Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1999-04-19
2004-07-20
Chan, Christina (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387300, C530S388100, C530S391100, C530S391500, C530S391700, C530S391900
Reexamination Certificate
active
06765087
ABSTRACT:
The invention relates to new isolated immunoglobulins which are devoid of light polypeptide chains. These immunoglobulins do not consist in the degradation products of immunoglobulins composed of both heavy polypeptide and light polypeptide chains but to the contrary, the invention defines a new member of the family of the immunoglobulins, especially a new type of molecules capable of being involved in the immune recognition. Such immunoglobulins can be used for several purposes, especially for diagnosis or therapeutical purposes including protection against pathological agents or regulation of the expression or activity of proteins.
Up to now the structure proposed for immunoglobulins consists of a four-chain model referring to the presence of two identical light polypeptide chains (light chains) and two identical heavy polypeptide chains (heavy chains) linked together by disulfide bonds to form a y- or T-shaped macromolecules. These chains are composed of a constant region and a variable region, the constant region being subdivided in several domains. The two heavy polypeptide chains are usually linked by disulphide bounds in a so-called “hinge region” situated between the first and second domains of the constant region.
Among the proteins forming the class of the immunoglobulins, most of them are antibodies and accordingly present an antigen binding site or several antigen binding sites.
According to the four-chain model, the antigen binding site of an antibody is located in the variable domains of each of the heavy and light chains, and requires the association of the heavy and the light chains variable domains.
For the definition of these four-chain model immunoglobulins, reference is made to Roitt. I et al (Immunology-second-Edition Gower Medical Publishing USA, 1989). Reference is especially made to the part concerning the definition of the four-chain immunoglobulins, their polypeptidic and genetic structures, the definition of their variable and constant regions and the obtention of the fragments produced by enzymatic degradation according to well known techniques.
The inventors have surprisingly established that different molecules can be isolated from animals which naturally produce them, which molecules have functional properties of immunoglobulins these functions being in some cases related to structural elements which are distinct from those involved in the function of four-chain immunoglobulins due for instance to the absence of light chains.
The invention relates to two-chain model immunoglobulins which neither correspond to fragments obtained for instance by the degradation in particular the enzymatic degradation of a natural four-chain model immunoglobulin, nor correspond to the expression in host cells, of DNA coding for the constant or the variable region of a natural four-chain model immunoglobulin or a part of these regions, nor correspond to antibodies produced in lymphopaties for example in mice, rats or human.
E. S. Ward et al (1) have described some experiments performed on variable domains of heavy polypeptide chains (V
H
) or/and light polypeptide chains (V
K
/F
V
) to test the ability of these variable domains, to bind specific antigens. For this purpose, a library of V
H
genes was prepared from the spleen genomic DNA of mice previously immunized with these specific antigens.
Ward et al have described in their publication that V
H
domains are relatively sticky, presumably due to the exposed hydrophobic surface normally capped by the V
K
or V
&lgr;
domains. They consequently envisage that it should be possible to design V
H
domains having improved properties and further that V
H
domains with binding activities could serve as the building blocks for making variable fragments (Fv fragments) or complete antibodies.
The invention does not start from the idea that the different fragments (light and heavy chains) and the different domains of these fragments of four-chain model immunoglobulin can be modified to define new or improved antigen binding sites or a four-chain model immunoglobulin.
The inventors have determined that immunoglobulins can have a different structure than the known four-chain model and that such different immunoglobulins offer new means for the preparation of diagnosis reagents, therapeutical agents or any other reagent for use in research or industrial purposes.
Thus the invention provides new immunoglobulins which are capable of showing functional properties of four-chain model immunoglobulins although their structure appears to be more appropriate in many circumstances for their use, their preparation and in some cases for their modification. Moreover these molecules can be considered as lead structures for the modification of other immunoglobulins. The advantages which are provided by these immunoglobulins comprise the possibility to prepare them with an increased facility.
The invention accordingly relates to immunoglobulins characterized in that they comprise two heavy polypeptide chains sufficient for the formation of a complete antigen binding site or several antigen binding sites, these immunoglobulins being further devoid of light polypeptide chains. In a particular embodiment of the invention, these immunoglobulins are further characterized by the fact that they are the product of the expression in a prokaryotic or in a eukaryotic host cell, of a DNA or of a cDNA having the sequence of an immunoglobulin devoid of light chains as obtainable from lymphocytes or other cells of Camelids.
The immunoglobulins of the invention can be obtained for example from the sequences which are described in FIG.
7
.
The immunoglobulins of the invention, which are devoid of light chains are such that the variable domains of their heavy chains have properties differing from those of the four-chain immunoglobulin V
H
. The variable domain of a heavy-chain immunoglobulin of the invention has no normal interaction sites with the V
L
or with the C
H
1 domain which do not exist in the heavy chain immunoglobulins. it is hence a novel fragment in many of its properties such as solubility and position of the binding site. For clarity reasons we will call it V
HH
in this text to distinguish it from the classical V
H
of four-chain immunoglobulins.
By “a complete antigen binding site” it is meant according to the invention, a site which will alone allow the recognition and complete binding of an antigen. This could be verified by any known method regarding the testing of the binding affinity.
These immunoglobulins which can be prepared by the technique of recombinant DNA, or isolated from animals, will be sometimes called “heavy-chain immunoglobulins” in the following pages. In a preferred embodiment of the invention, these immunoglobulins are in a pure form.
In a first embodiment, the immunoglobulins of the invention are obtainable in prokaryotic cells, especially in
E. coli
cells by a process comprising the steps of:
a) cloning in a Bluecript vector of a DNA or cDNA sequence coding for the V
HH
domain of an immunoglobulin devoid of light chain obtainable for instance from lymphocytes of Camelids,
b) recovering the cloned fragment after amplification using a 5′ primer containing an Xho site and a 3′ primer containing the Spe site having the following sequence TC TTA ACT AGT GAG GAG ACG GTG ACC TG SEQ ID NO: 51,
c) cloning the-recovered fragment in phase in the immuno PBS vector after digestion of the vector with Xho and Spe restriction enzymes,
d) transforming host cells, especially
E. coli
by transfection with the recombinant immuno PBS vector of step c,
e) recovering the expression product of the V
HH
coding sequence, for instance by using antibodies raised against the dromadary V
HH
domain.
In another embodiment the immunoglobulins are hetero-specific immunoglobulins obtainable by a process comprising the steps of:
obtaining a first DNA or cDNA sequence coding for a V
HH
domain or part thereof having a determined specificity against a given antigen and comprised between Xho and Spe sites,
obtaining a second DNA or cDNA s
Casterman Cecile
Hamers Raymond
Chan Christina
DiBrino Marianne
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Vrije Universiteit Brussel
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