AU727738B2 - Compositions and methods for eliciting CTL immunity - Google Patents

Description

r

I

S F Ref: 350274D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFCATION FOR A STANDARD PATENT

ORIGINAL

*.jt Name and Address of Applicant: Epimmune Inc.

6555 Nancy Ridge Drive Suite 200 San Diego California 92121 UNITED STATES OF AMERICA Maria A Vitiello, Robert W Esteban Cells, Howard Grey Actual Inventor(s): Chesnut, Alessandro D Sette, Address for Service: Invention Title: Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Compositions and Methods for Eliciting CTL Immunity The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845 1 Compositions and Methods for Eliciting CTL Immunity Cytotoxic T lymphocytes represent an important component of an animal's immune response against a variety of pathogens and cancers. CTL which have been specifically activated against a particular antigen are capable of killing the cell that contains or expresses the antigen. CTL are particularly important in providing an effective immune response against intracellular pathogens, such as a wide variety of viruses, and some bacteria and parasites. CTL responses are also believed to be capable of contributing to anti-tumor responses in afflicted or susceptible individuals.

The receptors on the surface of the CTL cannot recognize a foreign antigen directly, however. The CTL express an ca-P heterodimeric T cell receptor which is capable of recognizing foreign antigen fragments bound to major histocompatibility coInplex (MHC) class I molecules on the surface of the effected infected) cells. CTL also express the non-polymorphic CD8 antigen. This cell surface protein interacts with the third domain of the class I molecule on the antigen presenting cells [R:\LIBU]00227.doc:mcc 2 and plays a role in both stabilizing the interaction between the CTL and the antigen presenting cell and in CTL activation (Salter et al., Nature 345:41-46 (1990)).

There are a number of mechanisms by which CTL are thought to disrupt the infectious or tumorigenic process. Among these, one involves the production of lymphokines such as gamma interferon (IFNy) and tumor necrosis factor alpha (TNFa) which are known to act directly on infected cells to inhibit viral replication (Gilles et al., J. Virol.0 66:3955-3960 (1992)). In addition, IFNT causes increased expression of MHC class

I

molecules on the surface of virus infected cells and enhances their ability to be recognized by CTL and trigger immune intervention (Hayata et al. Hepatology 13: 1022-1028 (1991)) A second mechanism by which CTL combat infections or tumors is through direct killing of the afflicted cell, e.g. .those which are infected bythe tagtdvirus (oe ta.

bnn. Rev. Imunol. 10: 267-293 (1992) and Henkart et al, Ann±.

:Rev. Immunol. 3:31-58 (1985)). For example, since viruses must *.:replicate within the host cell the lysis of infected cells destroys virus production prior to the liberation of infectious *:.particles.. The exact mehns~)by which CTL kill infected target cells remains unclear. Once CTL recognized an antigen presenting cell, close contact between the cells is established over a large surface area. A "direct hit" is then delivered by translocating enzymes present in cytoplasmic vacuoles of CTL to the antigen presenting cell, which enzy-mes kill the cell or perhaps induce programmed cell death, "apoptosis". once

CTL

have delivered their "lethal hit" to the antigen presenting :cells, they can detach and go on to kill other antigen presenting cells through repetition of the antigen-specific recognition, lymphokine release and target cell killing mechanisms.

The means by which CTL distinguish infected from noninfected cells is through the T cell receptor and its ability to specifically recognize a peptide fragment of viral protein that 3 is bound to the peptide-binding cleft of the MHC class I molecule (Monaco et al., Immunol. Today 13:173-179 (1992) and Townsend et al., Ann. Rev. Immunol. 7:601-624 (1989)). Several viral fragments that can serve an antigenic peptides have been identified.

The biochemical events that take place in the cytoplasm of infected cells leading to CTL recognition are termed antigen processing and presentation. While not completely defined, it seems clear that during the synthesis and assembly of the infecting viral or bacterial proteins, some proteolysis takes place in the cytoplasm (Monaco et al., Immunol. Today 13:173-179 (1992)). Structures called proteosomes cleave the foreign proteins into peptide fragments. These fragments are then transported into the endoplasmic reticulum (ER) by means of 15 specific transporter proteins where newly synthesized MHC class SI molecules are present. Those peptides that are capable of specifically binding to a given MHC class I molecule do so in the ER. The non-polymorphic class I 0 chain, 02 microglobulin binds to the antigenic peptide-class I complex, thus forming a stable trimolecular complex that is transported to the cell surface and displayed as an integral membrane component.

The selection of which peptides bind to a particular MHC class I molecule is based on the ability of the peptide to bind within the binding pocket or cleft which resides at the outermost apex of the extra-cellular portion of the MHC molecule. For several MHC molecules, this peptide binding pocket has been precisely defined by X-ray crystallographic procedures allowing a visualization of the types and location of the chemical bonds that form to stabilize the interaction (Saper et al., J. Mol. Biol. 219:277-319 (1991)).

Because of the differences in the structure of the peptide binding pocket between the diverse set of histocompatibility alleles, the human HLA alleles, a distinct population of antigenic peptides is bound by each allele, although in some cases the population of antigenic peptides may overlap for closely related alleles. Thus, the specificity of the CTL for a foreign antigen resides at the level of the ability of MHC class I molecules to bind to a specific peptide as well as for the T cell receptor on the CTL to recognize the foreign protein fragment bound to that specific MHC class I allele.

In animals, CT8+, MHC class I-restricted cytotoxic

T

cells play an important role in the immune mediated clearance of viral infections Oldstone et al., Nature 321:239-243 (1986); Mackenzie et al., Immunol. 67:375 (1989); and Robertson et al., J. Virol. 66:3271-3277 (1992)). While similar studies have not been possible in humans, and thus direct proof is still lacking, all of the evidence points to a similar role for CTL.

1 The importance of CTL in viral clearance in animals is evidenced by lymphocytic choriomeningitis virus (LCMV) infection in mice (Oldstone et al., ature 321:239-243 (1986); Mackenzie et al., Immunol 67:375 (1989); Robertson et al., Virol.

66:3271-3277 (1992); and Ahmed et al., J. Virol 61:3920-3929 (1987)). When LCMV infects newborns or immune-suppressed adult animals, they become chronically infected and virus is expressed in nearly all tissues of the body. In contrast, adult mice infected with LCMV mount a vigorous cellular and humoral response against the virus and clear the infection within one to two weeks. When chronic carriers of LCMV are adoptively treated by transfer of CD8+LCMV-specific, MHC class I-restricted

CTL,

the viral infection is cleared and the mice become resistant to subsequent LCMV challenge. Additional studies have shown that "CTL are necessary and sufficient for LCMV clearance and that other aspects of the immune system need not be functioning (Oldstone et al., Nature 321:239-243 (1986) and Schulz et al.

Proc. Natl. Acad. Sci. USA 88:991-993 (1991)).

In addition to mediating the clearance of virus from chronically infected animals, studies have demonstrated that CTL generated in vivo against a synthetic peptide which presents an antigenic epitope of LCMV are able to protect mice against acute infection (Schulz et al., Proc. Nati. Acad. Sci. USA 88:991-993 (1991)). Mice injected with 100 pg of a synthetic 15 amino acid peptide in complete Freund's adjuvant were fully protected from a lethal LCMV challenge.

With regard to the role of CTL in other viral infections, studies with influenza virus and respiratory syncytial virus in mice have similarly demonstrated the importance of CTL activation in the rapid and effective recovery from these infections.

Strong evidence from animal studies indicates that an acute infection can become chronic when there is an inadequate immune response to clear the infection (Ahmed, Concepts in Viral Pathogenesis III, Notkins and Oldstone eds., Springer-Verlag New York, 304-310 (1989)). Once the chronic infection has been 15 established, it appears to be more or less "tolerated" by the host's immune system. Tolerance appears to be organism-specific rather than a result of general immunosuppression (Fields et Sal., Fields Virology, Raven Press, New York, NY 2:2137-2236 S(1990)). Studies examining which cells in the immune system are anergic or tolerant to the infecting organism suggest that the CD4+, class II-restricted T "helper" cells are dysfunctional S: (Schwartz, Cell 57:1073-1081 (1989)). Since class II-restricted T helper cells play a critical role in the initial priming of class I-restricted CTL (Cassell et al., Ann. NYAcad Sci.

532:51-60 (1988) and Fayolle et al., J. Immunol. 174:4069 (1991)), diminished CD4 cell function may impair the capacity of the immune system to respond adequately, and may thus clear the way for chronic infection.

Decreased T helper cell activity has been shown in the case of chronic hepatitis B infection in humans, although the fact that some CD4+ T helper function is seen suggests that these cells are not completely dysfunctional (see, Ahmed et al., J. Virol. 61:3920-3929 (1987); Alberti et al., Lancet 1:1421-1424 (1988); Neurath et al., Nature 315:154-156 (1985); Celis et al., J. Immunol. 132:1511-1516 (1984); and Ferrari et 6 al., J. Immunol. 139:2050-2055 (1987)). Class I-restricted

CTL

can be detected in patients with chronic HBV infection (Barnaba et al., J. Immunol 143:2650-2654 (1989)).

The requirement for lymphokines such as IL-2 in the generation of CD8+ CTL is well established, although the need for activation of CD4+ T helper cells to provide these lymphokines remains somewhat controversial. While the concept of linked T helper-B cell recognition for antibody production has been firmly defined, there is no compelling evidence for linked T helper-CTL recognition for the in vivo induction of CD8+ CTL. See, Reg., Buller et al., Nature 328:77-79 (1987); Sarobe et al., Eur. J. Imunol. 21:1555-1558 (1991); and Cassell and Forman, Annals N.Y. Acad. Sci. :51-60 (1991).

Thus, the data available suggest that CD8+ class I- 15 restricted cytotoxic T cells specific for foreign antigens such 006. as viral proteins play a critical part in prevention of disease o and clearance of an established disease process. Therefore, the challenge is to induce a sufficiently potent, antigen-specific, *cell-mediated immune response in humans and other mammals which, by itself or in conjunction with chemotherapeutic agents or the like, will either prevent a disease process such as an infection or tumor from becoming established, or will eliminate or at least ameliorate an infection or tumor which has already become established in the host. Quite surprisingly, the present 25 invention fulfills these and other related needs.

Summnary of the Invention S The present invention provides compositions for inducing a cytotoxic T lymphocyte response to an antigen of interest in a mammal. The compositions comprise a peptide that induces a CTL response to the antigen and a peptide that induces a HTL response, wherein the HTL-inducing peptide is lipidated. The HTL-inducing peptide is optionally linked to the CTL-inducing peptide or not linked. When linked, the HTL-inducing peptide may be separated from the CTL peptide by a spacer, such as Ala- Ala-Ala. The HTL-inducing peptide will usually be linked at its C-terminal end to the CTL-inducing peptide. Typically, the lipid is linked to the N-terminus of the HTL-inducing peptide, where the linkage can optionally include a spacer, such as Lys- Ser-Ser or the like.

The antigen to which the cytotoxic T lymphocyte response is induced is selected from a viral, bacterial, parasitic or tumor antigen. Among the viral antigens to which the CTL responses are effectively induced are antigens of hepatitis

B

(such as envelope, core or polymerase antigens), hepatitis C or human papilloma virus. A particularly effective hepatitis

B

antigen is HBcl8-27. Typically the CTL inducing peptide will be S 15 from seven to fifteen residues, and more usually from nine to :eleven residues. The immunogenic composition can further comprise a carrier, such as physiologic saline, and an adjuvant, such as incomplete freunds adjuvant, alum or montanide. When the peptide is lipidated, it may be modified or unmodified. The lipid is preferably a linear alkyl chain of 6-22 carbons, and p referably is a linear alkyl chain of 16 carbons. In some embodiments of the present invention the lipid is comprised of palmitic acid attached to epsilon and alpha amino groups of a Lys residue, wherein the Lys is linked to the amino terminus of the HTL-inducing peptide by means of a linker.

In other embodiments the present invention comprises methods for inducing a cytotoxic T lymphocyte response in a mammal against an antigen such as a viral, bacterial, parasitic, or tumor antigen. The method comprises administering to the mammal a peptide that induces a CTL response to the antigen, and administering, either separately or together, a lipidated peptide that induces a HTL response. The HTL-inducing peptide is optionally linked to the CTL-inducing peptide or unlinked.

When unlinked, the HTL-inducing peptide can be admixed with the CTL-inducing peptide. The HTL-inducing peptide and the

I,

8 CTL-inducing peptide are typically administered to the mammal in a regimen of two or more administrations. These boosters are spaced a sufficient interval apart to optimize development of a CTL response to the antigen of interest, e.g., a second administration may be approximately four weeks after the initial administration. In representative embodiments described herein the antigen is hepatitis B antigen, such as HBc18-27 and the mammal is a human, of the HLA-A2.1 histocompatibility type for the HBc18-27 CTL inducing peptide.

In yet other embodiments the invention provides methods for treating or preventing a disease that is susceptible to treatment by a CTL response by administering a CTL-inducing peptide to an antigen associated with said disease, and a HTLinducing peptide conjugated to a lipid. The induction of a CTL response can be used in the treatment or prevention of viral Sinfection hepatitis B, hepatitis C or human papilloma virus), bacterial or parasitic infection or tumors. When the Sdisease is hepatitis B infection, for example, the methods can be used to treat or prevent chronic or acute infection.

In yet other embodiments the invention provides methods Sfor inducing a cytotoxic T lymphocyte response in a human against an antigen of interest. The methods comprise administering a composition which comprises a peptide that induces a CTL response to said antigen in a human and an adjuvant. The method may further comprise administering a peptide that induces a HTL response, and in some embodiments the HTL inducing peptide is linked to the CTL inducing peptide.

In other aspects the invention provides methods for S0 inducing a CTL response in a human against an antigen of interest by administering a peptide that induces a CTL response to the antigen and a peptide that induces a HTL response, where the CTL inducing and/or the HTL inducing peptide is lipidated.

The CTL and HTL inducing peptides may be linked or unlinked.

The HTL inducing peptide is preferably lipidated. The lipidated HTL inducing peptide can be combined with a cocktail of at least two CTL inducing peptides to optimize coverage of individuals of different HLA types or, in some instances, different antigen strains.

In a further aspect of the invention methods are described for inducing an effective CTL response in a human against an antigen of interest. According to these methods one or more peptides that induce a CTL response to the antigen, such as a viral, bacterial, parasitic or tumor antigen, is administered to a human together or separately with a peptide that induces a HTL response, where at least the CTL inducing and/or the HTL inducing peptide is lipidated. In representative embodiments of such a method described herein the CTL response is induced is to a viral antigen, such as hepatitis B antigen.

Pharmaceutical composition for the treatment of hepatitis B infection are also provided. These compositions comprise a peptide that induces a CTL response to hepatitis B and a peptide that induces a HTL response, where the HTL-inducing peptide is conjugated to a lipid, together with a pharmaceutically acceptable carrier. The carrier can be a liposome, for example, and the pharmaceutical composition may further comprise an is adjuvant, such as incomplete Freund's adjuvant, alum or montanide.

According to a first embodiment of the invention, there is provided an immunogenically effective pharmaceutical composition comprising: a first peptide comprising an epitope, wherein the first peptide binds to an MHC class I molecule to form an epitope-MHC complex recognized by a cytotoxic S 20 T cell, and wherein the first peptide is selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV; (ii) a second peptide comprising an epitope, wherein the second peptide binds to an MHC class II molecule to form an epitope-MHC complex recognized by a helper T cell, wherein the first peptide and/or the second peptide is linked to a lipid and further 25 wherein the first peptide or the second peptide are covalently linked or are unlinked; and (iii) a pharmaceutically acceptable carrier.

According to a second embodiment of the invention, there is provided a method for stimulating an immune response in a mammal against an epitope, comprising the steps of: providing a first peptide comprising an epitope, wherein the first peptide binds to an MHC class I molecule to form an epitope-MHC complex recognized by a cytotoxic T cell, wherein the first peptide is selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV; (ii) providing a second peptide comprising an epitope, wherein the second peptide [I:\DAYLIB\LIBFF]08640spec.doc:gcc 9a binds to an MHC class II molecule to form an epitope-MHC complex recognized by a helper T cell, wherein the first peptide and/or the second peptide is linked to a lipid; and (iii) administering the first peptide and the second peptide to the mammal.

According to a third embodiment of the invention, there is provided a first and second peptide, when used in stimulating an immune response in a mammal against an epitope, wherein: said first peptide comprises an epitope, and wherein the first peptide binds to an MHC class I molecule to form an epitope-MHC complex recognized by a cytotoxic T cell, and wherein the first peptide is selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV; (ii) said second peptide comprises an epitope, and wherein the second peptide binds to an MHC class II molecule to form an epitope-MHC complex recognized by a helper T cell, wherein the first peptide and/or the second peptide is linked to a lipid.

According to a fourth embodiment of the invention, there is provided the use of a first and second peptide, in the preparation of a medicament for stimulating an immune response in a mammal against an epitope, wherein: said first peptide comprises an epitope, and wherein the first peptide binds to an MHC class I molecule to form an epitope-MHC complex recognized by a cytotoxic T cell, and wherein the first peptide is selected from the group comprising KVAEFVHFL, .2 LWVTVYYGV and IVGAETFYV; (ii) said second peptide comprises an epitope, and wherein the second peptide binds to an MHC class II molecule to form an epitope-MHC complex recognized by a helper T cell, wherein the first peptide and/or the second peptide is linked to a lipid.

According to a fifth embodiment of the invention, there is provided a composition 25 comprising a peptide of fewer than 15 amino acid residues, wherein the peptide binds an M. I: MHC class I molecule to form a peptide-MHC complex recognized by a cytotoxic T-cell and wherein the peptide comprises a sequence selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV.

According to a sixth embodiment of the invention, there is provided a method for stimulating an immune response in a mammal against an epitope, comprising the steps of: providing a peptide of fewer than 15 amino acid residues, wherein the peptide binds to an MHC class I molecule to form a peptide-MHC complex recognized by a cytotoxic T cell and wherein peptide comprises a sequence selected from the group [I :\DAYLIB\LI B FF] 08 64ospec.doc:gc 9b comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV; and (ii) administering the peptide to the mammal.

According to a seventh embodiment of the invention, there is provided a peptide when used for stimulating an immune response in a mammal against an epitope, wherein said peptide is of fewer than 15 amino acid residues, and wherein the peptide binds to an MHC class I molecule to form a peptide-MHC complex recognized by a cytotoxic T cell and wherein peptide comprises a sequence selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV.

According to an eighth embodiment of the invention, there is provided the use of a to peptide for the preparation of a medicament for stimulating an immune response in a mammal against an epitope, wherein said peptide is of fewer than 15 amino acid residues, and wherein the peptide binds to an MHC class I molecule to form a peptide-MHC complex recognized by a cytotoxic T cell and wherein peptide comprises a sequence selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV.

Brief Description of the Drawings Fig. 1 depicts the results of induction of HBV peptide-specific A2.1-restricted CTL by priming A2.1/K b transgenic mice with syngeneic spleen cells "loaded" with HBV.

Panels A-D: Splenocytes from HBV-primed transgenic mice were restimulated in vitro with four mixtures of syngeneic LPS blasts each coated with one of 13 different peptides.

2 20 After 9 days effector cells were assayed for lytic activity against 'Cr labelled Jurkat A2.1/K' target cells in the presence or absence of the four different peptide mixtures used I for induction. Panels E-M: [I :\DAYLIB\LIBFF10864Ospec.doc:gcc

V

Effector cells raised against the four different peptide mixtures were restimulated in vitro against the same peptide mixtures and assayed for lytic activity against 51 Cr labelled Jurkat A2.1/Kb target cells in the presence or absence of the individual peptides.

Fig. 2 illustrates the HBV peptide specificity of A2.1 transgenic CTL. Transgenic CTL raised from HBV-primed transgenic mice and restimulated in vitro twice with one of the four different peptide mixtures were restimulated with individual HBV peptides and assayed for lytic activity on 51

CR

labelled Jurkat target cells in the presence or absence of the HBV peptides used for the restimulation.

Fig. 3 illustrates the results of induction of HBV S*e* peptide-specific

A

2 .1-restricted CTL by priming A2.1/Kb 15 transgenic mice with HBV in IFA. A. Splenocytes from HBV- .primed transgenic mice were restimulated in vitro with syngeneic LPS blasts coated with HBV peptides. After 6d, effector cells were assayed for lytic activity against 5Cr labelled Jurkat SA2.1/Kb target cells in the presence or absence of the appropriate HBV peptide. Each panel represents the CTL activity induced by the indicated target peptide.

Fig 4. The effector CTL of Fig. 3 were restimulated with peptide coated LPS blasts followed at a one week interval by restimulation with peptide coated Jurkat A2.1/Kb cells. Six days after the last restimulation, effector cells were assayed for cytolytic activity against 5 1 Cr labelled Jurkat A2.1/Kb target cells in the absence or presence of the peptide used for the restimulation, plus related peptides. Each panel represents the CTL activity induced by the peptide indicated in the corresponding panel of Fig. 3. The target peptides are indicated in each panel.

Fig. 5 illustrates that no HBcl8-27-specific

CTL

response is detected in mice primed with the HBc 875.23 T helper epitope alone. Animals were primed subcutaneously with 100 pg of 875.23 (T helper epitope) in Complete Freund's Adjuvant

(CFA)

11 followed 9 days later (subcutaneously) with IFA alone.

Splenocytes were removed 3 weeks later, cultured for 6 days in the presence of LPS-blasts that had been incubated with the CTL epitope (875.15), 100 pg for 2 hrs before being washed and added to the culture as a source of antigen presenting cells. The presence of HBc 18-27 87 5 .15)-specific CTL was determined using a standard 6 hr 15 Cr release assay with Jurkat A2.1/Kb cells as targets.

Fig. 6 illustrates that no HBc 1 8 2 7 -specific

CTL

response was detected when mice were primed with HBcl8-27 (875.15) in IFA. Experimental protocol was similar to that described in Fig. 5, except that mice received 100 pg of peptide 875.15 subcutaneously in IFA rather than IFA alone for in vivo CTL priming.

15 Fig. 7 illustrates that HBcl8-27-specific CTL response was detected in 50% of the mice primed with HBc T helper peptide (875.23) mixed with HBc CTL inducing peptide (875.15) at a 1 to 1 ratio. The experimental protocol was similar to that S" described in Figs. 5 and 6.

Fig. 8 illustrates that HBc-specific (875.15)

CTL

activity was detected in mice primed with peptide 902.01 in which the HBc T helper and CTL inducing peptide were linked via a peptide bond. Experimental protocol was similar to that in Figs. 5 and 6.

Fig. 9 illustrates that the greatest HBcl8-27 (875.15)specific CTL activity was detected in mice primed with peptide 902.02 in which the HBc T helper and CTL epitopes were linked via peptide bonds using an exemplary spacer such as alaninealanine-alanine. Protocol was similar to that in Figs. 5 and 6.

Fig. 10 illustrates that previous priming of helper T cells was not required for in vivo priming of HBc 18-27-specific CTL responses using peptide 902.01 and 902.02. CTL response is shown from animals primed subcutaneously with peptide 902.01 (Fig. 10A) or 902.02 (Fig. 10B) alone without the previous priming with peptide 875.23 in CFA.

12 Fig. 11 illustrates the induction of HBenv 3 6 0 3 6 8 specific CTL response. A2.Kb transgenic mice were injected with 100 microliters of an emulsion (IFA) of 100 mg HBenv360-368 and 100 mg HBcl28-140. Three weeks later, splenocytes were restimulated with syngeneic LPS blasts coated with peptide HBenv360-368. Effector cells were assayed for cytotoxicity against 51 Cr labeled Jurkat A2/Kb target cells in the presence or absence of HBenv 360-368.

Fig. 12 illustrates the induction of a CTL response specific for HBc 18-27 by priming with a peptide containing HBc 18-27 linked to tetanus toxoid 830-843 (human helper T cell epitope). Effector cells were assayed against 51Cr labeled Jurkat A2-1/Kb target cells in the present or absence of HBc 18-27; Jy target cells in the presence or absence of HBc 18-27 o0. 15 and Jy cells that had been transfected with HBV core.

Fig. 13 illustrates the minimal sequence for CTL recognition within HBV env 329-348 peptide (799.09). CTL lines S. 110 and 113 were derived from splenocytes obtained from A2Kb transgenic mice primed subcutaneously with HBV virus in IFA and in vitro activated with 799.09 coated stimulator cells. 799.09 specific CTL lines 110 and 113 were assayed for lytic activity S. in a 6 hr 51Cr release assay using JA2Kb cells as targets in the presence of 799.09 peptide truncations (Panel A 799.09 N-terminus truncations; Panel B 799.09 overlapping 9 mers and 10 mers).

Fig. 14 shows the HBcl8-27 specific CTL response (d7 assay) from subjects immunized with placebo or CY-1899. The CTL response against HBcl8-27 was assessed by culturing 4 x 106 PBMC/well in 24 well plates in the presence of HBcl8-27 peptide.

On day three and 6 after initiation cultures were fed with U/ml IL-2 (final concentration). On day 7, part of the wells were harvested and CTL activity was measured using 51 Crlabeled .221 A 2 target cells in the absence or presence of HBcl8-27 peptide and in the presence of a 20 fold excess of K562 cells (K562 cells were added in order to decrease background 13 lysis caused by NK cells). The data are expressed in lytic cells were one lytic unit is defined as the number of lymphocytes required to achieve 30% lysis of 10000 .221 A2 during a 6 hour assay. Each bar represents the specific

CTL

activity in the present of peptide in the absence of peptide).

Fig. 15 shows the HBc18-27 specific CTL response (d14 assay) from subjects immunized with placebo or CY-1899. On day 7 after initiation of cultures (see Fig. the remaining wells were harvested and cells were restimulated with HBc18-27 peptide-coated autologous adherent cells. Cultures were fed with 10 U/ml IL-2 on day 9 and thereafter as needed.

CTL

activity was assayed on d14 using the procedure described in Fig. 1.

15 Fig. 16 shows the mean and standard deviation of peak CTL activity: the mean and standard deviation of peak CTL activity after the first and second injections of different doses of CY-1899.

Fig. 17 shows HBcl8-27 specific CTL from subjects injected with CY-1899 recognize endogenous processed antigen.

Effector CTL obtained from subjects 302 and 304 after 14 days of culture were restimulated as described in Fig. 2 CTL activity was assayed 7 days later as described in Fig. 1 using as targets .221 A 2 cells in the absence or presence of HBc18-27 peptide and .221 A 2 cells transfected with the HBV core protein Fig. 18 shows the proliferation response to T cells specific for the TT 830-843 helper peptide from subjects immunized with placebo or CY-1899. T cell proliferation response against the helper peptide epitope was measured by culturing 1.5 x 105 PBMC from each sample in flat-bottom 9 6 /plate wells with or without 10 pg/ml TT peptide. Seven days later, cultures were fed with medium containing recombinant IL-2 U/ml final concentration) to induce further proliferation of T cells which had been stimulated against the peptide. On day 9 14 after initiation of culture, 1 pCi of 3 H-thymidine was added to each well and 18 hr. later, cells in each well were harvested onto glass fiber mats and counted for 3 H-thymidine incorporation into DNA. Each bar represents the difference in cpm 3Hthymidine incorporation obtained from wells which received peptide minus those which did not receive peptide.

Description of the Specific Embodiments The present invention provides compositions and methods for inducing an effective CTL-mediated response to an antigen of interest in humans and other mammals. The composition is comprised of peptides that are capable of inducing MHC class

I-

restricted CTL responses to the antigen of interest

("CTL

15 peptide") and an adjuvant. Another embodiment to the present invention is directed to a composition comprised of said CTL peptide and a peptide capable of eliciting a helper T lymphocyte (HTL) response. Another embodiment of the invention is directed to either or both the CTL and HTL peptide by lipidated, linked or unlinked and administered with or without an adjuvant .preparation. In particularly preferred embodiments either the :CTL peptide or the HTL peptide is lipidated, linked or unlinked and administered without an adjuvant. In a preferred embodiment the HTL epitope is lipidated and linked to the CTL epitope and administered without an adjuvant. In another preferred embodiment the lipidated HTL peptide is admixed with, but not linked to, at least one CTL peptide.

By administering the compositions of the present invention an effective CTL response is stimulated in the recipient mammal to the antigen of interest. The cells which are targeted by the CTL response can be involved in a wide variety of disease or potential disease states, cells which are infected by viruses, bacteria or parasites, cells which express certain tumor antigens, and cells which express autoimmune antigens or other antigens that are capable of being recognized as self by the mammal's CTL. The specifically stimulated CTL attack the target cells by secreting lymphokines gamma interferon) and liberating products proteolytic enzymes such as serine esterases) that inhibit replication of the infecting organism in the cells and/or kill the cells which express the antigen of interest, and thus are able to interrupt or substantially prevent the disease of interest, a viral infection, parasite or bacterial infection, a tumor or an autoimmune disease process.

The CTL inducing peptides which are useful in the compositions and methods of the present invention can be selected from a variety of sources, depending of course on the targeted antigen of interest. The CTL inducing peptides are 1typically small peptides that are derived from selected epitopic 15 regions of target antigens associated with an effective

CTL

response to the disease of interest. Thus, by "CTL inducing peptide" or "CTL peptide" of the present invention is meant a chain of at least four amino acid residues, preferably at least six, more preferably eight to ten, sometimes eleven to fourteen residues, and usually fewer than about thirty residues, more usually fewer than about twenty-five, and preferably fewer than fifteen, eight to fourteen amino acid residues derived from selected epitopic regions of the target antigen(s) Peptides that induce CTL responses are used in the methods and compositions of the present invention irrespective of the method or methods used to identify the epitope recognized by CTL. The CTL epitope(s) contained in the CTL peptides can be identified in one of several ways. In those cases where antigen-specific CTL lines or clones are available, for example tumor-infiltrating lymphocytes (TIL) or virus-specific

CTL,

these cells can be used to screen for the presence of the relevant epitopes using target cells prepared with specific antigens. Such targets can be prepared using random, or selected synthetic peptide libraries, which would be utilized to sensitize the target cells for lysis by the CTL. Another 16 approach to identify the relevant CTL epitope when CTL are available is to use recombinant DNA methodologies. Gene, or preferably cDNA, libraries from CTL-susceptible targets are first prepared and transfected into non-susceptible target cells. This allows the identification and cloning of the gene coding the protein precursor to the peptide containing the CTL epitope. The second step in this process is to prepare truncated genes from the relevant cloned gene, in order to narrow down the region that encodes for the CTL epitope. This step is optional if the gene is not too large. The third step is to prepare synthetic peptides of approximately 10-20 amino acids of length, overlapping by 5 residues, which are used to sensitize targets for the CTL. When a peptide, or peptides, are shown to contain the relevant CTL epitope, smaller peptides can be prepared to establish the peptide of minimal size that cotains the CTL epitope. These epitopes are usually contained within 9-10 residues. Examples of peptides containing known CTL :epitopes identified in this way are listed below.

ANTIGEN SOURCE SEQUENCE SEQ.

HLA-

ID NO. RESTRICTION MAGE-l EAkDPTGHSY 1 Al HIV nef84-94 AVDLSHFLK 2 All EBNA4 416-424 I TFSVIK 3Al Influenza A, Ml58-66 GLFFL7A.

p5S6-22ATLGPNE~VTR 14 A.1A6 NP 383-391 SRWITR1 2 *RWIT 15 B2 HIV gag p24 265-274 KRWI ILGLNK 16B2 P.falciparum circumsp.368-375 KPKDELDY 17 P-falciparum circumsp.368-37 5 KSKDLEDY 18 835 P-falCiparum liverAgl850olas 7 KPNDKSLY 19 HIV-2 TPYDINQML 20B5 P.falciparum liverAgl786.l7 94 KPIVQYDNP 21 B53 853 self peptide YPAEITLI 22 B53 HIV 9P41 586-593 YLKDQQLL 23 8 NP 380-388 ELRSRYWAI 24 ]38 **EBV EBNA-3 FLRGRAYGI 25 B88 HIV gag26l-269 G E IYKRwI1 26 8 HIV gag331-339 DCKTILKAL 27 8 HIV p01185-193 DPKVYQWPL 28 8 IV gp41 586-593 YLKDQQLYL 29 B8 HIV gap p17.3 QGKYL 30 88 Another way of identifying a peptide Containing a CTL epitope, when CTLs are present, is to elute the peptide with an acid or base. The peptides associated with MHC molecules are present on the cells that are lysed by the CTL. The eluted peptides are separated using a purification method such as HPLC, and individual fractions are tested for their capacity to sensitize targets for CTL lysis. When a fraction has been identified as containing the CTL peptide, it is further purified and submitted to sequence analysis. The peptide sequence can also be determined using tandem mass spectrometry. A synthetic peptide is then prepared and tested with the CTL to corroborate that the correct sequence and peptide have been identified.

In some circumstances, where CTL are not available there are other means to identify potential CTL epitopes.

These methods rely in the identification of MHC-binding Speptides from known protein sequences. These methods have been 15 described in detail in pending patent applications

(U.S.

P: atent Applications Serial Nos. 08/159,339, 08/073,205 and EPO Patent Application No. 92201252.1, which are herein incorporated by reference). Briefly, the protein sequences for example from viral or tumor cell components are examined for the presence of MHC-binding motifs. These binding motifs which exist for each MHC allele, are conserved amino acid residues, usually at positions 2 (or 3) and 9 (or 10) in peptides of 9-10 residues long. Synthetic peptides are then prepared of those sequences bearing the MHC binding motifs, and subsequently are tested for their ability to bind to MHC molecules. The MHC binding assay can be done either using cells which express high number of empty MHC molecules "(cellular binding assay), or using purified MHC molecules.

Lastly, the MHC binding peptides are then tested for their capacity to induce a CTL response in naive individuals, either in vitro using human lymphocytes, or in vivo using

HLA-

transgenic animals. These CTL are tested using peptidesensitized target cells, and targets that naturally process the antigen, such as viral infected cells or tumor cells. For example, a HLA-Al-restricted CTL epitope for the tumorassociated antigen MAGE-3 has been identified using this approach and is the subject of a pending patent application 19 patent application Serial No. 08/186,266, which is herein incorporated by reference.

Desirably, the CTL peptide will be as small as possible while still maintaining substantially all of the biological activity of a larger peptide. When possible, it may be desirable to optimize peptides of the invention to a length of eight to twelve amino acid residues, more usually nine or ten amino acid residues, commensurate in size with endogenously processed antigen peptide that is bound to MHC class I molecules on the cell surface. See generally, Schumacher et al., Nature 350:703-706 (1991); Van Bleek et al., Nature 348:213-216 (1990); Rotzschke et al., Nature 348:252-254 (1990); and Falk et al., Nature 351:290-296 S(1991), which are incorporated herein by reference. By biological activity of a CTL inducing peptide is meant the ability to bind an appropriate MHC molecule and, in the case S*of peptides useful for stimulating CTL responses, induce a CTL S*response against the selected antigen or antigen mimetic. By a CTL response is meant a CD8 T lymphocyte response specific for an antigen of interest, wherein CD8 MHC class Irestricted T lymphocytes are activated. As noted above, the activated cytotoxic T lymphocytes will secrete a variety of products which inhibit and may or may not kill the targeted cell.

The compositions and methods of the present invention are particularly preferred for targeting host cells infected :by viruses. CTL responses are an important component of the immune responses of most mammals to a wide variety of viruses, and the present invention provides a means to effectively stimulate a CTL response to virus-infected cells and treat or prevent such an infection in a host mammal. Thus the compositions and methods of the present invention are applicable to any virus presenting protein and/or peptide antigens. Such viruses include but are not limited to the following, pathogenic viruses such as influenza A and B viruses (FLU-A, FLU-B), human immunodeficiency type I and II viruses (HIV-I, HIV-II), Epstein-Barr virus (EBV), human

T

lymphotropic (or T-cell leukemia) virus type I and type II (HTLV-I, HTLV-II), human papillomaviruses types 1 to 18 (HPV-1 to HPV-18), rubella varicella-zoster (VZV), hepatitis

B

(HBV), hepatitis C (HCV), adenoviruses and herpes simplex viruses(HV). In addition, cytomegalovirus

(CMV)

poliovirus, respiratory syncytial (RSV), rhinovirus, rabies, mumps, rotavirus and measles viruses.

In a like manner, the compositions and methods of the present invention are applicable to tumor-associated proteins, which could be sources for CTL epitopes. Such tumor proteins and/or peptides, include, but are not limited to, products of the MAGE-I, -2 and -3 genes, products of the c-ErbB2

(HER-

2/neu) proto-oncogene, tumor suppressor and regulatory genes which could be either mutated or overexpressed such as p53 15 ras, myc, and RBl. Tissue specific proteins to target

CTL

responses to tumors such as prostatic specific antigen

(PSA)

and prostatic acid phosphatase (PAP) for prostate cancer, and tyrosinase for melanoma. In addition viral related proteins associated with cell transformation into tumor cells such as EBNA-1, HPV E6 and E7 are likewise applicable. A large number of peptides from some of the above proteins have been identified for the presence of MHC-binding motifs and for their ability to bind with high efficiency to purified

MHC

molecules and are the subject of pending patent applications patent application Serial Nos. 08/159,339 and 08/073,205, previously incorporated herein by reference).

The peptides can be prepared "synthetically," as described hereinbelow, or by recombinant DNA technology.

Although the peptide will preferably be substantially free of other naturally occurring viral, bacterial, parasitic, tumor or self proteins and fragments thereof, in some embodiments the peptides can be synthetically conjugated to native fragments or particles. The term peptide is used interchangeably with polypeptide in the present specification to designate a series of amino acids connected one to the other by peptide bonds between the alpha-amino and alphacarboxy groups of adjacent amino acids. The polypeptides or 21 peptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications, subject to the condition that the modification not destroy the biological activity of the polypeptides as herein described.

The terms "homologous", "substantially homologous", and "substantial homology" as used herein denote a sequence of amino acids having at least 50% identity wherein one sequence is compared to a reference sequence of amino acids. The percentage of sequence identity or homology is calculated by comparing one to another when aligned to corresponding portions of the reference sequence.

The peptides useful in the present invention can be 15 optionally flanked and/or modified at one or both of the Nand C-termini, as desired, by amino acids from the naturally occurring HBV) sequences, amino acids added to facilitate linking to another peptide or to a lipid, other Nand C-terminal modifications, linked to carriers, etc., as further described herein. Additional amino acids can be added S* to the termini of a peptide to provide for modifying the physical or chemical properties of the peptide or the like.

Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, can be introduced at the C- or Nterminus of the peptide or oligopeptide. In addition, the peptide sequences can differ from the natural sequence by S"being modified by terminal-NH 2 acylation, by alkanoyl

(C-C

20 or thioglycolyl acetylation, terminal-carboxy amidation, ammonia, methylamine, etc. In some instances these modifications may provide sites for linking to a support or other molecule.

It will be understood that the peptides of the present invention or analogs thereof which have CTL stimulating activity may be modified to provide other desired attributes, improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide. For instance, 22 the peptides can be modified by extending, decreasing or substituting in the peptides amino acid sequences by, e.g., the addition or deletion of amino acids on either the amino terminal or carboxy terminal end, or both, of peptides derived from the sequences disclosed herein.

With respect to treatment or prevention of hepatitis

B

infection in humans, selection of a CTL inducing peptide(s) useful in the present invention can be as set forth in more detail in copending applications U.S. Serial Nos. 07/935,811, 07/935,898 and 08/024,120 which are incorporated herein by reference. These applications provide the ability to select one or more peptides that induce CTL response to a hepatitis

B

antigen, which response is capable of killing (or inhibiting) o cells which are infected by or otherwise express (in the case of transfected cells) the native HBV antigens. The HBV CTL inducing peptide will usually have at least four, sometimes six, often seven or more residues, or a majority of amino acids of that peptide, that are identical or homologous when compared to the corresponding portion of the naturally occurring HBV sequence. For example, those peptides which are preferred for stimulating HBV CTL responses include 23 SOURCE POSITION SEQUENCE SIZE SEQ.

BINDING

ID NO. A2 HBV POL 1117 LLAQFTSAI 9 31 9.6000 HBV ENV 338 LLVPVQWFV 10 32 1.6000 HBV ENV 335 WLSLLVPFV 9 33 0.9600 HBV ENV 1116 FLLAQFTSA 9 34 0.6600 SOL 1147 FLLSLGIHL 9 35 0.5200 HBV POL 1245 ALMPLYACI 9 36 0.5000 HBV ENV 249 ILLLCLIFLL 10 37 0.3000 HB OL 1092 KLHLYSHPI 9 38 0.2900 V ENV 259 VLLDYQGML 9 9 1100 H B V E N V r c9 p1t i0 HBV ENV 378 LLPIFFCLWV 10 40 0.1000 HBV 00 HBV ENV 177 VLQAGFFLL 9 41 0.0660 721 YLHTLWKAGI___ 10 42 0.0560 HBV POL -721 YLHTLWKAGV 10 43 0.1300 For exa a PLLPIFFCL 9i g Hc p e cs HBV NUC (CORE) t l t oe CTL e c s ad h Other HBV CTL stimulating peptides include HBenvomo peptionde 79 HBe3293n49 (peptide 799.09) or HBen A (peptide 799.10)e and the HBc region HB9is pti (peptide 802.03).

SFor example, a CTL inducing HBc peptide comprises from six to thirty amino acids and is derived from the region HBc18-27, contains at least one CTL epitopic site, and has at *least seven amino acids wherein a majority of amino acids of the peptide will be identical or substantially homologous when compared to the amino acids comprising the corresponding portion of the naturally occurring HBcl8-27 sequence.

A

representative peptide of this region is peptide HBcl8-27 which has the following sequence (for HBV subtype ayw): HBc18-27 [Seq. ID No. 4]

FLPSDFFPSV

1 24 With respect to treatment or prevention of hepatitis

C

infection in mammals, one or more peptides that induce a CTL response to a hepatitis C antigen may be selected. The HCV CTL-inducing peptide will usually have at least four, sometimes six, often seven or more residues, or a majority of amino acids of that peptide that are identical or homologous when compared to the corresponding portion of the naturally occurring HCV sequence. For example, those peptides which are preferred for stimulating HCV CTL responses include sequences contained within copending U.S patent applications (Serial No.

08/159,339 and 08/073,205, previously incorporated herein by reference), in particular peptides 1073.05 (LLFNILGGWV) [Seq ID No. 46], 1090.18 (FLLLADARV) [Seq ID No. 49), 939.20 (LLALLSCLTV) [Seq ID No. 47), 1073.07 (YLLPRRGPRL) [Seq. ID No. 52), 1013.10 (DLMGYIPLV) [Seq ID No. 51], 1073.10 (GVAGALVAFK) [Seq ID No. 61]. Also suitable are other peptides identified by other methods, such as STNPKPQK [Seq ID No. 62] and GPRLGVRAT [Seq ID No. 63] (Koziel et al., J.

Virol. 67:7522-7535, 1993), and YPWPLYGNEGLGWAGWLLSP (Seq ID 20 No. 64) (Kita et al., Abstract #631, 1993 Am. Assoc. For Study of Liver Diseases Meeting). Other HCV derived peptides for stimulating HCV CTL responses include the following: o SOURCE POSITION SEQUENCE SIZE SEQ.

BINDING

_ID NO. A2 HCV NS4 1807 LLFNILGGWV 10 46 3.5000 HCV CORE 178 LLALLSCLTV 10 47 0.6050 HCV NS4 1585 YLVAYQATV 9 48 0.2450 HCV NS1/ENV 725 FLLLADARV 9 49 0.2250 HCV NS4 1851 ILAGYGAGV 9 50 0.2150 HCV CORE 132 DLMGYIPLV 9 51 0.0835 HCV CORE 35 YLLPRRGPRL 10 52 0.0725 NS1/ENV2 686 ALSTGLIHL 9 53 0.0415 HCV CORE 178 LLALLSCLTI 10 54 0.0340 SHCV NS5 2578 RLIVFPDLGV 10 55 0.0320 15 HCV NS5 2885 RLHGLSAFSL 10 56 0.0200 SHCV NS4 1811 ILGGWVAAQL 10 57 0.0180 .HCV ENV 1 364 SMVGNWAKV9 58 0.0155 SHCV NS3 1131 YLVTRHADV 9 59 0.0109 HCV NS4 1666 VLAALAAYCL 10 60 0.0106 With respect to treatment or prevention of HPV infections in mammals, one or more peptides that induce a CTL response to a HPV may be selected. The HPV CTL-inducing peptide will usually have at least four, sometimes six, often seven or more residues, or a majority of amino acids of that peptide that are identical or homologous when compared to the corresponding portion of the naturally occurring HCV sequence.

For example, those peptides which are preferred for stimulating HPV CTL responses include sequences contained within copending U.S patent applications (Serial No.

08/159,339, 08/073,205 and EPO Patent Application 92201252.1, previously incorporated by reference), in particular the following peptides: 26 'SOURCE POSITION SEQUENCE SIZE SEQ. BINDING SEQUENCE |SIZE SEQ.

BINDING

ID NO. A2

HPV

1 6 E7 82 LLMGTLGIV 9 65 0.0240 HPV16 E7 11 YMLDLQPET 9 66 0.1400 HPV16 E6 52 FAFRDLCIV 9 67 0.0570 HPV16 E7 86 TLGIVCPIC 9 68 0.0750 HPV16 E7 7 TLHEYMLDL 9 69 0.0070

HPV

1 6 E7 85 GTLGIVCPI 9 70 0.0820 HPV16 E7 12 MLDLQPETT 9 71 0.0028

HPV

1 6 E6 29 TIHDIILECV 10 72 0.0210 With respect to treatment or prevention of human immunodeficiency virus 1 and 2 in humans, one or more peptides that induce a CTL response to a HIV 1 or 2 antigen may be selected. The HIV CTL-inducing peptide will usually have at least four, sometimes six, often seven or more residues, or a majority of amino acids of that peptide that are identical or Shomologous when compared to the corresponding portion of the naturally occurring HIV sequence. For example, those peptides which are preferred for stimulating HIV CTL responses include the following peptides: SOURCE POSITION SEQUENCE SIZE SEQ.

BINDING

S* h w c t t ID NO A2 HIV 367 VLAEAMSQV 9 73 0.1100 HIV 1496 LLWKGEGAW 10 74 0.0360 HIV 1496 LLWKGEGAV 9 75 0.0230 HIV 1004 ILKEPVHGV 9 76 0.0190 HIV 1129 IVGAETFYV 9 77 0.0099 HIV 1129 IIGAETFYV 9 78 0.0260 HIV 2182 LWVTVYYGV 9 79 0.0014 HIV 2182 LMVTVYYGV 9 80 0.4400 27 Several tumor associated antigens have also been correlated with CTL responses, including, but not limited to, renal cell carcinoma antigens, breast cancer antigens, carcinoembryonic antigen (CEA), melanoma (MAGE-1 and MAGE-3) antigens, prostate cancer specific antigen and others. For example, a HLA-Al-restricted CTL epitope for the tumorassociated antigen MAGE-3 has been identified using this approach and is the subject of a pending patent application patent application Serial No. 08/186,266, previously incorporated herein by reference.

Peptides which stimulate CTL responses to tumor antigens and which can be used in the methods and compositions of the present invention can be selected as described in, for 15 example, U.S. Patent Applications Serial Nos. 08/159,339 and 15 08/073,205, previously incorporated by reference. For example, representative peptide which are preferred for inducing MAGE-3 and -l CTL responses include the following: •e e e 28 SOURCE POSITION SEQUENCE SIZE SEQ.

BINDIN(

MAGE2 105 ID NO. A2 MAGE2 105 KVE81 0.5100 MAGE3ELHFLL 10 82 0.2200 MAGELVFGIELMEV 10 MAGE1 278 83 0.1100 KVLEYVIKV 9 MAGE1 105 84 0.0900 KVADLVGFLL 10 1 0 MAGE3 105 8 5 0.0560 KVAEFVHFL 9 MAGE1 92 VF 9 86 0.0550 CILESLFRA 9 MAGE1 CILSLRA 9 87 0.0460 264 FLWGPRALA 9 0.0 MAGE1 2 88 0.0420 MAGE 200 VMIAMEGGHA 10 .MAGE1 38 89 0.0360 HT. ry LVLGTLEEV e 1 5 MAGE1 90 0.0320 *301 ALREEEEGV 9 MAGE1 270 91 0.0210 hALAETSYVKV 10 9 uMAGE1 282 92 0.0150 MAGE 282 YVIKVSARV 9 *MAGE1 269 9 3 0.0140 RALAETS YV 9 94 0.0100 SThe present invention enhancesthe effectiveness of a CTL-inducing peptide by co-delivery with a sequence which contains at least one epitope that is capable of inducing HTL response. By a HTL response is meant a CD4 Tindu lymphcyteing a response wherein CD4' T lymphocytes are activated. The HTLs stimulated by the HTL-inducing peptide can be e the T-helper 1 Shelpe/or T-helper 2 phenotype, for example. The activated

T

helper lynphcytes will secrete a variety of products, including, for example, interleukin-2, which may facilitate atexpression of the T cell receptor and promote recognition by HTL-inducing epitopes can be provided by peptides which correspond substantially to the antigen targeted by the CTL-inducing peptide, or more preferably is a peptide to a CTL-inducing peptide or e ant i g e n trgeted by the more widely recognized antigen,d pref e r a b l y noeptide to a specific for a particular h o p r e f e r a b l y is not specific P fo e p t i d s icul a r h ocompatbility antigen restriction.

Peptides which are recognized by most individuals regardless 29 of their MHC class II phenotype ("Promiscuous") may be particularly advantageous. The HTL peptide will typically comprise from six to thirty amino acids and contain a HTLinducing epitope. For example, illustrative peptides useful in the present invention are those which contain HTL inducing epitopes within a HTL peptide from tetanus toxoid 830-843 having the sequence Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile- Gly-Ile-Thr-Glu (QYIKANSKFIGITE) [Seq. ID No. 95), malaria circumsporozoite 382-398 Lys-Ile-Ala-LysMet-Lys-Ala-Ser-Ser- Val-Phe-Asn-Val-Val-Asn-Ser (KIAKMEKASSVFNVVNS) (Seq. ID No.

96); malaria circumsporozoite 378-398 Asp-Ile-Glu-Lys-Lys-Ile- Ala-Lys-Met-Lys-Ala-S er-Ser-Va-Phe-Asn-Val-Val-Asn-Ser (DIEKKIAKMEKASSVFVVNS) [(Seq. ID No. 97], and ovalbumin 323- 3 36 Ile-Ser-Gln-Ala -Val-His -Al a-Ala-His-Ala-Glu-Ile-Asn-Glu [Seq. ID No. 98] and the influenza epitope 307-319 Pro-Lys-Tyr- V Lys-Gn-Asn-Thr-Leu-Lys-Leu-Aa-Thr Seq. ID No. 99). In addition suitable T helper peptides have been identified as S. :described in pending U.S. Patent Application Serial No 08/121,101, incorporated herein by reference.

Other examples of HTL-inducing peptides are those which are specific for the antigen (virus or other organism, tumor, etc.) being targeted by the CTL. For example, several H TL-inducing peptides specific for HBV have been described such as HBc h the 1-20, aving the sequence: Met-Asp-Ile-Asp-Pro-Tyrys-Glu-Phe-Gly-Ala-Thr-Val-Glu-Leu-Leu-Ser-Phe-Leu-Pro [Seq.

ID No. 100; peptides from the region HBc 5 0 6 9 which has the se e-Arg-Gln-Ala-Ile-Leu-Cys-Trp- Gly-Glu-eu-Met-Tyr-LeU-Ala (Seq. ID No. 101), and from the region of HBc 1 0 0 1 3 9 including HBc 1 0 0 1 1 9 having the sequence Leu-Leu-Trp-Phe-His-Ile-Ser-Cys-Leu-Thr-Phe-Gly-Arg-Glu- Thr- Val-Ile-Glu.Tyr-eu [Seq. ID No. 102) (where Ile 6 is Leu in the HBV adw subtype) HBc 1 1 7 1 3 1 having the sequence Glu-Tyr- Leu-Va1 -Ser-Phe-Gly-Va(-Trp-1e-Arg-Thr-Pro-ProAl a(Seq.

ID

No. 103), and peptide HBc 1 2 0 -139 having the sequence Val-Ser- Phe-Gly-Val-Trp-Ile-Arg-ThPro-Pro- Ala-Tyr-Arg-Pro-Pro-Asn- Ala-Pro-Ile [Seq. ID No. 104]). See, Ferrari et al., J. Clin.

Inveyst. 88:214-222 (1991), and U.S. Pat. No. 4,882,145, and U.S. Patent No. 5,143,726, each of which is incorporated herein by reference.

The CTL or HTL inducing peptides employed in the compositions and methods of the present invention need not be identical to specific peptides disclosed in aforementioned disclosures, and can be selected by a variety of techniques, for example, according to certain motifs as described above.

Therefore, the peptides may be subject to various changes, such as insertions, deletions, and substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use. By conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., 15 one hydrophobic residue for another, or one polar residue for 15 another. The substitutions include combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Usually, the portion of the sequence which is intended to substantially mimic a CTL or HTL stimulating epitope will not differ by more than about 20% from the corresponding sequence of a native antigen, when known, except Swhere additional amino acids may be added at either terminus for the purpose of modifying the physical or chemical properties of the peptide for, ease of linking or coupling, and the like. In those situations where regions of the peptide sequences are found to be polymorphic among antigen subtypes, it may be desirable to vary one or more particular amino acids to more effectively mimic differing

CTL

o or HTL epitopes of different antigen strains.

0 In some instances it may be desirable to combine two or more peptides which contribute to stimulating specific

CTL

responses in one or more patients or histocompatibility types.

The peptides in the composition can be identical or different, and together they should provide equivalent or greater biological activity than the parent peptide(s). For example, using the methods described herein, two or more peptides may define different or overlapping CTL epitopes from a particular region, the peptide region 799.08 (HBenv 309 328 peptide 31 region, 799.09 (HBenv 32 9 -349). 799.10 (HBenv 3 4 9 3 6 8 or peptide region 802.03 (HBc 9 1 1 1 0 which peptides can be combined in a "cocktail" to provide enhanced immunogenicity of CTL responses, and peptides can be combined with peptides having different MHC restriction elements. This composition can be used to effectively broaden the immunological coverage provided by therapeutic, vaccine or diagnostic methods and compositions of the invention among a diverse population.

In some embodiments the CTL inducing peptides of the invention are linked to the HTL inducing peptides.

CTL

inducing peptides T helper conjugates can be linked by a spacer molecule, or the CTL peptide may be linked to the HTL peptide without a spacer. When present, the spacer is ,typically comprised of relatively small, neutral molecules, 15 such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions and may have linear or branched side chains. The spacers are typically selected from, Ala, Gly, or other neutral S: spacers of nonpolar amino acids or neutral polar amino acids.

20 In certain preferred embodiments herein the neutral spacer is Ala. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. Preferred exemplary spacers are homo-oligomers of Ala. When present, the spacer will usually 25 be at least one or two residues, more usually three to six residues. When the HTL-inducing peptide is conjugated to the S. CTL-inducing peptide, in the present or absence of a spacer, preferably with the HTL peptide is positioned at the amino end of the conjugate.

The peptides of the invention can be combined via linkage to form polymers (multimers), or can be formulated in a composition without linkage, as an admixture. Where the same peptide is linked to itself, thereby forming a homopolymer, a plurality of repeating epitopic units are presented. When the peptides differ, a cocktail representing different antigen strains or subtypes, different epitopes within a subtype, different histocompatibility 32 restriction specificities, or peptides which contain

HTL

epitopes, heteropolymers with repeating units are provided.

In addition to covalent linkages, noncovalent linkages capable of forming intermolecular and intrastructural bonds are also contemplated.

Linkages for homo- or hetero-polymers or for coupling to carriers can be provided in a variety of ways. For example, cysteine residues can be added at both the amino- and carboxy-termini, where the peptides are covalently bonded via controlled oxidation of the cysteine residues. Also useful are a large number of heterobifunctional agents which generate Sa disulfide link at one functional group end and a peptide link at the other, including N-succidimidyl-3- 1 2 -pyridyldithio) proprionate (SPDP). This reagent creates a disulfide linkage between itself and a cysteine residue in one Sprotein and an amide linkage through the amino on a lysine or other free amino group in the other. A variety of such disulfide/amide forming agents are known. See, for example, I Rev. 62:185 (1982). Other bifunctional coupling agents t20 form a thioether rather than a disulfide linkage. Many of these thioether forming agents are commercially available and include reactive esters of 6 -maleimidocaproic acid, 2 bromoacetic acid, 2 -iodoacetic acid, 4 -(N-maleimido-methyl) cyclohexane- l-carboxylic acid and the like. The carboxyl groups can be activated by combining them with succinimide or l-hydroxy-2-nitro- 4 -sulfonic acid, sodium salt. A particularly preferred coupling agent is succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). Of course, it will be understood that linkage should not substantially interfere with either of the linked groups to function as described, to function as a CTL determinant or HTL determinant.

As a further aspect of the present invention the HTLinducing peptide(s) and CTL-inducing peptide(s) can be delivered to the patient in the presence of a lipid. The lipid residue, such as palmitic acid or the like (as described further below, which is attached to alpha and epsilon amino groups of a Lys residue

((PAM)

2 Lys), is attached to the amino terminus of the HTL-inducing peptide. The lipid can be attached directly to the HTL peptide, or, more typically, indirectly via a linkage, such as a Ser-Ser, Gly, Gly-Gly, Ser linkage or the like.

As another example of lipid-HTL priming of CTL responses, E. coi lipoprotein, such as tripalmitoyl-sglycerylcysteinly-seryl-serine

(P

3 CSS), can be used to prime specific CTL when covalently attached to an appropriate

HTL

peptide. See, Deres et al., Nature 342:561-564 (1989), incorporated herein by reference. The HTL peptides can be coupled to P 3 CSS, for example, and the lipopeptide administered in conjunction with the CTL inducing peptide to an mammal to specifically prime a CTL response to the antigen 15 of interest.

Yet another example of lipid priming of CTL response is achieved by conjugating the CTL/T helper-peptide-conjugate with uncharged fatty acid residues of different chain lengths and degrees of unsaturation, ranging from acetic to stearic 20 acid as well as to negatively charged succinyl residues via the appropriate carboxylic acid anhydrides.

The lipid may be linked to other peptides which present HTL epitopes which are then combined with the lipid.

When the HTL and CTL are linked in a conjugate, the arrangement of the components of the conjugate comprising the CTL inducing peptide/T helper peptide/lipid can be varied. In one case, the lipid moiety can be linked to the amino terminal end of the CTL inducing peptide, which in turn is linked at its carboxy terminal to the T helper peptide. In another case, the lipid is linked at the amino terminal end of the T helper peptide, which is linked at its carboxy terminal to the CTL inducing peptide. In each case, a spacer molecule can be selectively inserted between the lipid moiety and the CTL or T helper peptide, as well as between the T helper and the CTL inducing peptides. In the case of the spacer between the lipid and the T helper or CTL inducing peptide, a preferred example comprises Lys-Ser-Ser, although other spacers are 34 described herein. An example of a spacer between the T helper and CTL inducing peptides will be Ala-Ala-Ala, as also described in further detail herein.

As further described herein, the lipidated HTL peptide and CTL peptide can then be emulsified in an adjuvant, e.g., incomplete Freund's adjuvant, alum or montanide.

In an exemplary embodiment described below, a T helper peptide from substantially within TT830-843 was lipidated at its N-terminus (with (PAM) 2 via a linker (KSS) and then linked at its C-terminus (via a linker AAA) with a HBV CTL inducing peptide, HBcl8-27. Thus, the structure of the eptide was (PAM) 2 KSS-T helper-AAA-CTL and had the sequence of (PAM)2KSS-ISQAVHAAHAEINE-AAA-TYQRTRALV [Seq ID No. 105). This .conjugate, when administered to transgenic animals expressing the HLA2.1 antigen, was shown to induce specific CTL priming of animals. It was also established that the CTL induced by the peptide recognized endogenously synthesized HBcore antigens. When the same lipid-THL-CTL peptide conjugate was administered to humans an induction of specific CTL response 20 was observed, where the response dose dependent both in proportion of subjects exhibiting a positive response as well as in the magnitude of the response obtained.

The peptides of the invention can be prepared in a wide variety of ways. Because of their relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co. (1984); Tam et al., J. Am. Chem. Soc.

105:6442 (1983); Merrifield, Science 232:341-347 (1986); and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, pp. 1-284 (1979), each of which is incorporated herein by reference.

Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a CTL peptide and/or T helper peptide of interest is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression. These procedures are generally known in the art, as described generally in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1982), and Ausubel et al., (ed.) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York (1987), and U.S. Pat. Nos. 4,237,224, 4,273,875, 4,431,739, 4,363,877 and 4,428,941, for example, which disclosures are incorporated herein by reference. Thus, fusion proteins which comprise one or more peptide sequences can be used to present the CTL and HTL determinants. For example, a recombinant HBV surface antigen protein is prepared in which the HBenv amino acid sequence is altered so as to 15 more effectively present epitopes of peptide regions described herein to stimulate a CTL response. By this means a Spolypeptide is used which incorporates several CTL and HTL epitopes.

As the coding sequence for peptides of the length 20 contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al., J. Am. Chem. Soc. 103:3185 (1981), modification can be made simply by substituting the appropriate base(s) for those encoding the native peptide sequence. The coding sequence can 5 then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the *vectors used to transform suitable hosts to produce the desired fusion protein. A number of such vectors and suitable host systems are now available. For expression of the fusion proteins, the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in the desired cellular host. For example, promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vectors are transformed into suitable 36 bacterial hosts. Of course, yeast or mammalian cell hosts may also be used, employing suitable vectors and control sequences.

The peptides of the present invention and pharmaceutical and vaccine compositions thereof are useful for administration to mammals, particularly humans, to treat and/or prevent viral, bacterial, and parasitic infections. As the peptides are used to stimulate cytotoxic T-lymphocyte responses to HBV infected cells, the compositions can be used to treat or prevent acute and/or chronic HBV infection.

For pharmaceutical compositions, the peptides, i.e., the compositions of lipidated HTL/CTL peptides of the invention as described above will be administered to a mammal already suffering from or susceptible to the disease being treated. Those in the incubation phase or the acute phase of disease such as a viral infection, HBV, can be treated with the immunogenic peptides separately or in conjunction with other treatments, as appropriate. In therapeutic .applications, compositions are administered to a patient in an amount sufficient to elicit an effective CTL response to the disease and to cure or at least partially arrest its symptoms and/or complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, the peptide composition, the manner of administration, the stage and Sseverity of the disease being treated, the weight and general S. state of health of the patient, and the judgment of the prescribing physician, but generally range for the initial immunization (that is for therapeutic or prophylactic administration) from about 1.0 g to about 50 mg, preferably 1 pg to 500 pg, most preferably 1 pg to 250 pg followed by boosting dosages of from about 1.0 Mg to 50 mg, preferably 1 pg to 500 pg, and more preferably 1 pg to about 250 pg of peptide pursuant to a boosting regimen over weeks to months depending upon the patient's response and condition by measuring specific CTL activity in the patient's blood. It must be kept in mind that the peptides and compositions of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the peptides, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions.

Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of cytotoxic

T-

lymphocyte stimulatory peptides of the invention sufficient to effectively treat the patient.

.For therapeutic use, administration should begin at the first sign of disease HBV infection), to be followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. In cases of established or chronic disease, such as chronic HBV infection, S. loading doses followed by boosting doses may be required. The elicitation of an effective CTL response during early treatment of an acute disease stage will minimize the possibility of subsequent development of chronic disease such as hepatitis, HBV carrier stage, and ensuing hepatocellular carcinoma.

25 Treatment of an infected mammal with the compositions of the invention may hasten resolution of the disease in acutely afflicted mammals. For those mammals susceptible (or predisposed) to developing chronic disease the compositions of the present invention are particularly useful in methods for preventing the evolution from acute to chronic disease. Where the susceptible individuals are identified prior to or during infection, for instance, as described herein, the composition can be targeted to them, minimizing need for administration to a larger population.

The peptide compositions can also be used for the treatment of chronic or established disease such as viral hepatitis and to stimulate the immune system to eliminate virus-infected cells. Those with chronic hepatitis can be identified as testing positive for virus from about 3-6 months after infection. As individuals may develop chronic

HBV

infection because of an inadequate (or absent) CTL response during the acute phase of their infection, it is important to provide an amount of immuno-potentiating peptide compositions of the invention in a formulation and mode of administration sufficient to effectively stimulate a CTL response. Thus, for treatment of chronic hepatitis, a representative dose is in the range of about 1.0 pg to about 50 mg, preferably 1 pg to 500 pg, most preferably 1 pg to 250 pg followed by boosting dosages of from about 1.0 pg to 50 mg, preferably 1 pg to 500 9g, and more preferably 1 pg to about 250 pg per dose.

Administration should continue until at least clinical 15 symptoms or laboratory indicators indicate that the HBV infection has been eliminated or substantially abated and for a period thereafter. Immunizing doses followed by boosting doses at established intervals, from one to four weeks, may be required, possibly for a prolonged period of time, as 20 necessary to resolve the infection. For the treatment of chronic and carrier HBV infection it may also be desirable to combine the CTL and HTL peptides with other peptides or proteins that induce immune response to other HBV antigens, such as HBsAg.

'5 The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral or local S* administration. Preferably, the pharmaceutical compositions are administered parenterally, intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the HTL and CTL stimulatory peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, methanol, and dissolving agents such as DMSO, etc.

The concentration of HTL and CTL stimulatory peptides :.of the invention in the pharmaceutical formulations can vary widely, from less than about usually at or at least about 10% to as much as 20 to 50% or more by weight, and will S. 15 be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration So.. selected.

Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of 20 sterile Ringer's solution, and 50 mg of peptide. Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example, Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA (1985), which is incorporated herein by reference.

SThe peptides of the invention may also be administered Svia liposomes, which serve to target the peptides to a particular tissue, such as lymphoid tissue, or targeted selectively to infected cells, as well as increase the halflife of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes filled with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol,.such as cholesterol. The selection of lipids is generally guided by consideration of, liposome size and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, Szoka et al., Ann.

Rev. Biophys. Bioen. 9:467 (1980), U.S. Patent Nos.

*4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference. For targeting to the immune cells, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies 20 according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptide compositions of the invention, and more preferably at a concentration of 25%-75%.

For aerosol administration, the HTL and CTL stimulatory peptide compositions are preferably supplied in finely divided form along with a surfactant and propellant.

Typical percentages of peptides are 0.01%-20% by weight, preferably The surfactant must, of course, be nontoxic, and preferably soluble in the propellant.

Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, lecithin for intranasal delivery.

In another aspect the present invention is 15 directed to vaccines which contain as an active ingredient an immunogenically effective amount of a composition of HTL and S:CTL stimulating peptides as described herein. The peptide(s) may be introduced into a mammalian host, including humans, S: linked to its own carrier or as a homopolymer or heteropolymer 0 of active peptide units. Such a polymer has the advantage of increased immunological reaction and, where different peptides are used to make up the polymer, the additional ability to induce antibodies and/or cytotoxic T cells that react with different antigenic determinants of the virus. Useful carriers are well known in the art, and include, e.g., S. •thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly(D-lysine:D-glutamic acid), influenza protein and the like. The vaccines can also contain a physiologically tolerable (acceptable) diluent such as water, phosphate buffered saline, or saline, and further typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, alum, or montanide are materials well known in the art. Upon immunization with a peptide composition as described herein, via injection, aerosol, oral, transdermal or other route, the immune system of the host responds to the vaccine by producing large amounts of CTLs specific for the disease associated antigen, and the host becomes at least partially immune to the disease, HBV infection, or resistant to developing chronic disease.

Vaccine compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk of disease, viral infection, to enhance the patient's own immune response capabilities. Such an amount is defined to be a "immunogenically effective dose." In this use, the precise amounts depend on the patient's state 0 of health, age, the mode of administration, the nature of the formulation, etc.,. The peptides are administered to o. individuals of an appropriate HLA type, for vaccine se- compositions of peptide HBc 18-27, these will be administered to HLA-A2 individuals.

In some instances it may be desirable to combine the Speptide vaccines of the invention with vaccines which induce S: neutralizing antibody responses to the disease,

HBV

particularly to HBV envelope antigens, such as recombinant

HBV

.env-encoded antigens or vaccines prepared from purified plasma 20 preparations obtained from HBV-infected individuals.

A

variety of HBV vaccine preparations have been described, and are based primarily on HBsAg and polypeptide fragments thereof. For examples of vaccines which can be formulated with the peptides of the present invention, see generally, European Patent publications EP 154,902 and EP 291,586, and U.S. Patent Nos. 4,565,697, 4624918, 4,599,230, 4,599,231, ,918, 4,599,231 S4,803,164, 4,882,145, 4,977,092, 5,017,558 and 5,019,386, each of which is incorporated herein by reference. The vaccines can be combined and administered concurrently, or as separate preparations.y, or as separate The peptides may also find use as diagnostic reagents.

For example, a peptide of the invention may be used to determine the susceptibility of a particular individual to a treatment regimen which employs the peptide or related peptides, and thus may be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual. In addition, the peptides may also be 43 used to predict which individuals will be at substantial risk for developing chronic HBV infection.

The following examples are offered by way of illustration, not by way of limitation.

EXAMPLE 1 IDENTIFICATION OF CTL-8PECIFIC HBV EPITOPES merA line of transgenic mice which express a mouse-human chimeric class I molecule was used to define HBV core and surface antigen sequences that represent CTL-specific epitopes.

'The transgenic mouse line 66 obtained from Scripps Clinic and Research Foundation expresses a chimeric class

I

molecule composed of the 1l and a2 domains of human HLA-A2.1 Santigen and the 3 transmembrane and cytoplasmic domains of H- SKb. The transgenic mice were prepared as generally described in Vitiello et al., Exp. Med 173:1007-1015 (1991) which is incorporated herein by reference. When these mice are primed in ivo with the influenza virus, they generate a CTL response that is specific for virtually the same epitopes as Sthose recognized by human influenza-specific CTL. Thus, these transgenic animals can be used to determine HBV epitopes recognized by human T cells.

STo define which sequence regions within HBV surface Sand core proteins represented CTL epitopes, synthetic peptides derived from the two proteins were prepared and tested for their ability to bind to human HLA-A2.1. Binding was determined by the relative capacity of different peptide concentrations to inhibit recognition of A2.1 target cells in the presence of the influenza matrix peptide 57-68 by the CTL line 219, as determined by the inhibition of release of serine esterase from the cells. The 219 line was derived from A2.1 transgenic mice and is specific for the matrix peptide 57-68 in the context of HLA-A2.1.

Briefly, peptides to be assayed for CTL epitopes were dissolved in DMSO at a concentration of 20 mg/ml. Just before the assay, peptides were diluted in RPMI 1640 buffered with pM Hepes and containing .05% BSA (assay media). Fifty microliters of a 200 pg/ml, 66 pg/ml, or 22 pg/ml of peptide solution were added to wells of 96 round-bottomed plates containing 4x10 5 Jurkat A2.1/Kb cells in a volume of 50 p1 of assay media. Plates were incubated for 30 min. at 37C.

Fifty 1p of 2.5 pg/ml solution of the index peptide (matrix peptide 57-68 from PR8 influenza virus) were then added to the cells, followed by 50 1p containing 5x10 4 line 219 CTL, where the concentration of index peptide used was chosen as that which induced 75% serine esterase release from CTL 219, as determined by titration of the peptide. After 4 hours incubation at 37t, plates were centrifuged for 5 min. at 1000 RPM, and 20 pl supernatant transferred to flat-bottomed 96well plates. Esterase activity in the supernatant was measured by adding 180 p1 of a reaction mixture consisting of 0.2M TrisHl pH 8.1, 2.x N-benzyloxycarbonyl-L-Lysine thiobenzyl ester (BLT) and 2.2x10 4 M dithiobis (nitrobenzoic acid). Plates were incubated for 1 hour at 37C and absorbance read at 412 nm. Percent inhibition was calculated by the following formula:

A

412 (test index) peptide

A

412 test peptide alone V inhibition 100 x 100 x 100

A

412 index peptide

A

4 12 no peptide 0 Those peptides which bound to A2.1 and caused more than 24% inhibition of serine esterase release by the cells were assayed in vitro for the ability to restimulate a CTL response from splenocytes derived from HBV primed A2.1 transgenic mice. (Sette, A. et al., J. Immunol. 147:3893 (1991)). HBV priming was performed by injecting A2.1 spleen cells "loaded" with HBV virus as described by Carbone and Bevan, J. Exp. Med. 171:377-387 (1990).

Briefly, red blood cell depleted splenocytes were suspended in .4 ml of a solution composed of 200 1p of HBV purified virus and 200 pi of a 2 x hypertonic solution (0.5 M sucrose, 10% /v polyethylene glycol 00, 0 mM Hepes, pH 7.2, in RPMI 1640 medium), for 10 min. at 37C. The cell suspension was then rapidly diluted in prewarmed hypotonic media (60% HBSS and 40% water), incubated for 2 min. at 37, pelleted, washed twice in HBSS and irradiated (1,000 rad.) Mice were then injected with 5.0x10 6 loaded cells in a volume of 200 pl Mice were boosted with HBV-loaded spleen cells days later.

After about 2 weeks, spleen cells from primed mice (5x10 6 cells/well in 24 well plates) were cultured with 4 different mixtures of syngeneic irradiated (3000 rads)

LPS

Sblasts (2x10 6 cells/well) that had been independently coated with 13 different peptides. Coating was achieved by incubating aliquots of 25x10 6 LPS blasts in tubes each with .00 g of one of the 13 HBV synthetic peptides in one mL for S1-2 hrs at 37; the contents of the different tubes were then pooled to give 4 mixtures.

ixture No.tide No Peptide Location 1 800.04 HBenv47-63 802.01 HBcll-27 802.06 802.06 HBc162-17 6 80102 HBenv141-1 57 99.02 HBenv194-213 802.03 HBc91-110 S799.09 HBenv329-3 48 799.10 HBenv349-36 8 802.04 380204 HBclll-125 799.04 HBenv234-25 3 799.05 HBenv246-2 6 799.08 HBenv309-32 8 800.05 HBenv63-77 The mixture of cells was washed once, diluted at the required concentration and plated. The medium used for the cultures was RPMI 1640 supplemented with 10% FCS, 50g/ml gentamicin, Op c 0g/ml gentamicin, 46 2mM glutamine and 5x10- 5 M 2 -mercaptoethanol (R10). After nine days, effector cells were assayed for cytotoxicity against Jurkat

A

2 /kb target cells in the presence of different peptide mixtures corresponding to those used in the cultures.

The results obtained are shown in Fig. 1 panels, A through

D.

The effector cells (0.2x106 cells/well) obtained from these cultures were restimulated with irradiated (20,000 rads), peptide-coated Jurkat A2/Kb cells (.2x10 6 cells/well) if the presence of 3x10 6 feeder cells/well (C57BL/6 irradiated spleen cells) in Rio supplemented with 5%-rat ConA supernatant.

After 6 days, these effector cells were assayed for cytotoxicity against 51 Cr labeled Jurkat

A

2 /Kb target cells in the presence of the 13 individual peptides. Peptides that induced CTL lysis of Jurkat A 2 /Kb target cells above background (Fig. 1, panels E through H) HBenv 47-63, HBc 11-27 (panel E) HBenv 141-157, HBenv 194-213, HBc 91-110 (panel HBenv 329-348 and 349-368 (panel G) and HBenv 309- 328 (panel H) were independently used to restimulate the effector cells generated with the peptide mixtures. After 6d in culture, the effector cells were tested for cytotoxicity against 5 1 Cr Jurkat

A

2 /Kb cells in the presence of the peptide used for the restimulation (Fig. The set of experiments, outlined in this example allow us to determine that HBV peptides HBc 11-27 (Fig. 1 panels A,E; Fig. 2 panel J) HBc 91- 110 (Fig. 1 panels B,F; Fig. 2 panel HBenv 329-348 (Fig. 1 panels C,G; Fig. 2 panel N) HBenv 349-368 (Fig. 1 panels C,G; •Fig. 2 panel 0) and HBenv 309-328 (Fig. 1 panels D,H; Fig. 2 panel P) clearly represent CTL epitopes.

EXAMPLE 1A INDUCTION OF OVALBUMIN-SPECIFIC

CTL

RESPONSE IN MICE B6 mice were injected with 10, 50, or 200 pg of ovalbumin in HBSS intravenously, intraperitoneally and subcutaneously with 10, 50, 200 pg ovalbumin subcutaneously in IFA. Ten days later, splenocytes from primed animals were stimulated in vitro with irradiated EG7 cells (EL-4 cells transfected with OVA). Six days later, the effector cells were tested for cytolytic activity against 51 Cr labelled EL-4 and EG7 cells. No CTL activity was induced by injection of ovalbumin in HBSS either intravenously or interperitoneally.

Some CTL induction was seen at the 200 dg dose for subcutaneous injection of ovalbumin in HBSS. Strong

CTL

in vivo induction was seen when ovalbumin was administered with IFA, optimal induction occurred with the 0 g dose given subcutaneously. d o s e e EXAMPLE 2 INDUCTION OF A2.1-RESTRICTED

CTL

BY SUBCUTANEOUS PRIMING WITH PURIFIED

HBV

15 IN INCOMPLETE PREUND'S ADJUVANT (IFA) :Injection of ovalbumin (OVA) in IFA subcutaneously induces an ovalbumin-specific CTL response in mice, while injection of OVA either i.v. r i.p. generally does not lead to the generation of CTL. This technique was used to induce HBV-specific CTL in A2.1 transgenic mice.

SPriming and In Vitro Restimulation: A2.1/Kb transgenic mice were injected with 100 microliters of an emulsion of purified HBV virus in incomplete Freund's adjuvant (IFA). This emulsion was prepared by mixing purified HBV (1 mg protein/ml) diluted 1:5 in HBSS with an equal volume of IFA. Seven days after priming, splenocytes (5x10 6 cells/well in a 24 well plate) obtained from these animals were restimulated with syngeneic irradiated LPS blasts (2x10 6 /well) coated with each of the following peptides: 799.09 HBenv 329-348 802.03 HBc 91-110 875.20 HBenv 335-343 883.02 HBc 92-101 875.21 HBenv 338-347 883.03 HBc 93-102 799.10 HBenv 349-368 875.15 HBc 18-27 884.01 HBenv 348-357 875.18 HBc 107-115 884.02 HBenv 349-358 875.19 HBc 139-148 48 These peptides were chosen because: 1) They had been defined as containing CTL epitopes in Example I (peptides 799.10, 799.09, 802.03); 2) they represent truncations of peptides defined in Example I that are recognized by the CTL raised against the larger epitopes peptides 875.15, 884.02, 883.02, 883.03); or 3) they contain the A2.1 binding motif as described by Falk et al. (Nature 351:290-296 (1991)), leucine or methionine in position 2, and either leucine or valine in position 9 or valine in position 10, peptides 884.01, 875.20, 875.21, 875.18 and 875.19). Coating was achieved by incubating 50 pg of each individual peptide with 12x10 6 LPS blasts in a volume of 0.4 ml of RPMI medium supplemented with 10% FCS for lh at 37C. The cells were washed once. After 6 days, effector cells were assayed for 15 cytotoxicity against 51Cr labelled Jurkat A2/Kb cells in the presence of the appropriate peptides. The results are shown in Fig. 3.

These effector cells (.2x10 6 cells/well) were restimulated at weekly intervals. For the first restimulation, peptide-coated LPS blasts were used, followed by peptide-coated Jurkat A2.1/Kb cells. Six days after restimulation, effector cells were assayed for cytotoxicity S. against 51 Cr labelled Jurkat A2/K b target cells in the presence of the appropriate peptides. The results obtained are shown in Fig. 4.

Peptides clearly able to induce in vitro CTL from splenocytes of HBV-primed mice are Fig. 3 and 4, panel A: HBc S18-27; Fig. 3 and 4, panel B: HBenv 349-368; Fig. 3 and 4, panel D: HBenv 349-358; Fig. 3 and 4, panel F: HBenv 329-348; Fig. 3 and 4, panel I: HBc 91-110; Fig. 3 and 4, panel J: HBc 92-102; and Fig. 3 and 4, panel K: HBc 93-102. Truncation peptides recognized by CTL raised against the larger peptide and as such should contain at least part of a CTL epitope are: Fig. 3F, 4F: HBenv 335-343 and HBenv 338-347.

4 9 EXAMPLE 2A OTHER HBV CTL EPITOPEB The following peptides were identified following procedures disclosed in pending U.S. Patent Applications Serial No. 08/159,339 and 08/073,205, incorporated herein by reference.

CTL Epitopes Position Sequience Seci ID No.- HBV POL 561 FLLSLGIHL.C00H HBV POL 61 GLYSSTVPV.COOH 106 HBV POL 411 NLSWLSLDV.CO0H 107 HBV POL 491 HLYSHPIIL-COOH 108 EXAMPLE 3 .SYNTHESIS OP PEPTIDES Peptides were synthesized on an Applied Biosystems (Foster City, CA) 430A peptides synthesizer using Fmoc protected amino acids and 2 I(lH-Benzotriazol.l..yl).,1, 3 3 tetraethylroniu hexaluorpusphate (HBTU) esters foramn acid activation. Each amino acid was routinely triple coupled. Fmoc protected aioacids and H-ydroxybenzotriazole were purchased from Burdick and Jackson. H-BTU was purchased from Richelieu Biotechnologies (St-Hyacinthe, Canada).- Piperidine and trifluoroacetic acid, acetic anhydride, and .ethanedithiol were-purchased from Sigma Chemical Corporation.

a. Peptide Phe-LeuProSerAPhe-h-r-e-a-H(e.I No. 4] L-Valine coupled to SasrinO resin (Bachem Biosciences) was loaded into the peptide synthesis reaction vessel and washed one time with N-ehlyrldn (UNP). The following operations were then sequentially performed: 1. The Fmoc protecting group was removed by treatment of the resin bound amino acid with 25% piperidine in NMP.

2. The resin was washed 5 times with NMP.

3. A mixture containing Fmoc-serine, diisopropylethylamine, HBTU and NMP was added to the reaction vessel and allowed to react for 30 minutes, under vortex agitation.

4. The solvent was drained, and the resin was washed three times with

NMP.

Steps and were repeated two more times.

6. The resin was washed four more times with

NMP.

Steps 1-6 were repeated for each amino acid of the peptide.

Following the final coupling cycle, the resin-bound peptide was deproteced by reaction with 25% piperidine in NMP, washed :7 times with NMP, and washed 2 times with dichloromethane.

The resin was dried in vacuo for 24 hours. The peptide was cleaved from the Sasrin O resin by treatment with trifluoroacetic acid containing 2.5% ethanedithiol and water. The polystyrene resin was separated from the trifluoroacetic acid solution by filtration. Trifluoroacetic acid was removed by evaporation in vacuo. The crude peptide was triturated with diethylether and dissolved in water. The water was removed by lyophilization. The peptide was then purified by reverse phase HPLC on a C 8 column (VYDAC) using a gradient of acetonitrile, water, each containing 0.1% TFA as modifier.

b. Peptide (Pal)2LysSerSerPhe-LeuPro-Ser-Asp-he-Phe-Po- Ser-Val-OH [Seq. ID No. 109] SThe resin bound peptide described in section a was extended by the addition of two serine residues according to the above described procedure. The following operations were then performed: 1. The Fmoc protecting group was removed by treatment of the resin bound amino acid with 25% piperidine in NMP.

2. The resin was washed 5 times with NMP.

3. Bis-Fmoc-Lysine was converted to the corresponding symmetrical anhydride by treatment with 51 diisopropylcarbodiimide in NMP. The resin bound peptide was allowed to react with the resulting anhydride.

4. The resin was washed 5 times with NMP.

The Fmoc protecting group was removed by treatment of the resin bound amino acid with 25% piperidine in NMP.

6. Palmitic acid was reacted with hydroxybenzotriazole and diisopropylcarbodiimide in NMP. The resin bound peptide was allowed to react with the resulting solution.

7. The resin was washed 5 times with NMP.

Finally, the peptide was cleaved from the resin as described above.

c. Peptide Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile- Thr-Glu-Phe-Leu-Pro-Ser-Asp-Phe-Phe-Pro-Ser-Val-OH [Seq. ID No. 110). The resin bound peptide described in section a was 15 chain extended by the addition of Glu, Thr, Ile, Gly, Ile, Phe, Lys, Ser, Asn, Ala, Lys, Ile, Tyr, and Gin residues, according to the procedure described in Section a. Cleavage and purification were performed as described above.

d. Peptide Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile- 20 Thr-Glu-Ala-Ala-Ala-Phe-Leu-Pro-Ser-Asp-Phe-Phe-Pro-Ser-Val-OH [Seq. ID No. 111]. The resin bound peptide described in section a was chain extended by the sequential addition of Ala, Ala, Ala, Glu, Thr, Ile, Gly, Ile, Phe, Lys, Ser, Asn, Ala, Lys, Ile, Tyr, and Gln residues, according to the procedure described in section a. Cleavage and purification were performed as described above.

e. Peptide Ac-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile- Thr-Glu-Ala-Ala-Ala-Phe-Leu-Pro-Ser-Asp-Phe-Phe-Pro-Ser-Val-OH [Seq. ID No. 112]. The resin bound peptide described in Section d was acetylated by reaction with acetic anhydride in NMP. Cleavage and purification were performed as described above.

EXAMPLE 4 INDUCTION OF CTL BY COMBINING CTL AND T-HELPER

EPITOPES

This example describes experiments which define the relative in vivo HBV-specific CTL priming efficiency of peptides expressing HBV CTL epitopes alone, CTL epitopes mixed with peptides containing T helper epitopes or CTL epitopes physically linked to T helper epitopes.

Transgenic mice (HLA-A2.1/Kb) were primed subcutaneously (base of tail) with 100 pg of peptide 875.23 (Iab-restricted helper epitope HBc 128-140 TPPAYRPPNAPIL [Seq ID No. 113]) in complete Freund's adjuvant (CFA). Nine days later each of the following peptides were injected subcutaneously into two unprimed and two helper-primed mice, 100 pg/mouse in incomplete Freund's adjuvant

(IFA).

Peptide T Helper (HBc 128-140) CTL (HBc 18-27) Seq ID No.

0 20 1. 875.23 TPPAYRPPNAPIL 1 2. 875.15 113 2. 875.15 FLPSDFFPSV 4 3. 875.23+875.15 TPPAYRPPNAPIL FLPSDFFPSV 113 4 4. 902.01

TPPAYRPPNAPILFLPSDFFPSV-NH

2 114 902.02 TPPAYRPPNAPILAAAFLPSDFFPSV-NH2 115 6. No peptide Three weeks after priming with the CTL epitope, splenocytes were in vitro restimulated with LPS blasts coated with HBc 18-27 (coating was achieved by incubating 30 x 106 LPS blasts with 100 pg of HBcl8-27 in one ml of medium; after 1-2 hr at 37 0 C, the cells were washed). After 6 days, effector cells were assayed for lytic activity against s 5 Cr labelled Jurkat

A

2 /Kb target cells in the presence or absence of HBcl8-27.

The results showed that in 50% of the animals studied in which the T helper and CTL epitope peptides were simply mixed not linked) and administered in an immunizing dose, induction of some detectible antigen-specific

CTL

activity above the level of background killing was seen. An example of the response detected is shown in Fig. 7.

Surprisingly, when animals were primed with the T helper epitope linked to the CTL epitope, 100% showed evidence of specific CTL priming (Fig. the magnitude of which was greater than that detected when the epitopes were administered non-linked (Fig. Quite unexpectedly, as shown in Fig. 9, it was found that linking the T helper and CTL epitopes via an alanine-alanine-alanine spacer T helper-AAA-CTL) resulted in the induction of specific CTL activity greater than that detected by linking the T helper and CTL determinants alone. Priming with the T helper peptide or CTL peptide alone did not induce HBc-specific CTL (Figs. 5 and 6).

Also, prior immunization of animals to induce T helper- 15 specific immunity did not appear to be essential for priming for CTL using either the T helper and CTL mixture or the T helper-CTL conjugate, since immunization was detected when naive animals were primed with the appropriate conjugate (Figs. 10A and B).

EXAMPLE 4A RECOGNITION OF GENERATION OF ENDOGENOUS .PROCESSED ANTIGENS AFTER PRIMING This example discloses that CTL inducted by in vivo priming with peptide (as disclosed in Example 4) recognize endogenously synthesized antigens.

A fraction of the effector cells from the procedure disclosed in Example 4 whose CTL function is represented in Figs. 7, 8 and 9, were restimulated in vitro using HBcAg 18-27-coated JA2.1/K b stimulator cells. Six days later, effector cells were assayed for cytotoxicity and the cell lines that contained HBcAg 1 8 -27-specific cytotoxic activity were further restimulated. Six days later, these cell lines were tested for cytotoxic activity on 51 Cr labeled Jy Sl target cells in the absence or presence of HBcAg 18-27 and on 1 Cr labeled Jy core target cells, as demonstrated in the following table.

CTL lines obtained from animals primed with HBcAG 18-27 peptide derived recognize endogenously synthesized HBc antigen.

Animal No.

H21 H5-2 H6-3 H4-6 H4-2 In vivo Primed Day 0 Jv S 1 902.01 902.02 875.15 +875.23 0.1:1 0.5:1 2:1 8:1 0.3:1 1:1 4.5:1 18:1 0.4:1 1.5:1 6:1 24:1 Jy Core NoAg 875.15 43 66 73 74 78 77 82 67 78 79 29 51 63 62 74 77

S

a. 875.23 875.23 902.01 0.15:1 0.6:1 2.5:1 10:1 902.02 0.6:1 2.5:1 10:1 40:1 All CTL lines were specific for HBcAg 18-27 core albeit to a different extent. The much and recognized Jy hiaher level nf

S

a *5 lysis obtained in the presence of exogenously added peptide as compared to endogenously synthesized, is explained by the relatively greater concentration of peptide that can be added exogenously. Because CTL lines derived from both animals primed with 902.02 had the highest affinity for endogenous antigen (producing a 35% and 37% specific lysis of Jy core at the E:T of 1:1 and 0.6:1, respectively), it is demonstrated that this construct, T helper epitope AAA HBcAg 18-27 possesses immunogenic characteristics suitable for induction of cells that recognize HBV infected cells.

EXAMPLE INDUCTION OF A2.1-RESTRICTED

CTL-BPECIFIC

FOR HBenv 36 0 3 6 8 A2/Kb transgenic mice were injected with 100 microliters of an emulsion of 100 pg HBenv 360 36 8 and 100 pg HBc 128 140 helper epitope in incomplete Freund's adjuvant (IFA). (This emulsion was prepared by mixing 500 pg of both peptides in PBS with an equal volume of IFA.) Twenty-one days after priming, splenocytes (5x10 6 cells/well in a 24-well plate) obtained from these animals were restimulated with syngeneic LPS blasts (2x10 6 /well) coated with the peptide HBenv 360 368 These effector cells (0.2x10 6 /well) were restimulated at weekly intervals. For the first and second rs* 15 restimulations, HBenv 3 6 0- 3 6 8 coated LPS blasts were used, followed by HBenv 36 0- 368 coated Jurkat A2.1/K b cells. Six days after restimulation, effector cells were assayed for cytotoxicity against 51Cr labelled Jurkat A2/Kb target cells in the presence and absence of HBenv 360 368 (see Fig. 11) 20 Therefore, HBenv 360 _36 8 is a CTL specific epitope.

EXAMPLE 6 o TESTING OF LINKED TETANUS TOXOID T HELPER AND HBc CYTOTOXIC T CELL EPITOPES FOR in vivo PRIMING Transgenic mice (HLA-A2-l/kb) were primed subcutaneously (base of the tail) with 200 mg (0.07 mM) of peptide 934.02.

Tetanus Toxoid 830-843 HBV core 18-27 Seq Peptide T Helper Epitope Linker CTL Epitope ID No.

934.02 AC-QYIKANSKFIGITE AAA FLPSDFFPSV 112 Three weeks after priming, splenocytes were restimulated in vitro with LPS blasts coated with HBc 18-27 (as described in example After 7 days cells were restimulated with jurkat A2/Kb cells coated with HBc 18-27 (as described in Example I) After 6 days these effector cells were assayed for Cytotoxicity against 51Cr labeled jurkat A2/Kb target cells in the presence or absence of HBc 18-27, Jy target cells in the presence or absence of HBc 18-27 and Jy cells transfected with HBV core. The results, shown in Fig.

12, indicate that peptide 934.02 effectively induces

CTL

specific for HBc 18-27. Moreover, these CTL recognize and kill endogenously presented antigen (Jy core).

EXAMPLE 7 COMPARISON OF CTL IMMUNITY

INDUCED

BY PEPTIDE

IMMUNIZATION

Various modifications and formulations of an antigenic CTL peptide were tested in an effort to enhance its immunogenicity. BALD/c mice were primed subcutaneously in the base of the tail with one of the following peptides or peptide mixtures: .Peptide or Dose *.:Number Peptides ym/mouse Formulation 20 932.01 CTL (Flu NP 147-155) 0.1,0.01 Saline, Alum, IFA *TYQRTRALV [Seq ID No. 1161 *25 932.07 (PAM) KSS-CTL 0.1.0.01 Saline, Alum, IFA (PRi2KSSTYQTPALV [Seq ID No. 117) 932.01 CTL T helper (OVA 323-336) 0.1,0.01 ea. Saline, Alum, IFA +577.01 TYQRTRALV-ISQAVHAJJAEINE [Seq ID No. 1181 932 .07 (PAM) 2 KSS-CTL T helper 0.1,0.01 Saline, Alum, IFA +577.01 (PA) 2KSSTYQRTPLVIQAHAAEINE [Seq ID No. 119] 932.02 T helper-CTL 0.1.0.01 Saline, Alum, IFA ISQAVHAARAEINE.YQRTRALV [Seq ID No. 120] 932.04

(PAM)

2 KSS-T helper-CTL 0.1,0.01 Saline. Alum, IFA (P)2KSSIAVHAAHAE- E-YQRTRALV [Seq ID No. 1211 932 .03 T helper-AAJ-CTL 0.1,0.01 Saline, Alum, IFA ISQAVHAA1AEINE AA -TYQRTPALV [Seq ID No. 122) 932.05

(PAM)

2 KSS-T helper-AAACrL 0.1.0.01 Saline, Alum, IFA (PM S SAHAAEN A-TQT-L [Seq ID No. 1051 Three weeks after immunization splenocytes were removed and stimulated in vitro with the flu 147-155 peptide.

CTL activity was assayed one week later Using 5 1 Cr-labeled Blo.D2 fibroblasts as targets. Target cells were tested in the absence of antigen, in the presence of Flu 147-155 peptide or following infection with influenza PR8 virus.

Representative results obtained from one of four independently run experiments are summarized in the following table.

CTL Immunogenicity of Various Modifications and Formulations of PR8-NP 148-155 -Formulation Saline Alum

IFA

Peptide 0.1 0.01 0.1 0.01 0.1 932.01 -b 932.07 932.01 577.01 932.07 577.01 932.04. S 9320 932.03 932.05 25 a Dose (AM/mouse) b CTL immunogenicity of various modifications and Sformulations of NP 148-155 (nucleoprotein of PR8 influenza virus). Each symbol represents the result obtained from ".30 spleen cells derived from a single Balb/c mouse and reflects the effector to target ratio required to induce antigen specific lysis of 5 1Cr labeled B 1 0 D2 target cells in the presence of ND 1 4 8 1 5 5 peptide; E:T below 10:1; E+T between 10+1 and 30:1; E:T greater than 30:1; not achieved at any E:T tested.

Thus, the results were as follows: IFA formulation: The HTL-CTL linked peptides either lipidated 932.04 and 932.05) or unlipidated (932.02 and 932.03) were all very active, inducing good CTL activity in all animals at both injection doses. The mixture of HTL (577.01) and CTL (932.01) peptides also induced CTL activity in all animals injected. The CTL peptide (932.01) demonstrated good activity at the 100 nmoles dose, however, only one out of four animals responded at the 10 nmoles dose. The lipidated

CTL

peptide (932.07) was completely inactive but some activity, 58 albeit low, was observed when peptide 932.07 was mixed with the HTL peptide (577.01).

Baline formulation: The PAM 2 -HTL-CTL linked peptides (932.04 and 932.05) induced good CTL activity in all animals injected and were far superior to all other peptide or peptide combinations tested. The unlipidated versions of those peptides (932.02 and 932.03) were totally ineffective at the nmoles dose and only marginally active at the 100 nmoles dose underlying the importance of peptide lipidation for priming activity of saline formulations. For the other peptide combinations tested lipidation appeared to affect activity in the opposite direction as it did in IFA in that the CTL peptide (932.01) was completely inactive but its lipidated version (932.07) induced CTL activity in some of the animals injected. The activity of both peptides improved when mixed with the HTL peptide.

Alum formulation: The results obtained were similar to the ones obtained with peptides formulated in saline with the exception of peptide 932.07 that was inactive when injected in 0- alum.

Accordingly, the constructs

(PAM

2 KSS-T helper-CTL and

(PAM)

2 KSS-T helper-AAA-CTL were superior when injected in saline or Alum compared to all of the other combinations. The peptides T helper-CTL and T helper-AAA-CTL were superior to mixing the T helper CTL non-linked) and worked well in IFA, but not well in saline or Alum. Thus, for vaccine development, linking the (PAM) 2 KSS to a T helper peptide which is linked to the CTL peptide appears to be advantageous for inducing CTL immunity.

EXAMPLE 8 DEFINITION OF THE MINIMAL OPTIMAL

SEQUENCE

WITH THE CTL PEPTIDE EPITOPE 799.09 Transgenic mice (A2-1/Kb) were primed with HBV virus in IFA substantially as described in Example II. Seven days after priming, splenocytes obtained from these animals were restimulated with syngeneic irradiated LPS blasts coated with peptide 799.09 (as described in Example After 6 cycles of restimulation with 799.09 coated cells (as described in Example II) the effector cells were cloned by limiting dilution using syngeneic irradiated LPS blasts coated with peptide 799.09 (.2x10 6 cells/well of a 96 well plate) and media supplemented with 10% rat Con A supernatant. Two CTL lines were obtained, line 110 and 113 that killed JA2Kb target 15 cells coated with peptide 799.09. These lines were tested on a panel of 799.09 N-terminus truncated peptides and overlapping 9mers and 10mers covering the entire 799.09 sequence. As shown in Fig. 13 panel A the minimal N-terminus truncated peptide recognized by line 113 and 110 were respectively peptides HBV env. 333-348 (923-09) and 335-348 (923.07). Fig. 13 panel B shows that none of the 9mers or l0mers were recognized by line 113 implicating a longer peptide as the minimal sequence required for recognition by this CTL line. The minimal sequence recognized by line 110 is represented by peptides HBV env. 333-341 (923.26) and HBV env 335-343 (923.22) indicating that possibly two distinct but overlapping peptides can serve as antigenic determinants for 799.09 specific CTL.

EXAMPLE 9 PRIMING OF PEPTIDE 934.05 ("CY-1899") IN TRANSGENIC

MICE

This example discloses that peptide 934.02 tetanus toxoid 830 -843-AAA-HBcAg 18-27) when modified by linking

(PAM)

2 KSS to the tetanus toxoid end (resulting in "CY- 1899") and formulated in DMSO/saline induced CTLs in transgenic mice.

Lipopeptides were prepared by coupling the appropriate fatty acid to the amino terminus of the resin bound peptide.

A

typical procedure was as follows: A dichloromethane solution of a 4 fold excess of a pre-formed symmetrical anhydride of the appropriate fatty acid was added to the resin an the mixture was allowed to react for 2 hrs. The resin was washed with dichloromethane and dried. The resin was then treated with trifluoroacetic acid in the presence of appropriate scavengers 5% water] for 60 minutes at 20 0 C. After evaporation of excess trifluoroacetic acid, the crude peptide was washed with diethyl ether, dissolved in methanol and precipitated by the addition of water. The peptide was collected by filtration and dried.

Preparation of peptides for immunization: Peptides were routinely resuspended in DMSO at a concentration of mg/ml. Before use, peptides were prepared at the required concentration by dilution in saline or the appropriate medium.

For selected experiments, CY-1899 and 934.02 were also Sprepared as saline suspensions (in the complete absence of

DMSO).

Immunization procedures: Transgenic mice were primed subcutaneously (base of the tail) with 0.1 ml of the appropriate peptide formulated in saline, or DMSO/saline.

Media: a. RPMI-1640 supplemented with 10% fetal calf serum

(FCS)

2 mM Glutamine, 50 pg/ml Gentamicin and 5x10- 2 -mercaptoethanol served as culture medium and will be o* referred to as R10 medium.

Sb. RPMI-1640 containing 25 mM HEPES buffer and supplemented with 2% (FCS) was used as cell washing medium.

Cell lines: Jurkat A2.1/Kb is a stable transfectant of the human T cell leukemia line, Jurkat. Jurkat A2.1/Kb cells were routinely grown in R10 medium supplemented with 400 pg/ml of G418.

LPS-activated lymphoblasts: Splenocytes obtained from A2.1/Kb transgenic mice were resuspended at a concentration of 61 l-l.5x10 6 /ml in R10 medium supplemented with 25 pg/ml LPS and 7 pg/ml dextran sulfate in 75 cm 2 tissue culture flasks.

After 72 hr at 370C, the lymphoblasts were collected for use by centrifugation.

Peptide coating of lymphoblasts: Peptide coating of the LPS activated lymphoblasts was achieved by incubating 30x10 6 irradiated (3000 rads) lymphoblasts with 100 pg of peptide in 1 ml of R10 medium for 1 hr at 370C. Cells were then washed once and resuspended in R10 medium at the desired concentration.

In vitro CTL activation: One to four weeks after priming spleen cells (30x10 6 cells/flask) were co-cultured at 370C with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10x10 6 cells/flask) in 10 ml of R10 15 flask. After 6 days, the effector cells were harvested and assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0-1.5x10 6 were incubated at 37 0 C in the presence of 200 1p S" of sodium 51 Cr chromate. After 60 minutes, cells were washed three times and resuspended in RIO medium. Peptide 875.15 was added where required at a concentration of 1 pg/ml. For the assay, 10 4 51 Cr-labeled target cells were added to different concentrations of effector cells (final volume of 200 p1) in U-bottom 96-well plates. After a 6 hour incubation period at 370C, a 0.1 ml aliquot of supernatant was removed from each well and radioactivity was determined in a Micromedic .automatic gamma counter. The percent specific lysis was Sdetermined by the formula: percent specific release 100x S- (experimental release spontaneous release)/(maximum release spontaneous release). For the purpose of easy comparison between separate CTL assays run under the same conditions, Cr release data was expressed as lytic units/10 6 cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 Jurkat A2.1/K b target cells in a 6 hour 51 Cr release assay. To obtain specific lytic units/10 6 the lytic units/10 6 obtained in the absence of peptide is subtracted from the lytic units/10 6 obtained in the presence of peptide. For example, if 30% 51 Cr release is obtained at the E:T of 50:1 5x10 5 effector cells for 10,000 targets) in the absence of peptide and 5:1 5x10 4 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: (1Xl06+5xlO4)-(1X1O+5x10x5) 18LU/106.

RESULTS:

The results indicated that CY 1899 tetanus toxoid 8 3 0- 8 43-AAA-HBcAg 18-27) formulated in DMSO/saline is a strong immunogenic preparation inducing a good CTL response in all animals injected (six out of six).

CY 1899 in saline (without prior solubilization induced a poor CTL response in that only one of six animals 15 injected had detectible CTL activity.

Peptide 934.02 in saline induced a marginal

CTL

response in that only one of the four animals tested had detectable CTL activity.

S" No CTL activity was detected in any of the animals injected with peptide 934.02 in DMSO/saline.

CTL induced by CY-1899 were shown to be able to kill cells expressing endogenous HBc antigen.

EXAMPLE THE EFFECT OF LIPIDATION OF T HELPER EPITOPE ON CTL INDUCTION This example discloses that the desired CTL activity is induced in the absence of adjuvant (IFA or Alum) by lipidation of the T-Helper epitope where the T-Helper epitope and the CTL epitope are not linked.

The effect of lipidation of the HTL peptide epitope on the immunogenicity of PR8-NP 147-155 CTL peptide epitope was determined. Peptides used in this Example were as follows: 63 ID# Description Sequence Seq ID No.

932-01 CTL TYQRTRALV (PR8-NP 147-155) 116 932.07

(PAM)

2 -CTL

(PAM)

2 KSSTYQRTRALV 117 577.01 HTL ISQAVHAAHAEINE 98 932.06

(PAM)

2 -HTL (PAM) 2KSS ISQAVHAAHAEINE 123 932.04 or (PAM)2-HTL-CTL (PAM) 2KSSISQAVHAAHAEINETYQRTRALV 124 Balb/c mice were injected with the different combinations indicated above of HTL (100 nmoles/mouse) and CTL (10 nmoles/mouse) peptide epitope in saline. Four animals were injected with each different preparation. Ten days after immunization, splenocytes were stimulated in vitro with NP 147-155 peptide. CTL activity was assayed 6 days later using Cr-labeled B10.D2 fibroblasts as targets in the absence or 15 presence of NP 147-155 peptide. The data are expressed in lytic units/10 6 cells. One lytic unit is arbitrarily defined as the number of lymphocytes required to achieve 30% lysis of 5000 B10.D2 5 1 Cr-labeled target cells within 6 hours, in the absence or presence of NP 147-155 peptide. Each number 20 represents the specific CTL activity (LU 30/106 cells obtained in the presence of NP 147-155-LU 30/106 cells obtained in the absence of NP 147-0155) from an individual mouse.

64 Effect of HTL peptide epitope and peptide liquidation on the imiunogenicity of PR8-NP 147-155 CTL peptide epitope Priming Peptide Cytel ID Number 932.01 Description LU (Geometric Mean) CT

L

0 (a) lx/+1 .4) 932.01+ 577.01 CTL+ HTL 932.07 (PAMl) 2

-CTL

0 lx/+1 .7) 0 0 0 15X/+2 .2) a 932.07+ 577 .01 (PAN) 2

-CTL+HTL

1. 1 932.01+ 932.06 CTL+ (PAM) 2

-HTL

lX/+7 .2) 9.3 74.1 1.7 69. (16. 9x/+6. 1) 932.07+ 932.06 932.04 (PANM) 2 CTL+ PAM) 2

-I-TL

(PAM) 2

-HTL-CTL

11.7 3x/+6.1) 4.7 2 21 1.2 9x/+3.5) tI The results obtained indicate that, 1) unlipidated peptides (932.01 and 932.01 577.01) are ineffective at the concentrations tested when injected in saline; 2) the presence of both the HTL and the CTL peptide epitope and the lipidation of at least one peptide is necessary for CTL induction when the peptides are formulated in saline.

The preferred combination appeared to be the lipidated HTL peptides unlinked to the unlipidated

CTL

peptide. Thus, a titration of the CTL epitope and the lipidated HTL epitope in mixtures was performed the titration of (PAM) 2 -HTL peptide epitope and CTL peptide epitope in mixtures).

Balb/c mice were injected with 10 nmoles of the linked [(PAM) 2 -HTL-CTL] peptide immunogen and with mixtures 15 containing 0, 1, 10, or 100 nmoles CTL epitope (932.01) and 0, 1, 10, or 100 nmoles PAM-helper epitope (932.06) per mouse as indicated in the table. Four animals were injected with each different preparation. Fourteen days after immunization, splenocytes were removed and stimulated in vitro with the NP 147-155 peptide. CTL activity was assayed 6 days later using S:51Cr-labeled B10.D2 fibroblasts as targets. Target cells were tested in the absence or presence of antigen (NP 147-155 peptide). The data are expressed in lytic units/10 6 cells.

ne lytic unit is arbitrarily defined as the number of lymphocytes required to achieve 30% lysis of 5000 B10.D2 1 Cr-labeled target cells within 6 hours, in the absence or presence of antigen (NP 147-155 peptide). Each number Srepresents the specific CTL activity (LU 30/106 cells; obtained in the presence of NP 147-155 LU 30/106 cells obtained in the absence of NP 147 155) from an individual mouse.

-Titration of (PAM) 2 -HTL peptide epitope and CTL peptide epitope contained in mixtures ninoles (PAM) 2

HTL

epitope 932.06 0 LU 30 Geo Mean nmoles CTL epitope, 932.01 2. 10 LU 30 LU 30 Geo Mean Geo Mean 100 LU Geo Mean 3.8(a) 0.8 0.3 1.3 3.8 23.9 2.6 0.2 2 6X/+7. .2 >58 29. 5 2.2 53. 5 2 1. 2X/+4. .7 26 61 63 >54 48. 2X/+1. 5 2.5 4.4 0.2 4 .4 1. 8x/+4. 4 50 20.3 7.3 27.5 21. 2x/+2. .2 69 >58 >41 7.9 33 7 X/ +2 7 3 7.4 2.6 2.6 3. 5x/+1. 7 68 53 >64 >68 62. 9x/+1. 1 64 >64 73 62 65. 6x/+1. 1 100 1.5 0.8 0.7 0 0. 5x/+3. 2 17 .8 25.4 3x/+2 1 (PAM) 2

-HTL-

CTL65 ninoles 1053.05 The results obtained indicate that optimal

CTL

induction comparable to that achieved by immunization with 10 nmoles of the linked immunogen 1053.05) is achieved with mixtures containing 100 nmoles of the 932.06

[(PAM)

2

HTL]

peptide and 1, 10 or 100 nmoles of the 932.01 (CTL) peptide.

In the presence of 10 nmoles of 932.06, optimal CTL induction is achieved by the mixture containing 100 nmoles of 932.01 while a slight decrease was observed in the mixtures containing either 10 or 1 nmoles of the CTL peptide.

Lipidated T-Helper Epitopes with Multiple CTL Epitopes.

By following the procedures described in this 15 Example 10, a composition of matter comprising of a lipidated HTL admixed with multiple CTL epitopes can be prepared.

Optionally, the CTL epitopes can be packaged in different vials from the HTL epitope(s) e EXAMPLE 11 INDUCTION OF SPECIFIC CTL RESPONSE IN HUMANS The human clinical trial for CY-1899 was set up as an IND Phase I dose escalation study 50 and 500 yg) and was carried out in a single center as a randomized doubleblind placebo controlled trial.

A total of 27 subjects were enrolled and divided into 3 groups: Group I: 3 subjects were injected with placebo and 6 subjects were injected with 5 pg of CY-1899; Group II: 3 subjects were injected with placebo and 6 subjects were injected with 50 pg of CY-1899; Group III: 3 subjects were injected with placebo and 6 subjects were injected with 500 pg of CY-1899.

68 After 5 weeks (GI and GII) or 4 weeks (GIII) following the first injection, all subjects received a booster inoculation at the same dosage.

The endpoints measured in this study relate to the safety and tolerability of CY-1899 (also known as Theradi-m' HBV) as well as its immunogenicity. Cellular immune responses to CY-1899 are an index of the intrinsic activity of this drug, and can therefore be viewed as a measure of biological efficacy. The following summarized the clinical and laboratory data relating to safety and efficacy endpoints.

Safety: Adverse events were observed with similar frequency in the placebo and drug treatment groups. All adverse events were judged to be mild in degree and completely reversible. No relationship between adverse events and dose level were found. The most common adverse events were headache (4 subjects) and skin reaction at the site of inoculation (3 subjects).

,Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy subjects were bled before and after injection. Peripheral blood mononuclear cells were isolated from fresh heparinized blood by Ficoll-Hypaque density Sgradient centrifugation, aliquoted in freezing media and stored frozen. Samples were assayed for CTL and HTL activity.

Data: Injection of CY-1899 in normal volunteers resulted in induction of HBcl8-27 specific CTL is disclosed in Figs. 14 and 15. The CTL response observed was dose dependent both in the proportion of subjections exhibiting a positive S. response as well as in the magnitude of the response obtained.

The dose response effect is demonstrated in Fig. 16 where the mean peak CTL activity per each group of subjects after the first and second injections are shown.

The peptide-induced CTL responses were capable of killing cells expressing endogenous antigen (as disclosed in Fig. 17) validating the concept of peptide priming for induction of virus specific

CTL.

The proliferative T cell response against the helper TT peptide among the subjects receiving the three doses of CY- 69 1899 correlates with their CTL responses (as disclosed in Fig.

18). The most striking T cell responses against the TT helper peptide were observed in individuals receiving the highest vaccine dose. In three out of four subjects in this dose group a strong T cell proliferative response against

TT

peptide was correlated with high levels of CTL induction following vaccination with CY-1899. The only instance where a strong CTL response was observed in the absence of helper

T

cell induction was in the case of an individual who demonstrated a late CTL response 14 days after boosting.

EXAMPLE 12 SPECIFIC CTL INDUCING

VACCINE

FOR PREVENTING AND/OR TREATING HCV By following the procedures disclosed in Examples 1- 11 a therapeutic composition of matter useful for treating mammals with HCV infections by inducing specific CTLs can be made.

For example, following the procedures in Examples 1, and 2, or alternatively Example 2A, peptides suitable for inducing CTLs canb inducing CTLs can be identified, for example, the following pt peptides:

J

SOURCE POSITION SEQUENCE SIZE SEQ.

BINDING

ID NO. A2 HCV NS4 1807 LLFNILGGWV 10 125 3.5000 M12 125 .5000 HCV CORE 178 LLALLSCLTV 10 126 0.6050 HCV NS4 1585 YLVAYQATV 9- HCV S1/ENV 725 FLLLADARV 9 128 0.2250 HCV NS4 1851 I LAGYGAGV 9 129 0.2150 HCV CORE 132 DLMGYIPLV 9 130 0.0835 HCV CORE 35 YLLPRRGPRL 10 131 0.0725

NS

1 /ENV2 686 ALSTGLIHL 9 132 0.0415 HCV CORE-- HCV CORE 178 LLALLSCLTI 10 133 0.0340 HCV NS5 2578 RLIVFPDLGV 10 134 0.0320 HCV NS5 2885 RLHGLSAFSL 10 135 0.0200 H S4 1811 ILGGWVAAQL 10 136 0.0180 364 SMVGNWAKV 9 137 0.0155 HCV NS3 1131 YLVTRHADV 9 0.0109 138 0.0109 SHCV NS4 1666 VLAALAAYCL 10 139 0.0106 Once identified, such desired peptides can be obtained either commercially or prepared following the procedures disclosed in Example 3.

25 The preferability of the identified peptides is elaborated by optimization of the configuration of the composition of matter whether the T-Helper and CTL peptides are linked or unlinked, and whether the peptides are delivered in a saline, alum or IFA medium, lipidated T-Helper linked or unlinked to the CTL in saline, or linked and unlinked T-Helper with CTL in IFA).

By following the procedures disclosed above and in the preceding examples a vaccine capable of treating HCV can be obtained.

EXAMPLE 13 SPECIFIC CTL INDUCING

VACCINE

FOR PREVENTING AND/OR TREATING

MELANOMA

By following the procedures disclosed in Examples 1a therapeutic composition of matter useful for treating mammals with melanomas by inducing specific CTLs is made.

For example, following the procedures in Examples 1, and 2, or alternatively Example 2A, peptides suitable for inducing CTLs are identified, for example, the following peptides: SOURCE POSITION SEQUENCE SIZE SEQ. BINDING 5 15• B ND N 15 ID NO. A2 MAGE2 105 KMVELVHFL 9 140 0.5100 MAGE2 105 KMVELVHFLL 10 141 0.2200 MAGE3 153 LVFGIELMEV 0 1 0 00

MAGE

1 278 KVLEYVIKV 9 143 0.0900

MAGE

1 105 KVADLVGFLL 10 144 0.0560 M A G E 3 105 KVAEFVHFL 9 145 0.0550

MAGE

1 92 CILESLFRA 9 146 0.0460 146-0.0460

MAGE

1 264 FLWGPRALA 9 147 0.0420 MAGE1 200 safe-, MAGE1 200 VMIAMEGGHA 10 148 0.0360 MAGE1 38 LVLGTLEEV 9 149 0.0320

MAGE

1 301 ALREEEEGV 9 150 0.0210 MAGE1 270 ALAETSYVKV 10 151 0.0150 MAGE1 82YVIKVSARV 9 152 0.0140 MAGE1 269 RALAETSyV 9 153 0.0100 3015 0.01 The peptides can be obtained either commercially or prepared following the procedures disclosed in Example 3.

The preferability of the identified peptides is elaborated by optimization of the configuration of the 72 composition of matter whether the T-Helper and CTL peptides are linked or unlinked, and whether the peptides are delivered in a saline, alum or IFA medium, lipidated T-Helper linked or unlinked to the CTL in saline, or linked and unlinked T-Helper with CTL in IFA).

By following the procedures disclosed above and in the preceding examples a vaccine capable of treating melanomas is obtained.

EXAMPLE 14 SPECIFIC CTL INDUCING

VACCINE

FOR PREVENTING AND/OR TREATING

HPV

By following the procedures disclosed in Examples 1- 15 10 a therapeutic composition of matter useful for treating mammals with HPV infections by inducing specific CTLs is made.

For example, following the procedures in Examples 1, and 2, or alternatively Example 2A, peptides suitable for inducing CTLs are identified, for example, the following 20 peptides: DO SOURCE POSITION

SEQUENCE

HPV16 E7 82

LLMGTLGIV

HPV16 E7 11

YMLDLQPET

HPV16 E6 52

FAFRDLCIV

HPV16 E7 86

TLGIVCPIC

HPV16 E7 7

TLHEYMLDL

HPV16 E7 85

GTLGIVCPI

HPV16 E7 12

MLDLQPETT

HPV16 E6 29

TIHDIILEC

V

SIZE

9 9 9 9 9 9 9 10 SEQ. BINDING ID NO. A2 65 0.0240 66 0.1400 67 0.0570 68 0.0750 69 0.0070 70 0.0820 71 0.0028 72 0.0210 The peptides can be obtained either commercially or prepared following the procedures disclosed in Example 3.

The preferability of the identified peptides is elaborated by optimization of the configuration of the composition of matter whether the T-Helper and CTL peptides are linked or unlinked, and whether the peptides are delivered in a saline, alum or IFA medium, lipidated T-Helper linked or unlinked to the CTL in saline, or linked and unlinked T-Helper with CTL in IFA).

By following the procedures disclosed above and in the preceding examples a vaccine capable of treating HPV is obtained.

EXAMPLE SPECIFIC CTL INDUCING VACCINE SFOR PREVENTING AND/OR TREATING

HIV

20 By following the procedures disclosed in Examples 1- 20 10 a therapeutic composition of matter useful for treating humans with HIV infections by inducing specific CTLs is made.

For example, following the procedures in Examples i, and 2, or alternatively Example 2A, peptides suitable for S inducing CTLs are identified, for example, the following peptides: 74 SOURCE POSITION SEQUENCE SIZE SEQ. BINDING ID NO. A2 HIV 367 VLAEAMSQV 9 73 0.1100 7 4 0 .0 3 HIV 1496 LLWKGEGAWV 10 74 0.0360 HIV 1496 LLWKGEGAV 9 HIV 1004 ILKEPVHGV 9 76 0.0190 HIV 1129 IVGAETFYV 9 77 0.0099 HIV 1129 IIGAETF0 9 78 0.0260 HIV 2182 LWVTVYYGV 9 79 0.0014 HIV 2182 LMVTVYYGV 9 80 0.4400 1" The peptides are obtained either commercially or 15 prepared following the procedures disclosed in Example 3.

The preferability of the identified peptides is elaborated by optimization of the configuration of the composition of matter whether the T-Helper and CTL 20 peptides are linked or unlinked, and whether the peptides are delivered in a saline, alum or IFA medium, lipidated T-Helper linked or unlinked to the CTL in saline, or linked and unlinked T-Helper with CTL in IFA).

By following the procedures disclosed above and in the preceding examples a vaccine capable of treating HIV is obtained.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims (116)

1. An immunogenically effective pharmaceutical composition comprising: a first peptide comprising an epitope, wherein the first peptide binds to an MHC class I molecule to form an epitope-MHC complex recognized by a cytotoxic S T cell, and wherein the first peptide is selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV; (ii) a second peptide comprising an epitope, wherein the second peptide binds to an MHC class II molecule to form an epitope-MHC complex recognized by a helper T cell, wherein the first peptide and/or the second peptide is linked to a lipid and 0t fuither wherein the first peptide or the second peptide are covalently linked or are unlinked; and (iii) a pharmaceutically acceptable carrier.

2. The composition of claim 1, wherein the second peptide is covalently linked to the first peptide. is 3. The composition of claim 1, wherein the second peptide is not linked to the first peptide.

4. The composition of any one of claims 1-3, wherein the second peptide is linked to the lipid.

5. The composition of claim 4, wherein the second peptide is linked to the lipid 20 by a spacer.

6. The composition of claim 5, wherein the spacer molecule is Lys-Ser-Ser. S* 7. The composition of claim 4, wherein the lipid is linked to the N-terminus of the second peptide.

8. The composition of claim 7, wherein the second peptide is linked to the lipid 25 by a spacer.

9. The composition of claim 1, wherein the second peptide is linked at its S. C-terminal end to the first peptide.

10. The composition of claim 1, wherein the first peptide is linked to the second peptide by a spacer molecule. 1 1. The composition of claim 10, wherein the spacer molecule between the first peptide and the second peptide is Ala-Ala-Ala.

12. The composition of any one of claims 1-11, wherein the first peptide and/or the second peptide are each from six to thirty amino acid residues in length. [I :\DAYLIB\LIBFF0864Ospec.doc:gcc 76

13. The composition of any one of claims 1-12, wherein the first and/or the second peptide comprises a plurality of epitopic units.

14. The composition of any one of claims 1-13, wherein the physiologically acceptable carrier is physiologic saline or a liposome.

15. The composition of any one of claims 1-14, further comprising an adjuvant.

16. The composition of claim 15, wherein the adjuvant is incomplete Freund's adjuvant, alum, or aluminum hydroxide.

17. The composition of any one of claims 1-16, wherein the lipid comprises a linear alkyl chain of 6-22 carbons. i18. The composition of claim 17, wherein the lipid comprises a linear alkyl chain of 16 carbons.

19. The composition of claim 4, wherein the lipid comprises palmitic acid attached to epsilon and alpha amino groups of a Lys residue, wherein the Lys is linked to the amino terminus of the second immunogenic peptide by means of a linker.

20. A method for stimulating an immune response in a mammal against an epitope, comprising the steps of: providing a first peptide comprising an epitope, wherein the first peptide binds to an MHC class I molecule to form an epitope-MHC complex recognized by a cytotoxic T cell, wherein the first peptide is selected from the group comprising 20 KVAEFVHFL, LWVTVYYGV and IVGAETFYV; S. (ii) providing a second peptide comprising an epitope, wherein the second peptide binds to an MHC class II molecule to form an epitope-MHC complex recognized by a helper T cell, wherein the first peptide and/or the second peptide is linked to a lipid; and 25 (iii) administering the first peptide and the second peptide to the mammal.

21. The method of claim 20, wherein the second peptide is covalently linked to the first peptide.

22. The method of claim 20, wherein the second peptide is not linked to the first peptide.

23. The method of claim 20, wherein the second peptide is linked to the lipid.

24. The method of claim 23, wherein the second peptide linked to the lipid and the first peptide are administered prophylactically. The method of claim 20, wherein the second peptide is administered f STf concurrently with the first peptide. [I:\DAYLIB\LIBFF]08640spec.doc:gcc 77

26. The method of claim 20, wherein the second peptide is admixed with the first peptide.

27. The method of claim 20, wherein the second peptide and the first peptide are administered to the mammal in a regimen of two or more steps of administration, the administration steps spaced a sufficient interval apart to optimize development of said immune response to the epitope.

28. The method of claim 27, wherein step of administration comprises administering the second peptide and the first peptide approximately four weeks after the initial administration step. ,1 29. The method of any one of claims 20-28, wherein the mammal is a human. The method of claim 23, wherein the second peptide is linked to the lipid by a spacer.

31. The method of claim 30, wherein the spacer molecule is Lys-Ser-Ser.

32. The method of claim 23, wherein the lipid is linked to the N-terminus of the second peptide.

33. The method of claim 23, wherein the second peptide is linked at its C-terminal end to the first peptide.

34. The method of any one of claims 20-33, wherein the first peptide is linked to the second peptide by a spacer molecule. 2 35. The method of claim 34, wherein the spacer molecule is Ala-Ala-Ala.

36. The method of any one of claims 20-35, wherein the first peptide and/or the second peptide are each from six to thirty amino acid residues in length.

37. The method of any one of claims 20-36, wherein the first and/or the second peptide comprises a plurality of epitopic units. 25 38. A first and second peptide, when used in stimulating an immune response in a mammal against an epitope, wherein: i said first peptide comprises an epitope, and wherein the first peptide binds to an MHC class I molecule to form an epitope-MHC complex recognized by a cytotoxic T cell, and wherein the first peptide is selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV; (ii) said second peptide comprises an epitope, and wherein the second peptide binds to an MHC class II molecule to form an epitope-MHC complex recognized by a helper T cell, wherein the first peptide and/or the second peptide is linked to a lipid. [I:\DAYLIB\L BFF]O864Ospec.doc:gcc 78

39. The first and second peptide of claim 38, wherein the second peptide is covalently linked to the first peptide. The first and second peptide of claim 38, wherein the second peptide is not linked to the first peptide.

41. The first and second peptide of claim 38, wherein the second peptide is linked to the lipid.

42. The first and second peptide of claim 41, wherein the second peptide linked to the lipid and the first peptide are administered prophylactically.

43. The first and second peptide of claim 38, wherein the second peptide is administered concurrently with the first peptide.

44. The first and second peptide of claim 38, wherein the second peptide is admixed with the first peptide. The first and second peptide of claim 38, wherein the second peptide and the first peptide are administered to the mammal in a regimen of two or more steps of i administration, the administration steps spaced a sufficient interval apart to optimize development of said immune response to the epitope.

46. The first and second peptide of claim 45, wherein step of administration comprises administering the second peptide and the first peptide approximately four weeks after the initial administration step. 2 0

47. The first and second peptide of any one of claims 38-46, wherein the mammal is a human.

48. The first and second peptide of claim 41, wherein the second peptide is linked to the lipid by a spacer.

49. The first and second peptide of claim 48, wherein the spacer molecule is 25 Lys-Ser-Ser.

50. The first and second peptide of claim 41, wherein the lipid is linked to the N-terminus of the second peptide.

51. The first and second peptide of claim 41, wherein the second peptide is linked at its C-terminal end to the first peptide.

52. The first and second peptide of any one of claims 38-51, wherein the first peptide is linked to the second peptide by a spacer molecule.

53. The first and second peptide of claim 52, wherein the spacer molecule is Ala-Ala-Ala. [I:\DAYLIB\LIBFF0864Ospec.doc:gcc 79

54. The first and second peptide of any one of claims 38-53, wherein the first peptide and/or the second peptide are each from six to thirty amino acid residues in lehgth. The first and second peptide of any one of claims 38-54, wherein the first and/or the second peptide comprises a plurality of epitopic units.

56. Use of a first and second peptide, for the preparation of a medicament for stimulating an immune response in a mammal against an epitope, wherein: said first peptide comprises an epitope, and wherein the first peptide binds to an MHC class I molecule to form an epitope-MHC complex recognized by a It cytotoxic T cell, and wherein the first peptide is selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV; (ii) said second peptide comprises an epitope, and wherein the second peptide binds to an MHC class II molecule to form an epitope-MHC complex recognized by a helper T cell, wherein the first peptide and/or the second peptide is linked to a lipid.

57. The use of claim 56, wherein the second peptide is covalently linked to the first peptide.

58. The use of claim 56, wherein the second peptide is not linked to the first peptide. S59. The use of claim 56, wherein the second peptide is linked to the lipid. 2 60. The use of claim 59, wherein the second peptide linked to the lipid and the first peptide are administered prophylactically.

61. The use of claim 56, wherein the second peptide is administered concurrently with the first peptide.

62. The use of claim 56, wherein the second peptide is admixed with the first ltide. 25 pepticle.

63. The use of claim 56, wherein the second peptide and the first peptide are administered to the mammal in a regimen of two or more steps of administration, the administration steps spaced a sufficient interval apart to optimize development of said immune response to the epitope.

64. The use of claim 63, wherein step of administration comprises administering the second peptide and the first peptide approximately four weeks after the initial administration step. The use of any one of claims 56-64, wherein the mammal is a human. [I:\DAYLIB\LIBFF]08640spec.doc:gcc

66. The use of claim 59, wherein the second peptide is linked to the lipid by a spacer.

67. The use of claim 66, wherein the spacer molecule is Lys-Ser-Ser.

68. The use of claim 59, wherein the lipid is linked to the N-terminus of the second peptide.

69. The use of claim 59, wherein the second peptide is linked at its C-terminal end to the first peptide. The use of any one of claims 56-69, wherein the first peptide is linked to the second peptide by a spacer molecule.

71. The use of claim 70, wherein the spacer molecule is Ala-Ala-Ala.

72. The use of any one of claims 56-71, wherein the first peptide and/or the second peptide are each from six to thirty amino acid residues in length.

73. The use of any one of claims 56-72, wherein the first and/or the second peptide comprises a plurality of epitopic units.

74. A composition comprising a peptide of fewer than 15 amino acid residues, wherein the peptide binds an MHC class I molecule to form a peptide-MHC complex recognized by a cytotoxic T-cell and wherein the peptide comprises a sequence selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV.

75. The composition of claim 74, wherein the sequence is KVAEFVHFL. 20 76. The composition of claim 74, wherein the sequence is LWVTVYYGV.

77. The composition of claim 74, wherein the sequence is IVGAETFYV.

78. The composition of claim 74, comprising an adjuvant.

79. The composition of claim 78, wherein the adjuvant is incomplete Freund's adjuvant, alum, or aluminum hydroxide. 25 80. The composition of claim 74, wherein the peptide is linked to a lipid.

81. The composition of claim 80, wherein the lipid comprises a linear alkyl chain of 6-22 carbons.

82. The composition of claim 81, wherein the lipid comprises a linear alkyl chain of 16 carbons.

83. The composition of claim 80, wherein the lipid comprises palmitic acid attached to epsilon and alpha amino groups of a Lys residue, wherein the Lys is linked to the amino terminus of the second immunogenic peptide by means of a linker.

84. A method for stimulating an immune response in a mammal against an pitope, comprising the steps of: [I:\DAYLIB\LIBFF]O864Ospec.doc:gcc 81 providing a peptide of fewer than 15 amino acid residues, wherein the peptide binds to an MHC class I molecule to form a peptide-MHC complex recognized by a cytotoxic T cell and wherein peptide comprises a sequence selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV; and (ii) administering the peptide to the mammal. The method of claim 84, wherein the sequence is KVAEFVHFL.

86. The method of claim 85, wherein the peptide is administered to a human to treat or interrupt growth of a tumor.

87. The method of claim 84, wherein the sequence is LWVTVYYGV.

88. The method of claim 87, wherein the peptide is administered to treat a human immunodeficiency virus 1 or 2 infection in humans.

89. The method of claim 84, wherein the sequence is IVGAETFYV. The method of claim 89, wherein the peptide is administered to treat a human immunodeficiency virus 1 or 2 infection in humans.

91. The method of claim 84, wherein the peptide is administered to prevent a human immunodeficiency virus 1 or 2 infection in humans.

92. The method of any one of claims 84-91, comprising an adjuvant.

93. The method of claim 92, wherein the adjuvant is incomplete Freund's adjuvant, alum, or aluminum hydroxide. 20 94. The method of any one of claims 84-93, wherein the peptide is linked to a lipid. S S 95. The method of claim 94, wherein the lipid comprises a linear alkyl chain of 6-22 carbons. s

96. The method of claim 95, wherein the lipid comprises a linear alkyl chain of t 25 16 carbons. .97. The method of claim 94, wherein the lipid comprises palmitic acid attached to epsilon and alpha amino groups of a Lys residue, wherein the Lys is linked to the amino terminus of the second immunogenic peptide by means of a linker.

98. The method of any one of claims 84-97, wherein the first peptide and the so second peptide are administered prophylactically.

99. The method of any one of claims 84-98, wherein the second peptide and the first peptide are administered to the mammal in a regimen of two or more steps of Sadministration, the administration steps spaced a sufficient interval apart to optimize evelopment of said immune response to the epitope. [I:\DAYLIB\LIBFF]08640spec.doc:gcc 82

100. The method of claim 99, wherein step of administration comprises administering the second peptide and the first peptide approximately four weeks after the initial administration step.

101. The method of any one of claims 84-100, wherein the mammal is a human.

102. A peptide when used for stimulating an immune response in a mammal against an epitope, wherein said peptide is of fewer than 15 amino acid residues, and wherein the peptide binds to an MHC class I molecule to form a peptide-MHC complex recognized by a cytotoxic T cell and wherein peptide comprises a sequence selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV. t0 103. The peptide of claim 102, wherein the sequence is KVAEFVHFL.

104. The peptide of claim 103, wherein the peptide is administered to a human to treat or interrupt growth of a tumor.

105. The peptide of claim 102, wherein the sequence is LWVTVYYGV.

106. The peptide of claim 105, wherein the peptide is administered to treat a human immunodeficiency virus 1 or 2 infection in humans. .1 107. The peptide of claim 102, wherein the sequence is IVGAETFYV.

108. The peptide of claim 107, wherein the peptide is administered to treat a *..Goo human immunodeficiency virus 1 or 2 infection in humans.

109. The peptide of claim 102, wherein the peptide is administered to prevent a 20 human immunodeficiency virus 1 or 2 infection in humans.

110. The peptide of any one of claims 102-109, comprising an adjuvant.

111. The peptide of claim 110, wherein the adjuvant is incomplete Freund's adjuvant, alum, or aluminum hydroxide.

112. The peptide of any one of claims 102-111, wherein the peptide is linked to a 25 lipid.

113. The peptide of claim 112, wherein the lipid comprises a linear alkyl chain of 6-22 carbons.

114. The peptide of claim 113, wherein the lipid comprises a linear alkyl chain of 16 carbons. 1 15. The peptide of claim 112, wherein the lipid comprises palmitic acid attached to epsilon and alpha amino groups of a Lys residue, wherein the Lys is linked to the amino terminus of the second immunogenic peptide by means of a linker.

116. The peptide of any one of claims 102-115, wherein the first peptide and the '0f second peptide are administered prophylactically. [I :\DAYLIB\LIBFF1O864Ospec.doc:gcc 83

117. The peptide of any one of claims 102-116, wherein the second peptide and the first peptide are administered to the mammal in a regimen of two or more steps of administration, the administration steps spaced a sufficient interval apart to optimize development of said immune response to the epitope.

118. The peptide of 117, wherein step of administration comprises administering the second peptide and the first peptide approximately four weeks after the initial administration step.

119. The peptide of any one of claims 102-118, wherein the mammal is a human.

120. Use of a peptide for the preparation of a medicament for stimulating an In immune response in a mammal against an epitope, wherein said peptide is of fewer than amino acid residues, and wherein the peptide binds to an MHC class I molecule to tbrm a peptide-MHC complex recognized by a cytotoxic T cell and wherein peptide comprises a sequence selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV.

121. The use of claim 120, wherein the sequence is KVAEFVHFL.

122. The use of claim 121, wherein the peptide is administered to a human to treat or interrupt growth of a tumor.

123. The use of claim 120, wherein the sequence is LWVTVYYGV. 1* 24. The use of claim 123, wherein the peptide is administered to treat a human S immunodeficiency virus 1 or 2 infection in humans.

125. The use of claim 120, wherein the sequence is IVGAETFYV.

126. The use of claim 125, wherein the peptide is administered to treat a human immunodeficiency virus 1 or 2 infection in humans.

127. The use of claim 120, wherein the peptide is administered to prevent a human I 25 immunodeficiency virus 1 or 2 infection in humans.

128. The use of any one of claims 120-127, comprising an adjuvant.

129. The use of claim 128, wherein the adjuvant is incomplete Freund's adjuvant, alum, or aluminum hydroxide.

130. The use of any one of claims 102-129, wherein the peptide is linked to a lipid.

131. The use of claim 130, wherein the lipid comprises a linear alkyl chain of 6-22 carbons.

132. The use of claim 131, wherein the lipid comprises a linear alkyl chain of 16 carbons. [I:\DAYLIB\LIBFFO864Ospec.doc:gcc 84

133. The use of claim 130, wherein the lipid comprises palmitic acid attached to epsilon and alpha amino groups of a Lys residue, wherein the Lys is linked to the amino terminus of the second immunogenic peptide by means of a linker.

134. The use of any one of claims 120-133, wherein the first peptide and the second peptide are administered prophylactically.

135. The use of any one of claims 120-134, wherein the second peptide and the first peptide are administered to the mammal in a regimen of two or more steps of administration, the administration steps spaced a sufficient interval apart to optimize development of said immune response to the epitope. t0 136. The use of claim 135, wherein step of administration comprises administering the second peptide and the first peptide approximately four weeks after the initial administration step.

137. The use of any one of claims 120-136, wherein the mammal is a human.

138. An immunogenically effective pharmaceutical composition comprising: i a first peptide comprising an epitope, wherein the first peptide binds to an MHC class I molecule to form an epitope-MHC complex recognized by a cytotoxic T cell, and wherein the first peptide is selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV; (ii) a second peptide comprising an epitope, wherein the second peptide binds to an MHC class II molecule to form an epitope-MHC complex recognized by a helper T cell, wherein the first peptide and/or the second peptide is linked to a lipid, substantially as hereinbefore described with reference to any one of the examples.

139. A method for stimulating an immune response in a mammal against an epitope, wherein said method comprises administering an immunologically effective amount of the composition of any one of claims 1-19 or 138. S; 140. The composition of any one of claims 1-19 or 138, when used in stimulating an immune response in a mammal against an epitope.

141. Use of the composition of any one of claims 1-19 or 138, for the preparation of a medicament for stimulating an immune response in a mammal against an epitope.

142. A composition comprising a peptide of fewer than 15 amino acid residues, wherein the peptide binds an MHC class I molecule to form a peptide-MHC complex recognized by a cytotoxic T-cell and wherein the peptide comprises a sequence selected from the group comprising KVAEFVHFL, LWVTVYYGV and IVGAETFYV, substantially as hereinbefore described with reference to any one of the examples. [I:\DAYLIB\LIBFF]08640spec.doc:gcc

143. A method for stimulating an immune response to an epitope in a human, wherein said method comprises administering an immunologically effective amount of the composition of any one of claims 74-83 or 142.

144. The composition of any one of claims 74-83 or 142, when used for stimulating an immune response in a mammal against an epitope.

145. Use of the composition of any one of claims 74-83 or 142 for the preparation of a medicament for stimulating an immune response in a mammal against an epitope. Dated 12 October, 2000 Epimmune Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON a a [I:\DAYLB\LIB FF]O864Ospec.doc: gcc