Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-17
2004-02-24
Ponnaluri, Padmashri (Department: 1618)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S005000, C435S007100, C435S069100, C435S320100, C435S091500, C435S091500, C435S091500, C435S091500, C530S388100, C536S025300
Reexamination Certificate
active
06696245
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for selecting functional members of a repertoire of polypeptides using generic and target ligands. In particular, the invention describes a method for isolating a functional subset of a repertoire of antibody polypeptides with a generic ligand.
BACKGROUND OF THE INVENTION
The antigen binding domain of an antibody comprises two separate regions: a heavy chain variable domain (V
H
) and a light chain variable domain (V
L
, which can be either V
&kgr;
or V
&lgr;
). The antigen binding site itself is formed by six polypeptide loops: three from V
H
domain (H1, H2 and H3) and three from V
L
domain (L1, L2 and L3). A diverse primary repertoire of V genes that encode the V
H
and V
L
domains is produced by the combinatorial rearrangement of gene segments. The V
H
gene is produced by the recombination of three gene segments, V
H
, D and J
H
. In humans, there are approximately 51 functional V
H
segments (Cook and Tomlinson, 1995,
Immunol. Today,
16: 237-242), 25 functional D segments (Corbett et al., 1997,
J. Mol. Biol.,
268: 69) and 6 functional J
H
segments (Ravetch et al., 1981,
Cell,
27: 583-591), depending on the haplotype. The V
H
segment encodes the region of the polypeptide chain which forms the first and second antigen binding loops of the V
H
domain (H1 and H2), while the V
H
, D and J
H
segments combine to form the third antigen binding loop of the V
H
domain (H3). The V
L
gene is produced by the recombination of only two gene segments, V
L
and J
L
. In humans, there are approximately 40 functional V
&kgr;
segments (Schäble and Zachau, 1993,
Biol. Chem. Hoppe-Seyler,
374: 1001-1022), 31 functional V
&lgr;
segments (Williams et al., 1996,
J. Mol. Biol.,
264: 220-232; Kawasaki et al., 1997,
Genome Res.,
7: 250-261), 5 functional J
&kgr;
segments (Hieter et al., 1982,
J. Biol. Chem.,
257: 1516) and 4 functional J
&lgr;
segments (Vasicek and Leder, 1990,
J. Exp. Med.,
172: 609-620), depending on the haplotype. The V
L
segment encodes the region of the polypeptide chain which forms the first and second antigen binding loops of the V
L
domain (L1 and L2), while the V
L
and J
L
segments combine to form the third antigen binding loop of the V
L
domain (L3). Antibodies selected from this primary repertoire are believed to be sufficiently diverse to bind almost all antigens with at least moderate affinity. High affinity antibodies are produced by “affinity maturation” of the rearranged genes, in which point mutations are generated and selected by the immune system on the basis of improved binding.
Analysis of the structures and sequences of antibodies has shown that five of the six antigen binding loops (H1, H2, L1, L2, L3) possess a limited number of main-chain conformations or canonical structures (Chothia and Lesk, 1987,
J. Mol. Biol.,
196: 901-917; Chothia et al., 1989,
Nature,
342: 877-883). The main-chain conformations are determined by (i) the length of the antigen binding loop, and (ii) particular residues, or types of residue, at certain key position in the antigen binding loop and the antibody framework. Analysis of the loop lengths and key residues has enabled us to the predict the main-chain conformations of H1, H2, L1, L2 and L3 encoded by the majority of human antibody sequences (Chothia et al., 1992,
J. Mol. Biol.,
227: 799-817; Tomlinson et al., 1995,
EMBO J.,
14: 4628-4638; Williams et al., 1996, supra). Although the H3 region is much more diverse in terms of sequence, length and structure (due to the use of D segments), it also forms a limited number of main-chain conformations for short loop lengths which depend on the length and the presence of particular residues, or types of residue, at key positions in the loop and the antibody framework (Martin et al., 1996,
J. Mol. Biol.,
263: 800-815; Shirai et al., 1996,
FEBS Letters,
399: 1-8).
A similar analysis of side-chain diversity in human antibody sequences has enabled the separation of the pattern of sequence diversity in the primary repertoire from that created by somatic hypermutation. The two patterns are complementary: diversity in the primary repertoire is focused at the center of the antigen binding whereas somatic hypermutation spreads diversity to regions at the periphery that are highly conserved in the primary repertoire (Tomlinson et al., 1996,
J. Mol. Biol,
256: 813-817; Ignatovich et al., 1997,
J. Mol. Biol.,
268: 69-77). This complementarity seems to have evolved as an efficient strategy for searching sequence space, given the limited number of B cells available for selection an a given time. Thus, antibodies are first selected from the primary repertoire based on diversity at the centre of the binding site. Somatic hypermutation is then left to optimize residues at the periphery without disrupting favorable interactions established during the primary response.
The recent advent of phage-display technology (Smith, 1985,
Science,
228: 1315-1317; Scott and Smith, 1990,
Science,
249: 386-390; McCafferty et al., 1990,
Nature,
348: 552-554) has enabled the in vitro selection of human antibodies against a wide range of target antigens from “single pot” libraries. These phage-antibody libraries can be grouped into two categories: natural libraries which use rearranged V genes harvested from human B cells (Marks et al., 1991,
J. Mol. Biol.,
222: 581-597; Vaughan et al., 1996,
Nature Biotech.,
14: 309) or synthetic libraries whereby germline V gene segments are ‘rearranged’ in vitro (Hoogenboom and Winter, 1992,
J. Mol. Biol.,
227: 381-388; Nissim et al., 1994,
EMBO J.,
13: 692-698; Griffiths et al., 1994,
EMBO J.,
13: 3245-3260; De Kruif et al., 1995,
J. Mol. Biol.,
248: 97) or where synthetic CDRs are incorporated into a single rearranged V gene (Barbas et al., 1992,
Proc. Natl. Acad. Sci. USA,
89: 4457-4461). Although synthetic libraries help to overcome the inherent biases of the natural repertoire which can limit the effective size of phage libraries constructed from rearranged V genes, they require the use of long degenerate PCR primers which frequently introduce base-pair deletions into the assembled V genes. This high degree of randomization may also lead to the creation of antibodies which are unable to fold correctly and are also therefore non-functional. Furthermore, antibodies selected from these libraries may be poorly expressed and, in many cases, will contain framework mutations that may effect the antibodies immunogenicity when used in human therapy.
Recently, in an extension of the synthetic library approach it has been suggested (WO97/08320, Morphosys) that human antibody frameworks can be pre-optimized by synthesizing a set of ‘master genes’ that have consensus framework sequences and incorporate amino acid substitutions shown to improve folding and expression. Diversity in the CDRs is then incorporated using oligonucleotides. Since it is desirable to produce artificial human antibodies which will not be recognized as foreign by the human immune system, the use of consensus frameworks which, in most cases, do not correspond to any natural framework is a disadvantage of this approach. Furthermore, since it is likely that the CDR diversity will also have an effect on folding and/or expression, it would be preferable to optimize the folding and/or expression (and remove any frame-shifts or stop codons) after the V gene has been fully assembled. To this end, it would be desirable to have a selection system which could eliminate non-functional or poorly folded/expressed members of the library before selection with the target antigen is carried out.
A further problem with the libraries of the prior art is that, because the main-chain conformation is heterogeneous, three-dimensional structural modeling is difficult because suitable high resolution crystallographic data may not be available. This is a particular problem for the H3 region, where the vast majority of antibodies derived from natural or synthetic have medium length or long loops and therefore cannot be
Tomlinson Ian
Winter Greg
Domantis Limited
FitzGerald Mark J.
Palmer & Dodge LLP
Ponnaluri Padmashri
Williams Kathleen M.
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