Human procalcitonin and the preparation and use thereof

Details Human procalcitonin and the preparation and use thereof Human procalcitonin and the preparation and use thereof

2000-12-22

2003-05-13

Mosher, Mary E. (Department: 1648)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C435S007100, C435S188000, C435S967000, C436S501000, C530S333000, C930S060000

Reexamination Certificate

active

06562946

ABSTRACT:

The invention relates to human procalcitonin and the preparation, in particular by genetic engineering processes, and use thereof.
Procalcitonin (“pCT”) is a protein consisting of 116 amino acids and having a molecular weight of about 13,000 dalton. It is the prohormone of calcitonin which under normal metabolic conditions is produced and secreted by the C cells of the thyroid. pCT and calcitonin synthesis is initiated by translation of preprocalcitonin (“pre-pCT”), a precursor peptide comprising 141 amino acids. The amino acid sequence of human pre-pCT was described by Moullec et al. in FEBS Letters, 167:93-97 in 1984. pCT is formed after cleavage of the signal peptide (first 25 amino acids of pre-pCT). In healthy people the hormone calcitonin (amino acids 60-91 of the pCT amino acid sequence), and N-procalcitonin (amino acids 1-57 of the pCT amino acid sequence) and katacalcin (amino acids 96-116 of the pCT amino acid sequence) are produced intracellularly from pCT by specific proteolysis (see also Conlan et al. (1988) Biochem. J., 256:245-250). pCT and fragments thereof were detected in increased concentrations in the serum or plasma of patients, in particular in cases of certain neoplastic diseases (Ghillani et al. (1989) Cancer Research, 49:6845-6851) and sepsis (EP-B1-0 656 121) and SIRS (systemic inflammatory response syndrome) (Snider et al., (1997) J. Investig. Med., 45:552-560).
During the typical sepsis bacteria are released continuously or in phases from a focus into the bloodstream. Endotoxin or other pyrogenic and toxic substances interacting with body mechanisms cause the clinical manifestations. The acute onset triggers chills and in severe cases a shock reaction. Special forms of septic shock are Waterhouse-Friderichsen syndrome and toxic shock syndrome (TSS). TSS is known as an acute clinical picture in staphylococcal infections which is caused by a specific staphylococcal toxin. A severe sepsis quite frequently develops in patients with serious primary disorders such as, for example, neoplastic diseases, serious burns and traumas.
The importance for sepsis diagnosis of detecting pathogens in the blood (“positive blood culture, bacteremia”) has been pushed into the background, because in general the blood culture is positive only in 20 to 40% of sepsis cases. The term sepsis has therefore undergone a change. The modern term “sepsis” describes a clinical syndrome which in general comprises fever, leukocytosis, alterations of consciousness, a hyperdynamic circulation (“warm shock”) and a hypermetabolic state, a positive blood culture no longer being required as a prerequisite for sepsis diagnosis.
WO 98/33524 suggests employing antibodies binding to pCT for the therapy of sepsis and SIRS.
Over many years polyclonal antibodies were obtained from immunization by calcitonin and used for detecting so-called immunoreactive calcitonin which aside from calcitonin also comprises procalcitonin and further procalcitonin fragments. Immunization by synthetic peptides having amino acid sequences corresponding to the sequences of procalcitonin segments succeeded in producing various monoclonal antibodies binding to various calcitonin and katacalcin epitopes (Ghillani et al. (1988) J. Immunol., 141:3156-3163).
On the basis of these antibodies sandwich immunoassays for detecting pCT and calcitonin in serum samples were also developed. A combination of an anti-katacalcin antibody and an anti-calcitonin antibody was suggested for detecting calcitonin precursor molecules. A synthetic peptide suited to these antibodies was employed as standard material.
It is known that in immunochemical tests the measured signals for standards and samples need not necessarily be identical even if the amount of antigen is exactly the same. If standard and sample antigens are not really identical regarding their immunochemical reactivity, the antibodies employed in the test will recognize either the one or the other antigen better. This leads in the end to different measured signals for samples and standards.
It follows from this that the use as standard antigen of antigen fragments instead of the whole protein is often associated with disadvantages and can, in particular, lead to distorted measurements. Furthermore, it is in general not possible for the epitopes based on the three-dimensional structure of the correctly folded protein to be correctly represented by shorter peptides. This results in it not being possible to obtain antibodies against such conformation epitopes on use of peptides as immunogens. It is advantageous especially in competitive test formats if the substance to be detected has the same immunochemical reactivity as the corresponding solid phase or label-bound test reagent.
Although the complete amino acid sequence of human pCT has now been known since 1984, so far human pCT has not been prepared successfully, in particular not in relatively large amounts and reproducibly. So far only murine pCT could be expressed in
E. coli
by means of genetic engineering processes (Rehli et al. (1996) Biochem. Biophys. Res. Com., 226:420-425).
However, murine pCT differs from human pCT to such an extent (about 77% homology at the amino acid level) that it is still an object for the skilled worker to develop a process by which human pCT can be produced in relatively large amounts, cost effectively and in isolated form in order to be able to employ it particularly as an immunogen and/or standard and control sera antigen.
This object is achieved by providing the polypeptides according to the invention described in claims 1-3, the plasmids according to the invention described in claims 10 and 11, the cells according to the invention described in claim 12 and the preparation processes according to the invention described in claims 4-9. The polypeptides according to the invention, i.e. the polypeptides as claimed in one of claims 1-3 or the products of a process as claimed in claims 4-9, can be employed usefully in particular in the fields of diagnosis and therapy. Preferred embodiments of the invention are disclosed in claims 13-23. Furthermore, the polypeptides according to the invention can be used for immunization to obtain the antibodies according to the invention. A further embodiment of the invention is the pCT solutions described in claims 24-29.
It was not possible to predict the feasibility of human pCT expression according to the invention: pCT is expressed in the cell not as pCT, but originates from preprocalcitonin by proteolytic cleavage of the N-terminal signal peptide. It had to be assumed that in eukaryotes pCT without signal peptide is not expressed in the natural cell compartment and folds in a different way, potentially causing biological inactivation and possibly even instability. In addition, the heterologous expression in
E. coli
instead of the natural expression in animal cells and the expression attempted within the framework of the invention of a fusion protein of procalcitonin and the artificial sequence MRSHHHHHHGS (part of SEQ ID NO: 9) N-terminally therefrom could not be foreseen as prospectively successful. Although expression in
E. coli
of murine pCT as a poly-His fusion protein has been described in the literature (Rehli et al.), no conclusions can be drawn from this for the feasibility of human pCT expression since there is only about 77% identity at the amino acid level and therefore a completely different behavior must be expected. Furthermore, the murine pCT was not expressed directly after the putative signal peptide cleavage site (A25/V26) (Jakobs et al., 1981, Science, 213: 457-457), but only a murine procalcitonin fragment shortened by 7 amino acids was expressed, which again may have unforeseeable consequences for the expressibility. Finally, the publication contains neither the exact fermentation conditions nor the achievable yields after purification of the fusion protein.
In the following specific embodiments of the invention are described in more detail:
The invention relates preferably to an isolated polypeptide comprising the amino acid sequen

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