WO2001048205A2

WO2001048205A2 - Murine neu sequences and methods of use therefor

Info

Publication number: WO2001048205A2
Application number: PCT/US2000/035648
Authority: WO
Inventors: A. Gregory Spies
Original assignee: Corixa Corporation
Priority date: 1999-12-29
Filing date: 2000-12-29
Publication date: 2001-07-05
Also published as: WO2001048205A3; AU2744201A

Abstract

The present invention is directed toward murine homologues of human Her-2/neu, as well as pharmaceutical compositions and vaccines comprising such sequences. Murine neu sequences are useful for immunotherapy of Her-2/neu associated cancers.

Description

MURINE NEU SEQUENCES AND METHODS OF USE THEREFOR

Technical Field

The present invention relates generally to murine homologues of human Her-2/neu, and to methods employing such sequences for immunotherapy of malignancies associated with the Her-2/neu oncogene.

Background of the Invention

Breast cancer is a significant health problem for women in the United States and throughout the world. Although advances have been made in detection and treatment of the disease, breast cancer remains the second leading cause of cancer- related deaths in women, affecting more than 180,000 women in the United States each year. For women in North America, the life-time odds of getting breast cancer are now one in eight.

No vaccine or other universally successful method for the prevention or treatment of breast cancer is currently available. Management of the disease currently relies on a combination of early diagnosis (through routine breast screening procedures) and aggressive treatment, which may include one or more of a variety of treatments such as surgery, radiotherapy, chemotherapy and hormone therapy. The course of treatment for a particular breast cancer is often selected based on a variety of prognostic parameters, including an analysis of specific tumor markers. See, e.g., Porter- Jordan and Lippman, Breast Cancer 5:73-100, 1994. However, the use of established markers often leads to a result that is difficult to interpret, and the high mortality observed in breast cancer patients indicates that improvements are needed in the treatment, diagnosis and prevention of the disease.

One protein that has shown promise for the detection and therapy of breast cancer is Her-2/neu, the product of the Her-2/neu oncogene, also known as pi 85 or c-erbB2 (see, e.g., U.S. Patent No. 5,869,445). Her-2/neu is amplified, and the Her- 2/neu protein overexpressed, in a variety of cancers such as breast, ovarian, colon, lung and prostate cancer. Methods based on Her-2/neu for treating Her-2/neu-associated cancers are being developed. Such methods would, however, be improved by the identification of related sequences that exhibit improved therapeutic properties.

Accordingly, there remains a need in the art for improved methods for treating breast cancer and other Her-2/neu associated cancers. The present invention fulfills these needs and further provides other related advantages.

Summary of the Invention

The present invention provides compounds and methods for detecting, treating and preventing Her-2/neu associated malignancies. Within certain aspects, the present invention provides isolated polynucleotides comprising at least a portion of the murine neu sequence recited in SEQ ID NO:l. Certain such polynucleotides comprise at least 70, 200 or 1500 consecutive nucleotides of the murine neu sequence recited in SEQ ID NO:l, or the full sequence recited in SEQ ID NO:l. Expression vectors comprising such polynucleotides, and host cells transformed or transfected with such expression vectors, are also provided.

Within further aspects, isolated polypeptides comprising at least 500 or 800 consecutive amino acid residues of murine neu (SEQ ID NO:2) are provided. Certain such polypeptides may comprise the amino acid sequence recited in SEQ ID NO:2. Other polypeptides may comprise an immunogenic portion of murine neu, such as LCFVNTVPWDQLFR (SEQ ID NO:3). Also provided are fusion proteins, which comprise at least one such polypeptide, and polynucleotides encoding such fusion proteins.

The present invention further provides, within other aspects, pharmaceutical compositions comprising a polynucleotide, polypeptide or fusion protein as described above in combination with a physiologically acceptable carrier. Still further pharmaceutical compositions may comprise an antigen-presenting cell that expresses a polypeptide as described above, in combination with a physiologically acceptable carrier. Within further aspects, vaccines are provided, comprising a polynucleotide, polypeptide or fusion protein as described above in combination with a non-specific immune response enhancer. Still further vaccines may comprise an antigen-presenting cell that expresses a polypeptide as described above, in combination with a non-specific immune response enhancer.

The present invention further provides methods for inhibiting the development of a cancer, such as breast cancer, in a patient. Certain such methods comprise administering to a patient an effective amount of a polynucleotide, polypeptide, fusion protein or polynucleotide encoding a fusion protein as described above. Other such methods comprise administering to a patient an effective amount of an antigen-presenting cell that expresses a polypeptide as described above.

Within further aspects, the present invention provides methods for removing tumor cells from a biological sample, comprising contacting a biological sample with T cells that specifically react with murine neu protein (SEQ ID NO:2), wherein the step of contacting is performed under conditions and for a time sufficient to permit the removal of cells expressing Her-2/neu from the sample. Suitable biological samples include, but are not limited to, blood and fractions thereof.

Methods for inhibiting the development of a cancer in a patient are also provided, wherein the methods comprise administering to a patient a biological sample treated as described above.

The present invention further provides, within other aspects, methods for stimulating and/or expanding T cells specific for a Her-2/neu, comprising contacting T cells with one or more of: (i) a polypeptide as described above; (ii) a polynucleotide as described above; and (iii) an antigen presenting cell that expresses a polypeptide as described above; under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells. Isolated T cell populations, comprising T cells prepared as described above, are also provided.

Within further aspects, the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a T cell population as described above. The present invention further provides methods for inhibiting the development of a cancer in a patient, comprising the steps of: (a) incubating CD4⁺ and/or CD 8+ T cells isolated from a patient with at least one component selected from the group consisting of: (i) a polypeptide as described above; (ii) a polynucleotide as described above; and (iii) an antigen presenting cell that expresses a polypeptide as described above; such that T cells proliferate; and (b) administering to the patient an effective amount of the proliferated T cells, and thereby inhibiting the development of a cancer in the patient.

Within further aspects, the present invention provides methods for inhibiting the development of a cancer in a patient, comprising the steps of: (a) incubating CD4⁺ and/or CD8+ T cells isolated from a patient with at least one component selected from the group consisting of: (i) a polypeptide as described above; (ii) a polynucleotide as described above; and (iii) an antigen presenting cell that expresses a polypeptide as described above; such that T cells proliferate; (b) cloning at least one proliferated cell; and (c) administering to the patient an effective amount of the cloned T cells, and thereby inhibiting the development of a cancer in the patient.

These and other aspects of the invention will become evident upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each were individually noted for incorporation.

Brief Description of the Drawings

Figures 1A and IB present a cDNA sequence encoding murine neu isolated from C57B16 mice (SEQ ID NO:l). Figure 2 presents the predicted amino acid sequence of the protein encoded by the cDNA sequence in Figures 1 A and IB (SEQ ID NO:2).

Detailed Description of the Invention

As noted above, the present invention provides murine neu sequences, which may be used for detecting and treating Her-2/neu associated malignancies. The invention is based, in part, on the discovery of a novel murine neu cDNA, which is 94% identical to rat neu and 86% identical to human Her-2/neu. The encoded protein is 88% identical to the rat and human proteins. Murine neu sequences may be used, for example for the prevention and treatment of malignancies associated with Her-2/neu expression. In particular, murine neu, (or immunogenic portions thereof) may be superior to human Her-2/neu for such purposes. This may be due, for example, to an improved ability to prime T cells, as a result of increased affinity of a foreign epitope for T cell receptors and antibodies, better processing to peptides, the presence of other epitopes recognized as foreign by the human T cells and/or better antibody-mediated loading of antigen-presenting cells by extracellular protein.

MURINE NEU POLYNUCLEOTIDES

Any polynucleotide that encodes a murine neu protein, or a portion or other variant thereof as described herein, is encompassed by the present invention. Preferred polynucleotides comprise at least 15 consecutive nucleotides, preferably at least 27 consecutive nucleotides and more preferably at least 70 consecutive nucleotides, that encode a portion of a murine neu protein. Longer polypeptides are also preferred for some applications (e.g., at least 100, 200, 500, 1000 or 1500 polynucleotides). More preferably, a polynucleotide encodes an immunogenic portion of a murine neu protein. Polynucleotides complementary to any such sequences are also encompassed by the present invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

Polynucleotides preferably comprise a native sequence (i.e., an endogenous sequence that encodes a murine neu protein or a portion thereof), but may alternatively comprise a variant of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the immunogenicity of the encoded polypeptide is not diminished, relative to a native neu protein. The effect on the immunogenicity of the encoded polypeptide may generally be assessed as described herein. Variants preferably exhibit at least about 90% identity, more preferably at least about 95% identity to a polynucleotide sequence that encodes a native murine neu protein or a portion thereof.

The percent identity for two polynucleotide or polypeptide sequences may be readily determined by comparing sequences using computer algorithms well known to those of ordinary skill in the art, such as Megalign, using default parameters. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, or 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Optimal alignment of sequences for comparison may be conducted, for example, using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters. Preferably, the percentage of sequence identity is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the window may comprise additions or deletions (i.e., gaps) of 20 % or less, usually 5 to 15 %, or 10 to 12%, relative to the reference sequence (which does not contain additions or deletions). The percent identity may be calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

Variants may also, or alternatively, be substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native murine neu protein (or a complementary sequence). Suitable moderately stringent conditions include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-65°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS.

It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).

Polynucleotides may be prepared using any of a variety of techniques.

For example, polynucleotides may be amplified from cDNA prepared from cells expressing murine neu, such as brain cells. Such polynucleotides may be amplified via polymerase chain reaction (PCR). For this approach, sequence-specific primers may be designed based on the sequences provided herein, and may be purchased or synthesized.

An amplified portion may be used to isolate a full length gene from a suitable library (e.g., a murine brain cDNA library) using well known techniques. Within such techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5' and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5' sequences. For hybridization techniques, a partial sequence may be labeled (e.g., by nick-translation or end-labeling with ³²P) using well known techniques. A bacterial or bacteriophage library is then screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Hybridizing colonies or plaques are selected and expanded, and the DNA is isolated for further analysis. cDNA clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector. Restriction maps and partial sequences may be generated to identify one or more overlapping clones. The complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones. The resulting overlapping sequences are then assembled into a single contiguous sequence. A full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques. Alternatively, there are numerous amplification techniques for obtaining a full length coding sequence from a partial cDNA sequence. Within such techniques, amplification is generally performed via PCR. Any of a variety of commercially available kits may be used to perform the amplification step. Primers may be designed using, for example, software well known in the art. Primers are preferably 22-30 nucleotides in length, have a GC content of at least 50% and anneal to the target sequence at temperatures of about 68°C to 72°C. The amplified region may be sequenced as described above, and overlapping sequences assembled into a contiguous sequence.

One such amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res. 16:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region. Within an alternative approach, sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region. A variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591. Another such technique is known as "rapid amplification of cDNA ends" or RACE. This technique involves the use of an internal primer and an external primer, which hybridizes to a polyA region or vector sequence, to identify sequences that are 5' and 3' of a known sequence. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic 7:111-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res. 79:3055-60, 1991). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.

In certain instances, it is possible to obtain a full length cDNA sequence by analysis of sequences provided in an expressed sequence tag (EST) database, such as that available from GenBank. Searches for overlapping ESTs may generally be performed using well known programs (e.g., NCBI BLAST searches), and such ESTs may be used to generate a contiguous full length sequence. It may also be obtained by analysis of genomic fragments.

A sequence encoding murine neu isolated from C57B16 mice is provided in Figures 1A and IB and SEQ ID NO:l, with the predicted amino acid sequence of the encoded protein provided in Figure 2 and SEQ ID NO:2. Polynucleotide variants may generally be prepared by any method known in the art, including chemical synthesis by, for example, solid phase phosphoramidite chemical synthesis. Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide- directed site-specific mutagenesis (see Adelman et al., DNA 2:183, 1983). Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding a murine neu protein, or portion thereof, provided that the DNA is incorporated into a vector with a suitable RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to prepare an encoded polypeptide, as described herein. In addition, or alternatively, a portion may be administered to a patient such that the encoded polypeptide is generated in vivo (e.g., by transfecting antigen-presenting cells, such as dendritic cells, with a cDNA construct encoding a murine neu polypeptide, and administering the transfected cells to the patient).

A portion of a sequence complementary to a coding sequence (i.e., an antisense polynucleotide) may also be used as a probe or to modulate gene expression. cDNA constructs that can be transcribed into antisense RNA may also be introduced into cells or tissues to facilitate the production of antisense RNA. An antisense polynucleotide may be used, as described herein, to inhibit expression of a neu protein. Antisense technology can be used to control gene expression through triple-helix formation, which compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules (see Gee et al., In Huber and Carr, Molecular and Immunologic Approaches, Futura Publishing Co. (Mt. Kisco, NY; 1994)). Alternatively, an antisense molecule may be designed to hybridize with a control region of a gene (e.g., promoter, enhancer or transcription initiation site), and block transcription of the gene; or to block translation by inhibiting binding of a transcript to ribosomes.

A portion of a coding sequence or of a complementary sequence may also be designed as a probe or primer to detect gene expression. Probes may be labeled with a variety of reporter groups, such as radionuclides and enzymes, and are preferably at least 10 nucleotides in length, more preferably at least 20 nucleotides in length and still more preferably at least 30 nucleotides in length. Primers, as noted above, are preferably 22-30 nucleotides in length.

Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

Nucleotide sequences as described herein may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques. For example, a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors. In general, a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Other elements will depend upon the desired use, and will be apparent to those of ordinary skill in the art.

Within certain embodiments, polynucleotides may be formulated so as to permit entry into a cell of a mammal, and expression therein. Such formulations are particularly useful for therapeutic purposes, as described below. Those of ordinary skill in the art will appreciate that there are many ways to achieve expression of a polynucleotide in a target cell, and any suitable method may be employed. For example, a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g., avian pox virus). The polynucleotides may also be administered as naked plasmid vectors. Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.

Other formulations for therapeutic purposes include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

MURINE NEU POLYPEPTIDES

Within the context of the present invention, polypeptides may comprise at least an immunogenic portion of a murine neu protein or a variant thereof, as described herein. Polypeptides as described herein may be of any length, but are preferably at least seven amino acids in length. Within certain embodiments, a polypeptide comprises at least 500, preferably at least 800, consecutive amino acid residues of the murine neu sequence provided in SEQ ID NO:2. A polypeptide may, but need not, comprise the full murine neu sequence provided in SEQ ID NO:2. Additional sequences derived from the native protein and/or heterologous sequences may be present, and such sequences may (but need not) possess further immunogenic or antigenic properties.

An "immunogenic portion," as used herein is a portion of a protein that is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor. Such immunogenic portions generally comprise at least 5 amino acid residues, more preferably at least 9, and still more preferably at least 20 amino acid residues of a murine neu protein or a variant thereof. Certain preferred immunogenic portions include peptides in which an N-terminal leader sequence and/or transmembrane domain have been deleted. Other preferred immunogenic portions may contain a small N- and/or C-terminal deletion (e.g., 1-30 amino acids, preferably 5-15 amino acids), relative to the mature protein. In general, immunogenic portions that differ from the human Her-2/neu sequence are preferred. Such portions include, for example, the peptide sequences TYLPANAS (SEQ ID NO:4), RLRIVRGTQLFEDKYAL (SEQ ID NO:5), EVGSCTLVCPPNN (SEQ ID NO:6), TQRCEKCSKPCAGVCYGL (SEQ ID NO:7), KIFGSLAFLPESFDGN (SEQ ID NO:8), IHSLGLRSLRELG (SEQ ID NO:9), LCFVNTVPWDQLFR (SEQ ID NO:3), TCYGSEADQ (SEQ ID NO: 10) and/or IIATVVGVL (SEQ ID NO:l 1).

Further immunogenic portions may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones. As used herein, antisera and antibodies are "antigen- specific" if they specifically bind to an antigen (i.e., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins). Such antisera and antibodies may be prepared as described herein, and using well known techniques. An immunogenic portion of a native murine neu protein is a portion that reacts with such antisera and/or T-cells at a level that is not substantially less than the reactivity of the full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Such immunogenic portions may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide. Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, ^l25I-labeled Protein A.

As noted above, a composition may comprise a variant of a native murine neu protein. A polypeptide "variant," as used herein, is a polypeptide that differs from a native murine neu protein in one or more substitutions, deletions, additions and/or insertions, such that the immunogenicity of the polypeptide is not substantially diminished. In other words, the ability of a variant to react with antigen- specific antisera may be enhanced or unchanged, relative to the native protein, or may be diminished by less than 50%, and preferably less than 20%, relative to the native protein. Such variants may generally be identified by modifying one of the above polypeptide sequences and evaluating the reactivity of the modified polypeptide with antigen-specific antibodies or antisera as described herein. Preferred variants include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed. Other preferred variants include variants in which a small portion (e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N- and/or C-terminal of the mature protein.

Polypeptide variants preferably exhibit at least about 90%, more preferably at least about 95% and most preferably at least about 99%) identity to the native polypeptide. The percent identity may be determined as described above. Preferably, a variant contains conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, tip, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide. Within particularly preferred embodiments, a variant differs by no more than 6 amino acid residues over the length of the murine neu sequence (1256 amino acids).

As noted above, polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post- translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

Polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by DNA sequences as described above may be readily prepared from the DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells and plant cells. Preferably, the host cells employed are E coli, yeast or a mammalian cell line such as COS or CHO. Supematants from suitable host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.

Portions and other variants having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid- phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 55:2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied BioSystems Division (Foster City, CA), and may be operated according to the manufacturer's instructions.

Within certain specific embodiments, a polypeptide may be a fusion protein that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, such as a known tumor protein. A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the protein.

Fusion proteins may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion protein is expressed as a recombinant protein, allowing the production of increased levels, relative to a non-fused protein, in an expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.

A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 55:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference. The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5' to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.

Fusion proteins are also provided that comprise a polypeptide as described herein together with an unrelated immunogenic protein. Preferably, the immunogenic protein is capable of eliciting a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, e.g., Stoute et al., New Engl. J. Med. 336:86-91, 1997).

Within preferred embodiments, an immunological fusion partner is derived from protein D, a surface protein of the gram-negative bacterium Haemophilus influenza B (WO 91/18926). Preferably, a protein D derivative comprises approximately the first third of the protein (e.g., the first Ν-terminal 100-110 amino acids), and a protein D derivative may be lipidated. Within certain preferred embodiments, the first 109 residues of a Lipoprotein D fusion partner is included on the Ν-terminus to provide the polypeptide with additional exogenous T-cell epitopes and to increase the expression level in E. coli (thus functioning as an expression enhancer). The lipid tail ensures optimal presentation of the antigen to antigen present cells. Other fusion partners include the non-structural protein from influenzae virus, ΝS1 (hemaglutinin). Typically, the Ν-terminal 81 amino acids are used, although different fragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an Ν-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at the amino terminus has been described (see Biotechnology 70:795-798, 1992). Within a preferred embodiment, a repeat portion of LYTA may be incorporated into a fusion protein. A repeat portion is found in the C- terminal region starting at residue 178. A particularly preferred repeat portion incorporates residues 188-305.

In general, polypeptides (including fusion proteins) and polynucleotides as described herein are isolated. An "isolated" polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.

As noted above, peptide portions of a full length murine neu polypeptide may be used to elicit an immune response against cells expressing human her-2/neu. Such peptide portions generally comprise at least nine or ten consecutive amino acid residues present within murine her-2/neu. Preferred peptide portions display an increased binding affinity for its restricting MHC class I molecule, relative to the corresponding peptide portion of human Her-2/neu. Binding affinity may generally be assessed using well known assays, such as peptide competition assays.

For certain applications, a polypeptide may be used in the form of a peptide/Class I MHC tetramer. A tetramer is an association of four complexes of antigenic peptide and MHC protein. Such tetramers may be prepared using standard techniques (see, e.g., Ogg and McMichael, Current Opinion in Immunology 70:393- 396, 1998; Altman et al., Science 274:94-96, 1996). Briefly, a peptide may added to the carboxy terminus of HLA-A2 heavy chain. The fusion protein is folded in vitro in the presence of β2-microglobulin. MHC -peptide complex is then purified (e.g., by gel filtration) and biotinylated at the C-terminus of the heavy chain, and tetramers are produced by mixing the biotinylated peptide-MHC complex with phycoerythrin-labeled deglycosylated avidin at a molar ratio of 4:1. Human Her-2/neu-specific CTL may be tested for the ability to recognize a tetramer using assays similar to those described above. As an alternative to tetramer formation as described above, a peptide may be covalently linked to the MHC protein. Such linkage has been described for manufacture of a tetramer from one Class I MHC-restricted peptide (White et al., J. Immunol. 162:2671-2676, 1999).

T CELLS

Immunotherapeutic compositions may also, or alternatively, comprise T cells specific for murine neu. Such cells may generally be prepared in vitro or ex vivo, using standard procedures. For example, T cells may be isolated from bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood of a patient, using a commercially available cell separation system, such as the CEPRATE™ system, available from CellPro Inc., Bothell WA (see also U.S. Patent No. 5,240,856; U.S. Patent No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, T cells may be derived from related or unrelated humans, non-human mammals, cell lines or cultures. T cells may be stimulated with murine neu polypeptide, polynucleotide encoding a murine neu polypeptide and/or an antigen presenting cell (APC) that expresses such a polypeptide. Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide. Preferably, a murine neu polypeptide or polynucleotide is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells. Stimulation with murine neu may achieve a superior immune response, as compared to stimulation with Her-2/neu, due to the presence of foreign epitope(s). Such enhanced immune responses have been observed in several systems (see, for example, Fong et al., J Immunol. 759:3113-37, 1997; Concetti et al., Cancer Immunol. Immunother. 43:307- 15, 1996). T cells are considered to be specific for murine neu if the T cells kill target cells coated with the polypeptide or expressing a gene encoding the polypeptide. T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al., Cancer Res. 54:1065-1070, 1994. Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques. For example, T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA). Contact with a murine neu polypeptide (100 ng/ml - 100 μg/ml, preferably 200 ng/ml - 25 μg/ml) for 3 - 7 days should result in at least a two fold increase in proliferation of the T cells. Contact as described above for 2-3 hours should result in activation of the T cells, as measured using standard cytokine assays in which a two fold increase in the level of cytokine release (e.g., TNF or IFN-γ) is indicative of T cell activation (see Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells that have been activated in response to a murine neu polypeptide, polynucleotide or polypeptide-expressing APC may be CD4⁺ and/or CD8⁺. Murine neu-specific T cells may be expanded using standard techniques. Within preferred embodiments, the T cells are derived from a patient, or from a related or unrelated donor, and are administered to the patient following stimulation and expansion.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate in response to a murine neu polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways. For example, the T cells can be re-exposed to a murine neu polypeptide (e.g., a short peptide corresponding to an immunogenic portion of such a polypeptide) or, preferably, to a human Her-2/neu polypeptide, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize a murine neu or Her-2/neu polypeptide. Although murine neu is superior for activating T cells that cross-react with human Her-2/neu, Her-2/neu itself may be better for amplifying the number of murine neu-activated T cells that cross-react with Her-2/neu. Alternatively, one or more T cells that proliferate in the presence of murine neu can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution. Following expansion, the cells may be administered back to the patient as described, for example, by Chang et al., Crit. Rev. Oncol. Hematol. 22:213, 1996.

MURINE NEU POLYPEPTIDE AND POLYNUCLEOTIDE FORMULATIONS Within certain aspects, one or more polypeptides, polynucleotides, T cells and/or tetramers may be incorporated into pharmaceutical compositions or immunogenic compositions (i.e., vaccines). Pharmaceutical compositions comprise one or more polypeptides and/or tetramers and a physiologically acceptable carrier. Vaccines may comprise one or more polypeptides and a non-specific immune response enhancer. A non-specific immune response enhancer may be any substance that enhances an immune response to an exogenous antigen. Examples of non-specific immune response enhancers include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is incorporated; see e.g., Fullerton, U.S. Patent No. 4,235,877). Vaccine preparation is generally described in, for example, M.F. Powell and M.J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach)," Plenum Press (NY, 1995). Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other tumor antigens may be present, either incorporated into a fusion polypeptide or as a separate compound within the composition or vaccine.

A pharmaceutical composition or vaccine may contain a polynucleotide encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ. Such a polynucleotide may comprise DNA, RNA, a modified nucleic acid or a DNA/RNA hybrid. As noted above, a polynucleotide may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 75:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerri ) that expresses an immunogenic portion of the polypeptide on its cell surface or secretes such an epitope. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Suitable systems are disclosed, for example, in Fisher- Hoch et al., Proc. Natl. Acad. Sci. USA 5(5:317-321, 1989; Flexner et al., Ann. N Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 5:17-21, 1990; U.S. Patent Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 97:215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993; Guzman et al., Circulation 55:2838-2848, 1993; and Guzman et al., Cir. Res. 75:1202-1207, 1993. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. It will be apparent that a vaccine may comprise both a polynucleotide and a polypeptide component. Such vaccines may provide for an enhanced immune response.

It will be apparent that a vaccine may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides provided herein. Such salts may be prepared from pharmaceutically acceptable non-toxic bases, including organic bases (e.g., salts of primary, secondary and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium and magnesium salts).

While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactate polyglycolate) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.

Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives. Alternatively, compositions of the present invention may be formulated as a lyophilizate. Compounds may also be encapsulated within liposomes using well known technology.

Any of a variety of non-specific immune response enhancers may be employed in the vaccines of this invention. For example, an adjuvant may be included. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

Within the vaccines provided herein, the adjuvant composition is preferably designed to induce an immune response predominantly of the Thl type. High levels of Thl -type cytokines (e.g., IFN-γ, TNF-α, IL-2 and IL-12) tend to favor the induction of cell mediated immune responses to an administered antigen. In contrast, high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction of humoral immune responses. Following application of a vaccine as provided herein, a patient will support an immune response that includes Thl- and Th2-type responses. Within a preferred embodiment, in which a response is predominantly Thl -type, the level of Thl -type cytokines will increase to a greater extent than the level of Th2-type cytokines. The levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.

Preferred adjuvants for use in eliciting a predominantly Thl -type response include, for example, a combination of monophosphoryl lipid A, preferably 3- de-O-acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt. MPL adjuvants are available from Ribi ImmunoChem Research Inc. (see US Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). Also preferred is AS-2 (SmithKline Beecham). CpG-containing oligonucleotides (in which the CpG dinucleotide is unmethylated) also induce a predominantly Thl response. Such oligonucleotides are well known and are described, for example, in WO 96/02555. Another preferred adjuvant is a saponin, preferably QS21, which may be used alone or in combination with other adjuvants. For example, an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739. Other preferred formulations comprises an oil-in-water emulsion and tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

Other preferred adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Ribi ImmunoChem Research Inc., Hamilton, MT), RC-529 (Ribi ImmunoChem Research Inc., Hamilton, MT) and Aminoalkyl glucosaminide 4- phosphates (AGPs). The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Microspheres may be used within such formulations. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release. The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

Any of a variety of delivery vehicles may be employed within pharmaceutical compositions and vaccines to facilitate production of an antigen-specific immune response that targets tumor cells. Delivery vehicles include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs. Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and or maintenance of the T cell response, to have anti-tumor effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype). APCs may generally be isolated from any of a variety of biological fluids and organs, including tumor and peritumoral tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendritic cells or progenitors thereof as antigen-presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 592:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999). In general, dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up process and present antigens with high efficiency and their ability to activate naive T cell responses. Dendritic cells may, of course, be engineered to express specific cell- surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may be used within a vaccine (see Zitvogel et al., Nature Med. :594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and/or TNF to cultures of monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/or other compound(s) that induce maturation and proliferation of dendritic cells.

Dendritic cells are conveniently categorized as "immature" and "mature" cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which correlates with the high expression of Fcγ receptor and mannose receptor. The mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide encoding an murine neu polypeptide as provided herein, such that the polypeptide is expressed on the cell surface. Such transfection may take place ex vivo, and a composition or vaccine comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells, for example, may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997. Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently conjugated to an immunological partner that provides T cell help (e.g., a carrier molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated immunological partner, separately or in the presence of the polypeptide.

TREATMENT AND PREVENTION OF HER-2/NEU ASSOCIATED MALIGNANCIES

As noted above, the present invention provides methods for treating and preventing Her-2/neu associated malignancies, such as breast cancer, by administering to a patient a therapeutically effective amount of a polypeptide, polynucleotide or antigen-presenting cell as described herein. As used herein, a "patient" refers to any warm-blooded animal, preferably a human. A patient may or may not be afflicted with cancer. Accordingly, the above pharmaceutical compositions and vaccines may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer. Her- 2/neu associated malignancies include, but are not limited to, breast cancer, ovarian cancer, gastric cancer, colon cancer, uterine cancer, prostate cancer, lung cancer, leukemia, lymphoma and myeloma.

A Her-2/neu associated cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor. Pharmaceutical compositions and vaccines may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs. Administration may be by any suitable method, including administration by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal, topical and oral routes.

Certain preferred therapeutic methods involve the administration to a patient of full length murine neu, or one or more immunogenic portions thereof that differ from the corresponding human Her-2/neu sequence. Such administration should be performed, as described herein, so as to elicit an immune response to human Her- 2/neu. It will be apparent that other proteins or peptides, such as portions of human Her-2/neu, may be simultaneously or sequentially administered, or such sequence(s) may be linked to the murine neu sequence(s) in the form of a fusion protein. Polynucleotide(s) encoding any of the foregoing polypeptides may also, or alternatively, be administered. Well known techniques, such as those described herein, may be used to increase the immunogenicity of the murine neu sequence(s). As noted above, preferred immunogenic portions of murine neu include portions that comprise the peptide sequences TYLPANAS (SEQ ID NO:4), RLRIVRGTQLFEDKYAL (SEQ ID NO:5), EVGSCTLVCPPNN (SEQ ID NO:6), TQRCEKCSKPCAGVCYGL (SEQ ID NO:7), KIFGSLAFLPESFDGN (SEQ ID NO:8), IHSLGLRSLRELG (SEQ ID NO:9), LCFVNTVPWDQLFR (SEQ ID NO:3), TCYGSEADQ (SEQ ID NO: 10) or IIATVVGVL (SEQ ID NO:l 1).

Within certain embodiments, immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors with the administration of immune response-modifying agents (such as polypeptides and polynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established tumor-immune reactivity (such as effector cells or antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on an intact host immune system. Examples of effector cells include T cells as discussed above, T lymphocytes (such as CD8⁺ cytotoxic T lymphocytes and CD4⁺ T-helper tumor- infiltrating lymphocytes), killer cells (such as Natural Killer cells and lymphokine- activated killer cells), B cells and antigen-presenting cells (such as dendritic cells and macrophages) expressing a polypeptide provided herein. T cell receptors and antibody receptors specific for the polypeptides recited herein may be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy. The polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Patent No. 4,918,164) for passive immunotherapy .

Effector cells may generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein. Culture conditions for expanding single antigen-specific effector cells to several billion in number with retention of antigen recognition in vivo are well known in the art. Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells. As noted above, immunoreactive polypeptides as provided herein may be used to rapidly expand antigen-specific T cell cultures in order to generate a sufficient number of cells for immunotherapy. In particular, antigen-presenting cells, such as dendritic, macrophage or B cells, may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art. For example, antigen-presenting cells can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or other expression system. Cultured effector cells for use in therapy must be able to grow and distribute widely, and to survive long term in vivo. Studies have shown that cultured effector cells can be induced to grow in vivo and to survive long term in substantial numbers by repeated stimulation with antigen supplemented with IL-2 (see, for example, Cheever et al, Immunological Reviews 757: 177, 1997).

Alternatively, a vector expressing a polypeptide recited herein may be introduced into antigen presenting cells taken from a patient and clonally propagated ex vivo for transplant back into the same patient. Transfected cells may be reintroduced into the patient using any means known in the art, preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumor administration.

Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, will vary from individual to individual, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Preferably, between 1 and 10 doses may be administered over a 52 week period. Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients. A suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response, and is at least 10-50% above the basal (i.e., untreated) level. Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine- dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro. Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients as compared to non- vaccinated patients. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 1 μg to 5 mg, preferably 100 μg to 5 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients. Increases in preexisting immune responses to Her-2/neu generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.

ASSAYS FOR DETECTING HER-2/NEU SPECIFIC T CELLS

Tetramers as described above may be used to detect the presence of T cells bearing Her-2/neu specific T cell receptors using any of a variety of well known techniques, such as standard flow-cytometry (FACS) techniques. Using tetramers, the frequency, surface phenotype and cytokine profile of antigen-specific T cells may be directly and simultaneously measured. Further, the use of tetramers permits the detection of antigen-specific T cells present at frequencies below the detectable limit in most T cell bioassays, and allows assays to be performed using primary lymphocyte populations (e.g., blood or lymph nodes).

Briefly, a sample comprising T cells (CD4⁺ and/or CD8⁺ T cells) is contacted with a tetramer under conditions and for a time sufficient to permit binding of the tetramer to Her-2/neu-specific T cells. The level of tetramer binding may then be assessed by any suitable technique, such as FACS analysis (see, e.g., Ogg and McMichael, Current Opinion in Immunology 70:393-396, 1998; Altman et al., Science 274:94-96, 1996). This interaction between tetramer and Her-2/neu-specific T cells may further be used to purify Her-2/neu-specific T cells from a biological sample that comprises lymphocytes. Briefly, such a biological sample is contacted with a tetramer under conditions and for a time sufficient to permit binding of the tetramer to Her- 2/neu-specific T cells; and T cells bound to the tetramer are separated from the remainder of the biological sample, using any standard technique.

Suitable samples for such analysis include samples obtained from a patient, such as blood (and fractions thereof). Preferred samples comprise lymphocytes and may be obtained from sources such as blood, spleen, lymph nodes or tumor tissue from a patient. Isolated T cells may also be used. For example, T cells may be isolated from a patient by routine techniques (such as by Ficoll/Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells may be incubated in vitro for 2-9 days (typically 4 days) at 37°C with tetramer (e.g., 5 - 25 μg/ml). It may be desirable to incubate another aliquot of a T cell sample in the absence of tetramer to serve as a control.

Assays employing such tetramers may be used, for example, to determine the presence or absence of a Her-2/neu associated cancer in a patient. A Her- 2/neu associated malignancy may be one characterized by overexpression of Her-2/neu, or for which Her-2/neu expression is considered by those in the art to be a prognostic indicator. Such cancers include, for example, breast cancer, ovarian cancer, gastric cancer, colon cancer, uterine cancer, prostate cancer, lung cancer, leukemia, lymphoma and myeloma. For CD4⁺ T cells, activation is preferably detected by evaluating proliferation of the T cells. For CD8⁺ T cells, activation is preferably detected by evaluating cytolytic activity. A level of proliferation that is at least two fold greater and/or a level of cytolytic activity that is at least 20% greater than in disease-free patients indicates the presence of a Her-2/neu associated cancer in the patient.

In another embodiment, tetramers may be used to monitor the progression of Her-2/neu associated cancer in a patient. In this embodiment, assays as described above for the diagnosis of a cancer may be performed using samples isolated from a patient over time, and the change in the level of reactive T cells evaluated. For example, the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed. In general, a cancer is progressing in those patients in whom the level of reactive T cells detected increases over time. In contrast, the cancer is not progressing when the level of reactive T cells either remains constant or decreases with time.

Similarly, tetramers may be used to enrich and/or purify Her-2/neu- specific CTL from a biological sample. Briefly, a biological sample comprising lymphocytes is contacted with a tetramer as described above under conditions and for a time sufficient to permit binding of the tetramer to Her-2/neu-specific T cells. T cells bound to the tetramer are then separated from the remainder of the biological sample, using standard techniques. CTL isolated by such methods may be used, for example, for immunotherapeutic purposes in an autologous setting for treatment of Her-2/neu associated cancers.

The following Examples are offered by way of illustration and not by way of limitation.

Example 1 Isolation of Murine Neu cDNA This Example illustrates the identification and characterization of a murine neu cDNA. A blast search of the mouse EST database with the human Her-2/neu sequence identified five EST clones. Primers were designed to clone the full-length mNeu cDNA:

5 'primer: CCATGGAGCTGGCGGCCTGGTGCCGTTG (SEQ ID NO:12) 3 'primer: GGCCTTCTGGTTCATACTGGCACATCCAGGC (SEQ ID

NO:13)

Messenger RNA was isolated from C57/B16 mouse brain. When PCR amplification was performed using the mouse Neu primers and cDNA from mouse brain as a template, a single product was seen on an agarose gel migrating at about 3.8kb. This DNA product was purified and ligated into pCR-XL-TOPO using

Invitrogen' s TOPO XL PCR cloning kit.

Clones from four separate PCR reactions were sequenced were determined to have the sequence shown in Figures 1A and IB and SEQ ID NO:l . The 3,771 base pair cDNA encodes a protein of 1,256 amino acids (Figure 2; SEQ ID NO:2). The translated sequence differs from a previously published amino acid sequence (Nagata et al., J. Immunol. 159: 1336-1343, 1997) at 8 positions. All of the clones from the independent PCR reactions had these differences from the published sequence. This represents the first disclosure of the nucleic acid sequence encoding mouse Neu.

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

Claims

ClaimsWhat is claimed is:

1. An isolated polynucleotide comprising at least 70 consecutive nucleotides of SEQ ID NO: 1.

2. An isolated polynucleotide comprising at least 200 consecutive nucleotides of SEQ ID NO:l.

3. An isolated polynucleotide comprising at least 1500 consecutive nucleotides of SEQ ID NO:l.

4. An isolated polynucleotide comprising a sequence recited in SEQ ID NO:l.

5. An expression vector comprising a polynucleotide according to any one of claims 1-4.

6. A host cell transformed or transfected with an expression vector according to claim 5.

7. An isolated polypeptide comprising at least 500 consecutive amino acid residues of murine neu (SEQ ID NO:2).

8. A polypeptide according to claim 7, wherein the polypeptide comprises at least 800 consecutive amino acid residues of SEQ ID NO:2.

9. A polypeptide according to claim 7, wherein the polypeptide comprises the sequence recited in SEQ ID NO:2.

10. An isolated polypeptide comprising the sequence LCFVNTVPWDQLFR (SEQ ID NO:3).

11. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1-4, in combination with a physiologically acceptable carrier.

12. A vaccine comprising a polynucleotide according to any one of claims 1-4, in combination with a non-specific immune response enhancer.

13. A vaccine according to claim 12, wherein the non-specific immune response enhancer is an adjuvant.

14. A vaccine according to claim 12, wherein the non-specific immune response enhancer induces a predominantly Type I response.

15. A pharmaceutical composition comprising a polypeptide according to any one of claims 7-10, in combination with a physiologically acceptable carrier.

16. A vaccine comprising a polypeptide according to any one of claims 7-10, in combination with a non-specific immune response enhancer.

17. A vaccine according to claim 16, wherein the non-specific immune response enhancer is an adjuvant.

18. A vaccine according to claim 16, wherein the non-specific immune response enhancer induces a predominantly Type I response.

19. A pharmaceutical composition comprising an antigen-presenting cell that expresses a polypeptide according to any one of claims 7-10, in combination with a pharmaceutically acceptable carrier or excipient.

20. A pharmaceutical composition according to claim 19, wherein the antigen presenting cell is a dendritic cell or a macrophage.

21. A vaccine comprising an antigen-presenting cell that expresses a polypeptide according to any one of claims 7-10, in combination with a non-specific immune response enhancer.

22. A vaccine according to claim 21, wherein the non-specific immune response enhancer is an adjuvant.

23. A vaccine according to claim 21, wherein the non-specific immune response enhancer induces a predominantly Type I response.

24. A vaccine according to claim 21, wherein the antigen-presenting cell is a dendritic cell or a macrophage.

25. A method for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a polynucleotide according to any one of claims 1-4, and thereby inhibiting the development of a cancer in the patient.

26. A method for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a polypeptide according to any one of claims 7-10, and thereby inhibiting the development of a cancer in the patient.

27. A method for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of an antigen-presenting cell that expresses a polypeptide according to any one of claims 7-10, and thereby inhibiting the development of a cancer in the patient.

28. A method according to claim 27, wherein the antigen-presenting cell is a dendritic cell or a macrophage.

29. A method according to claim 25, wherein the cancer is breast cancer.

30. A method according to claim 26, wherein the cancer is breast cancer.

31. A method according to claim 27, wherein the cancer is breast cancer.

32. A method according claim 25, wherein the patient is afflicted with cancer.

33. A method according claim 26, wherein the patient is afflicted with cancer.

34. A method according claim 27, wherein the patient is afflicted with cancer.

35. A fusion protein comprising at least one polypeptide according to any one of claims 7-10.

36. A fusion protein according to claim 35, wherein the fusion protein comprises an expression enhancer that increases expression of the fusion protein in a host cell transfected with a polynucleotide encoding the fusion protein.

37. A fusion protein according to claim 35, wherein the fusion protein comprises a T helper epitope that is not present within the native murine neu protein.

38. A fusion protein according to claim 35, wherein the fusion protein comprises an affinity tag.

39. An isolated polynucleotide encoding a fusion protein according to claim 35.

40. A pharmaceutical composition comprising a fusion protein according to claim 35, in combination with a physiologically acceptable carrier.

41. A vaccine comprising a fusion protein according to claim 35, in combination with a non-specific immune response enhancer.

42. A vaccine according to claim 41, wherein the non-specific immune response enhancer is an adjuvant.

43. A vaccine according to claim 41, wherein the non-specific immune response enhancer induces a predominantly Type I response.

44. A pharmaceutical composition comprising a polynucleotide according to claim 39, in combination with a physiologically acceptable carrier.

45. A vaccine comprising a polynucleotide according to claim 39, in combination with a non-specific immune response enhancer.

46. A vaccine according to claim 45, wherein the non-specific immune response enhancer is an adjuvant.

47. A vaccine according to claim 45, wherein the non-specific immune response enhancer induces a predominantly Type I response.

48. A method for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a pharmaceutical composition according to claim 40 or claim 44.

49. A method for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a vaccine according to claim 41 or claim 45.

50. A method for removing tumor cells from a biological sample, comprising contacting a biological sample with T cells that specifically react with murine neu protein (SEQ ID NO:2), wherein the step of contacting is performed under conditions and for a time sufficient to permit the removal of cells expressing Her-2/neu from the sample, and thereby removing tumor cells from the sample.

51. A method according to claim 50, wherein the biological sample is blood or a fraction thereof.

52. A method for inhibiting the development of a cancer in a patient, comprising administering to a patient a biological sample treated according to the method of claim 50.

53. A method for stimulating and/or expanding T cells specific for a Her-2/neu, comprising contacting T cells with one or more of:

(i) a polypeptide according to any one of claims 7-10;

(ii) a polynucleotide according to any one of claims 1-4; and

(iii) an antigen presenting cell that expresses a polypeptide according to any one of claims 7-10; under conditions and for a time sufficient to permit the stimulation and/or expansion of T cells.

54. An isolated T cell population, comprising T cells prepared according to the method of claim 53.

55. A method for inhibiting the development of a cancer in a patient, comprising administering to a patient an effective amount of a T cell population according to claim 54.

56. A method for inhibiting the development of a cancer in a patient, comprising the steps of:

(a) incubating CD4⁺ and/or CD8+ T cells isolated from a patient with at least one component selected from the group consisting of:

(i) a polypeptide according to any one of claims 7-10; (ii) a polynucleotide according to any one of claims 1-4; and (iii) an antigen presenting cell that expresses a polypeptide according to any one of claims 7-10; such that T cells proliferate; and

(b) administering to the patient an effective amount of the proliferated T cells, and thereby inhibiting the development of a cancer in the patient.

57. A method for inhibiting the development of a cancer in a patient, comprising the steps of: (a) incubating CD4⁺ and/or CD8+ T cells isolated from a patient with at least one component selected from the group consisting of:

(i) a polypeptide according to any one of claims 7-10; (ii) a polynucleotide according to any one of claims 1-4; and (iii) an antigen presenting cell that expresses a polypeptide according to any one of claims 7-10; such that T cells proliferate;

(b) cloning at least one proliferated cell; and

(c) administering to the patient an effective amount of the cloned T cells, and thereby inhibiting the development of a cancer in the patient.