JP2008289482A - Composition and method for therapy and diagnosis of ovarian cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition and a method for the therapy and diagnosis of cancer, particularly ovarian cancer. <P>SOLUTION: Illustrative compositions comprise one or more ovarian tumor polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of diseases, particularly ovarian cancer. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates generally to the treatment of ovarian cancer. The present invention more particularly relates to polypeptides comprising at least a portion of ovarian carcinoma protein, and polynucleotides encoding such polypeptides, as well as antibodies and immune system cells that specifically recognize such polypeptides. . Such polypeptides, polynucleotides, antibodies and cells can be used in vaccines and pharmaceutical compositions for the treatment of ovarian cancer.

  Ovarian cancer is a serious health problem for women in the United States and around the world. Although progress has been made in detecting and treating this cancer, vaccines or other commonly successful methods for prevention or treatment are not currently available. The management of this disease currently relies on a combination of early diagnosis and aggressive treatment, which is a variety of treatments such as surgery, radiation therapy, chemotherapy, and hormone therapy. One or more may be included. The course of treatment for a particular cancer is often selected based on various prognostic parameters, including analysis of specific tumor markers. However, the use of established markers often leads to difficult results to interpret and high mortality continues to be observed in many cancer patients.

  Immunotherapy has the potential to substantially improve cancer treatment and survival. Such treatment may include the generation or enhancement of an immune response against the ovarian carcinoma antigen. However, to date, ovarian carcinoma antigens are relatively unknown and the generation of an immune response against such antigens has not been shown to be a therapeutic benefit.

  Thus, there is a need in the art for improved methods for identifying ovarian tumor antigens and for using such antigens in the treatment of ovarian cancer. The present invention fulfills these needs and provides other related advantages.

  Briefly stated, the present invention provides compositions and methods for the treatment of cancer (eg, ovarian cancer).

In one aspect, the present invention provides a polynucleotide composition. The polynucleotide composition comprises a sequence selected from the group consisting of:
(A) SEQ ID NO (SEQ ID NO, SEQ ID) 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206 and 208 The sequence provided in
(B) SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206 and 208 ;
(C) SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206 and 208, at least A sequence of 20 consecutive residues;
(D) SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206 and 208, Sequences that hybridize under stringent conditions;
(E) SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206, 208 and 210-214. A sequence having at least 75% identity to the sequence;
(F) SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206, 208 and 210-214. A sequence having at least 90% identity to the sequence; and (g) SEQ ID NO: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193 Degenerate variants of the sequences provided in ~ 199, 203-206, 208 and 210-214.

  In one preferred embodiment, the polynucleotide composition of the invention is present in at least about 20%, more preferably at least about 30%, and most preferably at least about 50% of the ovarian tumor samples tested. Expressed at a level that is at least about 2-fold, preferably at least about 5-fold, and most preferably at least about 10-fold higher than the level of normal tissue.

  In one aspect, the invention provides a polypeptide comprising an immunogenic portion of an ovarian carcinoma protein, or one or more such that the ability of the variant to react with an ovarian carcinoma protein-specific antiserum is not substantially reduced. Variants of ovarian carcinoma proteins that differ in substitutions, deletions, additions and / or insertions are provided. In certain embodiments, the ovarian carcinoma protein is SEQ ID NO: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-205, 208 and 210-214 and sequences encoded by polynucleotide sequences selected from the group consisting of the complements of such polynucleotides

  The present invention further provides polynucleotides that encode the above polypeptides or portions thereof, expression vectors comprising such polynucleotides, and host cells transformed or transfected with such expression vectors.

  The present invention further provides a polypeptide composition comprising an amino acid sequence selected from the group consisting of the sequences listed in SEQ ID NOs: 200-202, 207, 209 and 215.

  In certain preferred embodiments, the polypeptides of the invention are immunogenic, i.e., they are immune responses, particularly humoral immune responses and / or cellular immunity, as further described herein. Can elicit a response.

  The present invention further provides fragments, variants, and / or derivatives of the disclosed polypeptide sequences, wherein these fragments, variants, and / or derivatives are represented by SEQ ID NOs: 1-185, 187- At least about 50% of the immunogenic activity level of an ovarian carcinoma protein comprising an amino acid sequence encoded by a polynucleotide comprising a sequence listed in any one of 199, 203-206, 208 and 210-214, preferably Preferably has a level of immunogenic activity that is at least about 70% and more preferably at least about 90%.

  In another aspect, the present invention provides a pharmaceutical composition comprising a polypeptide and / or polynucleotide as described above and a physiologically acceptable carrier.

  In a related aspect of the invention, pharmaceutical compositions (eg, vaccine compositions) are provided for prophylactic or therapeutic applications. Such compositions generally comprise an immunogenic polypeptide or polynucleotide of the invention and an immunostimulant (eg, an adjuvant).

  The invention further provides a pharmaceutical composition comprising: (a) an antibody or antigen-binding fragment thereof that specifically binds to a polypeptide of the invention or fragment thereof; and (b) physiologically acceptable. Career.

  In a further aspect, the present invention provides a pharmaceutical composition comprising: (a) an antigen presenting cell that expresses the above polypeptide, and (b) a pharmaceutically acceptable carrier or excipient. Exemplary antigen presenting cells include dendritic cells, macrophages, monocytes, fibroblasts and B cells.

  In a related aspect, a pharmaceutical composition is provided comprising: (a) an antigen-presenting cell that expresses the above polypeptide, and (b) an immunostimulant.

  In other aspects, the invention further comprises a fusion protein comprising at least one of the above-described polypeptides and a polynucleotide encoding such a fusion protein, typically a physiologically acceptable carrier and / or immunostimulatory. Provided in the form of a pharmaceutical composition (eg, vaccine composition) containing the drug. The fusion protein can comprise a plurality of immunogenic polypeptides, or portions / variants thereof, as described herein, and the expression, purification, and / or immunogen of these polypeptides. One or more polypeptide segments for promoting sex may further be included.

  In a further aspect, the present invention provides a method for stimulating an immune response in a patient, preferably a T cell response in a human patient, the method comprising administering a pharmaceutical composition as described herein. . The patient can suffer from ovarian cancer, in which case the method can provide treatment for the disease, or patients suspected of being at risk for such disease can be treated prophylactically.

  In a further aspect, the present invention provides a method for inhibiting the development of cancer in a patient, the method comprising administering to the patient a pharmaceutical composition as listed above. The patient can suffer from ovarian cancer, in which case the method can provide treatment for the disease, or patients suspected of being at risk for such disease can be treated prophylactically.

  In another aspect, the present invention further provides a method for removing tumor cells from a biological sample. The method includes contacting a biological sample with a T cell that specifically reacts with a polypeptide of the invention, wherein the contacting step removes cells expressing the protein from the sample. It is carried out under conditions and for a time sufficient to make it possible.

  In a related aspect, a method is provided for inhibiting cancer development in a patient, the method comprising administering to the patient a biological sample treated as described above.

  In another aspect, there is further provided a method for stimulating and / or expanding T cells specific for a polypeptide of the invention, wherein the method comprises treating the T cells with one or more of the following: The step of contacting under conditions and for a time sufficient to allow stimulation and / or proliferation of: (i) an ovarian cancer species polypeptide as described above; (ii) a polypeptide encoding such a polypeptide; Nucleotides; and / or (iii) antigen-presenting cells that express such polypeptides. Also provided is an isolated T cell population comprising T cells prepared as described above.

  In a further aspect, the present invention provides a method for inhibiting cancer development in a patient, the method comprising administering to the patient an effective amount of the T cell population described above.

  The present invention further provides a method for inhibiting the development of cancer in a patient comprising the following steps: (a) CD4 + T cells and / or CD8 + T cells isolated from a patient are Incubating with one or more of: (i) a polypeptide comprising at least an immunogenic portion of a polypeptide disclosed herein; (ii) a polynucleotide encoding such a polypeptide; and (iii) Antigen presenting cells that express such polypeptides; and (b) administering to the patient an effective amount of this expanded T cell, thereby inhibiting the patient's cancer development. The expanded cells can be cloned before being administered to the patient, but need not.

  In a further aspect, the present invention provides a method for determining the presence or absence of cancer (preferably ovarian cancer) in a patient. The method includes the following steps: (a) contacting a biological sample obtained from a patient with a binding agent that binds to a polypeptide listed above; (b) the binding in the sample. Detecting the amount of polypeptide that binds to the agent; and (c) comparing the amount of this polypeptide with a predetermined cutoff value, thereby determining the presence or absence of cancer in the patient. In a preferred embodiment, the binding agent is an antibody, more preferably a monoclonal antibody.

  In another aspect, the present invention also provides a method of monitoring cancer progression in a patient. Such a method includes the following steps: (a) contacting a biological sample obtained from a patient at an initial time point with a binding agent that binds to the polypeptide peptide listed above; ) Detecting the amount of polypeptide that binds to this binding agent in the sample; (c) repeating steps (a) and (b) using the biological sample obtained from the patient at the next time point And (d) comparing the amount of polypeptide detected in step (c) with the amount detected in step (b), thereby monitoring the progression of cancer in the patient.

In another aspect, the invention further provides a method for determining the presence or absence of a patient's cancer. The method includes the following steps: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide encoding a polypeptide of the invention; Detecting the level of a polynucleotide (preferably mRNA) that hybridizes with the oligonucleotide in the sample; and (c) comparing the level of the polynucleotide that hybridizes with the oligonucleotide to a predetermined cutoff value. And thereby detecting the presence or absence of the patient's cancer.
In certain embodiments, the amount of mRNA uses, for example, at least one oligonucleotide primer that hybridizes to a polynucleotide encoding a polypeptide as listed above or the complement of such a polynucleotide. Detected by polymerase chain reaction. In other embodiments, the amount of mRNA uses a hybridization technique using an oligonucleotide probe that hybridizes to a polynucleotide encoding a polypeptide as listed above or the complement of such a polynucleotide. Is detected.

  In a related aspect, a method for monitoring cancer progression in a patient is provided. The method includes the following steps: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide encoding a polypeptide of the invention; Detecting in the sample the amount of polynucleotide that hybridizes to the oligonucleotide; (c) at a later time, using the biological sample obtained from the patient, steps (a) and (b) Repeating; and (d) comparing the amount of polynucleotide detected in step (c) with the amount detected in step (b), thereby monitoring the progression of cancer in the patient.

  In a further aspect, the present invention provides an antibody (eg, a monoclonal antibody) that binds to a polypeptide as described above, as well as a diagnostic kit comprising such an antibody. Also provided are diagnostic kits comprising one or more oligonucleotide probes or oligonucleotide primers as described above.

  These and other aspects of the invention will be apparent upon reference to the following detailed description. All references disclosed herein are hereby incorporated by reference in their entirety as if each was individually incorporated.

(Short description of sequence identifier)
Sequence number 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69- 72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, and 193-199 are described in Tables III-VII below.

  SEQ ID NO: 200 is the amino acid sequence of the polypeptide encoded by the polynucleotide set forth in SEQ ID NO: 182.

  SEQ ID NO: 201 is the amino acid sequence of the polypeptide encoded by the polynucleotide set forth in SEQ ID NO: 182.

  SEQ ID NO: 202 is the amino acid sequence of the polypeptide encoded by the polynucleotide set forth in SEQ ID NO: 182.

  SEQ ID NO: 203 is the cDNA sequence determined and extended relative to SEQ ID NO: 197.

  SEQ ID NO: 204 is the extended cDNA sequence determined relative to SEQ ID NO: 198.

  SEQ ID NO: 205 is the cDNA sequence determined and extended relative to SEQ ID NO: 199.

  SEQ ID NO: 206 is the cDNA sequence determined for the coding region of O568S fused to the N-terminal His tag.

  SEQ ID NO: 207 is the amino acid sequence of the polypeptide encoded by the polynucleotide set forth in SEQ ID NO: 206.

  SEQ ID NO: 208 is the cDNA sequence determined for the coding region of GPR39 downloaded from High Throughput Genomics Database.

  SEQ ID NO: 209 is an amino acid sequence encoded by the cDNA sequence shown in SEQ ID NO: 208.

  SEQ ID NO: 210 is the nucleotide sequence of O1034C, an ovary-specific EST clone discovered using electronic subtraction.

  SEQ ID NO: 211 is the full length nucleotide sequence of O591S.

  SEQ ID NO: 212 is the sequence BF345141 showing sequence homology with O1034C / O591S that allows for the extension of O591S.

  SEQ ID NO: 213 is the sequence BE336607 showing sequence homology with O1034C / O591S allowing O591S extension.

  SEQ ID NO: 214 is the consensus nucleotide sequence for O1034C / O591S containing 1897 base pairs.

  SEQ ID NO: 215 is a putative translation of the open reading frame (nucleotides 260-682) identified in SEQ ID NO: 214.

  The present invention thus relates to a composition and its use in the treatment and diagnosis of cancer (particularly ovarian cancer). As described further below, exemplary compositions of the invention include, but are not limited to: polypeptides (particularly immunogenic polypeptides), polynucleotides encoding such polypeptides, antibodies And other binding factors, antigen presenting cells (APC) and immune system cells (eg, T cells).

  The practice of the present invention, unless otherwise indicated, is conventional for virology, immunology, microbiology, molecular biology, and recombinant DNA technology, many of which are exemplified by those skilled in the art. As described below for purposes). Such techniques are explained fully in the literature. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Vol. ); Oligonucleotide Synthesis (N. Gait, 1984); Nucleic Acid Hybridization (B. Hames and S. Higgins, 1985); Transcribion and C. gal, S. Hig. re (R.Freshney ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) see.

  All publications, patents and patent applications cited herein, whether above or below, are hereby incorporated by reference in their entirety.

  As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. .

(Polypeptide composition)
As used herein, the term “polypeptide” is used in its conventional sense (ie, as a sequence of amino acids). These polypeptides are not limited to products of a particular length; therefore, peptides, oligopeptides, and proteins are included in the definition of this polypeptide, and such terms are not particularly so Can be used interchangeably herein unless otherwise indicated.
The term also includes post-expression modifications (eg, glycosylation, acetylation, phosphorylation, etc.) of the polypeptide, as well as other modifications known in the art (both naturally occurring and non-naturally occurring modifications). Do not refer to or exclude them. A polypeptide can be an entire protein or a partial sequence thereof. A polypeptide of particular interest in the context of the present invention is an amino acid subsequence that contains an epitope (ie, an antigenic determinant that is substantially responsible for the immunogenic properties of the polypeptide and can elicit an immune response).

  Particularly exemplified polypeptides of the present invention include SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206, The polynucleotide sequence shown in any one of 208 and 210-214, or under moderately stringent conditions or highly stringent conditions Comprises a polypeptide encoded by hybridizing sequence to the polynucleotide sequence. Certain other exemplary polypeptides of the invention comprise the amino acid sequence set forth in any one of SEQ ID NOs: 200-202, 207, 209, and 215.

Since the polypeptides of the present invention show that their identification is based, at least in part, on the increased level of their expression in ovarian tumor samples, sometimes as ovarian tumor protein or ovarian tumor polypeptide herein. To be mentioned.
Thus, an “ovarian tumor polypeptide” or “ovarian tumor protein” generally refers to a polypeptide sequence of the invention, or a polynucleotide sequence encoding such a polypeptide, which are provided herein. As determined using a representative assay, at a level that is at least 2-fold, preferably 5-fold greater than the level of expression in normal tissue, is substantially proportional to the ovarian tumor sample (eg, tested Of ovarian tumor samples, preferably greater than about 20%, more preferably greater than about 30%, and most preferably greater than about 50%). The ovarian tumor polypeptide sequences of the present invention have particular utility as both diagnostic markers as well as therapeutic targets, as described further below, based on increased expression levels in the tumor cells.

In certain preferred embodiments, the polypeptides of the invention are immunogenic.
That is, they react detectably in immunoassays (eg, ELISA or T cell stimulation assays) using antisera and / or T cells from patients with ovarian cancer. Screening for immunogenic activity can be performed using techniques well known to those skilled in the art. For example, such screening can be performed using methods such as those described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one illustrative example, the polypeptide can be immobilized on a solid support and contacted with the patient's serum, allowing the antibody in the serum to bind to the immobilized polypeptide. Unbound serum is then removed and bound antibody can be detected using, for example, 125I-labeled protein A.

  As will be appreciated by those skilled in the art, immunogenic portions of the polypeptides disclosed herein are also encompassed by the present invention. As used herein, an “immunogenic moiety” is immunologically reactive with a B cell and / or T cell surface antigen receptor that itself recognizes a polypeptide (ie, specifically A fragment of an immunogenic polypeptide of the invention that binds). Immunogenic portions generally use well-known techniques (eg, techniques summarized in Paul, Fundamental Immunology, 3rd edition, 243-247 (Raven Press, 1993) and references cited therein). Can be identified. 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 those that specifically bind to an antigen (ie, they react with this protein in an ELISA or other immunoassay and are unrelated proteins) It is “antigen specific” if it does not react detectably). Such antisera and antibodies can be prepared as described herein and using well-known techniques.

  In one preferred embodiment, the immunogenic portion of a polypeptide of the invention has an anti-antigenic activity at a level that is not substantially lower than the reactivity of the full-length polypeptide (eg, in an ELISA and / or T cell reactivity assay). The part that reacts with serum and / or T cells. Preferably, the level of immunogenic activity of the immunogenic portion is at least about 50%, preferably at least about 70%, and most preferably greater than 90% of the immunogenicity of the full-length polypeptide. In some instances, preferred immunogenic moieties are identified that have a level of immunogenic activity that is greater than the corresponding full-length polypeptide (eg, having an immunogenic activity of about 100% or 150% or greater). The

  In certain other embodiments, exemplary immunogenic portions can include peptides lacking the N-terminal leader sequence and / or transmembrane domain. Other exemplary immunogenic portions include small N-terminal and / or C-terminal deletions (eg, 1-30 amino acids, preferably 5-15 amino acids) compared to the mature protein.

  In another embodiment, the polypeptide composition of the present invention can also be a T cell and / or a polypeptide of the present invention (particularly a polypeptide having the amino acid sequence disclosed herein) or an immunogenic fragment thereof. Alternatively, one or more polypeptides that are immunologically reactive with antibodies raised against the variant may be included.

In another embodiment of the invention, one or more polypeptides capable of eliciting T cells and / or antibodies that are immunologically reactive with one or more polypeptides described herein, or One or more polypeptides encoded by contiguous nucleic acid sequences contained in the polynucleotide sequences disclosed herein, or immunogenic fragments or variants thereof, or of moderate to high stringency Polypeptides comprising one or more polypeptides encoded by one or more nucleic acid sequences that hybridize to one or more of these sequences under conditions are provided.

In another aspect, the invention provides at least about 5, 10, 15, 20, 25, 50, or 100 consecutive amino acids or more (all intermediate lengths) of the polypeptide compositions set forth herein. Polypeptide fragments (e.g., polypeptide fragments shown in SEQ ID NOs: 200-202, 207, 209, and 215, or SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23). 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 14 -151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206, 208, and 210-214, encoded by the polynucleotide sequence shown in any one of 210-214 Polypeptide fragments).

  In another aspect, the present invention provides variants of the polypeptide compositions described herein. In general, polypeptide variants encompassed by the present invention will typically be at least about 70%, 75%, 80% along their length relative to the polypeptide sequences presented herein. 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity (determined as described below ).

  In one preferred embodiment, the polypeptide fragments and variants provided by the present invention are immunologically reactive with antibodies and / or T cells that react with the full-length polypeptides detailed herein. is there.

  In another preferred embodiment, the polypeptide fragments and variants provided by the invention are at least about 50%, preferably at least about 50% of the level of immunogenic activity exhibited by the full-length polypeptide sequences detailed herein. Exhibits a level of immunogenic activity of at least about 70%, and most preferably at least about 90% or more.

  A polypeptide “variant”, when the term is used herein, typically represents one or more substitutions, deletions, additions, and, with the polypeptides disclosed in detail herein. And / or polypeptides that differ in insertion. Such variants can occur naturally or, for example, modify one or more of the above-described polypeptide sequences of the invention and have their immunological properties as described herein. It can be made synthetically by assessing activity and / or using any of a number of techniques well known in the art.

  For example, certain exemplary variants of the polypeptides of the invention include variants in which one or more moieties (eg, N-terminal leader sequence or transmembrane domain) have been removed. Other exemplary variants include variants in which a small portion (eg, 1-30 amino acids, preferably 5-15 amino acids) has been removed from the N-terminus and / or C-terminus of the mature protein.

  In many instances, the variant contains a conservative substitution. A “conservative substitution” is one in which an amino acid is replaced with another amino acid having similar properties, so that those skilled in the art of peptide chemistry have substantially altered the secondary structure and hydrophobic hydrophilic properties of the polypeptide. It is a substitution that expects nothing. As noted above, modifications can be made in the structure of the polynucleotides and polypeptides of the invention, and functional molecules encoding variant or derivative polypeptides that still have the desired properties (eg, immunogenic properties). You can win. If it is desired that the amino acid sequence of a polypeptide be altered to produce an equivalent of the polypeptide of the invention (or even an improved immunogenic variant or part of the polypeptide of the invention) One skilled in the art typically alters one or more codons of the coding DNA sequence according to Table 1.

  For example, a particular amino acid may interact with other amino acids in a protein structure without appreciably losing its ability to interact with structures such as, for example, an antigen binding region of an antibody or a binding site on a substrate molecule. Can be substituted. Because the ability and nature of a protein to interact define the biological functional activity of the protein, certain amino acid sequence substitutions can be made in the protein sequence and, of course, the DNA coding sequence underlying it, and Nevertheless, proteins with similar properties are obtained. Accordingly, it is contemplated that various changes can be made in the peptide sequence of the disclosed composition or the corresponding DNA sequence encoding that peptide without appreciable loss of the biological utility or activity of the peptide. The

このような変化を作製するために、アミノ酸の疎水性親水性指標が考慮され得る。タンパク質に相互作用的な生物学的機能を付与する際の疎水性親水性アミノ酸指標の重要性は、一般的に当該分野において理解されている（ＫｙｔｅおよびＤｏｏｌｉｔｔｌｅ、１９８２、本明細書中に参考として援用される）。アミノ酸の相対的な疎水性親水性的性質は、得られるタンパク質の二次構造に寄与し、これは次には、そのタンパク質の他の分子（例えば、酵素、基質、レセプター、ＤＮＡ、抗体、抗原など）との相互作用を規定することが受け入れられている。
各アミノ酸は、その疎水性および電荷特性に基づいて疎水性親水性指標を割り当てられている（ＫｙｔｅおよびＤｏｏｌｉｔｔｌｅ、１９８２）。これらの値は以下である：イソロイシン（＋４．５）；バリン（＋４．２）；ロイシン（＋３．８）；フェニルアラニン（＋２．８）；システイン／シスチン（＋２．５）；メチオニン（＋１．９）；アラニン（＋１．８）；グリシン（−０．４）；スレオニン（−０．７）；セリン（−０．８）；トリプトファン（−０．９）；チロシン（−１．３）；プロリン（−１．６）；ヒスチジン（−３．２）；グルタミン酸（−３．５）；グルタミン（−３．５）；アスパラギン酸（−３．５）；アスパラギン（−３．５）；リジン（−３．９）；およびアルギニン（−４．５）。

To make such changes, the hydrophobic hydrophilicity index of amino acids can be considered. The importance of hydrophobic hydrophilic amino acid indices in conferring interactive biological functions on proteins is generally understood in the art (Kyte and Doolittle, 1982, as a reference herein). ). The relative hydrophobic and hydrophilic nature of amino acids contributes to the secondary structure of the resulting protein, which in turn is another molecule of the protein (eg, enzyme, substrate, receptor, DNA, antibody, antigen Etc.) is accepted.
Each amino acid has been assigned a hydrophobic hydrophilicity index based on its hydrophobicity and charge properties (Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9). ); Alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine ( -3.9); and arginine (-4.5).

  Certain amino acids can be replaced by other amino acids with similar hydrophobic hydrophilicity indices or scores, and still produce proteins with similar biological activity (ie, biologically functionally equivalent proteins It is well known in the art. In making such changes, substitution of amino acids whose hydrophobicity index is within ± 2 is preferred, amino acid substitutions whose hydrophobicity index is within ± 1 are particularly preferred, and their hydrophobicity Even more particularly preferred is the substitution of amino acids whose hydrophilicity index is within ± 0.5. It is also understood in the art that similar amino acid substitutions can be made on the basis of hydrophilicity. US Pat. No. 4,554,101, specifically incorporated herein by reference in its entirety, is governed by the hydrophilicity of its adjacent amino acids that correlate with the biological properties of the protein, Reference to the maximum local average hydrophilicity of the protein.

  As detailed in US Pat. No. 4,554,101, the following hydrophilicity values were assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be replaced with another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent protein (especially an immunologically equivalent protein). In such changes, the substitution of amino acids whose hydrophilicity value is within ± 2 is preferred, the substitution of amino acids whose hydrophilicity value is within ± 1 is particularly preferred, and the hydrophilicity value is within ± 0.5 Even more particularly preferred are amino acid substitutions.

  As outlined above, amino acid substitutions are therefore generally based on the relative similarity of amino acid side chain substituents (eg, their hydrophobicity, hydrophilicity, charge, size, etc.). Typical substitutions taking into account the various features described above are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

  Furthermore, any polynucleotide can be further modified to increase in vivo stability. Possible modifications include, but are not limited to: addition of flanking sequences at the 5 ′ and / or 3 ′ ends; phosphorothioate or 2′O-methyl rather than phosphodiester linkages in the backbone Use; and / or the inclusion of unusual bases (eg, inosine, kyeosin, and wybutosine, and acetyl, methyl, thio, and other modified forms of adenine, cytidine, guanine, thymine, and uridine).

  Amino acid substitutions can further be made based on the similarity of residue polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphiphilic properties. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids having an uncharged polar head with 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 exhibit conservative changes include (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val Ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also or alternatively include non-conservative changes. In a preferred embodiment, the variant polypeptide differs from the native sequence by no more than 5 amino acid substitutions, deletions or additions. Variants can also (or alternatively) be modified, for example, by amino acid deletions or additions that have minimal effect on the immunogenicity, secondary structure and hydrophobic hydrophilic properties of the polypeptide.

  As described above, a polypeptide can include a signal (or leader) sequence at the N-terminus of a protein, which directs the movement of the protein simultaneously with or after translation. The polypeptide can also be used to facilitate the synthesis, purification or identification of the polypeptide (eg, poly-His), or to enhance the binding of the polypeptide to a solid support, Can be conjugated to a sequence. For example, the polypeptide can be conjugated to an immunoglobulin Fc region.

  When comparing polypeptide sequences, two sequences are said to be “identical” when the sequence of amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a “comparison window” refers to a segment of at least about 20, usually 30 to about 75, 40 to about 50, where two sequences are optimally aligned. After being done, the sequence can be compared to the same number of reference sequences in consecutive positions.

  Optimal alignment of sequences for comparison can be performed using default parameters using the Megalign program (DNASTAR, Inc., Madison, WI) in the Lasergene package lift of bioinformatics software. This program integrates several alignment schemes described in the following references: Dayhoff, M. et al. O. (Eds.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Supplement 3, pages 345-358, Dayhoff, M .; O. (1978) A model of evolutionary change in proteins-Matrics for detecting distant relationships. Hein J .; (1990) Unified Approach to Alignment and Phylogenes, pages 626-645, Methods in Enzymology vol. 183, Academic Press, Inc. San Diego, CA; Higgins, D .; G. And Sharp, P .; M.M. (1989) CABIOS 5: 151-153; Myers, E .; W. And Muller W. et al. (1988) CABIOS 4: 11-17; Robinson, E .; D. (1971) Comb. Theor 11: 105; Santou, N .; Nes, M .; (1987) Mol. Biol. Evol. 4: 406-425; H. A. And Sokal, R .; R. (1973) Numeric Taxonomy-the Principles and Practice of Numeric Taxonomy, Freeman Press, San Francisco, Calif .; Wilbur, W .; J. et al. And Lipman, D .; J. et al. (1983) Proc. Natl. Acad. , Sci. USA 80: 726-730.

  Alternatively, optimal alignment of sequences for comparison can be found in Smith and Waterman (1981) Add. APL. Math 2: 482, by the local identity algorithm, Needleman and Wunsch (1970) J. MoI. Mol. Biol. 48: 443 by the alignment algorithm of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444 similarity search method, computerized implementation of these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI, GAP, BEST, AST, TFASTA) or by visual inspection.

  One preferred example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25: 3389-3402 and Altschul et al. (1990) J. MoI. Mol. Biol. 215: 403-410. BLAST and BLAST 2.0 can be used, for example, using the parameters described herein to determine percent sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information. For amino acid sequences, a scoring matrix can be used to calculate a cumulative score. Word hit extension in each direction stops when: The cumulative alignment score drops by an amount X from its maximum reached value; the cumulative score is cumulative with a negative score of one or more residue alignments. If it is less than zero due to this; or the end of any sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.

  In one preferred approach, the “percent sequence identity” is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polypeptide sequence in the comparison window is: Compared to a reference sequence (which does not include additions or deletions), no more than 20 percent, usually 5-15 percent, or 10-12 percent additions or deletions for optimal alignment of the two sequences (ie , Gap). This percentage determines the number of positions where identical amino acid residues occur in both sequences to obtain the number of matching positions, which is the total number of positions in the reference sequence (ie, window size). Calculated by dividing and multiplying this result by 100 to get the percentage of sequence identity.

  In other exemplary embodiments, the polypeptide comprises a plurality of polypeptides described herein, or at least one polypeptide described herein and unrelated sequences (eg, known tumors). Protein). For example, the fusion partner can assist in providing a T helper epitope, preferably a T helper epitope recognized by humans (immunological fusion partner), or express the protein in a higher yield than the native recombinant protein. (Expression enhancer). Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners can be selected to increase the solubility of the polypeptide or to allow the polypeptide to be targeted to the desired intracellular fraction. Still further fusion partners include affinity tags, which facilitate polypeptide purification.

  Fusion polypeptides can generally be prepared using standard techniques, including chemical conjugation. Preferably, the fusion polypeptide is expressed as a recombinant polypeptide in the expression system, allowing an increased level of production compared to the non-fusion polypeptide. Briefly, DNA sequences encoding this polypeptide component can be assembled separately and ligated into a suitable expression vector. The 3 ′ end of the DNA sequence encoding one polypeptide component has the reading frame of these sequences at the 5 ′ end of the DNA sequence encoding the second polypeptide component, with or without a peptide linker. Connected to be in the same phase. This allows translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.

  Peptide linker sequences can be used to separate the first and second polypeptide components by a sufficient distance to ensure that each polypeptide is folded into its secondary and tertiary structure. Such peptide linker sequences are incorporated into the fusion protein using standard techniques well known in the art. Appropriate peptide linker sequences can be selected based on the following factors: (1) ability to adopt a flexible extended conformation; (2) interact with functional epitopes on the first and second polypeptides. Inability to adopt a secondary structure that can act; and (3) No hydrophobic or charged residues capable of reacting with a functional epitope of the polypeptide. Preferred peptide linker sequences include Gly, Asn and Ser residues. Other near neutral amino acids such as Thr and Ala can also be used in the linker sequence. Amino acid sequences that can be usefully used as linkers are described in Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83: 8258-8262, 1986; including the amino acid sequences disclosed in US Pat. No. 4,935,233 and US Pat. No. 4,751,180. The linker sequence can generally be from 1 to about 50 amino acids in length. A linker sequence is not required if the first and second polypeptides have a nonessential N-terminal amino acid region that can be used to separate functional domains and to prevent steric interference.

  The ligated DNA sequence is operably linked to appropriate transcriptional or translational regulatory elements. The regulatory elements responsible for DNA expression are located only 5 'to the DNA sequence encoding the first polypeptide. Similarly, the stop codon required to terminate translation and transcription termination signals is present only 3 'to the DNA sequence encoding the second polypeptide.

  The fusion polypeptide can include a polypeptide as described herein, along with an unrelated immunogenic protein, such as an immunogenic protein that can elicit a recall response. Examples of such proteins include tetanus protein, tuberculosis protein and hepatitis protein (see, eg, Stoute et al., New Engl. J. Med., 336: 86-91 (1997)).

  In one preferred embodiment, the immunological fusion partner is Mycobacterium sp., Such as a Ra12 fragment from Mycobacterium tuberculosis. Is from. Methods for using Ra12 in enhancing expression and / or immunogenicity of Ra12 compositions and heterologous polynucleotide / polypeptide sequences are described in US patent application 60 / 158,585 (the disclosure of which is herein incorporated by reference). The entirety of which is incorporated by reference). Briefly, Ra12 refers to a polynucleotide region that is a subsequence of Mycobacterium tuberculosis MTB32A nucleic acid. MTB32A is an M.M. It is a serine protease having a molecular weight of 32 KD encoded by genes of toxic and non-toxic strains of tuberculosis. The nucleotide and amino acid sequences of MTB32A have been described (see, eg, US patent application 60 / 158,585; see also Skekiy et al., Infection and Immun. (1999) 67: 3998-4007. Incorporated herein by reference). The C-terminal fragment of the MTB32A coding sequence is expressed at high levels and remains as a soluble polypeptide throughout the purification process. In addition, Ra12 can enhance the immunogenicity of the heterologous immunogenic polypeptide to which it is fused. One preferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other preferred Ra12 polynucleotides are generally at least about 15 contiguous nucleotides that encode a portion of the Ra12 polypeptide, at least about 30 nucleotides, at least 60 nucleotides, at least about 100 nucleotides, At least about 200 nucleotides, or at least about 300 nucleotides. A Ra12 polynucleotide may comprise a native sequence (ie, an endogenous sequence encoding a Ra12 polypeptide or a portion thereof), or may comprise a variant of such a sequence. A Ra12 polynucleotide variant may comprise one or more substitutions, additions, deletions and / or insertions such that the biological activity of the encoded fusion polypeptide comprises a native Ra12 polypeptide. Compared with, it does not decrease substantially. Preferably, the variant has at least about 70% identity, more preferably at least about 80% identity, and most preferably at least about 90% to the polynucleotide sequence encoding the native Ra12 polypeptide or portion thereof. % Identity.

  In another preferred embodiment, the immunological fusion partner is derived from protein D (WO 91/18926), a surface protein of the gram-negative bacterium Haemophilus influenza B. Preferably, the protein D derivative comprises approximately the first third of the protein (eg, the first N-terminal 100-110 amino acids), and the protein D derivative can be lipidated. In certain preferred embodiments, the first 109 residues of the lipoprotein D fusion partner provide a polypeptide having additional exogenous T cell epitopes and E. coli. Included at the N-terminus (thus functioning as an expression enhancer) to increase the expression level in E. coli. The lipid tail ensures optimal presentation of antigen to antigen presenting cells. Other fusion partners include NS1 (hemagglutinin), an unstructured protein from influenza virus. Typically, the N-terminal 81 amino acids are used, although different fragments containing T helper epitopes can be used.

  In another embodiment, the immunological fusion partner is a protein known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is derived from Streptococcus pneumoniae which synthesizes an N-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 specific bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for affinity for choline or some choline analogs (eg, DEAE). This property is useful for the expression of E. coli useful for the expression of fusion proteins. has been used for the development of E. coli C-LYTA expression plasmids. Purification of hybrid proteins containing a C-LYTA fragment at the amino terminus has been described (see Biotechnology 10: 795-798, 1992). In preferred embodiments, the repeat portion of LYTA can be incorporated into a fusion polypeptide. The repeat is found in the C-terminal region starting at residue 178. Particularly preferred repeat moieties incorporate residues 188-305.

  Yet another exemplary embodiment includes fusion polypeptides, and polynucleotides encoding them, wherein the fusion partner converts the polypeptide to an endosome / as described in US Pat. No. 5,633,234. Contains a targeting signal that can be directed to the lysosomal fraction. The immunogenic polypeptides of the present invention more efficiently bind to MHC class II molecules when fused to this targeting signal, thereby enhancing in vivo stimulation of CD4 + T cells specific for this polypeptide Is provided.

The polypeptides of the present invention are prepared using any of a variety of well-known synthetic and / or recombinant techniques. The latter of these techniques is further described below.
Polypeptides, moieties and other variants, generally less than about 150 amino acids, can be made by synthetic means using techniques well known to those skilled in the art. In one exemplary embodiment, such polypeptides are synthesized using any of the commercially available solid phase techniques (eg, Merrifield solid phase synthesis methods), where the amino acids are growing. It is continuously added to the amino acid chain. Merrifield, J.M. Am. Chem. Soc. 85: 2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer / Applied BioSystems Division (Foster City, Calif.) And can be operated according to the manufacturer's instructions.

  In general, the polypeptide compositions (including fusion polypeptides) of the invention are isolated. An “isolated” polypeptide is one that is removed from its original environment. For example, a naturally occurring protein or polypeptide is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such polypeptides are also purified, eg, at least about 90% pure, more preferably at least about 95% pure, and most preferably at least about 99% pure.

(Polynucleotide composition)
In another aspect, the present invention provides a polynucleotide composition. The terms “DNA” and “polynucleotide” are used herein interchangeably and refer to an isolated DNA molecule that does not contain the total genomic DNA of a particular species. As used herein, “isolated” means that the polynucleotide is substantially separated from other coding sequences and the DNA molecule is predominantly unrelated to coding DNA (eg, Large chromosomal fragments or other functional genes or polypeptide coding regions). Of course, this refers to a DNA molecule that was originally isolated and does not exclude genes or coding regions that were later artificially segmented.

  As will be appreciated by those skilled in the art, the polynucleotide compositions of the invention express proteins, polypeptides, peptides, etc., or are encoded by genomic sequences, extragenomic sequences and plasmids that can be adapted to express. It may contain sequences as well as smaller engineered gene segments. Such segments can be isolated in nature or can be synthetically modified by the hand of man.

  As will also be appreciated by those skilled in the art, the polynucleotides of the invention can be single stranded (coding or antisense) or double stranded and can be a DNA molecule (genomic, cDNA or synthetic) or an RNA molecule. . RNA molecules can include HnRNA molecules (which contain introns and correspond in a one-to-one manner to DNA molecules) and mRNA molecules (which do not contain introns). Additional coding or non-coding sequences may be present in the polynucleotides of the invention, but need not be, and polynucleotides may but need not be linked to other molecules and / or support materials.

  A polynucleotide may comprise a native sequence (ie, an endogenous sequence encoding a polypeptide / protein of the invention, or portion thereof), or a variant or derivative of such a sequence, and preferably immunogenic It may contain sequences encoding variants or derivatives.

  Thus, according to another aspect of the present invention, SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41-50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117, 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199, 203-206, 208 and 210-214, the complement of the polynucleotide sequence shown above, and the polynucleotide shown above. Polynucleotide composition comprising some or all of the degenerate variants of plastid sequences are provided. In certain preferred embodiments, the polynucleotide sequences set forth herein encode immunogenic polypeptides as described above.

  In other related embodiments, the invention relates herein to SEQ ID NOs: 1, 2, 5, 9, 10, 13, 16, 19, 23, 27, 28, 32, 33, 35, 38, 41. -50, 52, 53, 56, 57, 63, 65, 69-72, 75, 78, 80-82, 84, 86, 89-93, 95, 97-100, 103, 107, 111, 114, 117 120, 121, 125, 128, 132-134, 136, 137, 140, 143-146, 148-151, 156, 158, 160-162, 166-168, 171, 174-183, 185, 193-199 , 203-206, 208 and 210-214, polynucleotide variants having substantial identity to the sequences disclosed. For example, using the methods described herein (eg, BLAST analysis using standard parameters as described below), at least 70% sequence compared to the polynucleotide sequence of the invention Polynucleotide variants that contain identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. Those skilled in the art will appreciate that these values are appropriate for determining the corresponding identity of the proteins encoded by the two nucleotide sequences by considering codon degeneracy, amino acid similarity, reading frame positioning, etc. Recognize that can be adjusted to.

  Typically, a polynucleotide variant comprises one or more substitutions, additions, deletions and / or insertions, preferably so that the immunogenicity of the polypeptide encoded by the variant polynucleotide is: There is no substantial reduction relative to the polypeptide encoded by the polynucleotide sequences specifically set forth herein. It should be understood that the term “variant” also encompasses homologous genes of heterologous origin.

  In further embodiments, the present invention provides polynucleotide fragments comprising consecutive stretches of various lengths of sequences that are identical or complementary to one or more of the sequences disclosed herein. . For example, at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein As well as intermediate lengths of contiguous nucleotides in between are provided by the present invention. In this situation, the “intermediate length” is any length between the indicated values (eg, 16, 17, 18, 19 etc .; 21, 22, 23 etc .; 30, 31, 32 etc .; 50 51, 52, 53, etc .; 100, 101, 102, 103, etc .; 150, 151, 152, 153, etc .; including all integers between 200-500; 500-1,000, etc.) Easy to understand.

  In another embodiment of the invention, a polynucleotide composition capable of hybridizing under moderate to high stringency conditions to a polynucleotide sequence provided herein or a fragment thereof or a complementary sequence thereof is provided. Provided. Hybridization techniques are well known in the art of molecular biology. For illustrative purposes, moderately stringent conditions suitable for testing hybridization of other polynucleotides to the polynucleotides of the invention are 5 × SSC, 0.5% SDS, 1.0 mM. Pre-wash in a solution of EDTA (pH 8.0); 50 ° C.-60 ° C., 5 × SSC overnight hybridization; followed by 2 ×, 0.5 × containing 0.1% SDS and Includes two washes for 20 minutes at 65 ° C., each with 0.2 × SSC. Those skilled in the art will appreciate that the stringency of hybridization can be readily manipulated, for example, by changing the salt content of the hybridization solution and / or the temperature at which the hybridization is performed. For example, in another embodiment, suitable high stringency hybridization conditions include those described above, except that the temperature of hybridization is increased to, for example, 60-65 ° C or 65-70 ° C. Can be mentioned.

  In certain preferred embodiments, the above polynucleotides (eg, polynucleotide variants, fragments, and hybridizing sequences) are immunologically cross-reactive with the polypeptide sequences specifically set forth herein. Encodes a polypeptide. In other preferred embodiments, such polynucleotides are at least about 50%, preferably at least about 70%, and more preferably, of the immunogenic activity of the polypeptide sequences specifically set forth herein. It encodes a polypeptide having an immunogenic activity level of at least about 90%.

  The polynucleotide of the present invention, or a fragment thereof, may have other DNA sequences (eg, promoter, polyadenylation signal, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc.) regardless of the length of the coding sequence itself. So that its overall length can vary considerably. Thus, it is contemplated that almost any length nucleic acid fragment can be used, the full length of which is preferably limited by ease of preparation and use in the intended recombinant DNA protocol. For example, about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pair lengths etc. (including all intermediate lengths) Exemplary polynucleotide segments having a full length are contemplated to be useful in many implementations of the invention.

  When comparing polynucleotide sequences, the two sequences are said to be "identical" if the sequence of nucleotides in the two sequences is the same when aligned for maximum match as described below. Is called. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a “comparison window” refers to a segment of at least about 20, usually 30 to about 75, 40 to about 50 consecutive positions, where the two sequences are optimally aligned. Afterwards, the sequence can be compared to the same number of consecutive positions of the reference sequence.

  Optimal alignment of sequences for comparison can be performed using default parameters using the Megalign program (DNASTAR, Inc., Madison, Wis.) In the Lasergene suite of bioinformatics software. This program implements several alignment schemes described in the following references: Dayhoff, M. et al. O. (Eds.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Supplement 3, pages 345-358, Dayhoff, M. et al. O. (1978) A model of evolutionary change in proteins-Matrics for detecting distant relationships. Hein J .; (1990) Unified Approach to Alignment and Phylogenes, pages 626-645, Methods in Enzymology vol. 183, Academic Press, Inc. San Diego, CA; Higgins, D .; G. And Sharp, P .; M.M. (1989) CABIOS 5: 151-153; Myers, E .; W. And Muller W. et al. (1988) CABIOS 4: 11-17; Robinson, E .; D. (1971) Comb. Theor 11: 105; Santou, N .; Nes, M .; (1987) Mol. Biol. Evol. 4: 406-425; H. A. And Sokal, R .; R. (1973) Numeric Taxonomy-the Principles and Practice of Numeric Taxonomy, Freeman Press, San Francisco, Calif .; Wilbur, W .; J. et al. And Lipman, D .; J. et al. (1983) Proc. Natl. Acad. , Sci. USA 80: 726-730.

  Alternatively, optimal alignment of sequences for comparison can be found in Smith and Waterman (1981) Add. APL. Math 2: 482, by the local identity algorithm, Needleman and Wunsch (1970) J. Mol. Mol. Biol. 48: 443 by the alignment alignment algorithm of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444 similarity search method, computerized implementation of these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI, GAP, BST, ST) FASTA, and TFASTA) or by examination.

  One preferred example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25: 3389-3402 and Altschul et al. (1990) J. MoI. Mol. Biol. 215: 403-410. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein to determine percent sequence identity for polynucleotides of the invention. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information. In one illustrative example, the cumulative score is, for nucleotide sequences, parameters M (reward score for matched residue pairs; always> 0) and N (penalty score for mismatched residues; always <0) can be used to calculate. The expansion of word hits in each direction stops when: The cumulative alignment score drops by an amount X from its maximum reached value; the cumulative score is cumulative in the residue alignment of negative scores of 1 or more If it is less than zero due to this; or the end of any sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses word length (W) 11 and expectation (E) 10 as defaults and BLOSUM62 scoring matrix (Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA. 89: 10915) The alignment uses (B) 50, expected value (E) 10, M = 5, N = -4, and a comparison of both strands.

  Preferably, the “percent sequence identity” is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein a portion of the polynucleotide sequence in the comparison window is referenced Compared to a sequence (which does not include additions or deletions), no more than 20 percent, usually 5-15 percent, or 10-12 percent additions or deletions (ie, no optimal alignment of the two sequences) Gap). This percentage determines the number of positions where identical nucleobases occur in both sequences to obtain the number of matching positions, and this number of matching positions is the total number of positions in the reference sequence (ie, window size). Calculated by dividing and multiplying this result by 100 to get the percentage of sequence identity.

It will be appreciated by those skilled 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 have minimal homology to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that change due to differences in codon usage are specifically contemplated by the present invention. Furthermore, alleles of a gene comprising a polynucleotide sequence provided herein are within the scope of the invention. Alleles are endogenous genes that change as a result of one or more mutations (eg, nucleotide deletions, additions and / or substitutions). The resulting mRNA and protein may have an altered structure or function, but need not. Alleles can be identified using standard techniques (eg, hybridization, amplification and / or database sequence comparison).

  Accordingly, in another embodiment of the invention, a mutagenesis approach (eg, site-directed mutagenesis) is used for the preparation of immunogenic variants and / or derivatives of the polypeptides described herein. Is done. By this approach, specific alterations in the polypeptide sequence can be made through mutagenesis of the underlying polynucleotide encoding them. These techniques provide a direct approach to preparing and testing sequence variants that embodies one or more of the above considerations, for example, by introducing one or more nucleotide sequence changes into the polynucleotide. provide.

  Site-directed mutagenesis allows the generation of variants through the use of a specific oligonucleotide sequence encoding the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, on either side of the opposing deletion junction. Provide sufficient size primer sequence and sequence complexity to form a stable duplex. A mutation improves, alters, reduces, alters, or otherwise alters the properties of the polynucleotide itself and / or properties, activity, composition, stability, or of the encoded polypeptide or Can be used in selected polynucleotide sequences to alter the primary sequence.

  In certain embodiments of the invention, the inventors may use one of the disclosed polynucleotide sequences to alter one or more properties of the encoded polypeptide (eg, immunogenicity of the polypeptide vaccine). Intended for mutagenesis. Site-directed mutagenesis techniques are well known in the art and are used extensively to make both polypeptide and polynucleotide variants. For example, site-directed mutagenesis is often used to alter specific parts of a DNA molecule. In such embodiments, primers comprising a length typically on the order of about 14 nucleotides to about 25 nucleotides in length are used, and about 5 to about 10 residues on either side of the sequence junction are altered. Is done.

As will be appreciated by those skilled in the art, site-directed mutagenesis techniques have often used phage vectors that exist in both single-stranded and double-stranded forms.
Exemplary vectors useful in site-directed mutagenesis include vectors such as M13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely used in site-directed mutagenesis that eliminates the step of transferring the gene of interest from the plasmid to the phage.

In general, site-directed mutagenesis according to the present specification involves first obtaining a single-stranded vector containing a DNA sequence encoding the desired peptide in that sequence, or of a double-stranded vector containing this sequence. This is performed by melting and separating the two strands. Oligonucleotide primers carrying the desired mutated sequence are generally prepared synthetically. This primer is then annealed with a single stranded vector and subjected to DNA polymerase (eg, E. coli polymerase I Klenow fragment) to complete synthesis of the strand carrying the mutation.
In this way a heteroduplex is formed, where one strand encodes the original strand with no mutation and the second strand carries the desired mutation. This heteroduplex vector is then used to transform appropriate cells (eg, E. coli cells) and a clone containing a recombinant vector carrying the mutated sequence arrangement is selected.

  The preparation of sequence variants of selected peptide-encoding DNA segments using site-directed mutagenesis provides a means to produce potentially useful species, and this is not meant to be limiting. This is because there are other ways in which sequence variants of the peptides and the DNA sequences encoding them can be obtained. For example, a recombinant vector encoding the desired peptide sequence can be treated with a mutagen (eg, hydroxylamine) to obtain a sequence variant. Specific details regarding these methods and protocols can be found in Malloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each of which is herein incorporated by reference for that purpose. In the teachings).

  As used herein, the term “oligonucleotide-specific mutagenesis procedure” refers to a template-dependent process and vector-mediated growth, which is compared to its initial concentration of a particular nucleic acid. This results in an increase in the concentration of the molecule or an increase in the concentration of detectable signal (eg amplification). As used herein, the term “oligonucleotide-specific mutagenesis procedure” is intended to refer to a process that involves template-dependent extension of a primer molecule. The term template-dependent process refers to nucleic acid synthesis of RNA or DNA molecules, where the sequence of a newly synthesized nucleic acid strand is dictated by the well-known rules of complementary base pairing (eg, (See Watson, 1987). Typically, vector-mediated methodologies include the introduction of nucleic acid fragments into DNA or RNA vectors, clonal amplification of vectors, and recovery of amplified nucleic acid fragments. Examples of these methodologies are provided by US Pat. No. 4,237,224, which is specifically incorporated herein by reference in its entirety.

  In another approach for the production of polypeptide variants of the invention, recursive sequence recombination can be used, as described in US Pat. No. 5,837,458. In this approach, a repetitive cycle of recombination and screening or selection is performed to “evolve” individual polynucleotide variants of the invention having, for example, enhanced immunogenic activity.

  In other embodiments of the invention, the polynucleotide sequences provided herein can be advantageously used as probes or primers for nucleic acid hybridization. Thus, a nucleic acid segment comprising a sequence region of at least about 15 nucleotides in length that has the same sequence as or a sequence complementary to the 15 nucleotides in length of the sequence disclosed herein is identified. It is intended to find usefulness. For example, longer consecutive identical or complementary sequences up to about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and just full length sequences are also specified. Used in the embodiment.

  The ability of such nucleic acid probes to specifically hybridize to a sequence of interest allows them to be used in detecting the presence of complementary sequences in a given sample. However, other uses are also contemplated, such as the use of sequence information to prepare primers for use in preparing mutant primers or other genetic constructs.

  A sequence region consisting of a stretch of nucleotides on the order of 10-14, 15-20, 30, 50, or 100-200 nucleotides (also including intermediate lengths), and is disclosed herein. A polynucleotide molecule identical or complementary to a nucleotide sequence is specifically contemplated as a hybridization probe for use in, for example, Southern blotting or Northern blotting. This allows analysis of the gene product or fragment thereof in both a variety of cell types and also various bacterial cells. The overall size of the fragments, as well as the size of the complementary stretch, will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments generally find use in hybridization embodiments, where the length of consecutive complementary regions can be varied (eg, between about 15 and about 100 nucleotides), but of the complementary sequence desired to be detected. Depending on the length, longer continuous complementary stretches can be used.

  The use of hybridization probes about 15-25 nucleotides in length allows for the formation of stable and selective duplex molecules. However, molecules having a stretch of contiguous complementary sequences longer than 15 bases are generally preferred to increase the stability and selectivity of the hybrid, thereby improving the quality and extent of the resulting specific hybrid molecule. . It is generally preferred to design nucleic acid molecules having 15-25 contiguous nucleotides, or longer gene complementary stretches if desired.

  Hybridization probes can be selected from any portion of any of the sequences disclosed herein. All that is needed is the sequence described herein, or any contiguous sequence of sequences from about 15-25 nucleotides in length to the full length sequence, including the full length sequence, that is desired for use as a probe or primer. It is to reexamine the part. The choice of probe and primer sequences can be governed by various factors. For example, it may be desirable to use a primer towards the end of the entire sequence.

  Small polynucleotide segments or fragments can be readily prepared, for example, by directly synthesizing the fragments by chemical means (as this is usually done using an automated oligonucleotide synthesizer). Fragments can also be combined with selectable sequences for recombinant products by application of nucleic acid replication techniques (eg, PCRTM techniques of US Pat. No. 4,683,202, incorporated herein by reference). It can be obtained by introduction into a replacement vector and by other recombinant DNA techniques generally known to those skilled in the field of molecular biology.

  The nucleotide sequences of the present invention can be used for complementary stretches of either the complete gene or gene fragment of interest and its ability to selectively form duplex molecules. Depending on the envisaged application, it is typically desirable to use different conditions of hybridization to achieve different degrees of probe selectivity for the target sequence. For applications that require high selectivity, it is typically desirable to use relatively stringent conditions to form a hybrid. For example, relatively low concentrations of salt, and / or high temperature conditions (eg, as provided by salt concentrations of about 0.02 M to about 0.15 M salt at a temperature of about 50 ° C. to about 70 ° C.) Is selected. Such selection conditions, when present, have little tolerance for mismatches between the probe and the template or target strand, and are particularly suitable for the isolation of related sequences.

Of course, for some applications (eg, where it is desirable to prepare mutants using mutant primer strands that hybridize to the underlying template), lower stringency (reduced stringency) ) Hybridization conditions are typically required to allow heteroduplex formation. In these situations, it may be desirable to use salt conditions, such as salt conditions of about 0.15M to about 0.9M at a temperature range of about 20 ° C to about 55 ° C. This allows cross-hybridizing species to be readily identified as positive hybridizing signals for control hybridization.
In any case, the addition of an increased amount of formamide (which acts to destabilize the hybrid duplex in the same manner as the elevated temperature) may make the condition more stringent. Generally understood. Thus, hybridization conditions can be easily manipulated and are therefore generally a selection method that depends on the desired result.

  In accordance with another embodiment of the present invention, a polynucleotide composition comprising an antisense oligonucleotide is provided. Antisense oligonucleotides have been demonstrated to be effective and targeted inhibitors of protein synthesis, resulting in therapeutic approaches where disease can be treated by inhibiting the synthesis of proteins that contribute to the disease. provide. The efficacy of antisense oligonucleotides for inhibiting protein synthesis is well established. For example, the synthesis of polygalactauronase and muscarinic type 2 acetylcholine receptors is inhibited by antisense oligonucleotides specific for their respective mRNA sequences (US Pat. Nos. 5,739,119 and US Pat. 5,759,829). In addition, examples of antisense inhibition have been shown using the nucleoprotein cyclin, multidrug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatum GABAA receptor and human EGF (Jaskulski) Science 1988 Jun 10; 240 (4858): 1544-6; Vasanthakumar and Ahmed, Cancer Commun. 1989; 1 (4): 225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun 15; 57 (2 310-20; U.S. Pat. Nos. 5,801,154; 5,789,573; 5,718,709 and 5,610,288). Antisense constructs have also been described (US Pat. Nos. 5,747,470; 5,591, which can be used to inhibit and treat various abnormal cell growths, such as cancer). 317 and 5,783,683).

  Accordingly, in certain embodiments, the present invention provides an oligonucleotide sequence comprising all or part of any sequence or its complement that can specifically bind to a polynucleotide sequence described herein. To do. In one embodiment, the antisense oligonucleotide comprises DNA or a derivative thereof. In another embodiment, the oligonucleotide comprises RNA or a derivative thereof. In a third embodiment, the oligonucleotide is a modified DNA comprising a phosphorothioate modified backbone. In a fourth embodiment, the oligonucleotide sequence comprises a peptide nucleic acid or derivative thereof. In each case, preferred compositions are complementary, more preferably substantially complementary, and even more preferably fully complementary to one or more portions of the polynucleotides disclosed herein. A sequence region. Selection of an antisense composition specific for a given gene sequence is based on analysis of the selected target sequence and based on determination of secondary structure, Tm, binding energy, and relative stability. Antisense compositions can be selected based on their ability to relatively not form dimers, hairpins, or other secondary structures that reduce or prevent specific binding to a target mRNA in a host cell. A highly preferred target region of mRNA is a sequence substantially complementary to the region of or near the AUG translation initiation codon and the 5 'region of the mRNA. These secondary structure analysis and target site selection considerations are discussed, for example, in the OLIGO primer analysis software v. 4 and / or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25 (17): 3389-402).

  The use of antisense delivery methods using short peptide vectors (referred to as MPG (27 residues)) is also contemplated. This MPG peptide contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T antigen (Morris et al., Nucleic Acids Res. 1997 Jul 15; 25 (14) : 2730-6). Several molecules of this MPG peptide have been shown to coat antisense oligonucleotides and can be delivered to cultured mammalian cells within 1 hour with relatively high efficiency (90%). Furthermore, interaction with MPG strongly increases both the stability of the oligonucleotide to nucleases and its ability to cross the plasma membrane.

  In accordance with another embodiment of the present invention, the polynucleotide compositions described herein are used in the design and preparation of ribbonzyme molecules to inhibit the expression of tumor polypeptides and proteins of the present invention in tumor cells. Is done. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, Proc Natl Acad Sci US A. 1987 Dec; 84 (24): 8788-92; Forster and Symons, Cell. 1987 Apr 24; 49 (2) 211-20). For example, many ribozymes promote phosphoester transfer reactions with a high degree of specificity and often cleave only one of several phosphate esters in an oligonucleotide substrate (Cech et al., Cell. 1981 Dec; 27 (3 Pt 2): 487-96; Michel and Westoff, J Mol Biol. 1990 Dec 5; 216 (3): 585-610; Reinhold-Hurek and Shub, Nature. 1992 May 14; ): 173-6). This specificity was due to the requirement that this substrate binds through specific base-pairing interactions with the ribozyme internal guide sequence ("IGS") prior to chemical reaction.

  Six basic variants of naturally occurring enzymatic RNA are now known. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of the enzymatic nucleic acid that is held in close proximity to the enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes the target RNA, then binds to the target RNA via complementary base pairing and, once bound to the correct site, enzymatically cleaves the target RNA. Acts as follows. Such strategic cleavage of the target RNA destroys the target RNA's ability to directly synthesize the encoded protein. After the enzymatic nucleic acid binds to and cleaves the RNA target, the enzymatic nucleic acid can leave the RNA and repeatedly bind to and cleave the new target to search for another target.

  The enzymatic properties of ribozymes are advantageous over many technologies, such as antisense technology, where a nucleic acid molecule simply binds to a nucleic acid target and blocks translation. This is because the concentration of ribozyme required to affect therapeutic treatment is lower than the concentration of antisense oligonucleotide. This advantage reflects the ability of ribozymes to act enzymatically. Thus, a single ribozyme molecule can cleave many molecules of target RNA. Furthermore, this ribozyme is a highly specific inhibitor, and the specificity of this inhibition depends not only on the mechanism of base pairing that binds to the target RNA, but also on the mechanism of cleavage of the target RNA. A single mismatch or base substitution near the cleavage site can completely eliminate the catalytic activity of the ribozyme. Similar mismatches within the antisense molecule do not prevent their action (Woolf et al., Proc Natl Acad Sci US A. 1992 Aug 15; 89 (16): 7305-9). Therefore, the specificity of ribozyme action is higher than that of antisense oligonucleotides that bind to the same RNA site.

  This enzymatic nucleic acid molecule can be formed into a hammerhead motif, hairpin motif, hepatitis delta virus motif, group I intron motif or RNaseP RNA motif (associated with an RNA guide sequence) or Neurospora VS RNA motif. Examples of hammerhead motifs are described by Rossi et al., Nucleic Acids Res. 1992 Sep 11; 20 (17): 4559-65. Examples of hairpin motifs are described in Hampel et al. (European Patent Application Publication No. EP 0360257), Hampel and Tritz (Biochemistry 1989 Jun 13; 28 (12): 4929-33), Hampel et al., Nucleic Acids Res. 1990 Jan 25; 18 (2): 299-304 and US Pat. No. 5,631,359. Examples of hepatitis δ virus motifs are found in Perrotta and Bean, Biochemistry. 1992 Dec 1; 31 (47): 11843-52; examples of RNaseP motifs are described in Guerrier-Takada et al., Cell. 1983 Dec; 35 (3 Pt 2): 849-57; the Neurospora VS RNA ribozyme motif is described in Collins (Saville and Collins, Cell. 1990 May 18; 61 (4): 685-96; Saville and Collins, Proc Natl Acad Sci US A. 1991 Oct 1; 88 (19): 8826-30; Collins and Olive, Biochemistry. 1993 Mar 23; 32 (11): 2795-9); An example is described in US Pat. No. 4,987,071. What is important in the enzymatic nucleic acid molecule of the present invention is that it has a specific substrate binding site that is complementary to one or more target gene RNA regions, and confers RNA cleavage activity on this molecule within the substrate binding site Or a nucleotide sequence surrounding this substrate binding site. Thus, ribozyme constructs are not necessarily limited to the specific motifs described herein.

  Ribozymes can be designed as described in International Patent Application Publication Number WO 93/23569 and International Patent Application Publication Number WO 94/02595, each of which is specifically incorporated herein by reference, and As described, it can be synthesized for testing in vitro and in vivo. Such ribozymes can also be optimized for delivery. Although specific examples are provided, those of skill in the art will recognize that equivalent RNA targets in other species can be utilized when needed.

  Ribozyme activity may be altered by altering the length of the ribozyme binding arm or by modifications that prevent degradation by serum ribonucleases (eg, International Patent Application Publication No. WO 92/07065; International Patent Application Publication No. WO 93/15187; International Patent Application Publication No. WO 91/03162; European Patent Application Publication No. 92110298.4; US Patent No. 5,334,711, and International Patent Application Publication No. WO 94/13688 (these are in the sugar part of the enzymatic RNA molecule). (Describe various chemical modifications that can be made to)), modifications that enhance the efficacy of ribozymes in cells, and to shorten RNA synthesis time and reduce chemical requirements By chemically synthesizing ribozymes by removal of stem II bases. It can be optimized Te.

  Sullivan et al. (International Patent Application Publication No. WO 94/02595) describe a general method for the delivery of enzymatic RNA molecules. Ribozymes can be administered to cells by various methods known to those skilled in the art, including encapsulation in liposomes, iontophoresis, or other vehicles (eg, hydrogels, cyclodextrins, biodegradable nanocapsules). And those by incorporation into bioadhesive microspheres), but are not limited thereto. For some guidance, ribozymes can be delivered directly to cells or tissues ex vivo, with or without the use of the vehicles described above. Alternatively, the RNA / vehicle combination can be delivered locally by direct inhalation, direct injection, or use of a catheter, infusion pump or stent. Other delivery routes include intravascular injection, intramuscular injection, subcutaneous or joint injection, aerosol inhalation, oral delivery (tablet or pill form), local delivery, systemic delivery, ocular delivery, intraperitoneal delivery and / or medulla Examples include, but are not limited to intracavitary delivery. More detailed descriptions of ribozyme delivery and administration are provided in International Patent Application Publication No. WO 94/02595 and International Patent Application Publication No. WO 93/23569, each of which is specifically incorporated herein by reference. Is done.

  Another means of accumulating high concentrations of ribozymes in the cell is to incorporate ribozyme coding sequences into the DNA expression vector. Transcription of the ribozyme sequence is driven by a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from the pol II promoter or pol III promoter are expressed at high levels in all cells; the level of a given pol II promoter in a given cell type depends on nearby gene regulatory sequences (enhancers, silencers, etc.) ) Depending on the nature of Prokaryotic RNA polymerase promoters can also be used if the prokaryotic RNA polymerase enzyme is expressed in a suitable cell. Ribozymes expressed from such promoters have been shown to function in mammalian cells. Such a transcription unit can be a variety of vectors (plasmid DNA vectors, viral DNA vectors (eg, adenoviral vectors or adeno-associated vectors), or viral RNA vectors (eg, retroviral vectors) for introduction into mammalian cells. , Semliki Forest virus vector, Sindbis virus vector), but not limited thereto.

  In another embodiment of the invention, a peptide nucleic acid (PNA) composition is provided. PNA is a DNA mimic where nucleobases are attached to a pseudopeptide backbone (Good and Nielsen, Antisense Nucleic Drug Drug Dev. 1997 7 (4) 431-37). PNA can be utilized in a number of ways conventionally using RNA or DNA. Often, PNA sequences are performed technically better than the corresponding RNA or DNA sequences and have utility that does not exist in RNA or DNA. A review of PNA, including methods of production, features, and methods of use, is provided by Corey (Trends Biotechnol 1997 Jun; 15 (6): 224-9). Thus, in certain embodiments, PNA sequences can be prepared that are complementary to one or more portions of the ACE mRNA sequence, and such PNA compositions regulate and alter the translation of ACE-specific mRNA. Can be used to reduce, reduce, or reduce the level of ACE activity in a host cell to which such a PNA composition has been administered.

  PNA has a 2-aminoethyl-glycine linkage that replaces the normal phosphodiester backbone of DNA (Nielsen et al., Science 1991 Dec 6; 254 (5037): 1497-500; Hanvey et al., Science. 1992 Nov 27; 258 (5087): 1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 Jan; 4 (1): 5-23). This chemistry has three important consequences. First, in contrast to DNA or phosphorothioate oligonucleotides, PNA is a neutral molecule; second, PNA is achiral (this necessitates the development of stereoselective synthesis) And third, PNA synthesis uses the standard Boc or Fmoc protocol for solid phase peptide synthesis. On the other hand, other methods are used, including the improved Merrifield method.

PNA monomers or off-the-shelf oligomers are commercially available from PerSeptive Biosystems (Framingham, Mass.). Synthesis of PNA by either Boc or Fmoc protocols is easily performed using manual or automated protocols (Norton et al., Bioorg Med Chem. 1995 Apr; 3 (4): 437-45).
This manual protocol by itself leads to the production of chemically modified PNAs or the simultaneous synthesis of closely related families of PNAs.

  With respect to peptide synthesis, the success of a particular PNA synthesis depends on the characteristics of the selected sequence. For example, in theory, PNA can incorporate any combination of nucleotide bases, but the presence of adjacent purines can lead to the deletion of one or more residues in the product. In anticipation of this difficulty, we propose that coupling of residues that are added inefficiently in producing PNAs with adjacent purines should be repeated. This is followed by purification of the PNA by reverse phase high pressure liquid chromatography to provide a yield and purity of the product that is similar to the yield and purity observed during peptide synthesis.

Modification of PNA for a given application can be accomplished by coupling an amino acid during solid phase synthesis or by adding a compound containing a carboxylic acid group to the exposed N-terminal amine. Alternatively, PNA can be modified after synthesis by coupling to an introduced lysine or cysteine.
The convenience with which PNA can be modified facilitates better solubility or optimization of specific functional requirements. Once synthesized, the identity of PNA and their derivatives can be confirmed by mass spectrometry. Several studies have made and utilized modifications of PNA (eg, Norton et al., Bioorg Med Chem. 1995 Apr; 3 (4): 437-45; Petersen et al., J Pept Sci. 1995 May-Jun. 1 (3) 175-83; Orum et al., Biotechniques. 1995 Sep; 19 (3): 472-80; Footer et al., Biochemistry. 1996 Aug 20; 35 (33): 10673-9; Griffith et al., Nucleic Acid Res. 1995 Aug 11; 23 (15): 3003-8; Pardridge et al., Proc Natl Acad Sci USA 1995 Jun 6; 92 (12): 5592-6; Boffa et al., Proc Natl Acad S i USA 1995 Mar 14; 92 (6): 1901-5; Gambacorti-Passerini et al., Blood, 1996 Aug 15; 88 (4): 1411-7; Armitage et al., Proc Natl Acad Sci USA, 1997 Nov11; 94 ( 23): 12320-5; Seeger et al., Biotechniques, 1997 Sep; 23 (3): 512-7). US Pat. No. 5,700,922 discusses PNA-DNA-PNA chimeric molecules and their use in diagnosis, protein regulation in organisms, and treatment of conditions sensitive to therapy.

  Methods for characterizing the antisense binding properties of PNA are discussed in Rose (Anal Chem, 1993 Dec 15; 65 (24): 3545-9) and Jensen et al. (Biochemistry, 1997 Apr 22; 36 (16): 5072-7). Is done. Rose determines the binding of PNA to their complementary oligonucleotides by measuring the relative binding kinetics and stoichiometry using capillary gel electrophoresis. A similar type of measurement has been made by Jensen et al. Using BIAcore ™ technology.

  Other applications of PNA that are described and apparent to those skilled in the art include DNA strand invasion, antisense inhibition, mutation analysis, transcription enhancers, nucleic acid purification, transcription active gene isolation, transcription factor binding blocking, Examples include use in genome cleavage, biosensors, in situ hybridization, and the like.

(Identification, characterization and expression of polynucleotides)
The polynucleotide compositions of the present invention can be identified, prepared and / or manipulated using any of a variety of well-established techniques (generally Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor). Laboratories, Cold Spring Harbor, NY, 1989, and other similar references). For example, a polynucleotide may be expressed in tumor-associated expression (ie, as determined using representative assays provided herein, as described in detail below, in normal tissues). Can also be identified by screening a microarray of cDNA for (at least 2-fold higher expression in tumors). Such screening is performed, for example, according to the manufacturer's instructions at Affymetrix, Inc. (Santa Clara, CA) using microarray technology (and essentially Schena et al., Proc. Natl. Acad. Sci. USA 93: 10614-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA. 94: 2150-2155, 1997).
Alternatively, the polynucleotide can be amplified from cDNA prepared from cells (eg, tumor cells) that express the proteins described herein.

  A number of template dependent processes are available to amplify the target sequence of interest present in the sample. One of the best known amplification methods is the polymerase chain reaction (PCRTM), which is described in US Pat. Nos. 4,683,195, 4,683,202, and 4,800, 159, each of which is incorporated herein by reference in its entirety. Briefly, in PCRTM, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the target sequence. Excess deoxynucleoside triphosphate is added to the reaction mixture along with a DNA polymerase (eg, Taq polymerase). If a target sequence is present in the sample, the primer binds to its target and the polymerase extends the primer along the target sequence by adding nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primer dissociates from the target to form a reaction product, the excess primer binds to the target and reaction product, and the process is repeated . Preferably, reverse transcription and PCRTM amplification procedures can be performed to quantify the amount of amplified mRNA. Polymerase chain reaction methodologies are well known in the art.

  Any number of other template-dependent processes, many of which are variations of the PCRTM amplification technique, are readily known and available in the art. By way of example, some such methods include ligase chain reaction (also referred to as LCR) (for example, described in European Patent Application Publication No. 320,308 and US Pat. No. 4,883,750); Qβ replicase (described in PCT International Patent Application Publication No. PCT / US87 / 00880); Strand Displacement Amplification (SDA) and Repair Chain Reaction (RCR). Still other amplification methods are described in British Patent Application No. 2 202 328 and PCT International Patent Application Publication No. PCT / US89 / 01025. Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (PCT International Patent Application Publication No. WO 88/10315), which include nucleic acid sequence-based amplification (NASBA) and 3SR. European Patent Application Publication No. 329,822 describes a nucleic acid propagation process that includes the step of cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA). PCT International Patent Application Publication No. WO 89/06700 describes a nucleic acid sequence amplification scheme based on hybridization of a promoter / primer sequence to target single-stranded DNA (“ssDNA”), followed by transcription of many RNA copies of this sequence. To do. Other amplification methods such as “RACE” (Frohman, 1990) and “one-sided PCR” (Ohara, 1989) are also well known to those skilled in the art.

  The amplified portion of the polynucleotide of the invention can be used to isolate a full-length gene using a well-known technique from an appropriate library (eg, a tumor cDNA library). In such techniques, a library (cDNA library or genomic library) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, the library is selected in size to contain larger molecules. Randomly prepared libraries may also be preferred for identifying the 5 'region and upstream region of genes. Genomic libraries are preferred for obtaining introns and extending 5 'sequences.

With respect to hybridization techniques, subsequences can be labeled using well known techniques (eg, nick translation or end labeling with 32P).
The bacterial library or bacteriophage library is then generally screened by hybridizing a labeled probe and a filter containing denatured bacterial colonies (or a flora containing phage plaques) (Sambrook et al., Molecular Cloning). : A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Hybridizing colonies or plaques are selected, extended, and the DNA is isolated for further analysis. cDNA clones can be analyzed, for example, by PCR using primers derived from the partial sequence and primers derived from the vector to determine the amount of additional sequence. Restriction maps and subsequences can be generated to identify one or more overlapping clones. The complete sequence can then be determined using standard techniques, which can involve creating a series of deletion clones. The resulting overlapping sequences can then be constructed in one continuous sequence.
Well-known techniques can be used to generate full-length cDNA molecules by ligation to appropriate fragments.

  Alternatively, amplification techniques such as those described above can be useful for obtaining full-length coding sequences from partial cDNA sequences. One such amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res. 16: 8186, 1988). This technique uses restriction enzymes to generate fragments within known regions of the gene. This fragment is then circularized by intramolecular ligation and used as a template for PCR with a variety of primers derived from known regions. In an alternative approach, sequences adjacent to the partial sequence can be removed by amplification using primers for the linker sequence and primers specific for the known region. This amplified sequence is typically subjected to a second round of amplification using the same linker primer and a second primer specific for the known region. A modification to this procedure using two primers that initiate extension in the opposite direction from the known sequence is described in WO 96/38591. Another such technique is known as “rapid cDNA end amplification” or RACE. This technique involves the use of inner and outer primers (which hybridize to a polyA region or vector sequence) to identify sequences that are 5 'and 3' of known sequences. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods App. 1: 11: 19-19, 1991) and walking PCR (Parker et al., Nucl. Acids. Res. 19: 3055-60, 1991). Other methods that utilize amplification can also be used to obtain full-length cDNA sequences.

  In certain cases, full-length cDNA sequences can be obtained by analysis of sequences provided in an expressed sequence tag (EST) database (eg, available from GenBank). Searches for duplicate ESTs can generally be performed using well-known programs (eg, NCBI BLAST searches), and such ESTs can be used to generate contiguous full-length sequences. Full-length DNA sequences can also be obtained by analysis of genomic fragments.

  In other embodiments of the invention, a polynucleotide sequence encoding a polypeptide of the invention or a fusion protein or functional equivalent thereof, or a fragment thereof, is recombinant so as to direct expression of the polypeptide in a suitable host cell. Can be used in DNA molecules. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially the same or functionally equivalent amino acid sequences can be produced, and these sequences can be used to define a given polypeptide Can be cloned and expressed.

  As will be appreciated by those skilled in the art, in some cases it may be advantageous to create polypeptide-encoding nucleotide sequences that possess non-naturally occurring codons. For example, a preferred codon for a particular prokaryotic or eukaryotic host may be used to increase the rate of protein expression or to have a desired property (eg, the half-life of a transcript generated from a naturally occurring sequence). Can also be selected to produce recombinant RNA transcripts with a long half-life).

  Furthermore, the polynucleotide sequences of the present invention may be used to alter the polypeptide coding sequence for a variety of reasons including, but not limited to, modifications that alter the cloning, processing, and / or expression of the gene product. In addition, it can be manipulated using methods generally known in the art. For example, DNA shuffling by random cleavage and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to manipulate nucleotide sequences. In addition, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, or splice modification. The body can be made, or a mutation can be introduced.

  In another embodiment of the invention, the natural nucleic acid sequence, modified nucleic acid sequence, or recombinant nucleic acid sequence can be linked to a heterologous sequence to encode a fusion protein. For example, to screen a peptide library for inhibitors of polypeptide activity, it may be useful to encode a chimeric protein that can be recognized by commercially available antibodies. The fusion protein can also be engineered to include a cleavage site located between the polypeptide coding sequence and the heterologous protein sequence so that the polypeptide can be cleaved and removed from the heterologous portion and purified. .

  Sequences encoding the desired polypeptides can be synthesized in whole or in part using chemical methods well known in the art (Caruthers, MH et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. Alternatively, the protein itself can be produced using chemical methods to synthesize the amino acid sequence of the polypeptide or a portion thereof. For example, peptide synthesis can be performed using a variety of solid phase techniques (Roberge, J. Y. et al. (1995) Science 269: 202-204), and automated synthesis can be performed, for example, by ABI 431A Peptide Synthesizer ( Perkin Elmer, Palo Alto, CA).

  Newly synthesized peptides can be obtained by preparative high performance liquid chromatography (eg, Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, NY) or in the art. It can be substantially purified by other equivalent techniques available. The composition of the synthetic peptide can be confirmed by amino acid analysis or sequencing (eg, Edman degradation procedures). In addition, the amino acid sequence of the polypeptide or any portion thereof can be modified during direct synthesis and / or combined with sequences from other proteins or any portion thereof using chemical methods, Variant polypeptides can be produced.

  In order to express the desired polypeptide, the nucleotide sequence encoding the polypeptide or functional equivalent is a suitable expression vector (ie, a vector containing the elements necessary for transcription and translation of the inserted coding sequence). Can be inserted into. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding the polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N .; Y. , And Ausubel, F .; M.M. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York. N. Y. It is described in.

  A variety of expression vector / host systems may be utilized to contain and express the polynucleotide sequences. These include, but are not limited to: a microorganism (eg, a bacterium transformed with a recombinant bacteriophage, plasmid, or cosmid DNA expression vector); a yeast transformed with a yeast expression vector; a viral expression vector. Insect cell lines infected with (eg baculovirus); plant cells transformed with viral expression vectors (eg cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (eg Ti or pBR322 plasmid) System; or animal cell system.

  “Control elements” or “regulatory sequences” present in expression vectors are those untranslated regions (enhancers, promoters, 5 ′ and 3 ′) of those vectors that interact with host cell proteins to effect transcription and translation. Untranslated region). Such elements can change their strength and specificity. Depending on the vector system and host utilized, a number of suitable transcription and translation elements (including constitutive and inducible promoters) can be used. For example, when cloning in bacterial systems, a hybrid lacZ promoter such as an inducible promoter (eg, PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) Or PSPORT1 plasmid (Gibco BRL, Gaithersburg, MD)) can be used. In the system, promoters derived from mammalian genes or mammalian viruses are generally preferred: SV40 or EBV bases if required to generate cell lines containing multiple copies of the sequence encoding the polypeptide. These vectors can be advantageously used with an appropriate selectable marker.

In bacterial systems, any of a number of expression vectors can be selected depending upon the use intended for the polypeptide being expressed. For example, if needed in large quantities (eg, for antibody induction), vectors that direct high level expression of easily purified fusion proteins can be used. Such vectors include, but are not limited to, the following: multifunctional E. coli. E. coli cloning and expression vectors (eg BLUESCRIPT (Stratagene), where the sequence encoding the polypeptide of interest is in-frame with the amino-terminal Met of β-galactosidase and the subsequent 7 residues in frame with the sequence Can be ligated, resulting in the production of a hybrid protein); pIN vectors (Van Heeke, G. and SM Schuster (1989) J. Biol. Chem. 264: 5503-5509) and the like.
The pGEX vector (Promega, Madison, Wis.) can also be used to express foreign polypeptides as fusion proteins with glutathione S transferase (GST). In general, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption to glutathione-agarose beads and then elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can optionally be released from the GST moiety. .

  In yeast (Saccharomyces cerevisiae), a number of vectors containing constitutive or inducible promoters (eg, alpha factor, alcohol oxidase and PGH) can be used. For reviews, see Ausubel et al. (Supra) and Grant et al. (1987) Methods Enzymol. 153: 516-544.

  In cases where plant expression vectors are used, the expression of sequences encoding polypeptides can be driven by any of a number of promoters. For example, viral promoters (eg, the CaMV 35S and 19S promoters) can be used alone or in combination with an omega leader sequence derived from TMV (Takamatsu, N. (1987) EMBO J. 6: 307- 311). Alternatively, a plant promoter (eg, a small subunit of RUBISCO or a heat shock promoter) can be used (Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science. 224: 838-843; and Winter, J et al. (1991) Results Probl. Cell Differ. 17: 85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in numerous publicly available reviews (eg, Hobbs, S. or Murry, LE, McGraw Hill Year of Science and Technology (1992) McGraw Hill, New York). N.Y .; see pages 191-196).

  Insect systems can also be used to express a polypeptide of interest. For example, in one such system, the Autographa california nuclear polyhydrosis virus (AcNPV) causes the Spodoptera frugiperda cell or the Trichoplusia gene to be used as a foreign gene in a spopodera frugiperda cell. The sequence encoding the polypeptide can be cloned into a nonessential region of the virus (eg, a polyhedrin gene) and placed under the control of the polyhedrin promoter. Successful insertion of the polypeptide coding sequence inactivates the polyhedrin gene and produces a recombinant virus lacking the coat protein. This recombinant virus can then be used, for example, to express the polypeptide of interest, S. cerevisiae. Frugiperda cells or Trichoplusia larvae can be infected (Engelhard, EK et al. (1994) Proc. Natl. Acad. Sci. 91: 3224-3227).

  A number of viral-based expression systems are generally available in mammalian host cells. For example, when an adenovirus is used as an expression vector, the sequence encoding the polypeptide of interest can be ligated into an adenovirus transcription / translation complex composed of the late promoter and tripartite leader sequence. Viable viruses can be obtained that can express polypeptides in infected host cells using insertions in the non-essential E1 or E3 region of the viral genome (Logan, J. and Shenk, T. (1984) Proc. Natl.Acad.Sci.81: 3655-3659). In addition, transcription enhancers (eg, the Rous sarcoma virus (RSV) enhancer) can be used to increase expression in mammalian host cells.

Certain initiation signals can also be used to achieve more efficient translation of the sequence encoding the polypeptide of interest. Such signals include the ATG start codon and adjacent sequences. In cases where sequences encoding a polypeptide, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be required.
However, if only the coding sequence, or only part of it, is inserted, an exogenous translational control signal including the ATG start codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translation elements and initiation codons can be derived from a variety of sources, both natural and synthetic. The efficiency of expression depends on the inclusion of an enhancer appropriate for the particular cell line used (eg, enhancers described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162)). Can be increased by

  In addition, a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves the “prepro” form of the protein can also be used to facilitate correct insertion, folding, and / or function. Different host cells (eg, CHO, COS, HeLa, MDCK, HEK293 and WI38) (which have specific cellular and characteristic mechanisms for such post-translational activity) are accurate Can be selected to ensure proper modification and processing of foreign proteins.

  For long term, high yield production of recombinant proteins, stable expression is generally preferred. For example, a cell line that stably expresses the polynucleotide of interest is transformed using an expression vector that may contain a viral origin of replication and / or an endogenous expression element and a selectable marker gene on the same or a separate vector. obtain. After the introduction of the vector, the cells can be grown in enriched medium for 1-2 days before they are switched to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate to the cell type.

  A number of selection systems may be used to recover the transformed cell line. These selection systems include herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11: 223-32) and adenine phosphoribosyltransferase (Lowy), which can be used in tk- or aprt-cells, respectively. I. et al., (1990) Cell 22: 817-23) genes, but is not limited to these. Also, antimetabolite resistance, antibiotic resistance or herbicide resistance can be used as a basis for selection; for example, dhfr (which provides resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77: 3567-70) ;; npt (which confers resistance to aminoglycosides, neomycin and G-418 (Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14)); and als or pat (which confer resistance to chlorsulfuron and phosphinothricin acetyltransferase, respectively (Murry, supra)). Additional selection genes have been described. For example, trpB (which allows cells to utilize indole instead of tryptophan) or hisD (which allows cells to utilize histinol instead of histidine) (Hartman, S .; C. and RC Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047-51). Markers such as anthocyanin, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin are not only used for identifying transformants, but also for specific vectors. It has also been used extensively to quantify the amount of transient or stable protein expression that can be attributed to the system (Rhodes, CA et al. (1995) Methods Mol. Biol. 55: 121-131).

The presence / absence of marker gene expression suggests that the gene of interest is also present, but its presence and expression may need to be confirmed.
For example, if a sequence encoding a polypeptide is inserted within a marker gene sequence, recombinant cells containing the sequence can be identified by the absence of marker gene function. Alternatively, the marker gene can be placed in tandem with the polypeptide coding sequence under the control of one promoter. The expression of a marker gene in response to induction or selection usually indicates the expression of its tandem gene as well.

  Alternatively, host cells containing and expressing the desired polynucleotide sequence can be identified by various procedures known to those skilled in the art. These procedures include, but are not limited to: including, for example, membrane-based techniques, solution-based techniques, or chip-based techniques for the detection and / or quantification of nucleic acids or proteins, DNA-DNA hybridization or DNA-RNA hybridization and protein bioassay technology or immunoassay technology.

  Various protocols for detecting and measuring expression of a product using either polyclonal or monoclonal antibodies specific for the product encoded by the polynucleotide are known in the art. Examples include enzyme linked immunoassay (ELISA), radioimmunoassay (RIA) and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay that utilizes monoclonal antibodies that are reactive against two non-interfering epitopes on a given polypeptide may be preferred for some applications, but competitive binding Assays can also be utilized. These assays and other assays are notably described in Hampton, R .; (1990; Serial Methods, a Laboratory Manual, APS Press, St Paul. Minn.) And Maddox, D. et al. E. (1983; J. Exp. Med. 158: 1211-1216).

  A wide variety of labeling and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for generating labeled hybridization or PCR probes for detecting sequences associated with polynucleotides include oligo labeling, nick translation, end labeling or PCR amplification using labeled nucleotides. It is done. Alternatively, the sequence or any part thereof can be cloned into a vector for the production of mRNA probes. Such vectors are known in the art, are commercially available, and synthesize RNA probes in vitro by adding appropriate RNA polymerase (eg, T7, T3, or SP6) and labeled nucleotides. Can be used to These procedures can be performed using various commercial kits. Suitable reporter molecules or labels that may be used include radionuclides, enzymes, fluorescent agents, chemiluminescent agents, or chromogenic agents and substrates, cofactors, inhibitors, magnetic particles, and the like.

  Host cells transformed with the polynucleotide sequence of interest can be cultured under conditions suitable for recovering the protein from expression and cell culture. Proteins produced by recombinant cells can be secreted or contained within the cell, depending on the sequence and / or vector used. As will be appreciated by those skilled in the art, an expression vector comprising a polynucleotide of the invention is designed to contain a signal sequence that directs secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. obtain. Other recombinant constructs can be used to link the sequence encoding the polypeptide of interest to a nucleotide sequence encoding a polypeptide domain that facilitates purification of the soluble protein. Such purification facilitating domains include, but are not limited to: metal chelating peptides that allow purification on immobilized metals (eg, histidine-tryptophan module), immobilized immunoglobulins. Protein A domain that allows purification above and domains utilized in the FLAGS extension / affinity purification system (Immunex Corp., Seattle, Wash.). Using the inclusion of a cleavable linker sequence between the purification domain and the encoded polypeptide (eg, a sequence specific for factor XA or enterokinase (Invitrogen. San Diego, Calif.)) , Can facilitate purification. One such expression vector provides for the expression of a fusion protein comprising a polypeptide of interest and a nucleic acid encoding six histidine residues before the thioredoxin or enterokinase cleavage site. Histidine residues are described in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263-281), which facilitates purification on IMIAC (immobilized metal ion affinity chromatography), while the enterokinase cleavage site is fused. Means are provided for purifying the desired polypeptide from the protein. A discussion of vectors containing fusion proteins can be found in Kroll, D .; J. et al. (1993; DNA Cell Biol. 12: 441-453).

  In addition to recombinant production methods, the polypeptides and fragments thereof of the present invention can be synthesized directly by peptide synthesis using solid phase technology (Merrifield J. (1963) J. Am. Chem. Soc. 85: 2149-2154). Can be produced. Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be accomplished, for example, using an Applied Biosystems 431A peptide synthesizer (Perkin Elmer). Alternatively, the various fragments can be chemically synthesized separately and combined using chemical methods to produce the full-length molecule.

(Antibody compositions, fragments thereof and other binding agents)
In accordance with another aspect, the present invention further provides binding agents (eg, antibodies and antigen binding thereof) that exhibit immunological binding to the tumor polypeptides disclosed herein, or portions, variants or derivatives thereof. Fragment). An antibody, or antigen-binding fragment thereof, can react with a polypeptide of the invention at a detectable level (eg, in an ELISA assay) and does not detectably react with an irrelevant polypeptide under similar conditions. It is said to “specifically bind”, “immunologically bind” and / or “immunologically reactive” to a polypeptide of the invention.

  As used in this context, immunological binding generally refers to a type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be expressed in terms of the dissociation constant (Kd) of the interaction, where a smaller Kd indicates a greater affinity. The immunological binding properties of the selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rate of antigen binding site / antigen complex formation and dissociation, where these rates are determined by the concentration of the complex partner, the affinity of the interaction, and Depends on geometric parameters that affect the velocity in both directions equally. Thus, both “on rate constant” (Kon) and “off rate constant” (Koff) can be determined by calculating the concentration and the actual rate of association and dissociation. . The ratio of Koff / Kon allows the elimination of all parameters not related to affinity and is therefore equal to the dissociation constant Kd. See generally Davies et al. (1990) Annual Rev. Biochem. 59: 439-473.

  The “antigen-binding site” or “binding portion” of an antibody refers to the portion of an immunoglobulin molecule that participates in antigen binding. This antigen binding site is formed by amino acid residues in the N-terminal variable (“V”) region of the heavy chain (“H”) and light chain (“L”). Three highly branched stretches in the V and V regions of the heavy and light chains are referred to as “hypervariable regions”, which are more conserved adjacent stretches known as “framework regions” or “FRs”. Inserted between. Thus, the term “FR” refers to an amino acid sequence that is naturally found between and adjacent to hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in a three-dimensional space to form an antigen binding surface. This antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity determining regions” or “CDRs”.

  The binding agent may further distinguish between patients with and without cancer (eg, ovarian cancer) using the representative assays provided herein. For example, an antibody or other binding agent that binds to a tumor protein preferably produces a signal indicative of the presence of cancer in at least about 20% of patients with the disease, more preferably at least about 30% of patients. Alternatively, or in addition, the antibody produces a negative signal indicating the absence of the disease in at least about 90% of individuals without cancer. To determine whether a binding agent meets this requirement, biological samples from patients with and without cancer (as determined by standard clinical trials) (eg, blood, Serum, sputum, urine and / or tumor biopsy) can be assayed for the presence of a polypeptide that binds to this binding agent as described herein. Preferably, a statistically significant number of samples with and without disease is assayed. Each binding agent should satisfy the above criteria; however, those skilled in the art will recognize that binding agents can be used in combination to improve sensitivity.

  Any factor that satisfies the above requirements can be a binding factor. For example, the binding agent can be a ribosome, RNA molecule or polypeptide with or without a peptide component. In a preferred embodiment, the binding agent is an antibody or antigen binding fragment thereof. Antibodies can be prepared by any of a variety of techniques known to those skilled in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies are produced by suitable bacterial or mammalian cell hosts, either by cell culture techniques (including the production of monoclonal antibodies as described herein) or to allow the production of recombinant antibodies. Can be generated via transfection of antibody genes into In one technique, an immunogen comprising this polypeptide is first injected into any of a wide variety of mammals (eg, mice, rats, rabbits, sheep or goats). In this step, the polypeptide of the present invention can function as an immunogen without modification. Alternatively, particularly for relatively short polypeptides, an excellent immune response can be elicited when the polypeptide is bound to a carrier protein (eg, bovine serum albumin or keyhole limpet hemocyanin). The immunogen is preferably injected into the animal host according to a predetermined schedule incorporating one or more boost immunizations, and these animals are bled regularly. Polyclonal antibodies specific for this polypeptide can then be purified from such antisera, for example, by affinity chromatography using the polypeptide bound to a suitable solid support.

  Monoclonal antibodies specific to the antigenic polypeptide of interest are described, for example, in Kohler and Milstein, Eur. J. et al. Immunol. 6: 511-519, 1976, and improved versions thereof. Briefly, these methods involve the preparation of immortal cell lines that can generate antibodies with the desired specificity (ie, reactivity with the polypeptide of interest). Such cell lines can be generated, for example, from splenocytes obtained from animals immunized as described above. The splenocytes are then immortalized, for example, by fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. Various fusion techniques can be used. For example, splenocytes and myeloma cells can be referred to as nonionic detergents for several minutes and then plated at low density in a selective medium that supports hybrid cell growth but does not support myeloma cell growth. Can be done. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After sufficient time, usually after about 1-2 weeks, hybrid colonies are observed. Single colonies are selected and their culture supernatants are tested for binding activity against this polypeptide. Hybridomas with high reactivity and specificity are preferred.

  Monoclonal antibodies can be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques can be used to increase yield, such as injection of hybridoma cell lines into the peritoneal cavity of a suitable vertebrate host (eg, a mouse), for example. Monoclonal antibodies can then be recovered from ascites or blood. Contaminants can be removed from the antibody by conventional techniques such as chromatography, gel filtration, precipitation, and extraction. The polypeptides of the present invention can be used, for example, in a purification process in an affinity chromatography step.

  Many therapeutically effective molecules containing an antigen binding site that can exhibit the immunological binding properties of antibody molecules are known in the art. This proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, each of two of these (“F (ab) fragments”), which are intact antigen binding sites. Including covalent heterodimers. The enzyme pepsin can cleave IgG molecules to provide several fragments, including “F (ab ′) 2” fragments that contain both antigen binding sites. “Fv” fragments can be generated by preferential proteolytic cleavage of IgM and, in rare cases, IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art. The Fv fragment contains a non-covalent VH :: VL heterodimer, which contains an antigen binding site that retains most of the antigen recognition and antigen binding capabilities of the native antibody molecule. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69: 2659-2622; Hochman et al. (1976) Biochem 15: 2706-2710; and Ehrlich et al. (1980) Biochem 19: 4091-4096.

  A single chain Fv (“sFv”) polypeptide is a covalently linked VH :: VL heterodimer that includes a gene encoding VH and a gene encoding VL linked by a linker encoding the peptide. Expressed from a gene fusion comprising Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85 (16): 5879-5883. A number of methods have been described for identifying chemical structures for converting naturally aggregated (but chemically separated) light and heavy chain polypeptides from antibody V regions into sFv molecules. This sFv molecule is folded into a three-dimensional structure substantially similar to the structure of the antigen binding site. See, for example, US Pat. Nos. 5,091,513 and 5,132,405 (Huston et al.); And US Pat. No. 4,946,778 (Ladner et al.).

  Each of the above molecules comprises heavy and light chain CDR sets, which provide support for the CDRs and define the spatial relationship of the CDRs with respect to each other between the heavy and light chain FR sets. Each inserted. As used herein, the term “CDR set” refers to the three hypervariable regions of the heavy or light chain V region. Proceeding from the N-terminus of the heavy or light chain, these regions are labeled “CDR1”, “CDR2” and “CDR3”, respectively. Thus, the antigen binding site contains six CDRs, which contain a set of CDRs from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (eg, CDR1, CDR2, or CDR3) is referred to herein as a “molecular recognition unit”. Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the CDR amino acid residues form extensive contacts with the bound antigen, where the most extensive antigen contacts are Contact with chain CDR3. Thus, the molecular recognition unit is the main cause of the specificity of the antigen binding site.

  As used herein, the term “FR set” refers to the four contiguous amino acid sequences that make up the CDRs of a CDR set of a heavy or light chain V region. Some FR residues can contact the binding antigen; however, FR is the primary cause of folding the V region into the antigen binding site, particularly the FR residues immediately adjacent to the CDRs. In FR, certain amino acid residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of about 90 amino acid residues. When the V region is folded into the binding site, this CDR is displayed as a protruding loop motif that forms the antigen binding surface. Regardless of the exact CDR amino acid sequence, it is generally recognized that there are conserved structural regions of FR that affect the shape of the CDR loops that fold into a particular “canonical” structure. In addition, certain FR residues are known to be involved in non-covalent interdomain contacts that stabilize antibody heavy and light chain interactions.

  A number of “humanized” antibody molecules have been described that contain antigen binding sites derived from non-human immunobloglin, including antibody rodents fused to rodent V regions and human constant domains. Group V region association CDRs (Winter et al. (1991) Nature 349: 293-299; Lobglio et al. (1989) Proc. Nat. Acad. Sci. USA 86: 4220-4224; Shaw et al. (1987) J Immunol. 138: 4534 -4538; and Brown et al. (1987) Cancer Res. 47: 3577-3583), rodent CDRs implanted in human support FRs (Riechmann et al. (1988) Nature) prior to fusion with appropriate human antibody constant domains. 332: 323-327; Verhoey en et al. (1988) Science 239: 154-1536; and Jones et al. (1986) Nature 321: 522-525), and rodent CDRs supported by recombinant veneered rodent FRs ( And chimeric antibodies having European Patent Publication No. 519,596 (published December 23, 1992) These “humanized” molecules are the duration and efficacy of therapeutic application of these parts in human recipients. Designed to minimize undesired immunological responses to rodent anti-human antibody molecules that limit

  As used herein, the terms “veneered FR” and “recombinant veneered FR” comprise an antigen binding site that retains substantially all of the native FR polypeptide folded structure. Refers to the selective substitution of, for example, FR residues from the V region of a rodent heavy or light chain with a human FR residue to provide a heterologous molecule. Veneering technology is based on the understanding that the ligand binding characteristics of the antigen binding site are mainly determined by the structure and relative arrangement of the heavy and light chain CDR sets within the antigen binding surface. Davies et al. (1990) Ann. Rev. Biochem. 59: 439-473. Thus, antigen binding specificity can be preserved only in humanized antibodies in which CDR structures, interactions with each other, and interactions with the rest of the V region domain are carefully maintained. By using veneering techniques, external (eg, solvent accessible) FR residues (which are easily encountered in the immune system) are selectively replaced with human residues, resulting in weak immunogens. Hybrid molecules comprising either a sex veneering surface or a substantially non-immunogenic veneering surface are provided.

  The processing of veneering was performed by Kabat et al. (Sequences of Proteins of Immunological Interest, 4th Edition (edited by US Dept. of Health and Human Services, US Sovereign Printing Office, 198). Use available sequence data for variable domains and update to Kabat database and other US and foreign accessible databases (both nucleic acids and proteins). The solvent accessibility of V region amino acids can be deduced from the known three-dimensional structure for human and mouse antibody fragments. There are two general steps in veneering a mouse antigen binding site. First, the FR of the variable domain of the antibody molecule of interest is compared to the corresponding FR sequence of the human variable domain obtained from the above source. The most homologous human V region is then compared residue by residue with the corresponding mouse amino acid. Residues in the mouse FR that differ from the human counterpart are replaced by residues present in the human moiety using recombinant techniques well known in the art. Residue switching is performed only with moieties that are at least partially exposed (solvent accessible) and can have a significant effect on the tertiary structure of the V region domain (eg, Attention is paid to the substitution of amino acids, proline, glycine and charged amino acids.

  Thus, in this manner, the resulting “veneered” mouse antigen binding site is designed to retain: mouse CDR residues, residues substantially adjacent to the CDRs, buried Residues identified as being or mostly buried (solvent inaccessible), residues that are believed to be involved in non-covalent (eg, electrostatic and hydrophobic) contact between the heavy and light chain domains , As well as residues from the conserved structural region of FR that are thought to affect the “canonical” tertiary structure of the CDR loops. These design criteria are then used to convert both heavy and light chain CDRs of the mouse antigen binding site into mammalian cells for expression of human-like FRs (recombinant human antibodies that exhibit the antigen specificity of mouse antibody molecules). Recombinant nucleotide sequences are prepared that can be used to transfect

  In another embodiment of the invention, the monoclonal antibodies of the invention can be conjugated to one or more therapeutic agents. Suitable agents in this regard include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides include 90Y, 123I, 125I, 131I, 186Re, 188Re, 211At, and 212Bi. Preferred drugs include methotrexate and pyrimidine and purine analogs. Preferable differentiation inducers include phorbol ester and butyric acid. Preferred toxins include ricin, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.

  The therapeutic agent can be coupled (eg, covalently coupled) to a suitable monoclonal antibody either directly or indirectly (eg, via a linker group). A direct reaction between an agent and an antibody is possible when each has substituents that can react with each other. For example, nucleophilic groups (eg amino groups or sulfhydryl groups) can react on the one hand with carbonyl-containing groups (eg anhydrides or acid halides) or, on the other hand, good leaving groups (eg , Halides) can be reacted.

  Alternatively, it may be desirable to couple the therapeutic agent and the antibody via a linker group. The linker group may function as a spacer to separate the antibody from the drug, avoiding hindering the possibility of conjugation. The linker group may also serve to increase the chemical reactivity of the substituent on the drug or antibody, thus increasing the coupling efficiency. Increased chemical reactivity may also facilitate the use of drugs or functional groups on drugs that are not possible otherwise.

  A variety of bifunctional or polyfunctional reagents (both homofunctional and heterofunctional (eg, those described in the catalog of Pierce Chemical Co., Rockford, IL)) are used as linker groups. It will be apparent to those skilled in the art to obtain. Coupling can be effected, for example, via amino groups, carboxyl groups, sulfhydryl groups, or oxidized carbohydrate residues. There are many references describing such methodologies (eg, US Pat. No. 4,671,958 to Rodwell et al.).

  A linker that is cleavable during or during internalization into a cell if the therapeutic agent is more potent in the absence of the antibody portion of the immunoconjugate of the invention It may be preferred to use a group. A number of different cleavable linker groups have been described. Mechanisms for intracellular release of drugs from these linker groups include reduction of disulfide bonds (eg, US Pat. No. 4,489,710 to Spitler), irradiation of Photolabile bonds (eg, Center). U.S. Pat. No. 4,625,014), hydrolysis of derivatized amino acid side chains (eg, U.S. Pat. No. 4,638,045 to Kohn et al.), Serum complement-mediated hydrolysis ( For example, cleavage by Rodwell et al., US Pat. No. 4,671,958) and cleavage by acid-catalyzed hydrolysis (eg, US Pat. No. 4,569,789 to Blattler et al.).

  It may be preferred to couple more than one agent to the antibody. In one embodiment, multiple molecules of an agent are coupled to one antibody molecule. In another embodiment, more than one type of agent can be coupled to one antibody. Regardless of the particular embodiment, immunoconjugates having more than one agent can be prepared in a variety of ways. For example, more than one agent can be coupled directly to an antibody molecule or a linker can be used that provides multiple sites for attachment. Alternatively, a carrier can be used.

  The carrier may carry the drug in a variety of ways, including covalent bonding either directly or via a linker group. Suitable carriers include proteins such as albumin (eg, US Pat. No. 4,507,234 to Kato et al.), Polysaccharides such as peptides and aminodextrans (eg, US Pat. 699,784). The carrier may also carry the drug, eg, in liposome vesicles, either non-covalently or by encapsulation (eg, US Pat. Nos. 4,429,008 and 4,873,088). Carriers specific for radionuclide drugs include radiohalogenated small molecules and chelate compounds. For example, US Pat. No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis. Radionuclide chelates can be formed from chelate compounds containing nitrogen and sulfur atoms as donor atoms for binding metals, or metal oxides, radionuclides. For example, US Pat. No. 4,673,562 to Davison et al. Discloses representative chelating compounds and their synthesis.

(T cell composition)
In another aspect, the present invention provides T cells specific for the tumor polypeptides disclosed herein, or variants or derivatives thereof. Such cells can generally be prepared in vitro or ex vivo using standard procedures. For example, T cells can be obtained from commercially available cell separation systems such as the Isolex ™ system (Nexell Therapeutics, Inc. (Irvine, Calif.); US Pat. No. 5,240,856; US Pat. No. 5,215,926. WO 89/06280; see also WO 91/16116 and WO 92/07243), which can be isolated from the bone marrow, peripheral blood, or a fraction of bone marrow or a fraction of peripheral blood of the patient. The cells can be derived from related or unrelated humans, non-human mammals, cell lines or cultures.

  T cells can be stimulated with polypeptides, polynucleotides encoding the polypeptides, and / or antigen presenting cells (APCs) that express such polypeptides. Such stimulation is performed under conditions and for a time sufficient to allow generation of T cells that are specific for the polypeptide of interest. Preferably, a tumor polypeptide or polynucleotide of the invention is present in a delivery vehicle (eg, a microsphere) to facilitate the generation of specific T cells.

  A T cell kills a target cell that specifically proliferates, secretes a cytokine, or is coated with the polypeptide or expresses a gene encoding the polypeptide, It is considered specific for the polypeptides of the invention. T cell specificity can be assessed using any of a variety of standard techniques. For example, a stimulation index of more than 2-fold increase in lysis and / or proliferation compared to a negative control in a chromium release assay or proliferation assay indicates T cell specificity. Such assays are described, for example, in Chen et al., Cancer Res. 54: 1065-1070, 1994. Alternatively, detection of T cell proliferation can be achieved by various known techniques. For example, T cell proliferation is measured by measuring the rate of increase in DNA synthesis (eg, by pulse labeling a T cell culture with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into the DNA). Can be detected. Contact with tumor polypeptides (100 ng / ml to 100 μg / ml, preferably 200 ng / ml to 25 μg / ml) for 3-7 days typically results in at least a 2-fold increase in T cell proliferation . 2-3 hours of contact as described above should result in T cell activation as measured using standard cytokine assays. In this assay, a 2-fold increase in the level of cytokine (eg, TNF or IFN-γ) release indicates T cell activation (Coligan et al., Current Protocols in Immunology, Volume 1, Wiley Interscience (Greene 1998)). checking). T cells activated in response to tumor polypeptide, polynucleotide or polypeptide-expressing APC can be CD4 + and / or CD8 +. Tumor polypeptide-specific T cells can be expanded using standard techniques. In preferred embodiments, T cells are derived from a patient, an associated donor or an unrelated donor, and are administered to the patient after stimulation and expansion.

  For therapeutic purposes, CD4 + T cells or CD8 + T cells that proliferate in response to tumor polypeptides, polynucleotides or APCs can be expanded in large quantities either in vitro or in vivo. Such proliferation of T cells in vitro can be accomplished in various ways. For example, T cells can be tumor polypeptides, or short peptides corresponding to immunogenic portions of such polypeptides, with or without the addition of T cell growth factors (eg, interleukin-2), and It may be re-exposed to stimulator cells that synthesize tumor polypeptides. Alternatively, one or more T cells that proliferate in the presence of a tumor polypeptide can be expanded in large quantities by cloning. Methods for cloning cells are well known in the art, and these include limiting dilution.

(Pharmaceutical composition)
In further embodiments, the present invention provides a method for administering a cell or animal to a cell or animal, either alone or in combination with one or more other modes of therapy, in a pharmaceutically acceptable carrier. It relates to formulations of one or more polynucleotide, polypeptide, T cell, and / or antibody compositions disclosed herein.

  It is also understood that the compositions disclosed herein can be administered in combination with other agents (eg, other proteins or polypeptides, or various pharmaceutically active agents), if desired. . In fact, if the additional agent does not cause a significant detrimental effect upon contact with the target cell or host tissue, there is virtually no limit to other components that may be included as well. Thus, these compositions can be delivered with a variety of other agents as required in a particular case. Such compositions can be purified from the host cell or other biological source, or can be chemically synthesized as described herein. Similarly, such a composition may further comprise a substituted or derivatized RNA or DNA composition.

  Accordingly, another aspect of the invention includes one or more polynucleotides, polypeptides, antibodies, and / or T cell compositions described herein in combination with a physiologically acceptable carrier. A pharmaceutical composition is provided. In certain preferred embodiments, the pharmaceutical compositions of the invention comprise the immunogenic polynucleotide compositions and / or polypeptide compositions of the invention for use in prophylactic or therapeutic vaccine applications. Vaccine preparations are generally described in, for example, M.P. F. Powell and M.W. J. et al. Newman, “Vaccine Design (the subunit and adjuvant approach)”, Plenum Press (NY, 1995). In general, such compositions comprise one or more polynucleotide and / or polypeptide compositions of the present invention in combination with one or more immunostimulatory agents.

  It will be apparent that any pharmaceutical composition described herein may comprise pharmaceutically acceptable salts of the polynucleotides and polypeptides of the invention. Such salts include, for example, organic bases (eg, primary, secondary, and tertiary amines and basic amino acid salts) and inorganic bases (eg, sodium, potassium, lithium, ammonium, calcium, and From pharmaceutically acceptable non-toxic bases, including salts of magnesium).

  In another embodiment, an exemplary immunogenic composition (eg, vaccine composition) of the invention comprises DNA encoding one or more of the polypeptides as described above, wherein the polypeptide is in situ. Include as generated. As noted above, the polynucleotide can be administered by any of a variety of delivery systems known to those skilled in the art. Indeed, many gene delivery techniques are well known in the art (eg, those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15: 143-198, 1998, and references cited therein). ). Suitable polynucleotide expression systems will of course contain regulatory DNA regulatory sequences (eg, appropriate promoters and termination signals) necessary for expression in the patient. Alternatively, a bacterial delivery system can include administration of a bacterium (eg, Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on the cell surface or secretes such an epitope.

  Thus, in certain embodiments, a polynucleotide encoding an immunogenic polypeptide described herein is suitable for expression using any of a number of known virus-based systems. Into any mammalian host cell. In one exemplary embodiment, retroviruses provide a convenient and effective platform for gene delivery systems. A selected nucleotide sequence encoding a polypeptide of the present invention can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject. A number of exemplary retroviral systems have been described (eg, US Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7: 980-990; Miller, AD (1990) Human. Gene Therapy 1: 5-14; Scarpa et al. (1991) Virology 180: 849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90: 8033-8037; Opin.Genet.Develop.3: 102-109).

In addition, a number of exemplary adenovirus-based systems have also been described.
Unlike retroviruses that integrate into the host genome, adenoviruses remain extrachromosomal and thus minimize the risk associated with insertional mutagenesis (Haj-Ahmad and Graham (1986) J. Virol. 57. Bett et al. (1993) J. Virol. 67: 5911-5921; Mittereder et al. (1994) Human Gene Therapy 5: 717-729; Seth et al. (1994) J. Virol. 68: 933-940; (1994) Gene Therapy 1: 51-58; Berkner, KL (1988) BioTechniques 6: 616-629; and Rich et al. (1993) Human Gene Therapy 4: 461-476).

  Various adeno-associated virus (AAV) vector systems have also been developed for polynucleotide delivery. AAV vectors can be readily constructed using techniques well known in the art. See, for example, US Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8: 3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. et al. J. et al. (1992) Current Opinion in Biotechnology 3: 533-539; Muzyczka, N .; (1992) Current Topics in Microbiol. and Immunol. 158: 97-129; Kotin, R.A. M.M. (1994) Human Gene Therapy 5: 793-801; Shelling and Smith (1994) Gene Therapy 1: 165-169; and Zhou et al. (1994) J. MoI. Exp. Med. 179: 1867-1875.

  Additional viral vectors useful for delivering a polynucleotide encoding a polypeptide of the invention by gene transfer include those derived from poxvirus families (eg, vaccinia virus and avian poxvirus). As an example, a vaccinia virus recombinant expressing a novel molecule can be constructed as follows. The DNA encoding the polypeptide is first inserted into an appropriate vector so as to be adjacent to the vaccinia promoter and flanking vaccinia DNA sequences (eg, a sequence encoding thymidine kinase (TK)). This vector is then used to transfect cells that are simultaneously infected with vaccinia. By homologous recombination, the gene encoding the vaccinia promoter and the polypeptide of interest is inserted into the viral genome. The resulting TK. sup. (-) Recombinants can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking up virus plaques resistant to 5-bromodeoxyuridine.

  Vaccinia based infection / transfection systems are advantageously used to provide inducible transient or co-expression of one or more polypeptides described herein in an organism host cell. Can be done. In this particular system, cells are first infected in vitro with a vaccinia virus recombinant encoding bacteriophage T7 RNA polymerase. This polymerase exhibits exquisite specificity in that it only transcribes a template carrying the T7 promoter. Following infection, the cells are transfected with a polynucleotide of interest driven by a T7 promoter. A polymerase expressed in the cytoplasm from a vaccinia virus recombinant transcribes the transfected DNA into RNA, which is then translated into a polypeptide by the host translation machinery. This method provides high-level, transient cytoplasmic production of large amounts of RNA and its translation products. See, for example, Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87: 6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83: 812-8126.

  Alternatively, avipoxviruses (eg fowlpox virus and canarypox virus) can also be used to deliver the coding sequence of interest. Recombinant avipoxviruses that express immunogens from mammalian pathogens are known to confer protective immunity when administered to non-avian species. The use of avipox vectors is particularly desirable in humans and other mammalian species. This is because members of the genus avipox can replicate productively only in susceptible bird species and are therefore not infectious in mammalian cells. Methods for producing recombinant avipox viruses are known in the art and use genetic recombination as described above for the production of vaccinia virus. See, for example, WO 91/12882; WO 89/03429; and WO 92/03545.

  A number of optional alphavirus vectors can also be used for delivery of the polynucleotide compositions of the present invention, including US Pat. Nos. 5,843,723; 6,015,686; Examples include the vectors described in 6,008,035 and 6,015,694. Certain vectors based on Venezuelan equine encephalomyelitis (VEE) can also be used, illustrative examples of which can be found in US Pat. Nos. 5,505,947 and 5,643,576.

  In addition, molecular conjugation vectors (see, for example, Michael et al., J. Biol. Chem. (1993) 268: 6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89: 6099-6103). Adenovirus chimeric vectors) can also be used for gene delivery in the present invention.

  Additional exemplary information regarding these and other known virus-based delivery systems can be found, for example, in: Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86: 317-321, 1989; Flexner et al., Ann. N. Y. Acad. Sci. 569: 86-103, 1989; Flexner et al., Vaccine 8: 17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330 and 5,017,487; WO89 / US Pat. 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 91: 215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90: 11498-11502, 1993; Guzman et al., Circulation 88: 2838-2848, 1993; and Guzman et al., Cir. Res. 73: 1202-1207, 1993.

  In certain embodiments, the polynucleotide may be integrated into the genome of the target cell. This integration can be in a specific position and orientation via homologous recombination (gene replacement) or can be integrated at random non-specific positions (augmentation). In still further embodiments, the polynucleotide can be stably maintained in the cell as another episomal segment of DNA. Such polynucleotide segments or “episomes” encode sequences sufficient to be maintained and replicated independently of or in synchronization with the host cell cycle. The manner in which the expression construct is delivered to the cell and the manner in which the polynucleotide remains in the cell depends on the type of expression construct used.

  In another embodiment of the invention, the polynucleotide may be “naked,” as described, for example, in Ulmer et al., Science 259: 1745-1749, 1993 and outlined by Cohen, Science 259: 1691-1692,1993. Administered / delivered as DNA. Naked DNA uptake can be increased by coating the DNA on biodegradable beads, which are efficiently transported to cells.

  In yet another embodiment, the compositions of the invention can be delivered via a particle gun approach, many of which have been described. In one representative example, gas-driven particle acceleration is performed by Powder Pharmaceuticals PLC (Oxford, UK) and Powder Vaccines Inc. (Madison, WI) can be achieved with a device (apparatus) as manufactured. Some examples are U.S. Pat. Nos. 5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP 0500 799. In the issue. This approach provides a needle-free delivery approach. Here, a dry powder formulation of microscopic particles (eg, polynucleotide particles or polypeptide particles) is accelerated at high speed in a helium gas jet generated by a handheld device, and the particles are introduced into the target tissue of interest. Spray.

  In related embodiments, other devices and methods that may be useful for gas-driven non-injection needle injection of the compositions of the present invention are described in Bioject, Inc. (Portland, OR), some examples of which are U.S. Pat. Nos. 4,790,824; 5,064,413; 5,312,335; Nos. 5,383,851; 5,399,163; 5,520,639; and 5,993,412.

  In accordance with another embodiment, the pharmaceutical compositions described herein can comprise one or more immunizations in addition to the immunogenic polynucleotides, polypeptides, antibodies, T cells and / or APC compositions of the invention. Contains activator. An immunostimulant essentially refers to any substance that increases or enhances the immune response (antibody and / or cell-mediated) to an exogenous antigen. One preferred type of immunostimulant includes an adjuvant. Many adjuvants are substances designed to protect antigens from rapid catabolism (eg, aluminum hydroxide or mineral oil) and stimulators of immune responses (eg, proteins from lipid A, Bortadella pertussis or Mycobacterium tuberculosis) including. Certain adjuvants, such as Freund's incomplete adjuvant and Freund's complete adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beeheim) Aluminum salts (eg aluminum hydroxide gel (alum) or aluminum phosphate); calcium, iron or zinc salts; insoluble suspensions of acylated tyrosine; acetylated sugars; cationic derivatized polysaccharides or anionic derivatizations; Polyphosphazenes; biodegradable microspheres: monophosphoryl lipid A and monophosphori Quill (quil) A, are commercially available. Cytokines such as GM-CSF, interleukin-2, interleukin-7, interleukin-12 and growth factors can also be used as adjuvants.

  In certain embodiments of the invention, the adjuvant composition is preferably one that induces predominantly a Th1-type immune response. High levels of Th1-type cytokines (eg, IFN-γ, TNFα, IL-2 and IL-12) tend to support the induction of cell-mediated immune responses to the administered antigen. In contrast, high levels of Th2-type cytokines (eg, IL-4, IL-5, IL-6 and IL-10) tend to support the induction of humoral immune responses. Following application of a vaccine as provided herein, the patient supports an immune response that includes a Th1-type response and a Th2-type response. In preferred embodiments where the response is predominantly Th1-type, the level of Th1-type cytokines is increased to a higher degree than the level of Th2-type cytokines. The levels of these cytokines can be easily assessed using standard assays. For a review of the family of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7: 145-173, 1989.

  Certain preferred adjuvants for inducing a Th1-type dominant response include, for example, monophosphoryl lipid A, preferably a combination of 3-de-O-acylated monophosphoryl lipid A and an aluminum salt. MPL® adjuvant is available from Corixa Corporation (Seattle, WA; eg, US Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 912, 094). CpG-containing oligonucleotides (where CpG dinucleotides are not methylated) also induce a Th1-dominant response. Such oligonucleotides are well known and are described, for example, in WO96 / 02555, WO99 / 33488 and US Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273: 352, 1996. Another preferred adjuvant is a saponin (eg Quil A), or a derivative thereof (including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA)); Escin; Digitonin; Other preferred formulations are more than one in the combination of adjuvants of the invention (eg, at least two of the groups comprising QS21, QS7, Quil A, β-escin, or digitonin below) Contains saponin.

  Alternatively, the saponin formulation is a vaccine vehicle composed of chitosan or other polycationic polymer, polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine based polymer matrix, polysaccharide or chemically. It can be combined with particles composed of modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, and the like. Saponins can also be formulated in the presence of cholesterol to form a liposome or a particle structure such as ISCOM. In addition, saponins can be formulated with polyoxyethylene ethers or esters, either in non-particulate solutions or suspensions, or in particulate structures such as fractional liposomes or ISCOMs. Saponins can also be formulated with excipients such as Carbopol (R) to increase viscosity, or can be formulated in dry powder form with powder excipients such as lactose.

  In one preferred embodiment, the adjuvant system comprises a combination of monophosphoryl lipid A and a saponin derivative (eg, a combination of QS21 and 3D-MPL® adjuvant, as described in WO94 / 00153, or WO96 A low-reactive composition, wherein QS21 is quenched with cholesterol, as described in US Pat. Other preferred formulations include an oil-in-water emulsion and tocopherol. Another particularly preferred adjuvant formulation utilizing QS21, 3D-MPL® adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210.

  Another enhanced adjuvant system includes a combination of a CpG-containing oligonucleotide and a saponin derivative, in particular a combination of CpG and QS21 is disclosed in WO 00/09159. Preferably, the formulation further comprises an oil-in-water emulsion and tocophenol.

  Further representative adjuvants for use in the pharmaceutical compositions of the present invention include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron). Adubaund's SBAS series (eg, SBAS-2 or SBAS-4 (available from SmithKline Beecham, Rixensart, Belgium)), Detox (Enhanzyn®) (Corixa, Hamilton, MT), RC-529 (or , Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphates (AGP) (eg, pending US application registration number 08). 853,826 and 09 / 074,720 (the disclosures of which are incorporated herein by reference in their entirety), as well as the polyoxyethylene ether adjuvants described in WO 99 / 52549A1 Such adjuvants.

Other preferred adjuvants include an adjuvant molecule of general formula (I): HO (CH2CH2O) n-A-R,
Here, n is 1 to 50, A is a bond or —C (O) —, and R is C1 to 50 alkyl or phenyl C1 to 50 alkyl.

  One embodiment of the invention consists of a vaccine formulation comprising a polyoxyethylene ether of general formula (I), where n is between 1 and 50, preferably 4 to 24, most preferably 9 And the R component is C1-50, preferably C4 to C20 alkyl, and most preferably C12 alkyl, and A is a bond. The concentration of polyoxyethylene ether should be in the range of 0.1-20%, preferably 0.1-10%, and most preferably 0.1-1%. Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxy Ethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12th edition, 7717 items). These adjuvant molecules are described in WO99 / 52549.

  The polyoxyethylene ethers according to the general formula (I) above can be combined with another adjuvant, if desired. For example, a preferred adjuvant combination is preferably a combination with CpG as described in pending UK patent application GB9820956.2.

  In accordance with another embodiment of the invention, the immunogenic compositions described herein are antigen presenting cells (APCs) (eg, dendritic cells, macrophages, B cells, monocytes, and effective APCs). Delivered to the host via other cells) that can be manipulated as such. Such cells may increase their ability to present antigen, improve activation and / or maintenance of T cell responses, have their own anti-tumor effects, and / or immunize with recipients. It may be genetically modified to be genetically compatible (ie, a matching HLA haplotype), but need not be modified. APCs may generally be isolated from any of a variety of biological fluids and organs (including tumors and peritumoral tissues), and autologous, allogeneic, syngeneic, or xenogeneic cells It may be.

  Certain preferred embodiments of the invention use dendritic cells or their progenitor cells as antigen presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392: 245-251, 1998) and are effective as physiological adjuvants to elicit prophylactic or therapeutic anti-tumor immunity It has been shown to be (see Timerman and Levy, Ann. Rev. Med. 50: 507-529, 1999). In general, dendritic cells take up, process, and present antigens in their typical shape (stars in situ, with a pronounced cytoplasmic process (dendrites) visible in vitro), high efficiency Based on their ability and their ability to activate a naïve T cell response can be identified. Of course, dendritic cells can be engineered to express specific cell surface receptors or ligands not normally 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 vesicle antigen-loaded dendritic cells (called exosomes) can be used in vaccines (Zitvogel et al., Nature Med. 4: 594-). 600, 1998).

  Dendritic cells and precursors can be obtained from peripheral blood, bone marrow, tumor infiltrating cells, peritumoral tissue infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood, or any other suitable tissue or fluid. For example, dendritic cells are differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13 and / or TNFα to monocyte cultures collected from peripheral blood. obtain. Alternatively, CD34 positive cells collected from peripheral blood, umbilical cord blood or bone marrow can be cultured in culture medium with GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and / or dendritic cell differentiation, maturation, and It can be differentiated into dendritic cells by adding combinations of other compounds that induce proliferation.

  Dendritic cells are conveniently classified as “immature” and “mature” cells, which provides a simple way to distinguish between two well-characterized phenotypes. However, this name should not be construed to exclude any possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, and this capacity correlates with high expression of Fcγ and mannose receptors. The mature phenotype is typically characterized by lower expression of these markers, class I MHC and class II MHC, adhesion molecules (eg CD54 and CD11) and costimulatory molecules (eg CD40, CD80, Characterized by the high expression of cell surface molecules responsible for T cell activation such as CD86 and 4-1BB).

  APC can generally be transfected with a polynucleotide (or portion or other variant thereof) of the invention so that the encoded polypeptide or immunogenic portion thereof is expressed on the cell surface. The Such transfection can occur ex vivo, and then a pharmaceutical composition comprising such transfected cells can be used for therapeutic purposes, as described herein. Alternatively, gene delivery vehicles that target dendritic cells or other antigen presenting cells can be administered to a patient, resulting in transfection occurring in vivo. In vivo and ex vivo transfection of dendritic cells can be performed using such methods as the method described in WO 97/24447 or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75: 456-460, 1997, for example. It can generally be performed using any method known in the art. Dendritic cell antigen loading can involve dendritic cells or progenitor cells, tumor polypeptides, DNA (naked or in plasmid vectors) or RNA; or antigen-expressing recombinant bacteria or viruses (eg, cowpox, fowlpox, Incubating with adenovirus or lentiviral vectors). Prior to loading, the polypeptide can be covalently linked to an immunological partner (eg, a carrier molecule) that provides T cell assistance. Alternatively, dendritic cells can be pulsed with an unbound immunological partner, either alone or in the presence of a polypeptide.

  Although any suitable carrier known to those skilled in the art can be used in the pharmaceutical compositions of the invention, the type of carrier typically varies based on the mode of administration. The composition of the present invention may be any suitable mode of administration (including, for example, topical administration, oral administration, nasal administration, transmucosal administration, intravenous administration, intracranial administration, intraperitoneal administration, subcutaneous administration and intramuscular administration). Can be prescribed for.

  Carriers for use in such pharmaceutical formulations are biocompatible and can also be biodegradable. In certain embodiments, preferably the formulation provides a relatively constant level of active ingredient release. However, in other embodiments, a faster release rate immediately after administration may be desired. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques. Exemplary carriers useful in this regard include microparticles such as poly (lactide-co-glucolide), polyacrylate, latex, starch, cellulose, dextran. Other exemplary delayed release carriers include supramolecular biovectors comprising a non-liquid hydrophilic core (eg, a crosslinked polysaccharide or oligosaccharide), and optionally amphiphilic compounds Supramolecular biovectors comprising an outer layer (eg, phospholipids) comprising (see, eg, US Pat. No. 5,151,254 and PCT applications WO 94/20078, WO / 94/23701 and WO 96/06638) Can be mentioned. The amount of active compound contained in the sustained release formulation will depend on the site of implantation, the rate of release and the expected duration, and the nature of the condition to be treated or prevented.

  In another exemplary embodiment, biodegradable microspheres (eg, polylactic acid polyglycolate) are used as carriers for the compositions of the present invention. Suitable biodegradable microspheres are, for example, U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344; 5,407,609 and 5,942,252. Modified hepatitis B core protein carrier systems (eg, systems as described in WO / 99 40934 and references cited therein) are also useful for many applications. Another exemplary carrier / delivery system uses a carrier-containing particle-protein complex (eg, the complex described in US Pat. No. 5,928,647), which is a class I restricted cell in the host. Injury T lymphocyte responses can be induced.

  The pharmaceutical compositions of the present invention often contain one or more buffering agents (eg, neutral buffered saline or phosphate buffered saline), carbohydrates (eg, glucose, mannose, sucrose or dextran). ), Mannitol, proteins, polypeptides or amino acids (eg glycine), antioxidants, bacteriostatic agents, chelating agents (eg EDTA or glutathione), adjuvants (eg aluminum hydroxide), formulations of the recipient's blood It further includes solutes, suspending agents, thickeners and / or preservatives that render it isotonic, hypotonic or weakly hypertonic. Alternatively, the composition of the present invention can be formulated as a lyophilizate.

  The pharmaceutical compositions described herein can be present in single dose containers or multi-dose containers (eg, sealed ampoules or vials). Such containers are typically sealed to maintain the sterility and stability of the formulation until use. In general, formulations can be stored as suspensions, solutions or emulsions in oily or aqueous vehicles. Alternatively, the pharmaceutical composition can be stored in a lyophilized state requiring only the addition of a sterile aqueous carrier just prior to use.

  Appropriate dosing and treatment regimens (eg, oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation for use of the particular compositions described herein in various treatment regimes. The development of this is well known in the art and some of these are briefly discussed below for general illustration purposes.

  In certain applications, the pharmaceutical compositions disclosed herein can be delivered to animals by oral administration. Thus, these compositions can be formulated with an inert diluent or with an assimilable edible carrier, or these compositions can be placed in hard or soft shell gelatin capsules, or these compositions The product can be compressed into tablets, or these compositions can be incorporated directly into the dietary food.

The active compounds can be further incorporated into excipients and used in the form of orally ingested tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, cachets and the like (eg, Mathiowitz et al., Nature 1997 Mar 27; 386 (6623): 410-4; Hwang et al., Crit Rev The Drug Carrier Sys 1998; 15 (3): 243-84; US Pat. No. 5,641,515; US Pat. No. 5,580, 579 and US Pat. No. 5,792,451). Tablets, troches, pills, capsules, etc. may also contain any of a variety of additional ingredients such as binders (eg, gum tragacanth, gum arabic, corn starch or gelatin); excipients (eg, dicalcium phosphate ); Disintegrants (eg, corn starch, potato starch, alginic acid, etc.); lubricants (eg, magnesium stearate); and sweeteners (eg, sucrose, lactose or saccharin) or flavors (eg, peppermint, winter green oil, Or cherry flavor). Where the dosage unit form is a capsule, it may contain a liquid carrier in addition to the types of materials described above. A variety of other materials may be present as coatings or otherwise modify the physical form dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. Of course, any material used in preparing any dosage unit form must be pharmaceutically pure and substantially non-toxic in the amounts employed.
In addition, the active compound can be incorporated into sustained-release preparations and formulations.

  Typically, these formulations contain at least about 0.1% or more of the active compound, but the percentage of active ingredient can, of course, vary and conveniently is the weight or volume of the total formulation. Between about 1 or 2% and about 60% or more and 70% or more. Of course, the amount of active compound in each of the therapeutically useful compositions can be prepared in such a way that an appropriate dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product expiration date, and other pharmacological considerations are contemplated by those skilled in the art of preparing such pharmaceutical formulations. As such, various dosages and treatment regimens may themselves be desirable.

  For oral administration, the compositions of the invention may alternatively be incorporated with one or more excipients in the form of mouthwashes, dentifrices, buccal tablets, oral sprays, or sublingual oral dosage formulations. Alternatively, the active ingredient can be incorporated into an oral solution (eg, a solution comprising sodium borate, glycerin and potassium bicarbonate) or dispersed in a dentifrice, or in a therapeutically effective amount, water, binder , Abrasives, perfumes, foaming agents, and wetting agents can be added to the composition. Alternatively, these compositions can be formed into tablet or solution forms that can be placed under the tongue or otherwise dissolved in the mouth.

  In certain situations, it may be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly or even intraperitoneally. Such approaches are well known to those skilled in the art, some of which are described, for example, in, for example, US Pat. No. 5,543,158; US Pat. No. 5,641,515 and US Pat. No. 5,399,363. Further described in the issue. In certain embodiments, solutions of the active compounds as the free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.

  Exemplary pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (see, eg, US Pat. No. 5,466,577). , 468). In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. Prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use of agents that delay absorption, for example, aluminum monostearate and gelatin, in the composition.

  In one embodiment, for parenteral administration of an aqueous solution, the solution should be appropriately buffered if necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose. To be. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those of skill in the art in light of the present disclosure. For example, a dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or can be infused at the site of infusion (eg, “Remington” 's Pharmaceutical Sciences', 15th edition, pages 1035-1038 and pages 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. Furthermore, for human administration, the preparation will, of course, preferably meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biology standards.

  In another embodiment of the invention, the compositions disclosed herein can be formulated in neutral or salt form. Exemplary pharmaceutically acceptable salts include acid addition salts (formed with the free amino group of the protein), and these salts include inorganic acids (eg, hydrochloric acid or phosphoric acid), or acetic acid It is formed with organic acids such as oxalic acid, tartaric acid and mandelic acid. Salts formed with free carboxyl groups are also inorganic bases (eg, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide) and organics such as isopropylamine, trimethylamine, histidine, procaine It can be derived from a base. Upon formulation, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.

  The carrier may contain any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. May be included. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional vehicle or agent is incompatible with the active ingredient, its use in a therapeutic composition is contemplated. Supplementary active ingredients can also be incorporated into these compositions. The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an allergic reaction or similar annoying reaction when administered to a human.

  In certain embodiments, the pharmaceutical composition can be delivered by intranasal sprays, inhalation, and / or other aerosol delivery vehicles. Methods for delivering gene, nucleic acid and peptide compositions directly to the lungs via nasal aerosol spray are described, for example, in US Pat. No. 5,756,353 and US Pat. No. 5,804,212. ing. Similarly, drugs using intranasal microparticle resins (Takenaga et al., J Controlled Release 1998 Mar 2; 52 (1-2): 81-7) and lysophosphatidyl-glycerol compounds (US Pat. No. 5,725,871) Delivery of is also well known in the pharmaceutical arts. Similarly, representative transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is described in US Pat. No. 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles, lipid particles, vesicles, and the like are used for introduction of the compositions of the invention into suitable host cells / organisms.
In particular, the compositions of the invention can be formulated for delivery in an encapsulated form such as lipid particles, liposomes, vesicles, nanospheres, or nanoparticles. Alternatively, the compositions of the present invention can be attached to the surface of such carrier vehicles either covalently or non-covalently.

  The formation and use of liposomes and liposome-like preparations as potential drug carriers is generally known to those skilled in the art (eg, Lasic, Trends Biotechnol 1998 Jul; 16 (7): 307-21; Takakura, Nippon Rinsho 1998). Mar; 56 (3): 691-5; Chandran et al., Indian J Exp Biol. 1997 Aug; 35 (8): 801-9; Margalit, Crit Rev The Drug Carrier Syst. 1995; 12 (2-3): 233. -61; U.S. Patent No. 5,567,434; U.S. Patent No. 5,552,157; U.S. Patent No. 5,565,213; U.S. Patent No. 5,738,868 and U.S. Patent No. 5,795. 587 (Each of which in its entirety is detailed incorporated by reference herein) see).

Liposomes have been used successfully with many cell types that are usually difficult to transfect by other procedures including T cell suspensions, primary hepatocyte cultures and PC12 cells (Reneisen et al., J Biol Chem. 1990 Sep 25; 265 (27): 16337-42; Muller et al., DNA Cell Biol. 1990 Apr; 9 (3): 221-9). Furthermore, liposomes do not have the DNA length limitations typical of virus-based delivery systems.
Liposomes are effectively used to introduce genes, various drugs, radiotherapeutic agents, enzymes, viruses, transcription factors, allosteric effectors, and the like into various cultured cell lines and animals. Furthermore, the use of liposomes does not appear to be related to autoimmune responses or unacceptable toxicity after systemic delivery.

  In certain embodiments, liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multi-layer concentric bilayer vesicles (also referred to as multi-layer vesicles (MLV)).

  Alternatively, in other embodiments, the present invention provides pharmaceutically acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally supplement compounds in a stable and reproducible manner (see, eg, Quintanar-Guerrero et al., Drug Dev Ind Pharm. 1998 Dec; 24 (12): 1113-28). In order to avoid the side effects due to intracellular polymer overload, such ultrafine particles (size of about 0.1 μm) can be designed using polymers that can be degraded in vivo. Such particles are described, for example, in Couvreur et al., Crit Rev The Drug Carrier Sys. 1988; 5 (1): 1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 Mar; 45 (2): 149-55; Zambaux et al., J Controlled Release. 1998 Jan 2; 50 (1-3): 31-40; and US Pat. No. 5,145,684.

(Cancer therapy)
In a further aspect of the invention, the pharmaceutical compositions described herein can be used for the treatment of cancer, in particular for ovarian cancer immunotherapy. In such methods, the pharmaceutical compositions described herein are administered to a patient, typically a warm-blooded animal, preferably a human. The patient may or may not have cancer. Thus, the pharmaceutical composition described above can be used to prevent the development of cancer or to treat a patient suffering from cancer. The pharmaceutical compositions and vaccines can be administered either before or after treatment such as surgical removal of the primary tumor and / or administration of radiotherapy or conventional chemotherapeutic agents. As noted above, administration of the pharmaceutical composition is any suitable method, including intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, intradermal, anal, vaginal, topical and oral routes. Can be.

  In certain embodiments, the immunotherapy can be active immunotherapy, where treatment uses administration of immune response modifiers (eg, polypeptides and polynucleotides provided herein), Relies on in vivo stimulation of the endogenous host immune system that responds to the tumor.

  In other embodiments, the immunotherapy can be passive immunotherapy in which the treatment is a factor (eg, an effector cell or antibody) with established tumor immunoreactivity (which has an anti-tumor effect). Delivery) which can be mediated directly or indirectly and does not necessarily depend on the intact host immune system. Examples of effector cells include T cells as described above, T lymphocytes (eg, CD8 + cytotoxic T lymphocytes and CD4 + T helper tumor infiltrating lymphocytes) that express the polypeptides provided herein. Killer cells (eg, natural killer cells and lymphokine activated killer cells), B cells and antigen presenting cells (eg, dendritic cells and macrophages). T cell receptors and antibody receptors specific for the polypeptides listed herein can be cloned, expressed and transferred into other vectors or effector cells for adoptive immunotherapy. The polypeptides provided herein can also be used to generate antibodies or anti-idiotype antibodies (described above and in US Pat. No. 4,918,164) for passive immunotherapy.

  Effector cells can generally be obtained in an amount sufficient for adoptive immunotherapy by in vitro growth, as described herein. Culture conditions for growing single antigen-specific effector cells to billions while retaining antigen recognition in vivo are well known in the art. Such in vitro culture conditions typically employ intermittent stimulation with antigen, often in the presence of cytokines (eg, IL-2) and non-dividing feeder cells. As noted above, the immunoreactive polypeptides provided herein can be used to rapidly expand antigen-specific T cell cultures to generate a sufficient number of cells for immunotherapy. Can do. Specifically, antigen presenting cells (eg, dendritic cells, macrophages, monocytes, fibroblasts, and / or B cells) are pulsed with immunoreactive polypeptides using standard techniques well known in the art. Or can be transfected with one or more polynucleotides. For example, antigen presenting cells can be transfected with a polynucleotide having a suitable promoter to increase expression in a recombinant virus or other expression system. Cultured effector cells for use in therapy must be able to grow in vivo and be widely distributed and survive for long periods of time. Studies have shown that cultured effector cells can be induced to survive in large numbers for long periods of time by in vivo proliferation and repeated stimulation with antigen supplemented with IL-2 (eg, Cheever). Et al., Immunological Reviews 157: 177, 1997).

  Alternatively, vectors that express the polypeptides listed herein can be introduced into antigen-presenting cells obtained from a patient and can be clonally propagated ex vivo for transplantation back to the same patient. Transfected cells can be reintroduced into the patient using any means known in the art, preferably a sterile form by intravenous, intracavitary, intraperitoneal, or intratumoral administration.

  The route and frequency of administration and dosage of the therapeutic compositions described herein will vary from person to person and can be readily established using standard techniques. In general, pharmaceutical compositions and vaccines can be administered by injection (eg, intradermal, intramuscular, intravenous, or subcutaneous), nasally (eg, by inhalation) or orally. Preferably, between 1 and 10 doses may be administered over 52 weeks. Preferably, 6 doses are administered at 1 month intervals and booster (additional) vaccinations can then be given periodically. Alternative protocols may be appropriate for individual patients. An appropriate dose is that amount of the compound that, when administered as described above, can promote an anti-tumor immune response and is at least 10-50% above basal (ie, untreated) levels. Such a response can be monitored by measuring anti-tumor antibodies in the patient or by vaccine-dependent in vitro generation of cytolytic effector cells that can kill the patient's tumor cells. Such vaccines also have improved clinical outcome (e.g., more frequent relief of symptoms, complete or partial disease, or more in vaccinated patients compared to non-vaccinated patients). It should be able to generate an immune response that leads to long disease-free survival). In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 25 μg to 5 mg per kg body weight of the host. Suitable dose sizes vary with the size of the patient but typically range from about 0.1 mL to about 5 mL.

  In general, a suitable dosage and treatment regimen provides a sufficient amount of active compound to provide therapeutic and / or prophylactic benefits. Such a response may result in improved clinical outcome (eg, more frequent remission, complete or partial, or no longer disease in treated patients compared to untreated patients. Can be monitored by establishing survival). Increases in existing immune responses against tumor proteins are generally associated with improved clinical outcome. Such an immune response can generally be assessed using standard proliferation assays, cytotoxicity assays or cytokine assays, which are performed using samples obtained from the patient before and after treatment. Can be broken.

(Compositions, methods and kits for cancer detection and diagnosis)
In general, cancer involves one or more ovarian tumor proteins and / or such proteins in a biological sample obtained from a patient (eg, blood, serum, sputum, urine, and / or tumor biopsy). It can be detected in a patient based on the presence of the encoding polynucleotide. In other words, such a protein can be used as a marker to indicate the presence or absence of a cancer such as ovarian cancer. In addition, such proteins may be useful for the detection of other cancers. The binding agents provided herein generally allow detection of the level of antigen that binds to the agent in a biological sample. Polynucleotide primers and probes can be used to detect the level of mRNA encoding ovarian tumor protein, which also indicates the presence or absence of cancer. In general, ovarian tumor sequences are present at a level at least three times higher in tumor tissue than in normal tissue.

  There are a variety of assay formats known to those of skill in the art for using binding agents to detect polypeptide markers in a sample. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of cancer in a patient is determined by: (a) contacting a biological sample obtained from the patient with a binding agent; (b) detecting the level of polypeptide bound to the binding agent in the sample. And (c) comparing the level of the polypeptide with a predetermined cutoff value.

  In a preferred embodiment, the assay involves the use of a binding agent immobilized on a solid support to bind and remove the polypeptide from the rest of the sample. The bound polypeptide can then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent / polypeptide complex. Such detection reagents include, for example, binding agents that specifically bind to polypeptides or antibodies, or other agents that specifically bind to binding agents (eg, anti-immunoglobulin, protein G, protein A or lectin). May be included. Alternatively, a competition assay can be used, wherein the polypeptide is labeled with a reporter group and can bind to its immobilized binding agent after incubation of the sample and binding agent. The degree to which the sample components inhibit binding of the labeled polypeptide to the binding agent indicates the reactivity of the sample with the immobilized binding agent. Polypeptides suitable for use in such assays include full length ovarian tumor proteins and portions of the polypeptides to which the binding agent binds, as described above.

  The solid support can be any material known to those of skill in the art to which tumor proteins can be attached. For example, the solid support can be a test well in a microtiter plate or nitrocellulose or other suitable membrane. Alternatively, the support can be a bead or disk (eg, glass, fiberglass, latex, or plastic material (eg, polystyrene or polyvinyl chloride)). The support can also be a magnetic particle or fiber optic sensor (eg, as disclosed in US Pat. No. 5,359,681). The binder can be immobilized on the solid support using a variety of techniques known to those skilled in the art, which are well described in the patent and scientific literature. In the context of the present invention, the term “immobilization” refers to non-covalent association (eg adsorption) and covalent attachment (which is directly linked between the drug and a functional group on the support). Or can be linked using a crosslinker). Immobilization by adsorption to a well of a microtiter plate or a membrane is preferred. In such cases, adsorption can be accomplished by contacting the binder with the solid support in a suitable buffer for a suitable time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. Generally, contacting a well of a plastic microtiter plate (eg, polystyrene or polyvinyl chloride) with an amount of binder in the range of about 10 ng to about 10 μg, and preferably about 100 ng to about 1 μg, is appropriate. It is sufficient to immobilize the amount of binder.

  Covalent attachment of a binder to a solid support generally involves first reacting the support with a bifunctional reagent that reacts with both the support and a functional group (eg, a hydroxyl or amino group) on the binder. Can be achieved. For example, the binder can be covalently attached to a support having a suitable polymer coating using benzoquinone or by condensation of amines and active hydrogens on the binding partner with aldehyde groups on the support ( (See, for example, Pierce Immunotechnology Catalog and Handbook, 1991, A12-A13).

  In certain embodiments, the assay is a two antibody sandwich assay. The assay is performed by first contacting the antibody immobilized on a solid support (usually a well of a microtiter plate) with the sample and allowing the polypeptide in the sample to bind to the immobilized antibody. obtain. The unbound sample is then removed from the immobilized polypeptide-antibody complex, and a detection reagent (preferably a second antibody that can bind to a different site on the polypeptide) (containing a reporter group) is added. The The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the particular reporter group.

  More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent (eg, bovine serum albumin or Tween 20 ™ (Sigma Chemical Co., St. Louis, MO)) is known to those skilled in the art. The immobilized antibody is then incubated with the sample and the polypeptide is allowed to bind to the antibody. Prior to incubation, the sample can be diluted with an appropriate diluent, such as phosphate buffered saline (PBS). In general, a suitable contact time (ie, incubation time) is a time sufficient to detect the presence of a polypeptide in a sample obtained from an individual having an ovarian tumor. Preferably, the contact time is sufficient to achieve a binding level that is at least about 95% of the level achieved at equilibrium between the bound and unbound polypeptides. One skilled in the art will recognize that by assaying the level of binding that occurs over a period of time, the time required to reach equilibrium can be readily determined. At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound sample can then be removed by washing the solid support with a suitable buffer (eg, PBS containing 0.1% Tween 20 ™).
A second antibody comprising a reporter group can then be added to the solid support. Preferred reporter groups include those described above.

  The detection reagent is then incubated with the immobilized antibody-polypeptide complex for an amount of time sufficient to detect the bound polypeptide. An appropriate amount of time can generally be determined by assaying the level of binding that occurs over a period of time. Unbound detection reagent is then removed and bound detection reagent is detected using a reporter group. The method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopy can be used to detect dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin coupled to different reporter groups (generally radioactive or fluorescent groups or enzymes). Enzyme reporter groups can generally be detected by the addition of a substrate (generally for a specific time) followed by spectroscopic or other analysis of the reaction product.

  To determine the presence or absence of cancer (eg, ovarian cancer), the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal corresponding to a predetermined cutoff value. . In one preferred embodiment, the cutoff value for detection of cancer is the average signal value obtained when the immobilized antibody is incubated with a sample from a patient without cancer. In general, samples that produce a signal that is 3 standard deviations above a given cutoff value are considered positive for cancer. In an alternative preferred embodiment, this cutoff value is determined by Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co. , 1985, pp. 106-7, using the Receiver Operator Curve (Receiver Operator Curve). Briefly, in this embodiment, this cutoff value is the true positive rate (ie sensitivity) and false positive rate (100% -specificity) corresponding to each possible cutoff value for the diagnostic test result. It can be determined from a paired plot. The cutoff value closest to the upper left hand corner on the plot (ie, the value surrounding the largest region) is the most accurate cutoff value, and positive for samples that yield a signal higher than the cutoff value determined by the method Can be considered. Alternatively, the cutoff value can be shifted to the left along the plot to minimize the false positive rate, or can be shifted to the right to minimize the false negative rate. In general, a sample that produces a signal that is higher than the cut-off value determined by the present method is considered positive for cancer.

  In related embodiments, the assay is performed in a flow-through test format or a strip test format (where the binding agent is immobilized on a membrane such as nitrocellulose). In the flow-through test, the polypeptide in the sample binds to the immobilized binding agent as the sample passes through the membrane. The second labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane. Detection of the bound second binding agent can then be performed as described above. In the strip test format, one end of the membrane to which the binding agent is bound is immersed in a solution containing the sample. The sample travels along the membrane, through the area containing the second binder, and to the area of immobilized binder. The concentration of the second binding agent in the area of immobilized antibody indicates the presence of cancer. Typically, the concentration of the second binding agent at that site produces a pattern (eg, a line) that can be read visually. Not showing such a pattern indicates a negative result. Generally, the amount of binding agent immobilized on this membrane comprises a level of polypeptide that is sufficient for the biological sample to produce a positive signal in a two-antibody sandwich assay in the format discussed above. If selected, it is selected to produce a visually identifiable pattern. Preferred binding agents for use in such assays are antibodies and antigen binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably from about 50 ng to about 500 ng. Such a test can typically be performed with a very small amount of biological sample.

  Of course, there are numerous other assay protocols suitable for use with the tumor proteins or binding agents of the invention. The above description is intended to be exemplary only. For example, it will be apparent to one of skill in the art that the above protocol can be readily modified to use tumor polypeptides to detect antibodies that bind to such polypeptides in a biological sample. Detection of such tumor protein specific antibodies can be correlated with the presence of cancer.

  Cancer can also be detected based on the presence of T cells that react specifically with tumor proteins in a biological sample. In certain methods, a biological sample comprising CD4 + T cells and / or CD8 + T cells isolated from a patient is a tumor polypeptide, a polynucleotide encoding such a polypeptide and / or at least such a polypeptide. Incubate with APC expressing the immunogenic portion and detect the presence or absence of specific activation of T cells. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells can be isolated from patients by conventional techniques (eg, by Ficoll / Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells can be incubated in vitro with polypeptides (eg, 5-25 μg / ml) at 37 ° C. for 2-9 days (typically 4 days). It may be desirable to incubate another aliquot of the T cell sample in the absence of tumor polypeptide to serve as a control. For CD4 + T cells, activation is preferably detected by assessing T cell proliferation. For CD8 + T cells, activation is preferably detected by assessing cytolytic activity. A level of proliferation that is at least 2-fold higher and / or a level of cytolytic activity that is at least 20% higher than in a patient without disease indicates the presence of cancer in the patient.

  As noted above, cancer can also be detected based on the level of mRNA encoding the tumor protein in the biological sample. For example, at least two oligonucleotide primers can be used in a polymerase chain reaction (PCR) -based assay to amplify a portion of tumor cDNA from a biological sample, wherein at least one of the oligonucleotide primers One is specific (ie, hybridizes) to a polynucleotide that encodes a tumor protein. The amplified cDNA is then separated and detected using techniques well known in the art (eg, gel electrophoresis). Similarly, oligonucleotide probes that specifically hybridize to a polynucleotide encoding a tumor protein can be used in a hybridization assay to detect the presence of a polynucleotide encoding this tumor protein in a biological sample.

  In order to allow hybridization under assay conditions, oligonucleotide primers and probes are part of a polynucleotide encoding a tumor protein of the invention that is at least 10 nucleotides and preferably at least 20 nucleotides in length. Should contain oligonucleotide sequences having at least about 60% identity, preferably at least about 75%, and more preferably at least about 90% identity. Preferably, the oligonucleotide primers and / or probes hybridize to a polynucleotide encoding a polypeptide described herein under moderately stringent conditions, as defined above. . Oligonucleotide primers and / or probes that can be usefully used in the diagnostic methods described herein are preferably at least 10-40 nucleotides in length. In preferred embodiments, the oligonucleotide primer comprises at least 10 contiguous nucleotides of a DNA molecule having a sequence as disclosed herein, more preferably at least 15 contiguous nucleotides. Techniques for both PCR-based and hybridization assays are well known in the art (eg, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263, 1987; edited by Errich, PCR Technology, Stockton). Press, NY, 1989).

  One preferred assay uses RT-PCR, where PCR is applied in combination with reverse transcription. Typically, RNA is extracted from a biological sample (eg, biopsy tissue) and reverse transcribed to produce a cDNA molecule. PCR amplification using at least one specific primer produces a cDNA molecule that can be separated and visualized using, for example, gel electrophoresis. Amplification can be performed in biological samples taken from test patients and individuals not afflicted with cancer. The amplification reaction can be performed on several dilutions of cDNA ranging in magnitude by two orders of magnitude. If the increase in expression in several dilutions of the test patient sample is more than 2-fold compared to the same dilution of a sample without cancer, this is typically considered positive.

  In another embodiment, the compositions described herein can be used as markers for cancer progression. In this embodiment, assays such as those described above for cancer diagnosis can be performed over time and can assess changes in levels of reactive polypeptides or polynucleotides. For example, the assay can be performed every 24-72 hours over a period of 6 months to 1 year, and thereafter performed as needed. In general, cancer has progressed in patients whose levels of detected polypeptide or polynucleotide increase over time. In contrast, cancer is not progressing when the level of reactive polypeptide or polynucleotide either remains constant or decreases with time.

Certain in vivo diagnostic assays can be performed directly on the tumor. One such assay involves contacting tumor cells with a binding agent. The bound binding agent can then be detected directly or indirectly via a reporter group.
Such binders can also be used in histological applications. Alternatively, polynucleotide probes can be used in such applications.

  As described above, multiple oncoprotein markers can be assayed within a given sample to improve sensitivity. It will be apparent that binding agents specific for the various proteins provided herein can be combined in a single assay. In addition, multiple primers or probes can be used simultaneously. The selection of oncoprotein markers can be based on routine experimentation to determine the combination that results in optimal sensitivity. Additionally or alternatively, the assays for tumor proteins provided herein can be combined with assays for other known tumor antigens.

  The present invention further provides kits for use in any of the above diagnostic methods. Such a kit typically comprises two or more components necessary to perform a diagnostic assay. The component can be a compound, reagent, container and / or instrument. For example, one container in the kit may contain a monoclonal antibody or fragment thereof that specifically binds to a tumor protein. Such antibodies or fragments can be provided attached to a support material as described above. One or more additional containers may contain elements (eg, reagents or buffers) used in the assay. Such a kit may alternatively comprise a detection reagent as described above comprising a reporter group suitable for direct or indirect detection of antibody binding.

  Alternatively, the kit can be designed to detect mRNA levels encoding oncoproteins in a biological sample. Such kits generally comprise at least one oligonucleotide probe or primer as described above that hybridizes to a polynucleotide encoding a tumor protein. Such oligonucleotides can be used, for example, in PCR or hybridization assays. Additional components that may be present in such kits include a second oligonucleotide and / or a diagnostic reagent or container that facilitates detection of the polynucleotide encoding the tumor protein.

  The following examples are provided for purposes of illustration and not for limitation.

(Example)
(Example 1: Identification of representative ovarian cancer cDNA sequence)
Primary and metastatic ovarian tumor cDNA libraries were constructed from pools of three different ovarian tumor RNA samples, each in a kanamycin resistant pZErOTM-2 vector (Invitrogen). For the primary ovarian tumor library, the following RNA samples were used: (1) moderately differentiated papillary serous carcinoma of a 41 year old person, (2) stage IIIC ovarian tumor, and (3) 50 year old Caucasian papillary serous adenocarcinoma. For the metastatic ovarian tumor library, the RNA samples used were a network derived from: (1) a 73-year-old person with poorly differentiated metastatic papillary adenocarcinoma with a sarcoma, (2 A) 74-year-old person with little differentiated metastatic adenocarcinoma, and (3) a 68-year-old person with little differentiated metastatic papillary adenocarcinoma.

  The number of clones in each library was estimated by plating serial dilutions of the unamplified library. Insert data was determined from 32 primary ovarian tumor clones and 32 metastatic ovarian tumor clones. The results of library characterization are shown in Table I.

４つのサブトラクションライブラリーを、アンピシリン耐性ｐｃＤＮＡ３．１ベクター（Ｉｎｖｉｔｒｏｇｅｎ）中に構築した。２つのライブラリーは、原発性卵巣腫瘍由来であり、そして２つは、転移性卵巣腫瘍由来である。各々の場合、挿入物内の制限酵素切片の数を最小化して、全長サブトラクションライブラリーを生成した。サブトラクションを、以下により詳細に記載されるように、わずかに異なるプロトコルを用いて各々行った。

Four subtraction libraries were constructed in the ampicillin resistant pcDNA3.1 vector (Invitrogen). Two libraries are from primary ovarian tumors and two are from metastatic ovarian tumors. In each case, the number of restriction enzyme sections in the insert was minimized to generate a full length subtraction library. Subtraction was each performed using a slightly different protocol, as described in more detail below.

(A. POTS 2 library: primary ovarian tumor subtraction library)
Tracer: primary ovarian tumor library 10 μg, Not I digested driver: normal pancreas 35 μg in pcDNA3.1 (+)
20 μg normal PBMC in pcDNA3.1 (+)
10 μg normal skin in pcDNA3.1 (+)
35 μg normal bone marrow in pZErOTM-2
Digested with Bam HI / Xho I / Sca I.

  Two hybridizations were performed and Not I-cleaved pcDNA3.1 (+) was the cloning vector for the subtracted library. The sequence results for previously unidentified clones picked at random from the subtracted library are shown in Table II.

（Ｂ．ＰＯＴＳ ７ライブラリー：原発性卵巣腫瘍サブトラクションライブラリー）
トレーサー：原発性卵巣腫瘍ライブラリー１０μｇ、Ｎｏｔ Ｉで消化した
ドライバー：ｐｃＤＮＡ３．１（＋）中の正常膵臓３５μｇ
ｐｃＤＮＡ３．１（＋）中の正常ＰＢＭＣ２０μｇ
ｐｃＤＮＡ３．１（＋）中の正常皮膚１０μｇ
ｐＺＥｒＯＴＭ−２中の正常骨髄３５μｇ
Ｂａｍ ＨＩ／Ｘｈｏ Ｉ／Ｓｃａ Ｉで消化した
約２５μｇのｐＺＥｒＯＴＭ−２、Ｂａｍ ＨＩおよびＸｈｏ Ｉ で消化した。

(B. POTS 7 library: primary ovarian tumor subtraction library)
Tracer: primary ovarian tumor library 10 μg, Not I digested driver: normal pancreas 35 μg in pcDNA3.1 (+)
20 μg normal PBMC in pcDNA3.1 (+)
10 μg normal skin in pcDNA3.1 (+)
35 μg normal bone marrow in pZErOTM-2
Digested with Bam HI / Xho I / Sca I
Digested with approximately 25 μg of pZErOTM-2, Bam HI and Xho I.

  Two hybridizations were performed and Not I-cleaved pcDNA3.1 (+) was the cloning vector for the subtracted library. The sequence results for previously unidentified clones randomly picked from the subtracted library are shown in Table III.

（Ｃ．ＯＳ１Ｄライブラリー：転移性卵巣腫瘍サブトラクションライブラリー）
トレーサー：ｐＺＥｒＯＴＭ−２中の転移性卵巣ライブラリー１０μｇ、 Ｎｏｔ Ｉで消化した
ドライバー：ｐｃＤＮＡ３．１中の正常膵臓２４．５μｇ
ｐｃＤＮＡ３．１中の正常ＰＢＭＣ１４μｇ
ｐｃＤＮＡ３．１中の正常皮膚１４μｇ
ｐＺＥｒＯＴＭ−２中の正常骨髄２４．５μｇ
ｐＺＥｒＯＴＭ−２ ５０μｇ、Ｂａｍ ＨＩ／Ｘｈｏ Ｉ／ Ｓｆｕ Ｉで消化した。

(C. OS1D library: metastatic ovarian tumor subtraction library)
Tracer: 10 μg of metastatic ovary library in pZErOTM-2, driver digested with Not I: 24.5 μg of normal pancreas in pcDNA3.1
14 μg normal PBMC in pcDNA3.1
14 μg of normal skin in pcDNA3.1
24.5 μg normal bone marrow in pZErOTM-2
pZErOTM-2 50 μg, digested with Bam HI / Xho I / Sfu I.

  Three hybridizations were performed and the last two hybridizations were performed with an additional 15 μg biotinylated pZErOTM-2 to remove contaminating pZErOTM-2 vector. The cloning vector for the subtracted library was pcDNA3.1 / Not I cut. The sequence results for previously unidentified clones randomly picked from the subtracted library are shown in Table IV.

（Ｄ．ＯＳ１Ｆライブラリー：転移性卵巣腫瘍サブトラクションライブラリー）
トレーサー：転移性卵巣腫瘍ライブラリー１０μｇ、Ｎｏｔ Ｉで消化した
ドライバー：ｐｃＤＮＡ３．１中の正常膵臓１２．８μｇ
ｐｃＤＮＡ３．１中の正常ＰＢＭＣ７．３μｇ
ｐｃＤＮＡ３．１中の正常皮膚７．３μｇ
ｐＺＥｒＯＴＭ−２中の正常骨髄１２．８μｇ
ｐＺＥｒＯＴＭ−２ ２５μｇ、Ｂａｍ ＨＩ／Ｘｈｏ Ｉ／ Ｓｆｕ Ｉで消化した。

(D. OS1F library: metastatic ovarian tumor subtraction library)
Tracer: metastatic ovarian tumor library 10 μg, Not I digested driver: normal pancreas 12.8 μg in pcDNA3.1
7.3 μg normal PBMC in pcDNA3.1
7.3 μg normal skin in pcDNA3.1
12.8 μg normal bone marrow in pZErOTM-2
pZErOTM-2 25 μg, digested with Bam HI / Xho I / Sfu I.

  One hybridization was performed. The cloning vector for the subtracted library was pcDNA3.1 / Not I cut. The sequence results for previously unidentified clones picked at random from the subtracted library are shown in Table V.

公知の配列に対応する表ＶＩ中の配列もまた、上記ライブラリー中で同定された。

The sequences in Table VI corresponding to the known sequences were also identified in the library.

上記ライブラリーより同定されたなおさらなる卵巣癌のポリヌクレオチドおよび／またはポリペプチドの配列を、以下の表ＶＩＩに提供する。配列Ｏ５７４Ｓ（配列番号１８３および１８５）、Ｏ５８４Ｓ（配列番号１９３）およびＯ５８５Ｓ（配列番号１９４）は、新規配列を示す。残りの配列は、既知のゲノム配列および／またはＥＳＴ配列と少なくともいくらかの相同性を示した。

The sequences of still further ovarian cancer polynucleotides and / or polypeptides identified from the library are provided in Table VII below. The sequences O574S (SEQ ID NO: 183 and 185), O584S (SEQ ID NO: 193) and O585S (SEQ ID NO: 194) represent the novel sequence. The remaining sequences showed at least some homology with known genomic and / or EST sequences.

配列番号１８２（Ｏ５７３Ｓともいわれる）のクローンに対するさらなる研究は、配列番号２００〜２０２のアミノ酸配列をコードする複数のオープンリーディングフレームの同定を導いた。

Further work on the clone of SEQ ID NO: 182 (also referred to as O573S) led to the identification of multiple open reading frames encoding the amino acid sequences of SEQ ID NOs: 200-202.

(Example 2: Analysis of cDNA expression using microarray technology)
In further studies, the sequences disclosed herein were found to be overexpressed in certain tumor tissues as determined by microarray analysis. Using this approach, cDNA sequences are PCR amplified, and their mRNA expression profiles in tumor and normal tissues are essentially described using cDNA microarray technology as described (Shena et al., 1995). test. Briefly, clones are aligned on a glass slide as multiple replicas at each position corresponding to a unique cDNA clone (as many as 5500 clones can be aligned on a single slide or chip). . Each chip is hybridized with a pair of cDNA probes fluorescently labeled with Cy3 and Cy5, respectively. Typically, 1 μg of poly A + RNA is used to generate each cDNA probe. After hybridization, the chip is scanned and the fluorescence intensity is recorded for both the Cy3 and Cy5 channels. There are multiple built-in quality control processes. First, the quality of the probe is monitored using a panel of ubiquitously expressed genes. Second, the control plate can also contain yeast DNA fragments, and the complementary RNA of this yeast DNA fragment can be spiked into probe synthesis to measure probe quality and analytical sensitivity. Currently, this technique shows a sensitivity of 1 in 100,000 copies of mRNA. Finally, the reproducibility of this technique can be confirmed by including duplicate control cDNA elements at different positions.

  The microarray results for clones 57885 (SEQ ID NO: 197), 57886 (SEQ ID NO: 198) and 57887 (SEQ ID NO: 199) are as follows.

  The ovarian tumor of clone 57885: 16/38 (42%) showed an expression signal value of> 0.4. The average value for all ovarian tumors is 0.662 (the average value for all normal tissues is 0.187) and 3.64 times overexpression in ovarian tumors compared to essential normal tissues Bring a level. Normal tissue expression was elevated in the peritoneum, skin and thymus (> 0.4).

  The ovarian tumor of clone 57886: 16/38 (42%) showed an expression signal value of> 0.4. The average value for all ovarian tumors is 0.574 (the average value for all normal tissues is 0.166) and 3.46 times overexpression in ovarian tumors compared to essential normal tissues Bring a level. Normal tissue expression was elevated in the heart, pancreas and small intestine (> 0.4).

  The ovarian tumor of clone 57887: 17/38 (44%) showed an expression signal value of> 0.4. The average value for all ovarian tumors is 0.744 (average value for all normal tissues is 0.184), 4.04 times overexpression in ovarian tumors compared to essential normal tissues Bring a level. Normal tissue expression was elevated in the esophagus (> 0.4).

(Example 3: Expression of recombinant antigen O568S in E. coli)
This example is described in E.I. The expression of recombinant antigen O568S (SEQ ID NO: 177) in E. coli is described. This sequence was identified in Example 1 from the POTS 7 subtraction library using primary ovarian tumor cDNA as a tracer. PCR primers specific for the open reading frame of O568S were designed and used in the specific amplification of O568S. The PCR product was enzymatically digested with EcoRI and ligated into pPDM, a modified pET28 vector that had been cut with the restriction enzymes EcoRI and Eco72I. The sequence and orientation of this construct was confirmed via sequence analysis. This sequence is shown in SEQ ID NO: 206. This vector was then transformed into pLysS of the expression hosts BLR (DE3) and HMS174 (DE3). Protein expression was confirmed. This sequence is provided in SEQ ID NO: 207.

Example 4: Further sequences obtained for clone O591S
The sequence of O591S (clone identifier 57887) was used to search the public sequence database. It was found that the reverse strand showed some degree of identity to the C-terminus of GPR39. The cDNA for the coding region of GPR39 is disclosed in SEQ ID NO: 208 and the corresponding amino acid sequence is disclosed in SEQ ID NO: 209. The GPR39 coding region contains two exons. Both O591S and GPR39 encoded by the complementary strand of O591S are located on chromosome 2.

Example 5: Further characterization of O591S and identification of extended sequences
O1034C is an ovary-specific gene that has been identified by electronic subtraction. Briefly, electronic subtraction involves analyzing EST database sequences to identify ovary-specific genes. In the electronic subtraction method used to identify O1034C, the sequences of EST clones derived from ovarian libraries (normal and tumor) were obtained from the GenBank public human EST database. Each ovarian sequence was used as a “seed” query in a BLASTN search of the total human EST database to identify other EST clones (clones potentially derived from the same mRNA) that shared sequence with the seed sequence. EST clones with shared sequences were grouped into clusters, and clusters that shared sequences with other clusters were grouped into superclusters. The tissue source of each EST within each supercluster was noted and the superclusters were ranked based on the tissue distribution from which the EST was derived. Superclusters containing EST clones from the ovary library predominantly or alone were considered to represent genes that were differentially expressed in ovarian tissues compared to all other normal adult tissues.

  This clone was identified from the public EST database as Integrated Molecular Analysis of Genomics and the Expression (IMAGE) clone number 595449 (IMAGE Association is the depositary of EST clones and cDNA clones). This clone is disclosed as SEQ ID NO: 210. Accession number AA173737 and accession number AA173383 represent the sequences of ESTs identified in Genebank. This clone was identified as Unigene cluster HS. (Unigene is an experimental system for automatically distributing Genbank sequences into a non-overlapping set of clusters to which genes are directed). And this clone was annotated as encoding a neurotensin-like G protein coupled receptor (GRP39). However, the inventors have found that IMAGE # 595449 encodes a novel protein derived from the complementary strand to the strand encoding potential GPR39.

  Microarray analysis of a clone using a series of ovarian tumor-specific probes showed that this clone was 4.95 times overexpressed in the ovarian tumor sample and normal ovary sample groups compared to the essential normal tissue sample group. It showed that.

  An electronic full-length clone contig (O1034C) was created by subjecting IMAGE # 59449 to the EST database and Genbank's Blast A search and extending IMAGE # 595449 and its resulting contigs to completion. This process was repeated and completed when no further EST sequences were identified when extending the consensus sequence. This electronically derived clone was identified as encoding the previously described clone O591S (this sequence is disclosed in SEQ ID NO: 211). The discovery of this ovary specific candidate is described in more detail in Example 4.

  The consensus sequence for O1034C extended 5 'further than O591S due to additional sequences derived from two EST clones (registration number BF345141 and accession number BE336607). The sequences for the two EST clones are disclosed in SEQ ID NO: 212 and SEQ ID NO: 213, respectively. Although BF345141 differs from the O1034C / O591S consensus at its 3 'end (possibly representing a different splice form) and BE336607 at several bases at its 5' end, the two ESTs are available. , Compared to matching chromosomal sequences. These were found in human chromosome 2 clone RP11-159N20: htgs database accession number AC010974. These sequences were used to extend O1034C / O591S to form the final consensus sequence of 1897 base pairs for O1034C / O591S (disclosed in SEQ ID NO: 214).

  An open reading frame (ORF) was identified within the O1034C / O591S consensus sequence (nucleotides 260-682). This putative translation is disclosed in SEQ ID NO: 215. A BLASTx database search against the Genbank database showed that this ORF is not identical to any known human protein (E value <1e-25). The only match was with the G protein coupled receptors (including GPR39) which were shown by the inventors to be encoded at the 3 'end of O1034C / O591S in the complementary strand. However, this ORF encodes a protein with 93% similarity (131/141 amino acids) and 91% identity (129/141 amino acids) to the anonymous mouse product (registration # BAA95101), which means that It was suggested to be a genuine translation product representing a novel human ovary-specific antigen.

  The novelty of O1034C / O591S was confirmed by Northern blot analysis using a single stranded probe complementary to either GRP39 or O1034C / O591S. The strand-specific O1034C / O591S probe hybridized specifically to ovarian tumor samples probed on Northern blots, while all samples were negative when probed with GPR39. In addition, real-time PCR was performed using primers specific for either GPR39 or O1034C / O591S. These results further demonstrated the differential expression profile of these two sequences. This protein is a putative membrane protein as determined by Corixa's Tmpred protein estimation algorithm.

Example 6: Expression analysis and further characterization of ovarian sequence O568S
The ovarian sequence O568S was first identified as cDNA clone 24742 (SEQ ID NO: 118). Using clone 24742 as a query sequence to search the public sequence database, this sequence was found to have a high degree of homology with KIAA0762 (SEQ ID NO: 177) and VSGF. The DNA sequence for VSGF is provided in SEQ ID NO: 184 and the VSGF protein sequence is provided in SEQ ID NO: 186.

  Real-time PCR (see Gibson et al., Genome Research 6: 995-1001, 1996; Heid et al., Genome Research 6: 986-994, 1996) is a technique for assessing the level of PCR product accumulation during amplification. . This technique allows quantitative assessment of mRNA levels in multiple samples. Briefly, mRNA is extracted from tumor tissue and normal tissue, and cDNA is prepared using standard techniques. Real-time PCR is performed, for example, using a Perkin Elmer / Applied Biosystems (Foster City, Calif.) 7700 Prism instrument. Matching primers and fluorescent probes are designed for the gene of interest using, for example, a primer expression program (provided by Perkin Elmer / Applied Biosystems (Foster City, Calif.)). Optimal concentrations of primers and probes are initially determined by those skilled in the art, and control (eg, β-actin) primers and probes are commercially available from, for example, Perkin Elmer / Applied Biosystems (Foster City, Calif.). In order to quantify the amount of specific RNA in the sample, a standard curve is generated using a plasmid containing the gene of interest. A standard curve is generated using Ct values determined in real-time PCR. The Ct value is related to the initial cDNA concentration used in this assay. Standard dilutions of the gene of interest ranging from 10 to 106 copies are generally sufficient. In addition, a standard curve is generated for the control sequence. This allows the normalization of the initial RNA content of the tissue sample relative to the amount of control for comparison purposes.

  By real-time PCR analysis, O568 was highly overexpressed in the majority of ovarian tumors and ovarian tumor metastases tested compared to normal ovarian tissue and compared to a number of normal tissue panels. Expression is normal esophagus, spinal cord, bladder, colon, liver, PBMC (activated or paused), lung, skin, small intestine, stomach, skeletal muscle, pancreas, dendritic cells, heart, spleen, bone marrow, thyroid, trachea, Little or no observation was found in the thymus, bronchus, cerebellum, ureter, uterus and peritoneal epithelium. Some low level expression was observed in normal breast, brain, bone, kidney, adrenal gland and salivary glands, but expression levels in these normal tissues are generally higher than those observed in ovarian tumors overexpressing O568S. Was at least several times lower.

  In addition, a series of Northern blots were performed, which also demonstrated that the ORF region of O568S was specifically overexpressed in ovarian tumors. The first blot contained a series of normal tissue-derived RNA as well as ovarian tumor-derived RNA. This blot was probed with O568S-derived DNA corresponding to the 3'UTR of the VSGF sequence disclosed in SEQ ID NO: 184 as a labeled probe. This blot revealed an ovarian tumor-specific 5.0 Kb message as well as a potential 3.5 Kb brain-specific message and a ubiquitously expressed 1.35 Kb message.

  Another Northern blot was performed with RNA from a number of different brain tissues and probed with the 3'UTR region described above. Five brain samples out of 11 showed 3.5 Kb message overexpression. To determine whether the O568S ORF region was specifically overexpressed in ovarian tumors, a series of three blots were performed using three separate probes designed from within the O568S VSGF ORF. The results from these experiments clearly showed that only a 5.0 Kb message was expressed in ovarian tumors.

(Example 7: Synthesis of polypeptide)
Polypeptides were synthesized on a Perkin Elmer / Applied Biosystems Division 430A peptide synthesizer using FMOC chemistry with HPTU (O-benzotriazole-N, N, N ′, N′-tetramethyluronium hexafluorophosphate) activation. Synthesize. A Gly-Cys-Gly sequence is attached to the amino terminus of a peptide to provide a conjugation method, a method for binding a peptide to an immobilized surface, or a method for labeling a peptide. Cleavage of the peptide from the solid support is carried out using the following cleavage mixture: trifluoroacetic acid: ethanedithiol: thioanisole: water: phenol (40: 1: 2: 2: 3). After cleavage for 2 hours, the peptide is precipitated in cold methyl-t-butyl-ether. The peptide pellet is then dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized before being purified by C18 reverse phase HPLC. The peptide is eluted using a gradient of 0% to 60% acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA). After lyophilization of the pure fractions, the peptides are characterized using electrospray or other types of mass spectrometry and by amino acid analysis.

(Example 8: Northern blot analysis of O568S)
As described in Example 6, Northern blot analysis demonstrated that the ORF region of O568S was specifically overexpressed in ovarian tumors. The original probe used corresponded to the 3′UTR of the VSGF sequence disclosed in SEQ ID NO: 184. Results from these Northern blots revealed an ovarian tumor-specific 5.0 Kb message as well as a potential 3.5 Kb brain-specific message. Two additional probes were designed to confirm that the entire area covered by the ORF produced a single 5.0 Kb ovarian tumor-specific message.
These probes were located in the 5 ′ and 3 ′ regions of this ORF. Northern blot analysis using these two probes demonstrated that both probes hybridize to a 5.0 Kb product present only in ovarian tumor samples. Both probes did not hybridize to RNA derived from multiple brain samples.

  Example 9 Real-Time PCR and Northern Blot Analysis of O590S Real-time PCR analysis of the ovarian tumor antigen O590S was performed essentially as described in Example 6. O590S specific primers and probes were designed and quantitative real-time PCR was performed on a panel of cDNA and a panel of normal tissue prepared from various tissues including ovarian tumor samples. This analysis shows that O590S specific mRNA is approximately 65% of ovarian tumor samples tested, 100% of tumor samples derived from SCID mice, and of ovarian tumor cell lines tested, when compared to normal ovarian tissue. It was shown to be overexpressed at 100%. No detectable expression was observed in normal tissues.

  In addition to real-time PCR, Northern blot analysis was performed to determine the size of the O590S transcript. Northern blots were probed with a 537 bp PCR product specific for O590S. This PCR product was designed to avoid regions of repetitive sequences. This probe revealed a blurred band with a size of about 9.0 Kb. This band was present in the majority of ovarian tumor samples tested.

  While particular embodiments of the present invention have been described herein for purposes of illustration, it will be appreciated from the foregoing that various modifications can be made without departing from the spirit and scope of the invention. Is done. Accordingly, the invention is not limited except as by the appended claims.

Claims (18)

An isolated polypeptide comprising:
(A) the sequence provided in SEQ ID NOs: 215, 200-202, 207 and 209;
(B) a sequence having at least 70% identity to the sequence provided in SEQ ID NOs: 215, 200-202, 207 and 209; and (c) provided in SEQ ID NOs: 215, 200-202, 207 and 209 A sequence having at least 90% identity to the sequence being determined;
A polypeptide comprising an amino acid sequence of an ovarian tumor protein selected from the group consisting of: An isolated polynucleotide comprising:
(A) the sequences provided in SEQ ID NOs: 214, 203-206, 208 and 210-213;
(B) the complement of the sequence provided in SEQ ID NOs: 214, 203-206, 208 and 210-213;
(C) a sequence consisting of at least 20 consecutive residues of the sequences provided in SEQ ID NOs: 214, 203-206, 208 and 210-213;
(D) a sequence that hybridizes under moderately stringent conditions to the sequences provided in SEQ ID NOs: 214, 203-206, 208, and 210-213;
(E) a sequence having at least 75% identity to the sequences provided in SEQ ID NOs: 214, 203-206, 208 and 210-213;
(F) a sequence having at least 90% identity to the sequences provided in SEQ ID NOs 214, 203-206, 208 and 210-213; and (g) SEQ ID NOs 214, 203-206, 208 and 210- 213, a degenerate variant of the sequence provided in 213;
A polynucleotide comprising a sequence selected from the group consisting of: An isolated polypeptide comprising:
(A) a sequence encoded by the polynucleotide of claim 2;
(B) a sequence having at least 70% identity to a sequence encoded by the polynucleotide of claim 2; and (c) for a sequence encoded by the polynucleotide of claim 2. A polypeptide comprising an amino acid sequence of an ovarian tumor protein selected from the group consisting of sequences having at least 90% identity. An expression vector comprising the polynucleotide of claim 2 operably linked to an expression control sequence. A host cell transformed or transfected with the expression vector of claim 4. An isolated antibody, or antigen-binding fragment thereof, that specifically binds to the polypeptide of claim 1 or 3. A method for detecting the presence of ovarian cancer in a patient comprising the following steps:
(A) obtaining a biological sample from the patient;
(B) contacting the biological sample with a binding agent that binds to the polypeptide of claim 1 or claim 3;
(C) detecting in the sample the amount of polypeptide that binds to the binding agent; and (d) comparing the amount of the polypeptide to a predetermined cut-off value, thereby causing cancer in the patient Determining the presence of;
Including the method. A fusion protein comprising at least one polypeptide according to claim 1 or claim 3. An oligonucleotide that hybridizes under moderately stringent conditions to the sequences provided in SEQ ID NOs: 214, 203-206, 208, and 210-213. A method for stimulating and / or expanding T cells specific for an oncoprotein, wherein the method is under conditions and for a time sufficient to allow the T cells to be stimulated and / or expanded,
Less than:
(A) the polypeptide of claim 1 or claim 3;
(B) a polynucleotide according to claim 2; and (c) at least one component selected from the group consisting of antigen-presenting cells that expresses the polypeptide according to claim 1 or claim 3 and a T cell. A method comprising the step of contacting. 11. An isolated T cell population comprising T cells prepared according to the method of claim 10. A composition comprising a first component selected from the group consisting of a physiologically acceptable carrier and an immunostimulant, and a second component comprising:
Wherein the second component is:
(A) the polypeptide of claim 1 or claim 3;
(B) the polynucleotide of claim 2;
(C) the antibody according to claim 6;
(D) the fusion protein of claim 8; and (e) an antigen-presenting cell that expresses the polypeptide of claim 2 or claim 3;
A composition selected from the group consisting of: A method of stimulating an immune response in a patient comprising:
13. A method comprising administering the composition of claim 12 to the patient. 13. A method for the treatment of cancer in a patient, the method comprising administering to the patient the composition of claim 12. A method for determining the presence of cancer in a patient comprising the following steps:
(A) obtaining a biological sample from the patient;
(B) contacting the biological sample with the oligonucleotide according to claim 9;
(C) detecting the amount of polynucleotide hybridized to the oligonucleotide in the sample; and (d) comparing the amount of oligonucleotide hybridized to the oligonucleotide with a predetermined cutoff value. , Thereby determining the presence of cancer in the patient;
Including the method. A diagnostic kit comprising at least one oligonucleotide according to claim 9. A diagnostic kit comprising at least one antibody according to claim 6 and a detection reagent, wherein the detection reagent comprises a reporter group. A method of inhibiting the development of cancer in a patient comprising the following steps:
(A) (i) a polypeptide according to claim 1 or claim 3; (ii) a polynucleotide according to claim 2; and (iii) a polypeptide according to claim 1 or claim 3. Incubating CD4 + T cells and / or CD8 + T cells isolated from a patient with at least one component selected from the group consisting of antigen presenting cells, so that T cells proliferate;
(B) administering to the patient an effective amount of the expanded T cells and thereby inhibiting the development of cancer in the patient;
Including the method.