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

Abstract

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.

Description

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 (>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 DRAWINGS

FIGS. 1A-1F show IFN.gamma. C-terminal peptide induction of Ia expression on the murine macrophage line P388D.sub.1. Cells were incubated for 24 hours with IFN.gamma. peptides at a final concentration of 100 .mu.M as follows: (1A) medium alone, (1B) muIFN.gamma. (95-133) (SEQ ID NO. 2), (1C) huIFN.gamma. (95-134) (SEQ ID NO. 1), (1D) muIFN.gamma. (95-133)S (SEQ ID NO. 3), (1E) muIFN.gamma. (95-125) (SEQ ID NO. 4), and (1F) muIFN.gamma. (1-39) (SEQ ID NO. 5).

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. peptidesPEPTIDE SEQUENCE__________________________________________________________________________huIFN.gamma.(95-134) SEQ ID NO. 1: LTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMmuIFN.gamma.(95-133) SEQ ID NO. 2: AKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRCmuIFN.gamma.(95-133)S* SEQ ID NO. 3: PSCRENQNAVKIQKLSVVLRREQKHRVERLAFRNQSLPFmuIFN.gamma.(95-125) SEQ ID NO. 4: AKFEVNNPQVQRQAFNELIRVVHQLLPESSLmuIFN.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 yieldPeptide and concentration (PFU/ml).sup.a Fold reduction______________________________________muIFN.gamma.(95-133) (SEQ ID NO. 2) <10 >8.0 .times. 10.sup.11100 .mu.MmuIFN.gamma.(95-133) (SEQ ID NO. 2) 6.0 .times. 10.sup.6.sup. 1.3 .times. 10.sup.625 .mu.MhuIFN.gamma.(95-134) (SEQ ID NO. 1) 1.0 .times. 10.sup.6.sup. 8.0 .times. 10.sup.6100 .mu.MhuIFN.gamma.(95-134) (SEQ ID NO. 1) 1.0 .times. 10.sup.11 8.025 .mu.MmuIFN.gamma.(95-133)S* (SEQ ID NO. 3) 1.6 .times. 10.sup.10 5.0 .times. 10.sup.2100 .mu.MmuIFN.gamma.(95-133)S (SEQ ID NO. 3) 6.0 .times. 10.sup.12 1.325 .mu.MmuIFN.gamma.(95-125) (SEQ ID NO. 4) 4.0 .times. 10.sup.12 2.0100 .mu.MmuIFN.gamma.(1-39) (SEQ ID NO. 5) 8.0 .times. 10.sup.12 0.0100 .mu.MmuIFN.gamma. 200 U/ml (1.3 nM) 1.2 .times. 10.sup.8.sup. 6.7 .times. 10.sup.4muIFN.gamma. 50 U/ml (0.33 nM) 1.6 .times. 10.sup.10 5.0 .times. 10.sup.2Virus 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.

References

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Immunol. 131:2814-2820. __________________________________________________________________________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:LeuThrAsnTyrSerValThrAspLeuAsnValGlnArgLysAlaIle151015HisGluLeuIleGlnValMetAlaGluLeuSerProAlaAlaLysThr202530GlyLysArgLysArgSerGlnMet3540(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:AlaLysPheGluValAsnAsnProGlnValGlnArgGlnAlaPheAsn151015GluLeuIleArgValValHisGlnLeuLeuProGluSerSerLeuArg202530LysArgLysArgSerArgCys35(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:ProSerCysArgGluAsnGlnAsnAlaValLysIleGlnLysLeuSer151015ValValLeuArgArgGluGlnLysHisArgValGluArgLeuAlaPhe202530ArgAsnGlnSerLeuProPhe35(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:AlaLysPheGluValAsnAsnProGlnValGlnArgGlnAlaPheAsn151015GluLeuIleArgValValHisGlnLeuLeuProGluSerSerLeu202530(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:HisGlyThrValIleGluSerLeuGluSerLeuAsnAsnTyrPheAsn151015SerSerGlyIleAspValGluGluLysSerLeuPheLeuAspIleTrp202530ArgAsnTrpGlnLysAspGly35(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:ArgLysArgLysArg15(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:GlnAspProTyrValLysGluAlaGluAsnLeuLysLysTyrPheAsn151015AlaGlyHisSerAspValAlaAspAsnGlyThrLeuPheLeuGlyIle202530LeuLysAsnTrpLysGluGluSerAspArgLysIleMetGlnSerGln354045IleValSerPheTyrPheLysLeuPheLysAsnPheLysAspAspGln505560SerIleGlnLysSerValGluThrIleLysGluAspMetAsnValLys65707580PhePheAsnSerAsnLysLysLysArgAspAspPheGluLysLeuThr859095AsnTyrSerValThrAspLeuAsnValGlnArgLysAlaIleHisGlu100105110LeuIleGlnValMetAlaGluLeuSerProAlaAlaLysThrGlyLys115120125ArgLysArgSerGlnMetLeuPheArgGlyArgArgAlaSerGln130135140(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:HisGlyThrValIleGluSerLeuGluSerLeuAsnAsnTyrPheAsn151015SerSerGlyIleAspValGluGluLysSerLeuPheLeuAspIleTrp202530ArgAsnTrpGlnLysAspGlyAspMetLysIleLeuGlnSerGlnIle354045IleSerPheTyrLeuArgLeuPheGluValLeuLysAspAsnGlnAla505560IleSerAsnAsnIleSerValIleGluSerHisLeuIleThrThrPhe65707580PheSerAsnSerLysAlaLysLysAspAlaPheMetSerIleAlaLys859095PheGluValAsnAsnProGlnValGlnArgGlnAlaPheAsnGluLeu100105110IleArgValValHisGlnLeuLeuProGluSerSerLeuArgLysArg115120125LysArgSerArgCys130__________________________________________________________________________

Claims

1. A peptide having an amino acid sequence consisting of the following components:

optionally, an N-terminal amino acid residue or sequence of residues of from 2 to about 15 amino acids;

the sequence of a fragment of a mammalian interferon-gamma protein other than murine interferon-gamma, the fragment having about 39 or 40 amino acid residues and corresponding to the C-terminal of the interferon protein, or a sequence which differs therefrom by the deletion of up to 15 amino acid residues from the N-terminus, the C-terminus, or both; and

optionally, a C-terminal amino acid residue or sequence of residues of from 2 to about 15 amino acids,

wherein the peptide binds to the cytoplasmic domain of the interferon-gamma receptor, induces antiviral activity, or induces MHC class II antigen expression on a target cell, and wherein a peptide having only the amino acid sequence of said fragment binds to the cytoplasmic domain of the interferon-gamma receptor, induces antiviral activity, or induces MHC class II antigen expression on a target cell.

2. A composition comprising the peptide according to claim 1 encapsulated in a liposome.

3. A method for modulating an immune response in a subject, comprising the step of administering to the subject an amount of a peptide according to claim 1 effective to induce antiviral activity or to induce MHC class II antigen expression on a target cell.

4. A method according to claim 3, wherein the mammalian interferon-gamma protein is human interferon-gamma.

5. A method according to claim 4, wherein the fragment has the amino acid sequence shown as SEQ ID NO: 1.

6. A method according to claim 5, wherein the peptide has the amino acid sequence shown as SEQ ID NO: 1.

7. A method for modulating an immune response in a subject, comprising the step of administering to the subject an amount of a composition according to claim 2 effective to induce antiviral activity or to induce MHC class II antigen expression on a target cell.

8. A method according to claim 7, wherein the mammalian interferon-gamma protein is human interferon-gamma.

9. A method according to claim 8, wherein the fragment has the amino acid sequence shown as SEQ ID NO: 1.

10. A method according to claim 9, wherein the peptide has the amino acid sequence shown as SEQ ID NO: 1.

11. A peptide having an amino acid sequence consisting of the following components:

optionally, an N-terminal amino acid residue or sequence of residues of from 2 to about 15 amino acids;

a fragment of the sequence of human interferon-gamma from about amino acid residue 95 to about amino acid residue 134; and

optionally, a C-terminal amino acid residue or sequence of residues of from 2 to about 15 amino acids,

wherein the peptide binds to the cytoplasmic domain of the interferon-gamma receptor, induces antiviral activity, or induces MHC class II antigen expression on a target cell.

12. The peptide according to claim 11, wherein said N- and C-terminal amino acid residues are the same as the amino acid residues immediately adjacent to said fragment within the sequence of human interferon-gamma.

13. The peptide according to claim 11, consisting of an amino acid sequence shown as SEQ ID NO: 1.

14. A pharmaceutical composition comprising an antiviral effective amount of the peptide of claim 11 and a pharmaceutically acceptable carrier or diluent.

15. A composition comprising the peptide according to claim 11, encapsulated in a liposome.

16. A composition comprising a peptide encapsulated in a liposome, wherein said peptide has an amino acid sequence consisting of the following components:

optionally, an N-terminal amino acid residue or sequence of residues of from 2 to about 15 amino acids;

a fragment of the sequence of human or murine interferon-gamma selected from the group consisting of:

a) a sequence from about amino acid residue 95 to about amino acid residue 134 of human interferon-gamma or a sequence which differs therefrom by the deletion of up to 15 amino acid residues from the N-terminus, the C-terminus, or both, and

b) a sequence from about amino acid residue 95 to about amino acid residue 133 of murine interferon-gamma or a sequence which differs therefrom by the deletion of up to 15 amino acid residues from the N-terminus, the C-terminus, or both; and

optionally, a C-terminal amino acid residue or sequence of residues of from 2 to about 15 amino acids,

wherein the peptide binds to the cytoplasmic domain of the interferon-gamma receptor, induces antiviral activity, or induces MHC class II antigen expression on a target cell, and wherein a peptide having only the amino acid sequence of said fragment binds to the cytoplasmic domain of the interferon-gamma receptor, induces antiviral activity, or induces MHC class II antigen expression on a target cell.

17. The composition according to claim 16, wherein said fragment consists of an amino acid sequence from about amino residue 95 to about amino acid residue 134 of human interferon-gamma, and wherein said N- and C-terminal amino acid residues are the same as the amino acid residues immediately adjacent to said fragment within the sequence of human interferon-gamma.

18. The composition according to claim 16, wherein said fragment consists of an amino acid sequence from about amino residue 95 to about amino acid residue 133 of murine interferon-gamma, and wherein said N- and C-terminal amino acid residues are the same as the amino acid residues immediately adjacent to said fragment within the sequence of murine interferon-gamma.

19. The composition according to claim 16, wherein the peptide consists of an amino acid sequence selected from the group consisting of the amino acid sequences shown as SEQ ID NO: 1 and SEQ ID NO: 2.