of IL-4 and IL-10 in 50 ~l of medium per well
in a standard microtiter plate. Medium consists of RPMI 1640, 10% fetal
calf serum, 50 ~LM 2-mercaptoethanol, 2 mM glutamine, penicillin (100 U/L)
10 and streptomycin (100 ~lg/L). Add IL-4, 25 ,ul/well of 1600 U/ml (400 U/ml
final) diluted in medium and incubate overnight, e.g. 20-24 hours. Add
3H-thymidine (e.g. 50 IlCi/ml in medium) at 0.~-1.0 IlCi/well and again
incubate the cells overnight; thereafter harvest the cells and measure the
incorporated radioactivity.
VQriants and analogs of the IL-10 de.,cribed herein can be made by -
recombinant means as described in, e.g., Sambrook et al~ (1989)~
Mol~h~ory M~nua! Cold Spring Harbor Press, Cold
Spring Harbor, New York; or Ausubel (1987 and periodic supplements)
Current ProtQcols in Molecular Biology Greene/Wiley, New York; or by
20 synthetic techniques, as described, e.g., in Atherton et al. ~1989) ~lid
Pb~Peptide Synthesis- A Praçtical Approach IRL Press, Oxford.
II. Purifi~iQ~ ~d Pharmaceuti~ Compo~itions
When polype,oti~es of the present invention are expressed in
soluble form~ for example as a secreted product of transformed yeast or
25 mammali n celis, they can be puri~ied according to standard procedures of
the art, including steps of ammonium sulfate precipitation, ion exchange
chromatography, gel filtration, electrophoresis, affinity chromatography,
and/or the like: e.g. ~Enzyme Purification and Related Techniques,n
Methods in EnzymoJogy, 22:233-577 (1977); and Scopes, R., Protein
30 Purification: Principles and Practice (Springer-Verlag, New York, 1982)
provide guidance in Such purifications. Likewise, when polypeptides of the
invention are expressed in insoluble form, for example as aggregates,
inclusion bodies, or the like, they can be purified by standard procedures in
the art, including separating the inclusion bodies from disrupted hosl cells
~V0 93/1769X ~ PC~/US93/0166
by centrifugation, solubilizing the inclusion bodies with chaotropic and
reducing agents, diluting the solubilized mixture, and lowering the
concentration of chaotropic agent and reducing agent so that the
polypeptide takes on a biologically active conformation. The latter
procedures are disclosed in the following references, which are
incorporated by reference: Winkler et al., Biochemistry, 25: 4041-404~
(1986); Winkleret al., Biotechnology, 3:992-998 (1985); Koths et al., U.S.
patent 4,569,790; and European patent applications 86306917.5 and
86306353.3.
1 0 As used herein "effective amount" means an amount sufficient to
reduce or prevent graft-vs.-host disease or tissue rejection. See, e.g., Paul
(1989) Fundamental Immunoioay Raven Press, New York. The effective
amount for a particular patient may vary depending on such factors as the
state, type, and amount of tissue transplanted, the overall health of the
1 5 patient, method of administration, the severity of side-effects, and the like.
Generallyl IL-1û is administered as a pharmaceutical composition
comprising an effective amount of IL-10 and a pharmaceu~ical carrier. A
pharmaceutical carrier can be any compatible, non-toxic substance
suitable for delivering the compositions of the invention to a patient.
Generally, compositions useful for parenteral administration of such drugs
are well known, e.g. Remington's Pharmaceutical Science, 15th Ed. ~Mack
Publishing Company, Easton, PA 1980). Alternatively, compositions of the
invention may be introduced into a patient's body by implantable or
injectable drug delivery system, e.g. Urquhart et al., Ann. Rev. Pharmacol.
Toxicol., Vol. 24, pgs. 199-236 (1984); Lewis, ed. Controlled Release of
Pesticides and Pharmaceuticals (Plenum Press, New York, 1981); U.S.
patent 3,773,919; U.S. patent 3,270,960; and lhe like.
When administered parenterally, the IL-10 is formulated in a unit
dosage injectable form (solution, suspension, emulsion) in association with
a pharmaceutical carrier. Examples of such carriers are norrnal saline,
Ringer's solution, dextrose solution, and Hank's solution. Nonaqueous
carriers such as fixed oils and ethyl oleate may also be used. A preferred
carrier is 5% dextrose/saline. The carrier may contain minor amounts of
additives such as substances that enhance isotonicity and chemical
3~ stability, e.g., buffers and preservatives. The IL-10 is preferably formulated
in purified form substantially free of aggregates and other proteins at a
Wo 93/17~9X PCl~US93/~ ~`5 - .
`.2~; ;` '''''~
, ~ " ~ ~ 8
concentration in the range of about ~ to 20 ~g/ml. Preferably, IL-10 is
administered by continuous infusion so that an amount in the range of
about 50-8û0 ,ug is delivered per day (i.e. about 1-16 ~lg/kg/day~. The daily
infusion rate may be varied based on monitoring of sid~ effects and blood
5 cell counts.
EXAMPLES
The following examples serve to illustrate the present invention.
The selected vectors and hosts, the concentration of reagents, the
temperatures, and the values of other variables are only to exemplify
10 application of ~he present invention and are not to be consider~d
Iimitations thereof.
Example 1. Ex~ression of human ÇSIF in a ba~terial ho~
A synthetic human CSIF ~ene is assembled from a plurality of
chemically synthesized double-stranded DNA fragments to form an
1 S expression vector desisnated TAC-RBS-hCSlF. Clonin3 and expression
are carried out in a standard bacte-ial system, for example E. coD K-12
strain JM101, JM103, or the like, d~scribed by Viera and Messing, in G~r~e,
Vol. 19, pgs. 259-268 (1982). Restriction endonuclease digestions and
ligase reactions are performed using standard protocols, e.g. Maniatis ~t
?0 al., Molecular Cloning: A Labor~tory Manual ~Cold Spring Harbor
Laboratory, New York, 1982~.
The alkaiine method (Maniatis et al., cited above) is used for small
scale plasmid preparations. For large scale prep~rations a modification of
the alkaline method is used in which an equal volume of isopropanol is
25 used to precipitate nucleic acids from the cleared Iysate. Precipitatiorl with
cold 2.~ M ammonium acetate is used to remove RNA prior to cesium -~
chloride equilibrium density centrifugation and detection with ethidium
bromide.
For filter hybridizations Whatman 540 filter circles are used to lift
30 colonies which are then Iysed and fixed by successive treatments with
0.5M NaOH, 1.5M NaCI; 1 M Tris.HCI pH8.0, 1 .5M NaCI (2 min each); and
heating at 80C for 2 hours. Hybridizations are in 6xSSPE, 50%
formamide, 0.1% sodium dodecylsulphate (SDS), 100 llg/ml E. CDii tRNA
` WO 93/1769~ ~ . ` , Pl`/US93J0166:-
at 42C for 6 hours using 32P-labelled (kinased) synthetic DNAs.
(20xSSPE is prepared by dissolving 174 g of NaCI, 27.6 g of
NaH2PO4-9H2O, and 7.4 g of EDTA in 800 ml of H2O. pH is adjusted to 7.4
with NaOH, volume is adjusted to 1 liter, and the whole is sterilized by
autoclaving.) Filters are washed twice (15 min, room temperature) with
1xSSPE, 0.1% SDS. After autoradiography (Fuji RX film), positive
colonies are located by aligning the regrown colonies with the blue-stained
colonies on the filters. DNA is sequenced by the dideoxy method of
Sanger et al. Proc. N~t/. Acad. Sci., Vol. 74, pg. 5463 (1977). Templates `!
for the dideoxy reactions are either single-stranded Dl~lAs of relevant
regions recloned into M13mp vectors, e.g. Messing et al. Nucleic Acids
Res., Vol. 9, pg. 309 (1981 ); or double-stranded DNA prepared by the
minialkaline method and denatured with 0.2M NaOH (5 min, room
temperaturo) and precipitated from 0.2M NaOH, 1.43M ammonium a~etate ~
by the addition of 2 volumes of ethanol. DNA is synthesized by ;~`ph~sphoramidite chemistry using Applied Biosystems 380A synthesizers.
Synthesis, deprotection, cleavage and purification (7M urea PAGE,
elution, DEAE-cellulose chromatography) are done as described in the
380A synthesizer manual.
2û Complementary strands of synthetic ONAs to be cloned (400 ng
each) are mixed and phosphorylated with polynucleotide kinase in a
reaction volume of 50 ~I. This DNA is ligated with 1 ~19 of vector DNA
digested with appropriate restriction enzymes, and ligations are in a
volume of 50 ~l at room temperature for 4 to 12 hours. Conditions for
2~ phosphorylation, restriction enzyme digestions, polymerase reactions, and
ligation have been described (Maniatis et al., cited above). Colonies are
scored for lacZ+ (when desired) by plating on L agar supplemented with
ampicil!in, isopro,oyl-1-thio-beta-D-galactoside (IPTG) (0.4 mM) and
5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (x-gal) (40 mg/ml).
The TAC-RBS vector is constructed by filling-in with DNA
polymerase the single Bam HI site of the tacP-bearing plasmid pDR540
(Pharmacia). This is then ligated to unphosphorylated synthetic oligo-
nucleotides (Pharmacia) which form a double-stranded fragment encoding
a consensus ribosome binding site as given in SEQ. ID. NO. 5 herein and
3~ designated RBS. After ligation, the mixture is phosphorylated and
religated with the SstI linker ATGAGCTCAT. This complex is then cleaved
WO 93/17698 - ~ PCI/US93/(~
- 10 -
with SstI and Eco RI, and the 173 base pair (bp) fragment isolated by
polyacrylamide gel electrophoresis (PAGE) and cloned into EcoRI-SstI-
restricted pUC19 (Pharmacia) (as described below). The sequence of the
RBS-ATG-po.y.inker regions of the final construction (called TAC-RBS) is
5 shown in Figure 2.
The synthetic IL-10 gene is assemb.ed into a pUC19 plasmid in
eight steps. At each step inserts free of deletions and/or inserts can be
detected after cloning by maintaining the .acZ(a) gene of pUC19 in trame
with the ATG start codon inserted in step 1. Clones containing deletion
10 and/or insertion changes can be filtered out by scoring for blue co.onies on
L-ampicillin platss containing x-gal and IPTG. Alternatively, at each step
sequences of inserts can be readily confirmed using a universal
- sequencing primer on small scale plasmid DNA preparations, e.g.
available from Boehringer Mannheim.
15ln step 1, the TAC-RBS vector is digested with ~stI, treated with T4
DNA polymerase (whose 3'-exonuclease ac~ivity digests the 3'-profruding ~-
strands of the SstI cuts to form blunt-end fragments), and after d~activation
of T4 DNA polymerase, treated with Eco RI to form a 173 bp fragment con-
taining the TAC-RBS region and having a blunt end at the ATG start codon
20 and the Eco RI cut at the opposite end. Finally, the 173 bp TAC-RBS
fragment is isolated.
In step 2, the isolated TAC-RBS fragment of step 1 is mixed with
Eco RI/Kpn I-digested plasmid pUC19 and synthetic fragment 1A/B whose
nuoleio acid sequences are shown in SEQ. ID. NOs. 6 and 7 herein, whi~h
25 has a blunt end at its upstream terminus and a staggered end
corresponding to a Kpn I out at its downstream terminus. This Kpn I end is
adjacent to and downstream of a Bst EII site. The fragments are ligated to
form the pUC19 of step 2.
In step 3, synthetic fragments 2AJB and 3AJB are mixed with
30 BstEII/SmaI-digested pUC19 of step 2 (after amplification and
puri~ication) and ligated to form pUC19 of step 3~ The nucleic acid
sequences of synthetic fragment 2A/B are shown in SEQ. ID. NOs. 8 and 9
herein and the nucleic acid sequences of synthetic fragment 3A/B are -~
shown in SEQ. ID. NOs. 10 and 11 herein. Note that the downstream
35 terminus of fragment 3AIB contains extra bases which form the Sma I
blunt-end. These extra bases are cleaved in step 4. Also~ fragments 2A/B
~ I~ ::
wo 93/1769~ . 1 PCr/US93/0166
and 3A/B have complementary 9-residue single-stranded ends which
anneal upon admixture, leaving ~he upstream Bs~ EII cut of 2A/B and the
downstream blunt ~nd of 3A/B to ligate to the pUC19.
In step 4, the pUC19 of step 3 is diges~ed with AnII/XbaI, amplifiedl
purified, repurified, mixed with synthetic fragment 4AIB whose nucleic acid :sequences are shown in SEQ. ID. NOs. 12 and 13 h~rein, and ligated to
form pUC19 of step 4.
In st~p 5, the pUC19 of step 4 is digested with Xba I/SalI, amplified
and purified, and mixed with synthetic fragment 5A/B whose nucleic acid
sequences are shown in SEQ. ID. NOs. 14 and 1~ herein and ligated to
form the pUC19 of step 5. Note that the SalI-staggered end of fragmcnt
~A/B is eliminated by digestion with Hpa I in step 6.
ln step 6, the pUC19 of step 5 is digested with HpaI/PstI, amplified
and purified, and mixed with synthetic fragment 6A/B whose nucleic acid
sequences are shown in SEQ. ID. NOs. 16 and 17 herein and ligated to
form the pUC19 of step 6.
ln step 7, the pUC19 of step 6 is digested with Cla I/Sph I, amplified
and purified, and mixed with synthetic fragrnent 7AlB whose nucleic acid
~equences are shown in SEQ. ID. NOs. 18 and 19 herein and ligated to
form the pUC19 of step 7.
ln step 8, the pUC19 of step 7 is digested with MluI/Hin drl~,
amplified and purified, and mixed with synthetic fragments 8A/B and 9A/B
and ligated to form the final construction, which is then inserted into E col
K-12 strain JM101, e.g. available from the ATCC under accession number
25 - 33876, by standard techniques. The nucleic acid sequences of synthetic
fragment 8A/B are shown in SEQ. ID. NOs. 20 and 21 herein and the
nucleic acid sequences of synthetic fragment 9A/B are shown in SEQ. ID.
NOs. 22 and 23 herein. Af~er ~ultivation, protein is sxtracted from the
JM101 cells and diluticns of the extrac~s are tested for biological activity.
Example 2. Expression of vlL-10 in C0~ 7 MonkQy cells
A gene encoding the open reading frame of vlL-10 was amplified by
polymerase chain reaction using primers that allowed later insertion of the
amplified fragment into an Eco RI-digested pcD(SRo~) vector (Figure 1).
wo 93tl769~ pcr/us93/Q
~ `; ? '~L
~ ~3 ~ ~
I~d -- -- 1 2
The coding strand of the inserted fragment is shown in SEQ. ID. NO. 1
herein.
Clones carrying the insert in the proper orientation were identified
by expression of vlL-10 and/or the electrophoretic pattern of restriction
5 digests. One such vector carrying the vlL-10 gene was designated
pBCRF1 (SRoc) and was deposited with the ATCC under accession number ~:
68193. pBCRF1(SRa) was amplified in E. coli MC1û61, isolated by
standard techniques, and used to transfect COS 7 monkey cells as follows: -
One day prior to transfection, approximately 1.5 x 1 o6 cos 7 monkey cells
were seeded onto individual 100 mm plates in Dulbecco's modified Eagle
medium (DME) containîng 5% tetal calf serum (FCS) and 2 mM glutamine.
To perform the transfection, COS 7 cells were removed from the dishes by
incubation with trypsin, washed tw`ice in serum-free DME, and suspended
to 107 cells/ml in serum-free DME. A 0.75 ml aliquot was mix~d with 20
DNA and ~ransterred to a sterile 0.4 cm electroporation cuvette. After 10
minutes, the cells were pulsed at 2û0 volts, 960 ~lF in a BioRad Gene
Pulser unit. Atter another 10 minutes, the cells were removed from the
cuvette and added to 20 ml ot DME containing 5% FCS, 2mM glutamine,
penicillin, streptomycin, and gentamycin. The mixture was aliquoted to four
100 mm tissue culture dishes. After 12-24 hours at 37C, 5% C02, the
medium was replaced with similar medium containing only 1% FCS and
the incubation continued for an additional 72 hours at 37~C, 5% CO2, atter
which the medium was collected and assayed for its ability to inhibit IFN-y
synthesis.
10 ml aliquots of freshly isolated PBLs (about 2X106 cells/ml) were
incubated at 37C with PHA (100 ng/ml) in medium consisting oi (i) 90%
DME supplemented with 5% FCS and 2 mM glutamine, and (ii) 10%
supernatant from COS 7 cells previously trans~ected with pBCRF1 (SRa).
A~ter 24 hours the cells and supernatants were harvested to assay for the
presence of either IFN-y mRNA or IFN-y protein, respectively. Controls
were treated identically, except that the 10% supernatant was from COS 7
cultures previously trans~ected with a plasmid carrying an unrelated cDNA
insert. The vlL-10-treated samples exhibited about a 50% inhibition ot
IFN-ysynthesis relative to the contrQls.
`WO 93/1769X PC~/US93/01665
--13 --
Example 3 Ex~ression of vlL-1Q in Escherlchia coli
A gene encoding the mature vlL-10 shown in SEQ. ID. NO. 4 herein
may be expressed in E coli.
The cDNA insert of pBCRF1 (SRa) is recloned into an M13 plasmid
5 where it is altered twice by site-directed mutagenesis: first to form a ClaI
site at the 5'-end of the coding region for the mature vlL-10 polypeptide,
and se~ond to form a Bam HI site at the 3'-end of the coding region for the
mature vlL-10 polypeptide. The mutated sequence is then readily inserted
into the TRPC11 expression vector described below.
10The TRPC11 vector was constructed by ligating a synthetic
consensus RBS tragment to Cla I linkers (ATGCAT) and by cloning the
resulting fragments into Cla I-restricted pMT11 hc (which had been
previously modified to contain the Cla I site). pMT1 1 hc is a small (2.3
kilobase) high copy, AMPR, TETS derivative of pBR322 that bears the ~VX
15 plasmid Eco RI-Hin dm polylinker region. (JrVX is described by Mahiatis et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, 1982). This was modified to contain the ClaI site by restricting
pMT11 hc with Eco RI and Bam HI, filling in the resulting sticky ends and
ligating with Cla I linker (CATCGATG), thereby r~storing the Eco RI and -
20 Bam HI sites and replacing the Sm~ I sile with a CJa I site. One
transformant from the TRPC11 construction had a tandem RBS sequence
flanked by C/aI sites. One of the Cla I sites and part of the seoond copy of
the RBS sequenca were removed by digesting lhis plasmid with PstI,
treating with Bal31 nuclease, restricting with Eco RI and treating with T4
25 DNA polymerase in the presence o~ all four deoxynucleotide triphosphates.
The resulting 30-40 bp fragments were recovered by PAGE and cloned
into SmaI~restricted pUC12. A 248 bp E. coli trpP-bearing Eco RI
fragment derived from pKC101 (described by Nichols et al. in Methods In
Enzymology, Vol. 101, pg. 155 (Academic Press, N.Y. 1983)) was then
30 cloned into the Eco RI site to complete the TRPC11 construction~ which is
illustrated in Figure 2. TRPC11 is employed as a vector for vlL-10 by first
digesting it with Cla I and Bam HI, purifying it, and then mixing it in a
standard ligation solution with the Cla I-Bam HI fragment of the M13
containing the nucleotide sequence coding for the mature BCRF1. The -~
35 insert-containing TRPC11, referred to as TRPC1 1-BCRF1, is propagatsd in
: `
WO 93/17698 PCI/US93t~
` ;~ '~`:t.
14 - :
E. coli K12 strain JM101, e.g. available ~rom the ATCC under accession
number 33876.
Example 4
This Example was disclosed after the priority date of this document
5 by 8ejarano et al. (1992) Intern~tional Imrnunology 4:1389-1397. It
provides in vitr~ evidence for the effectiveness of IL-10 treatment in
appropriate circumstances. More particularly, it demonstrates that IL-10
inhibits allogeneic proiiferative and cytotoxic T cell responses generated in
primary mi%ed Iymphocyte cultures (MLC).
- 10 This Example shows how IL-10 inhibited the alloantigen-induced
proliferative responses in a dose-dependent fashion. The suppressive ;~
effect was optimal when IL-10 was added at the beginning of the cultures,
suggesting that it acts on the early stages of T cell activation. The
proliferative responses were enhanced in the presenee of anti-lL-1Q mAb,
15 indicating that endogenously produced IL-10 suppresses prolif~ration in
primary MLC. Whether the stimulator cells were irradiated allogeneic
peripheral blood mononuclear cells ~PBMC), purified monocytes, or B
~ells, the inhibitory effects of IL-10 wer~ still observed. Th~ reduced
proliferative responses were not restored by high concentrations of
20 exogenous IL-2, indicating that the effects of IL-10 are not only related to
inhibition of IL-2 synthesis. Furthermore, the production of IL-2, IFN-* IL-6,
GM-CSF, and TNF-~ in primary MLC was dirninished by IL-10 and
enhanced in the presence of anti-lL-10 mAb. The strongest effects were
observed on the production of IFN-y. Although IL-10 reduces the prolifer-
25 ative responses, the ratio of CD3+CD4+ and CD3+CD8f T cells remainedthe same in IL-10-treated cultures and in control oultures. However, the
percentages of activated CD3~ T c~lls as judged by CD25+ and HLA-DR+
expression were consistently reduced in the presence of IL-10.
IL-10 inhibits allospecifio proliferative responses and cytokine
30 production. In addition, it was demonstrated that the reduced proliferative
responses could not be restored by exogenous IL-12, suggesting that
~L-10 inhibits allospeoific proliferative T cell responses predominantly by
reducing the stimulatory capacity o~ the stimulator cells.
WO 93/1769~ PCJ/US93/~1665
. ' ! I ~
--15 --
These data indicate that IL-10 has important regulatory effects on
allogeneic responses in vitro.
Medium and Reagents.
Cells w~re cultured in Yssel's medium supplemented with 10%
5 pooled heat-inactivated human AB sen!m. See Yssel et al. (1986) ElJr. J.
Immunol. 16:1187- _.
The neutralizing anti-lL-10 mAb 19F1 was raised against vlL-10 and
efficiently neutralized hlL-10 and vlL-10. See Bejarano et al. (1985
Ç~ns~ 35:327; and ATCC deposit HB10487, deposited June 28, 1990.
10 The BB10 mAb, which recognizes the IL-2R p~ chain, was a kind gift of
Dr. J. Wijdenes (CRTS, Besançon, France; see Herve et al. ~19~ ~QQ~I
75:1017-1023). Murine anti-CD3 (anti-Leu-4, IgG1), anti-CD4 (anti-Leu-
3a, 19(31), anti-CD8 (anti-Leu-2a, IgG2a), anti-CD14 (anti-Leu-M3, IgG2b),
anti-CD19 (anti-Leu-1~, IgG1~, anti-CD25 (anti-lL2R p55, IgG1), anti-CD56
15 (anti-Leu-19, IgG1), anti-HLA-DR (clone L243, IgG2a) mAb and control
mAb of appropriate isotypes were purchased from Becton-C)ickinson
(Mountain View, CA).
Cell Preparations.
Buffy coat preparations were obtained from the Blood Bank of
20 Stanford l)niversity Hospital. PBMC were isolated by density gradient
centrifugation over Flcollhypaque (Pharmacia, Uppsala, Sweden).
For purification of T cells, PBMC were depleted of monocytes by
plastic adherence and iron phagocytosis. See Bejarano et al. (1985) Lnt. J.
Cancçr 35:327- _ . Non-adherent cells were passed through nylon wool
25 (Julius et al. (1973) ~r. J. ImmunQI. 3:64~- ), and then NK cells w~re
removed by depletion with magnetic beads. Briefly, following stainln~ with
saturating concentrations of anti-CD56 mAb for 30 min at 4C, cells were
washed twice with Hanks's balanced salt solution ~HBSS), and
subsequently rosetted with magnetic beads coated with sheep anti-mouse
30 IgG (Dynbeads M-4~0 sheep anti-mouse IgG, Dynal AS, Oslo, Norway~ at
a bead:cell ratio of 40:1. The mixture was incubated for 30 min at 4C
under gentle shaking before removal of rosetted cells with the magnetic
particle concentrator according to the manufacturer's recommendations.
~ ~ "i, ~
~VO 93/1769X PCrtUS93/~
" ~
, . - 16 -
The resulting cell preparations were >g9% CD3+, c1% CD14+, <1%
CD19+, <1% CD56~.
For isolation of CD14+ monocytes, PBMC were stained with
PE-conjugated CD14 mAb (Becton-Dickinson, Mountain View, CA),
washed twice in HBSS and thereafter sort~d into CD14 1 and CD14-
populations using a FACStar-Plus (Becton-Diekinson, Sunnyvale, CA3.
Reanalysis of the sorted populations showed that more than 99.5% of the
purified cells were CD14~. In some experiments monocytes were isolated
trom peripheral blood by density centrifugation in a blood component
separator, followed by centrifugal elutriation (see Figdor et al. (1984)
Llm~nunol. Methods 68:68- ). These monocyte preparations were
~95% pure, as judged by nonspecific esterase staining.
Purified B Iymphocytes were obtained by magnetic-bead depletion.
Briefly, non-adherent PBMC were incubated with saturating concentrations
of anti-CD3, anti-CD4, anti-CD8, anti-CD14 and anti-CD56 mAbsfor30
min at 4C. The cells were washed twice in HBSS and therea~ter rosetted
with magnetic beads coated with sheep anti-mouse IgG (Dynal AS, Oslo,
Norway) at a 40:1 bead:cell ratio. Subsequently, the rosetted cells were ~
depleted as described above. The resulting population consisted of >98% ~;
CD19+ cells.
Proliferation Assay.
PBMC or highly purified T cells (1 x 105 cells/well), were stimulated
by various irradiated (4000 rad) allogeneic s~imulator cells. PBMCI CD14+
- mono~ytes, monocytes separated by centrifugal elutriation, and purified B
Iymphocytes, were used as stimuiator cells at R:S ratios of 1:1, 5:1, 5:1,
and 3:1, respectively. Cultures were carried out in triplicate in 96-well flat- -
bottomed microtiter plates in the absence (solid bars; Figures 5-7) or in the
presence (hatched barsj of IL-10 in 200111 medium.
Cultures were pulsed with ~3H]TdR during the last 10 hours of a
5-day incubation period and harvested onto fiberglass filters, and the
radioacti~ity was determined by liquid scintillation counting. The results
are expressed in Fig. 4 as c.p.m of l3H]TdR incorporation and represent the
means of triplicate cultures.
wo s3~176sx pcr/us93to166
-- 17 --
~ulk Cultures.
PBMC or highly purified T cells were cultured with irradiated
allogeneic cells at the P~:S ratios described above in 50 ml flasks at a
concentration of 1 x 1 o6 responder cells/ml in the presence or in the
5 absence of 100 U/ml of IL-10. Five to six days later the supernatants were
collected and frozen at -20C for determination of their cytokine contents,
whereas the cells were recovered for phenotype analysis.
Fluorescence Analysis
Cells (105) recovered from the bulk cultures were incubated in
10 V-bottomed microtiter plates (Flow Laboratories, McLean, Va) with 10~11 of
purified PE-conjugated mAb for 30 min at 4C. In the double-labeling
experiments, the cells were washed twice in 1% normal mouse serum after
the FITC labeling, and a PE-conjugated mAb was added. The cells were
washed twice with HBSS containing 1% BSA and 0.02M NaN3 and
15 thereafter analyzed on a FACScan.
Lymphokine Determinations.
Supernatants collected from bulk cultures at day 5 or 6 were
assayed for the content of GM-CSF, IFN-~, TNF-c~, IL-2, IL-4, IL-5, and IL-6
by Iymphokine-specific ELISA (Bacchetta et al. (1989) ~. Imrnunol.
20 144:902- ). For the quantification of IL-2 production, cultures were
carried out in the presence of 1 Omg/ml of the anti-lL-2 receptor antibody
BB10, in order to minimize IL-2 consumption. Supernatants were
harvested after 7~ hours and the IL^2 levels were determined by specific
ELISA. The sensitivity of the various ELISA were: 40 pg/ml for IL-4; 20
2~ pg/mi for IL-2, 11 -5 and IL-6; 5C pg/ml for GM-CSF; and 100 pg/mi for
TNF-a and IFN-y.
IL-10 inhibits proliferative responses in MLC.
To determine the effects of IL-10 on the proliferative responses in
classical one-way primary MLC, PBMC were stimulated with allogeneic
30 PBMC in the absence or presence of various concentrations of IL-10.
Figure 4 shows that IL-10 inhibited the proliferative responses in a dose-
dependent fashion. Significant inhibitory effects wers already observed al
IL-10 concentrations as low as 1 U/ml, whereas maximal inhibitory effects
(ranging from 33 to 95% inhibition in different experiments) were obtained -`
~VO 93/1 769~ . t PCI /US93/(1' ~ :
~ -- 18
at IL-10 concentrations of 100 Utml. These inhibitory effects of iL-10 were
completely neutralized by the anti-lL-10 mAb indicating the specificity of
the inhibition (Figure 4c). In fact, the proliferative responses in MLC
carried out in the presence of the neutralizing anti-lL-10 mAb were
significantly enhanced, indicating that endogenously produced IL-10 is
responsible for suppressing proliferative responses in primary MLC.
Effect of monocytes on the inhibitory effects of IL-10 in MLC.
It is known that IL-10 strongly reduces the Ag-presenting (AP)
capacity of monocytes through down-regulation of class II MHC antigens.
In contrast, class II MHC expression ano AP-capacity of Eps~ein-Barr Virus
(EBV)-transformed B cells (EBV-transformed Iymphoblastoid cell line;
EBV-LCL) are not affected by IL-10.
In this experiment, highly enriched T cells obtained by negative
selection were used as responder cells. P~rified monocyte populations
enriched either by centritugal elutriation or by direct sorting of CD14+ cells
from PBMC, purified B Iymphocytes, and EBV-LCL were used as stimulator
cells. IL-10 strongly inhibited the proliferative responses induced by
allogeneic monocytes independently of whether the monocytes were
obtained by centrifugal elutriation (Figure Sa) or were positively sorted by
the FA(~S (Figure Sb); whereas the proliferative responses towards
allogeneic EBV-LCL remained unaffected (Figure 5d3 As observed with
spedfic proliferative responses to soluble antigens, these results indicate
that allospecific proliferation is blocked when allogeneic monocytes, but
not when allogeneic EBV-LCL, are used as stimulator cells The
2~ prolifsrative responses induced by freshly isolated highly purified -`
allogeneic B celis were also inhibited by IL-10 (Figure ~c), indicating that
the sup~ressive effect of IL-10 is also present when B cells are used as
stimulators, despîte the fact that IL-10 has no measurable effect on class I `
or class II MHC expression on these cells.
Kinetic experiments revealed that the effect of IL-10 on MLC-
inducsd proliferation decreased gradually with time. IL-10 was most
effective when added at the beginning of the primary cultures; if added at
day 2 or 3 after the onset of the cultures, the effects were only marginal and
no clear dose-response effects were observed (Figure 6). These results
3~ indicated that IL-10 acts on the early stages of activation of T cells in MLC.
~'0 93/1769X PCI/US93/0166
- 19 -
IL-10 prevents cytokine production in ~ILC.
IL-10 has been shown to reduce IFN-~ and GM-CSF production by
PBMC activated by anti-CD3 or PHA. In addition, IL-10 inhibits the
producticn of cytokines by monocytes. To determine the effect of IL-10 on
cytokine production in one-way MLC, allogeneic PBMC were used as
responder and as stimulator cells. The cultures were carried out in the
absence or in the presence of IL-10 or anti-lL-10 mAb, and supernatants
were collected at day 5 and assayed for their cytokine content. Table 1
shows that IFN-y, IL-6, GM-CSF, and TNF-o~ were produced in MLC, and
that IL-10 inhibited the production of these cytokines to various extents. No
significant IL-4 production was detected, and the levels of IL~5 were below
100 pg/ml. The production of IL-10 ranged from 1000 to 3000 pg/ml in
different experiments. The strongest inhibitory effects of exogenous IL-10
were cbserved on the production of IFN-y, wh~reas the weakest inhibitory
effectswere obs2rvedon IL-6 production.
Increased IFN-y, GM-CSF, and TNF-a levels were observed in
supernatants of MLC carried out in the presence of anti-lL-10 mAb (Table
1). These enhancing effects of anti-lL-10 mAb on cytokine production were --
dose-dependent. Taken ~cgether, these results indicate that both - ~-
endogenous and xog~nous IL-10 reduce the produ~ion of the cytokines ~:
tested. To evaluate the effect of IL-10 on IL-2 production in MLC, and to
minimize IL-2 consumption by activated T cells, the cultures were carried
out in the presence or in the absence of IL-10 and of the an~i-lL-2 receptor
mAb BB10. In these experiments, IL-10 prevented IL-2 production in a
dose-dependent manner ~Figure 7). No measurable levels of IL-2 could
be detected when IL-10 was added at 100 U/ml.
WO 93/17698 PCr/US93~0 'S
20-
TABLE 1.
EFFECT OF~EXQGENOUS AND ENDOGENOUS IL-10 ON
CYTOKINE PRODUC~TION BY ALLOANTI~N-STIMI)LAT~
LYMPHOCYTES
_ .
Condition _ Cytokine
,
IL-10 alL-10 IL-6 IL-10 GM-CSF TNF-a IFN-~
~U/ml) (~lg/ml~ (ng/ml) ~ng/ml)_(pg/ml) (pg/ml) (ng/mlL
A 0 22.2 572 109 79 <1
20.~ 41 61 ~1 -
13.0 1 1 38 <1
100 12.1 16 47 ~1
0.05 22.9 144 144 ~ 1
0.5 25.8 165 111 c1
24.9 172 51 ~1 :
A+B 0 3~.41473 1744 141 29.5
3~.3 928 1~1 20.8
23.9 784 82 18.1 ~;
100 24.7 61 2 ~ 66 1 1 .4 `
0.0~ 36.9 2372 137 33.1
0.~ 39.1 23~ 1 37 4~.3
~ luman PBMC ~A) were eultured for ~ days alone or with altogeneic
irradiated PBMC ~B) in the absence and in the presence of IL-10 or the anti
IL-10 mAb 19F1. Production of cytokines was determined in the
supernatants by cytokine-specific ELISA~
Table 1 presents the results of one Experiment out of four.
The inhibitory effects of IL-10 cannot be restored by exogenous IL-2.
To investigate whether the inhibitory effects of IL-10 were observed
in the presence of exogenous IL-2, MLC were carried out with various
concentrations of IL-2. Figure 8 shows that, when increasing amounts of
15 IL-2 were added to MLC in which purified T cells were used as responders
and PBMC or purified B cells as stimulators, the proliferation was
WO 93/17698 PCI`/US93/0166~ .
t ~
-21 -
enhanced both in the absence and in the presence of IL-10. However, the
inhibitory effects of IL-10 were still present when IL-2 was added at
concentrations up to 100 U/ml; and it should be noted that 10 U/ml are
sufficient to saturate high affinity IL-2R. Similarly, addition of 400 U/ml of
5 IL-4, which has T cell growth factor activity, tailed to restore the reduced
proliferative responses induced by IL-10. Taken together these results
indicate that the lack of IL-2 is not the limiting tactor responsible for the -
reduced proliferative responses observed when MLC are carried out in the
presence of IL-10.
IL-10 decreases the proportion of activated T cells in MLC. `
In order to determine whether the reduced proliferative responses in
MLC in the presence of IL-10 differentially affected CD4+ or CD8+ T cell
subsets, the propor~ions of CD3+CD4+ and CD3+CD8+ cells were
determined. In Table 2 it is shown that the total T cell number decreased
15 by 30 to 60% when the T ceils were stimulated with allogeneic PBMC,
purified monocytes, or B cells in the presence of IL-10. However, the
1~ ~ proportion of CD4+ and~CD8+ T cells remained the same~ indicating that
- ~ ~ IL-10 has no preferential effect on each of these T cells subsets.
In contrast. the proportion of activated T cells expressing CD25 and
20 HLA-DR antigens was consistently reduced in the IL-10-containing
cultures. The strongest reduction was usually observed in MLC where
purified CD3+ T cells were stimulated with purified B cells or monocytes.
,
:.~
~`'.
wo 93/17698 Pcr/US93t~
22 --
., ~ .
TABLE 2.
EFFECT OF IL-10 ON ALLOANTIGEN-STIMULATED T CELLS
T cell count % posinve cells
(X10 6/ml) CD3+CD4~ CD3+CD8+ CD3~CD25+ CD3+DR+
~ .
~a) IL-10 -- IL-10 -- L-10 -- IL-10 ~ IL-10 :-
EXPT. 1
T~PBMC ¦ 1.3 0.76 ¦ 69 73 ¦ 21 24 ¦ 4 2 ¦ 3
T+Bcells ¦ 1.2 0.72 ¦ ~5 62 ¦ 28 28 ¦24 18 ¦19 8
EXPT. 2
T + PBMC 1.4 1.0 NI)b) ND ND ND 39 27 36 32
T+B cells 0~90 0.68 ND ND ND ND 20 8 18 4
T + mon~ 1.2 0.40 ND ND ND ND 52 24 43 25
cytesC~ .~
EXPT. 3 :
T+PBMC ¦ 1.02 0.37 ¦ 77 78 ¦ 18 16 ¦16 13 ¦15 9
T + mon~ 0.~0 0.17 76 79 19 16 7 4 8 5
cy~esd) , . _ , . _ :
a3 Indicates absence of IL-10
5 b~ ND=Not Done
c) Ne~atively sorted monocytes
d) Positively sorted monocytes (CD14+).
Purified T cells were cultured with allogeneic irradiated PE~MC,
purified ~ cQlls or monocytes in the absence or presence of IL-10 (100
10 U/ml). Six days iater the recovered T cells were counted and phenotyped
by indirect immunofluorescence.
The prQsent results show that IL-10 reduced in a dose-dependent
fashion the proliferation of alloresponsive T cells in ciassical one-way
primary MLC in which allogeneic PBMC of two different donors were used
15 as responder and irradiated stimulator cells, respectively. These inhibitory
effects w~re completely neutralized by an anti-lL-10 mAb, demonstrating
~ wo 93/1769X ~ PCl`/US93/01665
" . . ,~
--23 --
the specificity of the inhibition. In addition, it was shown that the
proliferative responses were considerably enhanced in the presence of the
anti-lL-10 mAb, indicating that endogenous IL-10 production is responsible
for suppression of proli~erative responses in MLC.
IL-10 also reduced the proliferative responses in MLC where highly
purified T cells were used as responders and puri~ied monocytes as
stimulators. Interestingly, IL-10 was ineffective when purified T cells were
stimulated by irradiated allogeneic EBV-LCL. See Fig 5b. These data are
consistent with previous findings that IL-10 strongly blocked the sp~cific
proliferative responses of T cells or T cell clones towards soluble antigens
or antigenic peptides when monocytes, but not when EBV-LCL, were used
as antigen presenting cells (APC). This reduced antigen-presenting
capacity was found to be associated with the down-regulatory effect of
IL-10 on ~lass II MHC expression on monocytes. In contrast, IL-10 did not
affect clas~ II MHC expression on EBV-LCL. From these data it was
concluded that the reduced antigen-specific proliferative T cell responses
reflected prevention of activation of the responder cells, rather than a direct
suppressiv~ effect on T cell proliferation. This conclusion was ~urther
supported by the reduced Ca2+ fiuxes in the respondsr T cell clones
activated in the presenoe of IL-10.
MLC-induced proliferation was inhibited by IL-10 not only when -~monocytes were used as stirnulators, but also when purified B cells were
used. Several studies have shown lhat T cell recognition- of MHC
alloantigens is mechanistically similar to recognitisn of viral, bacterial, or
2~ other foreign pro~ein antigens. Recently, it has been shown that a
significant proportion of MHC class II ailoreactive T cell ciones recognize
processed determinants from human serum proteins in association with
allogeneic class II molecules. In contrast to the situation where new MHC-
peptide complexes have to be formed to activate antigen-specific T cell
clones, there is no evidence to indicate that new allo-MHC-peptide
complexes must be formed on monocytes and ~ cells to stimulate T cells in
a MLC. Moreover, IL-10 does not affect class II MHC membrane
expression.on human B cells. Therefore, it is unlike~ly that the inhibitory
effects of IL-10 on MLC-induced T ceil proliferation c~n be solely anributed
to a down-regulation of MHC class II expression on the monocytes. It is
Wo 93~1769~ C PCI~US93~
.~. .
-24 -
possible that other mechanisms, yet to be defined, are responsible for the
reduced stimulatory capacity of B cells in the presence o~ IL-10.
It has been demonstrated that, in addition to cross-linking of the
TCR/CD3 complex by specific ailoantigen, LFA-1 - ICAM-1 interactions are
5 required for cytokine production by allospecific T cells. Furthermore,
CD28 - B7/BB1 interactions have been sh~wn to be necessary ~or
induction of alloantigen-specific activation of resting T cells, resulting in
cytokine production, proliferation and cytotoxic activity. B7 is weakly
expressed on resting B cells and monocytes, but is elevated following
10 activation of these cells. However, it could be ruled out that the reduced
prsliferative and cytotoxic alloresponses were due to down-regulatory
effects of IL-10 on the expression of either TCRICD3 or these accessory
molecules. IL-10 did not affect TCR/CD3, LFA-1 expression on the
responder T cells, or ICAM-1 and B7 expression on B cells or monocytes
15 used as stimulators.
The data reported here indicate, moreover, that the reduced
proliferative responses towards alloantigens also reflect prevention of
activation of the responder T cells. IL-10 had ~o be present from the onset
of the cuitures to exert its maximal inhibitory effects. In addition, the
20 proportion of activated T cells, as judged by the expression o~ CD25 and
HLA DR antigens, was considerably lower in the ll -10-containing cultures
than in the control ML~. Although the total nurnber of CD3+ T cells
generated in MLC carried out in the presence of IL-10 was reduced, IL-10
did not preferentially affect the responses of CD4+ or GD8+ T cells, sinc~
25 the proportions of ~hese T cell subsets were comparable to those in oontrol
MLC carried out in the absence of IL-10. The reduced expression of CD25
indicates tha~ the inhibitory e~fect on T cS-Sll proliferation in a MLC is not amere consequence of the cytokine inhibitory activity of IL-10; and this-is
supported by the finding that IL-10 also reduces the proliferatiYe responses
30 when exogenous IL-2 is added at concentrations that are sufficient to
- saturate high affinity IL-~ receptors (Figure 8). Collectively, these data
suggest that IL-10 reduces the stimulatory capacity of Pi3MC, monocytes
and normal B cells in MLC. The possibiiity that IL-10 also has direct effects
- on the T cells cannot be completely excluded. However~ the fact that MLC
35 generated with EBV-LCLs as stimulators are not inhibited by IL-10 argues
Wo 93/17698 PCr/US93/01665
- 25 - -
against this notion, unless the EBV-LCL can override the inhibitory effect of
IL-1 0.
The levels of cytokines produced in MLC in which total allogeneic
PBMC were used as responder and stimulator were also significantly
reduced in the presence of exogenous IL-10. The amounts of IL-2, IFN-y,
TNF-a, and GM-CSF were about two- to three-foid lower than those of
control MLC carried out in the absence of IL-10. IL-6 production was much
less affected; this may be due to the fact that monocytes present in these
cultures already produce considerable amounts of this cytokine very early
10 after activation, before the suppressor activity of IL-10 becomes effective.
The present results therefore clearly indlcate that IL-10 plays an
important role in down-regulating alloresponsiveness in vitro. From these
in vitro observations, and considering that alloantigens are the major
targets for specific immunological rejection of transplanted tissue, one can
15 expect IL-10 to play a role in the induGtion or maintenance of tolerance
~oilowing allogeneic transplantation in vivo. ~ :
Example 5
The following Example was published aSter the priority dat~ of the -
present application by Roncarolo and Bacchetta (1992) in ~Qne Marrow
20 Transplantation: Procee~ln s of Foetal and NeQna~l C~ll Transform~tion
~od RetroviralGene Ihe~py vol. 9, supplement 1. It pr~vides in YiVo
evidence for the importance of T cell repertoire in tolerance after fetal stem ~ -
cell transplantation.
The T cell repertoire and the mechanism of tolerance was studied in
25 two patients with severe combined immunodeficiency transplanted with
HLA mismatched fetal liver stem cells. They are 18 and 6 years old (as of
1993) and healthy, and show normal immunoresponses to recall antigens.
Their T cells are of donor origin, whereas monocytes and B cells remained
of the host. The NK cells have different sources since in one patient they
30 derive from the donor and in the other one from the host. :)espite the HLA
mismatch between donor and host cells, no acute or chronic graft-versus^
host disease was observed. In vitro experiments with PBMC showed .`
specific nonresponsiveness for the HLA antigens expressed by the host
...
WO 93/1769X P~/US~3/0~ 5
,
J
;,_! '` 26 --
cells. However, an extensive clonal analysis showed that CD4+ and CD8+
host-reactive T cell clones recognizing class II and class I HLA molecules
of the host, respectively, were present in the peripheral blood of both
patients. Limiting dilution experiments indicated that the frequency of
5 CD8+ host-reactive cells was in the same range as that observed for
alloreactive T cells. In contrast, no donor-reactive CD8+ T cells could be
isolated. Host-reactive CD4+ and CD8+ T cell clones were normal in their
capacity to produce IL-2, IFN-y, GM-CSF, and IL-5, but they failPd
completely to synthesize IL-4. In addition, CD4+ T cell clones from patient
10 RV secreted very high levels of IL-10. Interestingly, exogenous IL-10 was
able to inhibit the proliferative responses of the CD4~ host-reactive T cell
clones. These data demonstrate that host-reactive cells are not deleted
from the donor T cell repertoire following allogeneic fetal liver stem cell
transplantation. Therefore, in vivo tolerance between the host and the
donor is maintained by a peripheral autnregulatory mechanism in which ~
cytokines may play a role. ~ -
Bone marrow transplantation from an HLA-identical donor is the
therapy of choice for children with severe combined immunodeficieney
(SCID). However, transplantation of hemopoietic fetal liver cells (FLT) from
20 a mismatched donor can give sustained engraftment and offer a possible
cure in the absence of an Hl A-identical marrow donor. Seel e.g.,
Touraine et al. (1987) Ihymus 10:75-_. In such patients,
immunocompetent T Iymphocytes of donor origin, as determined by HLA
typing, could be identified within 2-3 months post-transplantation.
25 Although peripheral T Iymphocyte chimerism is routinely detected in SCID
childr n transplanted with fetal liver stem cells, lack of B Iymphocyte
chimerism is not uncommon. The immunologic evaluation of these
patients offers a unique possibility to study human T cell differentiation and
selUnonself discrimination under in vivo conditions of tlLA mismatch
30 between T precursor cells and differentiation environment.
This report on two SCID children transplanted 18 and 6 years ago
with fetal liver stem cells from fully HLA-disparate donors provides
evidence for in vivo activity of IL-10 in regulating the immune system, and
WO 93tl769X PCr/lJS93tO166
--27 --
in particular that IL-10 produced by host-reactive cells may play an
important role in down-regulating their responses in vivo.
Patient SP received two fetal liver stem cell transplan~ations with
simultaneous injection of syngeneic fetal thymus. Although standard HLA
5 typing showed engraftment of cells only from the second donor, a more
precise cytcfluorometric analysis, using monoclonal antibodies specific for
polymorphic HLA determinants, indicated ~hat 10-20% of the T ~ ~-
lymphocytes were actually from the first donor. The second patient, RV, ~-
received seven fetal liver stem cell transplantations, but only one donor-
10 cell population could be identified in the peripheral blood. See Roncarolo
et al. (1986) J. CJin. Investia. 77:673-
TABLE 3
HL~ TYPING
A C B DR OQ
SP
Recipient 3-33 6 14-47 4-5 3
1st donor 2-11 4 27-62 1-8
2nd donor 1-2 7 8-18 3-9 3
RV
Recipient 2-13 4-7 x-62 8-10 - 4-5
Donor 2-30 4 8-35 11-13 6-7
In such patients, sustained engraftment of donor T cel!s was ~ -
1~ observed after transplantation, whereas B cells and monocytes were of
host origin.
:
, ~
. ~
.,''.:
~vo 93/17698 Pcr/uss3~0~
I ~ ~,t~
~;J ~ 28 -
TABLE 4. CHIMEP~ISM
a~TCR+ T cells
y~TCR+ T cells Donor origin
Monocytes -
Host origin
B cells
NK cells Hostorigin-> Patient SP
Donor origin^> Patient RV
-
Despite this state of split chimerism within cells of the immune ~ -
system, compiete reconstitution was achieved and normal in vivo and in
5 vitro antibody r~sponses to recall antigens were observed. This is due to
the ability of donor T cells to cooperate with the antigen-presenting c811s
(APC) of the host, across the allogeneic barrier. In particular, tetanus
toxoid specific T cell clones of donor origin, isolated from the peripheral
blood of patient SP, could recognize the antigen (Ag) processed and
10 presented by host E3 cells~ EBV-~ransformed B cell lines, and NK cell
clonss. In contrast, none of the Ag-specific T cell clones test~d so far were
restricted by the class II HLA antigens expressed by the donor cells. See
Roncarolo et al. (1988) J. Expt'! Med. 167:1523- _; Roncarolo et al.
~1991 ) J. Immunoi. 147:781- .
The chimerism in the NK population differed in the two palients
(Table 4). In one patient fresh NK cells and NK cell clones showed the
HLA phenotype of the host; in the other patient they were of donor origin.
These NK cells expressed the CD16 CD~6 antigens and displayed norrnal
cytotoxic activity against a variety of NK sensitive targets. These findings
20 suggest that the presence of host or donor functional NK cells does not
prevent slable engraftment of donor T cells atter fetal stem cell
transplantation. Despite the coexistence of Iymphoid cells with major
WO g3/1769~ PCr/US93/0166~
. . .`. .~ , ., ~;
--29 --
and/or minor histocompatibility antigen differences, complete tolerance
was achieved in vivo in these two patients, and no signs of acute or
chronicgraft-versus-hostdisease were observed. Furthermore, in vitro
studies showed that specific nonresponsiveness by the donor T cells
S towards the HLA antigens expressed by the host was present in a primary
mixed leucocyte culture, whereas thc proliferative responses against
allogeneic cells were normal. At the clonal level, however, the findings -have differed. Host-reactive cytotoxic T cell clones of donor origin
recognizing either HL~ class I or HLA class II antigens have been derived ~ - -
10 from the peripheral blood of both patients. In contrast to what has been - -
reported in SCID patients transplanted with marrow from HLA-haplo-
identical parental donors, no donor-reactive T cell clones could be isolated
in these two patients. Furthermore, in patient RV no T cell clones specific
for the HLA class I locus A antigens that were shared by the host could be :
15 identified. Frequency analysis using a modified limiting di'ution assay (see
- Vandekerckhove et al. (1992) .). Expl. Med. 175:1033-1043) confirmed the
lack of donor reactivity and demonstrated that the frequency of CD8+ host~
reactive T cells was in the same range as the frequency of T cells reacting
against third party HLA antigens. These findings demonstrate that host-
20 reactive cells are not clonally deleted Srom the donor T cell repertoire.
Evidently, such host-reactive T cells are under regulation, since clinical ,
manifestations of grafl-versus-host disease were not evident in these -
patients. lt is possible that the host-reactive cells are anergic in vivo and
that in vitro stimulation in the presence of lL~2 can break this anergy.
Host-reactive T cells display a peculiar pattern of Iymphokine
production after polyclonal and antigen-specific stimulation. None of the -
CD4+ or CD8~ T cell clones are able to secrete IL-4, whereas they ~ -
synthesize normal levels of IL-2, IL-~, and GM-CSF. IFN-y production by
these clones is usually very high. In addition, IL-10 production by CD4+
30 host-reactive clones of patient RV is extremely high after antigen-specific
stimuiation and seems to be inversely correlated to the low lL-2 synthesis~
Furthermore, addition of exogenous IL-10 can significantly suppress the
proliferative responses of CD4+ host-reactive T cell clones in vitro~ This
provides evidence that lL-10 production by host-reactive cells may play an
35 important role in down-regulating their responses in vivo. ~:~
~ ,
WO 93/1769X PCI`/US93/0- `~
.', ,t~ ~:
Example 6
This example investigates IL-10 production in SCID patients
transplanted with allogeneic stem cells. Children with Severe Combined
Immunodeficiency (SCID) can be transplanted with HLA mismatched fetal
5 stem cells and immunological reconstitution is obtained also when only the
T cells of the donor engraft. Despite tolerance to the host, host-reactive T
cells are still present at high frequencies in the peripheral blood of these
patients suggesting that an autoregulatory suppressor meohanism may be
responsible for the in vivo homeostasis. IL-10 has reccntly been described
10 as a suppressive cytokine able to prevent activation and proliferation of
(allo)antigen specific T cells. Semiquantitative PCR on total peripheral
blood mononuclear cells from two patient indicated that the IL-10 mRNA
levels were much higher than those of normal donors, whereas IFN-y and
GM-CSF mRNAs were comparable. PCR analysis on purified monocytes,
1~ B and T cells demonstrated that the monocytes of host origin were,
responsible for the enhanced IL-10 production. In one patient, high levels
of IL-10 mRNA were also observed in purified total T cells. Furthermore,
high levels of IL-10 production by CD4+ host reactive T cells suppressed in
an autoregulatory fashion the proliferation of these cells in response to
20 host cells. These results su~gest that high endogenous IL-10 produc~ion
may account for the suppression of allo reactivity after stem cell
transplantation .
On December 20th 1989, Applicants deposited separate cultures of
E. coli MC1061 carrying pH5C, pH1 ~C, and pBCP~F1 (SRa) with the
25 American Type Culture Collection, Rockville, MD, USA (ATCC), under
accession numbers 68191, 68192, and 68193, respectively. These
deposits were made under conditions as provided under ATCC's
agreement for Culture Deposit for Patent Purposes, which assures that the
deposit will be made available to the US Commissioner of Patents and
30 Trademarks pursuant to 35 USC 122 and 37 CFR 1.14, and will be made
available to the public upon issue of a U.S. patent, which requires that the
deposit be maintained. Availability o~ the deposited strain is not to be
construed as a license to practice the invention in contravention of the ~ - -
WO 93/1769X ~ PCI/IJS93/01665
- 31 ~
rights granted under the authority of any government in accordance with its
patent laws.
The Deposits have been modified to satisfy the requirements of the
Budapest Convention. ;
The descriptions of the ~oregoing embodiments of the invention
have been presented for purpose of illustration and description. They are
not intended to be exhaustive or to limit the invention to the precis~ forms
disclosed, and obviously many modifications and variations are possible in
light of the above teaching. The embodiments were chosen and described
in order to best explain the principles of the invention t~ thereby enable
others skilled in the art to best utili2e the invention in various embodiments -
and with various modifications as are suited to the particular use
contemplated. It is intended that the scope of the invention be defined by
the claims appended hereto.
-:.
~'0 93/1 769X ~ PCl`/US93tQ~
--32 --
SEOUENCE LI STING
~2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 178 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:
~A~ ORGANISM: Homo sapièns
(ix) FEATURE:
(D) OTHER INFO~MATION: Human IL-10 (human cytokine
synthesis inhibitory factor, human CSIF~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly
Val Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys
Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Ars
. 35 40 45
Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln
~0
Leu Asp Asn Leu Leu Leu Lys Glu Ser ~eu Leu Glu Asp Phe Lys
~; 65 70 75
Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr
Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile
100 105
Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg
110 115 120
Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys
125 130 135
Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln
3~ 140 145 150
Glu Lys Gly Ile Tyr Lys Ala Me~ Ser Glu Phe Asp Ile Phe Ile
lS5 160 1~5
Asn Tyr Ile Glu Ala Tyr Met Th- Met Lys I le Arg Asn :~
170 175
WO93/17698 ~ r PCT/US93/01665 ~`~
. -, ; ",
- 33 -
,:' :'
(2) INFORMATION FOR SEQ ID NO: 2:
~i) SEQUENCE CHARACTERISTICS~
(A) LENGTH: 170 amino acids
5(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(~i) ORIGINAL SOURCE:
~A) ORGANISM: B95-8 Epstein-Barr Virus -
10(ix) FEATURE: -~-
(D) OTHER INFORMATION: Viral IL-10 (BCRF1)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Glu Arg Arg Leu Val Val Thr Leu Gl~ Cys Leu Val Leu Leu
15 Tyr Leu Ala Pro Glu Cys Gly Gly Thr Asp Gln Cys Asp Asn Phe ;:
20 25 30 :
Pro Gln Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys
35 40 45
Thr Phe Phe Gln Thr Lys Asp Glu Val Asp Asn Leu Leu Leu Lys
2050 55 ~0 ~:~
Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala
65 70 75 :~
Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln
~0
Ala Glu Asn Gln Asp Pro Glu Ala Lys Asp His Val Asn Ser Leu
95 100 105
Gly Glu Asn Leu Lys Thr Leu Ars ~eu Arg Leu Arg Arg Cys His
110 115 120
Arg Phe Leu Pro Cys Glu As~ Lys Ser ~ys Ala Va~ Glu Gln Ile
30125 130 13S ;
Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala ;
140 145 150
Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met
155 160 165
Thr Ile Lys Ala Arg
170
.
~2) INFORMATION FQR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERIST CS~
'~'`'
.
.~.
W093/17698 PCT/US93/Ot''`~
34 -
(A) LENGTH: 160 am~no acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
tii) MOLECULE TYPE: protein
(ix) FEATURE:
(D) OTHER INFORMATION: Mature human IL-10 (human
cytokine synthesis inhibitory factor, human
CSIF)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Se- Cys Thr His Phe
S 10 15
Pro Gly Asn Leu Pro Asn Met Leu Arg As~ Leu Arg Asp Ala Phe
Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn
1~ 35 40 45
Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly_Tyr Leu
~0
` Gly Cys Gln Ala Leu Ser Glu Me~ Ile Gln Phe Tyr Leu Glu Glu
Val Met Pro Gln Ala Glu Asn Gln Asp P-o As- Ile Lvs Ala P.is
Val Asn Ser Leu Gly Glu Asn Leu Lys Th~ Lel~ Arg Le~ Arg Le
100 10~
~rg Arg Cys His Arg Phe Lel1 Pro Cys Glu Asn Lys Se- Lys Alz
110 11~ 120
Vâl Glu Gln Val Lys Asn Al2 Phe Asn Lys Le~ Gln Glu Lys Gly
125 13Q 1~5
Ile Tvr Lys Ala Met Se~ Glu Phe As~ Ile Phe Ile Asn Tyr Ile
140 14~ 150
Glu Ala Tyr Met Thr Met Lys Ile Arg P.sn
155 160
(2) IN-ORMATION FOR SEQ I3 NO: 4:
(i) SEQUENCE CHARACTFRTSTICS:
(A) LENGTH: 147 am~no ac~ds
~B) Typr: amino 2c~d
(D) TOPOLOGY: linea~
(i ) MOLECULE TV^E~ tei-.
~ .TU?~
WO93/1769R r', ~ PCT/US93/0166~ ~
- 35 - : .
(D) OTHER INFORMATION: Mature viral IL-10 (BCRF1) :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: :
Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg
Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu
Val Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys
~0 45
Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr
1050 5~ 60 ~:.
Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ala
65 70 75 ~:
Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg -
1~ Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn ~ys
100 105
Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn Lys_Leu Gln
110 115 120 :
Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile ~-~
125 130 135
Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg
140 195
~2) INFORMATION FOR SEQ ID NO: 5: .
25(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPF.: nucleic acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE~
~D) OTHER INFORMATION: As double-stranded -
fragmen~, encodes a consensus ribosome binding
site.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
.
35 GTAAGGAGGT TTAAC 15 ::
~2) INFORMATION FOR SEQ ID NO: 6: :~
~i) SEQUENCE CHARACTERISTICS:
WO 93/1769$ PC'r/US93/Ot ' ~
.~
36 -
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
tC) STRANDEDNESS: single
~D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: T and GGTAC at positions 51
and 56-60 differ from those of the native
sequence; together with SEQ ID NO: 7, SEQ ID
NO: 6 forms double-stranded Fragment lA/B of
synthetic CSIF gene with 4-base sticky end at
positions 57-60.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: .:
AGCCCAGGCC AGGGCACCCA GTCTGAGAAC AGCTGCACCC ACTTCCCAGG 50
TAACCGGTAC 60
- ':
(2) INFORMATION FOR SE~ ID NO: 7: .
~i) SEQUENCE CHARACTERISTICS~
(A) LENGTH: 56 base pairs
~B) TYPE: nucleic acid
(C) STRANDEDNESS: single -~
(D) TOPOLOGY: linear
(ix~ FEATURE: . .
~D) OTHER INFORMATION: C and A at positions 1 and :
6 differ from those of the native sequence;
2~ together with SEQ ID NO: 6, SEQ ID NO: 7 forms
double-stranded Fragment lA/B of synthetic CSIF
gene .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
CGGTTACCTG GGAAGTGGGT GCAGCTGTTC TCAGACTGGG TGCCCTGGCC 50
TG5GCT S6
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs
WO 93t17698 " ~ " .~., PCr/US93/()1665
- 37 - : :
(B) TYPE: nucleic acid :
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: T position 2 differs from
that of the native sequence; together with SEQ .
ID NO: 9, SEQ ID NO: 8 forms double-stranded .:.
Fragment 2A/B of synthetic CSIF gene with 5- and
9-base sticky ends at positions 1-5 and 54-62.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GTAACCTGCC TAACATGCTT CGAGATCTCC GAGATGCCTT CAGCAGAGTG ~0
AAGACTTTCT TT 62 ~.
~2~ INFORMATION FOR SEQ ID NO: 9: :
(i) SEQUENCE CHARACTERISTICS: ~
(A~ LENGTH: 48 base pairs ~-.
(B) TYPE: nucleic acid ~
IC) STRANDEDNESS: single ~:
(D) TOPOLOGY: linear
(ix) FEATURE: -.
(D) OTHER INFORMATION: Together with SEQ ID NO~
SEQ ID NG: 9 forms double-stranded Fragment ~A/B of ~-
synthetic CSIF gene.
~xi) SEQUENCE DESCRIPTION: SEQ I~ NO: 9:
CTTCACTCTG CTGAAGGCAT CTCGGAGATC TCGAAGCATG TTAGGCAG 48
(2) INFORMATION FOR SEQ ID NO: 10: .
(i) SEQUENCE CHARACTE~ISTICS:
(A) LENGTH: 35 base pairs
(B) TY?E: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY~ line r .
Iix) FEATURE: -~
WO93/17698 PCT/US93/09 5
_
(D) OTHER INFORMATION: C and T at positions 30 and
32 differ from those of the native sequence; together
with SEQ ID NO: 11, SEQ ID NO: 10 forms double-stranded
Fragment 3A/B of synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CAAATGAAGG ATCAGCTGGA CAACTTGTTC TTAAG 35
;
(2) INFORMATION FOR SEQ ID NO: 11: :
- ~-
(i) SEQUENCE CHARACTERISTICS: ~
:- '
(A) LENGTH: 44 base pairs ~
, -
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single --~
(D) TOPOLOGY: linear
~
(ix) FEATURE:
(D) OTHER INFORM~TION: A and G at positions 4 and
6 differ from those of the native sequence; together with
SEQ ID NO: 10, SEQ ID NO: 11 forms double-stranded
Fragment 3A/B of synthetic CSIF gene with 9-base sticky
end a~ positions 36-44.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO~
.
30 CTTAAGAACA AGTTGTCCAG CTGATCCTTC ATTTGAAAGA AAGT 44
(2) INFORMATION FOR SEQ ID NO: 12:
:
~i) SEQUENCE CHARACTERISTICS: ~ -~
~:
WO93/1769X ~ 1 PCT/US93/01665
- 39 - :
(A) ~ENGTH: 69 base pairs .
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D~ TVPOLOGY; linear
(ix) FEATURE:
(D) OTHER INFORMATION: T at position 69 differs .
from that of the native sequence; together with SEQ ID
NO: 13, SEQ ID NO: 12 forms double-stranded Fragment 4A/B -
of synth~tic CSIF gene. -~:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
GAGTCCTTGC TGGAGGACTT TAAGGGTTAC CTGGGTTGCC AAGCCTTGTC 50
TGAGATGATC CAGTTTTAT 69
~"
(2) INFORMATION FOR SEQ ID NO: 13: :~
(i) SEQUENCE CHARACTERISTICS: ..
(A) LENGTH: 73 base pairs
2~
~B~ TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) IOPOLOGY: linea~
(ix) FEATURE:
(D) OTH~R INFORMATION: T and A a~ poC_tions 2 and
3~ 5 diffe- fro~ those of the native sea~ence; toqethe~ with .,~
WO 93/1 769X PCl /US93/(\ ~5
, ~
40 _ .;
SEQ ID NO: 12, SEQ ID NO: 13 forms double-stranded
Fragment 4A/B of synthetic CSIF gene with 4-base sticky
end at positions 1-4.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CTAGATAAAA CTGGATCATC TCAGACAAGG CTTGGCAACC CAGGTAACCC 50
TTAAAGTCCT CCAGCAAGGA CTC 73
: .
(2) INFORMATION FOR SEQ ID NO: 14: .;
`
ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs
(B) TYPE: nucleic acid -~
tC) STRANDEDNESS: single
(D) TOP010GY: linear -`
(ix) FEATURE:
~ D) OTHER INFORMATION: A, T and G at positions 3,
57 and 61 dif~er from those of the native sequence;
together with SEQ ID NO: 15, SEQ ID NO: 14 forms double-
stran~ed Fragment 5A/B of synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
CTAGAGGAGG TGATGCCCCA AGCTGAGAAC CAAGACCCAG ACATCAAGGC 5
GCATGTTAAC G 61
t2) INFORMATION FOR SEQ ID NO: 15: :~
(i) SEQUENCE CHARACTERISTICS:
',..
W093/17698 PCT/US93/01665
, ..... .'.. ~ ;,:
--41 --
(A) LENGTH: 65 base pairs ~:~
(B) TYPE: nucleic acid .~;
.;
(C) STRANDEDNESS: single
tD) TOPOLOGY: linear
(ix) FEATURE: ;;
(D) OTHER INFORMATION: TCGAC, A and T at positions
1-5, 9 and 63 differ from those of the native sequence;
together ~ith SEQ ID NO: 14, SEQ ID NO: 15 forms double~
1~ stranded Fragment SAtB of synthetic CSIF gene with 4-base
sticky end at positions 1-4.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: ~.
TCGACGTTAA CATGCGCCTT GATGTCTGGG TCTTGGTTCT CAGCTTGGGG 50
20 CATCACCTCC TCTAG 65
(2) INFORMATION FOR SEQ ID NO: 16: -
(i~ SEQUENCE CHAR~CTERISTICS:
(A) LENGTH: 63 base pairs ~
(B) TYPE: nucleic acid ~-
(C) STRANDEDNESS: single
(D) TOPOLOGY: linea~
(ix) F r ATURE~
3~ ..
WO93/17698 ..',~ PCT/US93/0
J ~-
- 42 - :
(D) OTHER INFORMATION: CTGCA at positions 58-63
differ from those of the native sequence; together with
SEQ ID NO: 17, SEQ ID NO: 16 forms double-stranded
Fragment 6A/B of synthetic CSIF gene with 4-base sticky
end at positions 59-63.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
AACTCCCTGG GGGAGAACCT GAAGACCCTC AGGCTGAGGC TACGGCGCTG 50
TCATCGATCT GCA 63
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear :
'~
(ix) FEATURE:
(D) OTHER INFORMATION: G at position 1 differs
from that of the native sequence; together with SEQ ID ~:
NO: 16, SEQ ID NO: 17 forms double-stranded Fragment 6A/B -
of synthetic CSIF gene.
txi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
GATCGATGAC AGCGCCGTAG CCTCAGCCTG AGGGTCTTCA GGTTCTCCCC 50
CAGGGAGTT $9
. .
t2) INFORMATION FOR SEQ ID NO: 18: -~
,,-
: ' '
W093/t7698 . ~ PCl~/US93/01665 : ~:
--43 --
( i ) SEQUENCE CHAR~C TER I ST I C S:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic a~id
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ix) FEATURE:
(D) OTHER INFORMATION: C, G and GCATG at positions
51, 54 and 56-60 differ from those of the native sequence;
together with SEQ ID NO: 19, SEQ ID NO: 18 forms double- :~
stranded Fra~ment 7A/B of synthetic CSIF gene with 2- and
4-base sticky ends at positions 1-2 and 57-60. ~:~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
CGATTTCTTC CCTGTCAAAA CAAGAGCAAG GCCGTGGAGC AGGTGAAGAA 50
CGCGTGCATG - 60
~2) INFORMATION FOR SEQ ID NO: 19:
~i) SEQUENCE CHARACTERISTICS: :~
' ~
~A) LENGTH: 54 base pairs
~B) TYPE: nucleic acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
WO93/1769X PCT/US93/0
~, ...
-44 -
(ix) FEATURE:
(D) OTHER INFORMATION: C, C and G at positions 1,
3 and 6 differ from those of the native sequence;
together with SEQ ID NO: 18, SEQ ID NO: 19 forms double~
stranded Fragment 7A/B of synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:.
CACGCGTTCT TCACCTGCTC CACGGCCTTG CTCTTGTTTT GACAGGGAAG 50
0 AAAT 54
~2) INFORMATION FOR SEQ ID NO: 20:
(i3 SEQUENCE CHARACTERISTICS:
:~
(A) LENGTH: 58 base pairs -:~
(B) TYPE: nucleic acid ~
(C~ STRANDEDNESS: single .
(D) TOPOLOGY: linear - - ~
(ix) FEATURE: -
2~ ;`.
(D) OTHER INFORM~TION: Together with SEQ ID NO: :
21, SEQ ID NO: 20 forms double-stranded Fragment 8A/B of
synthetic CSIF gene with 4- and 9-base sticky ends at
positions 1~4 and 50-58.
~xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 20:
CGCGTTTAAT AATAAGCTCC AAGACAAAGG CATCTACAAA GCCATGAGTG 50 `~
AGTTTGAC 58
' '
WO93/17698 ~ PCT/US93/0166~ :
- 45 -
(2) INFORMATION FOR SEQ ID NO: 21:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
: .,
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear ~ `
.' :
(ix) FEATURE:
~D) OTHER INFORMATION: Together with SEQ ID NO: .:
20, SEQ ID NO: 21 forms double-stranded Fxagment 8A/B of
synthetic CSIF gene.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
20 ACTCATGGCT TTGTAGATGC CTTTGTCTTC GAGCTTATTA TTAAA 45
(2) INFORMATION FOR SEQ ID NO: 22:
~i) SEQUENCE CHARACTERISTICS:
2~
(A) LENGTH: 51 base pairs
.
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linea~
(ix) FEATURE:
~.
WO 93/17698 , PCI`/l~S~3/01 ;
" '~, . `
46 -
(D3 OTHER INFORMATION: Together with SEQ ID NO:
23, SEQ ID NO: 22 forms double-stranded Fragment 9A/B of
synthetic CSIF gene.
5lxi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: ~;
ATCTTCATCA ACTACATAGA AGCCTACATG ACAATGAAGA TACGAAACTG A 51 ::;
(2) INFORMATION FOR SEQ ID NO: 23:
10(i) SEQUENCE CHARACTERISTICS:
: :
~A) LENGTH: 64 base pairs - -
(B) TYPE: nucleic acid
1 5
(C) STRANDEDNESS: double .;
(D) TOPOLOGY: linear
20(ix) FEATURE: ;:.
'
(D) OTHER INFORMATION: AGCT at positions 1-4
differ from those of the native sequence; together with
SEQ ID NO: 22/ SEQ ID NO: 23 forms double-stranded ~.
Fragment 9A/B of synthetic CSIF gene with 4- and 9-base
sticky ~nds at positions 1-4 and 56-64. ~ -
(xi) SEQUENCE DESCRIPTION: SFQ ID NC: 23:
AGCTTCAGTT TCGTATCTTC ATTGTCATGT AGGCTTCTAT GTAGTTGATG 50
30 AAGATGTCAA ACTC 64 :~
(2) INFORMATION FOR SEQ ID NO: 24: `
(i) SEQU~NCE CHARACTERISTICS: ;~
WO93/17698 .~ PCT/US93/01665
-47 -
(A) LENGTH: 519 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear ~.
(ix) FEATURE:
(D) OTHER INFORMATION: Encodes viral IL-10.
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
AATTC ATG GAG CGA AGG TTA GTG GTC ACT CTG CAG TGC ~TG GTG 44
CTG CTT TAC CTG GCA CCT GAG TGT GGA GGT ACA GAC CAA TGT 86
GAC AAT TTT CCC CAA ATG TTG AGG GAC CTA AGA GAT GCC TTC 128
AGT CGT GTT ~AA ACC TTT TTC CAG ACA ~AG GAC GAG GTA GAT 170
~AC CTT TTG CTC AAG GAG TCT CTG CTA GAG GAC TTT ~AG GGC 212
TAC CTT GGA TGC CAG GCC CTG TCA GAA ATG ATC CAA TTC TAC 254
CTG GAG GAA GTC ATG CCA CAG GCT GAA AAC CAG GAC CCG G~G 296
GCT AAG GAC CAT GTC AAT TCT TTG GGT GAA AAT CTA AAG ACC 338
CTA CGG CTC CGC CTG CGC AGG TGC CAC AGG TTC CTG CCG TGT 380
GAG A~C AAG AGT AAA GCT GTG GAA CAG ATA AAA AAT GCC TTT 422
2~ AAC AAG CTG CAG GAA ~AA GGA ATT TAC ~AA GCC ATG AGT GAA ~64
TTT GAC ATT TTT ATT AAC TAC ATA GAA GCA TAC ATG ACA ATT S06
AAA GCC AGG TGA G 519
