Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Binds hormone or other secreted growth regulatory factor,...
Reexamination Certificate
2000-12-22
2002-09-17
Chan, Christina (Department: 1644)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Binds hormone or other secreted growth regulatory factor,...
C424S009100, C424S141100, C424S142100, C424S145100, C424S178100, C530S387100, C530S388100, C530S388150, C530S388240, C530S391100, C530S391300, C530S391900, C435S070210, C435S252800, C435S326000, C435S331000, C435S346000, C435S366000, C435S410000, C435S455000, C435S810000
Reexamination Certificate
active
06451311
ABSTRACT:
The invention relates to anti-procalcitonin antibodies, their preparation and use.
BACKGROUND OF THE INVENTION
Procalcitonin (“pCT”) is a protein consisting of 116 amino acids and having a molecular weight of about 13,000 Daltons. 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 elimination 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 sequence 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 (EP-B1-0 656 121). However, the disadvantage of such a method is that pCT detection requires at least two antibodies having to bind to pCT segments which are as far apart as possible.
It is therefore an object for the skilled worker to provide other specific binding partners which allow pCT detection even by just one specific binding partner.
This object is achieved by providing the antibodies according to the invention which bind to procalcitonin but not to free calcitonin which consists of amino acids 1-57 of procalcitonin, free katacalcin and free N-procalcitonin. In accordance with the invention, antibodies binding to a peptide having the amino acid sequence Asp-Ser-Pro-Arg-Ser-Lys-Arg-Cys-Gly-Asn-Leu-Ser (SEQ ID NO: 2) and antibodies binding to a peptide having the amino acid sequence Val-Gly-Ala-Pro-Gly-Lys-Lys-Arg-Asp-Met-Ser-Ser (SEQ ID NO: 4)are preferred.
In contrast to antibodies known so far which bind pCT but also at the same time bind free calcitonin or free katacalcin or free N-procalcitonin, the antibodies according to the invention may also be employed on their own for specific pCT detection in competitive assays or immunohistochemical methods. In addition, the antibodies according to the invention are particularly suitable for purifying pCT from a sample also containing pCT fragments by affinity chromatography.
Although Ghillani et al. in their immunization experiments employed peptides comprising the amino acid sequence Pro-Gly-Lys-Lys-Arg-Asp (SEQ ID NO: 3) or the amino acid sequence Val-Gly-Ala-Pro-Gly-Lys-Lys-Arg-Asp-Met-Ser-Ser (SEQ ID NO: 4), they only succeeded in providing antibodies recognizing either “mature” calcitonin or pCT together with calcitonin or pCT together with katacalcin, despite intensive efforts to identify the pCT epitopes.
BRIEF SUMMARY OF THE INVENTION
Surprisingly, antibodies have now been successfully created which bind to procalcitonin but not to free calcitonin, free katacalcin and free N-procalcitonin.
Specific embodiments of the invention are described in more detail below:
The invention relates preferably to such antibodies which bind procalcitonin but not free calcitonin, free katacalcin and free N-procalcitonin.
DETAILED DESCRIPTION OF THE INVENTION
The term “antibody” in accordance with this invention means an immunoglobulin, for example an immunoglobulin of the class or subclass IgA, IgD, IgE, IgG
1
, IgG
2a
, IgG
2b
, IgG
3
, IgG
4
, IgM. An antibody comprises at least one binding site (often called paratope) for an epitope (often also called antigenic determinant) on an antigen or hapten. Such an epitope is characterized, for example, by its three-dimensional structure and/or the presence of polar and/or apolar groups. The antibody binding site is complementary to the epitope. The antigen-antibody reaction or hapten-antibody reaction works according to the so-called “lock-and-key principle” and is, in general, specific to a high degree, i.e. the antibodies are capable of distinguishing small differences in primary structure, charge, three-dimensional configuration and steric arrangement of the antigen or hapten. In particular the so-called complementarity determining regions of the antibody contribute to the binding of the antibody to the antigen or hapten.
The term “antigens” comprises monovalent and polyvalent antigens. A polyvalent antigen is a molecule or a molecule complex to which more than one immunoglobulin can bind simultaneously, whereas only a single antibody can bind to a monovalent antigen at any one time. A hapten usually denotes a molecule which on its own is not immunogenic but is usually bound to a carrier for immunization purposes.
The term antibodies in accordance with this invention means not only complete antibodies but expressly also antibody fragments such as, for example, Fab, Fv, F(ab′)
2
, Fab′; and also chimeric, humanized, bi- or oligo-specific or single-chain antibodies; furthermore also aggregates, polymers and conjugates of immunoglobulins and/or fragments thereof as long as the binding properties to the antigen or hapten are maintained. Antibody fragments can be prepared for example by enzymatic cleavage of antibodies using enzymes such as pepsin or papain. Antibody aggregates, polymers and conjugates can be generated by a multiplicity of methods, for example by heat treatment, reaction with substances such as glutaraldehyde, reaction with immunoglobulin-binding molecules, biotinylation of antibodies and subsequent reaction with streptavidin or avidin, etc.
An antibody in accordance with this invention can be a monoclonal or a
Althaus Harald
Walter Götz
Chan Christina
Dade Behring Marburg GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Huynh Phuong N.
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