WO2009002418A2 - T-cell peptide epitopes from carcinoembryonic antigen, immunogenic analogs, and uses thereof - Google Patents

Abstract

The present invention provides vaccines and pharmaceutical compositions which can, when administered to a patient, elicit an immune response to the carcinoembryonic antigen (CEA). Immunogenic peptide epitopes of human CEA are provided, as well as immunogenic peptide analogs which can induce an immune response cross-reactive against CEA which response is superior in quality to that induced by the corresponding wild-type epitope. Also disclosed herein are pharmaceutical compositions and vaccines comprising one or more of said peptides and analogs for prophylaxis and/or treatment of cancer. This invention further provides polynucleotides encoding at least one of the CEA analogs described herein, and vectors and host cells comprising said polynucleotides. Methods of inducing an immune response in a patient, in addition to methods of treatment using the immunogenic peptides, peptide analogs and polynucleotides disclosed herein are also disclosed.

Description

TITLE OF THE INVENTION

T-CELL PEPTIDE EPITOPES FROM CARCINOEMBRYONIC ANTIGEN, IMMUNOGENIC

ANALOGS, AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/936,973 filed June 21, 2007, the contents of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to the therapy of cancer. More specifically, the present invention relates to novel isolated immunogenic T-cell peptide epitopes from carcinoembryonic antigen (CEA), to CEA peptides and that have been modified to induce increased immunogenicity ("analogs"), and to variant CEA proteins comprising the analogs. This invention also relates to polynucleotides encoding said immunogenic CEA epitopes, analogs, and variant proteins. Also provided are prophylactic and therapeutic vaccines comprising the peptides, analogs, modified proteins, and nucleic acids described herein, and methods of use.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of mortality worldwide. Despite an abundance of cancer-related research, conventional therapies, which combine surgery, radiation, and chemotherapy, often fail to effectively treat established cancers. Although vaccination has become standard procedure for the prevention of infectious diseases, the development of efficacious vaccines for the treatment and/or prevention of cancer remains a challenge. The development of a cancer vaccine capable of eliciting a clinically-relevant immune response is partly dependent on the choice of a target antigen that is preferentially expressed on tumor cells compared to normal cells. However, because many tumor-associated antigens (TAAs) are expressed in normal cells, albeit at lower levels, cancer vaccines must be designed to overcome self-tolerance. Additionally, the immune response generated by therapeutic vaccines is often not of sufficient magnitude to lead to tumor regression in a clinical setting.

Human CEA is one TAA that has been implicated in the pathogenesis of cancer. CEA is normally expressed during fetal development and in adult colonic mucosa. Aberrant CEA expression has long been correlated with many types of cancers, with the first report describing CEA overexpression in human colon tumors published over thirty years ago (Gold and Freedman, J. Exp. Med. 121:439-462 (1965)). Overexpression of CEA has since been detected in nearly all colorectal tumors, as well as in a high percentage of adenocarcinomas of the pancreas, liver, breast, ovary, cervix, and lung. Moreover, it was demonstrated in transgenic mice immunized with a recombinant vaccinia vector expressing CEA that anti-CEA immune responses could be elicited without inducing autoimmunity, making CEA a particularly attractive target for active and passive cancer immunotherapy (Kass et al. Cancer Res. 59: 676-83 (1999)). Therapeutic strategies targeting CEA have included the use of CEA-based DNA and protein vaccines, and dendritic cell-based vaccines (for review, see Berinstein, supra; and Sarobe et al. Current Cancer Drug Targets 4: 443-54 (2004)). Antigenic peptide or epitope- based vaccines have also been investigated as a means of promoting the destruction of cancerous cells overexpressing CEA by an individual's immune system.

The T-lymphocyte cellular-mediated immune response forms a critical component of the immune response and plays a crucial role in the eradication of tumor cells by the mammalian immune system. T cell-mediated immune responses require the activation of cytotoxic (CD8+) and helper (CD4+) T lymphocytes. Cytotoxic T lymphocytes (CTL) and their T-cell receptors (TCR) recognize small peptides presented by major histocompatibility complex (MHC) class I molecules on the cell surface (Bjorkman P J., Cell 89:167-170 (1997); Garcia et al, Science 274:209-219 (1996)). The peptides are derived from intracellular antigens via the endogenous antigen processing and presentation pathway (Germain R N., Cell 76:287-299 (1994); Pamer et al., Annu Rev Immunol 16:323-358(1998)). Peptides for human CD8+ epitopes range from 7 to 14 amino acids, and typically are 9-10 amino acids in length. TCR recognition of the peptide-MHC class I molecule complexes on the cell surface triggers the cytolytic activity of CTL, resulting in the death of cells presenting the peptide-MHC class I complexes (Kagi et al. , Science 265: 528-530 (1994)). MHC class I restricted epitope vaccines have been shown to confer protection in some animal models. The development of epitope vaccines encoding human HLA-restricted CTL epitopes capable of conferring broad, effective, and non-ethnically biased population coverage is highly desirable. Epitope-based vaccines offer a number of advantageous features compared to vaccines based on full-length TAAs, including ease and low cost of peptide synthesis. In addition, peptide vaccines can induce immune responses to subdominant epitopes when there is tolerance to a dominant epitope, and anchor-modified or heteroclitic peptide analogs can be constructed that can break tolerance and/or further increase immunogenicity relative to native peptides (for review, see Lazoura and Apostolopoulos, Current Medicinal Chemistry 12: 1481- 94 (2005)). The use of peptides as immunogens also minimizes safety risks associated with the use of intact proteins.

The identification of novel CEA epitopes and epitope analogs that generate effective anti-tumor immune responses without causing autoimmunity will allow the development of epitope-based cancer vaccines that elicit a clinically relevant prophylactic and/or therapeutic immune response against CEA. SUMMARY OF THE INVENTION

The present invention provides isolated immunogenic peptides ("epitopes") of human carcinoembryonic antigen (hCEA) or analogs thereof; which are selected based on their binding affinity for a Class I MHC allele, specifically, HLA-A*0201. The peptides and analogs described herein were selected based on their ability to elicit a maximum tumor-specific immune response in a tolerized setting, as well as for their minimal potential for eliciting off-target autoimmune activity. More specifically, the CEA protein (SEQ ID NO:1) was scanned using a proprietary software package called EI Suite that ranks protein fragments based on binding affinity for HLA-A* 0201, similarity to fragments of other human proteins, and amenability to immunogenic enhancement.

In addition to wild-type CEA epitopes, in some embodiments of the invention described herein, modifications were introduced at specific amino acid positions within the naturally occurring sequence of the corresponding wild-type immunogenic CEA peptide to form anchor-modified analogs. Said analogs provide an increased benefit as a vaccine component due to their capacity to induce an immune response cross-reactive against CEA which is superior in quality to that induced by the corresponding wild-type epitope. The immunogenic peptide analogs of the present invention comprise a sequence of amino acids selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, and SEQ ID NO: 13. Also provided herein are variant CEA proteins which comprise one or more of the analog sequences described herein.

The present invention further provides polynucleotides encoding said immunogenic CEA peptides, peptide analogs, and variant CEA proteins, as well as recombinant expression vectors, including but not limited to, adenovirus and plasmid vectors, comprising said polynucleotides. In some embodiments of this portion of the invention, the vector is an adenovirus vector, which, in preferred embodiments, is selected from the group consisting of: Ad5, Ad6, and Ad24. In further exemplary embodiments of the invention, the polynucleotides comprise a sequence of nucleotides that is operably linked to a promoter. Also provided are recombinant host cells comprising the expression vectors described herein.

Alternative embodiments of the invention relate to pharmaceutical compositions comprising one or more of the immunogenic CEA peptides, analogs, variant CEA proteins, or nucleic acids encoding said peptides, analogs, and proteins, together with a pharmaceutically acceptable carrier. In preferred embodiments, the pharmaceutical composition comprises a plurality of immunogenic peptides or analogs thereof. In some embodiments, the pharmaceutical composition further comprises an adjuvant. Also provided are vaccine compositions comprising one or more of the CEA peptide epitopes, analogs, variant proteins or comprising one or more polynucleotides encoding said epitopes, analogs, or variant proteins disclosed throughout the specification. In preferred embodiments, the vaccine compositions comprise a plurality of isolated polynucleotides, encoded peptides, or analogs thereof.

A further embodiment of the present invention is a method of eliciting an immune response to CEA in a patient in need thereof, said method comprising introducing into the patient the pharmaceutical compositions or vaccines disclosed herein.

The present invention further provides methods for inhibiting the development of a cancer in a mammal, or treating or minimizing an existing cancer, by eliciting an immune response to CEA, such methods comprising administering a vaccine or pharmaceutical composition comprising one or more immunogenic CEA peptide described herein, or analog thereof, or polynucleotide encoding said peptide or analog, as described herein. In preferred embodiments of the methods herein, the immune response is enhanced relative to the response elicited by a wild-type CEA.

As used throughout the specification and in the appended claims, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise.

As used throughout the specification and appended claims, the following definitions and abbreviations apply:

The term "promoter" refers to a recognition site on a DNA strand to which the RNA polymerase binds. The promoter forms an initiation complex with RNA polymerase to initiate and drive transcriptional activity. The complex can be modified by activating sequences termed "enhancers" or inhibiting sequences termed "silencers".

The term "cassette" refers to a nucleotide or gene sequence that is to be expressed from a vector, for example, a nucleotide or gene sequence encoding one or more of the CEA peptide epitopes, analogs, or modified CEA proteins described herein. In general, a cassette comprises a gene sequence that can be inserted into a vector, which in some embodiments, provides regulatory sequences for expressing the nucleotide or gene sequence, hi other embodiments, the nucleotide or gene sequence provides the regulatory sequences for its expression, hi further embodiments, the vector provides some regulatory sequences and the nucleotide or gene sequence provides other regulatory sequences. For example, the vector can provide a promoter for transcribing the nucleotide or gene sequence and the nucleotide or gene sequence provides a transcription termination sequence. The regulatory sequences that can be provided by the vector include, but are not limited to, enhancers, transcription termination sequences, splice acceptor and donor sequences, introns, ribosome binding sequences, and poly(A) addition sequences.

The term "vector" refers to some means by which DNA fragments can be introduced into a host organism or host tissue. There are various types of vectors including plasmid, virus (including adenovirus), bacteriophages and cosmids. The term "first generation," as used in reference to adenoviral vectors, describes adenoviral vectors that are replication-defective. First generation adenovirus vectors typically have a deleted or inactivated El gene region, and preferably have a deleted or inactivated E3 gene region. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented.

A "disorder" is any condition that would benefit from treatment with the molecules of the present invention, including the CEA peptide epitopes, CEA epitope analogs, modified CEA proteins and nucleic acid molecules encoding said epitopes, analogs, and modified proteins. Encompassed by the term "disorder" are chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. The molecules of the present invention are intended for use as treatments for disorders or conditions characterized by aberrant cell proliferation, including, but not limited to, pancreatic cancer, liver cancer, breast cancer, colorectal cancer, ovarian cancer, cervical cancer, and lung cancer.

The term "effective amount" means sufficient vaccine composition is introduced to produce the adequate levels of the polypeptide, so that a clinically significant immune response results. One skilled in the art recognizes that this level may vary. "hCEA" refers to a human carcinoembryonic antigen.

The term "nucleic acid" or "nucleic acid molecule" is intended for ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), probes, oligonucleotides, fragment or portions thereof, and primers.

"Wild-type CEA" or "wild-type protein" or "wt protein" refers to a CEA protein comprising a naturally occurring sequence of amino acids as set forth in SEQ ID NO: 1 and shown in FIGURE IA, which sequence is encoded by the major allele of CEA found in the human population, free of induced mutations or modifications.

The term "variant protein" or "variant CEA" refers to a CEA protein comprising modifications to at least one specific amino acid residue of the CEA protein relative to the full- length wild-type CEA protein as defined in SEQ ID NO:1. The "variant proteins" of the present invention comprise one or more of the epitope analogs described herein, and elicit an immune response that it increased relative to the protein of SEQ ID NO:1 when introduced into a subject. In some embodiments of the invention, the variant CEA protein comprises more than one of the CEA analogs disclosed herein, e.g. SEQ ID NO: 15, which comprises the analogs set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9 (see FIGURE 1C). Variant CEA proteins, as defined herein, may be substantially the same length as the wild-type CEA protein disclosed in SEQ ID NO:1, or may be of shorter length, such as a CEA protein that is deleted of its C-terminal anchoring domain (AD) (e.g. SEQ ID NO:14, see FIGURE IB). Variant CEA proteins of the present invention are at least 50%, preferably at least 70%, more preferably 80%, and even more preferably 90% of the length of the wild-type CEA protein set forth in SEQ ED NO: 1.

The term "mammalian" refers to any mammal, including a human being. The abbreviation "Ag" refers to an antigen. The term "antigen" refers to any biologic or macromolecular substance that can be recognized by a T-cell or an antibody molecule.

The abbreviations "Ab" and "mAb" refer to an antibody and a monoclonal antibody, respectively. The terms "major histocompatibility complex (MHC)" and "human leukocyte antigen (HLA)" are used interchangeably to refer to a locus of genes that encode proteins which present a vast variety of peptides onto the cell surface for specific recognition by a T-cell receptor. A subclass of MHC genes, called Class I MHC molecules, present peptides to CD8⁺ T- cells. "Epitope" refers to a peptide which is a portion of an antigen, wherein the peptide comprises an amino acid sequence that is capable of stimulating an immune response. The MHC class I epitopes disclosed herein are useful in pharmaceutical compositions (e.g., vaccines) for stimulating an immune response directed to CEA. hi preferred embodiments, epitopes according to this definition represent peptides which are likely to be non-covalently bound to the binding cleft of a class I MHC molecule on the surface of antigen presenting cells in a manner which facilitates its interaction with T-cell receptors (TCR). The term "epitope" is used interchangeably herein with the term "immunogenic peptide."

The term "wild type epitope" refers to an epitope comprising a sequence of nine or ten amino acids that can be found in the naturally occurring wild-type CEA protein as set forth in SEQ ID NO: 1.

The terms "9-mer" and "10-mer" refer to a linear sequence of nine or ten amino acids that occur in a target antigen. It is generally understood that a collection of sequences that includes all possible 9-mers and 10-mers present in a parent sequence, comprise sequences which overlap by eight or nine residues, respectively. The term "anchor residues" refers to the amino acid residues of an immunogenic peptide fragment that provide a contact point between the peptide and the MHC molecule. The anchor residues comprise side chains that fit into the peptide-binding clefts of said MHC molecules.

"Binding motif refers to a specific pattern or combination of anchor residues within protein sequences which are correlated with the ability to bind to a specified HLA allele or serotypes.

"Immunogen" refers to specific antigens that are capable of inducing or stimulating an immune response. Not all antigens are immunogenic. "Enhanced immunogenicity" refers an increased ability to activate the immune system when compared to the immune response elicited by the wild-type peptide. A variant peptide or analog can be said to have "increased immunogenicity" if it induces a higher level of T-cell activation relative to the level of activation induced by the corresponding wild-type peptide as measured in a standard in-vitro T-cell activation assay. In a preferred embodiment, the frequency of vaccination-induced epitope-specific T-cells will be increased at least 10-fold by the administration of an immunoenhanced analogs relative to the level of T-cell activation (i.e., number of epitope-specific CTLs) induced by immunization with the parent peptide. A 50-fold increase in T-cell activity is an especially preferred level of immunoenhancement. "Immunogenic analog" or "epitope analog" refers to a peptide epitope with one or more residues of the wild-type amino acid sequence substituted with an alternative amino acid sequence identified by the immunoenhancement filter of EI Suite. Coordinated substitutions are often carried out to regulate or modify (e.g., increase) immunogenicity of a natural peptide. The terms "prediction" and "predicting" are used herein refer to the use of the present teachings to estimate properties (e.g., ability to bind to MHC class I allele, likelihood of being efficiently processed and presented by APC, uniqueness to target antigen, immunogenicity) of amino acid sequences representing putative T-cell epitopes.

The terms "MHC class I binder" and "MHC peptide" are used to refer to peptides having a high known or predicted binding affinity for a mammalian class I major histocompatibility complex (MHC) molecule.

"Immunogenic composition" refers to a composition that is capable of inducing an immune response, a reaction, an effect, and/or an event. In some embodiments, such responses, reactions, effects, and/or events can be induced in vitro or in vivo. For example, the induction, activation, or expansion of cells involved in cell mediated immunity, such as CTLs represents an immune response, effect or an event. Representative immunogenic compositions include an immunoenhanced full-length target antigen or a minigene vaccine.

"Vaccine" refers to an immunogenic composition that is capable of eliciting a clinically relevant prophylactic and/or therapeutic immune response that prevents, cures, or ameliorates disease. "Epitope vaccine" generally refers to a composition of several epitopes derived from one or more target proteins of the same, or different pathogen or tumor cell, specific to one or more alleles of interest. The list of epitopes used may include those optimized for natural processing, immunogenicity, uniqueness (e.g., lack of similarity to other self-antigens), population coverage, and predicted disease relevance. For example, an immunogenic composition comprising more than one putative T-cell epitope derived from at least one target antigen linked together, with or without additional amino acids ("spacers") between the epitopes can be used as an epitope vaccine. Thus, an epitope vaccine can stimulate immune responses directed to single or multiple epitopes of one or more antigens. BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1, panel A, shows the amino acid sequence of the human CEA protein (SEQ ID NO:1), as set forth in NCBI Genbank Accession No. Ml 7303. Panel B shows an exemplary CEA protein sequence which is deleted of its C-terminal anchoring domain (SEQ ID NO: 14). Panel C shows an exemplary variant CEA protein sequence (SEQ ID NO: 15) which comprises analogs SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9. Modifications to the wt CEA sequence are shown in bold and underlined.

FIGURE 2 summarizes results obtained when comparing the top-scoring CEA- proteins to the human proteome. Of the 150 top-scoring CEA peptides, 45 had no matches in other human proteins, 64 matched a fragment of another CEA-like cell adhesion molecule, 26 matched a fragment of a pregnancy-specific glycoprotein, and 15 were similar to a fragment of a protein outside the CEA family. Altogether, 105/150 peptides were rejected or flagged.

FIGURE 3 shows the binding affinity of CEA epitope candidates. The stability of the peptide-MHC complex was evaluated by fluorescent activated cell sorting (FACS). The mean fluorescence intensity MFI resulting from the FACS analysis is shown for each epitope.

FIGURE 4 shows the relative binding stability of exemplary CEA epitopes described herein. The stability of peptide-MHC complex was evaluated by FACS analysis over time. MFI= mean fluorescence intensity. FIGURE 5 shows E/SW/e-selected peptides and analogs are immunogenic in

HLA-A2.1 mice. HHD mice were immunized by subcutaneous injection of peptides. Two weeks later, cell mediated immune response was measured by ICS on pooled PBMCs.

FIGURE 6 shows analogs of immunogenic CEA peptides identified by EI Suite strongly increase immune reactivity against the corresponding wild type epitopes. HHD mice were immunized by SC injection of peptides. Two weeks later, cell mediated immune response against natural peptides was measured by ICS on individual mouse splenocytes.

FIGURE 7 shows the immunogenicity of additional E/Støte-selected peptides (EXAMPLE 9). Groups of 6 HHD mice were vaccinated subcutaneously with lOOμg of peptide in combination with HBVcorel28 peptide (140 μg), IFA (1 :1) and CpG (50μg). Mice received two injections two weeks apart and the immune response was measured by intracellular staining for IFNγ three weeks later. Each triangle represents the CMI of a single mouse; the geometric mean of each group is indicated by a round dot. Student's t-test between CE A310 and CEA310L2 -vaccinated groups is statistically significant (p=0.007). The vaccination protocol has been repeated with similar results. FIGURE 8 provides a summary of the immunogenic CEA peptides disclosed herein, as well as immunogenic CEA peptide analogs, which include 2 wild type peptides and 10 anchor-modified analogs. Modifications to the peptide analogs, relative to the wild type epitope, are shown in bold and underlined. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated and purified peptides which comprise an amino acid sequence of an immunogenic T-cell peptide epitope of human CEA. The CEA peptide epitopes of the present invention can effectively elicit an immune response to the associated CEA, overexpression of which is commonly correlated with the development of adenocarcinomas. Also provided by the present invention are immunogenic analogs of CEA, said analogs consisting of modified peptide epitopes that are more immunogenic than their wild- type counterparts. The isolated and purified CEA epitopes and analogs provided by the present invention are useful as immunogens in vaccines for the treatment and/or prevention of disorders associated with aberrant expression of CEA, including but not limited to carcinomas overexpressing CEA. Association of aberrant CEA expression with a carcinoma does not require that the CEA protein be expressed in tumor tissue at all timepoints of its development, as abnormal CEA expression may be present at tumor initiation and not be detectable late into tumor progression or vice-versa.

The CEA epitopes and analogs provided herein were selected based on their binding affinity for a class I MHC allele, specifically, HLA-A* 0201. Said peptides and analogs were additionally selected based on their ability to elicit a maximum tumor-specific immune response in a tolerized setting, as well as for their minimal potential for eliciting off-target autoimmune activity.

More specifically, the CEA epitopes and analogs of the present invention were initially selected by scanning the CEA protein sequence (SEQ ID NO:1) using a proprietary software package called EI Suite that ranks protein fragments based on binding affinity for a Class I MHC allele, in this case HLA-A*0201, similarity to fragments of other human and murine proteins, and amenability to immunogenic enhancement {see Fridman et al. , WO

2006/0257944). CEA epitopes and analogs predicted to be MHC class I binders were analyzed in vitro for binding affinity to T2 cells, which are HLA-A* 0201 positive, MHC class II negative and TAP deficient (see EXAMPLE 5). Moreover, the immunogenicity of the selected peptide epitopes and analogs was determined in HHD transgenic mice (see EXAMPLE 7). HHD mice are transgenic for the HHD complex (human β2-microglobulin fused to HLA- A2.1 αl and α2 domain, H-2D^b α3 domain) and are devoid of H-2D^b and murine β2-microglobulin. (Pascolo et al. J Exp. Med. 185(12):2043-51 (1997)). For this reason, the immune response elicited in these mice is specifically restricted to human HLA-A2.1, making this line a suitable model for epitope identification and optimization. The immunogenic CEA peptides and peptide analogs of the present invention can effectively elicit an immune response to the CEA protein, which, as stated above, has been implicated in the pathogenesis of cancer.

As stated above, the present invention provides isolated immunogenic CEA peptides that were predicted to be optimal vaccine candidates by EI Suite and determined to bind HLA-A*0201 and be immunogenic in HHD transgenic mice. CEA epitopes present in the major wild-type CEA allele and identified in this manner are disclosed in SEQ ID NOs: 2 and 8.

Class I MHC molecules are heterodimers of non-covalently bound MHC-encoded heavy (or alpha) chain, and a non-MHC-encoded B2-microglobulin light chain. There are four separate regions: 1) the peptide binding region, 2) the immunoglobulin-like region, 3) the transmembrane region and 4) a cytoplasmic region. The peptide-binding region is a groove which functions to accommodate a peptide ligand of 8-10 amino acid residues. To this end, the CEA epitopes and analogs of the present invention consist of a linear sequence of nine or ten amino acids, hi preferred embodiments of this aspect of the invention, the CEA epitopes and analogs are nine or ten amino acids in length.

The binding of peptide ligands to the MHC binding groove is specific and is stabilized at both ends by contacts between atoms in the free amino and carboxyl termini of the peptide and invariant sites that are found at each end of the cleft of all MHC class I molecules. Because the amino acid side chains at these positions insert into pockets in the MHC molecule and function to anchor the peptide to the MHC molecule they are commonly referred to as anchor residues. The bound peptide lies in an extended conformation along the groove. Anchor residues can be divided into primary and secondary. Primary anchor positions exhibit strong preferences for relatively well-defined sets of amino acid residues. Secondary positions show weaker and/or less well-defined preferences that can often be better described in terms of less favored, rather than more favored residues.

The anchor residues confer sequence selectivity and binding specificity to the interaction between the peptide ligand and the MHC molecule. The main anchor residues of human HLA class I molecules occur at positions 2 and the C-terminus of the peptides. Generally speaking, peptide-binding to a particular MHC molecule requires the peptide to have one or more specific amino acids at a fixed position, frequently the terminal or penultimate amino acid of the peptide. Since more stable binding will generally improve immunogenicity, anchor residues are preferably conserved or optimized in the design of analogs, regardless of their position.

As discussed above, it is known in the art that HLA class I binders modified at anchor positions (position 2 and the C-terminus position of the peptide), are often more immunogenic than the wild-type peptide due to improved binding to the HLA molecule (G. Lipford et. al., Immunology 84: 298-303 (1995)). At the same time, T-cells specific for the modified peptide generally also recognize the wild-type peptide, since the mutations are restricted to residues that do not make contact with the T-cell receptor. Based on this knowledge, the immunogenicity enhancement filter of EI Suite was utilized to identify anchor-modified analogs that comprise substitutions/mutations that optimize peptide/MHC binding interactions at the anchor positions. Said immunoenhancement filter identified substitutions within anchor residues of CEA T-cell epitope candidates to improve their immunogenicity. Immunoenhanced peptide analogs that are cross-reactive to the target antigen are beneficial for use in cancer vaccines targeting tumor-associated antigens to overcome tolerance and poor immunogenicity.

To this end, the present invention provides anchor-modified CEA peptide analogs which can elicit an immune response against CEA that is stronger than the immune response elicited by the corresponding wild-type epitope. The peptide analogs of the present invention comprise a sequence of amino acids selected from the group consisting of: SEQ HD NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, and SEQ ID NO: 13. Said CEA peptide analogs consist of a linear sequence of nine or ten amino acids. In a further aspect of the invention, the immunogenic CEA analogs described herein are comprised within a CEA protein that is longer than nine or ten amino acids, such as a variation of the full-length CEA protein disclosed in FIGURE IA. In this aspect of the invention, specific amino acid residues of the CEA protein are modified to produce a "variant CEA protein" which comprises one or more of the analogs identified herein. Such variant CEA proteins with engrafted immunogenic analogs elicit an immune response that it increased relative to full-length wild-type CEA protein as defined in SEQ ID NO:1. hi this aspect of the invention, the modified CEA proteins may be substantially the same length as the wild-type CEA protein disclosed in SEQ ID NO: 1 , or may be a CEA protein of shorter length, such as a human CEA that is deleted of its C-terminal anchoring domain (AD), which is located from about amino acid 679 to about amino acid 702 of full-length human CEA (SEQ ID NO: 1). An exemplary human CEA protein comprising an AD deletion (hCEAΔAD) that may be used as the basis for engraftment of the CEA analogs disclosed herein is shown in FIGURE IB, which amino acid sequence is set forth in SEQ ID NO:14. The modified CEA proteins of the present invention comprise at least one modification relative to the wild-type CEA protein disclosed in SEQ ID NO: 1 , and comprise at least one immunogenic analog of the present invention, said analog comprising a sequence of amino acids as set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12 and SEQ ID NO: 13. hi preferred embodiments of this aspect of the invention, the modified CEA protein comprises a plurality of the CEA analogs as set forth in SEQ ID NOs: 3-7 and 9-13.

Accordingly, the present invention relates to an immunogenic composition comprising a variant CEA protein, the variant CEA protein comprising one or more CEA epitope analogs, the CEA epitope analogs comprising a sequence of amino acids selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO:12 and SEQ ID NO:13. In one embodiment of the invention, the variant CEA protein comprises more than one of the CEA analogs disclosed herein, e.g. SEQ ID NO: 15, which comprises analogs SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:9, see FIGURE 1C. The CEA peptides, peptide analogs, and variant CEA proteins comprising engrafted analogs of the present invention may be used in immunogenic compositions or vaccines for the prevention and/or treatment of disorders associated with aberrant CEA expression. Accordingly, the present invention provides an immunogenic composition comprising at least one of the isolated CEA peptide epitopes, CEA epitope analogs, or variant CEA proteins comprising a CEA analog disclosed throughout the specification, together with a pharmaceutically acceptable carrier, excipient, diluent, stabilizer, buffer, or alternative designed to facilitate administration of the composition in an effective amount to a patient in need thereof. The compositions provided herein may also contain additional physiologically acceptable components, such as buffer, normal saline or phosphate buffered saline, sucrose, other salts and/or polysorbate. hi preferred embodiments of this aspect of the invention, the immunogenic compositions comprise a plurality of the isolated CEA peptides and/or CEA epitope analogs described herein and set forth as SEQ ID NOs:2-13 (see FIGURE 8). hi a specific embodiment of the invention, the immunogenic compositions comprise three or more CEA peptides or analogs in combination with a pharmaceutically acceptable carrier (e.g., SEQ ID NOs: 6, and 12). hi a further specific embodiment of the invention, the immunogenic compositions comprise three or more CEA peptides or analogs in combination with a pharmaceutically acceptable carrier (e.g., SEQ ID NOs: 6, 10, and 12). In an alternative embodiment, the immunogenic compositions comprise four or more CEA peptides or analogs (e.g., SEQ ID NOs: 5, 6, 10, and 12). hi a still further embodiment, the immunogenic compositions comprise five or more CEA peptides or analogs (e.g., SEQ ID NOs: 5, 6, 10, 11, and 12).

The present invention is further related to nucleotides encoding the immunogenic CEA epitopes, CEA epitope analogs, and variant CEA proteins described herein, which are useful either alone or in combination to construct DNA-based vaccines and minigenes targeting CEA. Said nucleotides are useful in genetic vaccines to elicit or enhance immunity to the protein product expressed by the CEA tumor-associated antigen.

Accordingly, the present invention relates to an isolated nucleic acid molecule or polynucleotide comprising a sequence of nucleotides encoding a CEA epitope analog, said analog comprising a sequence of amino acids as set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12 and SEQ ID NO:13. The nucleic acid molecules of the present invention are substantially free from other nucleic acids.

The present invention also relates to recombinant vectors and recombinant host cells, both prokaryotic and eukaryotic, which contain the nucleic acid molecules disclosed throughout this specification. The isolated DNA molecules, associated vectors, and hosts of the present invention are useful for the development of a cancer vaccine. As stated above, the present invention further relates to recombinant vectors that comprise the nucleic acid molecules encoding the CEA epitope analogs disclosed throughout this specification. These vectors may be comprised of DNA or RNA. For most cloning purposes, DNA vectors are preferred. Typical vectors include plasmids, modified viruses, baculovirus, bacteriophage, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA that can encode a CEA fusion protein. It is well within the purview of the skilled artisan to determine an appropriate vector for a particular gene transfer or other use.

An expression vector containing a CEA peptide-encoding nucleic acid molecule may be used for high-level expression of CEA peptides in a recombinant host cell. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. Also, a variety of bacterial expression vectors may be used to express recombinant CEA peptide sequences in bacterial cells if desired. In addition, a variety of fungal cell expression vectors may be used to express recombinant CEA peptide sequences in fungal cells. Further, a variety of insect cell expression vectors may be used to express recombinant peptides in insect cells.

The present invention also relates to host cells transformed or transfected with vectors comprising the nucleic acid molecules of the present invention. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of bovine, porcine, monkey and rodent origin; and insect cells including but not limited to Drosophila and silkworm derived cell lines. In one embodiment of the invention, the host cell is a yeast cell which is selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyvermyces fragilis, Kluveromyces lactis, and Schizosaccharomyces pombe. Such recombinant host cells can be cultured under suitable conditions to produce a CEA peptide epitope, epitope analog or variant CEA protein. In one embodiment of the present invention, the host cell is human. As defined herein, the term "host cell" is not intended to include a host cell in the body of a transgenic human being, human fetus, or human embryo.

The nucleic acids of the present invention may be assembled into an expression cassette which comprises sequences designed to provide for efficient expression of the peptide, analog, variant protein, or minigene in a human cell. The cassette preferably contains a CEA peptide epitope or analog-encoding gene, or, alternatively, a variant CEA-encoding gene or minigene, with related transcriptional and translations control sequences operatively linked to it, such as a promoter, and termination sequences. In a preferred embodiment, the promoter is the cytomegalovirus promoter without the intron A sequence (CMV), although those skilled in the art will recognize that any of a number of other known promoters, or other eukaryotic gene promoters may be used. A preferred transcriptional terminator is the bovine growth hormone terminator, although other known transcriptional terminators may also be used. The combination of CMV-BGH terminator is particularly preferred. In accordance with this invention, the CEA peptide expression cassette is inserted into a vector. The vector is preferably an adenoviral or plasmid vector, although linear DNA linked to a promoter, or other vectors, such as adeno-associated virus or a modified vaccinia virus, retroviral or lentiviral vector may also be used. In one embodiment of the invention, the vector is an adenovirus vector (used interchangeably herein with "adenovector"). Adenovectors can be based on different adenovirus serotypes such as those found in humans or animals. Examples of animal adenoviruses include bovine, porcine, chimp, murine, canine, and avian (CELO). Preferred adenovectors are based on human serotypes, more preferably Group B, C, or D serotypes. Examples of human adenovirus Group B, C, D, or E serotypes include types 2 ("Ad2"), 4 ("Ad4"), 5 ("Ad5"), 6 ("Ad6"), 24

("Ad24"), 26 ("Ad26"), 34 ("Ad34") and 35 ("Ad35"). In particularly preferred embodiments of the present invention, the expression vector is an adenovirus type 5 or 6 (Ad 5 or Ad6) vector.

If the vector chosen is an adenovirus, it is preferred that the vector be a so-called first-generation adenoviral vector. These adenoviral vectors are characterized by having a non- functional El gene region, and preferably a deleted adenoviral El gene region. Adenovectors do not need to have their El and E3 regions completely removed. Rather, a sufficient amount the El region is removed to render the vector replication incompetent in the absence of the El proteins being supplied in trans; and the El deletion or the combination of the El and E3 deletions are sufficiently large enough to accommodate a gene expression cassette. hi some embodiments, the expression cassette is inserted in the position where the adenoviral El gene is normally located. In addition, these vectors optionally have a nonfunctional or deleted E3 region. In some embodiments of the invention, the adenovirus genome used is deleted of both the El and E3 regions (ΔE1ΔE3). The adenoviruses can be multiplied in known cell lines which express the viral El gene, such as 293 cells, or PERC.6 cells, or in cell lines derived from 293 or PERC.6 cell which are transiently or stablily transformed to express an extra protein. For examples, when using constructs that have a controlled gene expression, such as a tetracycline regulatable promoter system, the cell line may express components involved in the regulatory system. One example of such a cell line is T-Rex-293; others are known in the art. For convenience in manipulating the adenoviral vector, the adenovirus may be in a shuttle plasmid form. This invention is also directed to a shuttle plasmid vector which comprises a plasmid portion and an adenovirus portion, the adenovirus portion comprising an adenoviral genome which has a deleted El and optional E3 deletion, and has an inserted expression cassette comprising a CEA epitope, analog, or variant CEA protein- encoding nucleotide sequence or CEA minigene. hi preferred embodiments, there is a restriction site flanking the adenoviral portion of the plasmid so that the adenoviral vector can easily be removed. The shuttle plasmid may be replicated in prokaryotic cells or eukaryotic cells.

In an exemplary embodiment of the invention, the expression cassette is inserted into an Ad6 (ΔE1ΔE3) adenovirus plasmid (See Emini et al., WO2003031588A2, which is hereby incorporated by reference). This vector comprises an Ad6 adenoviral genome deleted of the El and E3 regions. In some embodiments of the invention, the expression cassette is inserted into the pMRKAd5-HV0 adenovirus plasmid (See Emini et al.₃ WO 02/22080, which is hereby incorporated by reference). This plasmid comprises an Ad5 adenoviral genome deleted of the El and E3 regions. The design of the pMRKAd5-HV0 plasmid was improved over prior adeno vectors by extending the 5' cis-acting packaging region further into the El gene to incorporate elements found to be important in optimizing viral packaging, resulting in enhanced virus amplification. Advantageously, these enhanced adenoviral vectors are capable of maintaining genetic stability following high passage propagation. Standard techniques of molecular biology for preparing and purifying DNA constructs enable the preparation of the adenoviruses, shuttle plasmids, and DNA immunogens of this invention.

The vectors described above may be used in immunogenic compositions and vaccines for preventing or decreasing the likelihood of the development of adenocarcinomas associated with aberrant CEA expression and/or for treating existing cancers. The vectors of the present invention allow for vaccine development and commercialization by providing an immunogenic CEA peptide which can elicit an enhanced immune response, relative to full-length wild-type CEA when administered to a mammal such as a human being.

In accordance with the method described above, the vaccine vector may be administered for the treatment or prevention of a cancer in any mammal, including but not limited to: lung cancer, breast cancer, and colorectal cancer. In a preferred embodiment of the invention, the mammal is a human.

Further, one of skill in the art may choose any type of vector for use in the treatment and prevention method described. Preferably, the vector is an adenovirus vector or a plasmid vector. In a preferred embodiment of the invention, the vector is an adenoviral vector comprising an adenoviral genome with a deletion in the adenovirus El region, and an insert in the adenovirus El region, wherein the insert comprises an expression cassette comprising: a polynucleotide comprising a sequence of nucleotides that encodes at least one immunogenic CEA analog as described herein and as set forth in SEQ ID NOs:3-7 and 9-13, and a promoter operably linked to the polynucleotide. hi a preferred embodiment of this aspect of the invention, the adenovirus vector is an Ad 5 vector, an Ad6 vector, or an Ad 24 vector.

Also contemplated for use in the present invention is an adenovirus vaccine vector comprising an adenovirus genome that naturally infects a species other than human, including, but not limited to, chimpanzee adenoviral vectors. A preferred embodiment of this aspect of the invention is a chimp Ad 3 vaccine vector.

In another aspect, the invention relates to a vaccine plasmid comprising a plasmid portion and an expression cassette portion, the expression cassette portion comprising: (a) a sequence of nucleotides that encodes an immunogenic T-cell peptide epitope analog of CEA as set forth in SEQ ED NOs:3-7 and 9-13; and (b) a promoter operably linked to the polynucleotide.

The amount of expressible DNA or transcribed RNA to be introduced into a vaccine recipient will depend partially on the strength of the promoters used and on the immunogenicity of the expressed gene product. In general, an immunologically or prophylactically effective dose of about 1 ng to 100 mg, and preferably about 0.25-5mg of a plasmid vaccine vector is administered directly into muscle tissue. An effective dose for recombinant adenovirus is approximately 1 θ6 — 1012 particles and preferably about 10? — lOl lparticles. It may be desirable for the vaccine vectors of the present invention to be in a physiologically acceptable solution, such as, but not limited to, sterile saline or sterile buffered saline. Alternatively, it may be advantageous to administer an agent which assists in the cellular uptake of DNA, such as, but not limited to calcium ion. These agents are generally referred to as transfection facilitating reagents and pharmaceutically acceptable carriers. Those of skill in the art will be able to determine the particular reagent or pharmaceutically acceptable carrier as well as the appropriate time and mode of administration.

It is a common goal of vaccine development to augment the immune response to the desired antigen to induce long lasting protective and therapeutic immunity. Coadministration of vaccines with compounds that can enhance the immune response against the antigen of interest, known as adjuvants, has been extensively studied. In addition to increasing the immune response against the antigen of interest, some adjuvants may be used to decrease the amount of antigen necessary to provoke the desired immune response or decrease the number of injections needed in a clinical regimen to induce a durable immune response and to provide protection from disease and/or induce regression of disease. Therefore, the vaccines and immunogenic compositions described herein may be formulated with an adjuvant in order to primarily increase the immune response elicited by the isolated CEA peptides, analogs, variant CEA proteins and nucleic acid molecules described herein. Adjuvants which may be used in conjunction with the isolated peptides, analogs, and variant CEA proteins of the present invention, include, but are not limited to, adjuvants containing CpG oligonucleotides, or other molecules acting on toll-like receptors such as TLR9 (for review, see, Daubenberger, C.A., Curr. Opin. MoI. Ther. 9(l):45-52 (2007)), T-helper epitopes, lipid-A and derivatives or variants thereof, liposomes, cytokines, (e.g. granulocyte macrophage-colony stimulating factor (GMCSF)), CD40, CD28, CD70, IL-2, heat-shock protein (HSP) 90, CD 134 (OX40), CD 137, non ionic block copolymers, incomplete Freund's adjuvant, and chemokines. Additional adjuvants for use with the compositions described herein are adjuvants containing saponins (e.g. QS21), either alone or combined with cholesterol and phospholipid in the characteristic form of an ISCOM ("immune stimulating complex," for review, see Barr and Mitchell, Immunology and Cell Biology 74: 8-25 (1996); and Skene and Sutton, Methods 40: 53-59 (2006)). Additionally, aluminum-based compounds, such as aluminum hydroxide (Al(OH)₃), aluminum hydroxyphosphate (AlPO₄), amorphous aluminum hydroxyphosphate sulfate (AAHS) or so-called "alum" (KAl(SO₄)" 12H₂O), many of which have been approved for administration into humans by regulatory agencies worldwide, may be combined with the compositions provided herein.

As stated above, the epitopes and analogs of the present invention can be included in an immunogenic composition or vaccine targeting human CEA, which can then be administered to a human patient in need thereof to induce an immune response to CEA. Moreover, the administration of immunogenic compositions and vaccines comprising analogs of the present invention to a patient in need thereof can effectively elicit an enhanced cellular immune response that is cross-reactive to human CEA protein relative to native epitopes. To this end, the present invention provides a method for inducing an immune response to CEA in an individual in need thereof, said method comprising introducing into the individual a immunogenic composition comprising one or more CEA peptides or CEA epitope analogs of the present invention, or, alternatively the immunogenic composition comprises a polynucleotide encoding one or more CEA peptides or CEA epitope analogs described herein. In a preferred embodiment of this aspect of the invention, the immunogenic composition comprises a plurality of the CEA peptides and analogs disclosed herein, or a polynucleotide encoding a plurality of said peptides and analogs. The invention further provides a method of treating an individual suffering from

CEA-associated cancer, said method comprising introducing into the individual an immunogenic composition comprising at least one of the CEA peptides or analogs disclosed herein, or a polynucleotide encoding at least one of said CEA peptides and analogs; wherein introduction of the immunogenic composition leads to a clinically relevant immune response to CEA. In a preferred embodiment of this aspect of the invention, the immunogenic composition comprises a plurality of the CEA peptides and analogs disclosed herein, or a polynucleotide encoding a plurality of said epitopes and analogs. In a specific embodiment of the method described above, the immunogenic composition comprises three or more of the CEA peptides or analogs disclosed, hi a further specific embodiment of this method, the immunogenic composition comprises four or more of the CEA peptides or analogs disclosed.

Immunogenic compositions of the present invention may be used alone at appropriate dosages which allow for optimal induction of a cellular immune response against CEA with minimal potential toxicity. In addition, co-administration or sequential administration of other agents may be desirable. The compositions of the present invention may be administered to a patient by intramuscular injection, subcutaneous injection, intradermal introduction, or impression though the skin. Other modes of administration such as intraperitoneal, intravenous, or inhalation delivery are also contemplated. In preferred embodiments of the invention, the vaccines and pharmaceutical compositions are administered by intramuscular administration.

Also contemplated within the scope of this invention is administration of the immunogenic compositions described herein as part of a therapeutic regime, such as a therapeutic regime wherein two pharmaceutical compositions are administered in a "prime and boost" regimen. For example the first composition is administered one or more times, then after a predetermined amount of time, for example, 2 weeks, 1 month, 2 months, six months, or other appropriate interval, a second composition is administered one or more times. One of skill in the art can determine an appropriate interval of time that will lead to maximal durability of the immune response. Said therapeutic regime may include administration of a second immunogenic composition comprising the isolated CEA peptides or epitope analogs as described herein or, alternatively, administration of a genetic vaccine or other vaccine such as a cell-based, protein, or additional peptide-based vaccine. Said second immunogenic composition or vaccine may be any composition or vaccine targeting CEA, and/or a composition or vaccine targeting a different antigen, such as another TAA. The therapeutic regime could also comprise radiotherapy or chemotherapy.

All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing methodologies and materials that might be used in connection with the present invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

The following examples illustrate, but do not limit the invention.

EXAMPLE l Epitope Identification by EI Suite.

In order to identify epitope candidates that may elicit maximum tumor-specific immune response in a tolerized setting, while minimizing potential for off-target autoimmune activity, the entire CEA protein (SEQ ID NO:1 FIGURE IA) was scanned with several independent filters, using a proprietary software package called EI Suite (as described in WO 2006/124408). EI Suite ranks protein fragments based on binding affinity for a Class I MHC allele (in this case, HLA-A*0201), similarity to fragments of other human and murine proteins, and amenability to immunogenic enhancement. A total of 12 HLA-A* 0201 -restricted peptides and peptide analogs were identified by EI Suite as having the highest potential for use in an epitope-based vaccine or for in vitro monitoring of vaccine-induced CEA-specific CTL responses. The selected CEA peptide epitopes are shown in FIGURE 8 and include 2 wild-type CEA peptides and 10 peptide analogs. Examples 2-4 provide the details of peptide selection.

EXAMPLE 2 The A* 0201 binding affinity filter.

HLA binding affinity has been shown to correlate with T-cell recognition for peptides derived from viral antigens (Sette et al. J. Immunol. 153: 5586-92 (1994)). More recently, this relationship has also been observed for tumor epitopes (Keogh et al. J Immunol. 167: 787-796 (2001)). Selecting potential epitopes on the basis of HLA binding affinity is, therefore, an efficient alternative to large-scale epitope mapping or other T cell-dependent strategies. Therefore, using EI Suite, the sequence of the CEA protein (FIGURE 1 , SEQ ID

NO:1) was scanned for peptides that were predicted to be strong binders to the HLA class I allele A*0201 (referred to as A2.1 below). A total of 1267 peptides representing all possible 9- and 10-mer frames of the 702 amino acid protein were evaluated. Each peptide was scored based on the degree of adherence to a statistical motif inferred from several publicly available epitope databases, including: SYFPEITHI (Rammensee et al. Immunogenetics 50: 213-19 (1999)), MHCBN (Bhasin et al. Bioinformatics 19(5): 665-66 (2003), and FIMM (Schδnbach et al. Nucleic Acids Res. 28(1): 222-24 (2000)). 150 top-scoring fragments were retained for further analysis.

EXAMPLE 3

Comparison of CEA peptides with fragments of other human proteins.

A vaccine containing only CEA-specific epitopes may prevent the off-target T- cell response that sometimes occurs when immune tolerance is broken to a shared antigen. The CTL-mediated destruction of melanocytes (van Elsas et al. J. Exp. Med. 190: 355-366(1999)) and pancreatic islet β-cells (Ludewig et al. J. Exp. Med. 2000, 191 : 795-804 (2000)) following immunotherapy are two examples of such off-target immune response. Moreover, CEA peptides that are also found in other proteins that are expressed in multiple normal tissues are more likely to have been presented to T-cells in a tolerizing setting. Immune tolerance to such peptides may therefore be more difficult to overcome. For this reason, EI Suite was used to select, from the 150 top-scoring CEA peptides, those unique to the CEA protein (shown in FIGURE IA and set forth herein as SEQ ID NO:1).

It is known that T-cells can often recognize cognate epitopes comprising modifications at the HLA contact positions (positions 2 and 9/10 for HLA-A2.1) (Keogh (2001), supra). In contrast, mismatches within the TCR contact region (positions 1 and 3-8/9, for HLA- A2.1) are generally expected to lead to a loss of recognition. Therefore, to minimize the likelihood of vaccine-induced autoimmunity, CEA peptides whose TCR contact region was identical to a fragment of another human protein were rejected as epitope candidates even if they were predicted to be strong HLA binders and potentially immunogenic. Of 150 top-scoring CEA peptides, 49 were rejected because their TCR contact residues were identical to a fragment of another human protein.

However, a certain degree of degeneracy in peptide recognition exists with respect to changes in the TCR contact region (Sparbier et al. Curr. Opin. Imm. 11: 214-21 (1999)). In fact, depending on the peptide and the T-cell clone, changes in the TCR contact region may result in an enhanced recognition, loss of recognition, or an intermediate response (Scardino et al. Eur. J. Imm. 31 : 3261-3270 (2001)). In light of this fact, an expanded search of the human proteome was performed, allowing a single mismatch in the TCR binding region, in addition to a mismatch, if any, at positions 2 or 9/10. Of 150 top-scoring CEA peptides, 105 had sufficiently close matches with other proteins by this definition. These peptides were flagged to indicate that, if selected as vaccine candidates, they must undergo additional screening to rule out cross- recognition of proteins other than the target protein CEA. Interestingly, 15/150 (10%) top- scoring CEA peptides had matches outside the CEA family.

The output of the self-similarity filter for the 150 top-scoring CEA peptides is summarized in FIGURE 2. Most of the matches were within proteins in the CEACAM branch (CEA-like cell adhesion molecule) of the CEA family (Nomenclature Announcement, Exp. Cell Res. 252: 243-249 (1999)). In fact, 44/150 (29%) top-scoring CEA peptides were identical to a fragment of another CEACAM, and a few occurred in a majority of expressed human CEACAMs, including biliary glycoprotein (BGP, CEACAMl) and non-specific cross-reacting antigen (NCA, CEACAM6). BGP and NCA are both known to be expressed broadly in normal epithelia, including pancreas, lung, liver, kidney, and cervix (Hammarstrom et al. Semin Cancer Biol. 9: 67-81 (1999)). NCA is also known to be overexpressed in colorectal carcinoma (Koops et al. Eur J Biochem 253(3): 778-786(1998)).

17% (26/150) top-scoring CEA peptides matched a pregnancy-specific glycoprotein (PSG) (Hammarstrom et al. 1999, supra). CEA-like cell adhesion molecules

(CEACAM) and pregnancy-specific glycoproteins (PSG) form two branches of the CEA family, with CEA being in the CEACAM subset. PSGs have a unique expression profile compared to CEACAM members, with ubiquitous expression noted in the placenta during fetal development, and a more restrictive expression pattern seen in the normal adult tissues pancreas, salivary glands, and brain (Hammarstrom et al. 1999, supra). EXAMPLE 4 Anchor-modified peptide analogs.

It is known that the use of peptides modified at one or more HLA contact positions, called anchor-modified peptide analogs, may improve HLA binding affinity (See, e.g. Lipford et al. Immunology 84: 298-303 (1998)). As a result of improved HLA binding, the modified peptide is often more immunogenic than the original wild-type (wt) peptide. Furthermore, analog-specific T-cells generally recognize the wt peptide because of the identical amino acid residues in the TCR contact region.

Therefore, anchor-modified immunogenic analogs were identified for 11 of 14 CEA peptides that were not rejected by the self-similarity filter and were predicted to be moderate or better A2.1 binders (the score of 2 or higher). Most of the analogs identified comprise an I to V mutation at the C-terminus. Peptides with this mutation had a greater affinity for A2.1 and elicited T-cells that recognized the wt peptide more consistently than any other alteration in the HLA contact region (Keogh et al. J. Immunol. 167: 787-796 (2001)). Analog CEA.690L2 was not modified at the C-terminus because the wt peptide CEA.690 already comprises a valine in this position. Instead, a substitution to a leucine, a preferred amino acid at position 2 in the A*0201 binding motif, was made to improve binding affinity.

Anchor modifications for weaker HLA binders (binding score <2) were not investigated. These peptides are thought to be present at low density on the cell surface in complex with the HLA molecule, having been out-competed by stronger binding peptides (Pardoll, D.M. Nat Rev Imm 2: 227-238 (2002)).

EXAMPLE 5 Binding affinity of T-cell Epitopes to T2 cells. To determine the binding ability of EI Swztø-selected wild type peptides and analogs, an in vitro cellular binding assay was performed using the TAP-deficient cell line T2. T2 cells are HLA-A* 0201 positive, MHC class II negative and TAP deficient, meaning that they lack a functional transporter associated with antigen presentation, so that they accumulate empty unstable class I molecules. T2 cells were incubated with 50 μM peptide in serum-free RPMI 1640 supplemented with 5 μg/mL human β2m (Fluka) for 18 hours at 37°C. HLA-A*0201 expression was then measured by flow cytometry using the anti-HLA-A2.1 monoclonal antibody (mAb) BB7.2 followed by incubation with fluorescein isothiocyanate (FITC)-conjugated F(ab')2 goat antimouse Ig (Biosource). The results are expressed as fluorescence index (FI) defined as a ratio (median channel of fluorescence) between the sample and a control without any peptide. An increase over the control of at least 65% (FI > 2) was arbitrarily chosen as the cutoff point.

The binding assay consisted of the exogenous addition of peptides and β2 microglobulin protein: peptide binding up-regulates surface HLA expression and HLA-A2 molecules on the surface are measured using FACS by means of an antibody capable of recognizing the peptide-MHC complex (Kuzushima et al. Blood; 98:1872-81 (2001); Passoni et al. Blood 99:2100-06 (2002)).

Results of the binding assay are shown in FIGURE 3. Most of the modified epitopes (41 IVlO, 690L2, 589V10, 682V10, 307V10) showed a marked increase of binding to HLA-A* 0201 compared to their wild-type counterparts.

EXAMPLE 6 Measurement of HLA-A* 0201 /peptide complex stability To assess the HLA-A* 0201 /peptide complex stability, T2 cells (10⁶AnL) were incubated overnight with 50 μM of each peptide in serum-free RPMI 1640 supplemented with 100 ng/mL human β2m at 37°C. Cells were then washed 4 times to remove free peptides, incubated for 1 hour with 10 μg/mL Brefeldin A (Sigma- Aldrich) to block cell surface expression of newly synthesized HLA-A* 0201 molecules, washed, and incubated at 37°C for 0, 2, 4, 6, or 8 hours. Subsequently, cells were stained with anti-HLA-A2.1 mAb BB7.2. For each time point, peptide-induced HLA-A*0201 expression was calculated as mean fluorescence value of peptide incubated T2 cells/mean fluorescence value T2 cells in the absence of the peptide. Dissociation complex 50 (DC50) was defined as the time required for the loss of 50% of the HLA-A* 0201 /peptide complexes stabilized at t = 0. Binding stability of the peptide-MHC complex was also evaluated over time. The

CAP-I peptide was used as positive control. Again, results demonstrate an improved stability of peptide analogs, in particular for 41 IVlO, 589V 10 and 682V 10, compared to their wild-type counterparts (FIGURE 4).

EXAMPLE 7

Immunogenicity of T-cell epitopes in HHD transgenic mice.

HLA- A2.1 (HHD) transgenic mice were bred at Charles River Laboratories

(Lecco, Italy). These mice are transgenic for the HHD complex (human β2-microglobulin fused to HLA-A2.1 αl and α2 domain, H-2D^b α3 domain) and are devoid of H-2D^b and murine β2- microglobulin. (Pascolo et al. J. Exp. Med. 185(12):2043-51 (1997)). For this reason, the immune response elicited in these mice is specifically restricted to human HLA-A2.1, making this line a suitable model for epitope identification and optimization.

To determine the in vivo immunogenicity of CEA analogs and wild-type peptides,

HHD transgenic mice were co-immunized with 100 μg of the CEA peptide and 140 μg of the HBV core 128 helper T cell peptide (I-A^b-restricted, sequence TPP A YRPPNAPIL (SEQ ID

NO: 16). Both immunogens were emulsified in incomplete Freund's adjuvant (IFA) together with 50 μg of CpG TLR9 agonist and the emulsion was injected subcutaneously (s.c.) at the tail base of each animal. Groups of 4 HHD mice were injected subcutaneously with the following components: 1) wild type peptide or analog (100 meg); 2) HBV core peptide (140mcg); 3) CpG oligonucleotide (50mcg); 4) incomplete Freund adjuvant. Mice were given a second injection 15 days later with the same component. Two weeks after the last injection, mice were bled and peripheral blood lympho- monocytes (PBMC) and/or splenocytes were recovered for immunological assays. PBMC or splenocytes were analyzed by intracellular staining (ICS) for interferon gamma release upon stimulation with wild type or analog peptides (see EXAMPLE 8). Results demonstrate that the peptide analogs were more immunogenic than their wild type counterparts (FIGURES 5 and 6). The enhancement in immune response for the peptide analogs relative to the corresponding wt peptide ranged from 11.8- to 310-fold depending on the epitope. Importantly, the response elicited by analogs was fully cross reactive with corresponding natural peptides.

EXAMPLE 8 Intracellular Staining (ICS) for IFNy.

One to two millions mouse splenocytes or PBMC in 0.6ml RPMI 10% FCS were incubated with peptides (5 μg/ml final concentration of each peptide) and brefeldin A (1 μg/ml; BD PharMingen, Franklin Lakes, NJ) at 37°C and 5% CO₂ for 12-16 hours. Cells were then washed with FACS buffer (PBS 1% FBS, 0.01% NaN₃) and incubated with purified anti-mouse CDl 6/CD32 Fc block (BD PharMingen) for 15 min at 4°C. Cells were then washed and stained with surface antibodies: CD4-PE conjugated anti-mouse (BD PharMingen), PercP CD8 conjugated anti mouse (BD PharMingen) and APC- conjugated anti-mouse CD3e (BD PharMingen) for 30 minutes at room temperature in the dark. After the washing, cells were fixed and permeabilized with Cytofix-Cytoperm Solution (BD PharMingen) for 20 min at 4°C in the dark. After washing with Perm Wash Solution (BD PharMingen) cells were incubated with the IFNγ-FITC antibodies (BD PharMingen). Cells were then washed, fixed with formaldehyde 1% in PBS and analyzed on a FACS-Calibur flow cytometer, using CellQuest software (Becton Dickinson, Franklin Lakes, NJ).

EXAMPLE 9

Identification of additional immunogenic CEA epitopes

Additional HLA-A2.1 restricted peptides from CEA were identified by EI Suite as potential immunogenic epitopes for peptide and/or minigene vaccine, using the procedure described in EXAMPLES 1-3. These peptides were modified at specific positions as described in Example 4, to increase their binding affinity to MHC-I and consequently enhance their immunogenic potency.

In order to assess the immunogenicity of these peptides and correlate the immunogenic outcome with biochemical binding properties, groups of 6 HHD mice were immunized subcutaneously with lOOμg of each peptide admixed with HBVcorel28 helper epitope and a TLR9 agonist (Coley's CpG) in incomplete Freund adjuvant. Two weeks later, mice received a second injection with the same peptide mixture. After three weeks the immune response against the natural target epitope was analyzed by intracellular staining for EFNγ, as described in EXAMPLE 8. Results show that CEA691 and CEA605 were immunogenic, but little or no improvement was conferred by the corresponding analogs (FIGURE 7). On the other hand, CE A310 epitope was poorly immunogenic while CEA310L2 analog was extremely powerful in eliciting a cross reactive immune response, hi fact, this analog was 123 fold more immunogenic than the natural peptide. Similarly, CEA687 was found to be significantly immunogenic in mice, resulting in 4 out of 6 mice responding to the vaccination. However, the analog CEA687L2 was about 3.4 fold more immunogenic and 100% of the mice responded to the treatment. Importantly, a very good correlation between in vitro binding affinity (i-Topia assay) and in vivo immunogenicity data was found for these epitopes (data not shown)..

These data demonstrate that CEA691L2, CEA605V9, CEA310L2 and CEA687L2 would be useful for the development of a peptide-based vaccine targeting CEA, or for the construction of an epitope modified minigene (EMM) vaccine, or any other modality that incorporates these analogs, such as a genetic vaccine encoding CEA, in which positions 691-699, 310-318, etc, are replaced with the corresponding analog sequences, as described throughout the specification.

Claims

WHAT IS CLAIMED IS:

1. A carcinoembryonic antigen (CEA) peptide analog comprising a sequence of amino acids selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:5, SEQ ID

NO:6, SEQ ID NO:7, SEQ ID NO:9, and SEQ DD NO: 10; wherein the peptide analog consists of nine or ten amino acids.

2. An immunogenic composition comprising the peptide analog of claim 1 and a pharmaceutically acceptable carrier.

3. An immunogenic composition comprising a plurality of the isolated peptides of claim 1 and a pharmaceutically acceptable carrier.

4. The immunogenic composition of claim 3, further comprising an adjuvant.

5. A method of eliciting an immune response to CEA in an individual in need thereof, said method comprising introducing into the individual the immunogenic composition of claim 3.

6. A method of treating an individual suffering from CEA-associated cancer, said method comprising introducing into the individual an effective amount of the immunogenic composition of claim 3.

7. A variant carcinoembryonic antigen (CEA) protein comprising at least one modified sequence of amino acids, wherein the modified sequence is selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

8. The variant CEA protein of claim 7, wherein the protein contains a plurality of modified sequences.

9. An immunogenic composition comprising the variant CEA protein of claim 7 and a pharmaceutically acceptable carrier.

10. The immunogenic composition of claim 9, further comprising an adjuvant.

11. A method of eliciting an immune response to CEA in an individual in need thereof, said method comprising introducing into the individual the immunogenic composition of claim 9.

12. An isolated polynucleotide encoding an immunogenic peptide analog of human carcinoembryonic antigen (hCEA); wherein the analog comprises a sequence of amino acids selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ BD NO:9 and SEQ ID NO: 10, and wherein the peptide consists of nine or ten amino acids.

13. An expression vector comprising the polynucleotide of claim 12.

14. A host cell comprising the expression vector of claim 13.

15. The expression vector of claim 13, wherein the polynucleotide comprises a sequence of nucleotides which is operably linked to a promoter.

16. The expression vector of claim 13 , wherein the vector is an adenovirus vector or a plasmid vector.

17. The expression vector of claim 16, wherein the vector is an adenovirus vector selected from the group consisting of: Ad5, Ad6, and Ad24.

18. The expression vector of claim 17, wherein the vector is an Ad6 vector.

19. The expression vector of claim 16, wherein the vector is a plasmid vector.

20. A pharmaceutical composition comprising one or more of the polynucleotide of claim 12 and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, further comprising an adjuvant.

22. A vaccine comprising one or more of the isolated peptides of claim 1.

23. A method of eliciting an immune response to CEA in an individual suffering from or predisposed to cancer, said method comprising introducing into the individual the pharmaceutical composition of claim 20.

24. A method of treating an individual suffering from CEA-associated cancer, said method comprising introducing into the individual the pharmaceutical composition of claim 20.