Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
2000-01-06
2002-11-19
Nolan, Patrick J. (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C424S278100, C530S388220, C536S023100, C536S023400, C536S023500
Reexamination Certificate
active
06482925
ABSTRACT:
CROSS REFERENCE TO RELATED APPLICATION
The present application is the national stage under 35 U.S.C. 371 of PCT/FR97/02126, filed Nov. 25, 1997.
The invention relates to new polypeptides corresponding to mutant forms of the soluble LAG-3 protein or of its fragments.
The lymphocyte activation gene 3 (LAG-3) expressed in activated T cells and human NK cells encodes a type I membrane protein of 498 amino acids with four extracellular domains of the immunoglobulin superfamily (IgSF) (1). The analysis of its sequence has revealed numerous identities with series of amino acid sequences present at corresponding positions on the CD4 receptor, although the overall sequence homology between these two molecules is barely above a base level (approximately 20% sequence identity). Internal sequence homologies also exist in the LAG-3 molecule, between domains 1 (D1) and 3 (D3) as well as between domains 2 (D2) and 4 (D4), which suggests that LAG-3 has evolved, like CD4, through a gene duplication from a pre-existing structure composed of two IgSFs (1). LAG-3 and CD4 can therefore be considered as “first cousins” in the evolution within the IgSF family (2).
The authors of the present invention have shown in previous studies, using a quantitative test of cell adhesion, that the formation of rosettes between COS-7 cells transfected with LAG-3 and B lymphocytes expressing the major histocompatibility complex class II (MHC) molecules is specifically dependent on the interaction between LAG-3 and the MHC class II molecules (2). A direct and specific binding of LAG-3 to various human class II molecules (including various alleles and isotypes) as well as to murine and monkey class II molecules has also been observed with a LAG-3Ig fusion protein (3). This dimeric recombinant globulin LAG-3Ig binds to the monomorphic residue of the MHC class II molecules with a greater affinity (Kd=60 nM at 37° C.) than CD4-Ig (4); LAG-3Ig is in fact capable of blocking the class II molecule/CD4 interaction in a test of intracellular adhesion (4).
The role of the LAG-3/class II molecule interaction was studied using monoclonal antibodies specific for LAG-3 (5) and LAG-3Ig molecules (6). This interaction leads to a negative regulation of the activation of T cell clones. The production of a LAG-3/class II molecule interaction is induced by contacts between T cells, probably via a negative signal from the class II molecules in the T cell.
In general, LAG-3 is only expressed after lymphocyte activation both in vivo and in vitro (5) and does not therefore play a role in the response inducing phase, unlike CD4. Moreover, blocking experiments with monoclonal antibodies have shown that LAG-3 does not participate in the phase for recognition by restricted CD4 T cell clones by the class II molecules. The functional role of LAG-3 is therefore substantially different from that of the other ligands for the MHC molecules, CD4 and CD8.
In patent application PCT/FR95/00593, the authors of the present invention have shown that some soluble polypeptide fractions of the LAG-3 protein of SEQ ID NO:11 were also capable of binding to the class II molecules. They have also underlined the potential importance of the amino acids of the region between residue 46 and residue 77, and in particular positions 73, 75, 76 and 77.
The authors of the invention have now sought to characterize precisely the region(s) of LAG-3 specifically involved in the binding to the class II molecules, and, to do this, have synthesized several mutated forms of LAG-3 possessing targeted mutations in the first two domains of the extracytoplasmic region of the mature protein, that is to say the protein free of signal peptide, having the N-terminal sequence L-Q-P-G-A-E (residues 1-6 of SEQ ID NO. 2). The D1 domain extends from amino acid No. 1 (L), amino acid No. 149 (M); the D2 domain extends from amino acid No. 150 (T) to 239 (G). Surprisingly, the authors of the invention have shown that the substitution of a single amino acid was able to induce well-known modifications in the affinity of LAG-3 for the class II molecules, either by substantially increasing the binding of LAG-3 to these proteins, or on the contrary by inhibiting it partially or totally.
One aspect of the present invention is therefore to provide a purified polypeptide corresponding to a mutated form of the soluble LAG-3 protein or of one of its fragments comprising the extracellular domain D1 and D2 consisting:
either in an amino acid substitution at one of the positions selected from the group consisting of:
position 73 where arginine is replaced by glutamic acid (R73E),
position 75 where arginine is replaced by alanine (R75A) or by glutamic acid (R75E),
position 76 where arginine is replaced by glutamic acid (R76E), or a combination of two or three of the preceding substitutions,
or in an amino acid substitution at one of the positions selected from the group consisting of:
position 30 where aspartic acid is replaced by alanine (D30A),
position 56 where histidine is replaced by alanine (H56A),
position 77 where tyrosine is replaced by phenylalanine (Y77F),
position 88 where arginine is replaced by alanine (R88A),
position 103 where arginine is replaced by alanine (R103A),
position 109 where aspartic acid is replaced by glutamic acid (D109E),
position 115 where arginine is replaced by alanine (R115A);
or a deletion of the region between position 54 (P) and position 66 (A).
An other aspect is also the use of these mutants of soluble LAG-3 for the manufacture of medicaments, in particular medicaments which are peptidomimetic for the LAG-3 molecule, that is to say which bind in a similar manner to the MHC class II molecules and which have the same type of immunosuppressive activity as the LAG-3 molecule.
The invention also relates to pharmaceutical compositions comprising, as active ingredient, one of the mutated forms of the LAG-3 protein defined above.
In the text which follows, the polypeptides defined above will be called “mutants of LAG-3”.
This definition applies to the polypeptides corresponding to the mutated forms of the whole soluble LAG-3 protein or of fragments of LAG-3 comprising the extracellular domains Di and D2, in particular the fragment composed of the two domains D1 and D2.
The soluble LAG-3 protein corresponds to the sequence between the first N-terminal residue and residue 412 of the mature protein (free of signal peptide).
According to a first embodiment, the invention relates to purified polypeptide mutants of soluble LAG-3 having an affinity for binding to the MHC class II molecules which is greater than the affinity of the wild-type LAG-3 protein. The mutations induced in these peptides are preferably located at the level of the external loop of LAG-3, which is located between residue 46 and residue 77.
More precisely, the invention relates to a purified polypeptide corresponding to a mutated form of the soluble LAG-3 protein or of one of its fragments comprising the two extracellular domains D1 and D2, consisting in an amino acid substitution at one of the positions selected from the group consisting of:
position 73 where arginine is replaced by glutamic acid (R73E),
position 75 where arginine is replaced by alanine (R75A) or by glutamic acid (R75E),
position 76 where arginine is replaced by glutamic acid (R76E), or a combination of two or three of the preceding substitutions.
Preferably, the mutants of LAG-3 result from a combination of two or three substitutions selected from the group consisting of:
(R73E+R75A),
(R73E+R76E),
(R75A+R76E),
(R73E+R75A+R76E)
These different mutants will be called hereinafter “positive mutants”.
According to another embodiment, the invention provides a purified polypeptide corresponding to a mutated form of the soluble LAG-3 protein or of one of its fragments comprising the extracellular domain D1 and D2, consisting in an amino acid substitution at one of the positions selected from the group consisting of:
position 30 where aspartic acid is replaced by alanine (D30A),
position 56 where histidine is replaced by a
El Tayar Nabil
Huard Bertrand
Mastrangeli Renato
Triebel Frederic
Applied Research Systems ARS Holding N.V.
Browdy and Neimark
Ewoldt Gerald R.
Nolan Patrick J.
LandOfFree
Mutants of the LAG-3 proteins and nucleotides encoding LAG-3... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Mutants of the LAG-3 proteins and nucleotides encoding LAG-3..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mutants of the LAG-3 proteins and nucleotides encoding LAG-3... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2924242