Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Phosphoproteins – e.g. – phosvitin – vitellogenin – etc.
Reexamination Certificate
2000-11-28
2004-06-08
Mertz, Prema (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Phosphoproteins, e.g., phosvitin, vitellogenin, etc.
C435S069510, C435S069700, C435S325000, C435S471000, C435S252300, C435S320100, C435S071200, C424S001410, C536S023400, C536S023520, C530S351000, C530S352000, C530S399000
Reexamination Certificate
active
06747131
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of recombinant DNA technology, to means, methods of utilizing this technology to synthesize useful functional proteins or polypeptides which include one or more phosphate (or thiophosphate) groups which are radio-labelled, to these and various other products useful in biomedical, medical, biochemical applications including diagnostics, prophylatics and therapeutics.
More specifically the invention relates to new interferons, especially to leukocyte, or alpha interferon(s) and fibroblast or beta interferon which contain one or more radioactive phosphorylated groups, to DNA sequences encoding putative phosphorylatable sites, which code for these new interferons.
BACKGROUND OF THE INVENTION
Radio-labelled proteins have numerous medical, biological, clinical, scientific and other applications. Interferons, specifically, labelled with
125
I have been used for binding and crosslinking studies (1, 17, 27-32, 34-37).
1
Human IFN-&agr;'s, -&bgr;, and -gamma have all been radio-iodinated by various procedures (reviewed in Pestka et al (2)). However, proteins labelled with radioactive iodine have serious well-known disadvantages and hazards.
1
The scientific publications, patents or other literature (“publications”) to which reference is made herein are referenced by numerals and identified further towards the end of this text. All of these publications are incorporated herein by reference.
The study of cell surface receptors for the interferons requires radio-labelled interferons, such as interferons labelled with
125
I, with high biological and high radio-specific activity. Several years ago, it was found that interferon gamma
2
can be phosphorylated to very high radio-specific activity while retaining biological activity (3, 4). Thus, [
32
P]Hu- and Mu-IFN-gamma were used for studying the human and murine IFN-gamma receptors, respectively (5, 6, 9). These studies were carried out by phosphorylating human and murine interferon gamma (Hu- and Mu-IFN-gamma) with cyclic AMP-dependent protein kinase from bovine heart muscle and [gamma-
32
P]ATP (3). These phosphorylated and
32
P-labelled interferons have provided valuable reagents (3, 4) of high radio-specificity to study cell surface receptors (5, 6) and to identify the chromosome encoding the gene for Hu-IFN-gamma (7, 8) and Mu-IFN-gamma (9) receptors. For all of these studies and applications, interferons which are phosphorylated are most useful. Several reports identified the phosphorylation sites of Hu- and Mu-IFN-gamma as serine residues near the COOH termini (4, 5, 10, 11).
2
The abbreviations used have followed standard nomenclature as described in detail in Methods of Enzymology, Interferons, Vol. 119, Part C, Edited by Sidney Pestka, Section I, Introduction (Reference 25). In brief, interferon alpha, beta, and gamma are designated IFN-&agr;, IFN-&bgr;, and -gamma, respectively. The species of origin is designated by a prefix Hu, Mu, Bo, etc. for human, murine, or bovine species, respectively, as Hu-IFN-&agr;, Hu-IFN-&bgr;, or Hu-IFN-gamma, for example.
However, under conditions used for the phosphorylation of IFN-gamma, it was reported that Hu-IFN-&agr;A and Hu-IFN-&bgr; cannot be phosphorylated by the cyclic AMP (cAMP)-dependent protein kinase (2, 3). A review of the phosphorylation of the various classes or groups of interferons and other proteins (1, 3, 4, 20, 21, 22, 38, 39, 40, 64) confirms that researchers have not successfully phosphorylated Hu-IFN-&agr; or Hu-IFN-&bgr; under conditions under which gamma interferons have been phosphorylated. It has been reported indeed that recombinant IFN-&agr; and IFN-&bgr; were not phosphorylated (3) and as a consequence it was uncertain whether an available site was present.
In the light of problems with iodinated compounds and limitations for use of iodinated IFN-gamma, it is understandable that there is a keen interest and need in making available phosphorylated Hu-IFN-&agr; and -&bgr; which can be labelled for numerous practical, scientific and commercial applications.
Likewise, there is such interest and need for other phosphorylated—and labelled—polypeptides which are not available yet in such chemical configurations. For example, a phosphorylatable tumor necrosis factor (TNF) would be valuable to study the receptor for TNF. TNF is not phosphorylatable with the cAMP-dependent bovine heart kinase. Indeed, it has been reported that interest in protein phosphorylation has increased enormously over the past few years (38, 39).
The invention as will be described in detail hereinafter contributes to meeting these and other needs.
By way of further background to the invention, the term “interferon” describes a family of animal proteins which possess antiviral, antiproliferative and other potentially useful properties. There appear to be three major classes of interferons: leukocyte (or alpha interferon), fibroblast (or beta interferon) and immune (or gamma interferon) (1, 2). Detailed description of interferons is found in various publications including in references 1, 2, U.S. Pat. Nos. 4,727,138; 4,734,491; 4,737,462, and many others; various hybrid human leukocyte interferons are described in U.S. Pat. No. 4,414,150 and in reference 46. In general the standard class of human IFN-&agr;'s are polypeptides of 165-166 amino acids (see reference 1 for details of human and non-human interferon-&agr; species); some species have been isolated that lack the 10 COOH-terminal amino acid residues; and some species of IFN-&agr; are glycosylated. The amino acid sequences of Hu-IFN-&agr; species and of Hu-IFN-&bgr; derived from CDNA or genomic DNA sequences are described in (1, Section I). Recombinant DNA-derived interferons including Hu-IFN-&agr;, -&bgr;, and -gamma and corresponding interferons from other animal species are likewise well described (1, 2). Various modifications of human and murine interferons have been reported. New non-natural human and murine interferons with often markedly changed biological properties have been constructed (1, 24, 45). The terminology “non-natural” is a term of art which refers to recombinant DNA interferons obtained by altering the nucleotide sequence of coding cDNAs (45).
The term “Hu-IFN-&agr;” as used herein is intended to include all different species of alpha interferons. A large number of DNA sequences corresponding to the interferons from various species have been isolated and identified. Likewise various IFN-&bgr;s and IFN-gamma(s) are disclosed. The invention encompasses all of these members of the family (reference 1, pages 5-14).
The term “native” as used herein refers to the proteins, e.g., interferons, which proteins are naturally produced; “synthetic” and “non-natural” refers to proteins produced by synthetic or DNA-recombinant procedures, either type which do not contain a phosphorylatable site (or where the phosphorylatable site is inaccessible, for instance due to the configuration of the protein), which protein in accordance with the invention is to be phosphorylated.
This invention contemplates and includes all interferons native, natural, modified, or recombinant DNA interferon-like proteins which are modifiable by introduction of one or more phosphate or analog groups. All of these interferons and others known in the art or to be known are within the contemplation of the invention. The present invention is principally concerned with various modified proteins or polypeptides, and alpha and beta interferons.
When reference is made to IFN-alpha, the term is intended to cover and include the various alpha species.
The term “modified” is used in this invention broadly, and means for instance, when reference is made to proteins, a protein which has been provided with a phosphorylatable site or provided with a phosphorus label (or analog label). The nucleotide sequences which code for such amino acid sequences which contain a putative phosphorylation site are also designated as “modified”, when appropriate.
The term “unphosphorylatable” protein
Mertz Prema
Pestka Biomedical Laboratories, Inc.
Ropes & Gray LLP
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