Active C-terminal peptides of interferon--gamma and their use

The subject invention concerns novel peptides of gamma interferon (IFN.gamma.) and methods of use of these peptides. Specifically exemplified are peptides from the C-terminus of IFN.gamma.. The subject peptides, once internalized into a cell, have biological activity which is comparable to the full-length IFN.gamma. protein.

BACKGROUND OF THE INVENTION 
Interferon gamma (IFN.gamma.) is a pleiotropic cytokine product of 
lymphocytes (subtype Th-1) and natural killer (NK) cells which plays a 
critical role in a variety of immunological functions (Farrar, M. A. et 
al., (1993). The cDNA and amino acid sequences for both murine and human 
IFN.gamma. have been determined (Gray, P. W. et al., 1983); Rinderknecht, 
E. et al., 1984). Both the murine and human IFN.gamma. proteins exist as 
homodimers that are biologically active. 
Among IFN.gamma.'s many effects are the induction of a number of antiviral 
proteins, upregulation of class II MHC expression, a role in B cell 
maturation, activation of cells to cytotoxic states, and release of 
mediators of inflammation (Johnson, H. M., 1985). Thus, IFN.gamma. plays 
an important role in host defense, inflammation and autoimmunity. These 
activities are induced as the IFN.gamma. molecule interacts in a 
species-specific manner with a single class of cell surface receptor and 
an associated cofactor molecule (Pestka, S. et al., 1987). In both mice 
and in humans, the IFN.gamma. receptor is a single chain glycoprotein of 
approximately 85-90 kD which has fairly large (&gt;200 amino acids) 
extracellular and cytoplasmic domains. 
An understanding of the structural basis for IFN.gamma. binding to its 
receptor provides insight into the mechanism by which ligand binding 
activates signal transduction. Murine IFN.gamma. has been shown to bind to 
a soluble form of its receptor via both the N-terminus and the C-terminus 
of the protein (Russell, J. K. et al., 1986; Magazine, H. I. et al., 1988; 
Griggs, N. D. et al., 1992). While the N-terminus of murine IFN.gamma. 
binds to the extracellular domain of the receptor (amino acid residues 
95-120 of the receptor) (VanVolkenburg, M. A. et al., 1993), the 
C-terminus of murine IFN.gamma. does not bind to this region of the 
receptor. A C-terminal peptide of murine IFN.gamma., consisting of amino 
acid residues 95-133, binds to the membrane proximal region of the 
cytoplasmic domain of the murine IFN.gamma. receptor (amino acid residues 
253-287) and human IFN.gamma. receptor (amino acid residues 252-291) 
(Szente, B. E. et al, 1994(a); Szente, B. E. et al., 1994(b)). Previous 
studies have shown that intracellular IFN.gamma. can induce an antiviral 
state and upregulation of MHC class II molecules in a species nonspecific 
fashion (Sanceau, J. et al., 1987; Smith, M. R. et al., 1990). 
Although it has been known that certain deletions or mutations of amino 
acids in the C-terminus of the IFN.gamma. molecule can diminish the 
biological activity of the protein, the discovery of peptides that 
comprise a portion of the C-terminus sequence of the full-length 
IFN.gamma. and that retain biological activity was unexpected. 
BRIEF SUMMARY OF THE INVENTION 
The subject invention concerns the discovery that a peptide fragment of 
human IFN.gamma., designated as huIFN.gamma. (95-134) (SEQ ID NO. 1), 
corresponding to amino acid residues 95-134 of mature full-length 
IFN.gamma., binds to the cytoplasmic domain of the IFN.gamma. receptor. 
The subject invention also concerns the murine counterpart, the peptide 
designated as muIFN.gamma. (95-133) (SEQ ID NO. 2), which also binds to 
the cytoplasmic domain of the IFN.gamma. receptor. The IFN.gamma. peptides 
of the subject invention bind to IFN.gamma. receptor protein in a species 
nonspecific manner. 
The subject invention further concerns the discovery that both the 
huIFN.gamma. (95-134) (SEQ ID NO. 1) and muIFN.gamma. (95-133) (SEQ ID NO. 
2) peptides have biological activity similar to the activity observed with 
full-length IFN.gamma.. Internalization of both murine and human 
IFN.gamma. C-terminal peptides by mouse macrophage cell lines, independent 
of the extracellular domain of the IFN.gamma. receptor, results in the 
induction of an antiviral state, as well as induction of MHC class II 
expression on the target cell. Thus, the subject invention also concerns 
the use of the peptides described herein as agonists of IFN.gamma. 
biological activity. The subject peptides can be used to treat a variety 
of clinically relevant disease states in animals and humans.

BRIEF DESCRIPTION OF THE SEQUENCES 
SEQ ID NO. 1 is an amino acid sequence of an IFN.gamma. peptide designated 
huIFN.gamma. (95-134). 
SEQ ID NO. 2 is an amino acid sequence of an IFN.gamma. peptide designated 
muIFN.gamma. (95-133). 
SEQ ID NO. 3 is an amino acid sequence of a peptide designated muIFN.gamma. 
(95-133)S. The sequence shown for SEQ ID NO. 3 is a scrambled version of 
SEQ ID NO. 2. 
SEQ ID NO. 4 is an amino acid sequence of an IFN.gamma. peptide designated 
muIFN.gamma. (95-125). 
SEQ ID NO. 5 is an amino acid sequence of an IFN.gamma. peptide designated 
muIFN.gamma. (1-39). 
SEQ ID NO. 6 is a polycationic amino acid sequence present in the 
C-terminal region of mouse IFN.gamma.. 
SEQ ID NO. 7 is a polycationic amino acid sequence present in the 
C-terminal region of human IFN.gamma.. 
SEQ ID NO. 8 is an amino acid sequence of mature, full-length human 
IFN.gamma.. 
SEQ ID NO. 9 is an amino acid sequence of mature, full-length murine 
IFN.gamma.. 
DETAILED DESCRIPTION OF THE INVENTION 
The subject invention pertains to agonist peptides of IFN.gamma.. These 
peptides are based on the amino acid sequence of the C-terminus region of 
the IFN.gamma. molecule and are capable of binding to the cytoplasmic 
domain of the IFN.gamma. receptor. Surprisingly, these peptides were found 
to possess the same or similar biological activity as that associated with 
the full-length, mature IFN.gamma. protein, even though these peptides do 
not bind to the extracellular domain of the IFN.gamma. receptor. Specific 
embodiments of the subject peptides have been shown to effect increased 
resistance to viral infection, as well as increased expression of MHC 
class II molecules on the target cell surface. Full-length IFN.gamma. is 
known to induce resistance to infection and increased MHC class II 
expressions in target cells. 
In a preferred embodiment, the huIFN.gamma. (95-134) peptide (SEQ ID NO. 1) 
based on human IFN.gamma. has an amino acid sequence corresponding to 
amino acid residues 95 through 134 of the full-length human IFN.gamma. 
protein (SEQ ID NO. 8). The muIFN.gamma. (95-133) peptide (SEQ ID NO. 2) 
based on murine IFN.gamma. has an amino acid sequence corresponding to 
amino acid residues 95 through 133 of the full-length murine IFN.gamma. 
protein (SEQ ID NO. 9). These peptides are shown in Table 1, along with 
the amino acid sequence of the peptides designated muIFN.gamma. (95-133)S 
(SEQ ID NO. 3), muIFN.gamma. (95-125) (SEQ ID NO. 4), and muIFN.gamma. 
(1-39) (SEQ ID NO. 5). The muIFN.gamma. (95-133)S (SEQ ID NO. 3) peptide 
is a scrambled version of the muIFN.gamma. (95-133) (SEQ ID NO. 2) 
peptide. The muIFN.gamma. (1-39) (SEQ ID NO. 5) and muIFN.gamma. (95-125) 
(SEQ ID NO. 4) have an amino acid sequence corresponding to amino acid 
residues 1 through 39 and 95 through 125 of the full-length murine 
IFN.gamma. protein (SEQ ID NO. 9), respectively. 
TABLE 1 
__________________________________________________________________________ 
Sequence of murine and human IFN.gamma. peptides 
PEPTIDE SEQUENCE 
__________________________________________________________________________ 
huIFN.gamma.(95-134) 
SEQ ID NO. 1: LTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQM 
muIFN.gamma.(95-133) 
SEQ ID NO. 2: AKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC 
muIFN.gamma.(95-133)S* 
SEQ ID NO. 3: PSCRENQNAVKIQKLSVVLRREQKHRVERLAFRNQSLPF 
muIFN.gamma.(95-125) 
SEQ ID NO. 4: AKFEVNNPQVQRQAFNELIRVVHQLLPESSL 
muIFN.gamma.(1-39) 
SEQ ID NO. 5: HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDG 
__________________________________________________________________________ 
The peptides shown in Table 1 were tested for their ability to increase 
viral resistance in a murine macrophage cell line, P388D.sub.1, using a 
standard viral yield reduction assay. In order to get the peptides 
internalized within the cytoplasm of a cell, macrophage cell lines were 
used because of their ability to nonspecifically endocytose material. The 
murine macrophage line P388D.sub.1 is known to be actively 
phagocytic/endocytic, and also responds well to IFN.gamma. treatment 
(Zlotnik, A. et al., 1983; Fairchild, R. L. et al., 1985; Steeg, P. S. et 
al., 1982). Both the muIFN.gamma. (95-133) (SEQ ID NO. 2) and the 
huIFN.gamma. (95-134) (SEQ ID NO. 1) peptides produced significant 
reductions in viral yield as shown in Table 2. None of the other peptides 
tested, including the muIFN.gamma. (95-133)S (SEQ ID NO. 3) peptide, were 
able to effect a significant reduction in virus yield. Thus, the peptides 
of the subject invention possess one of the biological activities of 
IFN.gamma.. 
TABLE 2 
______________________________________ 
IFN.gamma. peptide reduction of VSV yield 
Virus yield 
Peptide and concentration 
(PFU/ml).sup.a 
Fold reduction 
______________________________________ 
muIFN.gamma.(95-133) (SEQ ID NO. 2) 
&lt;10 &gt;8.0 .times. 10.sup.11 
100 .mu.M 
muIFN.gamma.(95-133) (SEQ ID NO. 2) 
6.0 .times. 10.sup.6.sup. 
1.3 .times. 10.sup.6 
25 .mu.M 
huIFN.gamma.(95-134) (SEQ ID NO. 1) 
1.0 .times. 10.sup.6.sup. 
8.0 .times. 10.sup.6 
100 .mu.M 
huIFN.gamma.(95-134) (SEQ ID NO. 1) 
1.0 .times. 10.sup.11 
8.0 
25 .mu.M 
muIFN.gamma.(95-133)S* (SEQ ID NO. 3) 
1.6 .times. 10.sup.10 
5.0 .times. 10.sup.2 
100 .mu.M 
muIFN.gamma.(95-133)S (SEQ ID NO. 3) 
6.0 .times. 10.sup.12 
1.3 
25 .mu.M 
muIFN.gamma.(95-125) (SEQ ID NO. 4) 
4.0 .times. 10.sup.12 
2.0 
100 .mu.M 
muIFN.gamma.(1-39) (SEQ ID NO. 5) 
8.0 .times. 10.sup.12 
0.0 
100 .mu.M 
muIFN.gamma. 200 U/ml (1.3 nM) 
1.2 .times. 10.sup.8.sup. 
6.7 .times. 10.sup.4 
muIFN.gamma. 50 U/ml (0.33 nM) 
1.6 .times. 10.sup.10 
5.0 .times. 10.sup.2 
Virus control 8.0 .times. 10.sup.12 
-- 
Cell control 0.0 -- 
______________________________________ 
.sup.a P388D.sub.1 cells were infected with VSV at a MOI = 0.5, and virus 
produced was harvested and assayed on L929 cells in a standard yield 
reduction assay (Weigent, D. A. et al., 1981). 
*Denotes a scrambled version of peptide muIFN.gamma.(95-133) (SEQ ID NO. 
2). 
MuIFN.gamma. (95-133) (SEQ ID NO. 2) showed no antiviral activity on murine 
L cells due to their lack of phagocytic activity. Also, supernatants from 
P388D.sub.1 cells treated with 100,.mu.M muIFN.gamma. (95-133) (SEQ ID NO. 
2) or huIFN.gamma. (95-134) (SEQ ID NO. 1) did not exhibit antiviral 
activity on L cells. Thus, the antiviral activity of IFN.gamma. C-terminal 
peptides is due to a direct intracellular effect of the peptides as 
opposed to the induction of interferon production in these cells. 
The peptides of the subject invention were also tested for their ability to 
induce increased MHC class II antigen expression on a target cell. 
P388D.sub.1 cells were treated with the peptides shown in Table 1 and then 
the level of cell surface Ia antigen (a murine MHC class II antigen) 
expression was determined by fluorescence-activated cell sorting (FACS) 
analysis. As shown in FIGS. 1(A)-1(F), cells treated with the muIFN.gamma. 
(95-133) (SEQ ID NO. 2) or huIFN.gamma. (95-134) (SEQ ID NO. 1) peptides 
exhibited a ten-fold increase in the level of Ia expression above that of 
untreated cells. The other peptides tested failed to significantly 
increase Ia expression on the treated cells. Thus, the peptides of the 
subject invention, once internalized within the cytoplasm of a cell, 
exhibit biological activity associated with IFN.gamma.. 
The discovery of peptide agonists of IFN.gamma. is highly unexpected. Use 
of synthetic peptide agonists rather than the full-length IFN.gamma. 
molecule offers advantages such as targeting of specific cells and immune 
system components. Also, specific amino acid residues of the peptides can 
be easily and rapidly modified to allow for generation of more effective 
agonists or antagonists. 
As those skilled in the art can readily appreciate, there can be a number 
of variant sequences of a protein found in nature, in addition to those 
variants that can be artificially created by the skilled artisan in the 
lab. The peptides of the subject invention encompasses those specifically 
exemplified herein, as well as any natural variants thereof, as well as 
any variants which can be created artificially, so long as those variants 
retain the desired biological activity. 
The peptides contemplated in the subject invention include the specific 
peptides exemplified herein as well as equivalent peptides which may be, 
for example, somewhat longer or shorter than the peptides exemplified 
herein. For example, using the teachings provided herein, a person skilled 
in the art could readily make peptides having from 1 to about 15 or more 
amino acids added to, or removed from, either end of the disclosed 
peptides using standard techniques known in the art. Preferably, any added 
amino acids would be the same as the corresponding amino acids of a mature 
full-length IFN.gamma. protein. The skilled artisan, having the benefit of 
the teachings disclosed in the subject application, could easily determine 
whether a variant peptide retained the biological activity of the specific 
peptides exemplified herein. Such a longer or shorter peptide would be 
within the scope of the subject invention as long as said peptide does not 
encompass the entire full-length IFN.gamma. protein and said longer or 
shorter peptide retains substantially the same relevant biological 
activity as the peptides exemplified herein. For example, a longer or 
shorter variant of the huIFN.gamma. (95-134) (SEQ ID NO. 1) peptide would 
fall within the scope of the subject invention if the variant peptide had 
the ability to induce MHC class II antigen expression or to increase 
cellular resistance to viral infection. 
Also within the scope of the subject invention are peptides which have the 
same amino acid sequences of a peptide exemplified herein except for amino 
acid substitutions, additions, or deletions within the sequence of the 
peptide, as long as these variant peptides retain substantially the same 
relevant biological activity as the peptides specifically exemplified 
herein. For example, conservative amino acid substitutions within a 
peptide which do not affect the ability of the peptide to, for example, 
induce the expression of MHC class II molecules would be within the scope 
of the subject invention. Thus, the peptides disclosed herein should be 
understood to include variants and fragments, as discussed above, of the 
specifically exemplified sequences. 
The subject invention further includes nucleotide sequences which encode 
the peptides disclosed herein. These nucleotide sequence can be readily 
constructed by those skilled in the art having the knowledge of the 
protein and peptide amino acid sequences which are presented herein. As 
would be appreciated by one skilled in the art, the degeneracy of the 
genetic code enables the artisan to construct a variety of nucleotide 
sequences that encode a particular peptide or protein. The choice of a 
particular nucleotide sequence could depend, for example, upon the codon 
usage of a particular expression system. 
The subject invention contemplates the use of the peptides described herein 
in pharmaceutical compositions for administration to an animal or human 
for the treatment of clinically important disease conditions that are 
amenable to treatment with full-length IFN.gamma.. For example, using the 
teachings described herein, the skilled artisan can use the subject 
invention to modulate or stimulate the immune response of an animal or 
human. Similarly, the subject peptides can be used to treat certain viral 
infections, as well as to treat certain forms of cancer or tumors. The 
peptides of the subject invention can be prepared in pharmaceutically 
acceptable carriers or diluents for administration to humans or animals in 
a physiologically tolerable form. Materials and methods for preparing such 
compositions are known in the art. 
The peptides of the subject invention can be administered using a variety 
of techniques that are known in the art. The peptides can be encapsulated 
in liposomes that are targeted to specific cells or tissues and the 
liposome-encapsulated peptides delivered to the cells or tissue either in 
vivo or ex vivo. Procedures for preparing liposomes and encapsulating 
compounds within the liposome are well known in the art. See, for example, 
U.S. Pat. No. 5,252,348, which issued to Schreier et al. Peptides could 
also be conjugated or attached to other molecules, such as an antibody, 
that targeted a specific cell or tissue. Peptides could also be 
administered using a drug delivery system similar to that described in 
U.S. Pat. No. 4,625,014, which issued to Senter et al. 
As described herein, the peptide sequences of the subject invention can 
also be the basis for producing peptides that act as IFN.gamma. 
antagonists. These antagonists are also within the scope of the subject 
invention. Inhibition or antagonism of IFN.gamma. function without agonist 
activity can be accomplished through the use of anti-peptide antibodies or 
modification of residues within the peptide itself. An especially 
productive means for generation of peptide antagonists has been 
substitution of L-amino acids with D-amino acids. The efficacy of this 
approach has been well characterized in the generation of arginine 
vasopressin analogs with selectively enhanced antidiuretic antagonism by 
appropriate substitution of L-amino acids with D-amino acids (Manning, M. 
et al., 1985). Further, not only can antagonism be produced with D-amino 
acid substitutions, but this antagonism can be directed toward a specific 
function. Production of potent antagonist peptides can be of value in 
specifically manipulating immune function. 
Peptide antagonists to IFN.gamma. can also be derived using those portions 
of the cytoplasmic domain of the IFN.gamma. receptor which bind to 
IFN.gamma. protein or the peptides disclosed herein. For example, a 
peptide comprising amino acid residues 253 through 287 of the murine 
IFN.gamma. receptor or amino acid residues 252 through 291 of the human 
IFN.gamma. receptor could be used as IFN.gamma. antagonists. These 
peptides from the IFN.gamma. receptor have been shown to bind to 
IFN.gamma. protein and to the huIFN.gamma. (95-134) (SEQ ID NO. 1) and 
muIFN.gamma. (95-133) (SEQ ID NO. 2) peptides of the subject invention 
(Szente, B. E. et al., 1994(a); Szente, B. E. et al., 1994(b)). 
A further aspect of the claimed invention is the use of the claimed 
peptides to produce antibodies, both polyclonal and monoclonal. These 
antibodies can be produced using standard procedures well known to those 
skilled in the art. These antibodies may be used as diagnostic and 
therapeutic reagents. For example, antibodies that bind to the 
huIFN.gamma. (95-134) (SEQ ID NO. 1) peptide may be used as an antagonist 
to block the function of IFN.gamma.. Antibodies that are reactive with the 
peptides of the subject invention can also be used to purify the 
IFN.gamma. protein or peptides from a crude mixture. 
The subject peptides can also be used in the design of new drugs that bind 
to the cytoplasmic domain of the IFN.gamma. receptor. Knowledge of peptide 
sequences that induce IFN.gamma. biological activity upon binding of the 
peptide to a localized region on the IFN.gamma. receptor enables a skilled 
artisan to develop additional bioactive compounds using rational drug 
design techniques. Thus, the skilled artisan can prepare both agonist and 
antagonist drugs using the teachings described herein. 
Materials and Methods 
Synthetic peptides 
Peptides corresponding to portions of the IFN.gamma. protein were 
synthesized with a Biosearch 9500AT automated peptide synthesizer 
(Milligen/Biosearch, Burlington, Mass.) using 9-fluoroenylmethyl 
oxycarbonyl chemistry (Chang, C. D. et al., 1978). Peptides were cleaved 
from the resins using trifluoroacetic 
acid/ethanedithiol/thioanisole/crystalline phenol/distilled water at a 
ratio of 80/3/5/7/5. The cleaved peptides were then extracted in ether and 
ethyl acetate and subsequently dissolved in water and lyophilized. Reverse 
phase HPLC analysis of the crude peptides revealed one major peak in each 
profile. Amino acid analysis of the peptides showed that the amino acid 
composition corresponded closely to theoretical values. 
The one-letter symbol for the amino acids used in the sequences shown in 
the Tables is well known in the art. For convenience, the relationship of 
the three-letter abbreviation and the one-letter symbol for amino acids is 
as follows: 
______________________________________ 
Ala A 
Arg R 
Asn N 
Asp D 
Cys C 
Gln Q 
Glu E 
Gly G 
His H 
lle I 
Leu L 
Lys K 
Met M 
Phe F 
Pro P 
Ser S 
Thr T 
Trp W 
Tyr Y 
Val V 
______________________________________ 
Yield Reduction Assay 
5.times.10.sup.5 P388D.sub.1 cells were incubated with peptides at a 
concentration of 100 .mu.M in maintenance medium (medium containing 2% 
serum) for 18 to 24 hours. Cells were then rinsed 3 times with medium 
containing no peptide. Vesicular stomatitis virus (VSV) was added at a 
Multiplicity of Infection (MOI) of either 0.1 or 0.5 as indicated for a 45 
minute incubation. Cells were again washed 3 times. Fresh maintenance 
medium was added and the cells were incubated overnight at 37.degree. C. 
Virus was harvested and titered on L929 cells using a standard yield 
reduction assay (Weigent, D. A. et al., 1981). 
Induction of Ia expression 
10.sup.5 P388D.sub.1 cells were incubated for 24 hours with IFN.gamma. 
peptides under standard culture conditions, all at a final concentration 
of 100 .mu.M, as follows: (A) medium alone, (B) muIFN.gamma. (95-133) (SEQ 
ID NO. 2), (C) huIFN.gamma. (95-134) (SEQ ID NO. 1), (D) muIFN.gamma. 
(95-133)S (SEQ ID NO. 3), (E) muIFN.gamma. (95-125) (SEQ ID NO. 4) and (F) 
muIFN.gamma. (1-39) (SEQ ID NO. 5). After the time period, the cells were 
physically dislodged from culture plates, washed with FACS buffer 
(PBS/0.5% BSA+10 mM NaN.sub.3), and incubated with the biotinylated 
anti-Ia monoclonal antibody MKD6 (12), for 1 hour at 37.degree. C. Cells 
were then washed and incubated with streptavidin-phycoerythrin (TAGO Inc., 
Burlingame, Calif.) for 15 minutes at room temperature. FACS Analysis was 
performed on a FACScan (Becton-Dickinson, Mountain View, Calif.) as 10,000 
events per sample. 
Following are examples which illustrate procedures, including the best 
mode, for practicing the invention. These examples should not be construed 
as limiting. All percentages are by weight and all solvent mixture 
proportions are by volume unless otherwise noted. 
EXAMPLE 1 
Reduction of Viral Yield 
The peptides of the subject invention were tested to determine whether they 
could function as agonists of IFN.gamma. biological activity. The ability 
of endocytosed murine and human C-terminal IFN.gamma. peptides of the 
subject invention to induce an antiviral response in P388D.sub.1 cells was 
shown using a standard yield reduction assay. The muIFN.gamma. (95-133) 
peptide (SEQ ID NO. 2) at a concentration of 100 .mu.M reduced virus yield 
by a factor of greater than 8.0.times.10.sup.11 (Table 2). At a 
concentration of 25 .mu.M, the same peptide reduced the virus yield by a 
factor of 1.3.times.10.sup.6. A scrambled version of this peptide, 
muIFN.gamma. (95-133)S (SEQ ID NO. 3) was more than 10.sup.9 -fold less 
effective in inhibiting virus yield at a concentration of 100 .mu.M, and 
had no effect on virus replication at a concentration of 25 .mu.M. 
In addition to testing a scrambled version of muIFN.gamma. (95-133) (SEQ ID 
NO. 2), the effect of a truncated form of the peptide, muIFN.gamma. 
(95-125) (SEQ ID NO. 4), which lacks the polycationic amino acid sequence 
RKRKR (SEQ ID NO. 6) was also tested for biological activity. Human 
IFN.gamma. also has a polycationic sequence, KRKR (SEQ ID NO. 7), in the 
C-terminus portion of the protein. The RKRKR (SEQ ID NO. 6) sequence has 
previously been shown to be required for antiviral activity of the 
IFN.gamma. molecule (Russell, J. K. et al., 1986; Leinikki, P. O. et al., 
1987; Seelig, G. F. et al., 1988; Wetzel, R. et al., 1990; Lundell, D. et 
al., 1991). The muIFN.gamma. (95-125) (SEQ ID NO. 4) peptide did not 
inhibit virus replication. The N-terminal peptide of murine IFN.gamma., 
muIFN.gamma. (1-39) (SEQ ID NO. 5), which binds to the extracellular 
domain of the murine receptor, was also ineffective in inhibiting virus 
replication. The human C-terminal peptide, huIFN.gamma. (95-134) (SEQ ID 
NO. 1), similar to its murine counterpart, produced a significant 
reduction in virus yield (8.0.times.10.sup.6 fold at 100 .mu.M). Murine 
IFN.gamma. at 1.3 nM final concentration inhibited virus yield by 
approximately 7.0.times.10.sup.4 fold on P388D.sub.1 cells. Human 
IFN.gamma. at a similar concentration had no effect. 
EXAMPLE 2 
Induction of MHC Class II Expression 
A second parameter of the agonist properties of the IFN.gamma. peptides of 
the subject invention is their ability to increase the level of expression 
of MHC class II molecules on cells. As shown in FIGS. 1(A)-1(F), treatment 
of the P388D.sub.1 cell line with either the muIFN.gamma. (95-133) (SEQ ID 
NO. 2) or huIFN.gamma. (95-134) (SEQ ID NO. 1) peptide resulted in an 
increase in Ia expression tenfold greater than that of untreated cells. 
This effect is specific to the IFN.gamma. C-terminal peptides with the 
intact polycationic region (RKRKR or KRKR), as cells treated with either 
the truncated version of the murine C-terminal peptide, muIFN.gamma. 
(95-125) (SEQ ID NO. 4), or the scrambled version, muIFN.gamma. (95-133)S 
(SEQ ID NO. 3), failed to upregulate Ia expression. Similar effects of 
C-terminal IFN.gamma. peptides of the subject invention were observed 
using WEHI-3 cells. The time course of induction of Ia expression by the 
subject peptides is more rapid than that induced by intact IFN.gamma., 
with optimal results being achieved by 24 hours. Whereas Ia levels are 
significantly increased within 24 hours of treatment with 100 .mu.M 
IFN.gamma. C-terminal peptide, IFN.gamma. itself requires 2 to 3 days to 
induce similar expression (Steeg, P. S. et al., 1982). This is most likely 
due to the time required for receptor-dependent internalization and 
intracellular processing of the intact IFN.gamma.. The peptides of the 
subject invention are delivered, in effect, already processed, and are 
taken up via a receptor-independent endocytic mechanism. Thus, the 
C-terminal peptides of murine and human IFN.gamma. are potent modulators 
not only of antiviral responses, but also of MHC class II molecule 
expression. 
It should be understood that the examples and embodiments described herein 
are for illustrative purposes only and that various modifications or 
changes in light thereof will be suggested to persons skilled in the art 
and are to be included within the spirit and purview of this application 
and the scope of the appended claims. All references to publications and 
patents cited herein are hereby incorporated by reference. 
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__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 9 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
LeuThrAsnTyrSerValThrAspLeuAsnValGlnArgLysAlaIle 
151015 
HisGluLeuIleGlnValMetAlaGluLeuSerProAlaAlaLysThr 
202530 
GlyLysArgLysArgSerGlnMet 
3540 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 39 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
AlaLysPheGluValAsnAsnProGlnValGlnArgGlnAlaPheAsn 
151015 
GluLeuIleArgValValHisGlnLeuLeuProGluSerSerLeuArg 
202530 
LysArgLysArgSerArgCys 
35 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 39 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ProSerCysArgGluAsnGlnAsnAlaValLysIleGlnLysLeuSer 
151015 
ValValLeuArgArgGluGlnLysHisArgValGluArgLeuAlaPhe 
202530 
ArgAsnGlnSerLeuProPhe 
35 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
AlaLysPheGluValAsnAsnProGlnValGlnArgGlnAlaPheAsn 
151015 
GluLeuIleArgValValHisGlnLeuLeuProGluSerSerLeu 
202530 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 39 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
HisGlyThrValIleGluSerLeuGluSerLeuAsnAsnTyrPheAsn 
151015 
SerSerGlyIleAspValGluGluLysSerLeuPheLeuAspIleTrp 
202530 
ArgAsnTrpGlnLysAspGly 
35 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
ArgLysArgLysArg 
15 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
ArgLysArgLys 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 143 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
GlnAspProTyrValLysGluAlaGluAsnLeuLysLysTyrPheAsn 
151015 
AlaGlyHisSerAspValAlaAspAsnGlyThrLeuPheLeuGlyIle 
202530 
LeuLysAsnTrpLysGluGluSerAspArgLysIleMetGlnSerGln 
354045 
IleValSerPheTyrPheLysLeuPheLysAsnPheLysAspAspGln 
505560 
SerIleGlnLysSerValGluThrIleLysGluAspMetAsnValLys 
65707580 
PhePheAsnSerAsnLysLysLysArgAspAspPheGluLysLeuThr 
859095 
AsnTyrSerValThrAspLeuAsnValGlnArgLysAlaIleHisGlu 
100105110 
LeuIleGlnValMetAlaGluLeuSerProAlaAlaLysThrGlyLys 
115120125 
ArgLysArgSerGlnMetLeuPheArgGlyArgArgAlaSerGln 
130135140 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 133 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
HisGlyThrValIleGluSerLeuGluSerLeuAsnAsnTyrPheAsn 
151015 
SerSerGlyIleAspValGluGluLysSerLeuPheLeuAspIleTrp 
202530 
ArgAsnTrpGlnLysAspGlyAspMetLysIleLeuGlnSerGlnIle 
354045 
IleSerPheTyrLeuArgLeuPheGluValLeuLysAspAsnGlnAla 
505560 
IleSerAsnAsnIleSerValIleGluSerHisLeuIleThrThrPhe 
65707580 
PheSerAsnSerLysAlaLysLysAspAlaPheMetSerIleAlaLys 
859095 
PheGluValAsnAsnProGlnValGlnArgGlnAlaPheAsnGluLeu 
100105110 
IleArgValValHisGlnLeuLeuProGluSerSerLeuArgLysArg 
115120125 
LysArgSerArgCys 
130 
__________________________________________________________________________