U.S. patent application number 09/968744 was filed with the patent office on 2003-08-07 for method to screen phage display libraries with different ligands.
Invention is credited to Tomlinson, Ian, Winter, Greg.
Application Number | 20030148372 09/968744 |
Document ID | / |
Family ID | 10820800 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030148372 |
Kind Code |
A1 |
Tomlinson, Ian ; et
al. |
August 7, 2003 |
Method to screen phage display libraries with different ligands
Abstract
The present invention relates to methods for selecting
repertoires of polypeptides using generic and target ligands. In
particular, the invention relates to a library wherein the folded
members have binding sites for both generic and target ligands.
Inventors: |
Tomlinson, Ian; (Cambridge,
GB) ; Winter, Greg; (London, GB) |
Correspondence
Address: |
PALMER & DODGE, LLP
KATHLEEN M. WILLIAMS / STR
111 HUNTINGTON AVENUE
BOSTON
MA
02199
US
|
Family ID: |
10820800 |
Appl. No.: |
09/968744 |
Filed: |
October 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09968744 |
Oct 1, 2001 |
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09511939 |
Feb 24, 2000 |
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Current U.S.
Class: |
435/7.1 ;
436/518; 530/350; 530/387.1 |
Current CPC
Class: |
G01N 33/6854 20130101;
C07K 1/047 20130101; C12N 15/1037 20130101; C40B 40/02 20130101;
C07K 14/705 20130101; C40B 30/04 20130101; C07K 2317/21 20130101;
C07K 16/005 20130101; C07K 2317/622 20130101; C07K 16/00 20130101;
G01N 33/6845 20130101; C07K 14/70503 20130101; C07K 14/7051
20130101 |
Class at
Publication: |
435/7.1 ;
436/518; 530/350; 530/387.1 |
International
Class: |
G01N 033/53; C07K
014/705; C07K 016/22; G01N 033/543 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 20, 1997 |
GB |
9722131.1 |
Claims
1. A library wherein the folded members have binding sites for both
generic and target ligands.
2. A library designed for selection with both generic and target
ligands.
3. The library according to claim 1 or claim 2 comprising a
repertoire of polypeptides of the immunoglobulin superfamily.
4. The library according to claim 3 wherein the polypeptides are
antibody or T-cell receptor polypeptides.
5. The library according to claim 4, wherein the polypeptides are
V.sub.H or V.sub..beta. domains.
6. The library according to claim 4, wherein the polypeptides are
V.sub.L or V.sub..alpha. domains.
7. The library wherein a repertoire of polypeptides according to
claim 5 and a repertoire of polypeptides according to claim 6 are
contacted with generic ligands and the subsets thereby obtained are
then pooled.
8. The library according to claim 1, wherein the polypeptides are
varied at random positions.
9. The library according to claim 1, wherein the polypeptides are
varied at selected positions.
10. The library according to claim 9, wherein the selected
positions are those which form the binding site for the target
ligand.
11. The library according to claim 10, wherein the selected
positions are a subset of those which form the binding site for the
target ligand.
12. The library wherein a repertoire of polypeptides according to
claim 11 is first contacted with a target ligand in order to
isolate a subset of polypeptides specific for the target ligand,
the subset of polypeptides then being varied at a further subset of
residues in order to modify the function, specificity or affinity
of target ligand interaction.
13. The library according to any one of claims 9-12, wherein the
polypeptides are varied at selected positions by incorporating all
20 different amino acids at each position to be varied.
14. The library according to any one of claims 9-12, wherein the
polypeptides are varied at selected positions by incorporating some
but not all of the 20 different amino acids at each position to be
varied.
Description
[0001] The present invention relates to methods for selecting
repertoires of polypeptides using generic and target ligands. In
particular, the invention describes a method for selecting
repertoires of antibody polypeptides with generic ligand to isolate
functional subsets thereof.
INTRODUCTION
[0002] The antigen binding domain of an antibody comprises two
separate regions: a heavy chain variable domain (V.sub.H) and a
light chain variable domain (V.sub.L: which can be either
V.sub..kappa. or V.sub..lambda.). The antigen binding site itself
is formed by six polypeptide loops: three from V.sub.H domain (H1,
H2 and H3) and three from V.sub.L domain (L1, L2 and L3). A diverse
primary repertoire of V genes that encode the V.sub.H and V.sub.L
domains is produced by the combinatorial rearrangement of gene
segments. The V.sub.H gene is produced by the recombination of
three gene segments, V.sub.H, D and J.sub.H. In humans, there are
approximately 51 functional V.sub.H segments (Cook and Tomlinson
(1995) Immunol Today, 16: 237), 25 functional D segments (Corbett
et al. (1997) J. Mol. Biol., 268: 69) and 6 functional J.sub.H
segments (Ravetch et al. (1981) Cell, 27: 583), depending on the
haplotype. The V.sub.H segment encodes the region of the
polypeptide chain which forms the first and second antigen binding
loops of the V.sub.H domain (H1 and H2), whilst the V.sub.H, D and
J.sub.H segments combine to form the third antigen binding loop of
the V.sub.H domain (H3). The V.sub.L gene is produced by the
recombination of only two gene segments, V.sub.L and J.sub.L. In
humans, there are approximately 40 functional V.sub..kappa.
segments (Schble and Zachau (1993) Biol. Chem. Hoppe-Seyler, 374:
1001), 31 functional V.sub..lambda.segments (Williams et al. (1996)
J. Mol. Biol., 264: 220; Kawasaki et al. (1997) Genome Res., 7:
250), 5 functional J.sub..kappa. segments (Hieter et al. (1982) J.
Biol. Chem., 257: 1516) and 4 functional J.sub..lambda.segments
(Vasicek and Leder (1990) J. Exp. Med., 172: 609), depending on the
haplotype. The V.sub.L segment encodes the region of the
polypeptide chain which forms the first and second antigen binding
loops of the V.sub.L domain (L1 and L2), whilst the V.sub.L and
J.sub.L segments combine to form the third antigen binding loop of
the V.sub.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.
[0003] 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; Chothia et al. (1989) Nature, 342: 877). 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; Tomlinson et al.
(1995) EMBO J., 14: 4628; Williams et al. (1996) J. Mol. Biol.,
264: 220). 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; Shirai et al. (1996) FEBS Letters, 399: 1).
[0004] 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. It was found that the two patterns are
complementary: diversity in the primary repertoire is focused at
the centre 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; Ignatovich et al. (1997) J. Mol. Biol, 268:
69). This complementarity seems to have evolved as an efficient
strategy for searching sequence space, given the limited number B
cells available for selection at any 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 optimise residues at the periphery without disrupting
favourable interactions established during the primary
response.
[0005] The recent advent of phage-display technology (Smith (1985)
Science, 228: 1315; Scott and Smith (1990) Science, 249: 386;
McCafferty et al. (1990) Nature, 348: 552) 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; Vaughan et al. (1996) Nature
Biotech., 14: 309) or synthetic libraries whereby germline V gene
segments are `rearranged` in vitro (Hoogenboom & Winter (1992)
J. Mol. Biol., 227: 381; Nissim et al. (1994) EMBO J., 13: 692;
Griffiths et al. (1994) EMBO J., 13: 3245; 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). 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 randomisation 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.
[0006] Recently, in an extension of the synthetic library approach
it has been suggested (WO97/08320, Morphosys) that human antibody
frameworks can be pre-optimised by synthesising 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 recognised 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 is preferable to
optimise 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.
[0007] A further problem with the libraries of the prior art is
that, because the main-chain conformation is heterogeneous,
three-dimensional structural modelling 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
antibody libraries have medium length or long loops and therefore
cannot be modelled.
SUMMARY OF THE INVENTION
[0008] According to the first aspect of the present invention,
there is provided a method for selecting, from a repertoire of
polypeptides, a population of functional polypeptides which bind a
target ligand in a first binding site and a generic ligand in a
second binding site, which generic ligand is capable of binding
functional members of the repertoire regardless of target ligand
specificity, comprising the steps of:
[0009] a) contacting the repertoire with the generic ligand and
selecting functional polypeptides bound thereto; and
[0010] b) contacting the selected functional polypeptides with the
target ligand and selecting a population of polypeptides which bind
to the target ligand.
[0011] The invention accordingly provides a method by which a
repertoire of polypeptides is preselected, according to
functionality as determined by the ability to bind the generic
ligand, and the subset of polypeptides obtained as a result of
preselection is then employed for further rounds of selection
according to the ability to bind the target ligand. Although, in a
preferred embodiment, the repertoire is first selected with the
generic ligand, it will be apparent to one skilled in the art that
the repertoire may be contacted with the ligands in the opposite
order, i.e. with the target ligand before the generic ligand.
[0012] The invention permits the person skilled in the art to
remove, from a chosen repertoire of polypeptides, those
polypeptides which are non-functional, for example as a result of
the introduction of frame-shift mutations, stop codons, folding
mutants or expression mutants which would be or are incapable of
binding to substantially any target ligand. Such non-functional
mutants are generated by the normal randomisation and variation
procedures employed in the construction of polypeptide repertoires.
At the same time the invention permits the person skilled in the
art to enrich a chosen repertoire of polypeptides for those
polypeptides which are functional, well folded and highly
expressed.
[0013] Preferably, two or more subsets of polypeptides are obtained
from a repertoire by the method of the invention, for example, by
prescreening the repertoire with two or more generic ligands, or by
contacting the repertoire with the generic ligand(s) under
different conditions. Advantageously, the subsets of polypeptides
thus obtained are combined to form a further repertoire of
polypeptides, which may be further screened by contacting with
target and/or generic ligands.
[0014] Preferably, the library according to the invention comprises
polypeptides of the immunoglobulin superfamily, such as antibody
polypeptides or T-cell receptor polypeptides. Advantageously, the
library may comprise individual immunoglobulin domains, such as the
V.sub.H or V.sub.L domains of antibodies, or the V.sub..beta. or
V.sub..alpha. domains of T-cell receptors. In a preferred
embodiment, therefore, repertoires of, for example, V.sub.H and
V.sub.L polypeptides may be individually prescreened using a
generic ligand and then combined to produce a functional repertoire
comprising both V.sub.H and V.sub.L polypeptides. Such a repertoire
can then be screened with a target ligand in order to isolate
polypeptides comprising both V.sub.H and V.sub.L domains and having
the desired binding specificity.
[0015] In an advantageous embodiment, the generic ligand selected
for use with imunoglobulin repertoires is a superantigen.
Superantigens are able to bind to functional immunoglobulin
molecules, or subsets thereof comprising particular main-chain
conformations, irrespective of target ligand specificity.
Alternatively, generic ligands may be selected from any ligand
capable of binding to the general structure of the polypeptides
which make up any given repertoire, such as antibodies themselves,
metal ion matrices, organic compounds including proteins or
peptides, and the like.
[0016] In a second aspect, the invention provides a library wherein
the functional members have binding sites for both generic and
target ligands. Libraries may be specifically designed for this
purpose, for example by constructing antibody libraries having a
main-chain conformation which is recognised by a given
superantigen, or by constructing a library in which substantially
all potentially functional members possess a structure recognisable
by a antibody ligand.
[0017] In a third aspect, the invention provides a method for
detecting, immobilising, purifying or immunoprecipitating one or
more members of a repertoire of polypeptides previously selected
according to the invention, comprising binding the members to the
generic ligand.
[0018] In a fourth aspect, the invention provides a library
comprising a repertoire of polypeptides of the immunoglobulin
superfamily, wherein the members of the repertoire have a known
main-chain conformation.
[0019] In a fifth aspect, the invention provides a method for
selecting a polypeptide having a desired generic and/or target
ligand binding site from a repertoire of polypeptides, comprising
the steps of:
[0020] a) expressing a library according to the preceding aspects
of the invention;
[0021] b) contacting the polypeptides with generic and/or target
ligands and selecting those which bind the generic and/or target
ligand; and
[0022] c) optionally amplifying the selected polypeptide(s) which
bind the generic and/or target ligand.
[0023] d) optionally repeating steps a)-c).
[0024] Repertoires of polypeptides are advantageously both
generated and maintained in the form of a nucleic acid library.
Therefore, in a sixth aspect, the invention provides a nucleic acid
library encoding a repertoire of such polypeptides.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1: Bar graph indicating positions in the V.sub.H and
V.sub..kappa. regions of the human antibody repertoire which
exhibit extensive natural diversity and make antigen contacts (see
Tomlinson et al. (1996) J. Mol. Biol., 256: 813). The H3 and the
end of L3 are not shown in this representation although they are
also highly diverse and make antigen contacts. Although sequence
diversity in the human lambda genes has been thoroughly
characterised (see Ignatovich et al. (1997) J. Mol. Biol, 268: 69)
very little data on antigen contacts currently exists for
three-dimensional lambda structures.
[0026] FIG. 2: Sequence of the scFv that forms the basis of a
library according to the invention. There are currently two
versions of the library: a "primary" library wherein 18 positions
are varied and a "somatic" library wherein 12 positions are varied.
The six loop regions H1, H2, H3, L1, L2 and L3 are indicated. CDR
regions as defined by Kabat (Kabat et al. (1991). Sequences of
proteins of immunological interest, U.S. Department of Health and
Human Services) are underlined.
[0027] FIG. 3: Analysis of functionality in a library according to
the invention before and after selecting with the generic ligands
Protein A and Protein L. Here Protein L is coated on an ELISA
plate, the scFv supernatants are bound to it and detection of scFv
binding is with Protein A-HRP. Therefore, only those scFv capable
of binding both Protein A and Protein L give an ELISA signal.
[0028] FIG. 4: Sequences of clones selected from libraries
according to the invention, after panning with bovine ubiquitin,
rat BIP, bovine histone, NIP-BSA, FITC-BSA, human leptin, human
thyroglobulin, BSA, hen egg lysozyme, mouse IgG and human IgG.
[0029] Underlines in the sequences indicate the positions which
were varied in the respective libraries.
[0030] FIG. 5: 5a: Comparison of scFv concentration produced by the
unselected and preselected "primary" DVT libraries in host cells.
5b: standard curve of ELISA as determined from known standards.
[0031] FIG. 6: Western blot of phage from preselected and
unselected DVT "primary" libraries, probed with an anti-phage pIII
antibody in order to determine the percentage of phage bearing
scFv.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Definitions
[0033] Repertoire A repertoire is a population of diverse variants,
for example nucleic acid variants which differ in nucleotide
sequence or polypeptide variants which differ in ammo acid
sequence. A library according to the invention will encompass a
repertoire of polypeptides or nucleic acids. According to the
present invention, a repertoire of polypeptides is designed to
possess a binding site for a generic ligand and a binding site for
a target ligand. The binding sites may overlap, or be located in
the same region of the molecule, but their specificities will
differ.
[0034] Organism As used herein, the term "organism" refers to all
cellular life-forms, such as prokaryotes and eukaryotes, as well as
non-cellular, nucleic acid-containing entities, such as
bacteriophage and viruses.
[0035] Functional As used herein, the term "functional" refers to a
polypeptide which possesses either the native biological activity
of the naturally-produced proteins of its type, or any specific
desired activity, for example as judged by its ability to bind to
ligand molecules, defined below. Examples of "functional"
polypeptides include an antibody binding specifically to an antigen
through its antigen-binding site, a receptor molecule (e.g. a
T-cell receptor) binding its characteristic ligand and an enzyme
binding to its substrate. In order for a polypeptide to be
classified as functional according to the invention, it follows
that it first must be properly processed and folded so as to retain
its overall structural integrity, as judged by its ability to bind
the generic ligand, also defined below.
[0036] For the avoidance of doubt, functionality is not equivalent
to the ability to bind the target ligand. For instance, a
functional anti-CEA monoclonal antibody will not be able to bind
specifically to target ligands such as bacterial LPS. However,
because it is capable of binding a target ligand (i.e. it would be
able bind to CEA if CEA were the target ligand) it is classed as a
"functional" antibody molecule and may be selected by binding to a
generic ligand, as defined below. Typically, non-functional
antibody molecules will be incapable of binding to any target
ligand.
[0037] Generic ligand A generic ligand is a ligand that binds a
substantial proportion of functional members in a given repertoire.
Thus, the same generic ligand can bind many members of the
repertoire regardless of their target ligand specificities (see
below). In general, the presence of functional generic ligand
binding site indicates that the repertoire member is expressed and
folded correctly. Thus, binding of the generic ligand to its
binding site provides a method for preselecting functional
polypeptides from a repertoire of polypeptides.
[0038] Target Ligand The target ligand is a ligand for which a
specific binding member or members of the repertoire is to be
identified. Where the members of the repertoire are antibody
molecules, the target ligand may be an antigen and where the
members of the repertoire are enzymes, the target ligand may be a
substrate. Binding to the target ligand is dependent upon both the
member of the repertoire being functional, as described above under
generic ligand, and upon the precise specificity of the binding
site for the target ligand.
[0039] Subset The subset is a part of the repertoire. In the terms
of the present invention, it is often the case that only a subset
of the repertoire is functional and therefore possesses a
functional generic ligand binding site. Furthermore, it is also
possible that only a fraction of the functional members of a
repertoire (yet significantly more than would bind a given target
ligand) will bind the generic ligand. These subsets are able to be
selected according to the invention.
[0040] Subsets of a library may be combined or pooled to produce
novel repertoires which have been preselected according to desired
criteria. Combined or pooled repertoires may be simple mixtures of
the polypeptide members preselected by generic ligand binding, or
may be manipulated to combine two polypeptide subsets. For example,
V.sub.H and V.sub.L polypeptides may be individually prescreened,
and subsequently combined at the genetic level onto single vectors
such that they are expressed as combined V.sub.H-V.sub.L dimers,
such as scFv.
[0041] Library The term library refers to a mixture of
heterogeneous polypeptides or nucleic acids. The library is
composed of members, which have a single polypeptide or nucleic
acid sequence. To this extent, library is synonymous with
repertoire. Sequence differences between library members are
responsible for the diversity present in the library. The library
may take the form of a simple mixture of polypeptides or nucleic
acids, or may be in the form organisms or cells, for example
bacteria, viruses, animal or plant cells and the like, transformed
with a library of nucleic acids. Preferably, each individual
organism or cell contains only one member of the library.
Advantageously, the nucleic acids are incorporated into expression
vectors, in order to allow expression of the polypeptides encoded
by the nucleic acids. In a preferred aspect, therefore, a library
may take the form of a population of host organisms, each organism
containing one or more copies of an expression vector containing a
single member of the library in nucleic acid form which can be
expressed to produce its corresponding polypeptide member. Thus,
the population of host organisms has the potential to encode a
large repertoire of genetically diverse polypeptide variants.
[0042] Immunoglobulin superfamily This refers to a family of
polypeptides which retain the immunoglobulin fold characteristic of
imunoglobulin (antibody) molecules, which contains two .beta.
sheets and, usually, a conserved disulphide bond. Members of the
immunoglobulin superfamily are involved in many aspects of cellular
and non-cellular interactions in vivo, including widespread roles
in the immune system (for example, antibodies, T-cell receptor
molecules and the like), involvement in cell adhesion (for example
the ICAM molecules) and intracellular signalling (for example,
receptor molecules, such as the PDGF receptor). The present
invention is applicable to all immunoglobulin superfamily
molecules, since variation therein is achieved in similar ways.
Preferably, the present invention relates to immunoglobulins
(antibodies).
[0043] Main-chain conformation The main-chain conformation refers
to the C.alpha. backbone trace of a structure in three-dimensions.
When individual hypervariable loops of antibodies or TCR molecules
are considered the main-chain conformation is synonymous with the
canonical structure. As set forth in Chothia and Lesk (1987) J.
Mol. Biol., 196: 901 and Chothia et al. (1989) Nature, 342: 877,
antibodies display a limited number of canonical structures for
five of their six hypervariable loops (H1, H2, L1, L2 and L3),
despite considerable side-chain diversity in the loops themselves.
The precise canonical structure exhibited depends on the length of
the loop and the identity of certain key residues involved in its
packing. The sixth loop (H3) is much more diverse in both length
and sequence and therefore only exhibits canonical structures for
certain short loop lengths (Martin et al. (1996) J. Mol. Biol.,
263: 800; Shirai et al (1996) FEBS Letters, 399: 1). In the present
invention, all six loops will preferably have canonical structures
and hence the main-chain conformation for the entire antibody
molecule will be known.
[0044] Antibody polypeptide Antibodies are immunoglobulins that are
produced by B cells and form a central part of the host immune
defence system in vertebrates. An antibody polypeptide, as used
herein, is a polypeptide which either is an antibody or is a part
of an antibody, modified or unmodified. Thus, the term antibody
polypeptide includes a heavy chain, a light chain, a heavy
chain-light chain dimer, a Fab fragment, a F(ab')2 fragment, a Dab
fragment, or an Fv fragment, including a single chain Fv (scFv).
Methods for the construction of such antibody molecules are well
known in the art.
[0045] Superantigen Superantigens are antigens, mostly in the form
of toxins expressed in bacteria, which interact with members of the
immunoglobulin superfamily outside the conventional ligand binding
sites for these molecules. Staphylococcal enterotoxins interact
with T-cell receptors and have the effect of stimulating CD4+
T-cells. Superantigens for antibodies include the molecules Protein
G that binds the IgG constant region (Bjorck and Kronvall (1984) J.
Immunol, 133: 969; Reis et al. (1984) J. Immunol., 132: 3091),
Protein A that binds the the IgG constant region and the V.sub.H
domain (Forsgren and Sjoquist (1966) J. Immunol., 97: 822) and
Protein L that binds the V.sub.L domain (Bjorck (1988) J. Immunol.,
140: 1994).
[0046] Preferred Embodiments of the Invention
[0047] The present invention provides a selection system which
eliminates (or significantly reduces the proportion of)
non-functional or poorly folded/expressed members of a polypeptide
library whilst enriching for functional, folded and well expressed
members before a selection for specificity against a "target
ligand" is carried out. A repertoire of polypeptide molecules is
contacted with a "generic ligand", a protein that has affinity for
a structural feature common to all functional, for example complete
and/or correctly folded, proteins of the relevant class. Note that
the term "ligand" is used broadly in reference to molecules of use
in the present invention. As used herein, the term "ligand" refers
to any entity that will bind to or be bound by a member of the
polypeptide library.
[0048] A significant number of defective proteins present in the
initial repertoire fail to bind the generic ligand and are thereby
eliminated. This selective removal of non-functional polypeptides
from a library results in a marked reduction in its actual size,
while its functional size is maintained, with a corresponding
increase in its quality. Polypeptides which are retained by virtue
of binding the generic ligand constitute a `first selected pool` or
`subset` of the original repertoire. Consequently, this `subset` is
enriched for functional, well folded and well expressed members of
the initial repertoire.
[0049] The polypeptides of the first selected pool or subset are
subsequently contacted with at least one "target ligand", which
binds to polypeptides with a given functional specificity. Such
target ligands include, but are not limited to, either half of a
receptor/ligand pair (e.g. a hormone or other cell-signalling
molecule, such as a neurotransmitter, and its cognate receptor),
either of a binding pair of cell adhesion molecules, a protein
substrate that is bound by the active site of an enzyme, a protein,
peptide or small organic compound against which a particular
antibody is to be directed or even an antibody itself.
Consequently, the use of such a library is less labour-intensive
and more economical, in terms of both time and materials, than is
that of a conventional library. In addition, since, compared to a
repertoire which has not been selected with a generic ligand, the
first selected pool will contain a much higher ratio of molecules
able to bind the target ligand to those that are unable to bind the
target ligand, there will be a significant reduction of background
during selection with the "target ligand".
[0050] Combinatorial selection schemes are also contemplated
according to the invention. Multiple selections of the same initial
polypeptide repertoire can be performed in parallel or in series
using different generic and/or target ligands. Thus, the repertoire
can first be selected with a single generic ligand and then
subsequently selected in parallel using different target ligands.
The resulting subsets can then be used separately or combined, in
which case the combined subset will have a range of target ligand
specificities but a single generic ligand specificity.
Alternatively, the repertoire can first be selected with a single
target ligand and then subsequently selected in parallel using
different generic ligands. The resulting subsets can then be used
separately or combined, in which case the combined subset will have
a range of generic ligand specificities but a single target ligand
specificity. The use of more elaborate schemes are also envisaged.
For example, the initial repertoire can be subjected to two rounds
of selection using two different generic ligands, followed by
selection with the target ligand. This produces a subset in which
all members bind both generic ligands and the target ligand.
Alternatively, if the selection of the initial repertoire with the
two generic ligands is performed in parallel and the resulting
subsets combined and then selected with the target ligand the
resulting subset binds at least one of the two generic ligands and
the target ligand. Combined or pooled repertoires may be simple
mixtures of the subsets or may be manipulated to physically link
the subsets. For example, V.sub.H and V.sub.L polypeptides may be
individually selected in parallel by binding two different generic
ligands, and subsequently combined at the genetic level onto single
vectors such that they are expressed as combined V.sub.H-V.sub.L.
This repertoire can then be selected against the target ligand such
that the selected members able to bind both generic ligands and the
target ligand.
[0051] The invention encompasses libraries of functional
polypeptides selected or selectable by the methods broadly
described above, as well as nucleic acid libraries encoding
polypeptide molecules which may be used in a selection performed
according to these methods (preferably, molecules which comprise a
first binding site for a target ligand and a second binding site
for a generic ligand). In addition, the invention provides methods
for detecting, immobilising, purifying or immunoprecipitating one
or more members of a repertoire of functional polypeptides selected
using the generic or target ligands according to the invention.
[0052] The invention is particularly applicable to the enrichment
of libraries of molecules of the immunoglobulin superfamily. This
is particularly true as regards the generation of populations of
antibodies and T-cell receptors which are functional and have a
desired specificity, as is required for use in diagnostic,
therapeutic or prophylactic procedures. To this end, the invention
provides antibody and T-cell receptor libraries wherein all the
members have both natural frameworks and loops of known main-chain
conformation, as well as strategies for useful mutagenesis of the
starting sequence and the subsequent selection of functional
variants so generated. Such polypeptide libraries may comprise
V.sub.H or V.sub..beta. domains or, alternatively, it may comprise
V.sub.L or V.sub..alpha. domains, or even both V.sub.H or
V.sub..beta. and V.sub.L or V.sub..alpha. domains.
[0053] There is significant need in the art for improved libraries
of antibody or T-cell receptor molecules. For example, despite
progress in the creation of "single pot" phage-antibody libraries,
several problems still remain. Natural libraries (Marks et al.
(1991) J. Mol. Biol., 222: 581; Vaughan et al. (1996) Nature
Biotech., 14: 309) which use rearranged V genes harvested from
human B cells are highly biased due to the positive and negative
selection of the B cells in vivo. This can limit the effective size
of phage libraries constructed from rearranged V genes. In
addition, clones derived from natural libraries invariably contain
framework mutations which may effect the antibodies immunogenicity
when used in human therapy. Synthetic libraries (Hoogenboom &
Winter (1992) J. Mol. Biol., 227: 381; Barbas et al. (1992) Proc.
Natl. Acad. Sci. USA, 89: 4457; Nissim et al. (1994) EMBO J., 13:
692; Griffiths et al. (1994) EMBO J., 13: 3245; De Kruif et al.
(1995) J. Mol. Biol., 248: 97) can overcome the problem of bias but
they require the use of long degenerate PCR primers which
frequently introduce base-pair deletions into the assembled V
genes. This high degree of randomisation may also lead to the
creation of antibodies which are unable to fold correctly and are
also therefore non-functional. In many cases it is likely that
these non-functional members will outnumber the functional members
in a library. Even if the frameworks can be pre-optimised for
folding and/or expression (WO97/08320, Morphosys) by synthesising a
set of `master genes` with consensus framework sequences and by
incorporating amino acid substitutions shown to improve folding and
expression, there remains the problem of immunogenicity since, in
most cases, the consensus sequences do not correspond to any
natural framework. Furthermore, since it is likely that the CDR
diversity will also have an effect of folding and/or expression, it
is preferable to optimise the folding and/or expression (and remove
any frame-shifts or stop codons) after the V gene has been fully
assembled.
[0054] A further problem with existing libraries is that because
the main-chain conformation is heterogeneous, three-dimensional
structural modelling 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 antibody libraries have medium
length or long loops and therefore cannot be modelled.
[0055] Another problem with existing libraries is the reliance on
epitope tags (such as the myc, FLAG or HIS tags) for detection of
expressed antibody fragments. As these are usually located at the N
or C terminal ends of the antibody fragment they tend to be prone
to proteolytic cleavage. Superantigens, such as Protein A and
Protein L can be used to detect expressed antibody fragments by
binding the folded domains themselves but since they are V.sub.H
and V.sub.L family specific, only a relatively small proportion of
members of any existing antibody library will bind one of these
reagents and an even smaller proportion will bind to both.
[0056] To this end, it would be desirable to have a selection
system which could eliminate (or at least reduce the proportion of)
non-functional or poorly folded/expressed members of the library
before selection against the target antigen is carried out whilst
enriching for functional, folded and well expressed members all of
which are able to bind generic ligands such as the superantigens
Protein A and Protein L. In addition, it would be advantageous to
construct an antibody library wherein all the members have natural
frameworks and have loops with known main-chain conformations.
[0057] The invention accordingly provides a method by which a
polypeptide repertoire may be selected to remove non-functional
members. This results in a marked reduction in the actual library
size (and a corresponding increase in the quality of the library)
without reducing the functional library size. The invention also
provides a method for creating new polypeptide repertoires wherein
all the functional members are able to bind a given generic ligand.
The same generic ligand can be used for the subsequent detection,
immobilisation, purification or immunoprecipitation of any one or
more members of the repertoire.
[0058] Any `naive` or `immune` antibody repertoire can be used with
the present invention to enrich for functional members and/or to
enrich for members that bind a given generic ligand or ligands.
Indeed, since only a small percentage of all human germ-line
V.sub.H segments bind Protein A with high affinity and only a small
percentage of all human germline V.sub.L segments bind Protein L
with high affinity preselection with these superantigens is highly
advantageous. Alternatively, pre-selection with via the epitope tag
enables non-functional variants to be removed from synthetic
libraries. The libraries that are amenable to preselection include,
but are not limited to, libraries comprised of V genes rearranged
in vivo of the type described by Marks et al. (1991) J. Mol. Biol.,
222: 581 and Vaughan et al. (1996) Nature Biotech., 14: 309,
synthetic libraries whereby germline V gene segments are
`rearranged` in vitro (Hoogenboom & Winter (1992) J. Mol.
Biol., 227: 381; Nissim et al. (1994) EMBO J., 13: 692; Griffiths
et al. (1994) EMBO J., 13: 3245; 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) or into multiple master frameworks (WO97/08320,
Morphosys).
[0059] Selection of polypeptides according to the invention
[0060] Once a diverse pool of polypeptides is generated, selection
according to the invention is applied. Two broad selection
procedures are based upon the order in which the generic and target
ligands are applied; combinatorial variations on these schemes
involve the use of multiple generic and/or target ligands in a
given step of a selection. When a combinatorial scheme is used, the
pool of polypeptide molecules may be contacted with, for example,
several target ligands at once, or by each singly, in series; in
the latter case, the resulting selected pools of polypeptides may
be kept separate or may, themselves, be pooled. These selection
schemes may be summarized as follows:
[0061] a. Selection procedure 1:
[0062] Initial polypeptide selection using the generic ligand
[0063] In order to remove non-functional members of the library, a
generic ligand is selected, such that the generic ligand is only
bound by functional molecules. For example, the generic ligand may
be a metallic ion, an antibody (in the form of a monoclonal
antibody or a polyclonal mixture of antibodies), half of an
enzyme/ligand complex or organic material; note that ligands of any
of these types are, additionally or alternatively, of use as target
ligands according to the invention. Antibody production and metal
affinity chromatography are discussed in detail below. Ideally,
these ligands bind a site (e.g. a peptide tag or superantigen
binding site) on the members of the library which is of constant
structure or sequence, which structure is liable to be absent or
altered in non-functional members. In the case of antibody
libraries, this method is of use to select from a library only
those functional members which have a binding site for a given
superantigen or monoclonal antibody; such an approach is useful in
selecting functional antibody polypeptides from both natural and
synthetic pools thereof.
[0064] The superantigens Protein A and/or Protein L are of use in
the invention as generic ligands to select antibody repertoires,
since they bind correctly folded V.sub.H and V.sub.L domains (which
belong to certain V.sub.H and V.sub.L families), respectively,
regardless of the sequence and structure of the binding site for
the target ligand. In addition, Protein A or another superantigen
Protein G are of use as generic ligands to select for folding
and/or expression by binding the heavy chain constant domains of
antibodies. Anti-.kappa. and anti-.lambda. antibodies are also of
use in selecting light chain constant domains. Small organic
mimetics of antibodies or of other binding proteins, such as
Protein A (Li et al. (1998) Nature Biotech., 16: 190), are also of
use.
[0065] When this selection procedure is used, the generic ligand,
by its very nature, is able to bind all functional members of the
preselected repertoire; therefore, this generic ligand (or some
conjugate thereof) may be used to detect, immobilise, purify or
immunoprecipitate any member or population of members from the
repertoire (whether selected by binding a given target ligand or
not, as discussed below). Protein detection via immunoassay
techniques as well as immunoprecipitation of member polypeptides of
a repertoire of the invention may be performed by the techniques
discussed below with regard to the testing of antibody selection
ligands of use in the invention (see "Antibodies for use as ligands
in polypeptide selection"). Immobilization may be performed through
specific binding of a polypeptide member of a repertoire to either
a generic or target ligand according to the invention which is,
itself, linked to a solid or semi-solid support, such as a filter
(e.g. of nitrocellulose or nylon) or a chromatographic support
(including, but not limited to, a cellulose, polymer, resin or
silica support); covalent attachment of the member polypeptide to
the generic or target ligand may be performed using any of a number
of chemical crosslinking agents known to one of skill in the art.
Immobilization on a metal affinity chromatography support is
described below (see "Metallic ligands as use for the selection of
polypeptides"). Purification may comprise any or a combination of
these techniques, in particular immunoprecipitation and
chromatography by methods well known in the art.
[0066] Using this approach, selection with multiple generic ligands
can be performed either one after another to create a repertoire in
which all members bind two or more generic ligands, separately in
parallel, such that the subsets can then be combined (in this case,
members of the preselected repertoire will bind at least one of the
generic ligands) or separately followed by incorporation into the
same polypeptide chain whereby a large functional library in which
all members may be able to bind all the generic ligands used during
preselection. For example, subsets can be selected from one or more
libraries using different generic ligands which bind heavy and
light chains of antibody molecules (see below) and then combined to
form a heavy/light chain library, in which the heavy and light
chains are either non-covalently associated or are covalently
linked, for example, by using V.sub.H and V.sub.L domains in a
single-chain Fv context.
[0067] Secondary polypeptide selection using the target ligand
[0068] Following the selection step with the generic ligand, the
library is screened in order to identify members that bind to the
target ligand. Since it is enriched for functional polypeptides
after selection with the generic ligand, there will be an
advantageous reduction in non-specific ("background") binding
during selection with the target ligand. Furthermore, since
selection with the generic ligand produces a the marked reduction
in the actual library size (and a corresponding increase in the
quality of the library) without reducing the functional library
size, a smaller repertoire should elicit the same diversity of
target ligand specifities and affinities as the larger starting
repertoire (that contained many non-functional and poorly
folded/expressed members).
[0069] One or more target ligands may be used to select
polypeptides from the first selected polypeptide pool generated
using the generic ligand. In the event that two or more target
ligands are used to generate a number of different subsets, two or
more of these subsets may be combined to form a single, more
complex subset. A single generic ligand is able to bind every
member of the resulting combined subset; however, a given target
ligand binds only a subset of library members.
[0070] b. Selection procedure 2:
[0071] Initial selection of repertoire members with the target
ligand
[0072] Here, selection using the target ligand is performed prior
to selection using the generic ligand. Obviously, the same set of
polypeptides can result from either scheme, if such a result is
desired. Using this approach, selection with multiple target
ligands can be performed in parallel or by mixing the target
ligands for selection. If performed in parallel, the resulting
subsets may, if required, be combined.
[0073] Secondary polypeptide selection using the generic ligand
[0074] Subsequent selection of the target ligand binding subset can
then be performed using one or more generic ligands. Whilst this is
not a selection for function, since members of the repertoire that
are able to bind to the target ligand are by definition functional,
it does enable subsets that bind to different generic ligands to be
isolated. Thus, the target ligand selected population can be
selected by one generic ligand or by two or more generic ligands.
In this case, the generic ligands can be used one after another to
create a repertoire in which all members bind the target ligand and
two or more generic ligands or separately in parallel, such that
different (but possibly overlapping) subsets binding the target
ligand and different generic ligands are created. These can then be
combined (in this case, members will bind at least one of the
generic ligands).
[0075] Selection of immunoglobulin-family polypeptide library
members
[0076] The members of the repertoires or libraries selected in the
present invention advantageously belong to the immunoglobulin
superfamily of molecules, in particular, antibody polypeptides or
T-cell receptor polypeptides. For antibodies, it is envisaged that
the method according to this invention may be applied to any of the
existing antibody libraries known in the art (whether natural or
synthetic) or to antibody libraries designed specifically to be
preselected with generic ligands (see below).
[0077] Construction of libraries of the invention
[0078] a. Selection of the main-chain conformation
[0079] The members of the immunoglobulin superfamily all share a
similar fold for their polypeptide chain. For example, although
antibodies are highly diverse in terms of their primary sequence,
comparison of sequences and crystallographic structures has
revealed that, contrary to expectation, five of the six antigen
binding loops of antibodies (H1, H2, L1, L2, L3) adopt a limited
number of main-chain conformations, or canonical structures
(Chothia and Lesk (1987) supra; Chothia et al (1989) supra).
Analysis of loop lengths and key residues has therefore enabled
prediction of the main-chain conformations of H1, H2, L1, L2 and L3
found in the majority of human antibodies (Chothia et al. (1992)
supra; Tomlinson et al. (1995) supra; 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) supra; Shirai
et al. (1996) supra).
[0080] According to the present invention, libraries of antibody
polypeptides are designed in which certain loop lengths and key
residues have been chosen to ensure that the main- chain
conformation of the members is known. Advantageously, these are
real conformations of immunoglobulin superfamily molecules found in
nature, to minimize the chances that they are non-functional, as
discussed above. Germline V gene segments serve as one suitable
basic framework for constructing antibody or T-cell receptor
libraries; other sequences are also of use. Variations may occur at
a low frequency, such that a small number of functional members may
possess an altered main-chain conformation, which does not affect
its function.
[0081] Canonical structure theory is also of use in the invention
to assess the number of different main-chain conformations encoded
by antibodies, to predict the main-chain conformation based on
antibody sequences and to chose residues for diversification which
do not affect the canonical structure. It is now known that, in the
human V.sub..kappa. domain, the L1 loop can adopt one of four
canonical structures, the L2 loop has a single canonical structure
and that 90% of human V.sub..kappa. domains adopt one of four or
five canonical structures for the L3 loop (Tomlinson et al. (1995)
supra); thus, in the V.sub..kappa. domain alone, different
canonical structures can combine to create a range of different
main-chain conformations. Given that the V.sub..lambda. domain
encodes a different range of canonical structures for the L1, L2
and L3 loops and that V.sub..kappa. and V.sub..lambda. domains can
pair with any V.sub.H domain which can encode several canonical
structures for the H1 and H2 loops, the number of canonical
structure combinations observed for these five loops is very large.
This implies that the generation of diversity in the main-chain
conformation may be essential for the production of a wide range of
binding specificities. However, by constructing an antibody library
based on a single known main-chain conformation it was found,
contrary to expectation, that diversity in the main-chain
conformation is not required to generate sufficient diversity to
target substantially all antigens. Even more surprisingly, the
single main-chain conformation need not be a consensus structure--a
single naturally occurring conformation can be used as the basis
for an entire library. Thus, in a preferred aspect, the invention
provides a library in which the members encode a single known
main-chain conformation. It is to be understood, however, that
occasional variations may occur such that a small number of
functional members may possess an alternative main-chain
conformation, which may be unknown.
[0082] The single main-chain conformation that is chosen is
preferably commonplace among molecules of the immunoglobulin
superfamily type in question. A conformation is commonplace when a
significant number of naturally occurring molecules are observed to
adopt it. Accordingly, in a preferred aspect of the invention, the
natural occurrence of the different main-chain conformations for
each binding loop of an immunoglobulin superfamily molecule are
considered separately and then a naturally occurring immunoglobulin
superfamily molecule is chosen which possesses the desired
combination of main-chain conformations for the different loops. If
none is available, the nearest equivalent may be chosen. Since a
disadvantage of immunoglobulin-family polypeptide libraries of the
prior art is that many members have unnatural frameworks or contain
framework mutations (see above), in the case of antibodies or
T-cell receptors, it is preferable that the desired combination of
main-chain conformations for the different loops is created by
selecting germline gene segments which encode the desired
main-chain conformations. It is more preferable, that the selected
germline gene segments are frequently expressed and most preferable
that they are the most frequently expressed.
[0083] In designing antibody libraries, therefore, the incidence of
the different main-chain conformations for each of the six antigen
binding loops may be considered separately. For H1, H2, L1, L2 and
L3, a given conformation that is adopted by between 20% and 100% of
the antigen binding loops of naturally occurring molecules is
chosen. Typically, its observed incidence is above 35% (i.e.
between 35% and 100%) and, ideally, above 50% or even above 65%.
Since the vast majority of H3 loops do not have canonical
structures, it is preferable to select a main-chain conformation
which is commonplace among those loops which do display canonical
structures. For each of the loops, the conformation which is
observed most often in the natural repertoire is therefore
selected. In human antibodies, the most popular canonical
structures (CS) for each loop are as follows: H1-CS 1 (79% of the
expressed repertoire), H2-CS 3 (46%), L1-CS 2 of V.sub..kappa.
(39%), L2-CS 1 (100 %), L3-CS 1 of V.sub..kappa. (36%) (calculation
assumes a .kappa.:.lambda. ratio of 70:30, Hood et al. (1967) Cold
Spring Harbor Symp. Quant. Biol, 48: 133). For H3 loops that have
canonical structures, a CDR3 length (Kabat et al. (1991) Sequences
of proteins of immunological interest, U.S. Department of Health
and Human Services) of seven residues with a salt-bridge from
residue 94 to residue 101 appears to be the most common. There are
at least 16 human antibody sequences in the EMBL data library with
the required H3 length and key residues to form this conformation
and at least two crystallographic structures in the protein data
bank which can be used as a basis for antibody modelling (2cgr and
1tet). The most frequently expressed germline gene segments that
this combination of canonical structures are the V.sub.H segment
3-23 (DP-47), the J.sub.H segment JH4b, the V.sub..kappa. segment
02/012 (DPK9) and the J.sub..kappa. segment J.sub..kappa.1. These
segments can therefore be used in combination as a basis to
construct a library with the desired single main-chain
conformation.
[0084] Alternatively, instead of choosing the single main-chain
conformation based on the natural occurrence of the different
main-chain conformations for each of the binding loops in
isolation, the natural occurrence of combinations of main-chain
conformations is used as the basis for choosing the single
main-chain conformation. In the case of antibodies, for example,
the natural occurrence of canonical structure combinations for any
two, three, four, five or for all six of the antigen binding loops
can be determined. Here, it is preferable that the chosen
conformation is commonplace in naturally occurring antibodies and
most preferable that it observed most frequently in the natural
repertoire. Thus, in human antibodies, for example, when natural
combinations of the five antigen binding loops, H1, H2, L1, L2 and
L3, are considered, the most frequent combination of canonical
structures is determined and then combined with the most popular
conformation for the H3 loop, as a basis for choosing the single
main-chain conformation.
[0085] b. Diversification of the canonical sequence
[0086] Having selected several known main-chain conformations or,
preferably a single known main-chain conformation, the library of
the invention is constructed by varying the binding site of the
molecule in order to generate a repertoire with structural and/or
functional diversity. This means that variants are generated such
that they possess sufficient diversity in their structure and/or in
their function so that they are capable of providing a range of
activities. For example, where the polypeptides in question are
cell-surface receptors, they may possess a diversity of target
ligand binding specificities.
[0087] The desired diversity is typically generated by varying the
selected molecule at one or more positions. The positions to be
changed can be chosen at random or are preferably selected. The
variation can then be achieved either by randomization, during
which the resident amino acid is replaced by any amino acid or
analogue thereof, natural or synthetic, producing a very large
number of variants or by replacing the resident amino acid with one
or more of a defined subset of amino acids, producing a more
limited number of variants.
[0088] Various methods have been reported for introducing such
diversity. Error-prone PCR (Hawkins et al. (1992) J. Mol. Biol.,
226: 889), chemical mutagenesis (Deng et al. (1994) J. Biol. Chem.,
269: 9533) or bacterial mutator strains (Low et al. (1996) J. Mol.
Biol., 260: 359) can be used to introduce random mutations into the
genes that encode the molecule. Methods for mutating selected
positions are also well known in the art and include the use of
mismatched oligonucleotides or degenerate oligonucleotides, with or
without the use of PCR. For example, several synthetic antibody
libraries have been created by targeting mutations to the antigen
binding loops. The H3 region of a human tetanus toxoid-binding Fab
has been randomized to create a range of new binding specificities
(Barbas et al. (1992) supra). Random or semi-random H3 and L3
regions have been appended to germline V gene segments to produce
large libraries with unmutated framework regions (Hoogenboom and
Winter (1992) supra; Nissim et al. (1994) supra; Griffiths et al.
(1994) supra; De Kruif et al. (1995) supra). Such diversification
has been extended to include some or all of the other antigen
binding loops (Crameri et al. (1996) Nature Med., 2: 100; Riechmann
et al. (1995) Bio/Technology, 13: 475; Morphosys, WO97108320,
supra).
[0089] Since loop randomization has the potential to create
approximately more than 10.sup.15 structures for H3 alone and a
similarly large number of variants for the other five loops, it is
not feasible using current transformation technology or even by,
using cell free systems to produce a library representing all
possible combinations. For example, in one of the largest libraries
constructed to date, 6.times.10.sup.10 different antibodies, which
is only a fraction of the potential diversity for a library of this
design, were generated (Griffiths et al. (1994) supra).
[0090] In addition to the removal of non-functional members and the
use of a single known main-chain conformation, the present
invention addresses these limitations by diversifying only those
residues which are directly involved in creating or modifying the
desired function of the molecule. For many molecules, the function
will be to bind a target ligand and therefore diversity should be
concentrated in the target ligand binding site, while avoiding
changing residues which are crucial to the overall packing of the
molecule or to maintaining the chosen main-chain conformation;
therefore, the invention provides a library wherein the selected
positions to be varied may be those that constitute the binding
site for the target ligand.
[0091] Diversification of the canonical sequence as it applies to
antibodies
[0092] In the case of an antibody library, the binding site for the
target ligand is most often the antigen binding site. Thus, in a
highly preferred aspect, the invention provides an antibody library
in which only those residues in the antigen binding site are
varied. These residues are extremely diverse in the human antibody
repertoire and are known to make contacts in high-resolution
antibody/antigen complexes. For example, in L2 it is known that
positions 50 and 53 are diverse in naturally occurring antibodies
and are observed to make contact with the antigen. In contrast, the
conventional approach would have been to diversify all the residues
in the corresponding Complementarity Determining Region (CDR1) as
defined by Kabat et al. (1991, supra), some seven residues compared
to the two diversified in the library according to the invention.
This represents a significant improvement in terms of the
functional diversity required to create a range of antigen binding
specificities.
[0093] In nature, antibody diversity is the result of two
processes: somatic recombination of germline V, D and J gene
segments to create a naive primary repertoire (so called germline
and junctional diversity) and somatic hypermutation of the
resulting rearranged V genes. Analysis of human antibody sequences
has shown that diversity in the primary repertoire is focused at
the centre of the antigen binding site whereas somatic
hypermutation spreads diversity to regions at the periphery of the
antigen binding site that are highly conserved in the primary
repertoire (see Tomlinson et al. (1996) supra). This
complementarity has probably evolved as an efficient strategy for
searching sequence space and, although apparently unique to
antibodies, it can easily be applied to other polypeptide
repertoires according to the invention. According to the invention,
the residues which are varied are a subset of those that form the
binding site for the target ligand. Different (including
overlapping) subsets of residues in the target ligand binding site
are diversified at different stages during selection, if
desired.
[0094] In the case of an antibody repertoire, the two-step process
of the invention is analogous to the maturation of antibodies in
the human immune system. An initial `naive` repertoire is created
where some, but not all, of the residues in the antigen binding
site are diversified. As used herein in this context, the term
"naive" refers to antibody molecules that have no pre-determined
target ligand. These molecules resemble those which are encoded by
the immunoglobulin genes of an individual who has not undergone
immune diversification, as is the case with fetal and newborn
individuals, whose immune systems have not yet been challenged by a
wide variety of antigenic stimuli. This repertoire is then selected
against a range of antigens. If required, further diversity can
then be introduced outside the region diversified in the initial
repertoire. This matured repertoire can be selected for modified
function, specificity or affinity.
[0095] The invention provides two different naive repertoires of
antibodies in which some or all of the residues in the antigen
binding site are varied. The "primary" library mimics the natural
primary repertoire, with diversity restricted to residues at the
centre of the antigen binding site that are diverse in the germline
V gene segments (germline diversity) or diversified during the
recombination process (junctional diversity). Those residues which
are diversified include, but are not limited to, H50, H52, H52a,
H53, H55, H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93,
L94 and L96. In the "somatic" library, diversity is restricted to
residues that are diversified during the recombination process
(junctional diversity) or are highly somatically mutated). Those
residues which are diversified include, but are not limited to:
H31, H33, H35, H95, H96, H97, H98, L30, L31, L32, L34 and L96. All
the residues listed above as suitable for diversification in these
libraries are known to make contacts in one or more
antibody-antigen complexes. Since in both libraries, not all of the
residues in the antigen binding site are varied, additional
diversity is incorporated during selection by varying the remaining
residues, if it is desired to do so. It shall be apparent to one
skilled in the art that any subset of any of these residues (or
additional residues which comprise the antigen binding site) can be
used for the initial and/or subsequent diversification of the
antigen binding site.
[0096] In the construction of libraries according to the invention,
diversification of chosen positions is typically achieved at the
nucleic acid level, by altering the coding sequence which specifies
the sequence of the polypeptide such that a number of possible
amino acids (all 20 or a subset thereof) can be incorporated at
that position. Using the IUPAC nomenclature, the most versatile
codon is NNK, which encodes all amino acids as well as the TAG stop
codon. The NNK codon is preferably used in order to introduce the
required diversity. Other codons which achieve the same ends are
also of use, including the NNN codon, which leads to the production
of the additional stop codons TGA and TAA.
[0097] A feature of side-chain diversity in the antigen binding
site of human antibodies is a pronounced bias which favors certain
amino acid residues. If the amino acid composition of the ten most
diverse positions in each of the V.sub.H, V.sub..kappa. and
V.sub..lambda. regions are summed, more than 76% of the side-chain
diversity comes from only seven different residues, these being,
serine (24%), tyrosine (14%), asparagine (11%), glycine (9%),
alanine (7%), aspartate (6%) and threonine (6%). This bias towards
hydrophilic residues and small residues which can provide
main-chain flexibility probably reflects the evolution of surfaces
which are predisposed to binding a wide range of antigens and may
help to explain the required promiscuity of antibodies in the
primary repertoire.
[0098] Since it is preferable to mimic this distribution of amino
acids, the invention provides a library wherein the distribution of
amino acids at the positions to be varied mimics that seen in the
antigen binding site of antibodies. Such bias in the substitution
of amino acids that permits selection of certain polypeptides (not
just antibody polypeptides) against a range of target ligands is
easily applied to any polypeptide repertoire according to the
invention. There are various methods for biasing the amino acid
distribution at the position to be varied (including the use of
tri-nucleotide mutagenesis, WO97/08320, Morphosys, supra), of which
the preferred method, due to ease of synthesis, is the use of
conventional degenerate codons. By comparing the amino acid profile
encoded by all combinations of degenerate codons (with single,
double, triple and quadruple degeneracy in equal ratios at each
position) with the natural amino acid use it is possible to
calculate the most representative codon. The codons (AGT)(AGC)T,
(AGT)(AGC)C and (AGT)(AGC)(CT)--that is, DVT, DVC and DVY,
respectively using IUPAC nomenclature--are those closest to the
desired amino acid profile: they encode 22% serine and 11%
tyrosine, asparagine, glycine, alanine, aspartate, threonine and
cysteine. Preferably, therefore, libraries are constructed using
either the DVT, DVC or DVY codon at each of the diversified
positions.
[0099] As stated above, polypeptides which make up antibody
libraries according to the invention may be whole antibodies or
fragments thereof, such as Fab, F(ab').sub.2, Fv or scFv fragments,
or separate V.sub.H or V.sub.L domains, any of which is either
modified or unmodified. Of these, single-chain Fv fragments, or
scFvs, are of particular use. ScFv fragments, as well as other
antibody polypeptides, are reliably generated by antibody
engineering methods well known in the art. The scFv is formed by
connecting the V.sub.H and V.sub.L genes using an oligonucleotide
that encodes an appropriately designed linker peptide, such as
(Gly-Gly-Gly-Gly-Ser).sub.3 or equivalent linker peptide(s). The
linker bridges the C-terminal end of the first V region and
N-terminal end of the second V region, ordered as either
V.sub.H-linker-V.sub.L or V.sub.L-linker-V.sub.H. In principle, the
binding site of the scFv can faithfully reproduce the specificity
of the corresponding whole antibody and vice-versa.
[0100] Similar techniques for the construction of Fv, Fab and
F(ab').sub.2 fragments, as well as chimeric antibody molecules are
well known in the art. When expressing Fv fragments, precautions
should be taken to ensure correct chain folding and association.
For Fab and F(ab').sub.2 fragments, V.sub.H and V.sub.L
polypeptides are combined with constant region segments, which may
be isolated from rearranged genes, germline C genes or synthesised
from antibody sequence data as for V region segments. A library
according to the invention may be a V.sub.H or V.sub.L library.
Thus, separate libraries comprising single V.sub.H and V.sub.L
domains may be constructed and, optionally, include C.sub.H or
C.sub.L domains, respectively, creating Dab molecules.
[0101] c. Library vector systems according to the invention
[0102] Libraries according to the invention can be used for direct
screening using the generic and/or target ligands or used in a
selection protocol that involves a genetic display package.
[0103] Bacteriophage lambda expression systems may be screened
directly as bacteriophage plaques or as colonies of lysogens, both
as previously described (Huse et al. (1989) Science, 246: 1275;
Caton and Koprowski (1990) Proc. Natl. Acad. Sci. U.S.A., 87;
Mullinax et al. (1990) Proc. Natl. Acad. Sci. U.S.A., 87: 8095;
Persson et al. (1991) Proc. Natl. Acad. Sci. U.S.A., 88: 2432) and
are of use in the invention. Whilst such expression systems can be
used to screening up to 10.sup.6 different members of a library,
they are not really suited to screening of larger numbers (greater
than 10.sup.6 members). Other screening systems rely, for example,
on direct chemical synthesis of library members. One early method
involves the synthesis of peptides on a set of pins or rods, such
as described in WO84/03564. A similar method involving peptide
synthesis on beads, which forms a peptide library in which each
bead is an individual library member, is described in U.S. Pat. No.
4,631,211 and a related method is described in WO92/0009 1. A
significant improvement of the bead-based methods involves tagging
each bead with a unique identifier tag, such as an oligonucleotide,
so as to facilitate identification of the amino acid sequence of
each library member. These improved bead-based methods are
described in WO93/06121.
[0104] Another chemical synthesis method involves the synthesis of
arrays of peptides (or peptidomimnetics) on a surface in a manner
that places each distinct library member (e.g., unique peptide
sequence) at a discrete, predefined location in the array. The
identity of each library member is determined by its spatial
location in the array. The locations in the array where binding
interactions between a predetermined molecule (e.g., a receptor)
and reactive library members occur is determined, thereby
identifying the sequences of the reactive library members on the
basis of spatial location. These methods are described in U.S. Pat.
No. 5,143,854; WO90/15070 and WO92/10092; Fodor et al. (1991)
Science, 251: 767; Dower and Fodor (1991) Ann. Rep. Med. Chem., 26:
271.
[0105] Of particular use in the construction of libraries of the
invention are selection display systems, which enable a nucleic
acid to be linked to the polypeptide it expresses. As used herein,
a selection display system is a system that permits the selection,
by suitable display means, of the individual members of the library
by binding the generic and/or target ligands.
[0106] Any selection display system may be used in conjunction with
a library according to the invention. Selection protocols for
isolating desired members of large libraries are known in the art,
as typified by phage display techniques. Such systems, in which
diverse peptide sequences are displayed on the surface of
filamentous bacteriophage (Scott and Smith (1990) supra), have
proven useful for creating libraries of antibody fragments (and the
nucleotide sequences that encoding them) for the in vitro selection
and amplification of specific antibody fragments that bind a target
antigen. The nucleotide sequences encoding the V.sub.H and V.sub.L
regions are linked to gene fragments which encode leader signals
that direct them to the periplasmic space of E. coli and as a
result the resultant antibody fragments are displayed on the
surface of the bacteriophage, typically as fusions to bacteriophage
coat proteins (e.g., pIII or pVIII). Alternatively, antibody
fragments are displayed externally on lambda phage capsids
(phagebodies). An advantage of phage-based display systems is that,
because they are biological systems, selected library members can
be amplified simply by growing the phage containing the selected
library member in bacterial cells. Furthermore, since the
nucleotide sequence that encode the polypeptide library member is
contained on a phage or phagemid vector, sequencing, expression and
subsequent genetic manipulation is relatively straightforward.
[0107] Methods for the construction of bacteriophage antibody
display libraries and lambda phage expression libraries are well
known in the art (McCafferty et al. (1990) supra; Kang et al.
(1991) Proc. Natl. Acad. Sci. U.S.A., 88: 4363; Clackson et al.
(1991) Nature, 352: 624; Lowman et al. (1991) Biochemistry, 30:
10832; Burton et al. (1991) Proc. Natl. Acad. Sci U.S.A., 88:
10134; Hoogenboom et al. (1991) Nucleic Acids Res., 19: 4133; Chang
et al. (1991) J. Immunol., 147: 3610; Breitling et al. (1991) Gene,
104: 147; Marks et al. (1991) supra; Barbas et al. (1992) supra;
Hawkins and Winter (1992) J. Immunol., 22: 867; Marks et al., 1992,
J. Biol. Chem., 267: 16007; Lerner et al. (1992) Science, 258:
1313, incorporated herein by reference).
[0108] One particularly advantageous approach has been the use of
scFv phage-libraries (Huston et al., 1988, Proc. Natl. Acad. Sci
U.S.A., 85: 5879-5883; Chaudhary et al. (1990) Proc. Natl. Acad.
Sci U.S.A., 87: 1066-1070; McCafferty et al. (1990) supra; Clackson
et al. (1991) supra; Marks et al. (1991) supra; Chiswell et al.
(1992) Trends Biotech., 10: 80; Marks et al. (1992) supra). Various
embodiments of scFv libraries displayed on bacteriophage coat
proteins have been described. Refinements of phage display
approaches are also known, for example as described in WO96/06213
and WO92/01047 (Medical Research Council et al.) and WO97/08320
(Morphosys, supra), which are incorporated herein by reference.
[0109] Other systems for generating libraries of polypeptides or
nucleotides involve the use of cell-free enzymatic machinery for
the in vitro synthesis of the library members. In one method, RNA
molecules are selected by alternate rounds of selection against a
target ligand and PCR amplification (Tuerk and Gold (1990) Science,
249: 505; Ellington and Szostak (1990) Nature, 346: 818). A similar
technique may be used to identify DNA sequences which bind a
predetermined human transcription factor (Thiesen and Bach (1990)
Nucleic Acids Res., 18: 3203; Beaudry and Joyce (1992) Science,
257: 635; WO92/05258 and WO92/14843). In a similar way, in vitro
translation can be used to synthesise polypeptides as a method for
generating large libraries. These methods which generally comprise
stabilised polysome complexes, are described further in WO88108453,
WO90/05785, WO90/07003, WO91/02076, WO91/05058, and WO92/02536.
Alternative display systems which are not phage-based, such as
those disclosed in WO95/22625 and WO95/11922 (Affymax) use the
polysomes to display polypeptides for selection. These and all the
foregoing documents also are incorporated herein by reference.
[0110] The invention accordingly provides a method for selecting a
polypeptide having a desired generic and/or target ligand binding
site from a repertoire of polypeptides, comprising the steps
of:
[0111] a) expressing a library according to the preceding aspects
of the invention;
[0112] b) contacting the polypeptides with the generic and/or
target ligand and selecting those which bind the generic and/or
target ligand; and
[0113] c) optionally amplifying the selected polypeptide(s) which
bind the generic and/or target ligand.
[0114] d) optionally repeating steps a)-c).
[0115] Preferably, steps a)-d) are performed using a phage display
system.
[0116] Since the invention provides a library of polypeptides which
have binding sites for both generic and target ligands the above
selection method can be applied to a selection using either the
generic ligand or the target ligand. Thus, the initial library can
be selected using the generic ligand and then the target ligand or
using the target ligand and then the generic ligand. The invention
also provides for multiple selections using different generic
ligands either in parallel or in series before or after selection
with the target ligand.
[0117] Preferably, the method according to the invention further
comprises the steps of subjecting the selected polypeptide(s) to
additional variation (as described herein) and repeating steps a)
to d).
[0118] Since the generic ligand, by its very nature, is able to
bind all library members selected using the generic ligand, the
method according to the invention further comprises the use of the
generic ligand (or some conjugate thereof) to detect, immobilise,
purify or immunoprecipitate any functional member or population of
members from the library (whether selected by binding the target
ligand or not).
[0119] Since the invention provides a library in which the members
have a known main-chain conformation the method according to the
invention further comprises the production of a three-dimensional
structural model of any functional member of the library (whether
selected by binding the target ligand or not). Preferably, the
building of such a model involves homology modelling and/or
molecular replacement. A preliminary model of the main-chain
conformation can be created by comparison of the polypeptide
sequence to the sequence of a known three-dimensional structure, by
secondary structure prediction or by screening structural
libraries. Computational software may also be used to predict the
secondary structure of the polypeptide. In order to predict the
conformations of the side-chains at the varied positions, a
side-chain rotamer library may be employed.
[0120] In general, the nucleic acid molecules and vector constructs
required for the performance of the present invention are available
in the art and may be constructed and manipulated as set forth in
standard laboratory manuals, such as Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
USA.
[0121] The manipulation of nucleic acids in the present invention
is typically carried out in recombinant vectors. As used herein,
vector refers to a discrete element that is used to introduce
heterologous DNA into cells for the expression and/or replication
thereof. Methods by which to select or construct and, subsequently,
use such vectors are well known to one of moderate skill in the
art. Numerous vectors are publicly available, including bacterial
plasmids, bacteriophage, artificial chromosomes and episomal
vectors. Such vectors may be used for simple cloning and
mutagenesis; alternatively, as is typical of vectors in which
repertoire (or pre-repertoire) members of the invention are
carried, a gene expression vector is employed. A vector of use
according to the invention may be selected to accommodate a
polypeptide coding sequence of a desired size, typically from 0.25
kilobase (kb) to 40 kb in length. A suitable host cell is
transformed with the vector after in vitro cloning manipulations.
Each vector contains various functional components, which generally
include a cloning (or "polylinker") site, an origin of replication
and at least one selectable marker gene. If given vector is an
expression vector, it additionally possesses one or more of the
following: enhancer element, promoter, transcription termination
and signal sequences, each positioned in the vicinity of the
cloning site, such that they are operatively linked to the gene
encoding a polypeptide repertoire member according to the
invention.
[0122] Both cloning and expression vectors generally contain
nucleic acid sequences that enable the vector to replicate in one
or more selected host cells. Typically in cloning vectors, this
sequence is one that enables the vector to replicate independently
of the host chromosomal DNA and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2 micron plasmid origin is suitable for
yeast, and various viral origins (e.g. SV 40, adenovirus) are
useful for cloning vectors in mammalian cells. Generally, the
origin of replication is not needed for mammalian expression
vectors unless these are used in mammalian cells able to replicate
high levels of DNA, such as COS cells.
[0123] Advantageously, a cloning or expression vector may contain a
selection gene also referred to as selectable marker. This gene
encodes a protein necessary for the survival or growth of
transformed host cells grown in a selective culture medium. Host
cells not transformed with the vector containing the selection gene
will therefore not survive in the culture medium. Typical selection
genes encode proteins that confer resistance to antibiotics and
other toxins, e.g. ampicillin, neomycin, methotrexate or
tetracycline, complement auxotrophic deficiencies, or supply
critical nutrients not available in the growth media.
[0124] Since the replication of vectors according to the present
invention is most conveniently performed in E. coli, an E.
coli-selectable marker, for example, the .beta.-lactamase gene that
confers resistance to the antibiotic ampicillin, is of use. These
can be obtained from E. coli plasmids, such as pBR322 or a pUC
plasmid such as pUC18 or pUC19.
[0125] Expression vectors usually contain a promoter that is
recognised by the host organism and is operably linked to the
coding sequence of interest. Such a promoter may be inducible or
constitutive. The term "operably linked" refers to a juxtaposition
wherein the components described are in a relationship permitting
them to function in their intended manner. A control sequence
"operably linked" to a coding sequence is ligated in such a way
that expression of the coding sequence is achieved under conditions
compatible with the control sequences.
[0126] Promoters suitable for use with prokaryotic hosts include,
for example, the .beta.-lactamase and lactose promoter systems,
alkaline phosphatase, the tryptophan (trp) promoter system and
hybrid promoters such as the tac promoter. Promoters for use in
bacterial systems will also generally contain a Shine-Dalgarno
sequence operably linked to the coding sequence.
[0127] In the library according to the present invention, the
preferred vectors are expression vectors that enables the
expression of a nucleotide sequence corresponding to a polypeptide
library member. Thus, selection with the generic and/or target
ligands can be performed by separate propagation and expression of
a single clone expressing the polypeptide library member or by use
of any selection display system. As described above, the preferred
selection display system is bacteriophage display. Thus, phage or
phagemid vectors may be used. The preferred vectors are phagemid
vectors which have an E. coli. origin of replication (for double
stranded replication) and also a phage origin of replication (for
production of single-stranded DNA). The manipulation and expression
of such vectors is well known in the art (Hoogenboom and Winter
(1992) supra; Nissim et al. (1994) supra). Briefly, the vector
contains a P-lactamase gene to confer selectivity on the phagemid
and a lac promoter upstream of a expression cassette that consists
(N to C terminal) of a pelB leader sequence (which directs the
expressed polypeptide to the periplasmic space), a multiple cloning
site (for cloning the nucleotide version of the library member),
optionally, one or more peptide tag (for detection), optionally,
one or more TAG stop codon and the phage protein pIII. Thus, using
various suppressor and non-suppressor strains of E. coli and with
the addition of glucose, iso-propyl thio-.beta.-D-galactosi- de
(IPTG) or a helper phage, such as VCS M13, the vector is able to
replicate as a plasmid with no expression, produce large quantities
of the polypeptide library member only or produce phage, some of
which contain at least one copy of the polypeptide-pull fusion on
their surface.
[0128] Construction of vectors according to the invention employs
conventional ligation techniques. Isolated vectors or DNA fragments
are cleaved, tailored, and religated in the form desired to
generate the required vector. If desired, analysis to confirm that
the correct sequences are present in the constructed vector can be
performed in a known fashion. Suitable methods for constructing
expression vectors, preparing in vitro transcripts, introducing DNA
into host cells, and performing analyses for assessing expression
and function are known to those skilled in the art. The presence of
a gene sequence in a sample is detected, or its amplification
and/or expression quantified by conventional methods, such as
Southern or Northern analysis, Western blotting, dot blotting of
DNA, RNA or protein, in situ hybridization, immunocytochemistry or
sequence analysis of nucleic acid or protein molecules. Those
skilled in the art will readily envisage how these methods may be
modified, if desired.
[0129] Mutagenesis using the polymerase chain reaction (PCR)
[0130] Once a vector system is chosen and one or more nucleic acid
sequences encoding polypeptides of interest are cloned into the
library vector, one may generate diversity within the cloned
molecules by undertaking mutagenesis prior to expression;
alternatively, the encoded proteins may be expressed and selected,
as described above, before mutagenesis and additional rounds of
selection are performed. As stated above, mutagenesis of nucleic
acid sequences encoding structurally optimized polypeptides, is
carried out by standard molecular methods. Of particular use is the
polymerase chain reaction, or PCR, (Mullis and Faloona (1987)
Methods Enzymol., 155: 335, herein incorporated by reference). PCR,
which uses multiple cycles of DNA replication catalyzed by a
thermostable, DNA-dependent DNA polymerase to amplify the target
sequence of interest, is well known in the art.
[0131] Oligonucleotide primers useful according to the invention
are single-stranded DNA or RNA molecules that hybridize to a
nucleic acid template to prime enzymatic synthesis of a second
nucleic acid strand. The primer is complementary to a portion of a
target molecule present in a pool of nucleic acid molecules used in
the preparation of sets of arrays of the invention. It is
contemplated that such a molecule is prepared by synthetic methods,
either chemical or enzymatic. Alternatively, such a molecule or a
fragment thereof is naturally occurring, and is isolated from its
natural source or purchased from a commercial supplier. Mutagenic
oligonucleotide primers are 15 to 100 nucleotides in length,
ideally from 20 to 40 nucleotides, although oligonucleotides of
different length are of use.
[0132] Typically, selective hybridization occurs when two nucleic
acid sequences are substantially complementary (at least about 65%
complementary over a stretch of at least 14 to 25 nucleotides,
preferably at least about 75%, more preferably at least about 90%
complementary). See Kanehisa (1984) Nucleic Acids Res. 12: 203,
incorporated herein by reference. As a result, it is expected that
a certain degree of mismatch at the priming site is tolerated. Such
mismatch may be small, such as a mono-, di- or tri-nucleotide.
Alternatively, it may comprise nucleotide loops, which we define as
regions in which mismatch encompasses an uninterrupted series of
four or more nucleotides.
[0133] Overall, five factors influence the efficiency and
selectivity of hybridization of the primer to a second nucleic acid
molecule. These factors, which are (i) primer length, (ii) the
nucleotide sequence and/or composition, (iii) hybridization
temperature, (iv) buffer chemistry and (v) the potential for steric
hindrance in the region to which the primer is required to
hybridize, are important considerations when non-random priming
sequences are designed.
[0134] There is a positive correlation between primer length and
both the efficiency and accuracy with which a primer will anneal to
a target sequence; longer sequences have a higher melting
temperature (T.sub.M) than do shorter ones, and are less likely to
be repeated within a given target sequence, thereby minimizing
promiscuous hybridization. Primer sequences with a high G-C content
or that comprise palindromic sequences tend to self-hybridize, as
do their intended target sites, since unimolecular, rather than
bimolecular, hybridization kinetics are genererally favored in
solution; at the same time, it is important to design a primer
containing sufficient numbers of G-C nucleotide pairings to bind
the target sequence tightly, since each such pair is bound by three
hydrogen bonds, rather than the two that are found when A and T
bases pair. Hybridization temperature varies inversely with primer
annealing efficiency, as does the concentration of organic
solvents, e.g. formamide, that might be included in a hybridization
mixture, while increases in salt concentration facilitate binding.
Under stringent hybridization conditions, longer probes hybridize
more efficiently than do shorter ones, which are sufficient under
more permissive conditions. Stringent hybridization conditions
typically include salt concentrations of less than about 1M, more
usually less than about 500 mM and preferably less than about 200
mM. Hybridization temperatures range from as low as 0.degree. C. to
greater than 22.degree. C., greater than about 30.degree. C., and
(most often) in excess of about 37.degree. C. Longer fragments may
require higher hybridization temperatures for specific
hybridization. As several factors affect the stringency of
hybridization, the combination of parameters is more important than
the absolute measure of any one alone.
[0135] Primers are designed with these considerations in mind.
While estimates of the relative merits of numerous sequences may be
made mentally by one of skill in the art, computer programs have
been designed to assist in the evaluation of these several
parameters and the optimization of primer sequences. Examples of
such programs are "PrimerSelect" of the DNAStar.TM. software
package (DNAStar, Inc.; Madison, Wis.) and OLIGO 4.0 (National
Biosciences, Inc.). Once designed, suitable oligonucleotides are
prepared by a suitable method, e.g. the phosphoramidite method
described by Beaucage and Carruthers (1981) Tetrahedron Lett., 22:
1859) or the triester method according to Matteucci and Caruthers
(1981) J. Am. Chem. Soc., 103: 3185, both incorporated herein by
reference, or by other chemical methods using either a commercial
automated oligonucleotide synthesizer or VLSIPS.TM. technology.
[0136] PCR is performed using template DNA (at least 1 fg; more
usefully, 1-1000 ng) and at least 25 pmol of oligonucleotide
primers; it may be advantageous to use a larger amount of primer
when the primer pool is heavily heterogeneous, as each sequence is
represented by only a small fraction of the molecules of the pool,
and amounts become limiting in the later amplification cycles. A
typical reaction mixture includes: 2 .mu.l of DNA, 25 pmol of
oligonucleotide primer, 2.5 .mu.l of 10X PCR buffer 1
(Perkin-Elmer, Foster City, Calif.), 0.4 .mu.l of 1.25 .mu.M dNTP,
0.15 .mu.l (or 2.5 units) of Taq DNA polymerase (Perkin Elmer,
Foster City, Calif.) and deionized water to a total volume of 25
.mu.l. Mineral oil is overlaid and the PCR is performed using a
programmable thermal cycler.
[0137] The length and temperature of each step of a PCR cycle, as
well as the number of cycles, is adjusted in accordance to the
stringency requirements in effect. Annealing temperature and timing
are determined both by the efficiency with which a primer is
expected to anneal to a template and the degree of mismatch that is
to be tolerated; obviously, when nucleic acid molecules are
simultaneously amplified and mutagenized, mismatch is required, at
least in the first round of synthesis. In attempting to amplify a
population of molecules using a mixed pool of mutagenic primers,
the loss, under stringent (high-temperature) annealing conditions,
of potential mutant products that would only result from low
melting temperatures is weighed against the promiscuous annealing
of primers to sequences other than the target site. The ability to
optimize the stringency of primer annealing conditions is well
within the knowledge of one of moderate skill in the art. An
annealing temperature of between 30.degree. C. and 72.degree. C. is
used. Initial denaturation of the template molecules normally
occurs at between.92.degree. C. and 99.degree. C. for 4 minutes,
followed by 20-40 cycles consisting of denaturation (94-99.degree.
C. for 15 seconds to 1 minute), annealing (temperature determined
as discussed above; 1-2 minutes), and extension (72.degree. C. for
1-5 minutes, depending on the length of the amplified product).
Final extension is generally for 4 minutes at 72.degree. C., and
may be followed by an indefinite (0-24 hour) step at 4.degree.
C.
[0138] Structural analysis of repertoire members
[0139] Since the invention provides a repertoire of polypeptides of
known main-chain conformation, a three-dimensional structural model
of any member of the repertoire is easily generated. Typically, the
building of such a model involves homology modelling and/or
molecular replacement. A preliminary model of the main-chain
conformation is created by comparison of the polypeptide sequence
to a similar sequence of known three-dimensional structure, by
secondary structure prediction or by screening structural
libraries. Molecular modelling computer software packages are
commercially available, and are useful in predicting polypeptide
secondary structures. In order to predict the conformations of the
side-chains at the varied positions, a side-chain rotamer library
may be employed.
[0140] Antibodies for use as ligands in polypeptide selection
[0141] A generic or target ligand to be used in the polypeptide
selection according to the present invention may, itself, be an
antibody. This is particularly true of generic ligands, which bind
to structural features that are substantially conserved in
functional polypeptides to be selected for inclusion in repertoires
of the invention. If an appropriate antibody is not publicly
available, it may be produced by phage display methodology (see
above) or as follows:
[0142] Either recombinant proteins or those derived from natural
sources can be used to generate antibodies using standard
techniques, well known to those in the field. For example, the
protein (or "immunogen") is administered to challenge a mammal such
as a monkey, goat, rabbit or mouse. The resulting antibodies can be
collected as polyclonal sera, or antibody-producing cells from the
challenged animal can be immortalized (e.g. by fusion with an
immortalizing fusion partner to produce a hybridoma), which cells
then produce monoclonal antibodies.
[0143] a. Polyclonal antibodies
[0144] The antigen protein is either used alone or conjugated to a
conventional carrier in order to increases its immunogenicity, and
an antiserum to the peptide-carrier conjugate is raised in an
animal, as described above. Coupling of a peptide to a carrier
protein and immunizations may be performed as described (Dymecki et
al. (1992) J. Biol. Chem., 267: 4815). The serum is titered against
protein antigen by ELISA or alternatively by dot or spot blotting
(Boersma and Van Leeuwen (1994) J. Neurosci. Methods, 51: 317). The
serum is shown to react strongly with the appropriate peptides by
ELISA, for example, following the procedures of Green et al. (1982)
Cell, 28: 477.
[0145] b. Monoclonal antibodies
[0146] Techniques for preparing monoclonal antibodies are well
known, and monoclonal antibodies may be prepared using any
candidate antigen, preferably bound to a carrier, as described by
Arnheiter et al. (1981) Nature, 294, 278. Monoclonal antibodies are
typically obtained from hybridoma tissue cultures or from ascites
fluid obtained from animals into which the hybridoma tissue was
introduced. Nevertheless, monoclonal antibodies may be described as
being "raised against" or "induced by" a protein.
[0147] After being raised, monoclonal antibodies are tested for
function and specificity by any of a number of means. Similar
procedures can also be used to test recombinant antibodies produced
by phage display or other in vitro selection technologies.
Monoclonal antibody-producing hybridomas (or polyclonal sera) can
be screened for antibody binding to the immunogen, as well.
Particularly preferred immunological tests include enzyme-linked
immunoassays (ELISA), immunoblotting and immunoprecipitation (see
Voller, (1978) Diagnostic Horizons, 2: 1, Microbiological
Associates Quarterly Publication, Walkersville, Md.; Voller et al.
(1978) J. Clin. Pathol., 31: 507; U.S. Reissue Pat. No. 31,006; UK
Patent 2,019,408; Butler (1981) Methods Enzymol., 73: 482; Maggio,
E. (ed.), (1980) Enzyme Immunoassay, CRC Press, Boca Raton, Fla.)
or radioimmunoassays (RIA) (Weintraub, B., Principles of
radioimmunoassays, Seventh Training Course on Radioligand Assay
Techniques, The Endocrine Society, March 1986, pp. 1-5, 46-49 and
68-78), all to detect binding of the antibody to the immunogen
against which it was raised. It will be apparent to one skilled in
the art that either the antibody molecule or the immunogen must be
labeled to facilitate such detection. Techniques for labeling
antibody molecules are well known to those skilled in the art (see
Harlour and Lane (1989) Antibodies, Cold Spring Harbor Laboratory,
pp. 1-726).
[0148] Alternatively, other techniques can be used to detect
binding to the immunogen, thereby confirming the integrity of the
antibody which is to serve either as a generic antigen or a target
antigen according to the invention. These include chromatographic
methods such as SDS PAGE, isoelectric focusing, Western blotting,
HPLC and capillary electrophoresis.
[0149] "Antibodies" are defined herein as constructions using the
binding (variable) region of such antibodies, and other antibody
modifications. Thus, an antibody useful in the invention may
comprise whole antibodies, antibody fragments, polyfunctional
antibody aggregates, or in general any substance comprising one or
more specific binding sites from an antibody. The antibody
fragments may be fragments such as Fv, Fab and F(ab').sub.2
fragments or any derivatives thereof, such as a single chain Fv
fragments. The antibodies or antibody fragments may be
non-recombinant, recombinant or humanized. The antibody may be of
any immunoglobulin isotype, e.g., IgG, IgM, and so forth. In
addition, aggregates, polymers, derivatives and conjugates of
immunoglobulins or their fragments can be used where
appropriate.
[0150] The invention is further described, for the purposes of
illustration only, in the following examples.
[0151] Metallic ions as ligands for the selection of
polypeptides
[0152] As stated above, ligands other than antibodies are of use in
the selection of polypeptides according to the invention. One such
category of ligand is that of metallic ions. For example, one may
wish to preselect a repertoire for the presence of a functional
histidine (HIS) tag using a Ni-NTA matrix. Immobilized metal
affinity chromatography (IMAC; Hubert and Porath (1980) J.
Chromatography, 98: 247) takes advantage of the metal-binding
properties of histidine and cysteine amino acid residues, as well
as others that may bind metals, on the exposed surfaces of numerous
proteins. It employs a resin, typically agarose, comprising a
bidentate metal chelator (e.g. iminodiacetic acid, IDA, a
dicarboxylic acid group) to which is complexed metallic ions; in
order to generate a metallic-ion-bearing resin according to the
invention, agarose/IDA is mixed with a metal salt (for example,
CuCl.sub.2 2H.sub.2O), from which the IDA chelates the divalent
cations. One commercially available agarose/IDA preparation is
"CHELATING SEPHAROSE 6B" (Pharmacia Fine Chemicals; Piscataway,
N.J.). Metallic ion that are of use include, but are not limited
to, the divalent cations Ni.sup.2+, Cu.sup.2+, Zn.sup.2+ and
Co.sup.2+. A pool of polypeptide molecules is prepared in a binding
buffer which consists essentially of salt (typically, NaCl or KCl)
at a 0.1- to 1.0M concentration and a weak ligand (such as Tris or
ammonia), the latter of which has affinity for the metallic ions of
the resin, but to a lesser degree than does a polypeptide to be
selected according to the invention. Useful concentrations of the
weak ligand range from 0.01- to 0.1M in the binding buffer.
[0153] The polypeptide pool is contacted with the resin under
conditions which permit polypeptides having metal-binding domains
(see below) to bind; after impurities are washed away, the selected
polypeptides are eluted with a buffer in which the weak ligand is
present in a higher concentration than in the binding buffer,
specifically, at a concentration sufficient for the weak ligand to
displace the selected polypeptides, despite its lower binding
affinity for the metallic ions. Useful concentrations of the weak
ligand in the elution buffer are 10- to 50-fold higher than in the
binding buffer, typically from 0.1 to 0.3 M; note that the
concentration of salt in the elution buffer equals that in the
binding buffer. According to the methods of the present invention,
the metallic ions of the resin typically serve as the generic
ligand; however, it is contemplated that they may also be used as
the target ligand.
[0154] IMAC is carried out using a standard chromatography
apparatus (columns, through which buffer is drawn by gravity,
pulled by a vacuum or driven by pressure); alternatively, a
large-batch procedure is employed, in which the metal-bearing resin
is mixed, in slurry form, with the polypeptide pool from which
members of a repertoire of the invention are to be selected.
[0155] Partial purification of a serum T4 protein by IMAC has been
described (Staples et al., U.S. Pat. No. 5,169,936); however, the
broad spectrum of proteins comprising surface-exposed metal-binding
domains also encompasses other soluble T4 proteins, human serum
proteins (e.g. IgG, haptoglobin, hemopexin, Gc-globulin, Clq, C3,
C4), human desmoplasmin, Dolichos biflorus lectin, zinc-inhibited
Tyr(P) phosphatases, phenolase, carboxypeptidase isoenzymes, Cu,Zn
superoxide dismutases (including those of humans and all other
eukaryotes), nucleoside diphosphatase, leukocyte interferon,
lactoferrin, human plasma .alpha..sub.2-SH glycoprotein,
.beta..sub.2-macroglobulin, .alpha..sub.1-antitrypsin, plasminogen
activator, gastrointestinal polypeptides, pepsin, human and bovine
serum albumin, granule proteins from granulocytes, lysozymes,
non-histone proteins, human fibrinogen, human serum transferrin,
human lymphotoxin, calmodulin, protein A, avidin, myoglobins,
somatomedins, human growth hormone, transforming growth factors,
platelet-derived growth factor, .alpha.-human atrial natriuretic
polypeptide, cardiodilatin and others. In addition, extracellular
domain sequences of membrane-bound proteins may be purified using
IMAC. Note that repertoires comprising any of the above proteins or
metal-binding variants thereof may be produced according to the
methods of the invention.
[0156] Following elution, selected polypeptides are removed from
the metal binding buffer and placed in a buffer appropriate to
their next use. If the metallic ion has been used to generate a
first selected polypeptide pool according to the invention, the
molecules of that pool are placed into a buffer that is optimized
for binding with the second ligand to be used in selection of the
members of the functional polypeptide repertoire. If the metal is,
instead, used in the second selection step, the polypeptides of the
repertoire are transferred to a buffer suitable either to storage
(e.g. a 0.5% glycine buffer) or the use for which they are
intended. Such buffers include, but are not limited to: water,
organic solvents, mixtures of water and water-miscible organic
solvents, physiological salt buffers and protein/nucleic acid or
protein/protein binding buffers. Alternatively, the polypeptide
molecules may be dehydrated (i.e. by lyophilization) or immobilized
on a solid or semi-solid support, such as a nitrocellulose or nylon
filtration membrane or a gel matrix (i.e. of agarose or
polyacrylamide) or crosslinked to a chromatography resin.
[0157] Polypeptide molecules may be removed from the elution buffer
by any of a number of methods known in the art. The polypeptide
eluate may be dialyzed against water or another solution of choice;
if the polypeptides are to be lyophilized, water to which has been
added protease inhibitors (e.g. pepstatin, aprotinin, leupeptin, or
others) is used. Alternatively, the sample may be subjected to
ammonium sulfate precipitation, which is well known in the art,
prior to resuspension in the medium of choice.
[0158] Use of polypeptides selected according to the invention
[0159] Polypeptides selected according to the method of the present
invention may be employed in substantially any process which
involves ligand-polypeptide binding, including in vivo therapeutic
and prophylactic applications, in vitro and in vivo diagnostic
applications, in vitro assay and reagent applications, and the
like. For example, in the case of antibodies, antibody molecules
may be used in antibody based assay techniques, such as ELISA
techniques, according to methods known to those skilled in the
art.
[0160] As alluded to above, the molecules selected according to the
invention are of use in diagnostic, prophylactic and therapeutic
procedures. For example, enzyme variants generated and selected by
these methods may be assayed for activity, either in vitro or in
vivo using techniques well known in the art, by which they are
incubated with candidate substrate molecules and the conversion of
substrate to product is analyzed. Selected cell-surface receptors
or adhesion molecules might be expressed in cultured cells which
are then tested for their ability to respond to biochemical stimuli
or for their affinity with other cell types that express
cell-surface molecules to which the undiversified adhesion molecule
would be expected to bind, respectively. Antibody polypeptides
selected according to the invention are of use diagnostically in
Western analysis and in situ protein detection by standard
immunohistochemical procedures; for use in these applications, the
antibodies of a selected repertoire may be labelled in accordance
with techniques known to the art. In addition, such antibody
polypeptides may be used preparatively in affinity chromatography
procedures, when complexed to a chromatographic support, such as a
resin. All such techniques are well known to one of skill in the
art.
[0161] Therapeutic and prophylactic uses of proteins prepared
according to the invention involve the administration of
polypeptides selected according to the invention to a recipient
mammal, such as a human. Of particular use in this regard are
antibodies, other receptors (including, but not limited to T-cell
receptors) and in the case in which an antibody or receptor was
used as either a generic or target ligand, proteins which bind to
them.
[0162] Substantially pure antibodies or binding proteins thereof of
at least 90 to 95% homogeneity are preferred for administration to
a mammal, and 98 to 99% or more homogeneity is most preferred for
pharmaceutical uses, especially when the mammal is a human. Once
purified, partially or to homogeneity as desired, the selected
polypeptides may be used diagnostically or therapeutically
(including extracorporeally) or in developing and performing assay
procedures, immunofluorescent stainings and the like (Lefkovite and
Pernis, (1979 and 1981) Immunological Methods, Volumes I and II,
Academic Press, N.Y.).
[0163] The selected antibodies or binding proteins thereof of the
present invention will typically find use in preventing,
suppressing or treating inflammatory states, allergic
hypersensitivity, cancer, bacterial or viral infection, and
autoimmune disorders (which include, but are not limited to, Type I
diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus
erythematosus, Crohn's disease and myasthenia gravis).
[0164] In the instant application, the term "prevention" involves
administration of the protective composition prior to the induction
of the disease. "Suppression" refers to administration of the
composition after an inductive event, but prior to the clinical
appearance of the disease. "Treatment" involves administration of
the protective composition after disease symptoms become
manifest.
[0165] Animal model systems which can be used to screen the
effectiveness of the antibodies or binding proteins thereof in
protecting against or treating the disease are available. Methods
for the testing of systemic lupus erythematosus (SLE) in
susceptible mice are known in the art (Knight et al. (1978) J. Exp.
Med., 147: 1653; Reinersten et al. (1978) New Eng. J. Med., 299:
515). Myasthenia Gravis (MG) is tested in SJL/J female mice by
inducing the disease with soluble AchR protein from another species
(Lindstrom et al. (1988) Adv. Immunol., 42: 233). Arthritis is
induced in a susceptible strain of mice by injection of Type II
collagen (Stuart et al. (1984) Ann. Rev. Immunol., 42: 233). A
model by which adjuvant arthritis is induced in susceptible rats by
injection of mycobacterial heat shock protein has been described
(Van Eden et al. (1988) Nature, 331: 171). Thyroiditis is induced
in mice by administration of thyroglobulin as described (Maron et
al. (1980) J. Exp. Med., 152: 1115). Insulin dependent diabetes
mellitus (IDDM) occurs naturally or can be induced in certain
strains of mice such as those described by Kanasawa et al. (1984)
Diabetologia, 27: 113. EAE in mouse and rat serves as a model for
MS in human. In this model, the demyelinating disease is induced by
administration of myelin basic protein (see Paterson (1986)
Textbook of Immunopathology, Mischer et al., eds., Grune and
Stratton, N.Y., pp. 179-213; McFarlin et al. (1973) Science, 179:
478: and Satoh et al. (1987) J. Immunol., 138: 179).
[0166] The selected antibodies, receptors (including, but not
limited to T-cell receptors) or binding proteins thereof of the
present invention may also be used in combination with other
antibodies, particularly monoclonal antibodes (MAbs) reactive with
other markers on human cells responsible for the diseases. For
example, suitable T-cell markers can include those grouped into the
so-called "Clusters of Differentiation," as named by the First
International Leukocyte Differentiation Workshop (Bernhard et al.
(1984) Leukocyte Typing, Springer Verlag, N.Y.).
[0167] Generally, the present selected antibodies, receptors or
binding proteins will be utilized in purified form together with
pharmacologically appropriate carriers. Typically, these carriers
include aqueous or alcoholic/aqueous solutions, emulsions or
suspensions, any including saline and/or buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride and lactated Ringer's. Suitable
physiologically-acceptable adjuvants, if necessary to keep a
polypeptide complex in suspension, may be chosen from thickeners
such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and
alginates.
[0168] Intravenous vehicles include fluid and nutrient replenishers
and electrolyte replenishers, such as those based on Ringer's
dextrose. Preservatives and other additives, such as
antimicrobials, antioxidants, chelating agents and inert gases, may
also be present (Mack (1982) Remington's Pharmaceutical Sciences,
16th Edition).
[0169] The selected polypeptides of the present invention may be
used as separately administered compositions or in conjunction with
other agents. These can include various immunotherapeutic drugs,
such as cylcosporine, methotrexate, adriamycin or cisplatinum, and
immunotoxins. Pharmaceutical compositions can include "cocktails"
of various cytotoxic or other agents in conjunction with the
selected antibodies, receptors or binding proteins thereof of the
present invention, or even combinations of selected polypeptides
according to the present invention having different specificities,
such as polypeptides selected using different target ligands,
whether or not they are pooled prior to administration.
[0170] The route of administration of pharmaceutical compositions
according to the invention may be any of those commonly known to
those of ordinary skill in the art. For therapy, including without
limitation immunotherapy, the selected antibodies, receptors or
binding proteins thereof of the invention can be administered to
any patient in accordance with standard techniques. The
administration can be by any appropriate mode, including
parenterally, intravenously, intramuscularly, intraperitoneally,
transdermally, via the pulmonary route, or also, appropriately, by
direct infusion with a catheter. The dosage and frequency of
administration will depend on the age, sex and condition of the
patient, concurrent administration of other drugs,
counterindications and other parameters to be taken into account by
the clinician.
[0171] The selected polypeptides of this invention can be
lyophilized for storage and reconstituted in a suitable carrier
prior to use. This technique has been shown to be effective with
conventional immunoglobulins and art-known lyophilization and
reconstitution techniques can be employed. It will be appreciated
by those skilled in the art that lyophilization and reconstitution
can lead to varying degrees of antibody activity loss (e.g. with
conventional immunoglobulins, IgM antibodies tend to have greater
activity loss than IgG antibodies) and that use levels may have to
be adjusted upward to compensate.
[0172] The compositions containing the present selected
polypeptides or a cocktail thereof can be administered for
prophylactic and/or therapeutic treatments. In certain therapeutic
applications, an adequate amount to accomplish at least partial
inhibition, suppression, modulation, killing, or some other
measurable parameter, of a population of selected cells is defined
as a "therapeutically-effective dose". Amounts needed to achieve
this dosage will depend upon the severity of the disease and the
general state of the patient's own immune system, but generally
range from 0.005 to 5.0 mg of selected antibody, receptor (e.g. a
T-cell receptor) or binding protein thereof per kilogram of body
weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly
used. For prophylactic applications, compositions containing the
present selected polypeptides or cocktails thereof may also be
administered in similar or slightly lower dosages.
[0173] A composition containing a selected polypeptide according to
the present invention may be utilized in prophylactic and
therapeutic settings to aid in the alteration, inactivation,
killing or removal of a select target cell population in a mammal.
In addition, the selected repertoires of polypeptides described
herein may be used extracorporeally or in vitro selectively to
kill, deplete or otherwise effectively remove a target cell
population from a heterogeneous collection of cells. Blood from a
mammal may be combined extracorporeally with the selected
antibodies, cell-surface receptors or binding proteins thereof
whereby the undesired cells are killed or otherwise removed from
the blood for return to the mammal in accordance with standard
techniques.
[0174] The invention is further described, for the purposes of
illustration only, in the following examples.
EXAMPLE 1
[0175] Antibody library design
[0176] A. Main-chain conformation
[0177] For five of the six antigen binding loops of human
antibodies (L1, L2, L3, H1 and H2) there are a limited number of
main-chain conformations, or canonical structures ((Chothia et al.
(1992) J. Mol. Biol., 227: 799; Tomlinson et al. (1995) EMBO J.,
14: 4628; Williams et al. (1996) J. Mol. Biol., 264: 220). The most
popular main-chain conformation for each of these loops is used to
provide a single known main-chain conformation according to the
invention. These are: H1-CS 1 (79% of the expressed repertoire),
H2-CS 3 (46%), L1-CS 2 of V.sub..kappa. (39%), L2-CS 1 (100%),
L3-CS1 of V.sub..kappa. (36%). The H3 loop forms a limited number
of main-chain conformations for short loop lengths (Martin et al.
(1996) J. Mol. Biol., 263: 800; Shirai et al. (1996) FEBS Letters,
399: 1). Thus, where the H3 has a CDR3 length (as defined by Kabat
et al. (1991). Sequences of proteins of immunological interest,
U.S. Department of Health and Human Services) of seven residues and
has a lysine or arginine residue at position H94 and an aspartate
residue at position H101 a salt-bridge is formed between these two
residues and in most cases a single main-chain conformation is
likely to be produced. There are at least 16 human antibody
sequences in the EMBL data library with the required H3 length and
key residues to form this conformation and at least two
crystallographic structures in the protein data bank which can be
used as a basis for antibody modelling (2cgr and 1tet).
[0178] In this case, the most frequently expressed germline gene
segments which encode the desired loop lengths and key residues to
produce the required combinations of canonical structures are the
V.sub.H segment 3-23 (DP-47), the J.sub.H segment JH4b, the
V.sub..kappa. segment 02/012 (DPK9) and the J.sub..kappa. segment
J.sub..kappa.1. These segments can therefore be used in combination
as a basis to construct a library with the desired single
main-chain conformation. The V.sub..kappa. segment 02/012 (DPK9) is
member of the V.sub..kappa.1 family and therefore will bind the
superantigen Protein L. The V.sub.H segment 3-23 (DP47) is a member
of the V.sub.H3 family and therefore should bind the superantigen
Protein A, which can then be used as a generic ligand.
[0179] B. Selection of positions for variation
[0180] Analysis of human V.sub.H and V.sub..kappa. sequences
indicates that the most diverse positions in the mature repertoire
are those that make the most contacts with antigens (see Tomlinson
et al., (1996) J. Mol. Biol., 256: 813; FIG. 1). These positions
form the functional antigen binding site and are therefore selected
for side-chain diversification (FIG. 2). H54 is a key residue and
points away from the antigen binding site in the chosen H2
canonical structure 3 (the diversity seen at this position is due
to canonical structures 1, 2 and 4 where H54 points into the
binding site). In this case H55 (which points into the binding
site) is diversified instead. The diversity at these positions is
created either by germline or junctional diversity in the primary
repertoire or by somatic hypermutation (Tomlinson et al., (1996) J.
Mol. Biol., 256: 813; FIG. 1). Two different subsets of residues in
the antigen binding site were therefore varied to create two
different library formats. In the "primary" library the residues
selected for variation are from H2, H3, L2 and L3 (diversity in
these loops is mainly the result of germline or junctional
diversity). The positions varied in this library are: H50, H52,
H52a, H53, H55, H56, H58, H95, H96, H97, H98, L50, L53, L91, L92,
L93, L94 and L96 (18 residues in total, FIG. 2). In the "somatic"
library the residues selected for variation are from H1, H3, L1 and
the end of L3 (diversity here is mainly the result of somatic
hypermutation or junctional diversity). The positions varied in
this library are: H31, H33, H35, H95, H96, H97, H98, L30, L31, L32,
L34 and L96 (12 residues in total, FIG. 2).
[0181] C. Selection of amino acid use at the positions to be
varied
[0182] Side-chain diversity is introduced into the "primary" and
"somatic" libraries by incorporating either the codon NNK (which
encodes all 20 amino acids, including the TAG stop codon, but not
the TGA and TAA stop codons) or the codon DVT (which encodes 22%
serine and 11% tyrosine, asparagine, glycine, alanine, aspartate,
threonine and cysteine and using single, double, triple and
quadruple degeneracy in equal ratios at each position, most closely
mimics the distribution of amino acid residues for in the antigen
binding sites of natural human antibodies).
EXAMPLE 2
[0183] Library construction and selection with the generic
ligands
[0184] The "primary" and "somatic" libraries were assembled by PCR
using the oligonucleotides listed in Table 1 and the germline V
gene segments DPK9 (Cox et al. (1994) Eur. J. Immunol., 24: 827)
and DP-47 (Tomlinson et al. (1992) J. Mol. Biol., 227: 7768).
Briefly, first round of amplification was performed using pairs of
5' (back) primers in conjunction with NNK or DVT 3' (forward)
primers together with the corresponding germline V gene segment as
template (see Table 1). This produces eight separate DNA fragments
for each of the NNK and DVT libraries. A second round of
amplification was then performed using the 5' (back) primers and
the 3' (forward) primers shown in Table 1 together with two of the
purified fragments from the first round of amplification. This
produces four separate fragments for each of the NNK and DVT
libraries (a "primary" V.sub.H fragment, SA; a "primary"
V.sub..kappa. fragment, 6A; a "somatic" V.sub.H fragment, 5B; and a
"somatic" V.sub..kappa. fragment, 6B).
[0185] Each of these fragments was cut and then ligated into
pCLEANV.sub.H (for the V.sub.H fragments) or pCLEANVK (for the
V.sub..kappa. fragments) which contain dummy V.sub.H and
V.sub..kappa. domains, respectively in a version of pHEN1 which
does not contain any TAG codons or peptide tags (Hoogenboom &
Winter (1992) J. Mol. Biol., 227: 381). The ligations were then
electroporated into the non-suppressor E. Coli. strain HB2151.
Phage from each of these libraries was produced and separately
selected using immunotubes coated with 10 .mu.g/ml of the generic
ligands Protein A and Protein L for the V.sub.H and V.sub..kappa.
libraries, respectively. DNA from E. Coli. infected with selected
phage was then prepared and cut so that the dummy V.sub..kappa.
inserts were replaced by the corresponding V.sub..kappa. libraries.
Electroporation of these libraries results in the following insert
library sizes: 9.21.times.10.sup.8 ("primary" NNK),
5.57.times.10.sup.8 ("primary" DVT), 1.00.times.10.sup.9 ("somatic"
NNK) and 2.38.times.10.sup.8 ("somatic" DVT). As a control for
pre-selection four additional libraries were created but without
selection with the generic ligands Protein A and Protein L: insert
library sizes for these libraries were 1.29.times.10.sup.9
("primary" NNK), 2.40.times.10.sup.8 ("primary" DVT),
1.16.times.10.sup.9 ("somatic" NNK) and 2.17.times.10.sup.8
("somatic" DVT).
[0186] To verify the success of the pre-selection step, DNA from
the selected and unselected "primary" NNK libraries was cloned into
a pUC based expression vector and electroporated into HB2151. 96
clones were picked at random from each recloned library and induced
for expression of soluble scFv fragments. Production of functional
scFv is assayed by ELISA using Protein L to capture the scFv and
then Protein A-HRP conjugate to detect binding. Only scFv which
express functional V.sub.H and V.sub..kappa. domains (no
frame-shifts, stop codons, folding or expression mutations) will
give a signal using this assay. The number of functional antibodies
in each library (ELISA signals above background) was 5% with the
unselected "primary" NNK library and 75% with the selected version
of the same (FIG. 3). Sequencing of clones which were negative in
the assay confirmed the presence of frame-shifts, stop codons, PCR
mutations at critical framework residues and amino acids in the
antigen binding site which must prevent folding and/or
expression.
EXAMPLE 3
[0187] Library selection against target ligands
[0188] The "primary" and "somatic" NNK libraries (without
pre-selection) were separately selected using five antigens (bovine
ubiquitin, rat BIP, bovine histone, NIP-BSA and hen egg lysozyme)
coated on immunotubes at various concentrations. After 2-4 rounds
of selection, highly specific antibodies were obtained to all
antigens except hen egg lysozyme. Clones were selected at random
for sequencing demonstrating a range of antibodies to each antigen
(FIG. 4).
[0189] In the second phase, phage from the pre-selected NNK and DVT
libraries were mixed 1:1 to create a single "primary" library and a
single "somatic" library. These libraries were then separately
selected using seven antigens (FITC-BSA, human leptin, human
thyroglobulin, BSA, hen egg lysozyme, mouse IgG and human IgG)
coated on immunotubes at various concentrations. After 2-4 rounds
of selection, highly specific antibodies were obtained to all the
antigens, including hen egg lysozyme which failed to produce
positives in the previous phase of selection using the libraries
that had not been pre-selected using the generic ligands. Clones
were selected at random for sequencing, demonstrating a range of
different antibodies to each antigen (FIG. 4).
EXAMPLE 4
[0190] Effect of pre-selection on scFv expression and production of
phage bearing scFv
[0191] To further verify the outcome of the pre-selection, DNA from
the unselected and pre-selected "primary" DVT libraries is cloned
into a pUC based expression vector and electroporated into HB2151,
yielding 10.sup.5 clones in both cases. 96 clones are picked at
random from each recloned library and induced for expression of
soluble scFv fragments. Production of functional scFv is again
assayed using Protein L to capture the scFv followed by the use of
Protein A-HRP to detect bound scFv. The percentage of functional
antibodies in each library is 35.4% (unselected) and 84.4%
(pre-selected) indicating a 2.4 fold increase in the number of
functional members as a result of pre-selection with Protein A and
Protein L (the increase is less pronounced than with the equivalent
NNK library since the DVT codon does not encode the TAG stop codon.
In the unselected NNK library, the presence of a TAG stop codon in
a non-suppressor strain such as HB2151 will lead to termination and
hence prevent functional scFv expression. Pre-selection of the NNK
library removes clones containing TAG stop codons to produce a
library in which a high proportion of members express soluble
scFv.)
[0192] In order to assess the effect to pre-selection of the
"primary" DVT library on total scFv expression, the recloned
unselected and pre-selected libraries (each containing 10.sup.5
clones in a pUC based expression vector) are induced for polyclonal
expression of scFv fragments. The concentration of expressed scFv
in the supernatant is then determined by incubating two fold
dilutions (columns 1-12 in FIG. 5a) of the supernatants on Protein
L coated ELISA plate, followed by detection with Protein A-HRP,
ScFvs of known concentration are assayed in parallel to quantify
the levels of scFv expression in the unselected and pre-selected
DVT libraries. These are used to plot a standard curve (FIG. 5b)
and from this the expression levels of the unselected and
pre-selected "primary" DVT libraries are calculated as 12.9 .mu./ml
and 67.1 .mu.g/ml respectively i.e. a 5.2 fold increase in
expression due to pre-selection with Protein A and Protein L.
[0193] To assess the amount of phage bearing scFv, the unselected
and pre-selected "primary" DVT libraries are grown and polyclonal
phage is produced. Equal volumes of phage from the two libraries
are run under denaturing conditions on a 4-12% Bis-Tris NuPAGE Gel
with MES running buffer. The resulting gel is western blotted,
probed using an anti-pIII antibody and exposed to X-ray film (FIG.
6). The lower band in each case corresponds to pIII protein alone,
whilst the higher band contains the pIII-scFv fusion protein.
Quantification of the band intensities using the software package
NIH image indicates that pre-selection results in an 11.8 fold
increase in the amount of fusion protein present in the phage.
Indeed, 43% of the total pIIl in the pre-selected phage exists as
pIII-scFv fusion, suggesting that most phage particles will have at
least one scFv displayed on the surface.
[0194] Hence, not only does pre-selection using generic ligands
enable enrichment of functional members from a repertoire but it
also leads to preferential selection of those members which are
well expressed and (if required) are able to elicit a high level of
display on the surface of phage without being cleaved by bacterial
proteases.
1TABLE 1 PCR Primers for the Assembly of the "Primary" and
"Somatic" Antibody Libraries Template 1st round of amplification 1A
DP-47 5' (back) primer GAGGTGCAGCTGTTGGAGTC DVT 3' (forward)
GCCCTTCACGGAGTCTGCGTAMNNTG-
TMNNMNNACCMNNMNNMNNAATMNNTGAGACCCACTCCAGCCC primer NNK 3'(forward)
TABHTGAGACCCACTCCAGCCCGCCCTTCACGGAGTCTGCGTAABHTGTABHABHACCABH-
ABHABHAA primer 2A DP-47 5' (back) primer CGCAGACTCCGTGAAGGGC DVT
3' (forward) TCCCTGGCCCCAGTAGTCAAAMNNMNN-
MNNMNNTTTCGCACAGTAATATACGG primer NNK 3' (forward)
TCCCTGGCCCCAGTAGTCAAAABHABHABHABHTTTCGCACAGTAATATACGG primer 3A
DPK9 5' (back) primer GACATCCAGATGACCCAGTC DVT 3' (forward)
ATGGGACCCCACTTTGCAAMNNGGATGCMNNATAGATCAGGAGCTTAGGGG primer NNK 3'
(forward) ATGGGACCCCACTTTGCAAABHGGATGCABHATAGATCAG- GAGCTTAGGGG
primer 4A DPK9 5' (back) primer TTGCAAAGTGGGGTCCCAT DVT 3'
(forward) CTTGGTCCCTTGGCCGAACGTMNNAGG-
MNNMNNMNNMNNCTGTTGACAGTAGTAAGTTGC primer NNK 3' (forward)
CTTGGTCCCTTGGCCGAACGTABHAGGABHABHABHABHCTGTTGACAGTAGTAAGTTGC primer
1B DP-47 5' (back) primer GAGGTGCAGCTGTTGGAGTC DVT 3' (forward)
CTGGAGCCTGGCGGACCCAMNNCATM- NNATAMNNGCTAAAGGTGAATCCAGAG primer NNK
3' (forward) CTGGAGCCTGGCGGACCCAABHCATABHATAABHGCTAAAGGTGAATCCAGAG
primer 2B DP-47 5' (back) primer TGGGTCCGCCAGGCTCCAG DVT 3'
(forward) TCCCTGGCCCCAGTAGTCAAAMNNMNNMNNMNNTTTCGCACAGTAATATACGG
primer NNK 3' (forward) TCCCTGGCCCCAGTAGTCAAAABHABHABHABHTTTCGCA-
CAGTAATATACGG primer 3B DPK9 5' (back) primer GACATCCAGATGACCCAGTC
DVT 3' (forward) CTGGTTTCTGCTGATACCAMNNTAAM-
NNMNNMNNAATGCTCTGACTTGCCCGG primer NNK 3' (forward)
CTGGTTTCTGCTGATACCAABHTAAABHABHABHAATGCTCTGACTTGCCCGG primer 4B
DPK9 5' (back) primer TGGTATCAGCAGAAACCAGGG DVT 3' (forward)
CTTGGTCCCTTGGCCGAACGTMNNAGGGGTACTGTAACTCTGTTGACAGTAGTAAGTTGC primer
NNK 3' (forward) CTTGGTCCCTTGGCCGAACGTABHAGGGGTA-
CTGTAACTCTGTTGACAGTAGTAAGTTGC primer 2nd round of amplification 5A
1A/2A 5' (back) primer GTCCTCGCAACTGCGGCCCAGCCGGC-
CATGGCCGAGGTGCAGCTGTTGGAGTC 3' (forward) primer
GAACCGCCTCCACCGCTCGAGACGGTGACCAGGGTTCCCTGGCCCCAGTAGTCAAA 6A 3A/4A
5' (back) primer AGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTC-
GACGGACATCCAGATGACCCAGTC 3' (forward) primer
GAGTCATTCTCGACTTGCGGCCGCCCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACG 5B
1B/2B 5' (back) primer
GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTG- TTGGAGTC 3'
(forward) primer GAACCGCCTCCACCGCTCGAGACGGTGACCAGGGTT-
CCCTGGCCCCAGTAGTCAAA 6B 3B/4B 5' (back) primer
AGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTC
3' (forward) primer GAGTCATTCTCGACTTGCGGCCGCCCGTTTGATTTCCACCTTGGTC-
CCTTGGCCGAACG
* * * * *