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
1999-12-03
2004-09-14
Helms, Larry R. (Department: 1642)
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...
C530S387100, C530S388850, C424S130100
Reexamination Certificate
active
06790938
ABSTRACT:
DESCRIPTION
The invention relates to novel nucleic acid sequences which encode human autoantibodies against blood platelet membrane proteins and which encode antiidiotypic antibodies, to novel amino acid sequences of human antibodies, and to their use for the diagnosis and therapy of diseases.
Autoimmune thrombocytopenic purpura (AITP) is an immune disease which is defined by a low blood platelet count associated with normal or elevated megakaryocyto-poiesis. The destruction of platelets in the reticuloendothelial system (spleen, liver and bone marrow) is increased due to the presence of anti-platelet autoantibodies. These autoantibodies, which can be detected in about 75% of AITP patients, are predominantly directed against the platelet membrane glycoproteins (GP) IIb/IIIa and Ib/IX. Several different autoantibody specificities may be found in one and the same patient (cf., e.g., Berchtold and Wenger, Blood 81 (1993), 1246-1250; Kiefel et al., Br. J. Haematol. 79 (1991), 256-262; McMillan et al., Blood 70 (1987), 1040 and Fujisawa et al., Blood 79 (1991); 1441). However, it is still difficult to characterize binding epitopes and to ascertain the pathogenic significance of the autoantibodies due to the limited quantity of autoantibodies which can be obtained from AITP patients. It has only been possible to obtain a few human monoclonal antibodies from lymphocytes of AITP patients which react with GPIIb/IIIa AIPT using the hybridoma technique (Kunicki et al., Hum. Antibodies Hybridiomas 1(1990) 83-95).
Natural autoantibodies against various selfantigens, for example against intracellular and cytoskeletal components of human platelets, have also been reported to occur in healthy individuals (Guilbert et al., J. Immunol. 128 (1982), 2779-2787; Hurez et al., Eur. J. Immunol. 23 (1993), 783-789 and Pfueller et al., Clin. Exp. Immunol. 79 (1990), 367-373). Some of these autoantibodies which have been observed in sera from healthy individuals can also be directed against platelet-membrane proteins (Souberbielle, Eur. J. Haematol. 56 (1996), 178-180). However, the role of these natural autoantibodies, and there relationship to disease-associated autoantibodies, is still unknown.
Corticosteroids can be used for treating AITP. About half of the patients react within 4 weeks to an administration of prednisone; however long-term remissions are only rarely seen. The administration of high doses of intravenous immunoglobulin (IVIgG) is recommended as an emergency treatment for patients who are exhibiting severe bleeding or extremely low platelet counts. This treatment is followed in most patients by a rapid, but usually only transient, increase in the platelet count. The mechanisms by which corticosteroids and IVIgG act in the treatment of AITP are still unknown. Investigations carried out by Berchtold et al., (Blood 74 (1989), 2414-2417 and Berchtold and Wenger, Blood 81 (1993), 1246-1250) have disclosed that antiiodiotypic antibodies which are present in IVIgG can inhibit the binding of autoantibodies to platelet glycoproteins.
The problem underlying the present application is that of identifying novel DNA sequences which are responsible for autoantibodies binding to GPIIb/IIIa. This approach can be used for making available novel pharmaceutical preparations which can be employed for improving the diagnosis and therapy of AITP.
It was surprisingly possible to identify binding sequences from autoantibodies after using peripheral circulating B cells from a healthy human donor to prepare a combinatorial phagemid display library of human antibody heavy and light chains. Following the presentation of human heavy and light antibody Fab fragments on the surface of the filamentous phage M13, it was possible to identify phage clones which exhibit specific binding to GPIIb/IIIa.
For this, the phagemid library was brought consecutively into contact with thrombasthenic platelets lacking GPIIb/IIIa (negative selection) and normal platelets (positive selection). After several rounds of selection and amplification by infecting
E.coli
, 23 clones were obtained which were able to bind to the GPIIb/IIIa complex. Inhibition studies using pools of monoclonal antibodies directed against the GPIIb/IIIa yielded two groups of clones: both groups were inhibited by monoclonal antibodies which were specific for the GPIIb/IIIa complex and one group was also inhibited by a GPIIb-specific monoclonal antibody. These findings were confirmed by carrying out a DNA analysis of the clones which indicated the presence of 2 different anti-GPIIb/IIIa phage clones. These results demonstrate that 2 GPIIb/IIIa-specific phage clones, i.e. autoantibodies, can be cloned from the genome of a healthy individual and that these clones are able to recognize confirmational epitopes belonging to the GPIIb/IIIa complex. Inhibition studies furthermore established that both phage clones inhibit the binding of platelet-associated autoantibodies from AITP patients to purified GPIIb/IIIa and therefore presumably recognize GPIIb/IIIa epitopes which are AITP-associated. Since the phage clones contain the antigen-binding sequences of natural autoantibodies which are derived from the genome of a healthy individual, this finding can lead to new insights into the origin of platelet-associated autoantibodies in AITP.
In addition to this, it is possible to use the novel phage clones to produce recombinant antiidiotypic antibodies against anti-GPIIb/IIIa autoantibodies, with the anti-GPIIb/IIIa phage clones being used as antigen. The recombinant antiidiotypic antibodies which can be obtained in this way constitute an attractive clinical alternative to using IVIgG.
The nucleotide sequences of the identified phage clones, and the amino acid sequences which are deduced from these nucleotide sequences, are depicted in the sequencing listings SEQ ID No. 1 to 8 (autoantibodies) and SEQ ID No. 9 to 18 (antiidiotypic antibodies).
I. Autoantibodies
A first aspect of the present invention relates to nucleic acids which encode auto-antibodies. Part of the subject-matter of the invention is therefore a nucleic acid which encodes the heavy chain of a human antibody, or a functional derivative or a fragment thereof, and encompasses a CDR3 region, selected from:
(a) a nucleotide sequence which encodes the amino acid sequence:
VLPFDPISMDV, (SEQ ID NO:31)
(b) a nucleotide sequence which encodes the amino acid sequence:
ALGSWGGWDHYMDV, (SEQ ID NO:32)
(c) a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably at least 90%, with an amino acid sequence from (a) or (b), and
(d) a nucleotide sequence which encodes an amino acid sequence having an equivalent ability to bind to GPIIb/IIIa.
The novel nucleic acid furthermore preferably comprises a CDR1 region selected from:
(a) a nucleotide sequence which encodes the amino acid sequence: GYSWR, (SEQ ID NO:33)
(b) a nucleotide sequence which encodes the amino acid sequence: SYAMH, (SEQ ID NO:34)
and
(c) a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably at least 90%, with an amino acid sequence from (a) or (b).
The novel nucleic acid preferably furthermore comprises a CDR2 region selected from:
(a) a nucleotide sequence which encodes th amino acid sequence:
DISYSGSTKYKPSLRS, (SEQ ID NO:35)
(b) a nucleotide sequence which encodes the amino acid sequence:
VISYDGSNKYYADSVKG, (SEQ ID NO:36)
and
(c) a nucleotide sequence which encodes an amino acid sequence having an homology of at least 80%, and preferably of at least 90%, with an amino acid sequence from (a) or (b).
A second aspect of the present invention is a nucleic acid which encodes the light chain of a human antibody, or a functional derivative or a fragment thereof, and comprises a CDR3 region, selected from:
(a) a nucleotide sequence which encodes the amino acid sequence:
ATWDDGLNGPV, (SEQ ID NO:37)
(b) a nucleotide sequence which encodes the amino acid sequence:
AAWDDSLNGWV, (SEQ ID NO:38)
(c) a nucleotide sequence which encodes an amino acid sequence having an
Berchtold Peter
Escher Robert F. A.
ASAT AG Applied Science & Technology
Helms Larry R.
Rothwell Figg Ernst & Manbeck P.C.
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