Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
1995-06-09
2001-10-09
Mertz, Prema (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C424S085400, C424S085500, C424S085600, C424S085700, C514S002600, C514S008100, C514S012200, C435S069510, C435S071200, C435S325000, C435S252300, C435S320100, C435S471000
Reexamination Certificate
active
06300474
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of biotechnology and more particularly to polypeptides having antitumor, antiviral, immunomodulatory and other activities, DNA that codes for such polypeptides, recombinant vectors that include such DNA host organisms transformed with the recombinant vector that produces the polypeptide and therapeutic application of the polypeptide.
The publications and other materials hereof used to illuminate the background of the invention, and in particular cases, to provide additional details respecting its practice are hereby incorporated by reference, and for convenience are numerically referenced by the following text and respectively grouped in the appended bibliography.
BACKGROUND OF THE INVENTION
Human leukocyte interferon was first discovered and prepared in the form of very crude fractions by Isaacs and Lindenmann (1, 2). Efforts to purify and characterize the material have led to the preparation of relatively homogeneous leukocyte interferons derived from normal or leukemic (chronic myelogenous leukemia or “CML”) donors leukocytes These interferons are a family of proteins characterized by a potent ability to confer a virus-resistant state in their target cells. In addition, interferon can inhibit cell proliferation, modulate immune responses and alter expression of proteins. These properties have prompted the clinical use of leukocyte interferon as a therapeutic agent for the treatment of viral infections and malignancies.
With the advent of recombinant DNA technology, the controlled microbial production of an enormous variety of useful polypeptides has become possible. The workhorse of recombinant DNA technology is the plasmid, a non-chromosomal circle of double-stranded DNA found in bacteria and other microbes, oftentimes in multiple copies per cell. Included in the information encoded in the plasmid DNA is that required to reproduce the plasmid (i.e., an origin of replication) and ordinarily, one or more selection characteristics such as, in the case of bacteria, resistance to antibiotics which permit clones of the host cell containing the plasmid of interest to be recognized and preferentially grown in selective media. The utility of plasmids lies in the fact that they can be specifically cleaved by one or another restriction endonuclease or “restriction enzyme,” each of which recognizes a specific site in the DNA. Heterologous genes or gene fragments may be inserted into the plasmid at the cleavage site. To construct vectors with specific sequences inserted, DNA recombination is performed outside the cell, but the resulting “recombinant” plasmid can be introduced into cells by a process known as transformation and large quantities of the heterologous gene-containing recombinant plasmid obtained by growing the transformant. Moreover, where a promoter which governs the transcription of the encoded DNA message, is properly placed upstream (5′) of a coding sequence or a gene, the resulting expression vector can be used to produce the polypeptide sequence for which the inserted sequence or gene codes, a process referred to as expression.
Expression is initiated in a region known as the promoter which is recognized by and bound by RNA polymerase. In many cases promoter regions are overlapped by “control” regions such as the bacterial operators. Operators are DNA sequences which are recognized by so-called repressor proteins which serve to regulate the frequency of transcription initiation at a particular promoter. The polymerase travels along the DNA, transcribing the information contained in the coding strand from its 5′ to 3′ end into messenger RNA (mRNA) which is in turn translated into a polypeptide having the amino acid sequence for which the DNA codes. Each amino acid is encoded by a nucleotide triplet or “codon” within the coding sequence, i.e., that part which encodes the amino acid sequence of the expressed product. In bacteria (e.g.
Escherichia coli
) the mRNA contains a ribosome binding site, a translation initiation or “start” signal (ordinarily ATG in the DNA, which in the resulting mRNA becomes AUG), the nucleotide codons within the coding sequence itself, one or more stop codons, and an additional sequence of messenger RNA, the 3′ untranslated region. Ribosomes bind to the binding site provided on the messenger RNA, in bacteria ordinarily as the mRNA is formed, and produce the encoded polypeptide, beginning at the translation start signal and ending at the stop signal. The desired product is produced if the sequences encoding the ribosome binding site are positioned properly with respect to the AUG initiator codon and if all remaining codons follow the initiator codon in phase. The resulting product may be obtained from the host cell and recovered by appropriate purification. In other systems, proteins may be secreted from the host cells. A wide variety of expression vectors and host systems exist so that RNA and proteins may be expressed in prokaryotic and eukaryotic cells as well as in intact animals and plants.
During the past several decades a large number of human and animal interferons have been produced, identified, purified and cloned (see ref. 1-72). Several of the interferon preparations have been prepared for clinical trial in both crude form, for some of the original interferon preparations, as well as in purified form. Several individual recombinant interferon-&agr; species have been cloned and expressed. The proteins have then been purified by various procedures and formulated for clinical use in a variety of formulations (
73
). Most of the interferons in clinical use that have been approved by various regulatory agencies throughout the world are mixtures or individual species of human &agr; interferon (Hu-IFN-&agr;). In some countries Hu-IFN-&bgr; and &ggr; have also been approved for clinical trial and in some cases approved for therapeutic use (56,74). The major thesis underlying clinical use of these interferons was that they were natural molecules produced by normal individuals. Indeed, the specific thesis was that all the interferons prepared for clinical use, be they natural- or recombinant-generated products, represented interferons that were produced naturally by normal people. This is true for a large number of interferons as well as specific growth factors. lymphokines, cytokines, hormones, clotting factors and other proteins that have been produced (17, 21, 22, 25-27, 29-34, 39, 40, 45-51, 53-57, 62-64, 68-72).
Reports have suggested that Hu-IFN-&agr;A (also designated Hu-IFN-&agr;2a and by the trade name Roferon A) was not represented in interferons produced by a normal population of individuals (75-79). Believing that certain interferons (or, more generically, certain polypeptides) are uniquely found in diseased cells, the inventor of the present invention undertook to identify interferons which are so uniquely characterized. For convenience the inventor began by screening known interferons, in particular, the sources of the several variants of Hu-IFN-&agr;2 that have been described. As discussed more fully below, it was found that the source of two of the variants of Hu-IFN-&agr;2, Hu-IFN-&agr;2a and Hu-IFN-&agr;2c, are not present in normal individuals. Only Hu-IFN-&agr;2b is found in normal individuals (79).
DESCRIPTION OF THE DRAWINGS
FIG.
1
. Nucleotide and Amino Acid Sequence of Hu-IFN-&agr;001 (SEQ ID NOS: 1 and 2). The location of the AlwNI site is underlined. The signal peptide is shown as the 23 amino acids labeled −1 to −23.
FIG.
2
. Comparison of the Protein Sequence of Hu-IFN-&agr;001 (SEQ ID NO: 2)with that of Hu-IFN-&agr;J (SEQ ID NO: 3). The signal peptide represents the first 23 amino acids at the amino terminus.
FIG.
3
. Expression vector for Hu-IFN-&agr;001. The structure of the plasmid pHu-IFN-&agr;001 is shown. The NsiI site represents nucleotide position #1. The P
R
promoter drives expression of Hu-IFN-&agr;001.
FIG.
4
. SDS-Polyacrylamide Gel Electrophoresis of the Purified Hu-IFN-&agr;001. Hu-IFN-&agr;001 wa
Mertz Prema
PBL Biomedical Laboratories
Ropes & Gray
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