Nucleic acid and amino acid sequences

Details Nucleic acid and amino acid sequences Nucleic acid and amino acid sequences

2001-01-08

2004-05-25

Caputa, Anthony C. (Department: 1642)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S069100, C435S320100, C435S325000, C536S023500

Reexamination Certificate

active

06740516

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns novel nucleic acid sequences, vectors and host cells containing them, amino acid sequences encoded by said sequences, and antibodies reactive with said amino acid sequences, as well as pharmaceutical compositions comprising any of the above. The present invention further concerns methods for screening for candidate activator or deactivators utilizing said amino acid sequences.
BACKGROUND OF THE INVENTION
Prostate-specific antigen (PSA) is the most important tumor marker for early detection, staging, and monitoring of men with prostate cancer today. PSA testing has appreciable false-positive and false-negative results, particularly in the 2.5-10 ng/ml range. Measurement of the percentage of non-protein-bound (i.e. free) PSA in serum, which is lower in patients with prostate cancer, have been evaluated as a method for increasing the accuracy of PSA testing.
Thus measurement of PSA in serum, has been postulated as having potential clinical utility for increasing the sensitivity and specificity of PSA testing. Cutoff figures are affected by total PSA levels at prostate value. The prevalence rate of cancer in the screened population, depending on age, race, previous biopsy history etc., also influences the screening cutoffs. It has also been postulated that the percentage of free PSA may also correlate with a potential aggressiveness of early-stage prostate cancer. Thus, the level of free PSA may not only be used in order to diagnose prostate cancer, but also to predict the course of development of this cancer, and the patient's prognosis, and decide on a suitable treatment regime.
Human kallikrein-2 gene (termed herein after: “KLK” which is also known as KLK-2) is transcribed from the same locus as the PSA and is also known to be prostate specific. It has been speculated that both PSA and KLK have common expression control such as common enhancer and/or promoter and both function as serine proteases.
GLOSSARY
In the following description and claims use will be made, at times, with a variety of terms, and the meaning of such terms as they should be construed in accordance with the invention is as follows:
“Prostate specific antigen (PSA) variant”—the sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6, sequences having at least 70% identity to said sequence and fragments of the above sequences of least 20 b.p. long. SEQ ID NO: 1 to ID NO:5 are nucleic acid sequences which resulted from alternative splicing of the native and known PSA sequence appearing in HSPSAR and HUMPSANTIG (GenBank Acc. X05332 and M24543, respectively). It should be emphasized that the PSA variants of the invention are naturally occurring sequences resulting from the alternative splicing of the RNA transcribed from the PSA gene and not merely truncated or mutated forms of the gene. SEQ ID NO: 6 is an alternative splice variant of the human kallikrein-2 gene (KLK-2) appearing in GenBank as KLK2 under Accession Number NM
—
005551.
SEQ ID NO: 1—(PSAL
—
0): The nucleic acid sequence starting in position 4364 of the HUMPSANTIG up to position 7305, then a different sequence. The coded peptide (SEQ ID NO:7) starting identically to the original PSA for 16 aa, then a different sequence which is trancribed from the PSA intron between exons 1 and 2.
SEQ ID NO:2—(PSAL
—
1): Nucleic acid sequence identical to SEQ 1.
Peptide (SEQ ID NO: 8)—Starting in a Methionine 114 aa upstream from the original PSA, and has the same 16 aa identity and 3′ end as PSAL
—
0.
SEQ ID NO:3—(PSAL
—
2): Nucleic acid sequence which starts in same place as PSAL
—
0 but goes up to position 6336 of the HUMPSANTIG, then continues in a different sequence. Peptide (SEQ ID NO: 9)—Identical to PSAL
—
1 peptide.
SEQ ID NO: 4—(PSAL
—
5): Nucleic acid sequence which starts in same place as PSAL
—
0, goes up to position 6069 of HUMPSANTIG and end there (original intron). Peptide (SEQ ID NO:10)—Has same starting place as PSAL
—
1, the same 16 aa identity to PSA, then a different intron region translated.
SEQ ID NO: 5—(PSAL
—
6): Nucleic acid sequence starts in the same place as PSAL
—
0, goes up to position 5913 of HUMPSANTIG, then enter the original PSA exon # 2 and continues. Peptide (SEQ ID NO:11) has same starting place as PSAL
—
1, then enters the same identity region and continues as the original PSA until the end.
SEQ ID NO:6 is a splice variant of the KLK-2 that includes coding region from the original KLK-2 intron between exons 1 and 2. The term of “PSA variant” in the context of the present invention concerns splice variants of the known PSA gene as well as splice variants of the KLK-2 gene, which is also known to code for antigens specific to the prostate.
“Prostate specific antigen variant product (PSA variant product)—also referred at times as the “PSA variant protein” or “PSA variant polypeptide”—an amino acid sequence coded by said PSA variant nucleic acid sequence. The amino acid sequence may be a peptide, a protein, as well as peptides or proteins having chemically modified amino acids (see below) such as a glycopeptide or glycoprotein. An example of a PSA variant product is shown in any one of SEQ ID NO: 7 to SEQ ID NO: 12, and includes also analogues of said sequences in which one or more amino acids has been added, deleted, substituted (see below) or chemically modified (see below) as well as fragments of this sequence having at least 10 amino acids. The products may be membrane associated or present in a free form in body fluids, for example in the serum.
“Nucleic acid sequence”—a sequence composed of DNA nucleotides, RNA nucleotides or a combination of both types and may includes natural nucleotides, chemically modified nucleotides and synthetic nucleotides.
“Amino acid sequence”—a sequence composed of any one of the 20 naturally appearing amino acids, amino acids which have been chemically modified (see below), or composed of synthetic amino acids.
“Fragment of PSA variant product” a sequence which is the same as part of but not all of the amino acid sequence of the PSA variant product.
“Fragments of PSA variant nucleic acid sequence” a continuous portion, preferably of about 20 nucleic acid sequences of the PSA variant nucleic acid sequence (see below), which sequence does not appear in the original PSA.
“Conservative substitution”—refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. [Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gln, or Glu, is a conservative substitution.
“Non-conservative substitution”—refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gln.
“Chemically modified”—when referring to the product of the invention, means a product (protein) where at least one of its amino acid resides is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art. Among the numerous known modifications typical, but not exclusive examples include: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.
“Biologically active”—refers to a PSA variant product which has the ability to serve as a marker of cancer, of predisposition to cancer, or of malig

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