Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
1997-07-01
2001-11-06
Schwadron, Ronald B. (Department: 1644)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C424S133100, C424S135100, C424S152100, C424S143100, C424S800000, C424S801000, C435S320100, C435S326000, C435S328000, C435S334000, C435S348000, C530S387100, C530S387300, C530S388220, C530S866000, C530S867000, C530S388150
Reexamination Certificate
active
06312690
ABSTRACT:
The present invention relates to the production, in insect cells, of an anti-Rhesus D recombinant monoclonal antibody.
The subjects whose red blood cells are agglutinated by alloantibodies directed against the D antigen, (which is one of the antigens of the RH system) are commonly designated by the term “Rhesus positive”, or “Rh-positive”, and the subjects whose red blood cells are not agglutinated by these alloantibodies by the term “Rhesus negative”, or “Rh-negative”.
The haemolytic disease of newborn is due, in the majority of cases, to the presence, in an Rh-negative mother, of anti-Rhesus D alloantibodies (the alloimmunization against other antigens of the RH system is a lot more rare), which cause in a Rhesus positive foetus a haemolytic anaemia which will require either transfusions in utero, or an exchange transfusion at birth in severe cases.
Alloimmunization of the mother occurs generally during a previous childbirth; foetal red blood cells pass into the maternal blood stream and induce immunization if the child is Rh-positive.
The prevention of haemolytic disease of newborn consists in injecting anti-Rhesus D antibodies into Rhesus negative women immediately after childbirth or an abortion.
Currently, the anti-Rhesus antibodies used for this purpose are polyclonal immunoglobulins obtained from Rhesus negative volunteer donors immunized on several occasions against Rh-positive red blood cells.
This poses problems, on the one hand because of the need to have a sufficient number of volunteers to satisfy the needs, and on the other hand because of the risks of contamination by viruses or other pathogens which might be present in the immunoglobulin preparations obtained from the blood of volunteers.
However, this mode of production has at present no alternative. Indeed, although there are trials to culture lymphoblastoid lines, derived from B lymphocytes transformed by the Epstein-Barr virus and secreting monoclonal antibodies, no product obtained from this type of culture has received marketing authorization because of the risks involved in the use of the Epstein-Barr virus.
In order to obtain a source of anti-Rhesus D immunoglobulins not exhibiting the disadvantages of the immunoglobulins used up until now, the inventors had as objective the production of a recombinant anti-Rhesus human monoclonal antibody in insect cells, using an expression vector derived from a baculovirus.
With this aim in view, the inventors first selected a line producing an anti-Rhesus D monoclonal antibody called D7C2. This antibody belongs to the class of the IgM's, agglutinate the red blood cells with a common Rhesus phenotype, the weak Rhesus, and most of the partial Rhesus with the exception of D
VI
; this antibody binds to a 30 to 32 kDa of the membrane of the red blood cells.
The inventors then cloned the sequences encoding the variable regions of the heavy (H) and light (L) chains of D7C2, and constructed expression cassettes allowing the expression of each of the said sequences under the control of a strong baculovirus promoter.
The subject of the present invention is a DNA fragment, characterized in that it is chosen from the group consisting of:
a DNA fragment which comprises a sequence which encodes the variable region of the light chain of the D7C2 monoclonal antibody;
a DNA fragment which comprises a sequence which encodes the variable region of the heavy chain of the D7C2 monoclonal antibody.
“Sequence which encodes the variable region of the light chain of the D7C2 monoclonal antibody” is understood to mean, in particular, the sequence identified in the sequence listing in the annex under the number SEQ ID NO: 1, which encodes the polypeptide SEQ ID NO: 2.
“Sequence which encodes the variable region of the heavy chain of the D7C2 monoclonal antibody” is understood to mean, in particular, the sequence SEQ ID NO: 3 which encodes the polypeptide SEQ ID NO: 4.
However, it will appear to persons skilled in the art that this definition also covers sequences which are functionally equivalent to the sequences SEQ ID NO: 1 and SEQ ID NO: 3, namely, in particular:
any sequence encoding the polypeptides SEQ ID NO: 2 and SEQ ID NO: 4, and also
sequences encoding the polypeptides which may differ from the polypeptides SEQ ID NO: 2 and SEQ ID NO: 4 in a few amino acids, on the condition that this variation is not situated at the level of a peptide sequence involved in the recognition of the epitope.
This covers, for example, a sequence encoding a polypeptide whose sequence differs from that of the polypeptide SEQ ID NO: 4 in the replacement of the Thr residue at position 23, by a residue Ala, which results from the replacement of an A at position 67 in the sequence SEQ ID NO: 3, by a G.
Sequences which are functionally equivalent to the sequences SEQ ID NO: 1 and SEQ ID NO: 3, are also sequences resulting from a modification of the ends thereof for the purpose of creating restriction sites therein, or of modifying or suppressing those which are present therein. This covers, for example, a sequence encoding a polypeptide whose sequence differs from that of the polypeptide SEQ ID NO: 2 in the replacement of the first three residues, Asp-Ile-Glu, by the residues Gln-Ser-Val, which results from the replacement of an A at position 67 in the sequence SEQ ID NO: 3, by a G.
The subject of the present invention is also an expression cassette comprising a sequence encoding the variable region of the light chain of the D7C2 monoclonal antibody, or a sequence encoding the variable region of the heavy chain of the D7C2 monoclonal antibody, which sequence is placed under the transcriptional control of an appropriate promoter.
According to a preferred embodiment of the present invention, the said promoter is a baculovirus promoter.
By way of example of baculovirus promoters which can be used for carrying out the present invention, there may be mentioned the polyhedrin and P10 promoters of the baculoviruses AcMNPV or SlMNPV, or derivatives of baculovirus promoters, consisting of synthetic or recombinant promoters, obtained from a baculovirus promoter, and which are functional in insect cells, such as for example that described by WANG et al., [Gene, 100, 131-137, (1991)].
An expression cassette in accordance with this embodiment comprises, for example:
a baculovirus promoter as defined above;
a sequence encoding a secretory signal peptide;
a sequence encoding the variable domain of the light chain of the D7C2 monoclonal antibody and a sequence encoding the constant domain of the light chain of an immunoglobulin; or
a sequence encoding the variable domain of the heavy chain of the D7C2 monoclonal antibody and a sequence encoding the constant domain of the heavy chain of an immunoglobulin.
A large number of sequences encoding signal peptides which are functional in insect cells can be used to carry out the present invention. By way of nonlimiting example, there may be mentioned the sequences encoding the signal peptides of Drosophila acetylcholinesterase, of ovine trophoblastin, as well as the sequences encoding the signal peptides of the H and L chains of murine or human immunoglobulins and the like.
Each of the cassettes in accordance with the invention allows the expression, either of the light chain, or of the heavy chain, of a recombinant antibody, called hereinafter r-D7C2, having the specificity of the D7C2 monoclonal antibody.
According to a preferred feature of this embodiment, the sequences encoding the constant domains of the light and heavy immunoglobulin chains are sequences of human origin.
The sequence encoding the constant domain of the light chain may be chosen from the sequences encoding the constant domains of the kappa (&kgr;) and lambda (&lgr;) light chains.
The sequence encoding the constant domain of the heavy chain may be chosen from the sequences encoding the constant domains of the heavy chains &ggr;1, &ggr;2, &ggr;3, &ggr;4, &agr;, &egr;, and &mgr;. There may thus be obtained a recombinant antibody belonging to the desired immunoglobulin class (IgG1, IgG2, IgG3, IgG4,
Chaabihi Hassan
Edelman Léna
Kaczorek Michel
Margaritte Christel
Institut Pasteur
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Schwadron Ronald B.
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