U.S. patent application number 11/862791 was filed with the patent office on 2008-05-08 for novel fab fragment libraries and methods for their use.
This patent application is currently assigned to Dyax Corp., a Massachusetts corporation. Invention is credited to Hendricus Renerus Jacobus Mattheus Hoogenboom.
Application Number | 20080108514 11/862791 |
Document ID | / |
Family ID | 26153316 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080108514 |
Kind Code |
A1 |
Mattheus Hoogenboom; Hendricus
Renerus Jacobus |
May 8, 2008 |
NOVEL FAB FRAGMENT LIBRARIES AND METHODS FOR THEIR USE
Abstract
The present invention provides Fab libraries and methods for
using the Fab libraries to obtain antibodies against a target. The
Fab library of the invention contains at least 10.sup.9 different
Fabs, and in some embodiments, at least 10.sup.10 different Fabs.
The Fab libraries of the invention are used to isolate polyclonal
or monoclonal Fabs that bind with high specificity to targets.
Inventors: |
Mattheus Hoogenboom; Hendricus
Renerus Jacobus; (Maastricht, NL) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Dyax Corp., a Massachusetts
corporation
|
Family ID: |
26153316 |
Appl. No.: |
11/862791 |
Filed: |
September 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09988899 |
Nov 19, 2001 |
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11862791 |
Sep 27, 2007 |
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PCT/US00/13682 |
May 18, 2000 |
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09988899 |
Nov 19, 2001 |
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Current U.S.
Class: |
506/17 |
Current CPC
Class: |
C40B 50/06 20130101;
C07K 2317/55 20130101; C07K 16/18 20130101; C07K 16/00 20130101;
C07K 16/26 20130101 |
Class at
Publication: |
506/017 |
International
Class: |
C40B 40/08 20060101
C40B040/08 |
Foreign Application Data
Date |
Code |
Application Number |
May 18, 1999 |
EP |
99201558.6 |
Claims
1. A plurality of polynucleotides encoding a Fab library, the
library comprising a plurality of vectors wherein each vector of
the plurality of vectors comprises: a first cloning region and a
second cloning region, wherein each cloning region comprises at
least one, for the vector unique, restriction enzyme cleavage site,
each cloning region being 5' flanked by a ribosome binding site and
a signal sequence, a polynucleotide encoding an anchor region,
located 3' of the second cloning region, a member of a first
plurality of variable polynucleotides, said plurality of variable
polynucleotides encoding a first plurality of polypeptides, wherein
the member of the first plurality is cloned into the first cloning
region of the vector, and the member of the first plurality of
polypeptides encodes a polypeptide selected from the group
consisting of a complete antibody variable region, a complete
antibody variable region followed by a complete antibody constant
region, a complete antibody variable region followed by a part of
an antibody constant region, a part of an antibody variable region,
a part of an antibody variable region followed by a complete
antibody constant region or a part of an antibody variable region
followed by a part of an antibody constant region; a member of a
second plurality of variable polynucleotides, said plurality of
variable polynucleotides encoding a second plurality of
polypeptides, wherein the member of the second plurality is cloned
into the second cloning region of the vector, and the member of the
second plurality of polypeptides encodes a polypeptide selected
from the group consisting of a complete antibody variable region, a
complete antibody variable region followed by a complete antibody
constant region, a complete antibody variable region followed by a
part of an antibody constant region, a part of an antibody variable
region, a part of an antibody variable region followed by a
complete antibody constant region or a part of an antibody variable
region followed by a part of an antibody constant region; and a
polynucleotide encoding a tag; wherein the plurality of vectors
comprises the first plurality of variable polynucleotides and the
second plurality of variable polynucleotides.
2. The plurality of polynucleotides according to claim 1, wherein
the first plurality of variable polynucleotides are V.sub.L
polynucleotides, and the second plurality of variable
polynucleotides are V.sub.H polynucleotides.
3. The plurality of polynucleotides according to any one of the
preceding claims, wherein the plurality of polynucleotides encode a
Fab library of at least 10.sup.9 different Fabs.
4. The plurality of polynucleotides according to claim 3, wherein
the polynucleotides encode a Fab library of at least 10.sup.10
different Fabs.
5. The plurality of polynucleotides according to claim 4, wherein
the polynucleotides encode a Fab library of at least
3.7.times.10.sup.10 different Fabs.
6. A plurality of vectors, wherein each vector of the plurality of
vectors comprises a first cloning region and a second cloning
region, wherein each cloning region comprises at least one, for the
vector unique, restriction enzyme cleavage site, each cloning
region being 5' flanked by a ribosome binding site and a signal
sequence, a polynucleotide encoding an anchor region, located 3' of
the second cloning region, a member of a first plurality of
variable polynucleotides, said plurality of variable
polynucleotides encoding a first plurality of polypeptides, wherein
the member of the first plurality is cloned into the first cloning
region of the vector, and the member of the first plurality of
polypeptides encodes a polypeptide selected from the group
consisting of a complete antibody variable region, a complete
antibody variable region followed by a complete antibody constant
region, a complete antibody variable region followed by a part of
an antibody constant region, a part of an antibody variable region,
a part of an antibody variable region followed by a complete
antibody constant region or a part of an antibody variable region
followed by a part of an antibody constant region; a member of a
second plurality of variable polynucleotides, said plurality of
variable polynucleotides encoding a second plurality of
polypeptides, wherein the member of the second plurality is cloned
into the second cloning region of the vector, and the member of the
second plurality of polypeptides encodes a polypeptide selected
from the group consisting of a complete antibody variable region, a
complete antibody variable region followed by a complete antibody
constant region, a complete antibody variable region followed by a
part of an antibody constant region, a part of an antibody variable
region, a part of an antibody variable region followed by a
complete antibody constant region or a part of an antibody variable
region followed by a part of an antibody constant region; and a
polynucleotide encoding a tag, wherein the plurality of vectors
comprises the first plurality of polynucleotides and the second
plurality of variable polynucleotides.
7. The plurality of vectors according to claim 6, wherein the first
plurality of variable polynucleotides are V.sub.L polynucleotides,
and the second plurality of variable polynucleotides are V.sub.H
polynucleotides.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 09/988,899, filed on Nov. 19, 2001, which in a
continuation of international application no. PCT/US00/13682, filed
on May 18, 2000 which claims priority to European application no.
99201558.6, filed on May 18, 1999. The contents of these
applications are herein incorporated by reference in their
entireties.
[0002] This invention relates in general to phage display libraries
of human Fab fragments, and methods using the Fab fragment
libraries to isolate high affinity antibodies. Especially, the
invention relates to polynucleotides encoding a Fab library, Fab
libraries, and methods for designing, constructing and selecting
from Fab libraries.
[0003] Display on filamentous phage in combination with selection
forms a powerful tool for the identification of peptide- or
protein-based drugs (Winter et al., 1994; Clackson et al., 1994).
Of these, antibodies are especially of interest, due to their
capacity to recognize a variety of targets with high specificity
and affinity. Particularly the use of partial or complete human
antibodies, which elicit no or minimal immune response when
administered to patients, is yielding an increasing list of
FDA-approved protein-based drugs (Holliger et al. 1998). Phage
display technology enables the generation of large repertoires of
human antibodies (Marks et al., 1991, Hoogenboom et al., 1992;
Griffiths et al., 1993; Vaughan et al., 1996), and biopanning
procedures permit the selection of individual antibodies with a
desired specificity.
[0004] Key to the success of the technology were two critical
observations: (i) the expression of functional antibody fragments
by secretion into the periplasm of E. coli (Better et al., 1988;
Skerra et al., 1988), and, (ii) the rapid access to variable region
gene pools by the polymerase chain reaction (Larrick et al., 1989;
Ward et al., 1989; Marks et al., 1991). For the construction of
antibody libraries, V-genes are amplified from B-cell cDNA and
heavy and light chain genes are randomly combined and cloned to
encode a combinatorial library of single-chain Fv (scFv) or Fab
antibody fragments (Marks et al., 1991; Clackson et al. 1991;
Persson et al., 1991; Orum et al., 1993). The natural primary
(unselected) antibody repertoire within B-cells contains a large
array of antibodies that recognize a variety of antigens; this
array can be cloned as a `naVve` repertoire of rearranged genes, by
harvesting the V-genes from the IgM mRNA of B-cells of unimmunized
human donors, isolated from peripheral blood lymphocytes (Marks et
al., 1991), bone marrow or tonsils (Vaughan et al., 1996), or from
similar animal sources (Gram et al., 1992). This procedure provides
access to antibodies that have not yet encountered antigen,
although the frequency of those genuine `germline` antibodies will
depend heavily on the source of B-cells (Klein et al., 1997). A
single `naive` library, if sufficiently large and diverse, can
indeed be used to generate antibodies to a large panel of antigens,
including self, non-immunogenic and relatively toxic antigens
(Griffiths et al., 1993; Marks et al., 1991). In a different
approach, antibodies may be built artificially, by in vitro
assembly of V-gene segments and D/J segments, yielding `synthetic`
antibodies (Hoogenboom et al., 1992). A major drawback of these
procedures is that from the initial "naive" and "synthetic"
libraries, only moderate affinity antibodies were isolated (Marks
et al., 1991; Nissim et al., 1994). Over the last few years more
efficient techniques have been developed to build larger libraries
of antibody fragments, using sophisticated in vivo recombination
methods (Griffiths et al., 1993) or brute force cloning procedures
(Vaughan et al., 1996; Sheets et al., 1998). Such large libraries
have yielded a greater number of human antibodies per antigen
tested, with an average much higher affinity (up to sub-nanomolar).
However, technical restrictions on the size of libraries that may
be obtained or handled in selection, the loss of library diversity
upon library amplification, and the relatively long down-stream
analysis path of the selected antibodies, i.e., large scale
affinity analysis, have limited the spread of these libraries as
generic tools in antibody generation.
[0005] Most large libraries made to date use the single chain
format for display on phage (Vaughan et al., 1996; Sheets et al.
1998). One report described the use of a human naive Fab library on
phage (not permitting immediate screening of selected soluble Fab
fragments) (Griffiths et al., 1994). scFv's have the tendency to
form dimers and higher order multimers in a clone-dependent and
relatively unpredictable way (Weidner, et al. 1992; Holliger, et
al. 1993; Marks et al., 1993). As a consequence, the affinity assay
used (such as BIAcore analysis) often necessitates purification of
the selected antibody fragments. For example, ranking for off-rates
using BIAcore is not easily possible with unpurified scFv
fragments; the monomeric fraction of selected scFv clones first
needs to be purified by affinity chromatography and gel-filtration
(Sheets et al., 1998; Schier et al., 1996).
[0006] As was postulated and observed by Griffiths and colleagues
(Griffiths et al., 1994), the size of the antibody library dictates
the probability of the selection of high affinity antibodies to the
antigen. Comparison of the first naVve scFv repertoire containing
2.9.times.10.sup.7 clones (Marks et al., 1991), with a recently
constructed scFv repertoire of approximately 10.sup.10 clones
(Vaughan et al., 1996; Sheets et al. 1998), confirms this
postulation: increasing the library size 500-fold resulted in
approximately 100-fold higher affinities. This increase is caused
by lowering the off-rates from 10.sup.-1-10.sup.-2 s.sup.-1 for
fragments selected from the smaller sized library to
10.sup.-3-10.sup.-4 s.sup.-1 for those from the larger library.
[0007] It is an object of the invention to create a Fab library
that is a valuable source of antibodies for many different targets,
and which will play a vital role in target discovery and validation
in the area of functional genomics.
[0008] The invention provides a plurality of polynucleotides
encoding a Fab library comprising a plurality of vector wherein the
vector comprises: [0009] a first and second cloning region, wherein
[0010] each cloning region comprises at least one, for the vector
unique, restriction enzyme cleavage site, [0011] each cloning
region being 5' flanked by a ribosome binding site and a signal
sequence, [0012] a polynucleotide encoding an anchor region,
located 3' of the second cloning region, [0013] a first and a
second plurality of variable polynucleotides, [0014] each encoding
a complete antibody variable region or part of an antibody variable
region, possibly followed by a complete antibody constant region or
part of an antibody constant region, [0015] the first plurality of
variable polynucleotides being cloned into the vector at the
restriction enzyme cleavage site(s) of the first cloning region,
[0016] the second plurality of variable polynucleotides being
cloned into the vector at the restriction enzyme cleavage site(s)
of the second cloning region.
[0017] It is to be understood that the term "for the vector unique
restriction enzyme cleavage site" refers to the presence of one of
such a restriction site in the vector sequence, without taking into
account the possible presence of such a site on the above-mentioned
first and/or second polynucleotides encoding a complete antibody
variable region or part of an antibody variable region, possibly
followed by a complete antibody constant region or part of an
antibody constant region. The said first and second polynucleotides
may comprise restriction sites identical to the "unique" site. This
means that the said restriction site was "unique" before both first
and second polynucleotide sequences were cloned into the
vector.
[0018] The first and second variable polynucleotides are preferably
cloned in the cloning region in a predetermined orientation.
Therefore, in case the cloning region comprises a single unique
restriction site, this site is preferably of such a type that
non-identical restriction ends are generated, such as e.g,
generated by the restriction enzyme SfiI. However, the cloning
region may comprise two or more unique restriction sites, so that
the variable polynucleotides can be conveniently cloned as a
restriction fragment that has the corresponding ends.
[0019] Preferably, in the vector according to the invention, the
first and second cloning regions, both ribosomal binding sites,
signal sequences and the anchor sequence are part of a single
polylinker sequence. Both cloning regions may therefore be part of
a single cassette, comprising the first cloning region, 5' flanked
by a ribosomal binding site and a signal sequence, lying adjacent
to the second cloning region, also 5' flanked by its corresponding
ribosomal binding site and a signal sequence, and 3' flanked by the
anchor sequence.
[0020] Preferably, the first plurality of variable polynucleotides
are V.sub.L polynucleotides, and the second plurality of variable
polynucleotides are V.sub.H polynucleotides. More preferably, the
V.sub.L polynucleotides are V.sub.6 polynucleotides, V.sub.6C.sub.6
polynucleotides, V.sub.8 polynucleotides, V.sub.8C.sub.8
polynucleotides, a mixture of V.sub.6 and V.sub.8 polynucleotides,
or a mixture of V.sub.6C.sub.6 and V.sub.8C.sub.8
polynucleotides.
[0021] In another embodiment of the polynucleotides according to
the invention, the vector further comprises a tag for purification
or detection of an antibody, said tag for purification of the
antibody preferably comprising a poly-histidine tail; the tag for
detection of the antibody is preferably a c-myc-derived tag.
[0022] In another embodiment of the polynucleotides according to
the invention, the vector further comprises an amber stop codon
located between the second variable polynucleotide and the anchor
protein.
[0023] In still another embodiment of the polynucleotides according
to the invention, the vector further comprises a C.sub.H1 domain
located between the second variable polynucleotide and the anchor
protein, the C.sub.H1 domain preferably being a human gamma-1
C.sub.H1 domain.
[0024] "Anchor protein" is defined as a protein or part thereof
that can at least partially be accommodated in the outer coat of a
particle generated by an organism expressing the library, such as a
phage or virus particle, or in the outer coat of an organism
itself, in case the organism itself expresses the library. The
outer coat is herein defined as the structure of a cell, virus or
phage particle defining the outer surface thereof. In case of a
phage or phagemid expressing the library, the anchor protein may be
a coat protein, such as the gene III product. However, other
systems, known to the skilled person, may be used to obtain a
library according to the present invention. Therefore, e.g.,
transmembrane proteins, or the transmembrane domain thereof, may be
contemplated to be used as anchor protein in eukaryotic expression
systems. In the invention, the anchor protein may be fused to an
antibody variable region or part thereof, resulting in the
presentation of the said variable region to the outer environment
of the organism, the region being anchored in its outer coat. In a
preferred embodiment of the polynucleotides according to the
invention, the anchor protein is a minor coat protein III of a
filamentous phage f.sub.d.
[0025] In one embodiment of the invention, the polynucleotides
according to the invention, and therefore the Fab library, encodes
at least 10.sup.9 different Fabs. In another embodiment of the
invention, the Fab library of the invention encodes at least
10.sup.10 different Fabs. In still another embodiment of the
invention, the Fab library encodes at least 3.7.times.10.sup.10
different Fabs. In still another embodiment of the invention, the
Fab library encodes 10.sup.9 to 3.7.times.10.sup.10 different
Fabs.
[0026] Further, the invention provides a Fab library, comprising
[0027] a plurality of vectors as defined above, [0028] the second
cloning region in each vector forming a fusion polynucleotide
encoding a plurality of fusion proteins, [0029] a plurality of
capsid particles, wherein the plurality of vector containing the
first and second pluralities of variable polynucleotides is
packaged into the capsid particles, wherein [0030] at least some of
the capsid particles display the fusion protein encoded by the
vector packaged into the capsid on the surface of the capsid.
Further the invention relates to a method of making a plurality of
polynucleotides encoding a Fab library, comprising the steps of:
[0031] amplifying a first plurality of variable polynucleotides
with a first set of primers, [0032] amplifying a second plurality
of variable polynucleotides with a second set of primers, [0033]
wherein each set of primers comprises oligonucleotides designed to
be homologous to the 5' and 3' end of variable polynucleotides
encoding antibody variable regions or parts thereof, such that they
can be used to amplify variable polynucleotide pools from natural
or synthetic sources of genes while retaining all or part of the
antibody's antigen combining site; [0034] cloning the first and
second plurality of variable polynucleotides into a plurality of
vectors, [0035] wherein the vector comprises: [0036] a first and a
second cloning region, wherein [0037] each cloning region comprises
at least one, for the vector unique, restriction enzyme cleavage
site, [0038] each cloning region being 5' flanked by a ribosome
binding site and a signal sequence, [0039] a polynucleotide
encoding an anchor region, located 3' of the second cloning region,
[0040] wherein the first plurality of variable polynucleotides is
cloned into the restriction enzyme cleavage site(s) of the first
cloning region of the vector and the second plurality of variable
polynucleotides into the restriction enzyme cleavage site(s) of the
second cloning region of the vector.
[0041] In one embodiment, the method of constructing the Fab
library comprises the steps of: amplifying a plurality of variable
gene pools with a set of the primers, wherein the primers comprise
oligonucleotides designed to be homologous to the 5' and 3' end of
variable polynucleotides encoding antibody variable regions or
parts thereof, such that they can be used to amplify variable
polynucleotide pools from natural or synthetic sources of genes
while retaining all or part of the antibody's antigen combining
site; cloning the amplified variable gene pools into a vector with
a two-step procedure to obtain a Fab library; wherein the vector
comprises a phage or phagemid vector which will accommodate
expression of the cloned antibody variable polynucleotides as
antibody Fab fragments, wherein one of the two antibody chains is
fused to one of the phage coat proteins (e.g., geneIII
product).
[0042] In one embodiment, the BACK primers were designed to have at
the most three mutations in a total of twentyone to twentythree
nucleotides when compared to the human germline gene segment region
they would have to bind to, but with at least 3 homologous residues
towards the 3' site of the oligonucleotide. This set of
oligonucleotides will recognise approximately 90% of human germline
gene segments and as such provide access to most of the present
diversity of the B-cells in non-immunized sources. In another
embodiment, the heavy chain primers should end with `GG` to ensure
stable binding at high annealing temperatures (at least 55EC).
Similarly the VkappaBACK primers and most of the VlambdaBACK
primers will be designed to preferentially end in `CC`. In an
alternate embodiment, the primers consist of the sequences in FIG.
2.
[0043] The invention also provides methods for obtaining antibodies
specific to an antigen from the Fab library. In certain
embodiments, the methods of the invention allow a rapid initial
screen of off-rates using the Fab libraries of the invention. In
alternate embodiments, the methods of the patent are used to screen
off-rates for a large series of antigen specific Fabs using the Fab
libraries of the invention.
[0044] The present invention also relates to isolated antibodies
specific to an antigen of choice, and their corresponding nucleic
acids, that are isolated from the Fab libraries of the invention.
In an alternative embodiment, these isolated antibodies are high
affinity antibodies. The antibodies may be used as research
reagents or as therapeutic products. The antibodies of the
invention will be ideal for investigating the nature and
localization of their targets, and the antibodies can be used to
purify the target. Thus, the antibodies of the invention will be
important for target validation and target discovery in the area of
functional genomics.
[0045] The invention also relates to a vector as is defined above,
comprising one of the first and one of the second plurality of the
variable polynucleotides cloned into the first and second cloning
region respectively.
[0046] The present invention further relates to host cells
containing the Fab libraries of the invention or the
polynucleotides that encode the Fab libraries of the invention.
[0047] In one aspect the invention involves linking the desired
specific binding pair member, such as an antibody molecule, to a
phage coat protein. By then enriching for the specific binding pair
member, such as by affinity techniques, for example, the DNA which
encodes the specific binding pair member is also enriched and may
then be isolated. The DNA so obtained may then be cloned and
expressed in other systems, yielding potentially large quantities
of the desired specific binding pair member, or may be subjected to
sequencing and further cloning and genetic manipulations prior to
expression.
[0048] Typically the target for the specific binding pair member,
e.g., an antigen or hapten when the specific binding pair member is
an antibody, is known, and the methods herein provide a means for
creating and/or identifying a specific binding pair member which
specifically binds the target of interest. Thus, when the protein
is an antibody the present invention provides a novel means for
producing antibodies, particularly monoclonal antibodies, with
specificity for predetermined targets, thereby circumventing the
laborious, time-consuming and often unpredictable process of
conventional monoclonal antibody technology.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIG. 1. Phagemid vector pCES1 for display of antibody Fab
fragments. Schematic representation (A) and polylinker region (SEQ
ID NOS: 5-8) (B) of pCES1. The polylinker region comprises two
signal sequences (`S`; pelB and the geneIII leader sequence), the
C6 domain, ribosome binding site (rbs), CH1 domain, hexa histidine
tag (H6) and a c-myc derived sequence. Variable domain genes can be
cloned as ApaLI-XhoI or ApaLI-Asc fragments (for VL or VLCL
respectively) and SfiI/PstI-BstEII or SfiI-NotI fragments (for VH
or VHCH1 respectively. The amber stop codon (*) between the
antibody genes and bacteriophage gene III enables the production of
soluble Fab fragments in a non-suppressor strain of E. coli.
Expression of the bicistronic operon is under control of the LacZ
promotor (pLacZ).
[0050] FIG. 2. This figure describes oligonucleotides used in one
embodiment for PCR amplification of human heavy and light chain
V-regions (SEQ ID NOS: 9-71).
[0051] The term "active" refers to those forms of the polypeptide
which retain the biologic and/or immunologic activities of any
naturally occurring polypeptide.
[0052] The term "activated" cells as used in this application are
those which are engaged in extracellular or intracellular membrane
trafficking, including the export of neurosecretory or enzymatic
molecules as part of a normal or disease process.
[0053] The term `antibody` means an immunoglobulin whether natural
or partly or wholly synthetically produced. The term also covers
any protein or polypeptide having a binding domain which is
homologous to an immunoglobulin binding domain. These proteins can
be derived from natural sources, or partly or wholly synthetically
produced. Example antibodies are the immunoglobulin isotypes and
the Fab, scFv, Fv, dAab, VHH, Fd fragments.
[0054] The term `antibody polypeptide dimer` means an association
of two polypeptide chain components of an antibody, capable of
binding an antigen. Thus, it may be one arm of an antibody
consisting of a heavy chain and a light chain, it may be a Fab
fragment consisting of V.sub.L, V.sub.H, C.sub.L and C.sub.H1
antibody domains, or an Fv fragment consisting of a V.sub.L domain
and a V.sub.H domain.
[0055] The term `capsid` means a replicable genetic display
package, with or without the genetic information. The capsids
display a member of a specific binding pair at its surface. The
package may be a population of bacteriophages which display an
antigen binding domain, e.g., a Fab, at the surface of some or all
of the capsids within the population. This type of package has been
called a phage antibody (pAb).
[0056] The term `C.sub.H1 domain` means the first constant region
of the heavy chain of an antibody or part thereof or extended with
aminoacids from the hinge regions as to allow pairing of the
expressed (VH)CH1 fragment with the antibody's light chain, and
possible disulphide-bridge formation. This may be the CH1 domain of
a human antibody of isotype gamma-1.
[0057] A "component part of an antibody antigen-binding site" may
be or correspond to a polypeptide chain component, e.g., a V.sub.H
or a V.sub.L domain. However, it may be a CDR, or a V.sub.L
sequence plus CDR of a V.sub.H, a V.sub.H sequence plus CDR of a
V.sub.L, a V.sub.H plus V.sub.L sequence lacking only a CDR, and so
on. The proviso is that the first and second component parts of an
antigen-binding site of an antibody must in combination (together)
form an antigen-binding site. Thus, if the second component part of
an antigen-binding site of a non-human antibody specific for an
antigen of interest is a CDR, then the first component part of an
antigen-binding site of a human antibody will comprise the
remainder of a V.sub.H and V.sub.L region required to form a
antigen-binding site (with or without associated antibody constant
domains (in a Fab format), or with or without a linker peptide
sequence (in a Fv format). The second component part of an
antigen-binding site of a non-human antibody may comprise a V.sub.L
domain plus part of a V.sub.H domain, that part being one or more
CDRs, for instance, perhaps CDR3. In such case, the first component
part of an antigen-binding site of a human antibody would comprise
the remainder of a V.sub.H sequence which in combination with the
second component part forms an antigen-binding site. Of course, the
converse situation holds and the person skilled in the art will be
able to envisage other combinations of first and second component
parts which together form an antigen-binding site.
[0058] The term `conditionally defective` means a gene which does
not express a particular polypeptide under one set of conditions,
but expresses it under another set of conditions. An example, is a
gene containing an amber mutation expressed in non-suppressing or
suppressing hosts respectively. Alternatively, a gene may express a
protein or polypeptide which is defective under one set of
conditions, but not under another set. An example is a gene with a
temperature sensitive mutation.
[0059] The term "derivative" refers to polypeptides chemically
modified by such techniques as ubiquitination, labeling (e.g., with
radionuclides or various enzymes), pegylation (derivatization with
polyethylene glycol) and insertion or substitution by chemical
synthesis of amino acids such as ornithine, which do not normally
occur in human proteins.
[0060] The term `domain` means a part of a protein or polypeptide
that is folded within itself and independently of other parts of
the same protein or polypeptide and independently of a
complementary binding member.
[0061] The term `eluant` means a solution used to breakdown the
linkage between two molecules. The linkage can be a non-covalent or
covalent bond(s). The two molecules can be members of a sbp.
[0062] The term `expression modulating fragment,` EMF, means a
series of nucleotides which modulates the expression of an operably
linked ORF or another EMF.
[0063] As used herein, a sequence is said to `modulate the
expression of an operably linked sequence` when the expression of
the sequence is altered by the presence of the EMF. EMFs include,
but are not limited to, promoters, and promoter modulating
sequences (inducible elements). One class of EMFs are fragments
which induce the expression or an operably linked ORF in response
to a specific regulatory factor or physiological event.
[0064] The term "Fab" refers to antibody fragments including
fragments which comprise two N-terminal portions of the heavy chain
polypeptide joined by at least one disulfide bridge in the hinge
region and two complete light chain polypeptides, where each light
chain is complexed with one N-terminal portion of a heavy chain.
Fab also includes Fab fragments which comprise all or a large
portion of a light chain polypeptide (e.g., V.sub.LC.sub.L)
complexed with the N-terminal portion of a heavy chain polypeptide
(e.g., V.sub.HC.sub.H1).
[0065] The term "Fab library" refers to a collection of Fab
polynucleotide sequences within clones; or a genetically diverse
collection of Fab polypeptides displayed on rgdps capable of
selection or screening to provide an individual Fab polypeptide or
a mixed population of Fab polypeptides.
[0066] The term `folded unit` means a specific combination of an
alpha-helix and/or beta-strand and/or beta-turn structure. Domains
and folded units contain structures that bring together amino acids
that are not adjacent in the primary structure.
[0067] The term `genetically diverse population` means antibodies
or polypeptide components thereof, this is referring not only to
diversity that can exist in the natural population of cells or
organisms, but also diversity that can be created by artificial
mutation in vitro or in vivo. Mutation in vitro may for example,
involve random mutagenesis using oligonucleotides having random
mutations of the sequence desired to be varied. In vivo mutagenesis
may for example, use mutator strains of host microorganisms to
harbour the DNA (see Example 38 of WO 92/01047). The words "unique
population" may be used to denote a plurality of e.g., polypeptide
chains, which are not genetically diverse i.e., they are all the
same. A restricted population is one which is diverse but less so
than the full repertoire of an animal. The diversity may have been
reduced by prior selection, e.g., using antigen binding
specificity.
[0068] The term `helper phage` means a phage which is used to
infect cells containing a defective phage genome and which
functions to complement the defect. The defective phage genome can
be a phagemid or a phage with some function encoding gene sequences
removed. Examples of helper phages are M13KO7, M13K07 gene III no.
3; and phage displaying or encoding a binding molecule fused to a
capsid protein.
[0069] The term `homologs` means polypeptides having the same or
conserved residues at a corresponding position in their primary,
secondary or tertiary structure. The term also extends to two or
more nucleotide sequences encoding the homologous polypeptides.
Example homologous peptides are the immunoglobulin isotypes.
[0070] The term "host cell" refers to a prokaryotic or eukaryotic
cell into which the vectors of the invention may be introduced,
expressed and/or propagated. Typical prokaryotic host cells include
various strains of E. coli. Typical eukaryotic host cells are yeast
or filamentous fungi, or mammalian cells, such as Chinese hamster
ovary cells, murine NIH 3t3 fibroblasts, or human embryonic kidney
193 cells.
[0071] The term `immunoglobulin superfamily` means a family of
polypeptides, the members of which have at least one domain with a
structure related to that of the variable or constant domain of
immunoglobulin molecules. The domain contains two B-sheets and
usually a conserved disulphide bond (see A. F. Williams and A. N.
Barclay 1988 Ann. Rev Immunol. 6 381-405). Example members of an
immunoglobulin superfamily are CD4, platelet derived growth factor
receptor (PDGFR), intercellular adhesion molecule. (ICAM). Except
where the context otherwise dictates, reference to immunoglobulins
and immunoglobulin homologs in this application includes members of
the immunoglobulin superfamily and homologs thereof.
[0072] The term "infection" refers to the introduction of nucleic
acids into a suitable host cell by use of a virus or viral
vector.
[0073] The term "intermediate fragment" means a nucleic acid
between 5 and 1000 bases in length, and preferably between 10 and
40 bp in length.
[0074] The term "isolated" as used herein refers to a nucleic acid
or polypeptide separated not only from other nucleic acids or
polypeptides that are present in the natural source of the nucleic
acid or polypeptide, but also from polypeptides, and preferably
refers to a nucleic acid or polypeptide found in the presence of
(if anything) only a solvent, buffer, ion, or other component
normally present in a solution of the same. The terms "isolated"
and "purified" do not encompass nucleic acids or polypeptides
present in their natural source.
[0075] The term `mutator strain` means a host cell which has a
genetic defect which causes DNA replicated within it to be mutated
with respect to its parent DNA. Example mutator strains are
NR9046mutD5 and NR9046 mut T1 (See Example 38 of WO 92/01047).
[0076] The term "naturally occurring polypeptide" refers to
polypeptides produced by cells that have not been genetically
engineered and specifically contemplates various polypeptides
arising from post-translational modifications of the polypeptide
including, but not limited to, acetylation, carboxylation,
glycosylation, phosphorylation, lipidation and acylation.
[0077] The term `nucleotide sequence` refers to a heteropolymer of
nucleotides or the sequence of these nucleotides. The terms
`nucleic acid` and `polynucleotide` are also used interchangeably
herein to refer to a heteropolymer of nucleotides. Generally,
nucleic acid segments provided by this invention may be assembled
from fragments of the genome and short oligonucleotide linkers, or
from a series of oligonucleotides, or from individual nucleotides,
to provide a synthetic nucleic acid which is capable of being
expressed in a recombinant transcriptional unit comprising
regulatory elements derived from a microbial or viral operon, or a
eukaryotic gene.
[0078] The terms "oligonucleotide fragment" or a "polynucleotide
fragment", "portion," or "segment" is a stretch of polypeptide
nucleotide residues which is long enough to use in polymerase chain
reaction (PCR) or various hybridization procedures to identify or
amplify identical or related parts of mRNA or DNA molecules.
[0079] The terms "oligonucleotides" or "nucleic acid probes" are
prepared based on the polynucleotide sequences provided in the
present invention. Oligonucleotides comprise portions of such a
polynucleotide sequence having at least about 15 nucleotides and
usually at least about 20 nucleotides. Nucleic acid probes comprise
portions of such a polynucleotide sequence having fewer nucleotides
than about 6 kb, usually fewer than about 1 kb. After appropriate
testing to eliminate false positives, these probes may, for
example, be used to determine whether specific mRNA molecules are
present in a cell or tissue or to isolate similar nucleic acid
sequences from chromosomal DNA as described by Walsh et al. (Walsh,
P. S. et al., 1992, PCR Methods Appl 1:241-250).
[0080] The term "open reading frame," ORF, means a series of
nucleotide triplets coding for amino acids without any termination
codons and is a sequence translatable into protein.
[0081] The term `phage vector` means a vector derived by
modification of a phage genome, containing an origin of replication
for a bacteriophage, but not one for a plasmid.
[0082] The term `phagemid vector` means a vector derived by
modification of a plasmid genome, containing an origin of
replication for a bacteriophage as well as the plasmid origin of
replication.
[0083] The term "phenotype" refers to a physical (e.g., pigment, or
cell shape) and/or metabolic property of a cell which can be
measured or exploited in some fashion and which is effected by the
reporter gene.
[0084] The term `polylinker region` means a polynucleotide that
contains at least two restriction enzyme sites that are unique in
the vector that contains the polylinker region, i.e., these
restriction sites are easily used cloning sites in the vector.
[0085] A polypeptide "fragment," "portion," or "segment" is a
stretch of amino acid residues of at least about 5 amino acids,
often at least about 7 amino acids, typically at least about 9 to
13 amino acids, and, in various embodiments, at least about 17 or
more amino acids. To be active, any polypeptide must have
sufficient length to display biologic and/or immunologic
activity.
[0086] The term "probes" includes naturally occurring or
recombinant or chemically synthesized single- or double-stranded
nucleic acids. They may be labeled by nick translation, Klenow
fill-in reaction, PCR or other methods well known in the art.
Probes of the present invention, their preparation and/or labeling
are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel,
F. M. et al., 1989, Current Protocols in Molecular Biology, John
Wiley & Sons, New York N.Y., both of which are incorporated
herein by reference in their entirety.
[0087] The term "purified" as used herein denotes that the
indicated nucleic acid or polypeptide is present in the substantial
absence of other biological macromolecules, e.g., polynucleotides,
proteins, and the like. In one embodiment, the polynucleotide or
polypeptide is purified such that it constitutes at least 95% by
weight, more preferably at least 99.8% by weight, of the indicated
biological macromolecules present (but water, buffers, and other
small molecules, especially molecules having a molecular weight of
less than 1000 daltons, can be present).
[0088] The term "recombinant," when used herein to refer to a
polypeptide or protein, means that a polypeptide or protein is
derived from recombinant (e.g., microbial or mammalian) expression
systems. `Microbial` refers to recombinant polypeptides or proteins
made in bacterial or fungal (e.g., yeast) expression systems. As a
product, `recombinant microbial` defines a polypeptide or protein
essentially free of native endogenous substances and unaccompanied
by associated native glycosylation. Polypeptides or proteins
expressed in most bacterial cultures, e.g., E. coli, will be free
of glycosylation modifications; polypeptides or proteins expressed
in yeast will have a glycosylation pattern in general different
from those expressed in mammalian cells.
[0089] The term `recombinant expression vehicle or vector` refers
to a plasmid or phage or virus or vector, for expressing a
polypeptide from a DNA (RNA) sequence. An expression vehicle can
comprise a transcriptional unit comprising an assembly of (1) a
genetic element or elements having a regulatory role in gene
expression, for example, promoters or enhancers, (2) a structural
or coding sequence which is transcribed into mRNA and translated
into protein, and (3) appropriate transcription initiation and
termination sequences. Structural units intended for use in yeast
or eukaryotic expression systems preferably include a leader
sequence enabling extracellular secretion of translated protein by
a host cell. Alternatively, where recombinant protein is expressed
without a leader or transport sequence, it may include an
N-terminal methionine residue. This residue may or may not be
subsequently cleaved from the expressed recombinant protein or
polypeptide to provide a final product.
[0090] The term "recombinant expression system" means host cells
which have stably integrated a recombinant transcriptional unit
into chromosomal DNA or carry the recombinant transcriptional unit
extrachromosomally. Recombinant expression systems as defined
herein will express heterologous polypeptides or proteins upon
induction of the regulatory elements linked to the DNA segment or
synthetic gene to be expressed. This term also means host cells
which have stably integrated a recombinant genetic element or
elements having a regulatory role in gene expression, for example,
promoters or enhancers. Recombinant expression systems as defined
herein will express polypeptides or proteins endogenous to the cell
upon induction of the regulatory elements linked to the endogenous
DNA segment or gene to be expressed. The cells can be prokaryotic
or eukaryotic.
[0091] The term "recombinant variant" refers to any polypeptide
differing from naturally occurring polypeptides by amino acid
insertions, deletions, and substitutions, created using recombinant
DNA techniques. Guidance in determining which amino acid residues
may be replaced, added or deleted without abolishing activities of
interest, such as cellular trafficking, may be found by comparing
the sequence of the particular polypeptide with that of homologous
peptides and minimizing the number of amino acid sequence changes
made in regions of high homology.
[0092] Preferably, amino acid "substitutions" are the result of
replacing one amino acid with another amino acid having similar
structural and/or chemical properties, i.e., conservative amino
acid replacements. Amino acid substitutions may be made on the
basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues involved. For example, nonpolar (hydrophobic) amino
acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan, and methionine; polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine; positively charged (basic) amino acids
include arginine, lysine, and histidine; and negatively charged
(acidic) amino acids include aspartic acid and glutamic acid.
[0093] "Insertions" or "deletions" are typically in the range of
about 1 to 5 amino acids. The variation allowed may be
experimentally determined by systematically making insertions,
deletions, or substitutions of amino acids in a polypeptide
molecule using recombinant DNA techniques and assaying the
resulting recombinant variants for activity.
[0094] Alternatively, where alteration of function is desired,
insertions, deletions or non-conservative alterations can be
engineered to produce altered polypeptides. Such alterations can,
for example, alter one or more of the biological functions or
biochemical characteristics of the polypeptides of the invention.
For example, such alterations may change polypeptide
characteristics such as ligand-binding affinities, interchain
affinities, or degradation/turnover rate. Further, such alterations
can be selected so as to generate polypeptides that are better
suited for expression, scale up and the like in the host cells
chosen for expression. For example, cysteine residues can be
deleted or substituted with another amino acid residue in order to
eliminate disulfide bridges.
[0095] Alternatively, recombinant variants encoding these same or
similar polypeptides may be synthesized or selected by making use
of the "redundancy" in the genetic code. Various codon
substitutions, such as the silent changes which produce various
restriction sites, may be introduced to optimize cloning into a
plasmid or viral vector or expression in a particular prokaryotic
or eukaryotic system. Mutations in the polynucleotide sequence may
be reflected in the polypeptide or domains of other peptides added
to the polypeptide to modify the properties of any part of the
polypeptide, to change characteristics such as ligand-binding
affinities, interchain affinities, or degradation/turnover
rate.
[0096] The term `repertoire of artificially rearranged
immunoglobulin genes` means a collection of nucleotide e.g., DNA,
sequences derived wholly or partly from a source other than the
rearranged immunoglobulin sequences from an animal. This may
include for example, DNA sequences encoding VH domains by combining
unrearranged V segments with D and J segments and DNA sequences
encoding VL domains by combining V and J segments. Part or all of
the DNA sequences may be derived by oligonucleotide synthesis.
[0097] The term `repertoire of rearranged immunoglobulin genes`
means a collection of naturally occurring nucleotides e.g., DNA
sequences which encoded expressed immunoglobulin genes in an
animal. The sequences are generated by the in vivo rearrangement of
e.g., V, D and J segments for H chains and e.g., the V and J
segments for L chains. Alternatively the sequences may be generated
from a cell line immunised in vitro and in which the rearrangement
in response to immunisation occurs intracellularly. The word
"repertoire" is used to indicate genetic diversity.
[0098] The term `replicable genetic display package` (Rgdp) means a
biological particle which has genetic information providing the
particle with the ability to replicate. The particle can display on
its surface at least part of a polypeptide. The polypeptide can be
encoded by genetic information native to the particle and/or
artificially placed into the particle or an ancestor of it. The
displayed polypeptide may be any member of a specific binding pair
e.g., heavy or light chain domains based on an immunoglobulin
molecule, an enzyme or a receptor etc. The particle may be a virus
e.g., a bacteriophage such as fd or M13.
[0099] The term "reporter gene" refers to a nucleic acid which
encodes a protein or polypeptide that produces a phenotypic change
in the host cell that may be measured and/or used to separate host
cells. For example, the reporter gene may encode a protein or
polypeptide that has fluorescent properties, e.g.,
.beta.-galactosidase, auto-fluorescent protein GFP, etc.; or the
reporter gene may encode a selectable marker, e.g., antibiotic
resistance; or an epitope that is expressed on the surface of the
host cell.
[0100] The term `ribosome binding site` means a polyribonucleotide
that allows a ribosome to select the proper initiation codon during
the initiation of translation. In some prokaryotes, this
polyribonucleotide is called the Shine-Dalgarno sequence, and the
Shine-Delgarno sequence base pairs with the 16S RNA of the
ribosome.
[0101] The term "secreted" protein or polypeptide refers to a
protein or polypeptide that is transported across or through a
membrane, including transport as a result of signal sequences in
its amino acid sequence when it is expressed in a suitable host
cell. "Secreted" proteins or polypeptides include without
limitation proteins or polypeptides secreted wholly (e.g., soluble
proteins) or partially (e.g., receptors) from the cell in which
they are expressed. "Secreted" proteins or polypeptides also
include without limitation proteins or polypeptides which are
transported across the membrane of the endoplasmic reticulum.
[0102] The term "signal sequence" means an amino acid sequence that
is found at the amino terminus of a polypeptide and directs
transportation of the polypeptide across or through a membrane.
Signal sequences include amino terminal polypeptides that are 13-36
residues long, and have a 7 to 13 residue hydrophobic core flanked
by several hydrophilic residues that usually include one or more
basic residues near the N-terminus.
[0103] The term "stringent" is used to refer to conditions that are
commonly understood in the art as stringent. An exemplary set of
conditions include a temperature of 60-70.degree. C., (preferably
about 65.degree. C.) and a salt concentration of 0.70 M to 0.80 M
(preferably about 0.75M). Further exemplary conditions include,
hybridizing conditions that (1) employ low ionic strength and high
temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium
citrate/0.1% SDS at 50.degree. C.; (2) employ during hybridization
a denaturing agent such as formamide, for example, 50% (vol/vol)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM NaCl, 75 mM sodium citrate at 42.degree. C.; or (3) employ
50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M Sodium
pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm
DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree. C.,
with washes at 42.degree. C. in 0.2.times.SSC and 0.1% SDS.
[0104] In instances wherein hybridization of deoxyoligonucleotides
is concerned, additional exemplary stringent hybridization
conditions include washing in 6.times.SSC/0.05% sodium
pyrophosphate at 37EC (for 14-base oligos), 48EC (for 17-base
oligos), 55EC (for 20-base oligos), and 60EC (for 23-base
oligos).
[0105] As used herein, "substantially equivalent" can refer both to
nucleotide and amino acid sequences, for example a mutant sequence,
that varies from a reference sequence by one or more substitutions,
deletions, or additions, the net effect of which does not result in
an adverse functional dissimilarity between the reference and
subject sequences. Typically, such a substantially equivalent
sequence varies from one of those listed herein by no more than
about 20% (i.e., the number of individual residue substitutions,
additions, and/or deletions in a substantially equivalent sequence,
as compared to the corresponding reference sequence, divided by the
total number of residues in the substantially equivalent sequence
is about 0.2 or less). Such a sequence is said to have 80% sequence
identity to the listed sequence. In one embodiment, a substantially
equivalent, e.g., mutant, sequence of the invention varies from a
listed sequence by no more than 10% (90% sequence identity); in a
variation of this embodiment, by no more than 5% (95% sequence
identity); and in a further variation of this embodiment, by no
more than 2% (98% sequence identity). Substantially equivalent,
e.g., mutant, amino acid sequences according to the invention
generally have at least 95% sequence identity with a listed amino
acid sequence, whereas substantially equivalent nucleotide sequence
of the invention can have lower percent sequence identities, taking
into account, for example, the redundancy or degeneracy of the
genetic code. For the purposes of the present invention, sequences
having substantially equivalent biological activity and
substantially equivalent expression characteristics are considered
substantially equivalent. For the purposes of determining
equivalence, truncation of the mature sequence (e.g., via a
mutation which creates a spurious stop codon) should be
disregarded.
[0106] Nucleic acid sequences encoding such substantially
equivalent sequences, e.g., sequences of the recited percent
identities, can routinely be isolated and identified via standard
hybridization procedures well known to those of skill in the
art.
[0107] The term `suppressible translational stop codon` means a
codon which allows the translation of nucleotide sequences
downstream of the codon under one set of conditions, but under
another set of conditions translation ends at the codon. Example of
suppressible translational stop codons are the amber, ochre and
opal codons.
[0108] The term `tag` means an extension of the antibody Fab
fragment, for example expressed at the carboxyterminus of the heavy
chain, that comprises at least one amino acids but more typically
five to fifteen amino acids, and that can be specifically
recognised by an antibody or other binding ligand or binding matrix
for the sequence. Tags may be combined in the same Fab. Examples
are a stretch of five histidine residues that can be recognised by
specific antibodies and by defined immobilised metal ions, and a
stretch of the following 12 amino acids (EQKLISEEDLN) that are
recognised by the 9E10 antibody (Marks et al., 1991).
[0109] The term `target` means any molecule that is antigenic,
e.g., can be recognized with reasonably specificity by an antibody
from the Fab library.
[0110] The term `target element` refers to a nucleic acid sequence
that alters the expression of the target gene. Target elements
include, but are not limited to, promoters, and promoter modulating
sequences (inducible elements). One class of target elements are
fragments which induce the expression in response to a specific
regulatory factor or physiological event.
[0111] The term "transfection" refers to the taking up of an
expression vector by a suitable host cell, whether or not any
coding sequences are in fact expressed.
[0112] The term "transformation" means introducing DNA into a
suitable host cell so that the DNA is replicable, either as an
extrachromosomal element, or by chromosomal integration.
[0113] The term "universal set" refers to a set of nucleic acids,
most preferably a set of oligonucleotides, which represent all
possible combinations of sequence for a given length of
nucleotides, e.g., all 4096 insert oligonucleotides six nucleotides
in length. In a preferred embodiment, the term universal set refers
to the set of all possible oligonucleotides of a given length,
wherein one or more positions in the oligonucleotides are held
constant (i.e., the same nucleotide is present at this position in
all members of the set).
[0114] As used herein, an `uptake modulating fragment,` UMF, means
a series of nucleotides which mediate the uptake of a linked DNA
fragment into a cell. UMFs can be readily identified using known
UMFs as a target sequence or target motif with the computer-based
systems described below.
[0115] The presence and activity of a UMF can be confirmed by
attaching the suspected UMF to a marker sequence. The resulting
nucleic acid molecule is then incubated with an appropriate host
under appropriate conditions and the uptake of the marker sequence
is determined. As described above, a UMF will increase the
frequency of uptake of a linked marker sequence.
[0116] The term `vector` refers to a plasmid or phage or virus or
vector, for expressing a polypeptide from a DNA (RNA) sequence. The
vector can comprise a transcriptional unit comprising an assembly
of (1) a genetic element or elements having a regulatory role in
gene expression, for example, promoters or enhancers, (2) a
structural or coding sequence which is transcribed into mRNA and
translated into protein, and (3) appropriate translation initiation
and termination sequences. Structural units intended for use in
yeast or eukaryotic expression systems may include a leader
sequence enabling extracellular secretion of translated protein by
a host cell.
[0117] The term `V.sub.L polynucleotides` means polynucleotides
encoding the CDR containing domains of some or all of the light
chain genes from the V.sub.6- and/or V.sub.8-families.
[0118] The term `V.sub.H polynucleotides` means polynucleotides
encoding the CDR containing domains of some or all of the heavy
chain genes from the heavy chain gene family.
[0119] Each of the above terms is meant to encompasses all that is
described for each, unless the context dictates otherwise.
[0120] The recombinant constructs of the present invention comprise
a vector, such as a plasmid or viral vector, into which a nucleic
acid(s) of interest may be inserted. The vector may further
comprise regulatory sequences, including for example, a promoter,
operably linked to the nucleic acid(s) of interest. Large numbers
of suitable vectors and promoters are known to those of skill in
the art and are commercially available for generating the
recombinant constructs of the present invention. The following
vectors are provided by way of example. Bacterial: pBs,
phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a,
pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540,
pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
[0121] Methods which are well known to those skilled in the art can
be used to construct vectors containing a polynucleotide of the
invention and appropriate transcriptional/translational control
signals. These methods include in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination/genetic
recombination. See, for example, the techniques described in
Maniatis et al., Molecular Cloning A Laboratory Manual, Cold Spring
Harbor Laboratory, N.Y. (1989) and Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Associates and
Wiley Interscience, N.Y. (1989).
[0122] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lacI, lacZ, T3, T7,
gpt, lambda P, and trc. Eukaryotic promoters include CMV immediate
early, HSV thymidine kinase, early and late SV40, LTRs from
retrovirus, and mouse metallothionein-I.
[0123] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), a-factor, acid phosphatase, or heat shock proteins,
among others. The polynucleotide of the invention is assembled in
appropriate phase with translation initiation and termination
sequences, and preferably, a leader sequence capable of directing
secretion of translated protein into the periplasmic space or
extracellular medium. Optionally, the polynucleotide of the
invention can encode a fusion protein including an N-terminal
identification peptide imparting desired characteristics, e.g.,
stabilization or simplified purification of expressed recombinant
product.
[0124] Useful expression vectors for bacteria are constructed by
inserting a polynucleotide of the invention together with suitable
translation initiation and termination signals, optionally in
operable reading phase with a functional promoter. The vector will
comprise one or more phenotypic selectable markers and an origin of
replication to ensure maintenance of the vector and to, if
desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0125] As a representative but nonlimiting example, useful
expression vectors for bacteria can comprise a selectable marker
and bacterial origin of replication derived from commercially
available plasmids comprising genetic elements of the well known
cloning vector pBR322 (ATCC 37017). Such commercial vectors
include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,
Sweden) and GEM 1 (Promega Biotec, Madison, Wis., USA). These
pBR322 `backbone` sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0126] The present invention further provides host cells containing
the vectors of the present invention, wherein the nucleic acid has
been introduced into the host cell using known transformation,
transfection or infection methods. The host cell can be a higher
eukaryotic host cell, such as a mammalian cell, a lower eukaryotic
host cell, such as a yeast cell, or the host cell can be a
prokaryotic cell, such as a bacterial cell. Introduction of the
recombinant construct into the host cell can be effected, for
example, by calcium phosphate transfection, DEAE, dextran mediated
transfection, or electroporation (Davis, L. et al., Basic Methods
in Molecular Biology (1986)).
[0127] Any host/vector system can be used to identify one or more
of the target elements of the present invention. These include, but
are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell,
COS cells, and Sf9 cells, as well as prokaryotic host such as E.
coli and B. subtilis. The most preferred cells are those which do
not normally express the particular reporter polypeptide or protein
or which expresses the reporter polypeptide or protein at low
natural level.
[0128] The host of the present invention may also be a yeast or
other fungi. In yeast, a number of vectors containing constitutive
or inducible promoters may be used. For a review see, Current
Protocols in Molecular Biology, Vol. 2, Ed. Ausubel et al., Greene
Publish. Assoc. & Wiley Interscience, Ch. 13 (1988); Grant et
al., Expression and Secretion Vectors for Yeast, in Methods in
Enzymology, Ed. Wu & Grossman, Acad. Press, N.Y. 153:516-544
(1987); Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3
(1986); Bitter, Heterologous Gene Expression in Yeast, in Methods
in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y.
152:673-684 (1987); and The Molecular Biology of the Yeast
Saccharomyces, Eds. Strathern et al., Cold Spring Harbor Press,
Vols. I and II (1982).
[0129] The host of the invention may also be a prokaryotic cell
such as E. coli, other enterobacteriaceae such as Serratia
marescans, bacilli, various pseudomonads, or other prokaryotes
which can be transformed, transfected, infected, etc. (i.e., a
method exists for introducing nucleic acids to the host cell).
[0130] The present invention further provides host cells
genetically engineered to contain the polynucleotides of the
invention. For example, such host cells may contain nucleic acids
of the invention introduced into the host cell using known
transformation, transfection or infection methods. The present
invention still further provides host cells genetically engineered
to express the polynucleotides of the invention, wherein such
polynucleotides are in operative association with a regulatory
sequence heterologous to the host cell which drives expression of
the polynucleotides in the cell.
[0131] The host cell can be a higher eukaryotic host cell, such as
a mammalian cell, a lower eukaryotic host cell, such as a yeast
cell, or the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the recombinant construct into the
host cell can be effected by calcium phosphate transfection, DEAE,
dextran mediated transfection, or electroporation (Davis, L. et
al., Basic Methods in Molecular Biology (1986)). The host cells
containing one of polynucleotides of the invention, can be used in
conventional manners to produce the gene product encoded by the
isolated fragment (in the case of an ORF) or can be used to produce
a heterologous protein under the control of the EMF.
[0132] Any host/vector system can be used to express one or more of
the ORFs of the present invention. These include, but are not
limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS
cells, and Sf9 cells, as well as prokaryotic host such as E. coli
and B. subtilis. The most preferred cells are those which do not
normally express the particular polypeptide or protein or which
expresses the polypeptide or protein at low natural level. Mature
proteins can be expressed in mammalian cells, yeast, bacteria, or
other cells under the control of appropriate promoters. Cell-free
translation systems can also be employed to produce such proteins
using RNAs derived from the DNA constructs of the present
invention. Appropriate cloning and expression vectors for use with
prokaryotic and eukaryotic hosts are described by Sambrook, et al.,
in Molecular Cloning: A Laboratory Manual, Second Edition, Cold
Spring Harbor, N.Y. (1989), the disclosure of which is hereby
incorporated by reference.
[0133] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell tines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
also any necessary ribosome binding sites, polyadenylation site,
splice donor and acceptor sites, transcriptional termination
sequences, and 5' flanking nontranscribed sequences. DNA sequences
derived from the SV40 viral genome, for example, SV40 origin, early
promoter, enhancer, splice, and polyadenylation sites may be used
to provide the required nontranscribed genetic elements.
Recombinant polypeptides and proteins produced in bacterial culture
are usually isolated by initial extraction from cell pellets,
followed by one or more salting-out, aqueous ion exchange or size
exclusion chromatography steps. Protein refolding steps can be
used, as necessary, in completing configuration of the mature
protein. Finally, high performance liquid chromatography (HPLC) can
be employed for final purification steps. Microbial cells employed
in expression of proteins can be disrupted by any convenient
method, including freeze-thaw cycling, sonication, mechanical
disruption, or use of cell lysing agents.
[0134] A number of types of cells may act as suitable host cells
for expression of the protein. Mammalian host cells include, for
example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human
kidney 293 cells, human epidermal A431 cells, human Colo205 cells,
3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell strains derived from in vitro culture of
primary tissue, primary explants, HeLa cells, mouse L cells, BHK,
HL-60, U937, HaK or Jurkat cells.
[0135] Alternatively, it may be possible to produce the protein in
lower eukaryotes such as yeast or in prokaryotes such as bacteria.
Potentially suitable yeast strains include Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any yeast strain capable of expressing heterologous
proteins. Potentially suitable bacterial strains include
Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any
bacterial strain capable of expressing heterologous proteins. If
the protein is made in yeast or bacteria, it may be necessary to
modify the protein produced therein, for example by phosphorylation
or glycosylation of the appropriate sites, in order to obtain the
functional protein. Such covalent attachments may be accomplished
using known chemical or enzymatic methods.
[0136] In another embodiment of the present invention, cells and
tissues may be engineered to express an endogenous gene comprising
the polynucleotides of the invention under the control of inducible
regulatory elements, in which case the regulatory sequences of the
endogenous gene may be replaced by homologous recombination. As
described herein, gene targeting can be used to replace a gene's
existing regulatory region with a regulatory sequence isolated from
a different gene or a novel regulatory sequence synthesized by
genetic engineering methods. Such regulatory sequences may be
comprised of promoters, enhancers, scaffold-attachment regions,
negative regulatory elements, transcriptional initiation sites,
regulatory protein binding sites or combinations of said sequences.
Alternatively, sequences which affect the structure or stability of
the RNA or protein produced may be replaced, removed, added, or
otherwise modified by targeting, including polyadenylation signals.
mRNA stability elements, splice sites, leader sequences for
enhancing or modifying transport or secretion properties of the
protein, or other sequences which alter or improve the function or
stability of protein or RNA molecules.
[0137] The targeting event may be a simple insertion of the
regulatory sequence, placing the gene under the control of the new
regulatory sequence, e.g., inserting a new promoter or enhancer or
both upstream of a gene. Alternatively, the targeting event may be
a simple deletion of a regulatory element, such as the deletion of
a tissue-specific negative regulatory element. Alternatively, the
targeting event may replace an existing element; for example, a
tissue-specific enhancer can be replaced by an enhancer that has
broader or different cell-type specificity than the naturally
occurring elements. Here, the naturally occurring sequences are
deleted and new sequences are added. In all cases, the
identification of the targeting event may be facilitated by the use
of one or more selectable marker genes that are contiguous with the
targeting DNA, allowing for the selection of cells in which the
exogenous DNA has integrated into the host cell genome. The
identification of the targeting event may also be facilitated by
the use of one or more marker genes exhibiting the property of
negative selection, such that the negatively selectable marker is
linked to the exogenous DNA, but configured such that the
negatively selectable marker flanks the targeting sequence, and
such that a correct homologous recombination event with sequences
in the host cell genome does not result in the stable integration
of the negatively selectable marker. Markers useful for this
purpose include the Herpes Simplex Virus thymidine kinase (TK) gene
or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt)
gene.
[0138] The gene targeting or gene activation techniques which can
be used in accordance with this aspect of the invention are more
particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S.
Pat. No. 5,578,461 to Sherwin et al.; International Application No.
PCT/US92/09627 (WO93/09222) by Selden et al.; and International
Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al.,
each of which is incorporated by reference herein in its
entirety.
[0139] In general, techniques for preparing polyclonal and
monoclonal antibodies as well as hybridomas capable of producing
the desired antibody are well known in the art (Campbell, A. M.,
Monoclonal Antibodies Technology Laboratory Techniques in
Biochemistry and Molecular Biology, Elsevier Science Publishers,
Amsterdam, The Netherlands (1984); St. Groth et al., J. Immunol.
35:1-21 (1990); Kohler and Milstein, Nature 256:495-497 (1975)),
the trioma technique, the human B-cell hybridoma technique (Kozbor
et al., Immunology Today 4:72 (1983); Cole et al., in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), pp.
77-96).
[0140] Methods for immunization are well known in the art. Such
methods include subcutaneous or interperitoneal injection of the
polypeptide. One skilled in the art will recognize that the amount
of the protein encoded by the reporter gene of the present
invention used for immunization will vary based on the animal which
is immunized, the antigenicity of the peptide and the site of
injection.
[0141] The polypeptide or protein of the invention which is used as
an immunogen may be modified or administered in an adjuvant in
order to increase the polypeptide or protein's antigenicity.
Methods of increasing the antigenicity of a polypeptide or protein
are well known in the art and include, but are not limited to,
coupling the antigen with a heterologous protein (such as globulin
or .beta.-galactosidase) or through the inclusion of an adjuvant
during immunization.
[0142] For monoclonal antibodies, spleen cells from the immunized
animals are removed, fused with myeloma cells, such as SP2/0-Ag14
myeloma cells, and allowed to become monoclonal antibody producing
hybridoma cells.
[0143] Any one of a number of methods well known in the art can be
used to identify the hybridoma cell which produces an antibody with
the desired characteristics. These include screening the hybridomas
with an ELISA assay, western blot analysis, or radioimmunoassay
(Lutz et al., Exp. Cell Research. 175:109-124 (1988)).
[0144] Hybridomas secreting the desired antibodies are cloned and
the class and subclass is determined using procedures known in the
art (Campbell, A. M., Monoclonal Antibody Technology: Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science
Publishers, Amsterdam, The Netherlands (1984)).
[0145] For polyclonal antibodies, antibody containing antisera is
isolated from the immunized animal and is screened for the presence
of antibodies with the desired specificity using one of the
above-described procedures.
[0146] The present invention further provides the above-described
antibodies in detectably labeled form. Antibodies can be detectably
labeled through the use of radioisotopes, affinity labels (such as
biotin, avidin, etc.), enzymatic labels (such as horseradish
peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as
FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for
accomplishing such labeling are well-known in the art, for example,
see (Sternberger, L. A. et al., J. Histochem. CytoChem. 18:315
(1970); Bayer, E. A. et al., Meth. Enzym. 62:308 (1979); Engval, E.
et al., Immunol. 109:129 (1972); Goding, J. W. J. Immunol. Meth.
13:215 (1976)).
[0147] The labeled antibodies of the present invention can be used
for in vitro, in vivo, and in situ assays to identify cells or
tissues in which the polypeptide or protein of the invention is
expressed.
[0148] The present invention further provides the above-described
antibodies immobilized on a solid support. Examples of such solid
supports include plastics such as polycarbonate, complex
carbohydrates such as agarose and sepharose, acrylic resins and
such as polyacrylamide and latex beads. Techniques for coupling
antibodies to such solid supports are well known in the art (Weir,
D. M. et al., `Handbook of Experimental Immunology` 4th Ed.,
Blackwell Scientific Publications, Oxford, England, Chapter 10
(1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y.
(1974)). The immobilized antibodies of the present invention can be
used for immuno-affinity purification of host cells that are
expressing the polypeptide or protein of the invention.
[0149] Host cells are transfected or preferably infected or
transformed with the above-described vectors, and cultured in
nutrient media appropriate for selecting transductants or
transformants containing the vector.
[0150] The host cells which express the polypeptide or protein of
the invention product may be identified by at least four general
approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence
or absence of gene functions; (c) assessing the level of
transcription as measured by the expression of mRNA transcripts in
the host cell; and (d) detection of the gene product as measured by
immunoassay or by its biological activity.
[0151] In the first approach, the presence of the polypeptide or
protein of the invention inserted in the vector can be detected by
DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide
sequences that are homologous to the polypeptide or protein of the
invention, respectively, or portions or derivatives thereof.
[0152] In the second approach, the recombinant expression
vector/host system can be identified and selected based upon the
presence or absence of certain "marker" gene functions (e.g.,
thymidine kinase activity, resistance to antibiotics, resistance to
methotrexate, transformation phenotype, occlusion body formation in
baculovirus, etc.). For example, if the polypeptide or protein of
the invention is inserted within a marker gene sequence of the
vector, recombinant cells containing the polypeptide or protein of
the invention can be identified by the absence of the marker gene
function. Alternatively, a marker gene can be placed in tandem with
the polypeptide or protein of the invention under the control of
the same or different promoter used to control the expression of
the polypeptide or protein of the invention. Expression of the
marker in response to induction or selection indicates expression
of the polypeptide or protein of the invention.
[0153] In the third approach, transcriptional activity of the
polypeptide or protein of the invention can be assessed by
hybridization assays. For example, RNA can be isolated and analyzed
by Northern blot using a probe homologous to the polypeptide or
protein of the invention or particular portions thereof.
Alternatively, total nucleic acids of the host cell may be
extracted and assayed for hybridization to such probes.
[0154] In the fourth approach, the expression of a product from the
polypeptide or protein of the invention can be assessed
immunologically, for example by Western blots, immunoassays such as
radioimmuno-precipitation, enzyme-linked immunoassays and the
like.
[0155] The polynucleotides of the invention also provide
polynucleotides including nucleotide sequences that are
substantially equivalent to the polynucleotides of the invention.
Polynucleotides according to the invention can have at least about
80%, more typically at least about 90%, and even more typically at
least about 95%, sequence identity to a polynucleotide of the
invention. The invention also provides the complement of the
polynucleotides including a nucleotide sequence that has at least
about 80%, more typically at least about 90%, and even more
typically at least about 95%, sequence identity to a polynucleotide
encoding a polypeptide recited above. The polynucleotide can be DNA
(genomic, cDNA, amplified, or synthetic) or RNA. Methods and
algorithms for obtaining such polynucleotides are well known to
those of skill in the art and can include, for example, methods for
determining hybridization conditions which can routinely isolate
polynucleotides of the desired sequence identities.
[0156] A polynucleotide according to the invention can be joined to
any of a variety of other nucleotide sequences by well-established
recombinant DNA techniques (see Sambrook J et al. (1989) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY).
Useful nucleotide sequences for joining to polypeptides include an
assortment of vectors, e.g., plasmids, cosmids, lambda phage
derivatives, phagemids, and the like, that are well known in the
art. Accordingly, the invention also provides a vector including a
polynucleotide of the invention and a host cell containing the
polynucleotide. In general, the vector contains an origin of
replication functional in at least one organism, convenient
restriction endonuclease sites, and a selectable marker for the
host cell. Vectors according to the invention include expression
vectors, replication vectors, probe generation vectors, and
sequencing vectors. A host cell according to the invention can be a
prokaryotic or eukaryotic cell and can be a unicellular organism or
part of a multicellular organism.
[0157] The sequences falling within the scope of the present
invention are not limited to the specific sequences herein
described, but also include a representative fragment thereof, or a
nucleotide sequence at least 99.9% identical to a nucleic acid of
the invention. Furthermore, to accommodate codon variability, the
invention includes nucleic acid molecules encoding the polypeptide
sequences of the invention. In other words, in the coding region of
a polypeptide sequence of the invention, substitution of one codon
for another which encodes the same amino acid is expressly
contemplated. Any specific sequence disclosed herein can be readily
screened for errors by resequencing a particular fragment, such as
an ORF, in both directions (i.e., sequence both strands).
[0158] The present invention further provides recombinant
constructs comprising a nucleic acid of the invention, or a
fragment thereof. The recombinant constructs of the present
invention comprise a vector, such as a plasmid or viral vector,
into which a nucleic acid of the invention, or a fragment thereof
is inserted, in a forward or reverse orientation. In the case of a
vector comprising one of the ORFs of the present invention, the
vector may further comprise regulatory sequences, including for
example, a promoter, operably linked to the ORF. For vectors
comprising the EMFs and UMFs of the present invention, the vector
may further comprise a marker sequence or heterologous ORF operably
linked to the EMF or UMF. Large numbers of suitable vectors and
promoters are known to those of skill in the art and are
commercially available for generating the recombinant constructs of
the present invention. The following vectors are provided by way of
example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs
KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3,
pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat,
pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL
(Pharmacia).
[0159] The isolated polynucleotide of the invention may be operably
linked to an expression control sequence such as the pMT2 or pED
expression vectors disclosed in Kaufman et al., Nucleic Acids Res.
19, 4485-4490 (1991), in order to produce the protein or
polypeptide recombinantly. Many suitable expression control
sequences are known in the art. General methods of expressing
recombinant proteins are also known and are exemplified in R.
Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined
herein "operably linked" means that the isolated polynucleotide of
the invention and an expression control sequence are situated
within a vector or cell in such a way that the protein or
polypeptide is expressed by a host cell which has been transformed
(transfected) with the ligated polynucleotide/expression control
sequence.
[0160] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lacI, lacZ, T3, T7,
gpt, lambda P.sub.R, and trc. Eukaryotic promoters include CMV
immediate early, HSV thymidine kinase, early and late SV40, LTRs
from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art. Generally, recombinant expression
vectors will include origins of replication and selectable markers
permitting transformation of the host cell, e.g., the ampicillin
resistance gene of E. coli and S. cerevisiae TRP1 gene, and a
promoter derived from a highly-expressed gene to direct
transcription of a downstream structural sequence. Such promoters
can be derived from operons encoding glycolytic enzymes such as
3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or
heat shock proteins, among others. The heterologous structural
sequence is assembled in appropriate phase with translation
initiation and termination sequences, and preferably, a leader
sequence capable of directing secretion of translated protein or
polypeptide into the periplasmic space or extracellular medium.
Optionally, the heterologous sequence can encode a fusion protein
including an N-terminal identification peptide imparting desired
characteristics, e.g., stabilization or simplified purification of
expressed recombinant product. Useful expression vectors for
bacterial use are constructed by inserting a structural DNA
sequence encoding a desired protein or polypeptide together with
suitable translation initiation and termination signals in operable
reading phase with a functional promoter. The vector will comprise
one or more phenotypic selectable markers and an origin of
replication to ensure maintenance of the vector and to, if
desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0161] As a representative but non-limiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, Wis., USA).
These pBR322 `backbone` sections are combined with an appropriate
promoter and the structural sequence to be expressed. Following
transformation of a suitable host strain and growth of the host
strain to an appropriate cell density, the selected promoter is
induced or derepressed by appropriate means (e.g., temperature
shift or chemical induction) and cells are cultured for an
additional period. Cells are typically harvested by centrifugation,
disrupted by physical or chemical means, and the resulting crude
extract retained for further purification.
[0162] Included within the scope of the nucleic acid sequences of
the invention are nucleic acid sequences that hybridize under
stringent conditions to a polynucleotide of the invention, which
polynucleotide is greater than about 10 bp, preferably 20-50 bp,
and even greater than 100 bp. In accordance with the invention,
polynucleotide sequences of the invention, or functional
equivalents thereof, may be used to generate recombinant DNA
molecules that direct the expression of that polynucleotide, or a
functional equivalent thereof, in appropriate host cells.
[0163] The polynucleotides of the invention are further directed to
sequences which encode variants of the polypeptides or proteins of
the invention. These amino acid sequence variants may be prepared
by methods known in the art by introducing appropriate nucleotide
changes into a native or variant polynucleotide. There are two
variables in the construction of amino acid sequence variants: the
location of the mutation and the nature of the mutation. The amino
acid sequence variants of the nucleic acids are preferably
constructed by mutating the polynucleotide to give an amino acid
sequence that does not occur in nature. These amino acid
alterations can be made at sites that differ in the nucleic acids
from different species (variable positions) or in highly conserved
regions (constant regions). Sites at such locations will typically
be modified in series, e.g., by substituting first with
conservative choices (e.g., hydrophobic amino acid to a different
hydrophobic amino acid) and then with more distant choices (e.g.,
hydrophobic amino acid to a charged amino acid), and then deletions
or insertions may be made at the target site. Amino acid sequence
deletions generally range from about 1 to 30 residues, preferably
about 1 to 10 residues, and are typically contiguous. Amino acid
insertions include amino- and/or carboxyl-terminal fusions ranging
in length from one to one hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Intrasequence insertions may range generally from about 1 to 10
amino residues, preferably from 1 to 5 residues. Examples of
terminal insertions include the heterologous signal sequences
necessary for secretion or for intracellular targeting in different
host cells.
[0164] Amino acid sequence deletions generally range from about 1
to 30 residues, preferably about 1 to 10 residues, and are
typically contiguous. Amino acid insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one to one hundred
or more residues, as well as intrasequence insertions of single or
multiple amino acid residues. Intrasequence insertions may range
generally from about 1 to 10 amino residues, preferably from 1 to 5
residues. Examples of terminal insertions include the heterologous
signal sequences necessary for secretion or for intracellular
targeting in different host cells.
[0165] PCR may also be used to create amino acid sequence variants
of the polynucleotides of the invention. When small amounts of
template DNA are used as starting material, primer(s) that differs
slightly in sequence from the corresponding region in the template
DNA can generate the desired amino acid variant. PCR amplification
results in a population of product DNA fragments that differ from
the polynucleotide template encoding the polypeptide or protein at
the position specified by the primer. The product DNA fragments
replace the corresponding region in the plasmid and this gives the
desired amino acid variant.
[0166] In a preferred method, polynucleotides encoding the
polynucleotides of the invention are changed via site-directed
mutagenesis. This method uses oligonucleotide sequences that encode
the polynucleotide sequence of the desired amino acid variant, as
well as a sufficient adjacent nucleotide on both sides of the
changed amino acid to form a stable duplex on either side of the
site of being changed. In general, the techniques of site-directed
mutagenesis are well known to those of skill in the art and this
technique is exemplified by publications such as, Edelman et al.,
DNA 2:183 (1983). A versatile and efficient method for producing
site-specific changes in a polynucleotide sequence was published by
Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may
also be used to create amino acid sequence variants of the novel
nucleic acids. When small amounts of template DNA are used as
starting material, primer(s) that differs slightly in sequence from
the corresponding region in the template DNA can generate the
desired amino acid variant. PCR amplification results in a
population of product DNA fragments that differ from the
polynucleotide template encoding the polypeptide at the position
specified by the primer. The product DNA fragments replace the
corresponding region in the plasmid and this gives the desired
amino acid variant.
[0167] A further technique for generating amino acid variants is
the cassette mutagenesis technique described in Wells et al., Gene
34:315 (1985); and other mutagenesis techniques well known in the
art, such as, for example, the techniques in Sambrook et al.,
supra, and Current Protocols in Molecular Biology, Ausubel et al.
Due to the inherent degeneracy of the genetic code, other DNA
sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be used in the practice of the
invention for the cloning and expression of these novel nucleic
acids. Such DNA sequences include those which are capable of
hybridizing to the appropriate novel nucleic acid sequence under
stringent conditions.
[0168] The invention encompasses polypeptides or proteins encoded
by the polynucleotides of the invention. Fragments of the
polypeptides or proteins of the present invention which are capable
of exhibiting biological activity are also encompassed by the
present invention. Fragments of the protein or polypeptide may be
in linear form or they may be cyclized using known methods, for
example, as described in H. U. Saragovi, et al., Bio/Technology 10,
773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc.
114, 9245-9253 (1992), both of which are incorporated herein by
reference.
[0169] The present invention also provides both full-length and
mature forms of the polypeptides or proteins of the invention. The
full-length form of the such polypeptides or proteins can be
identified by translation of the nucleotide sequence of each
polynucleotide of the invention. The mature form of such
polypeptide or protein may be obtained by expression of the
full-length polynucleotide in a suitable mammalian cell or other
host cell. The sequence of the mature form of the polypeptide or
protein may also be determinable from the amino acid sequence of
the full-length form.
[0170] Where the protein or polypeptide of the present invention is
membrane-bound (e.g., is a receptor), the present invention also
provides for soluble forms of such protein or polypeptide. In such
forms part or all of the intracellular and transmembrane domains of
the protein or polypeptide are deleted such that the protein or
polypeptide is fully secreted from the cell in which it is
expressed. The intracellular and transmembrane domains of proteins
or polypeptides of the invention can be identified in accordance
with known techniques for determination of such domains from
sequence information.
[0171] The invention also relates to methods for producing a
polypeptide or protein of the invention comprising growing a
culture of the cells of the invention in a suitable culture medium,
and purifying the protein or polypeptide of the invention from the
culture. For example, the methods of the invention include a
process for producing a polypeptide or protein of the invention in
which a host cell containing a suitable expression vector that
includes a polynucleotide or protein of the invention is cultured
under conditions that allow expression of the encoded polypeptide
or protein. The polypeptide or protein can be recovered from the
culture, conveniently from the culture medium, and further
purified.
[0172] The invention further provides a polypeptide or protein of
the invention including an amino acid sequence that is
substantially equivalent to an amino acid sequence encoded by a
polynucleotide of the invention. Polypeptides or proteins according
to the invention can have at least about 95%, and more typically at
least about 98%, sequence identity to an amino acid sequence
encoded by a polynucleotide of the invention.
[0173] The present invention further provides isolated polypeptides
or proteins encoded by the polynucleotides of the present invention
or by degenerate variants of the polynucleotides of the present
invention. By `degenerate variant` is intended polynucleotides
which differ from a nucleic acid fragment of the present invention
(e.g., an ORF) by nucleotide sequence but, due to the degeneracy of
the genetic code, encode an identical polypeptide sequence.
Preferred polynucleotides of the present invention are the ORFs
that encode proteins or polypeptides. A variety of methodologies
known in the art can be utilized to obtain any one of the isolated
polypeptides or proteins of the present invention. At the simplest
level, the amino acid sequence can be synthesized using
commercially available peptide synthesizers. This is particularly
useful in producing small peptides and fragments of larger
polypeptides. Fragments are useful, for example, in generating
antibodies against the native polypeptide. In an alternative
method, the polypeptide or protein is purified from bacterial cells
which naturally produce the polypeptide or protein. One skilled in
the art can readily follow known methods for isolating polypeptides
and proteins in order to obtain one of the isolated polypeptides or
proteins of the present invention. These include, but are not
limited to, immunochromatography, HPLC, size-exclusion
chromatography, ion-exchange chromatography, and immuno-affinity
chromatography. See, e.g., Scopes, Protein Purification: Principles
and Practice, Springer-Verlag (1994); Sambrook, et al., in
Molecular Cloning: A Laboratory Manual; Ausubel et al., Current
Protocols in Molecular Biology.
[0174] The polypeptides and proteins of the present invention can
alternatively be purified from cells which have been altered to
express the desired polypeptide or protein. As used herein, a cell
is said to be altered to express a desired polypeptide or protein
when the cell, through genetic manipulation, is made to produce a
polypeptide or protein which it normally does not produce or which
the cell normally produces at a lower level. One skilled in the art
can readily adapt procedures for introducing and expressing either
recombinant or synthetic sequences into eukaryotic or prokaryotic
cells in order to generate a cell which produces one of the
polypeptides or proteins of the present invention. The purified
polypeptides or proteins can be used in in vitro binding assays
which are well known in the art to identify molecules which bind to
the polypeptides or proteins. These molecules include but are not
limited to, for e.g., small molecules, molecules from combinatorial
libraries, antibodies or other proteins or polypeptides. The
molecules identified in the binding assay are then tested for
antagonist or agonist activity in in vivo tissue culture or animal
models that are well known in the art. In brief, the molecules are
titrated into a plurality of cell cultures or animals and then
tested for either cell/animal death or prolonged survival of the
animal/cells.
[0175] In addition, the binding molecules may be complexed with
toxins, e.g., ricin or cholera, or with other compounds that are
toxic to cells. The toxin-binding molecule complex is then targeted
to the tumor or other cell by the specificity of the binding
molecule.
[0176] The protein or polypeptide of the invention may also be
expressed as a product of transgenic animals, e.g., as a component
of the milk of transgenic cows, goats, pigs, or sheep which are
characterized by somatic or germ cells containing a polynucleotide
encoding the protein or polypeptide of the invention.
[0177] The protein or polypeptide of the invention may also be
produced by known conventional chemical synthesis. Methods for
constructing the proteins or polypeptides of the present invention
by synthetic means are known to those skilled in the art. The
synthetically-constructed proteins or polypeptides, by virtue of
sharing primary, secondary or tertiary structural and/or
conformational characteristics with proteins or polypeptides may
possess biological properties in common therewith, including
protein activity. Thus, they may be employed as biologically active
or immunological substitutes for natural, purified proteins or
polypeptides of the invention in screening of therapeutic compounds
and in immunological processes for the development of
antibodies.
[0178] The proteins or polypeptides of the invention provided
herein also include proteins or polypeptides characterized by amino
acid sequences similar to those of purified proteins or
polypeptides of the invention but into which modifications are
naturally provided or deliberately engineered. For example,
modifications in the peptide or DNA sequences can be made by those
skilled in the art using known techniques. Modifications of
interest in the protein sequences may include the alteration,
substitution, replacement, insertion or deletion of a selected
amino acid residue in the coding sequence. For example, one or more
of the cysteine residues may be deleted or replaced with another
amino acid to alter the conformation of the molecule. Techniques
for such alteration, substitution, replacement, insertion or
deletion are well known to those skilled in the art (see, e.g.,
U.S. Pat. No. 4,518,584). Preferably, such alteration,
substitution, replacement, insertion or deletion retains the
desired activity of the protein or polypeptide.
[0179] Other fragments and derivatives of the polypeptides or
proteins of the invention which would be expected to retain protein
activity in whole or in part and may thus be useful for screening
or other immunological methodologies may also be easily made by
those skilled in the art given the disclosures herein. Such
modifications are encompassed by the present invention.
[0180] The protein or polypeptide of the invention may also be
produced by operably linking a polynucleotide of the invention to
suitable control sequences in one or more insect expression
vectors, and employing an insect expression system. Materials and
methods for baculovirus/insect cell expression systems are
commercially available in kit form from, e.g., Invitrogen, San
Diego, Calif., U.S.A. (the MaxBat.RTM. kit), and such methods are
well known in the art, as described in Summers and Smith, Texas
Agricultural Experiment Station Bulletin No. 1555 (1987),
incorporated herein by reference. As used herein, an insect cell
capable of expressing a polynucleotide of the present invention is
"transformed."
[0181] The protein or polypeptide of the invention may be prepared
by culturing transformed host cells under culture conditions
suitable to express the recombinant protein or polypeptide. The
resulting expressed protein or polypeptide may then be purified
from such culture (i.e., from culture medium or cell extracts)
using known purification processes, such as gel filtration and ion
exchange chromatography. The purification of the protein or
polypeptide may also include an affinity column containing agents
which will bind to the protein or polypeptide; one or more column
steps over such affinity resins as concanavalin A-agarose,
Heparin-Toyopearl.RTM. or Cibacrom blue 3GA Sepharose.RTM.; one or
more steps involving hydrophobic interaction chromatography using
such resins as phenyl ether, butyl ether, or propyl ether; or
immunoaffinity chromatography.
[0182] Alternatively, the protein or polypeptide of the invention
may also be expressed in a form which will facilitate purification.
For example, it may be expressed as a fusion protein, such as those
of maltose binding protein (MBP), glutathione-S-transferase (GST)
or thioredoxin (TRX). Kits for expression and purification of such
fusion proteins are commercially available from New England BioLab
(Beverly, Mass.), Pharmacia (Piscataway, N.J.) and In Vitrogen,
respectively. The protein or polypeptide can also be tagged with an
epitope and subsequently purified by using a specific antibody
directed to such epitope. One such epitope ("Flag") is commercially
available from Kodak (New Haven, Conn.).
[0183] Finally, one or more reverse-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify the protein or polypeptide. Some
or all of the foregoing purification steps, in various
combinations, can also be employed to provide a substantially
homogeneous isolated recombinant protein or polypeptide. The
protein or polypeptide thus purified is substantially free of other
mammalian proteins and is defined in accordance with the present
invention as an "isolated protein."
[0184] The polynucleotides and polypeptides of the present
invention are expected to exhibit one or more of the uses or
biological activities (including those associated with assays cited
herein) identified below. Uses or activities described for the
polypeptides or proteins of the present invention may be provided
by administration or use of such proteins or polypeptides or by
administration or use of polynucleotides encoding such proteins or
polypeptides (such as, for example, in gene therapies or vectors
suitable for introduction of DNA).
[0185] The polynucleotides provided by the present invention can be
used by the research community for various purposes. The
polynucleotides can be used to express recombinant protein or
polypeptide for analysis, characterization, diagnostic or
therapeutic use; as markers for tissues in which the target protein
is abnormally or normally expressed (e.g., constitutively or at a
particular stage of tissue differentiation or development or in
disease states); as molecular weight markers on Southern gels; as
chromosome markers or tags (when labeled) to identify chromosomes
or to map related gene positions; to compare with endogenous DNA
sequences in patients to identify potential genetic disorders; as
probes to hybridize and thus discover novel, related DNA sequences;
as a source of information to derive PCR primers for genetic
fingerprinting; as a probe to "subtract-out" known sequences in the
process of discovering other novel polynucleotides; for selecting
and making oligomers for attachment to a "gene chip" or other
support, including for examination of expression patterns; for
attachment to a substrate to make an antibody chip for examining
protein (target) expression patterns or target expression levels or
the presence of the target, and as an antigen to raise
anti-idiotype antibodies. When the target protein binds or
potentially binds to another protein or other factor, the
polynucleotides of the invention can also be used in interaction
trap assays (such as, for example, that described in Gyuris et al.,
Cell 75:791-803 (1993)) to identify polynucleotides encoding the
other protein or factor with which binding occurs or to identify
other factors or proteins involved in the binding interation.
[0186] The proteins or polypeptides provided by the present
invention can similarly be used to determine biological activity,
including in a panel of multiple proteins or polypeptides for
high-throughput screening; as a reagent (including the labeled
reagent) in assays designed to quantitatively determine levels of
the target protein in biological samples; as markers for tissues in
which the target protein of the invention is normally or abnormally
expressed (either constitutively or at a particular stage of tissue
differentiation or development or in a disease state); and, of
course, to isolate correlative receptors or ligands. Where the
target protein binds or potentially binds to another protein or
factor (such as, for example, in a receptor-ligand interaction),
the polypeptide of the invention can be used to identify the other
protein or factor with which binding occurs or to identify
inhibitors of the binding interaction. Proteins involved in these
binding interactions can also be used to screen for peptide or
small molecule inhibitors or agonists of the binding
interaction.
[0187] Any or all of these research utilities are capable of being
developed into reagent grade or kit format for commercialization as
research products.
[0188] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include without limitation "Molecular Cloning: A Laboratory
Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J.,
E. F. Fritsch and T. Maniatis eds., 1989, and "Methods in
Enzymology: Guide to Molecular Cloning Techniques", Academic Press,
Berger, S. L. and A. R. Kimmel eds., 1987.
[0189] Polynucleotides, proteins, and polypeptides of the present
invention can also be used as nutritional sources or supplements.
Such uses include without limitation use as a protein or
polypeptide or amino acid supplement, use as a carbon source, use
as a nitrogen source and use as a source of carbohydrate. In such
cases the protein, polypeptide, or polynucleotide of the invention
can be added to the feed of a particular organism or can be
administered as a separate solid or liquid preparation, such as in
the form of powder, pills, solutions, suspensions or capsules. In
the case of microorganisms, the protein, polypeptide, or
polynucleotide of the invention can be added to the medium in or on
which the microorganism is cultured.
[0190] A protein or polypeptide of the present invention may
exhibit cytokine, cell proliferation (either inducing or
inhibiting) or cell differentiation (either inducing or inhibiting)
activity or may induce production of other cytokines in certain
cell populations, or may be an antagonist or agonist of any of the
above. A polynucleotide of the invention can encode a polypeptide
exhibiting such attributes. Many protein factors discovered to
date, including all known cytokines, have exhibited activity in one
or more factor-dependent cell proliferation assays, and hence the
assays serve as a convenient confirmation of cytokine agonist or
antagonist activity. The activity of a protein or polypeptide of
the present invention is evidenced by any one of a number of
routine factor dependent cell proliferation assays for cell lines
including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,
BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2,
TF-1, Mo7e and CMK.
[0191] The activity of a protein or polypeptide of the invention
may, among other means, be measured by the following methods:
[0192] Assays for T-cell or thymocyte proliferation include without
limitation those described in: Current Protocols in Immunology, Ed
by J. E. coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 3, In vitro assays for Mouse Lymphocyte
Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai
et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J.
Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular
Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol.
149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761,
1994.
[0193] Assays for cytokine production and/or proliferation of
spleen cells, lymph node cells or thymocytes include, without
limitation, those described in: Polyclonal T cell stimulation,
Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in
Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John
Wiley and Sons, Toronto. 1994; and Measurement of mouse and human
interleukin gamma., Schreiber, R. D. In Current Protocols in
Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John
Wiley and Sons, Toronto. 1994.
[0194] Assays for proliferation and differentiation of
hematopoietic and lymphopoietic cells include, without limitation,
those described in: Measurement of Human and Murine Interleukin 2
and Interleukin 4, Bottomly, K., Davis, L. S, and Lipsky, P. E. In
Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.
6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al.,
J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature
336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci.
U.S.A. 80:2931-2938, 1983; Measurement of mouse and human
interleukin 6-Nordan, R. In Current Protocols in Immunology. J. E.
e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto. 1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A.
83:1857-1861, 1986; Measurement of human Interleukin 11-Bennett,
F., Giannotti, J., Clark, S. C. and Turner, K. J. In Current
Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.15.1
John Wiley and Sons, Toronto. 1991; Measurement of mouse and human
Interleukin 9-Ciarletta, A., Giannotti, J., Clark, S. C. and
Turner, K. J. In Current Protocols in Immunology. J. E. e.a.
Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.
1991.
[0195] Assays for T-cell clone responses to antigens (which will
identify, among others, proteins that affect APC-T cell
interactions as well as direct T-cell effects by measuring
proliferation and cytokine production) include, without limitation,
those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W
Strober, Pub. Greene Publishing Associates and Wiley-Interscience
(Chapter 3, In vitro assays for Mouse Lymphocyte Function; Chapter
6, Cytokines and their cellular receptors; Chapter 7, Immunologic
studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA
77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411,
1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol. 140:508-512, 1988.
[0196] In all the above assays, the polypeptide or protein of the
invention is added into the assay system and activity of a target
cytokine is determined in the presence and absence of the
polypeptide or protein of the invention.
[0197] Further, the polypeptides of the invention may be used to
examine the expression level or presence of a cytokine. In
alternate embodiments, the detection of a cytokine or of a the
level of a cytokine will be diagnostic for a disease state or
condition.
[0198] A protein or polypeptide of the present invention may also
exhibit immune stimulating or immune suppressing activity, or may
be antagonists or agonists of either activity, including without
limitation the activities for which assays are described herein. A
polynucleotide of the invention can encode a polypeptide or protein
exhibiting such activities. A protein or polypeptide of the
invention may be useful in the treatment and/or detection (e.g., a
diagnostic) of various immune deficiencies and disorders (including
severe combined immunodeficiency (SCID)), e.g., in regulating (up
or down) growth and proliferation of T and/or B lymphocytes, as
well as effecting the cytolytic activity of NK cells and other cell
populations. These immune deficiencies may be genetic or be caused
by viral (e.g., HIV) as well as bacterial or fungal infections, or
may result from autoimmune disorders. More specifically, infectious
diseases caused by viral, bacterial, fungal or other infections may
be treatable or detectable (e.g., a diagnostic test) using a
protein or polypeptide of the present invention, including
infections by HIV, hepatitis viruses, herpesviruses, mycobacteria,
Leishmania spp., malaria spp. and various fungal infections such as
candidiasis. Of course, in this regard, a protein or polypeptide of
the present invention may also be useful where a boost to the
immune system generally may be desirable, i.e., in the treatment of
cancer.
[0199] Autoimmune disorders which may be treated or detected using
a protein or polypeptide of the present invention include, for
example, connective tissue disease, multiple sclerosis, systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary
inflammation, Guillain-Barre syndrome, autoimmune thyroiditis,
insulin dependent diabetes mellitis, myasthenia gravis,
graft-versus-host disease and autoimmune inflammatory eye disease.
Such a protein or polypeptide of the present invention may also to
be useful in the treatment of allergic reactions and conditions,
such as asthma (particularly allergic asthma) or other respiratory
problems. Other conditions, in which immune suppression is desired
(including, for example, organ transplantation), may also be
treatable using a protein or polypeptide of the present
invention.
[0200] Using the proteins or polypeptides of the invention it may
also be possible to modulate immune responses, in a number of ways.
Down regulation may be in the form of inhibiting or blocking an
immune response already in progress or may involve preventing the
induction of an immune response. The functions of activated T cells
may be inhibited by suppressing T cell responses or by inducing
specific tolerance in T cells, or both. Immunosuppression of T cell
responses is generally an active, non-antigen-specific, process
which requires continuous exposure of the T cells to the
suppressive agent. Tolerance, which involves inducing
non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and
persists after exposure to the tolerizing agent has ceased.
Operationally, tolerance can be demonstrated by the lack of a T
cell response upon reexposure to specific antigen in the absence of
the tolerizing agent.
[0201] Down regulating or preventing one or more antigen functions
(including without limitation B lymphocyte antigen functions (such
as, for example, B7)), e.g., preventing high level lymphokine
synthesis by activated T cells, will be useful in situations of
tissue, skin and organ transplantation and in graft-versus-host
disease (GVHD). For example, blockage of T cell function should
result in reduced tissue destruction in tissue transplantation.
Typically, in tissue transplants, rejection of the transplant is
initiated through its recognition as foreign by T cells, followed
by an immune reaction that destroys the transplant. The
administration of a molecule which inhibits or blocks interaction
of a B7 lymphocyte antigen with its natural ligand(s) on immune
cells (such as a soluble, monomeric form of a peptide having B7-2
activity alone or in conjunction with a monomeric form of a peptide
having an activity of another B lymphocyte antigen (e.g., B7-1,
B7-3) or blocking antibody), prior to transplantation can lead to
the binding of the molecule to the natural ligand(s) on the immune
cells without transmitting the corresponding costimulatory signal.
Blocking B lymphocyte antigen function in this matter prevents
cytokine synthesis by immune cells, such as T cells, and thus acts
as an immunosuppressant. Moreover, the lack of costimulation may
also be sufficient to anergize the T cells, thereby inducing
tolerance in a subject. Induction of long-term tolerance by B
lymphocyte antigen-blocking reagents may avoid the necessity of
repeated administration of these blocking reagents. To achieve
sufficient immunosuppression or tolerance in a subject, it may also
be necessary to block the function of a combination of B lymphocyte
antigens.
[0202] The efficacy of particular blocking reagents in preventing
organ transplant rejection or GVHD can be assessed using animal
models that are predictive of efficacy in humans. Examples of
appropriate systems which can be used include allogeneic cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice,
both of which have been used to examine the immunosuppressive
effects of CTLA4Ig fusion proteins in vivo as described in Lenschow
et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl.
Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of
GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York,
1989, pp. 846-847) can be used to determine the effect of blocking
B lymphocyte antigen function in vivo on the development of that
disease. Further, the polypeptides of the invention can be used to
detect GVHD after organ transplant.
[0203] Blocking antigen function may also be therapeutically useful
for treating autoimmune diseases. Many autoimmune disorders are the
result of inappropriate activation of T cells that are reactive
against self tissue and which promote the production of cytokines
and autoantibodies involved in the pathology of the diseases.
Preventing the activation of autoreactive T cells may reduce or
eliminate disease symptoms. Administration of reagents which block
costimulation of T cells by disrupting receptor:ligand interactions
of B lymphocyte antigens can be used to inhibit T cell activation
and prevent production of autoantibodies or T cell-derived
cytokines which may be involved in the disease process.
Additionally, blocking reagents may induce antigen-specific
tolerance of autoreactive T cells which could lead to long-term
relief from the disease. The efficacy of blocking reagents in
preventing or alleviating autoimmune disorders can be determined
using a number of well-characterized animal models of human
autoimmune diseases. Examples include murine experimental
autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr
mice or NZB hybrid mice, murine autoimmune collagen arthritis,
diabetes mellitus in NOD mice and BB rats, and murine experimental
myasthenia gravis (see Paul ed., Fundamental Immunology, Raven
Press, New York, 1989, pp. 840-856). Further, polypeptides of the
invention can be used to diagnose an immune disorder and/or the
susceptibility of an organism for an immune disorder.
[0204] Upregulation of an antigen function (preferably a B
lymphocyte antigen function), as a means of up regulating immune
responses, may also be useful in therapy. Upregulation of immune
responses may be in the form of enhancing an existing immune
response or eliciting an initial immune response. For example,
enhancing an immune response through stimulating B lymphocyte
antigen function may be useful in cases of viral infection. In
addition, systemic viral diseases such as influenza, the common
cold, and encephalitis might be alleviated by the administration of
stimulatory forms of B lymphocyte antigens systemically.
[0205] Alternatively, anti-vital immune responses may be enhanced
in an infected patient by removing T cells from the patient,
costimulating the T cells in vitro with viral antigen-pulsed APCs
either expressing a peptide of the present invention or together
with a stimulatory form of a soluble peptide of the present
invention and reintroducing the in vitro activated T cells into the
patient. Another method of enhancing anti-viral immune responses
would be to isolate infected cells from a patient, transfect them
with a nucleic acid encoding a protein or polypeptide of the
present invention as described herein such that the cells express
all or a portion of the protein or polypeptide on their surface,
and reintroduce the transfected cells into the patient. The
infected cells would now be capable of delivering a costimulatory
signal to, and thereby activate, T cells in vivo.
[0206] The presence of a polypeptide or protein of the present
invention having the activity of a B lymphocyte antigen(s) on the
surface of the tumor cell provides the necessary costimulation
signal to T cells to induce a T cell mediated immune response
against the transfected tumor cells. In addition, tumor cells which
lack MHC class I or MHC class II molecules, or which fail to
reexpress sufficient mounts of MHC class I or MHC class II
molecules, can be transfected with nucleic acid encoding all or a
portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC
class I .alpha. chain protein and .beta..sub.2 microglobulin
protein or an MHC class II'' chain protein and an MHC class II
.beta. chain protein to thereby express MHC class I or MHC class II
proteins on the cell surface. Expression of the appropriate class I
or class II MHC in conjunction with a peptide having the activity
of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell
mediated immune response against the transfected tumor cell.
Optionally, a gene encoding an antisense construct which blocks
expression of an MHC class II associated protein, such as the
invariant chain, can also be cotransfected with a DNA encoding a
peptide having the activity of a B lymphocyte antigen to promote
presentation of tumor associated antigens and induce tumor specific
immunity. Thus, the induction of a T cell mediated immune response
in a human subject may be sufficient to overcome tumor-specific
tolerance in the subject.
[0207] The activity of a protein or polypeptide of the invention
may, among other means, be measured by the following methods:
[0208] Suitable assays for thymocyte or splenocyte cytotoxicity
include, without limitation, those described in: Current Protocols
in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.
Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 3, In vitro assays for
Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies
in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974,
1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al.,
I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.
140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974,
1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al.,
J. Immunol. 137:3494-3500, 1986; Bowman et al., J. Virology
61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et
al., J. Immunol. 153:3079-3092, 1994.
[0209] Assays for T-cell-dependent immunoglobulin responses and
isotype switching (which will identify, among others, proteins that
modulate T-cell dependent antibody responses and that affect
Th1/Th2 profiles) include, without limitation, those described in:
Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: In vitro antibody production, Mond, J. J. and Brunswick,
M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol
1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
[0210] Mixed lymphocyte reaction (MLR) assays (which will identify,
among others, proteins that generate predominantly Th1 and CTL
responses) include, without limitation, those described in: Current
Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D.
H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 3, In vitro assays for
Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies
in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et
al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
[0211] Dendritic cell-dependent assays (which will identify, among
others, proteins expressed by dendritic cells that activate naive
T-cells) include, without limitation, those described in: Guery et
al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal
of Immunology 154:5071-5079, 1995; Porgador et al., Journal of
Experimental Medicine 182:255-260, 1995; Nair et al., Journal of
Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine
169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical
Investigation 94:797-807, 1994; and Inaba et al., Journal of
Experimental Medicine 172:631-640, 1990.
[0212] Assays for lymphocyte survival/apoptosis (which will
identify, among others, proteins that prevent apoptosis after
superantigen induction and proteins that regulate lymphocyte
homeostasis) include, without limitation, those described in:
Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al.,
Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research
53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry
14:891-897, 1993; Gorczyca et al., International Journal of
Oncology 1:639-648, 1992.
[0213] Assays for proteins that influence early steps of T-cell
commitment and development include, without limitation, those
described in: Antica et al., Blood 84:111-117, 1994; Fine et al.,
Cellular Immunology 155:111-122, 1994; Galy et al., Blood
85:2770-2778, 1995; Toki et al., Proc. Nat. Acad. Sci. USA
88:7548-7551, 1991.
[0214] A protein or polypeptide of the present invention may be
useful in regulation of hematopoiesis (as an antagonist or agonist)
and, consequently, in the treatment and/or detection (e.g., a
diagnostic) of myeloid or lymphoid cell deficiencies. Even marginal
biological activity in support of colony forming cells or of
factor-dependent cell lines indicates involvement in regulating
hematopoiesis, e.g., in supporting the growth and proliferation of
erythroid progenitor cells alone or in combination with other
cytokines, thereby indicating utility, for example, in treating
and/or detecting (e.g., a diagnostic) various anemias or for use in
conjunction with irradiation/chemotherapy to stimulate the
production of erythroid precursors and/or erythroid cells; in
supporting the growth and proliferation of myeloid cells such as
granulocytes and monocytes/macrophages (i.e., traditional CSF
activity), for example, in conjunction with chemotherapy to prevent
or treat consequent myelo-suppression; in supporting the growth and
proliferation of megakaryocytes and consequently of platelets
thereby allowing prevention or treatment of various platelet
disorders such as thrombocytopenia, and generally for use in place
of or complimentary to platelet transfusions; and/or in supporting
the growth and proliferation of hematopoietic stem cells which are
capable of maturing to any and all of the above-mentioned
hematopoietic cells and therefore find therapeutic utility in
various stem cell disorders (such as those usually treated with
transplantation, including, without limitation, aplastic anemia and
paroxysmal nocturnal hemoglobinuria), as well as in repopulating
the stem cell compartment post irradiation/chemotherapy, either
in-vivo or ex-vivo (i.e., in conjunction with bone marrow
transplantation or with peripheral progenitor cell transplantation
(homologous or heterologous)) as normal cells or genetically
manipulated for gene therapy.
[0215] The activity of a protein or polypeptide of the invention
may, among other means, be measured by the following methods:
[0216] Suitable assays for proliferation and differentiation of
various hematopoietic lines are cited above.
[0217] Assays for embryonic stem cell differentiation (which will
identify, among others, proteins that influence embryonic
differentiation hematopoiesis) include, without limitation, those
described in: Johansson et al. Cellular Biology 15:141-151, 1995;
Keller et al., Molecular and Cellular Biology 13:473-486, 1993;
McClanahan et al., Blood 81:2903-2915, 1993.
[0218] Assays for stem cell survival and differentiation (which
will identify, among others, proteins that regulate
lympho-hematopoiesis) include, without limitation, those described
in: Methylcellulose colony forming assays, Freshney, M. G. In
Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.
265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al.,
Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive
hematopoietic colony forming cells with high proliferative
potential, McNiece, I. K. and Briddell, R. A. In Culture of
Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,
Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental
Hematology 22:353-359, 1994; Cobblestone area forming cell assay,
Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I.
Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York,
N.Y. 1994; Long term bone marrow cultures in the presence of
stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of
Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-179,
Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating
cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R.
I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New
York, N.Y. 1994.
[0219] A protein or polypeptide of the present invention also may
have utility in compositions used for bone, cartilage, tendon,
ligament and/or nerve tissue growth or regeneration, as well as for
wound healing and tissue repair and replacement, and in the
treatment of burns, incisions and ulcers (as an antagonist or
agonist).
[0220] A protein or polypeptide of the present invention, which
acts as an antagonist or agonist of cartilage and/or bone growth,
has application in the healing of bone fractures and cartilage
damage or defects in humans and other animals. Such a preparation
employing a protein or polypeptide of the invention may have
prophylactic use in closed as well as open fracture reduction and
also in the improved fixation of artificial joints. De novo bone
formation induced by an osteogenic agent contributes to the repair
of congenital, trauma induced, or oncologic resection induced
craniofacial defects, and also is useful in cosmetic plastic
surgery.
[0221] A protein or polypeptide of this invention may also be used
in the treatment and/or detection (e.g., a diagnostic) of
periodontal disease, and in other tooth repair processes. Such
agents may provide an environment to attract bone-forming cells,
stimulate growth of bone-forming cells or induce differentiation of
progenitors of bone-forming cells. A protein or polypeptide of the
invention may also be useful in the treatment of osteoporosis or
osteoarthritis, such as through stimulation of bone and/or
cartilage repair or by blocking inflammation or processes of tissue
destruction (collagenase activity, osteoclast activity, etc.)
mediated by inflammatory processes.
[0222] Another category of tissue regeneration activity that may be
attributable to the protein or polypeptide of the present invention
is tendon/ligament formation. A protein or polypeptide of the
present invention, which induces tendon/ligament-like tissue or
other tissue formation in circumstances where such tissue is not
normally formed, has application in the healing of tendon or
ligament tears, deformities and other tendon or ligament defects in
humans and other animals. Such a preparation employing a
tendon/ligament-like tissue inducing protein (as an antagonist or
agonist) may have prophylactic use in preventing damage to tendon
or ligament tissue, as well as use in the improved fixation of
tendon or ligament to bone or other tissues, and in repairing
defects to tendon or ligament tissue. De novo tendon/ligament-like
tissue formation induced by a composition of the present invention
contributes to the repair of congenital, trauma induced, or other
tendon or ligament defects of other origin, and is also useful in
cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The compositions of the present invention may provide
environment to attract tendon- or ligament-forming cells, stimulate
growth of tendon- or ligament-forming cells, induce differentiation
of progenitors of tendon- or ligament-forming cells, or induce
growth of tendon/ligament cells or progenitors ex vivo for return
in vivo to effect tissue repair. The compositions of the invention
may also be useful in the treatment of tendinitis, carpal tunnel
syndrome and other tendon or ligament defects. The compositions may
also include an appropriate matrix and/or sequestering agent as a
carrier as is well known in the art.
[0223] The protein or polypeptide of the present invention may also
be useful for proliferation of neural cells and for regeneration of
nerve and brain tissue, i.e., for the treatment and/or detection
(e.g., a diagnostic) of central and peripheral nervous system
diseases and neuropathies, as well as mechanical and traumatic
disorders, which involve degeneration, death or trauma to neural
cells or nerve tissue. More specifically, a protein or polypeptide
may be used in the treatment and/or detection (e.g., a diagnostic)
of diseases of the peripheral nervous system, such as peripheral
nerve injuries, peripheral neuropathy and localized neuropathies,
and central nervous system diseases, such as Alzheimer's,
Parkinson's disease, Huntington's disease, amyotrophic lateral
sclerosis, and Shy-Drager syndrome. Further conditions which may be
treated in accordance with the present invention include mechanical
and traumatic disorders, such as spinal cord disorders, head trauma
and cerebrovascular diseases such as stroke. Peripheral
neuropathies resulting from chemotherapy or other medical therapies
may also be treatable using a protein or polypeptide of the
invention.
[0224] Proteins or polypeptides of the invention may also be useful
to promote better or faster closure of non-healing wounds,
including without limitation pressure ulcers, ulcers associated
with vascular insufficiency, surgical and traumatic wounds, and the
like.
[0225] It is expected that a protein or polypeptide of the present
invention may also exhibit activity for generation or regeneration
of other tissues, such as organs (including, for example, pancreas,
liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac) and vascular (including vascular endothelium)
tissue, or for promoting the growth of cells comprising such
tissues. Part of the desired effects may be by inhibition or
modulation of fibrotic scarring to allow normal tissue to
regenerate. A protein or polypeptide of the invention may also
exhibit angiogenic activity.
[0226] A protein or polypeptide of the present invention may also
be useful for gut protection or regeneration and treatment of lung
or liver fibrosis, reperfusion injury in various tissues, and
conditions resulting from systemic cytokine damage.
[0227] A protein or polypeptide of the present invention may also
be useful for promoting or inhibiting differentiation of tissues
described above from precursor tissues or cells; or for inhibiting
the growth of tissues described above.
[0228] The activity of a protein or polypeptide of the invention
may, among other means, be measured by the following methods:
[0229] Assays for tissue generation activity include, without
limitation, those described in: International Patent Publication
No. WO95/16035 (bone, cartilage, tendon); International Patent
Publication No. WO95/05846 (nerve, neuronal); International Patent
Publication No. WO91/07491 (skin, endothelium).
[0230] Assays for wound healing activity include, without
limitation, those described in: Winter, Epidermal Wound Healing,
pps. 71-112 (Maibach, H. I. and Rovee, D. T., eds.), Year Book
Medical Publishers, Inc., Chicago, as modified by Eaglstein and
Mertz, J. Invest. Dermatol 71:382-84 (1978).
[0231] A protein or polypeptide of the present invention may also
exhibit agonist or antagonist activity against activin- or
inhibin-related activities. Inhibins are characterized by their
ability to inhibit the release of follicle stimulating hormone
(FSH), while activins and are characterized by their ability to
stimulate the release of follicle stimulating hormone (FSH). Thus,
a protein or polypeptide of the present invention that are agonists
of inhibin, may be useful as a contraceptive based on the ability
of inhibins to decrease fertility in female mammals and decrease
spermatogenesis in male mammals. Additionally, the proteins or
polypeptides of the invention that are antagonists of activin, may
be useful as a contraceptive based on the ability of activin
molecules in stimulating FSH release from cells of the anterior
pituitary. Alternatively, the protein or polypeptide of the
invention that are agonists of activin, may be useful as a
fertility inducing therapeutic, based upon the ability of activin
molecules in stimulating FSH release from cells of the anterior
pituitary. See, for example, U.S. Pat. No. 4,798,885. Further, a
proteins or polypeptides of the present invention that are
antagonists of inhibin, may be useful as a fertility inducing
therapeutic, based upon the ability of inhibins to decrease
fertility in female mammals and decrease spermatogenesis in male
mammals. A protein or polypeptide of the invention may also be
useful for advancement of the onset of fertility in sexually
immature mammals, so as to increase the lifetime reproductive
performance of domestic animals such as cows, sheep and pigs.
[0232] The activity of a protein or polypeptide of the invention
may, among other means, be measured by the following methods:
[0233] Assays for activin/inhibin activity include, without
limitation, those described in: Vale et al., Endocrinology
91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et
al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663,
1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095,
1986.
[0234] A protein or polypeptide of the present invention may be an
antagonist or agonist of chemotactic or chemokinetic activity
(e.g., act as a chemokine) for mammalian cells, including, for
example, monocytes, fibroblasts, neutrophils, T-cells, mast cells,
eosinophils, epithelial and/or endothelial cells. Chemotactic and
chemokinetic proteins can be used to mobilize or attract a desired
cell population to a desired site of action. Antagonists or
agonists of chemotactic or chemokinetic proteins provide particular
advantages in treatment of inflammation, or wounds and other trauma
to tissues, as well as in treatment of localized infections. For
example, attraction of lymphocytes, monocytes or neutrophils to
tumors or sites of infection may result in improved immune
responses against the tumor or infecting agent.
[0235] A protein or polypeptide or peptide is an agonist of
chemotactic activity for a particular cell population if it can
stimulate, directly or indirectly, the directed orientation or
movement of such cell population. vPreferably, the protein or
polypeptide or peptide has the ability to directly stimulate
directed movement of cells. Whether a particular protein has
chemotactic activity for a population of cells can be readily
determined by employing such protein or peptide in any known assay
for cell chemotaxis.
[0236] The activity of a protein or polypeptide of the invention
may, among other means, be measured by the following methods:
[0237] Assays for chemotactic activity (which will identify
proteins that induce or prevent chemotaxis) consist of assays that
measure the ability of a protein to induce the migration of cells
across a membrane as well as the ability of a protein to induce the
adhesion of one cell population to another cell population.
Suitable assays for movement and adhesion include, without
limitation, those described in: Current Protocols in Immunology, Ed
by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta
Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest.
95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et
al Eur. J. Immunol. 25:1744-1748; Gruber et al., J. of Immunol.
152:5860-5867, 1994; Johnston et al., J. of Immunol. 153:1762-1768,
1994.
[0238] A protein or polypeptide of the invention may also be an
antagonist or agonist of hemostatic or thrombolytic activity. Such
a protein or polypeptide is expected to be useful in treatment
and/or detection (e.g., a diagnostic) of various coagulation
disorders (including hereditary disorders, such as hemophilias) or
to enhance coagulation and other hemostatic events in treating
wounds resulting from trauma, surgery or other causes. A protein or
polypeptide of the invention may also be useful for dissolving or
inhibiting formation of thromboses and for treatment and prevention
of conditions resulting therefrom (such as, for example, infarction
of cardiac and central nervous system vessels (e.g., stroke).
[0239] The activity of a protein or polypeptide of the invention
may, among other means, be measured by the following methods:
[0240] Assay for hemostatic and thrombolytic activity include,
without limitation, those described in: Linet et al., J. Clin.
Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res.
45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991);
Schaub, Prostaglandins 35:467-474, 1988.
[0241] A protein or polypeptide of the present invention may also
demonstrate activity as receptors, receptor ligands or antagonists
or agonists of receptor/ligand interactions. Examples of such
receptors and ligands include, without limitation, cytokine
receptors and their ligands, receptor kinases and their ligands,
receptor phosphatases and their ligands, receptors involved in
cell-cell interactions and their ligands (including without
limitation, cellular adhesion molecules (such as selecting,
integrins and their ligands) and receptor/ligand pairs involved in
antigen presentation, antigen recognition and development of
cellular and humoral immune responses). Receptors and ligands are
also useful for screening of potential peptide or small molecule
inhibitors of the relevant receptor/ligand interaction. A protein
or polypeptide of the present invention (including, without
limitation, fragments of receptors and ligands) may themselves be
useful as inhibitors of receptor/ligand interactions.
[0242] The activity of a protein or polypeptide of the invention
may, among other means, be measured by the following methods:
[0243] Suitable assays for receptor-ligand activity include without
limitation those described in: Current Protocols in Immunology, Ed
by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion
under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl.
Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med.
168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160
1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994;
Stitt et al., Cell 80:661-670, 1995.
[0244] Proteins or polypeptides of the present invention may also
be antagonists or agonists of inflammation. Anti-inflammatory
activity may be achieved by providing a stimulus to cells involved
in the inflammatory response, by inhibiting or promoting cell-cell
interactions (such as, for example, cell adhesion), by inhibiting
or promoting chemotaxis of cells involved in the inflammatory
process, inhibiting or promoting cell extravasation, or by
stimulating or suppressing production of other factors which more
directly inhibit or promote an inflammatory response. Proteins or
polypeptides of the invention can be used to treat and/or detect
(e.g., a diagnostic) inflammatory conditions including chronic or
acute conditions, including without limitation intimation
associated with infection (such as septic shock, sepsis or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion
injury, endotoxin lethality, arthritis, complement-mediated
hyperacute rejection, nephritis, cytokine or chemokine-induced lung
injury, inflammatory bowel disease, Crohn's disease or resulting
from over production of cytokines such as TNF or IL-1. Proteins or
polypeptides of the invention may also be useful to treat
anaphylaxis and hypersensitivity to an antigenic substance or
material.
[0245] Nervous system disorders, which can be treated and/or
detected (e.g., a diagnostic) with the polypeptides or proteins of
the invention include but are not limited to nervous system
injuries, and diseases or disorders which result in either a
disconnection of axons, a diminution or degeneration of neurons, or
demyelination. Nervous system lesions which may be treated and/or
detected (e.g., a diagnostic) in a patient (including human and
non-human mammalian patients) according to the invention include
but are not limited to the following lesions of either the central
(including spinal cord, brain) or peripheral nervous systems:
[0246] (i) traumatic lesions, including lesions caused by physical
injury or associated with surgery, for example, lesions which sever
a portion of the nervous system, or compression injuries; [0247]
(ii) ischemic lesions, in which a lack of oxygen in a portion of
the nervous system results in neuronal injury or death, including
cerebral infarction or ischemia, or spinal cord infarction or
ischemia; [0248] (iii) malignant lesions, in which a portion of the
nervous system is destroyed or injured by malignant tissue which is
either a nervous system associated malignancy or a malignancy
derived from non-nervous system tissue; [0249] (iv) infectious
lesions, in which a portion of the nervous system is destroyed or
injured as a result of infection, for example, by an abscess or
associated with infection by human immunodeficiency virus, herpes
zoster, or herpes simplex virus or with Lyme disease, tuberculosis,
syphilis; [0250] (v) degenerative lesions, in which a portion of
the nervous system is destroyed or injured as a result of a
degenerative process including but not limited to degeneration
associated with Parkinson's disease, Alzheimer's disease,
Huntington's chorea, or amyotrophic lateral sclerosis; [0251] (vi)
lesions associated with nutritional diseases or disorders, in which
a portion of the nervous system is destroyed or injured by a
nutritional disorder or disorder of metabolism including but not
limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke
disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease
(primary degeneration of the corpus callosum), and alcoholic
cerebellar degeneration; [0252] (vii) neurological lesions
associated with systemic diseases including but not limited to
diabetes (diabetic neuropathy, Bell's palsy), systemic lupus
erythematosus, carcinoma, or sarcoidosis; [0253] (viii) lesions
caused by toxic substances including alcohol, lead, or particular
neurotoxins; and [0254] (ix) demyelinated lesions in which a
portion of the nervous system is destroyed or injured by a
demyelinating disease including but not limited to multiple
sclerosis, human immunodeficiency virus-associated myelopathy,
transverse myelopathy or various etiologies, progressive multifocal
leukoencephalopathy, and central pontine myelinolysis.
[0255] Therapeutics which are useful according to the invention for
treatment of a nervous system disorder may be selected by testing
for biological activity in promoting the survival or
differentiation of neurons. For example, and not by way of
limitation, therapeutics which elicit any of the following effects
may be useful according to the invention: [0256] (i) increased
survival time of neurons in culture; [0257] (ii) increased
sprouting of neurons in culture or in vivo; [0258] (iii) increased
production of a neuron-associated molecule in culture or in vivo,
e.g., choline acetyltransferase or acetylcholinesterase with
respect to motor neurons; or [0259] (iv) decreased symptoms of
neuron dysfunction in vivo.
[0260] Such effects may be measured by any method known in the art.
In preferred, non-limiting embodiments, increased survival of
neurons may be measured by the method set forth in Arakawa et al.
(1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons
may be detected by methods set forth in Pestronk et al. (1980, Exp.
Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci.
4:17-42); increased production of neuron-associated molecules may
be measured by bioassay, enzymatic assay, antibody binding,
Northern blot assay, etc., depending on the molecule to be
measured; and motor neuron dysfunction may be measured by assessing
the physical manifestation of motor neuron disorder, e.g.,
weakness, motor neuron conduction velocity, or functional
disability.
[0261] In a specific embodiments, motor neuron disorders that may
be treated and/or detected (e.g., a diagnostic) according to the
invention include but are not limited to disorders such as
infarction, infection, exposure to toxin, trauma, surgical damage,
degenerative disease or malignancy that may affect motor neurons as
well as other components of the nervous system, as well as
disorders that selectively affect neurons such as amyotrophic
lateral sclerosis, and including but not limited to progressive
spinal muscular atrophy, progressive bulbar palsy, primary lateral
sclerosis, infantile and juvenile muscular atrophy, progressive
bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis
and the post polio syndrome, and Hereditary Motorsensory Neuropathy
(Charcot-Marie-Tooth Disease).
[0262] A protein or polypeptide of the invention may also exhibit
one or more of the following additional activities or effects:
inhibiting the growth, infection or function of, or killing,
infectious agents, including, without limitation, bacteria,
viruses, fungi and other parasites; effecting (suppressing or
enhancing) bodily characteristics or plant characteristics,
including, without limitation, height, weight, hair color, eye
color, skin, fat to lean ratio or other tissue pigmentation, or
organ or body part size or shape (such as, for example, breast
augmentation or diminution, change in bone form or shape);
effecting biorhythms or caricadic cycles or rhythms; effecting the
fertility of male or female subjects; effecting the metabolism,
catabolism, anabolism, processing, utilization, storage or
elimination of dietary fat, lipid, protein, carbohydrate, vitamins,
minerals, co-factors or other nutritional factors or component(s);
effecting behavioral characteristics, including, without
limitation, appetite, libido, stress, cognition (including
cognitive disorders), depression (including depressive disorders)
and violent behaviors; providing analgesic effects or other pain
reducing effects; promoting differentiation and growth of embryonic
stem cells in lineages other than hematopoietic lineages; hormonal
or endocrine activity; in the case of enzymes, correcting
deficiencies of the enzyme and treating deficiency-related
diseases; treatment of hyperproliferative disorders (such as, for
example, psoriasis); immunoglobulin-like activity (such as, for
example, the ability to bind antigens or complement); and the
ability to act as an antigen in a vaccine composition to raise an
immune response against such protein or another material or entity
which is cross-reactive with such protein.
[0263] A protein or polypeptide of the present invention (from
whatever source derived, including without limitation from
recombinant and non-recombinant sources) may be administered to a
patient in need, by itself, or in pharmaceutical compositions where
it is mixed with suitable carriers or excipient(s) at doses to
treat or ameliorate a variety of disorders. Such a composition may
also contain (in addition to protein or polypeptide and a carrier)
diluents, fillers, salts, buffers, stabilizers, solubilizers, and
other materials well known in the art. The term "pharmaceutically
acceptable" means a non-toxic material that does not interfere with
the effectiveness of the biological activity of the active
ingredient(s). The characteristics of the carrier will depend on
the route of administration. The pharmaceutical composition of the
invention may also contain cytokines, lymphokines, or other
hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,
IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem cell factor, and erythropoietin. The
pharmaceutical composition may further contain other agents which
either enhance the activity of the protein or polypeptide or
compliment its activity or use in treatment. Such additional
factors and/or agents may be included in the pharmaceutical
composition to produce a synergistic effect with protein or
polypeptide of the invention, or to minimize side effects.
Conversely, protein or polypeptide of the present invention may be
included in formulations of the particular cytokine, lymphokine,
other hematopoietic factor, thrombolytic or anti-thrombotic factor,
or anti-inflammatory agent to minimize side effects of the
cytokine, lymphokine, other hematopoietic factor, thrombolytic or
anti-thrombotic factor, or anti-inflammatory agent. A protein or
polypeptide of the present invention may be active in multimers
(e.g., heterodimers or homodimers) or complexes with itself or
other proteins. As a result, pharmaceutical compositions of the
invention may comprise a protein or polypeptide of the invention in
such multimeric or complexed form.
[0264] Techniques for formulation and administration of the
compounds of the instant application may be found in "Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest
edition. A therapeutically effective dose further refers to that
amount of the compound sufficient to result in amelioration of
symptoms, e.g., treatment, healing, prevention or amelioration of
the relevant medical condition, or an increase in rate of
treatment, healing, prevention or amelioration of such conditions.
When applied to an individual active ingredient, administered
alone, a therapeutically effective dose refers to that ingredient
alone. When applied to a combination, a therapeutically effective
dose refers to combined amounts of the active ingredients that
result in the therapeutic effect, whether administered in
combination, serially or simultaneously.
[0265] In practicing the method of treatment or use of the present
invention, a therapeutically effective amount of protein or
polypeptide of the present invention is administered to a mammal
having a condition to be treated. Protein or polypeptide of the
present invention may be administered in accordance with the method
of the invention either alone or in combination with other
therapies such as treatments employing cytokines, lymphokines or
other hematopoietic factors. When co-administered with one or more
cytokines, lymphokines or other hematopoietic factors, protein or
polypeptide of the present effective amount of protein or
polypeptide of the present invention is administered to a mammal
having a condition to be treated. Protein or polypeptide of the
present invention may be administered in accordance with the method
of the invention either alone or in combination with other
therapies such as treatments employing cytokines, lymphokines or
other hematopoietic factors. When co-administered with one or more
cytokines, lymphokines or other hematopoietic factors, protein or
polypeptide of the preseoutes of administration may, for example,
include oral, rectal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections. Administration of protein or polypeptide of
the present invention used in the pharmaceutical composition or to
practice the method of the present invention can be carried out in
a variety of conventional ways, such as oral ingestion, inhalation,
topical application or cutaneous, subcutaneous, intraperitoneal,
parenteral or intravenous injection. Intravenous administration to
the patient is preferred.
[0266] Alternately, one may administer the compound in a local
rather than systemic manner, for example, via injection of the
compound directly into a arthritic joints or in fibrotic tissue,
often in a depot or sustained release formulation. In order to
prevent the scarring process frequently occurring as complication
of glaucoma surgery, the compounds may be administered topically,
for example, as eye drops. Furthermore, one may administer the drug
in a targeted drug delivery system, for example, in a liposome
coated with a specific antibody, targeting, for example, arthritic
or fibrotic tissue. The liposomes will be targeted to and taken up
selectively by the afflicted tissue.
[0267] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in a conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the
active compounds into preparations which can be used
pharmaceutically. These pharmaceutical compositions may be
manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes. Proper formulation is dependent upon the route of
administration chosen. When a therapeutically effective amount of
protein or polypeptide of the present invention is administered
orally, protein or polypeptide of the present invention will be in
the form of a tablet, capsule, powder, solution or elixir. When
administered in tablet form, the pharmaceutical composition of the
invention may additionally contain a solid carrier such as a
gelatin or an adjuvant. The tablet, capsule, and powder contain
from about 5 to 95% protein or polypeptide of the present
invention, and preferably from about 25 to 90% protein or
polypeptide of the present invention. When administered in liquid
form, a liquid carrier such as water, petroleum, oils of animal or
plant origin such as peanut oil, mineral oil, soybean oil, or
sesame oil, or synthetic oils may be added. The liquid form of the
pharmaceutical composition may further contain physiological saline
solution, dextrose or other saccharide solution, or glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When
administered in liquid form, the pharmaceutical composition
contains from about 0.5 to 90% by weight of protein or polypeptide
of the present invention, and preferably from about 1 to 50%
protein or polypeptide of the present invention.
[0268] When a therapeutically effective amount of protein or
polypeptide of the present invention is administered by
intravenous, cutaneous or subcutaneous injection, protein or
polypeptide of the present invention will be in the form of a
pyrogen-free, parenterally acceptable aqueous solution. The
preparation of such parenterally acceptable protein solutions,
having due regard to pH, isotonicity, stability, and the like, is
within the skill in the art. A preferred pharmaceutical composition
for intravenous, cutaneous, or subcutaneous injection should
contain, in addition to protein or polypeptide of the present
invention, an isotonic vehicle such as Sodium Chloride Injection,
Ringer's Injection, Dextrose Injection, Dextrose and Sodium
Chloride Injection, Lactated Ringer's Injection, or other vehicle
as known in the art. The pharmaceutical composition of the present
invention may also contain stabilizers, preservatives, buffers,
antioxidants, or other additives known to those of skill in the
art. For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0269] For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a patient to be treated.
Pharmaceutical preparations for oral use can be obtained solid
excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain tablets or dragee cores. Suitable excipients
are, in particular, fillers such as sugars, including lactose,
sucrose, mannitol, or sorbitol; cellulose preparations such as, for
example, maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate. Dragee cores are provided with suitable coatings. For
this purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0270] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration. For buccal
administration, the compositions may take the form of tablets or
lozenges formulated in conventional manner.
[0271] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch. The compounds may
be formulated for parenteral administration by injection, e.g., by
bolus injection or continuous infusion. Formulations for injection
may be presented in unit dosage form, e.g., in ampoules or in
multi-dose containers, with an added preservative. The compositions
may take such forms as suspensions, solutions or emulsions in oily
or aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents.
[0272] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions. Alternatively,
the active ingredient may be in powder form for constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0273] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides. In addition to the formulations described previously,
the compounds may also be formulated as a depot preparation. Such
long acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0274] A pharmaceutical carrier for the hydrophobic compounds of
the invention is a cosolvent system comprising benzyl alcohol, a
nonpolar surfactant, a water-miscible organic polymer, and an
aqueous phase. The cosolvent system may be the VPD co-solvent
system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the
nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol
300, made up to volume in absolute ethanol. The VPD co-solvent
system (VPD: 5 W) consists of VPD diluted 1:1 with a 5% dextrose in
water solution. This co-solvent system dissolves hydrophobic
compounds well, and itself produces low toxicity upon systemic
administration. Naturally, the proportions of a co-solvent system
may be varied considerably without destroying its solubility and
toxicity characteristics. Furthermore, the identity of the
co-solvent components may be varied: for example, other
low-toxicity nonpolar surfactants may be used instead of
polysorbate 80; the fraction size of polyethylene glycol may be
varied; other biocompatible polymers may replace polyethylene
glycol, e.g., polyvinyl pyrrolidone; and other sugars or
polysaccharides may substitute for dextrose. Alternatively, other
delivery systems for hydrophobic pharmaceutical compounds may be
employed. Liposomes and emulsions are well known examples of
delivery vehicles or carriers for hydrophobic drugs. Certain
organic solvents such as dimethylsulfoxide also may be employed,
although usually at the cost of greater toxicity. Additionally, the
compounds may be delivered using a sustained-release system, such
as semipermeable matrices of solid hydrophobic polymers containing
the therapeutic agent. Various of sustained-release materials have
been established and are well known by those skilled in the art.
Sustained-release capsules may, depending on their chemical nature,
release the compounds for a few weeks up to over 100 days.
Depending on the chemical nature and the biological stability of
the therapeutic reagent, additional strategies for protein
stabilization may be employed.
[0275] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene glycols.
Many of the proteinase inhibiting compounds of the invention may be
provided as salts with pharmaceutically compatible counterions.
Such pharmaceutically acceptable base addition salts are those
salts which retain the biological effectiveness and properties of
the free acids and which are obtained by reaction with inorganic or
organic bases such as sodium hydroxide, magnesium hydroxide,
ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino
acids, sodium acetate, potassium benzoate, triethanol amine and the
like.
[0276] The pharmaceutical composition of the invention may be in
the form of a complex of the protein(s) or polypeptide(s) of
present invention along with protein or peptide antigens. The
protein and/or peptide antigen will deliver a stimulatory signal to
both B and T lymphocytes. B lymphocytes will respond to antigen
through their surface immunoglobulin receptor. T lymphocytes will
respond to antigen through the T cell receptor (TCR) following
presentation of the antigen by MHC proteins. MHC and structurally
related proteins including those encoded by class I and class II
MHC genes on host cells will serve to present the peptide
antigen(s) to T lymphocytes. The antigen components could also be
supplied as purified MHC-peptide complexes alone or with
co-stimulatory molecules that can directly signal T cells.
Alternatively antibodies able to bind surface immunoglobulin and
other molecules on B cells as well as antibodies able to bind the
TCR and other molecules on T cells can be combined with the
pharmaceutical composition of the invention. The pharmaceutical
composition of the invention may be in the form of a liposome in
which protein or polypeptide of the present invention is combined,
in addition to other pharmaceutically acceptable carriers, with
amphipathic agents such as lipids which exist in aggregated form as
micelles, insoluble monolayers, liquid crystals, or lamellar layers
in aqueous solution. Suitable lipids for liposomal formulation
include, without limitation, monoglycerides, diglycerides,
sulfatides, lysolecithin, phospholipids, saponin, bile acids, and
the like. Preparation of such liposomal formulations is within the
level of skill in the art, as disclosed, for example, in U.S. Pat.
Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which
are incorporated herein by reference.
[0277] The amount of protein or polypeptide of the present
invention in the pharmaceutical composition of the present
invention will depend upon the nature and severity of the condition
being treated, and on the nature of prior treatments which the
patient has undergone. Ultimately, the attending physician will
decide the amount of protein or polypeptide of the present
invention with which to treat each individual patient. Initially,
the attending physician will administer low doses of protein or
polypeptide of the present invention and observe the patient's
response. Larger doses of protein or polypeptide of the present
invention may be administered until the optimal therapeutic effect
is obtained for the patient, and at that point the dosage is not
increased further. It is contemplated that the various
pharmaceutical compositions used to practice the method of the
present invention should contain about 0.01 .mu.g to about 100 mg
(preferably about 0.1 .mu.g to about 10 mg, more preferably about
0.1 .mu.g to about 1 mg) of protein or polypeptide of the present
invention per kg body weight. For compositions of the present
invention which are useful for bone, cartilage, tendon or ligament
regeneration, the therapeutic method includes administering the
composition topically, systematically, or locally as an implant or
device. When administered, the therapeutic composition for use in
this invention is, of course, in a pyrogen-free, physiologically
acceptable form. Further, the composition may desirably be
encapsulated or injected in a viscous form for delivery to the site
of bone, cartilage or tissue damage. Topical administration may be
suitable for wound healing and tissue repair. Therapeutically
useful agents other than a protein or polypeptide of the invention
which may also optionally be included in the composition as
described above, may alternatively or additionally, be administered
simultaneously or sequentially with the composition in the methods
of the invention. Preferably for bone and/or cartilage formation,
the composition would include a matrix capable of delivering the
protein-containing composition to the site of bone and/or cartilage
damage, providing a structure for the developing bone and cartilage
and optimally capable of being resorbed into the body. Such
matrices may be formed of materials presently in use for other
implanted medical applications.
[0278] The choice of matrix material is based on biocompatibility,
biodegradability, mechanical properties, cosmetic appearance and
interface properties. The particular application of the
compositions will define the appropriate formulation. Potential
matrices for the compositions may be biodegradable and chemically
defined calcium sulfate, tricalciumphosphate, hydroxyapatite,
polylactic acid, polyglycolic acid and polyanhydrides. Other
potential materials are biodegradable and biologically
well-defined, such as bone or dermal collagen. Further matrices are
comprised of pure proteins or extracellular matrix components.
Other potential matrices are nonbiodegradable and chemically
defined, such as sintered hydroxyapatite, bioglass, aluminates, or
other ceramics. Matrices may be comprised of combinations of any of
the above mentioned types of material, such as polylactic acid and
hydroxyapatite or collagen and tricalciumphosphate. The bioceramics
may be altered in composition, such as in
calcium-aluminate-phosphate and processing to alter pore size,
particle size, particle shape, and biodegradability. Presently
preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic acid in the form of porous particles having diameters
ranging from 150 to 800 microns. In some applications, it will be
useful to utilize a sequestering agent, such as carboxymethyl
cellulose or autologous blood clot, to prevent the protein or
polypeptide compositions from disassociating from the matrix.
[0279] A preferred family of sequestering agents is cellulosic
materials such as alkylcelluloses (including
hydroxyalkylcelluloses), including methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most
preferred being cationic salts of carboxymethylcellulose (CMC).
Other preferred sequestering agents include hyaluronic acid, sodium
alginate, poly(ethylene glycol), polyoxyethylene oxide,
carboxyvinyl polymer and poly(vinyl alcohol). The amount of
sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt
% based on total formulation weight, which represents the amount
necessary to prevent desorbtion of the protein or polypeptide from
the polymer matrix and to provide appropriate handling of the
composition, yet not so much that the progenitor cells are
prevented from infiltrating the matrix, thereby providing the
protein or polypeptide the opportunity to assist the osteogenic
activity of the progenitor cells. In further compositions, proteins
or polypeptides of the invention may be combined with other agents
beneficial to the treatment of the bone and/or cartilage defect,
wound, or tissue in question. These agents include various growth
factors such as epidermal growth factor (EGF), platelet derived
growth factor (PDGF), transforming growth factors (TGF-.alphA. and
TGF-.beta.), and insulin-like growth factor (IGF).
[0280] The therapeutic compositions are also presently valuable for
veterinary applications. Particularly domestic animals and
thoroughbred horses, in addition to humans, are desired patients
for such treatment with proteins or polypeptides of the present
invention. The dosage regimen of a protein-containing
pharmaceutical composition to be used in tissue regeneration will
be determined by the attending physician considering various
factors which modify the action of the proteins or polypeptides,
e.g., amount of tissue weight desired to be formed, the site of
damage, the condition of the damaged tissue, the size of a wound,
type of damaged tissue (e.g., bone), the patient's age, sex, and
diet, the severity of any infection, time of administration and
other clinical factors. The dosage may vary with the type of matrix
used in the reconstitution and with inclusion of other proteins in
the pharmaceutical composition. For example, the addition of other
known growth factors, such as IGF I (insulin like growth factor I),
to the final composition, may also effect the dosage. Progress can
be monitored by periodic assessment of tissue/bone growth and/or
repair, for example, X-rays, histomorphometric determinations and
tetracycline labeling.
[0281] Polynucleotides of the present invention can also be used
for gene therapy. Such polynucleotides can be introduced either in
vivo or ex vivo into cells for expression in a mammalian subject.
Polynucleotides of the invention may also be administered by other
known methods for introduction of nucleic acid into a cell or
organism (including, without limitation, in the form of viral
vectors or naked DNA).
[0282] Cells may also be cultured ex vivo in the presence of
proteins or polypeptides of the present invention in order to
proliferate or to produce a desired effect on or activity in such
cells. Treated cells can then be introduced in vivo for therapeutic
purposes.
[0283] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
More specifically, a therapeutically effective amount means an
amount effective to prevent development of or to alleviate the
existing symptoms of the subject being treated. Determination of
the effective amounts is well within the capability of those
skilled in the art, especially in light of the detailed disclosure
provided herein. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. For example, a dose can be
formulated in animal models to achieve a circulating concentration
range that includes the IC.sub.50 as determined in cell culture
(i.e., the concentration of the test compound which achieves a
half-maximal inhibition of the C-proteinase activity). Such
information can be used to more accurately determine useful doses
in humans.
[0284] A therapeutically effective dose refers to that amount of
the compound that results in amelioration of symptoms or a
prolongation of survival in a patient. Toxicity and therapeutic
efficacy of such compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio between LD.sub.50 and ED.sub.50. Compounds
which exhibit high therapeutic indices are preferred. The data
obtained from these cell culture assays and animal studies can be
used in formulating a range of dosage for use in human. The dosage
of such compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. The
exact formulation, route of administration and dosage can be chosen
by the individual physician in view of the patient's condition.
See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p.1. Dosage amount and interval may be
adjusted individually to provide plasma levels of the active moiety
which are sufficient to maintain the C-proteinase inhibiting
effects, or minimal effective concentration (MEC). The MEC will
vary for each compound but can be estimated from in vitro data; for
example, the concentration necessary to achieve 50-90% inhibition
of the C-proteinase using the assays described herein. Dosages
necessary to achieve the MEC will depend on individual
characteristics and route of administration. However, HPLC assays
or bioassays can be used to determine plasma concentrations.
[0285] Dosage intervals can also be determined using MEC value.
Compounds should be administered using a regimen which maintains
plasma levels above the MEC for 10-90% of the time, preferably
between 30-90% and most preferably between 50-90%. In cases of
local administration or selective uptake, the effective local
concentration of the drug may not be related to plasma
concentration.
[0286] The amount of composition administered will, of course, be
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration and
the judgment of the prescribing physician.
[0287] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. Compositions comprising a compound of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labelled for
treatment of an indicated condition.
[0288] In one application of this embodiment, a nucleotide sequence
of the present invention can be recorded on computer readable
media. As used herein, `computer readable media` refers to any
medium which can be read and accessed directly by a computer. Such
media include, but are not limited to: magnetic storage media, such
as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. A skilled artisan can readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide sequence of the present
invention. As used herein, `recorded` refers to a process for
storing information on computer readable medium. A skilled artisan
can readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide sequence information of the present
invention.
[0289] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide sequence of the present invention.
The choice of the data storage structure will generally be based on
the means chosen to access the stored information. In addition, a
variety of data processor programs and formats can be used to store
the nucleotide sequence information of the present invention on
computer readable medium. The sequence information can be
represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. A
skilled artisan can readily adapt any number of dataprocessor
structuring formats (e.g. text file or database) in order to obtain
computer readable medium having recorded thereon the nucleotide
sequence information of the present invention. Computer software is
publicly available which allows a skilled artisan to access
sequence information provided in a computer readable medium. The
examples which follow demonstrate how software which implements the
BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE
(Brutlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms
on a Sybase system is used to identify open reading frames (ORFs)
within a nucleic acid sequence. Such ORFs may be protein or
polypeptide encoding fragments and may be useful in producing
commercially important protein or polypeptides such as enzymes used
in fermentation reactions and in the production of commercially
useful metabolites.
[0290] As used herein, `a computer-based system` refers to the
hardware means, software means, and data storage means used to
analyze the nucleotide sequence information of the present
invention. The minimum hardware means of the computer-based systems
of the present invention comprises a central processing unit (CPU),
input means, output means, and data storage means. A skilled
artisan can readily appreciate that any one of the currently
available computer-based systems are suitable for use in the
present invention. As stated above, the computer-based systems of
the present invention comprise a data storage means having stored
therein a nucleotide sequence of the present invention and the
necessary hardware means and software means for supporting and
implementing a search means. As used herein, `data storage means`
refers to memory which can store nucleotide sequence information of
the present invention, or a memory access means which can access
manufactures having recorded thereon the nucleotide sequence
information of the present invention.
[0291] As used herein, `search means` refers to one or more
programs which are implemented on the computer-based system to
compare a target sequence or target structural motif with the
sequence information stored within the data storage means. Search
means are used to identify fragments or regions of a known sequence
which match a particular target sequence or target motif. A variety
of known algorithms are disclosed publicly and a variety of
commercially available software for conducting search means are and
can be used in the computer-based systems of the present invention.
Examples of such software includes, but is not limited to,
MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A skilled
artisan can readily recognize that any one of the available
algorithms or implementing software packages for conducting
homology searches can be adapted for use in the present
computer-based systems. As used herein, a `target sequence` can be
any nucleic acid or amino acid sequence of six or more nucleotides
or two or more amino acids. A skilled artisan can readily recognize
that the longer a target sequence is, the less likely a target
sequence will be present as a random occurrence in the database.
The most preferred sequence length of a target sequence is from
about 10 to 100 amino acids or from about 30 to 300 nucleotide
residues. However, it is well recognized that searches for
commercially important fragments, such as sequence fragments
involved in gene expression and protein or polypeptide processing,
may be of shorter length.
[0292] As used herein, `a target structural motif,` or `target
motif,` refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein or polypeptide target motifs include, but are not
limited to, enzyme active sites and signal sequences. Nucleic acid
target motifs include, but are not limited to, promoter sequences,
hairpin structures and inducible expression elements (protein or
polypeptide binding sequences).
[0293] The present invention further provides methods to identify
the presence or expression of one of the targets recognized by a
polypeptide or protein of the present invention, or homolog
thereof, in a test sample.
[0294] In general, methods for detecting a target recognized by a
polypeptide or protein of the invention can comprise contacting a
sample with a polypeptide or protein of the invention that binds to
and forms a complex with the target for a period sufficient to form
a complex, and detecting the complex, so that if a complex is
detected, a target of the invention is detected in the sample.
[0295] In detail, such methods comprise incubating a test sample
with one or more of the antibodies of the present invention and
assaying for binding of the antibodies to the target within the
test sample.
[0296] Conditions for incubating an antibody, including a Fab
fragment of the invention, with a test sample vary. Incubation
conditions depend on the format employed in the assay, the
detection methods employed, and the type and nature of the antibody
used in the assay. One skilled in the art will recognize that any
one of the commonly available amplification or immunological assay
formats can readily be adapted to employ the antibodies of the
present invention. Examples of such assays can be found in Chard,
T., An Introduction to Radioimmunoassay and Related Techniques,
Elsevier Science Publishers, Amsterdam, The Netherlands (1986);
Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic
Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985);
Tijssen, P., Practice and Theory of immunoassays: Laboratory
Techniques in Biochemistry and Molecular Biology, Elsevier Science
Publishers, Amsterdam, The Netherlands (1985); and Kuwata et al.,
BioChem. Biophys. Res. Commun. 245:764-73 (1998), Hillenkamp et
al., Anal. Chem. 63:1193-202 (1991), U.S. Pat. Nos. 5,111,937 and
5,719,060. The test samples of the present invention include cells,
protein or polypeptide or membrane extracts of cells, or biological
fluids such as sputum, blood, serum, plasma, or urine. The test
sample used in the above-described method will vary based on the
assay format, nature of the detection method and the tissues, cells
or extracts used as the sample to be assayed. Methods for preparing
protein or polypeptide extracts or membrane extracts of cells are
well known in the art and can be readily be adapted in order to
obtain a sample which is compatible with the system utilized.
[0297] In another embodiment of the present invention, kits are
provided which contain the necessary reagents to carry out the
assays of the present invention. Specifically, the invention
provides a compartment kit to receive, in close confinement, one or
more containers which comprises: (a) a first container comprising
one of the antibodies of the present invention; and (b) one or more
other containers comprising one or more of the following: wash
reagents, reagents capable of detecting presence of a bound
antibody.
[0298] In detail, a compartment kit includes any kit in which
reagents are contained in separate containers. Such containers
include small glass containers, plastic containers or strips of
plastic or paper. Such containers allows one to efficiently
transfer reagents from one compartment to another compartment such
that the samples and reagents are not cross-contaminated, and the
agents or solutions of each container can be added in a
quantitative fashion from one compartment to another. Such
containers will include a container which will accept the test
sample, a container which contains the antibodies used in the
assay, containers which contain wash reagents (such as phosphate
buffered saline, Tris-buffers, etc.), and containers which contain
the reagents used to detect the bound antibody or probe. Types of
detection reagents include labeled secondary antibodies, or in the
alternative, if the primary antibody is labeled, the enzymatic, or
antibody binding reagents which are capable of reacting with the
labeled antibody. One skilled in the art will readily recognize
that the disclosed probes and antibodies of the present invention
can be readily incorporated into one of the established kit formats
which are well known in the art.
[0299] Using the polypeptides or proteins of the invention, the
present invention further provides methods of obtaining and
identifying agents which bind to a target recognized by the
polypeptide or protein. In detail, said method comprises the steps
of: [0300] (a) contacting a target with an isolated protein or
polypeptide of the present invention; and [0301] (b) determining
whether the target binds to said protein or polypeptide.
[0302] In general, such methods for identifying compounds that bind
to a polypeptide of the invention can comprise contacting a
compound with a polypeptide of the invention for a time sufficient
to form a polypeptide/compound complex, and detecting the complex,
so that if a polypeptide/compound complex is detected, a compound
that binds to a polynucleotide of the invention is identified.
[0303] Methods for identifying compounds that bind to a polypeptide
of the invention can also comprise contacting a compound with a
polypeptide of the invention in a cell for a time sufficient to
form a polypeptide/compound complex, wherein the complex drives
expression of a receptor gene sequence in the cell, and detecting
the complex by detecting reporter gene sequence expression, so that
if a polypeptide/compound complex is detected, a compound that
binds a polypeptide of the invention is identified.
[0304] Compounds identified via such methods can include compounds
which modulate the activity of a target recognized by a polypeptide
or protein of the invention (that is, increase or decrease the
target's activity, relative to activity observed in the absence of
the compound). Alternatively, compounds identified via such methods
can include compounds which modulate the expression of a
polynucleotide of the invention (that is, increase or decrease
expression relative to expression levels observed in the absence of
the compound). Compounds, such as compounds identified via the
methods of the invention, can be tested using standard assays well
known to those of skill in the art for their ability to modulate
activity/expression.
[0305] The agents screened in the above assay can be, but are not
limited to, peptides, carbohydrates, vitamin derivatives, or other
pharmaceutical agents. The agents can be selected and screened at
random or rationally selected or designed using protein modeling
techniques.
[0306] For random screening, agents such as peptides,
carbohydrates, pharmaceutical agents and the like are selected at
random and are assayed for their ability to bind to the target
recognized by the polypeptide or protein of the present invention.
Alternatively, agents may be rationally selected or designed. As
used herein, an agent is said to be `rationally selected or
designed` when the agent is chosen based on the configuration of
the particular protein or polypeptide. For example, one skilled in
the art can readily adapt currently available procedures to
generate peptides, pharmaceutical agents and the like capable of
binding to a specific peptide sequence in order to generate
rationally designed antipeptide peptides, for example see Hurby et
al., Application of Synthetic Peptides: Antisense Peptides,` In
Synthetic Peptides, A User's Guide, W.H. Freeman, NY (1992), pp.
289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or
pharmaceutical agents, or the like.
[0307] In addition to the foregoing, one class of agents of the
present invention, as broadly described, can be used to control
gene expression through binding to one of the ORFs or EMFs of the
present invention. As described above, such agents can be randomly
screened or rationally designed/selected. Targeting the ORF or EMF
allows a skilled artisan to design sequence specific or element
specific agents, modulating the expression of either a single ORF
or multiple ORFs which rely on the same EMF for expression
control.
[0308] As choice of antibody format, we preferred the Fab format
above the scFv format, because the Fab format allows rapid high
through-put affinity-screening assays for crude antibody
preparations. Many scFv's indeed form higher molecular weight
species including dimers (Weidner, et al., (1992) J. Biol. Chem.
267, 10281-10288; Holliger, et al., (1993) Proc. Natl. Acad. Sci.
U.S.A. 90, 6444-6448) and trimers (Kortt et al., (1997) Protein
Eng. 10, 423-433), which complicate both selection and
characterisation. We chose the Fab display format in which a
variable domain from a heavy or light chain gene is linked to a
phage coat protein, and in some embodiments, also carries a tag for
detection and purification. The other chain is expressed as
separate fragment secreted into the periplasm, where it can pair
with the gene that is in a protein fusion with the phage coat
protein (Hoogenboom, et al., (1991) Nucleic Acids Res. 19,
4133-4137). In some embodiments, the phage coat protein a pIII coat
protein. In other embodiments, the variable domain from a heavy
chain gene is fused to the phage coat protein and the light chain
gene is expressed as a separate fragment.
[0309] The choice for the Fab format was based on the notion that
the monomeric appearance of the Fab permits the rapid screening of
large numbers of clones for kinetics of binding (off-rate) with
crude protein fractions. This reduces the time for post-selection
analysis dramatically when compared to that needed for selected
single-chain Fv (scFv) antibodies from phagemid libraries (Vaughan,
et al., (1996) Nat. Biotechnol. 14, 309-314; Sheets, et al., (1998)
Proc. Natl. Acad. Sci. U.S.A. 95, 6157-6162), or Fab fragments from
other phage libraries (Griffiths, et al., (1993) EMBO J. 12,
725-734).
[0310] The Fab library of the invention produced on average 14
different Fab's against 6 antigens that were tested. These include
tetanus toxoid, the hapten phenyl-oxazolone, the breast cancer
associated MUC1 antigen and three highly related glycoprotein
hormones: human Chorionic Gonadotropin (`hCG`), human Luteinizing
Hormone (`hLH`) and human Follicle Stimulating Hormone (`hFSH`).
For the glycoprotein hormones, the Fab library of the invention
produced a panel of either hormone-specific or cross-reactive
antibodies. Thus, without using sophisticated selection protocols,
hormone specific as well as cross-reactive Fab's were retrieved
against these highly homologous glycohormones, demonstrating that
the library is a rich source of antibody specificities. The
affinities of the anti-glycohormone antibodies varied between 2.7
and 38 nM. Finally, the Fab-format indeed permitted the rapid
screening and a reliable ranking of individual clones based on
off-rate using crude fractions.
[0311] Furthermore, the specificities of the antibodies obtained by
selections on the gonadotropins are unique: due to the high degree
of homology between hLH and hCG it has been very difficult to
isolate hCG specific monoclonal antibodies with the hybridoma
technology, whereas there are very few hLH specific antibodies
(Moyle, et al., (1990) J. Biol. Chem. 265, 8511-8518; Cole, (1997)
Clin. Chem. 43, 2233-2243). Using a straight forward selection
procedure, taking no precaution to avoid the selection of
cross-reactive Fab's, we have readily isolated fragments with all
possible specificities: Fab's specific for any of the three
hormones hCG, hLH and hFSH, and cross-reactive Fab's recognizing
the common .alpha.-chain or epitopes on the .beta.-chain shared by
hCG and hLH. These selections demonstrated that antibodies directed
against different epitopes within single antigen molecules can be
retrieved from the library. The Fab library of the invention
permits the monitoring of selections with polyclonal phage
preparations and large scale screening of antibody off-rates with
unpurified Fab fragments.
[0312] Overall, antibodies with off-rates in the order of 10.sup.-2
to 10.sup.-4 s.sup.-1 and affinities up to 2.7 nM, were recovered.
The kinetics of these phage antibodies are of the same order of
magnitude as antibodies associated with a secondary immune
response.
[0313] An indication that antibodies from the Fab library behave
similarly or better than antibodies from a scFv library with
regards to affinity comes from a comparison of selections of two
different libraries on the same two antigens under identical
conditions. Antibodies to MUC1 selected from a large naVve scFv
library (Henderikx et al., (1998) Cancer Res. 58, 4324-4332) have
faster off-rates then the equivalent Fab's isolated from the
library described in this study. Further, they show a very distinct
V-gene usage and have a different fine specificity. Similarly, when
comparing the off-rates of phage antibodies against the
pancarcinoma marker Epithelial Glycoprotein-2, one of the Fab's
selected from the present library appears to have a 10-fold slower
off-rate than the best scFv (Vaughan et al., (1996) Nat.
Biotechnol. 14, 309-314).
[0314] The affinities of the selected antibody fragments is,
however, very much dependent on the antigen used for selection.
Sheets and colleagues reported an affinity varying between 26 and
71 nM for the selected scFv fragments specific for the
anti-Clostridia botulinum neurotoxin type A fragments, whereas for
antibodies to the extracellular domain of human ErbB-2, K.sub.d's
between 0.22 and 4.03 nM were found (Sheets et al., (1998) Proc.
Natl. Acad. Sci. U.S.A. 95, 6157-6162). The affinities of the
gonadotropin specific Fab's selected from our library varied
between 2.7 and 38 nM, which is comparable to the protein binding
scFv's from the naVve library made by Vaughan et al. and Sheets et
al., and approaches the values of the best antibodies in their
kind.
[0315] The size of the Fab library of the invention is not only
important for affinity, but it also determines the success rate of
selection of antibodies against a large set of different antigens.
In this respect the Fab library of the invention performs very
well: over 24 antibodies to the hapten phOx, and on average 13
antibodies against the other antigens were selected.
[0316] In the limited set of 14 Fab clones that were sequenced, we
identify antibodies with variable region genes from all large
V-gene families, including V.sub.H1/3/4, V.sub.61/3, and
V.sub.81/2, but also less frequently used segments of family
V.sub.H6, V.sub.62/7 and V.sub.87 were retrieved. Most likely the
use of an extended set of variable region gene primers, designed on
the most recent sequence information of the germline V-regions,
and/or the separate PCRs, combined with partially separate cloning,
ensured access to a highly diverse sample of the human V-gene
repertoire.
[0317] According to the present invention, a library is prepared
from polynucleotides which are capable of encoding the desired
specific binding pair member. A variety of techniques exist for
preparing the library, which may be prepared, for example, from
either genomic DNA or cDNA. See, e.g., Sambrook et al., Molecular
Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989, which is
incorporated herein by reference. Cells may serve as the source of
the polynucleotides which encode the specific binding pair members
of interest. Enrichment procedures and means for amplifying the
regions containing the gene(s), may be employed. For instance, when
the desired specific binding pair member is an antibody, RNA and or
genomic DNA may be prepared, for example, from spleen cells
obtained from an unimmunized animal, from an animal immunized with
target(s) of interest, from hybridoma cells, or from lymphoblastoid
cells. The library of antibodies obtained from the unimmunized
animals contain an unbiased representation of the entire antibody
repertoire, while the library of antibodies obtained from the
immunized animals contain a biased population of antibodies
directed against epitopes of the target(s). Spleen cells, or immune
cells from other tissues or the circulatory system may be obtained
from a variety of animal species, such as human, mouse, rat,
equine, bovine, avian, etc.
[0318] Amplification of messenger RNA (mRNA) isolated from cells of
interest, such as spleen or hybridoma cells, may be performed
according to protocols outlined in, e.g., U.S. Pat. No. 4,683,202,
Orlandi, et al. Proc. Natl. Acad. Sci. USA 86:3833-3837 (1989),
Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728-5732 (1989), and
Huse et al. Science 246:1275-1281 (1989), Abelson, J. and Simon, M.
(eds), Methods in Enzymology, combinatorial chemistry, Vol. 267,
San Diego: Academic Press (1996), Kay, B. K., Winter, J.,
McCafferty, J. (eds), Phage Display of peptides and Proteins, a
Laboratory Manual, San Diego: Academic Press (1996), each
incorporated herein by reference. Oligonucleotide primers useful in
amplification protocols may be unique or degenerate or incorporate
inosine at degenerate positions. Thus, for multi-chain
immunoglobulins, primers would be generally used for amplification
of sequences encoding the variable regions of both the heavy and
light chains. Restriction endonuclease recognition sequences may be
incorporated into the primers to allow for the cloning of the
amplified fragment into a vector in a predetermined reading frame
for expression.
[0319] Expression libraries containing the amplified cDNA are
typically prepared in a vector such as a bacteriophage or phagemid.
The characteristics of the suitable bacteriophage or phagemid
depends on the specific embodiment employed, and will generally be
those which conveniently allow insertion of the recombinant
polynucleotides into host cells by in vitro packaging or
transformation.
[0320] Host cells are then infected with the phage or phagemid and
helper phage, and cultivated under conditions allowing for the
expression and assembly of phage particles. In one embodiment, the
appropriate host cells for the bacteriophage or phagemids of the
invention are various strains of E. coli, specific examples
depending on which of the several suitable vectors is chosen. Of
course, phage or phagemid having bacterial hosts other than E. coli
may also be used.
[0321] To enrich for and isolate phage particles or phage which
contain cloned library sequences that encode a desired specific
binding pair member, and thus to ultimately isolate the nucleic
acid sequences themselves, phage particles or phage harvested from
the host cells are affinity purified. A target or binding partner
for the desired specific binding pair member is used in the
affinity purification. For example, when the desired specific
binding pair member is an antibody which specifically binds a
particular target, the target is used to retrieve phage particles
or phage having the desired antibody on its outer surface. The
target is typically adsorbed to an insoluble substrate, such as a
particle or bead or plate. The phage particles or phage so obtained
may then be amplified by infecting into host cells (with helper
phage for the phage particles containing the phagemids). Additional
rounds of affinity enrichment and amplification may be employed
until the desired level of enrichment is reached or the desired
phage particles or phage are no longer enriched relative to the
background phage particles or phage.
[0322] The enriched antibody-phage particles or phage are also
screened with additional detection techniques such as expression
plaque (or colony) lift (see, e.g., Young and Davis, Science,
222:778-782 (1983), incorporated herein by reference) whereby the
same or another binding partner is used as a probe. Screening may
employ additional assays (for a catalytic activity, for example)
which are used to detect, in situ, plaques expressing specific
binding pair members having the desired characteristics. The phage
particles or phage obtained from the screening protocol are
infected into cells, propagated, and the phage particle or phage
DNA isolated and sequenced, and/or recloned into a vector intended
for gene expression in prokaryotes or eukaryotes to obtain larger
amounts of the selected specific binding pair member.
[0323] In another embodiment, the specific binding pair member
encoded in the library (or multiple chains comprising said specific
binding pair member) is transported to an extra-cytoplasmic
compartment of the host cell, usually the periplasmic space, to
facilitate processing and/or proper assembly. When
extra-cytoplasmic transport of the desired specific binding pair
member is employed, the sequences encoding the specific binding
pair member are cloned adjacent to appropriate transcriptional and
translational signals and signal peptide leaders that will direct
the mature chains to the periplasm. As above, at least one of the
chains is cloned as a fusion protein with a phage coat protein so
that the phage coat protein does not substantially interfere with
the ability of the specific binding pair member of interest to bind
a target which is used in the affinity enrichment protocol.
[0324] A preferred example of this embodiment is the placement of a
specific binding pair member in the N-terminus region of the minor
coat protein pIII of bacteriophage fd. Before incorporation into
the phage, pIII resides in the inner membrane of the host cell with
its N-terminus protruding into the periplasm. In this configuration
the polypeptide of a specific binding pair member in the N-terminus
of pIII is available for binding to other polypeptide chains that
make up the specific binding pair member of interest. This complex
is then incorporated into the mature phage particle or phage as it
exits the cell and the C-terminus embeds in the coat of the phage
particle or phage.
[0325] In this embodiment the synthesis and amplification of
polynucleotides is as described above, and then is cloned into or
near a vector sequence encoding a coat protein, where the vector
is, or is derived from, a filamentous phage, such as f1, fd, Pf1,
M13, etc. In a preferred embodiment the filamentous phage is
fd-tet. The phage vector is chosen to contain a cloning site
located in the 5' region of a gene encoding a phage coat protein,
such as, for example, the pIII coat protein. An appropriate vector
(e.g., fd-tet B1 which is described below) allows oriented cloning
of foreign sequences so that they are expressed at or near the
N-terminus of the mature coat protein.
[0326] A library is constructed by cloning the polynucleotides
(e.g., the V.sub.H region) from the donor cells into a coat protein
gene (e.g., gene III, "gIII") cloning site. The cloned sequences
of, for example, the V.sub.H domains are ultimately expressed as
polypeptides or proteins fused to the N-terminus of the mature coat
protein on the outer, accessible surface of the assembled phage
particles or phage.
[0327] When the desired protein is a multi-chain protein, such as
an antibody or binding fragment thereof, the polynucleotide
encoding the chain(s) not cloned into a phage coat protein may be
cloned directly into an appropriate site (as described below) of
the vector containing the first chain-coat protein library; or,
preferably, the subsequent chain(s) may be cloned as a separate
library in a different plasmid vector, amplified, and subsequently
the fragments installed in the first chain-coat protein library
vector. For example, when the first chain is an antibody heavy
chain or binding fragment thereof, the ultimate destination of
light chain V.sub.L cDNA sequence is in a vector that already
contains a V.sub.H sequence in a coat protein gene, thus randomly
recombining V.sub.H and V.sub.L sequences in a single vector.
[0328] The second or subsequent chain of the desired multi-chain
protein, such as V.sub.L, is cloned so that it is expressed with a
signal peptide leader sequence that will direct its secretion into
the periplasm of the host cell. For example, several leader
sequences have been shown to direct the secretion of antibody
sequences in E. coli, such as OmpA (Hsiung, et al., Biotechnology
4:991-995 (1986)), pelB (Better, et al., Science 240:1041-1043
(1988)), phoA (Skerra and Pluckthun, Science 240:1038-1043 (1988)),
beta-lactamase (Zemel-Dreasen and Zamir, Gene 27:315-322 (1984)),
and those described in Abelson, J. and Simon, M. (eds), Methods in
Enzymology, combinatorial chemistry, Vol. 267, San Diego: Academic
Press (1996), and Kay, B. K., Winter, J., McCafferty, J. (eds),
Phage Display of peptides and Proteins, a Laboratory Manual, San
Diego Academic Press (1996), each incorporated herein by
reference.
[0329] Generally, the successful cloning strategy utilizing a phage
coat protein, such as pIII of filamentous phage fd, will provide:
(1) expression of a protein chain (or a first polypeptide chain
when the desired protein is multichained, e.g., the V.sub.H chain)
fused to the N-terminus of a full sized (or nearly full sized) coat
protein (e.g., pIII) and transport to the inner membrane of the
host where the hydrophobic domain in the C-terminal region of the
coat protein anchors the fusion protein in the membrane, with the
N-terminus containing the chain protruding into the periplasmic
space and available for interaction with a second or subsequent
chain (e.g., V.sub.L to form an Fab fragment) which is thus
attached to the coat protein; and (2) adequate expression of a
second or subsequent polypeptide chain if present (e.g., V.sub.L)
and transport of this chain to the soluble compartment of the
periplasm.
[0330] In one embodiment for affinity enrichment of desired clones,
about 10.sup.3 to 10.sup.4 library equivalents (a library
equivalent is one of each recombinant--10.sup.4 equivalents of a
library of 10.sup.9 members is 10.sup.9.times.10.sup.4=10.sup.13
phage particles or phage) are incubated with target to which the
desired specific binding pair member (e.g., antibody) is sought.
The target is in one of several forms appropriate for affinity
enrichment schemes. In one example the target is immobilized on a
surface or particle, optionally anchored by a tether of enough
length (3 to 12 carbons, for example) to hold the target far enough
away from the surface to permit free interaction with the antibody
combining site. The library of phage particle or phage bearing
antibodies is then panned on the immobilized target generally
according to procedures well-known in the art, for example, those
described in Abelson, J. and Simon, M. (eds), Methods in
Enzymology, combinatorial chemistry, Vol. 267, San Diego: Academic
Press (1996), Kay, B. K., Winter, J., McCafferty, J. (eds), Phage
Display of peptides and Proteins, a Laboratory Manual, San Diego:
Academic Press (1996), each incorporated herein by reference.
[0331] A second example of target presentation is target attached
to a recognizable ligand (again optionally with a tether of some
length). A specific example of such a ligand is biotin. The target,
so modified, is incubated with the library of phage particles or
phage and binding occurs with both reactants in solution. The
resulting complexes are then bound to streptavidin (or avidin)
through the biotin moiety. The streptavidin may be immobilized on a
surface such as a plastic plate or on particles, in which case the
complexes are physically retained; or the streptavidin may be
labelled, with a fluorophore, for example, to tag the active
phage/antibody for detection and/or isolation by sorting
procedures, e.g., on a fluorescence-activated cell sorter.
[0332] In one embodiment, the phage particles or phage bearing
antibodies without the desired specificity are removed by various
means, for example, by washing. The degree and stringency of
washing required will be determined for each specific binding pair
member of interest. A certain degree of control can be exerted over
the binding characteristics of the antibodies recovered by
adjusting the conditions of the binding incubation and the
subsequent washing. The temperature, pH, ionic strength, divalent
cations concentration, and the volume and duration of the washing
will select for antibodies within particular ranges of affinity for
the hapten. Selection based on slow dissociation rate, which is
usually predictive of high affinity, is the most practical route.
This may be done either by continued incubation in the presence of
a saturating amount of free hapten, or by increasing the volume,
number, and length of the washes. In each case, the rebinding of
dissociated antibody-phage is prevented, and with increasing time,
antibody-phage of higher and higher affinity are recovered.
[0333] Antibodies with certain catalytic activities may be enriched
in groups of antibodies with high affinity for reactants
(substrates and intermediates) but low affinity for products. A
double screen to enrich for antibodies with these characteristics
may be useful in finding antibodies to catalyze certain reactions.
Further, catalytic antibodies capable of certain cleavage reactions
may also be selected. One category of such reactions is the
cleavage of a specific end group from a molecule. For example, a
catalytic antibody to cleave a specific amino acid from an end of a
peptide may be selected by immobilizing the peptide and panning the
antibody library under conditions expected to promote binding but
not cleavage (e.g., low temperature, particular ionic strength, pH,
cation concentration, etc., depending on the nature of the end
group and the cleavage reaction) and followed by a wash. This
allows antibodies that recognize the end group to bind and become
immobilized, and from this group will come those capable of
cleavage. To find those capable of cleavage, the conditions are
shifted to those favorable for cleavage. This step will release
those antibody-phage capable of cleaving themselves free of the
immobilized peptide.
[0334] An alternative way to accomplish this is to pan for
antibodies that bind to the specific end group by attaching that
end group to a bond different from that to be cleaved (a
non-peptide bond, for example). By subsequent panning (of the
positive phage from the first screen) on the end group attached via
the proper bond under cleavage conditions, the non-binding fraction
will be enriched for those with the desired catalytic activity.
[0335] To elute the active antibody-phage particle or phage from
the immobilized target, after washing at the appropriate
stringency, the bound (active) phage particle or phage can be
recovered by eluting with pH shift. For example, pH2 or pH11 may be
used, which is then neutralized and the eluted phage are amplified
by infecting or transforming the host cells. The cells are then
grown as tetracycline resistant colonies. The colonies are scraped
up and the extruded phage are purified by standard procedures as
before. These phage are then used in another round of affinity
enrichment (panning), and this cycle is repeated until the desired
level of enrichment is reached or until the target phage are no
longer enriched relative to the background phage particles or
phage. To isolate individual clones, phage particles or phage from
the final round of panning and elution are infected into cells or
their DNA is transformed into cells and grown on agar (usually
L-agar) and antibiotics (usually tet) to form well separated
individual colonies, each of which is a clone carrying vectors with
both V.sub.H and V.sub.L sequences. The single stranded DNA from
phage particles or phage extruded from each colony may be isolated
and DNA coding for the V.sub.H and V.sub.L fragments sequenced. The
replicative form of the phage DNA (double stranded) may be isolated
by standard means and the DNA in the cloning sites (V.sub.H and
V.sub.L sequences) recloned into a vector designed for gene product
expression in prokaryotes or eukaryotes to obtain larger amounts of
the particular antibodies selected in the screening process.
[0336] Phage identified as having an antibody recognized by the
target ligand are propagated as appropriate for the particular
phage vector used. For fd-tet this is done in a liquid culture of
rich medium (L-broth, for example) with antibiotic (Tet) selection.
The phage are harvested and DNA prepared and sequenced by standard
methods to determine the DNA and amino acid sequence of the
particular antibody.
[0337] The DNA may be recloned in a suitable eukaryotic or
prokaryotic expression vector and transfected into an appropriate
host for production of large amounts of protein. Antibody is
purified from the expression system using standard procedures. The
binding affinity of the antibody is confirmed by well known
immunoassays with the target antigen or catalytic activity as
described in Harlow and Lane, Antibodies, A Laboratory Manual, Cold
Spring Harbor, N.Y. (1988), Abelson, J. and Simon, M. (eds),
Methods in Enzymology, combinatorial chemistry, Vol. 267, San
Diego: Academic Press (1996), Kay, B. K., Winter, J., McCafferty,
J. (eds), Phage Display of peptides and Proteins, a Laboratory
Manual, San Diego: Academic Press (1996), each incorporated herein
by reference.
[0338] In another embodiment, phage particles or phage displaying
the desired specific binding pair member are affinity purified as
follows: approximately 10.sup.3-10.sup.4 library equivalents of
phage particles or phage are reacted overnight with 1 microgram
purified antibody at 4.degree. C. The mixture is panned by a
procedure as follows. A polystyrene petri plate is coated with 1 ml
of streptavidin solution (1 mg/ml in 0.1M NaHCO.sub.3, pH 8.6,
0.02% NaN.sub.3) and is incubated overnight at 4.degree. C. The
following day the streptavidin solution is removed. The plate is
filled with 10 ml blocking solution (30 mg/ml BSA, 3 micrograms/ml
streptavidin in 0.1M NaHCO.sub.3, pH 9.2, 0.02% NaN.sub.3) and
incubated for 2 hours at room temperature. Two micrograms of
biotinylated goat anti-mouse IgG (BRL) are added to the
antibody-reacted library and incubated for 2 hours at 4.degree. C.
Immediately before panning, blocking solution is removed from
streptavidin coated plate, and the plate is washed 3 times with
TBS/0.05% Tween 20. The antibody-reacted library is then added to
the plate and incubated for 30 minutes at room temperature.
Streptavidin coated agarose beads (BRL) may also be used for this
affinity purification. The library solution is removed and the
plate is washed ten times with TBS/0.05% Tween 20 over a period of
60 minutes. Bound phage are removed by adding elution buffer (1
mg/ml BSA, 0.1N HCl, pH adjusted to 2.2 with glycine) to the petri
plate and incubating for 10 minutes to dissociate the immune
complexes. The eluate is removed, neutralized with 2M Tris (pH
unadjusted) and used to infect log phase F'-containing bacterial
cells. These cells are then plated on LB agar plates containing
tetracycline (20.mu.g/ml), and grown overnight at 37.degree. C.
Phage-particles or phage are isolated from these plates as
described and the affinity purification process was repeated for
two to three rounds. After the final round of purification, a
portion of the eluate is used to infect cells and plated at low
density on LB tetracycline plates. Individual colonies are
transferred to culture tubes containing 2 ml LB tetracycline and
grown to saturation. Phage or phagemid DNA is isolated using a
method designed for the Beckman Biomek Workstation (Mardis and
Roe., Biotechniques, 7:840-850 (1989)) which employs 96-well
microtiter plates. Single stranded DNA is sequenced by the dideoxy
method using Sequenase (U.S. Biochemicals) and an oligonucleotide
sequencing primer (5'-CGATCTAAAGTTTTGTCGTCT-3' SEQ ID NO: 2) which
is complementary to the sequence located 40 nucleotides 3' of the
second BstXI site in fdTetB1.
[0339] We considered a number of variables to address in the
construction of a novel, very large antibody phage library: (i) the
primer design was optimised for amplification of variable gene
pools to maintain maximum diversity; (ii) a highly efficient
two-step cloning method was developed to obtain a very large naive
library; (iii) an antibody format and compatible cloning vector
were chosen, which should permit the rapid down-stream analysis of
selected clones.
[0340] In order to achieve access to as many different human heavy
and light chain V-region gene segments as possible, a new set of
oligonucleotide primers was developed (Table I), the design of
which was based on the most recent sequence information provided by
the V-base.
[0341] The primers were designed to be the or several consensus
sequences which would have at least a 70% homology to the
respective 5' or 3' end based coding region in the human germ line
gene segments of the specific V gene family they would have to
amplify. The primers would amplify at least one V-gene segment
using the PCR conditions described below, and in one embodiment are
appended with appropriate positioned restriction sites for cloning
into the vector for Fab expression.
[0342] The primers should allow efficient amplification of all
commonly used V-gene segments. Further, to obtain the large sized
Fab libraries of the invention (over 10.sup.10 in diversity), we
used a two-step cloning procedure: heavy and light chain variable
genes were first separately cloned as digested PCR products, and
were then combined by restriction fragment cloning to form a large
library of Fab fragments. This cloning procedure should be a more
efficient route for library construction than the relatively
inefficient direct cloning of digested PCR-products, while avoiding
the DNA instability often associated with in vivo recombination
systems (Griffiths, et al., (1994) EMBO J. 13, 3245-3260).
[0343] A new phagemid vector, pCES1 (FIG. 1), was constructed, that
allows the stepwise cloning of antibody fragments in Fab format. In
this vector system, the variable heavy chain region genes are
cloned as V.sub.H-gene fragments; the vector supplies all Fab's
with a human gamma-1 C.sub.H1 domain. The V.sub.HC.sub.H1 formed by
insertion of the V.sub.H-gene fragments to the vector is fused (in
the vector) to two tags for purification and detection (a histidine
tail for Immobilised Metal Affinity Chromatography (Hochuli, et
al., (1988) BioTechnology 6, 1321-1325) and a c-myc-derived tag
(Munro, et al., H. R. (1986) Cell 46, 291-300)), followed by an
amber stop codon (Hoogenboom, et al., (1991) Nucleic Acids Res. 19,
4133-4137) and the minor coat protein III of filamentous phage
f.sub.d. The antibody light chain is cloned as full V.sub.LC.sub.L
fragment, for directed secretion and assembly with the
V.sub.HC.sub.H1 on the phage particle.
[0344] In one embodiment, the vector comprises an expression
cassette with a bicistronic or double cistronic expression cassette
to allow linked (for the bicistronic) or independent (for the
double cistronic) expression of the antibody light and heavy chain
or their fusions, such expression cassette consisting of the
following elements: (1) a promoter suited for non-inducible and
inducible expression (e.g lacZ); (2) a ribosome binding site and
signal sequence preceeding the light and heavy chain cloning
regions; (3) possible, but not necessarily, a region following the
heavy or light chain cloning region that encodes a tag sequence
such as a stretch of 5-6 hsitidines or a sequence recognised by an
antibody and an amber codon; (4) a phage coat protein encoded as a
fusion to the 3' end of either the heavy or light chain.
[0345] This new phage library will be a valuable source of
antibodies to essentially any target. The antibodies may be used as
research reagents or as starting point for the development of
therapeutic antibodies or agricultural products. As the list of
sequenced genomes and disease-related gene products is expanding
rapidly, there will be a growing need for an in vitro and
eventually automated method for antibody isolation. As antibodies
have been and will be ideal probes for investigating the nature,
localization and purification of novel gene products, this library
is envisaged to play an important role in target validation and
target discovery in the area of functional genomics.
[0346] Protein variants expressed on the surface of bacteriophage
have been selected on the basis of their affinity for ligand
(antigen) using chromatography, panning or adsorption to cells.
Elution from affinity matrices has been achieved by specific
elution using the ligand (antigen or a related compound) or
non-specific elution using, for example, 100 mM triethylamine.
Washing procedures remove non-specifically bound phage. The phage
binds to and is eluted from the matrix according to the affinity or
the nature of the binding interaction. Specifically eluted phage
are then used to infect male E. coli cells expressing the F pilus,
allowing recovery of phage containing DNA encoding proteins with
the desired binding characteristics.
[0347] Selection can be made not only on the basis of specificity,
but also on the basis of affinity. Separation is readily attainable
by affinity chromatography between phage expressing an antibody
with a dissociation constant of 10.sup.-8 M and one with a
dissociation constant of 10.sup.-5 M. Clackson, T. et al. (1991).
Nature 352: 624-628. The isolation of the latter antibody from an
immune repertoire demonstrates that antibodies with affinities
characteristic of the primary immune response can be isolated using
phage technology.
[0348] Antibodies directed against cell surface antigens can also
be isolated by selective adsorption of phage on the surface of
cells. Similarly, it may be possible to incorporate negative
selection with cells to remove undesired cross-reactivities with
cell surface markers. As these are rather difficult and as yet
poorly understood methods, methods based on the selection on
purified antigen should be used whenever possible.
[0349] Any selection for binders within a population will
automatically tend to select for high affinity variants at the
expense of the lower, enriching the high affinity population. This
has been used to good effect recently in the isolation of high
affinity human antibodies from a naive repertoire. Marks, J. D. et
al. (1991) J. Mol. Biol. 222, 581-597. For optimal selection, the
antigen concentration should be less than the affinity constant.
This should be borne in mind when isolating an antibody with
pre-defined characteristics. Further details on various selection
methods is given in the reviews in this manual.
[0350] With such large panels of antibodies isolated, it is useful
to have methods available to readily determine the kinetic
parameters of each individual antibody-antigen interaction. We have
shown that it is feasible to rapidly and accurately determine the
off-rate of non-purified antibodies in periplasmic fractions
prepared from small scale cultures using surface plasmon resonance.
Using this method, a series of tetanus toxoid specific Fab's showed
a monophasic dissociation, which is expected for a truly monomeric
Fab-fragment binding to a low density antigen surface. Using this
off-rate screening assay, we determined the off-rates for the best
tetanus toxoid and MUC1 specific Fab's to be in the order of
10.sup.-2 to 10.sup.-4 s.sup.-1.
[0351] We tested the integrity of selected Fab's obtained from
periplasmic fractions using western blots. When incubated in
non-reducing sample buffer, two products were detected with the
9E10 antibody, which recognises the myc-tag at the end of the CH1
domain, the major product is the intact Fab-molecule, in which an
intermolecular disulfide bridge covalently links heavy and light
chain fragments; the low molecular product is most likely derived
from non disulfide bridge linked heavy chains. Analysis with
anti-light chain sera reveals a similar pattern and shows that the
clones use a nearly equal percentage of kappa and lambda chains
(found in six and seven clones respectively of a total of 13
tested). Upon reduction of purified, functional antigen-binding
Fabs, equal amounts of heavy and light chain are seen, while under
non-reducing conditions, the main product is represented by the
disulphide linked Fab-molecule, with an equal amount of the
non-covalently linked V.sub.HC.sub.H1 and V.sub.LC.sub.L products
visible. Production yields of selected hormone specific Fab's
varied between 160 .mu.g and 1.43 mg Fab per litre culture, which
was in the same range as was found for the unselected Fabs.
[0352] A panel of 14 antigen-specific Fab's was fully sequenced (3
anti-MUC1 antibodies; 11 anti-gonadotropin antibodies). The heavy
chain genes are derived from the four largest V.sub.H families
(V.sub.H1, V.sub.H3, V.sub.H4 and V.sub.H6); the V.sub.L genes
belong to one of four V.sub..kappa.-families or one of three
V.sub..lamda.-families. Chain promiscuity is seen for the
.alpha.-chain specific clone SC#4G, the .alpha./.beta.-LH specific
clones LH#2H and LH#3G, and .beta.-FSH specific clone FS#8B, which
all used a highly homologous V.sub..kappa.2 light chain gene
segment combined with different heavy chain fragments. The 3
anti-MUC1 antibodies use heavy and light chain genes derived from 2
different VH and V6 families; clone MUC#9 uses a VH with a
cross-over of 2 segments.
[0353] The present invention is further illustrated in the
following examples. Upon consideration of the present disclosure,
one of skill in the art will appreciate that many other embodiments
and variations may be made in the scope of the present invention.
Accordingly, it is intended that the broader aspects of the present
invention not be limited to the disclosure of the following
examples.
[0354] As source of lymphoid tissues we used peripheral blood
lymphocytes from 4 healthy donors and part of a tumor-free spleen
removed from a patient with gastric carcinoma. B lymphocytes were
isolated from 2-L of blood on a Ficoll-Pacque gradient. For RNA
isolation, the cell pellet was immediately dissolved in 50 ml 8 M
guanidinium thiocyanate/0.1 M 2-mercaptoethanol (Chirgwin, et al.,
(1979) Biochemistry 18, 5294-5299). Chromosomal DNA was sheared to
completion by passing through a narrow syringe (1.2/0.5 mm gauge),
and insoluble debris was removed by low speed centrifugation (15
min 2,934.times.g at room temperature). RNA was pelleted by
centrifugation through a CsCl-block gradient (12 ml supernatant on
a layer of 3.5 ml 5.7 M CsCl/0.1 M EDTA; in total 4 tubes) during
20 h at 125,000.times.g at 20.degree. C. in a SW41-rotor (Beckman).
The yield of total RNA was approx. 600 .mu.g. RNA was stored at
-20.sup.EC in ethanol.
[0355] From the spleen, 2 g of tissue was used for homogenisation
with a polytron in 20 ml 8 M guanidinium thiocyanate/0.1 M
2-mercaptoethanol. The total volume was increased to 80 ml with
guanidinium thiocyanate buffer, and after passage through a narrow
syringe for shearing and removal of debris, RNA was pelleted as
described before, except for 15 h at 85,000.times.g at 20EC in a
SW28.1 rotor (12 ml supernatant on 3.5 ml 5.7 M CsCl/0.1 M EDTA in
5 SW28.1 tubes). From 2 g of tissue, 3 mg of total RNA was
extracted.
[0356] Random primed cDNA was prepared with 250 .mu.g PBL RNA,
while in a separate reaction 300 .mu.g spleen RNA was used as
template. RNA was heat denatured for 5 min at 65.degree. C. in the
presence of 20 .mu.g random primer (Promega), subsequently buffer
and DTT were added according to the suppliers instructions
(Gibco-BRL), as well as 250 .mu.M dNTP (Pharmacia), 800 U RNAsin
(40 U/.mu.l; Promega) and 2,000 U MMLV-RT (200 U/.mu.l; Gibco-BRL)
in a total volume of 500 .mu.l. After 2 h at 42.degree. C., the
incubation was stopped by a phenol/chloroform extraction; cDNA was
precipitated and dissolved in 85 .mu.l water.
[0357] Oligonucleotides used for PCR amplification of human heavy
and light chain V-regions are described in FIG. 2. IgM-derived
heavy chain variable regions were obtained by a primary PCR with an
IgM constant region primer. All primary PCRs were carried out with
separate BACK primers and combined FOR primers, to maintain maximal
diversity. The PCR-products were reamplified with a combination of
JHFOR-primers, annealing to the 3' end of V.sub.H, and Sfi-tagged
VHBACK-primers, annealing to the 5' end, and subsequently cloned as
V.sub.H-fragments. The light chain V-genes of the kappa and lambda
families were obtained by PCR with a set of CKFOR- or
C.lamda.OR-primer annealing to the 3' end of the constant domain
and BACK-primers, priming at the 5' end of the V-regions. The
DNA-segments were reamplified with primers tagged with restriction
sites and cloned as V.sub..kappa.C.sub..kappa.- and
V.sub..lamda.C.sub..lamda.-fragments.
[0358] PCR was performed in a volume of 50 .mu.l using AmpliTaq
polymerase (Cetus) and 500 pM of each primer for 28 cycles (1 min
at 94.sup.EC, 1 min at 55.sup.EC and 2 min at 72.sup.EC), 9
separate IgM derived VH-amplifications were generated with 2 .mu.l
random primed cDNA (equivalent to 6 .mu.g PBL RNA or to 7 .mu.g
spleen RNA) as template for each reaction. For the light chain
families, 6 different V.sub..kappa.C.sub..kappa.-products and 11
V.sub..lamda.C.sub..lamda.-products (C.sub..lamda.2- and
C.sub..lamda.7-primers combined in each reaction) were obtained.
All products were purified from agarose gel with the QIAex-II
extraction kit (Qiagen). As input for reamplification to introduce
restriction sites, 100-200 ng purified DNA-fragment was used as
template in a 100 .mu.l reaction volume. The large amount of input,
ensuring the maintenance of variability, was checked by analysis of
4 .lamda.l of the "unamplified" PCR-mixture on agarose gel.
[0359] For the construction of the primary heavy chain and the two
primary light chain repertoires, the PCR-products, appended with
restriction sites, were gel purified prior to digestion and the
different V.sub.H-, V.sub..kappa.- and V.sub..lamda.-families
combined into three groups. The V.sub..kappa.C.sub..kappa.- and
V.sub..lamda.C.sub..lamda.-fragments were digested with ApaLI and
AscI, and cloned into the phagemid vector pCES1. The
V.sub.H-fragments, 1.5 .mu.g in total, were digested with SfiI and
BstEII and ligated in a 100-200 .mu.l reaction mixture with 9 U
T.sub.4-DNA ligase at room temperature to 4 .mu.g, gel-purified
vector pUC119-CES1 (similar to vector pCES1, but with the pIII gene
deleted). The desalted ligation mixture for light or heavy chain
pools was used for electroporation of the E. coli strain TG1, to
create the one-chain libraries.
[0360] The Fab library was obtained by cloning of V.sub.H
fragments, digested from plasmid-DNA prepared from the heavy chain
repertoires, into the plasmid collection containing the light chain
repertoires. Plasmid DNA isolated from at least 3.times.10.sup.9
bacteria of the V.sub.H library was digested with SfiI and BstEII
for cloning in the vector that already contained .lamda. and
.kappa. light chain libraries. To retain clones with internal
BstEII site in the V.lamda._ (this site is relatively frequent in
some .lamda. germline V-segments (Persic, et al., (1997) Gene 187,
9-18), and also in the constant domain of one of the .lamda.
families), the cloning of V.sub.HC.sub.H1 in the .lamda. light
chain repertoire containing vector was also carried out using SfiI
and NotI cloning sites, to create a less restriction-biased
V.sub..lamda.--library.
[0361] The rescue of phagemid particles with helper phage M13-KO7
was performed according to (Marks, et al., (1991) J. Mol. Biol.
222, 581-597) on 10-L scale, using representative numbers of
bacteria from the library for inoculation, to ensure the presence
of at least 10 bacteria from each clone in the start inoculum. For
selections, 10.sup.13 cfu's (colony forming units) were used with
antigens immobilised in immunotubes (Maxisorp tubes, Nunc) (Marks,
et al., (1991) J. Mol. Biol. 222, 581-597) or with soluble
biotinylated antigens (Hawkins, et al., (1992b) J. Mol. Biol. 226,
889-896). The amount of the immobilised antigens tetanus toxoid and
the hapten phenyl-oxazolone (conjugated to BSA in a ratio of 17 to
1) was reduced 10-fold during subsequent selection rounds, starting
at 100 .mu.g/ml at round 1. Capture with biotinylated antigen in
solution was used for a 100-mer peptide encoding five copies of the
tandem repeat of MUC1 (Henderikx, et al., (1998) Cancer Res. 58,
4324-4332), or with human Chorionic Gonadotropin (hCG), human
Luteinizing Hormone (hLH), human Follicle Stimulating Hormone
(hFSH) and its chimeric derivative (hFSH-CTP, containing the
carboxy terminal peptide from the hCG .beta.-subunit fused to the
.beta.-subunit of hFSH). Antigens were biotinylated at a ratio of
ten to twenty molecules NHS-Biotin (Pierce) per molecule antigen
according to the suppliers recommendations. Unless stated
otherwise, the antigens were used for selection at concentrations
of 100 nM, 30 nM and 10 nM during round 1, 2 and 3 respectively.
For hFSH-CTP 50, 15 and 10 nM was used respectively; for MUC1
peptide, 500, 100, 20 and 5 nM was used.
[0362] Soluble Fab was produced from individual clones as described
before (Marks, et al., (1991) J. Mol. Biol. 222, 581-597). Culture
supernatants were tested in ELISA with directly coated antigen or
indirectly captured biotinylated antigen via immobilised
biotinylated BSA-streptavidin. Tetanus toxoid and phOx-BSA were
coated at 10 .mu.g/ml in 0.1 M NaHCO.sub.3 pH 9.6 for 16 h at
4.degree. C. For coating of hCG and hFSH-CTP a concentration of 4
.mu.g/ml in 50 mM NaHCO.sub.3 pH 9.6 was used. For capture of
biotinylated antigens, biotinylated BSA was coated at 2 .mu.g/ml in
PBS during 1 h at 37.degree. C. After 3 washes with PBS-0.1% (v/v)
Tween 20 (PBST), plates were incubated during 1 h with streptavidin
(10 .mu.g/ml in PBS/0.5% gelatin) (Henderikx, et al., (1998) Cancer
Res. 58, 4324-4332). Following washing as above, biotinylated
antigen was added for an overnight incubation at 4.degree. C. at a
concentration of 0.5 .mu.g/ml for MUC-1 peptide, 3 .mu.g/ml for
hLH, and 0.6 .mu.g/ml for hFSH (binding to hCG was tested with
directly coated antigen). The plates were blocked during 30 min at
room temperature with 2% (w/v) semi-skimmed milk powder (Marvel) in
PBS. The culture supernatant was diluted 1 or 5-fold in 2% (w/v)
Marvel/PBS and incubated 2 h; bound Fab was detected with anti-myc
antibody 9E10 (5 .mu.g/ml) recognising the myc-peptide tag at the
carboxyterminus of the heavy Fd chain, and rabbit anti-mouse-HRP
conjugate (DAKO) (Marks, et al., (1991) J. Mol. Biol. 222,
581-597). Following the last incubation, staining was performed
with tetramethylbenzidine (TMB) and H.sub.2O.sub.2 as substrate and
stopped by adding half a volume of 2 NH.sub.2SO.sub.4; the optical
density was measured at 450 nm. Clones giving a positive signal in
ELISA (over 2.times. the background), were analysed by
BstNI-fingerprinting of the PCR-products obtained by amplification
with the oligonucleotide primers M13-reverse and geneIII-forward
(Marks, et al., (1991) J. Mol. Biol. 222, 581-597).
[0363] Large-scale induction of soluble Fab fragments from
individual clones was performed on 50 ml scale in 2.times.TY
containing 100 .mu.g/ml ampicillin and 2% glucose. After growth at
37.degree. C. to an OD.sub.600 of 0.9, the cells were pelleted (10
min at 2,934.times.g) and resuspended in 2.times.TY with ampicillin
and 1 mM IPTG. Bacteria were harvested after 3.5 h growing at
30.degree. C. by centrifugation (as before); periplasmic fractions
were prepared by resuspending the cell pellet in 1 ml ice cold PBS.
After 2 to 16 h rotating head-over-head at 4.degree. C., the
spheroplasts were removed by two centrifugation steps: after
spinning during 10 min at 3,400.times.g, the supernatant was
clarified by an additional centrifugation step during 10 min at
13,000.times.g in an eppendorf centrifuge. The periplasmic fraction
obtained was directly used for determination of fine specificities
by surface plasmon resonance or for western blot studies.
[0364] For sequencing, plasmid DNA was prepared from 50 ml cultures
grown at 30.degree. C. in LB-medium, containing 100 .mu.g/ml
ampicillin and 2% glucose, using the QIAGEN midi-kit (Qiagen).
Sequencing was performed with the thermocycling kit (Amersham) with
CY5-labeled primers CH1FOR (5'-GTC CTT GAC CAG GCA GCC CAG
GGC-3'-SEQ ID NO: 3) and M13REV (5'-CAG GAA ACA GCT ATG AC-3'-SEQ
ID NO: 4); samples were run on the ALF-Express (Pharmacia). V-gene
sequences were aligned to V-base (Tomlinson et al., V-BASE, MRC
Centre for Protein Engineering, 1997,
http://www.mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html) or the
Sanger Centre (Sanger Centre Germline Query, 1997,
http//www.sanger.ac.uk/Data Search/gq-search.html).
[0365] An hCG-preparation purified from urine and immuno-affinity
purified recombinant hLH, hFSH and hFSH-CTP produced in CHO-cells
(Matzuk, et al., (1989) J. Cell. Biol. 109, 1429-1438; Muyan, et
al., (1996) Mol. Endocrinol. 10, 1678-1687) were used for western
blot studies as was described (Moyle, et al., (1990) J. Biol. Chem.
265, 8511-8518). Between 0.5 and 1 .mu.g of each hormone was loaded
per lane; proteins were diluted in non-reducing sample buffer and
boiled during 5 min or directly applied on gel without
heat-treatment; proteins were transferred to blotting membrane by
electrotransfer. Blots were subsequently incubated for 16 h at room
temperature with a 10-fold diluted periplasmic fraction in PBS/4%
Marvel. Bound Fab was detected with anti-myc antibody 9E10 (5
.mu.g/ml) and 4.000-fold diluted anti-mouse alkaline
phosphatase-conjugate (Promega), using the substrates
5-bromo-1-chloro-3-indolyl phosphate (BCIP) and nitro blue
tetrazolium (NBT) (Boehringer Mannheim) for visualisation.
[0366] The specificity of the Fab's was further characterised by
surface plasmon resonance (BIAcore 2000, Biacore). Recombinant hLH,
hFSH and the urinary hCG were immobilised on the flow-cells of a
CM-chip using the NHS/EDC-kit (Pharmacia), yielding a surface of
1906 RU for hLH, 1529 RU for hFSH and 1375 RU for hCG. Periplasmic
fractions were diluted three-fold in Hepes Buffered Saline (HBS; 10
mM Hepes, 3.4 mM EDTA, 150 mM NaCl, 0.05% (v/v) surfactant P20, pH
7.4) and analysed using a flow rate of 10 .mu.l/min.
[0367] Fab's were obtained by refolding of the total bacterial
proteins from a 50 ml culture (de Haard, et al., (1998) Protein
Eng., 11:1267-1276). Briefly, the pelleted cells from a 50 ml
induced bacterial culture were resuspensed in 8 ml 8 M urea (in
PBS). After sonication, the mixture was rotated head over head for
30 min and insoluble material was removed by centrifugation for 30
min at 13,000.times.g. The supernatant was dialysed against PBS
with four buffer changes. Insoluble proteins were removed by
centrifugation and the flow through fraction, obtained by
filtration through a 0.2 .mu.m membrane, was immediately loaded on
an hCG column (bed volume 0.3 ml). The column material was prepared
by coupling 8.4 mg protein to one gram Tresyl sepharose according
to the suppliers instructions (Pierce). The column (1 ml column
material) was washed with 10 volumes 100 mM Tris, 500 mM NaCl pH
7.5, subsequently with 10 volumes 100 mM Tris/500 mM NaCl pH 9.5
and with 2 volumes 0.9% NaCl, bound Fab was eluted with two volumes
0.1 M TEA and immediately neutralised with 0.5 volume 1M Tris pH
7.5. The Fab fraction was dialysed against PBS using a Microcon 30
spin dialysis filter (Amicon). Finally, a gel-filtration analysis
was carried out on a Superdex 75HR column (Pharmacia). The yield
was determined by measuring the optical density at 280 nm (using a
molar extinction coefficient of 13 for Fab's).
[0368] The kinetics of binding were analysed by surface plasmon
resonance on three different hCG surfaces (303 RU, 615 RU and 767
RU immobilised, with 4955 RU BSA on a separate flow cell as a
negative control). Fab present in crude periplasmic extracts was
quantified on a high density surface of purified anti-human-Fab
polyclonal antibody (Pierce) as described (Kazemier, et al., (1996)
J. Immunol. Methods 194, 201-209). Anti-hCG Fab's controls were
purified by affinity chromatography on hCG columns as described
above and used to calibrate the system.
[0369] The Fab library was constructed in two-steps. In the first
step, variable region gene pools were amplified from approx.
4.times.10.sup.8 B-cells from the PBLs of four healthy donors, and,
as a source of possibly more heavily mutated IgM antibodies, from a
segment of a (tumor-free) spleen removed from a patient with
gastric carcinoma, containing approximately 1.5.times.10.sup.8
B-cells (Roit, et al., (1985) Immunology, Gower Medical Publishing,
Ltd., London). Only IgM-derived V.sub.H segments were amplified by
using an amplification with an oligonucleotide primer located in
the first constant domain of this isotype. These products were
cloned into phagemid vector pCES1 for V.sub.L, and in pUC119-CES1
for V.sub.H (cloning was more efficiently in the smaller sized
vector, in which gene III was deleted). The PBL and spleen derived
V.sub.H, V.sub..kappa. and V.sub..lamda.-libraries were cloned
separately to maintain diversity, to yield one-chain libraries in
size typical for libraries made by cloning of PCR-fragments (Marks,
et al., (1991) J. Mol. Biol. 222, 581-597): 1.75.times.10.sup.8
individual clones for the heavy chain, 9.4.times.10.sup.7 clones
for V.sub..kappa., and 5.2.times.10.sup.7 clones for V.sub..lamda..
In the second step, the heavy chain fragments were digested from
plasmid DNA isolated from the primary V.sub.H repertoire, and
cloned into the vector containing the light chain repertoires
(again separately for PBL and spleen derived repertoire). The
libraries were combined using this efficient cloning procedure, to
create a naVve Fab repertoire with 3.7.times.10.sup.10 individual
clones (4.3.times.10.sup.10 recombinant clones, 86% of which have a
full-length Fab insert), with 70% of clones harbouring a kappa
light chain, 30% a lambda chain. All of 20 clones with full length
Fab insert tested scored positive in dot-blot analysis with the
9E10 antibody to indicating an expression level of soluble Fab of
at least 0.2 mg/L.
[0370] We evaluated the library by selection with different
antigens. First, the results from three model antigens, the protein
tetanus toxoid, the hapten 2-phenyloxazol-5-one (phOx) (Griffiths,
et al., (1984) Nature 312, 271-275, and the peptide MUC1, are
discussed. Three rounds of biopanning on tetanus toxoid yielded a
diverse set of ELISA positive Fab's, in a series of 47 tetanus
toxoid binding Fab's, at least 21 were different with regard to
BstNI-fingerprint. Similarly, an extensive panel of phOx-specific
Fab's was retrieved after three rounds of panning: at least 24
different clones were identified in a series of 50 ELISA positive
clones. Solution capture with biotinylated MUC1 peptide resulted in
the selection of 14 different antibody fragments out of 37
ELISA-positive clones selected after 3 rounds.
[0371] As a more stringent test panel of antigens to assay the
performance of the library, we chose to derive antibodies to three
structurally related glycoproteins: human Chorionic Gonadotropin
(hCG), human Luteinizing Hormone (hLH) and human Follicle
Stimulating Hormone (hFSH) (reviewed in (Cole, (1997) Clin. Chem.
43, 2233-2243)). These hormones are heterodimers sharing an
identical .alpha.-chain with 92 amino acid residues, but have
.beta.-subunits of different composition and length. The
.beta.-chain of hCG contains 145 amino acid residues, and the one
from hLH only 121 residues, the latter showing 85% homology to
.beta.-hCG. The .beta.-chain of hFSH is only 111 amino acids and
shares 36% of the residues with hCG. Antibodies that specifically
detect hCG have been used extensively in pregnancy tests (Cole,
(1997) Clin. Chem. 43, 2233-2243) and for cancer diagnosis (Masure,
et al., (1981) J. Clin. Endocrinol. Metab. 53, 1014-1020;
Papapetrou, et al., (1980) Cancer 45, 2583-2592). A large set of
antibodies to these targets would extend the limited number of
hormone specific antibodies (especially against hLH), obtained
using the hybridoma technology (Cole, (1997) Clin. Chem. 43,
2233-2243). The human origin of the antibodies might be beneficial
when using these for imaging or therapy of testicular and bladder
cancer (Masure, et al., (1981) J. Clin. Endocrinol. Metab. 53,
1014-1020; Papapetrou, et al., (1980) Cancer 45, 2583-2592).
[0372] Selections were thus performed on biotinylated urinary hCG,
recombinant hLH, hFSH and hFSH-CTP (the latter is a chimeric
molecule containing the carboxy terminal peptide of .beta.-hCG
fused to the .beta.-chain of FSH (Fares, et al., (1992) Proc. Natl.
Acad. Sci. U.S.A. 89, 4304-4308)). The highest degree of enrichment
in respect to the increase in the number of eluted phage particles
in round 3 versus round 1 was found for hCG (10.000-fold), followed
by hFSH-CTP (1.000-fold), hFSH (300-fold) and hLH (150-fold).
Polyclonal phage of selected populations were tested for binding
using sensorchips containing immobilised hormones (Schier, et al.,
(1996) Hum. Antibodies Hybridomas 7, 97-105). Polyclonal phage
selected with hCG showed binding after rounds two and three of
selection to all three proteins, i.e., hCG, hLH, and hFSH, with the
strongest signal visible for hCG. Similar analysis of the
polyclonal phage populations selected for three rounds on hFSH
showed a dominance of hFSH-specific binding, while selections on
hFSH-CTP yielded binders to both hFSH and hCG. Selections on hLH
yielded antibodies reactive with hFSH and hCG. Thus, this
polyclonal phage screening provides a rapid test to check the
overall quality of the clones in the selected repertoire, and may
also be used to guide the choice of the conditions for the next
selection round (Schier, et al., (1996) Hum. Antibodies Hybridomas
7, 97-105).
[0373] ELISA of monoclonal phage antibodies revealed that three
rounds of selection with hCG indeed resulted in the isolation of a
high percentage (74%) of clones positive for the gonadotropin. 27%
of these clones were hLH cross-reactive; none were reactive against
streptavidin. BstNI-fingerprint analysis of the ELISA-positive
clones revealed a high degree of diversity (8 different patterns).
From a representative hCG-specific (coded CG#4F) and hLH
cross-reactive (CG#5C) clone, the specificity was tested in BIAcore
using unpurified soluble Fab fragments. Clone CG#4F gave a high
response on hCG, with no visible binding to either hLH or hFSH-CTP.
In contrast, clone CG#5C bound to hCG and hLH, but not to hFSH-CTP.
Western blots, with the different hormones in non-reduced form,
showed the specific recognition of the .beta.-subunit of hCG by
clone CG#4F, while the cross-reactive clone CG#5C reacted with the
.beta.-subunit of both hCG and hLH.
[0374] Selection with the hormone hLH resulted in the isolation of
hLH-specific and hCG cross-reactive clones. Examination of
individual clones from selection round three in ELISA revealed a
large fraction of hLH specific clones (69%), and a minor group of
cross-reactive clones (16%); no streptavidin reactive clones were
selected. Within the group of specific clones, a large array of
different species (>21) could be discriminated with fingerprint
analysis; however, all cross-reactive species had a single pattern.
The unique hLH specificity was confirmed for representative clones
LH#2H and LH#3G, shown in surface plasmon resonance; and on western
blot. LH#3G only recognises the intact .alpha./.beta.-heterodimer
of hLH. Two representative clones of a pan-reactive antibody in
ELISA, coded LH#1C and LH#3F, reacted in BIAcore with hFSH-CTP, hCG
and hLH, and in western blot analysis with the .alpha.-chains from
all three hormones.
[0375] When hFSH was used as antigen during selection, 6 different
antibodies were isolated from the library, with one type,
represented by clone FS#8B, dominating the selected population.
This Fab only recognised hFSH in BIAcore, and, as western blot
analysis demonstrated, in particular its .beta.-unit. Further, the
specificity of an .alpha.-chain binding clone, SC#2B, was confirmed
in BIAcore and western blot.
[0376] Upon selection with FSH-CTP 7 different .alpha.-chain
specific Fab's were identified by fingerprint analysis, from which
the clones coded SC#2B, SC#2F, SC#2G and SC#4G were examined in
more detail. Immunoblot analysis with the recombinant Fab as
detecting antibody confirmed the .alpha.-chain specificity.
[0377] The affinities and off-rates of affinity purified hCG
reactive Fab's LH#1C, SC#2B, LH#3F and CG#5C were determined. The
off-rates for most Fab's were in the order of 10.sup.-2 and
10.sup.-3 s.sup.-1. The off-rate values obtained using crude
periplasmic fractions were in good agreement with the values found
for the purified Fab's, validating the utility of the off-rate
screen with unpurified Fab fragments. The affinities, 23 nM and 38
nM for the .alpha.-subunit specific antibody LH#1C and the
.beta.-subunit hCG/hLH-cross reactive antibody CG#5C respectively,
are comparable to the affinity of antibodies selected from a murine
immune phage antibody library (H.d.H., B. Kazemier, et al.,
unpublished); the top affinity, 2.7 nM for the .alpha.-chain
specific Fab SC#2B, approaches the values of the best anti-hCG
monoclonal antibodies (H.d.H., B. Kazemier, et al.,
unpublished).
[0378] The aim of this procedure is to select and enrich for
phage-antibodies to an antigen coated on the surface of
immunotubes. The antigen is coated to the immunotube (e.g., a
Nunc-immunotube) and incubated with the phage library. Non-bound
phage are washed away and the binding phage are eluted, therefore
the phage library becomes enriched for phage antibodies that
specifically bind the antigen.
[0379] The aim of this procedure is to biotinylate proteins or
peptides. At neutral pH or above, primary amine-groups react with
NHS-SS-Biotin, and N-hydroxysulfosuccimide is released. The
N-terminal free NH.sub.2-groups as well as lysines (K) of the
protein react with NHS-S-S-Biotin, in this pH range.
[0380] NHS-SS-Biotin is a unique biotin analog with an extended
spacer arm of approximately 24.3 .ANG. in length, the spacer arm of
NHS-LC-Biotin is 22.4 .ANG.. These long chain analogs reduce steric
hindrances associated with binding of biotinylated molecules to
avidin or streptavidin.
[0381] The presence of the S-S linker in NHS-S-S-Biotin enables
disruption of binding using reducing agents (DTT, DTE,
B-mercaptoethanol). NHS-LC-Biotin is used when biotinylated
protein/peptide is needed that is not sensitive to reducing
agents.
[0382] The aim of this procedure is to select phage antibodies
against a biotinylated antigen. The selection is done in solution,
and can be used to select phage antibodies against antigens that
are prone to denaturation when coated onto solid surfaces.
[0383] First the biotinylated antigen is incubated with the phage
antibody library. After addition of the Dynabeads (Dynal) coated
with streptavidin, the biotin of the antigen-antibody-complex will
bind to the streptavidin. This Dynabead-antigen-antibody-complex is
pulled out with a magnet (e.g., a Dynal magnet) and therefore
should contain the specific antibodies.
[0384] The aim of this procedure is to select for those antibodies
out of a library that bind to antigens present in the cell
membrane, using adherent growing cells or cells in suspension. The
method can be used for selection of antibodies against targets
expressed on (tumor) cell lines.
[0385] By incubating whole cells, organelles, or membrane fractions
with a high variety phage antibody repertoire, such as the Fab
Libraries of the invention (concentrated by PEG precipitation),
only (or preferentially) relevant antibodies, to one of the
molecules exposed on the surface of the cellular membrane(s), will
be retained while not binding phage antibodies are separated from
the antibodies bound to the cells, organelles or membrane fractions
(by methods well known in the art for separating cells, organelles
or membrane fractions from molecules in solution). The retained
phage population is enriched for those clones which are specific
for cell related molecules. In principle the following factors will
positively influence the enrichment of individual clones: Affinity,
antigen abundance, and low toxicity of the antibody construct to
TG1 host.
[0386] The aim of this procedure is to prepare soluble antibody
fragments from the periplasm of E. coli. In the periplasm there is:
less protease activity, less contaminating proteins than in the
cytoplasm or supernatants, and the antibody is more concentrated.
Therefore, periplasmic preparations are more stable and more pure
than culture supernatants.
[0387] As a consequence of induction of phagemid containing
bacterial cultures in low glucose medium with IPTG, soluble
antibody fragments are produced and directed to the periplasm where
they are concentrated within 4 hours. Overnight culturing in these
circumstances will make the bacterial membrane leaky and antibodies
will be found in the supernatant. For preparation of periplasmic
fractions, the bacterial cell wall is first lysed by cold osmotic
shock (icecold TES) and then rapidly diluted in a chilled solution
of low osmotic strength (TES/H.sub.2O). The EDTA makes the outer
membrane more permeable, and the cold inhibits protease activity.
Subsequently, the bacterial cells are spun down and the supernatant
then contains the periplasmic proteins.
[0388] The antibodies in the periplasmic fraction can be used as a
`crude extract` or the antibodies can be purified by conventional
means well known in the art, for e.g., those recited in Section 5.6
and 5.7.
[0389] The aim of this procedure is to purify antibodies labeled
with a His6 tag from periplasmic fractions of Fabs made as
described in Example 6.18.
[0390] Immobilized metal affinity chromatography (IMAC) for the
purification of recombinant 6.times.His-tagged proteins under
native conditions: Recombinant histidine tagged proteins are
captured on a chelated metal containing resin through coordination
of free N-atoms of the histidines to the metal (mostly Ni.sup.2+ or
Co.sup.2+). After washing away contaminating proteins and other
cell constituents, the his-tagged protein is specifically eluted
from the resin with imidazol which competes for the binding of
histidine-residues to the metal ion.
[0391] The present invention is not to be limited in scope by the
exemplified embodiments which are intended as illustrations of
single aspects of the invention, and compositions and methods which
are functionally equivalent are within the scope of the invention.
Indeed, numerous modifications and variations in the practice of
the invention are expected to occur to those skilled in the art
upon consideration of the present preferred embodiments.
Consequently, the only limitations which should be placed upon the
scope of the invention are those which appear in the appended
claims.
[0392] All references cited within the body of the instant
specification are hereby incorporated by reference in their
entirety.
Sequence CWU 1
1
71 1 11 PRT Artificial Sequence Description of Artificial Sequence
Illustrative peptide 1 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn
1 5 10 2 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 2 cgatctaaag ttttgtcgtc t 21 3 24 DNA Artificial
Sequence Description of Artificial Sequence Primer 3 gtccttgacc
aggcagccca gggc 24 4 17 DNA Artificial Sequence Description of
Artificial Sequence Primer 4 caggaaacag ctatgac 17 5 6 PRT
Artificial Sequence Description of Artificial Sequence 6X-His tag 5
His His His His His His 1 5 6 349 DNA Artificial Sequence
Description of Artificial Sequence Synthetic polylinker of pCES1
CDS (1)..(99) CDS (140)..(340) CDS (344)..(349) 6 tta ttc gca att
cct tta gtt gtt cct ttc tat tct cac agt gca cag 48 Leu Phe Ala Ile
Pro Leu Val Val Pro Phe Tyr Ser His Ser Ala Gln 1 5 10 15 gtc caa
ctg cag gtc gac ctc gag atc aaa cgt gga act gtg gga gag 96 Val Gln
Leu Gln Val Asp Leu Glu Ile Lys Arg Gly Thr Val Gly Glu 20 25 30
tgt taataaggcg cgccaattct atttcaagga gacagtcata atg aaa tac cta 151
Cys Met Lys Tyr Leu 35 ttg cct acg gca gcc gct gga ttg tta tta ctc
gcg gcc cag ccg gcc 199 Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu
Ala Ala Gln Pro Ala 40 45 50 atg gcc cag gtg cag ctg cag gag agc
ggg gtc acc gtc tca agc gcc 247 Met Ala Gln Val Gln Leu Gln Glu Ser
Gly Val Thr Val Ser Ser Ala 55 60 65 tcc acc aaa tct tgt gcg gcc
gca cat cat cat cat cat cac ggg gcc 295 Ser Thr Lys Ser Cys Ala Ala
Ala His His His His His His Gly Ala 70 75 80 85 gca gaa caa aaa ctc
atc tca gaa gag gat ctg aat ggg gcc gca tag 343 Ala Glu Gln Lys Leu
Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala 90 95 100 act gtt 349 Thr
Val 7 33 PRT Artificial Sequence Description of Artificial Sequence
Synthetic peptide 7 Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser
His Ser Ala Gln 1 5 10 15 Val Gln Leu Gln Val Asp Leu Glu Ile Lys
Arg Gly Thr Val Gly Glu 20 25 30 Cys 8 67 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 8 Met Lys Tyr
Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala
Gln Pro Ala Met Ala Gln Val Gln Leu Gln Glu Ser Gly Val Thr 20 25
30 Val Ser Ser Ala Ser Thr Lys Ser Cys Ala Ala Ala His His His His
35 40 45 His His Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp
Leu Asn 50 55 60 Gly Ala Ala 65 9 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 9 tggaagaggc acgttctttt
cttt 24 10 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 10 acactctccc ctgttgaagc tctt 24 11 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 11
tgaacattct gtaggggcca ctg 23 12 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 12 agagcattct gcaggggcca
ctg 23 13 23 DNA Artificial Sequence Description of Artificial
Sequence Primer 13 cagrtgcagc tggtgcartc tgg 23 14 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 14
saggtccagc tggtrcagtc tgg 23 15 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 15 cagrtcacct tgaaggagtc
tgg 23 16 23 DNA Artificial Sequence Description of Artificial
Sequence Primer 16 saggtgcagc tggtggagtc tgg 23 17 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 17
gaggtgcagc tggtggagwc ygg 23 18 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 18 caggtgcagc tacagcagtg
ggg 23 19 23 DNA Artificial Sequence Description of Artificial
Sequence Primer 19 cagstgcagc tgcaggagtc sgg 23 20 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 20
gargtgcagc tggtgcagtc tgg 23 21 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 21 caggtacagc tgcagcagtc
agg 23 22 23 DNA Artificial Sequence Description of Artificial
Sequence Primer 22 gacatccagw tgacccagtc tcc 23 23 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 23
gatgttgtga tgactcagtc tcc 23 24 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 24 gaaattgtgw tgacrcagtc
tcc 23 25 23 DNA Artificial Sequence Description of Artificial
Sequence Primer 25 gatattgtga tgacccacac tcc 23 26 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 26
gaaacgacac tcacgcagtc tcc 23 27 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 27 gaaattgtgc tgactcagtc
tcc 23 28 23 DNA Artificial Sequence Description of Artificial
Sequence Primer 28 cagtctgtgc tgactcagcc acc 23 29 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 29
cagtctgtgy tgacgcagcc gcc 23 30 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 30 cagtctgtcg tgacgcagcc
gcc 23 31 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 31 cartctgccc tgactcagcc t 21 32 23 DNA Artificial
Sequence Description of Artificial Sequence Primer 32 tcctatgwgc
tgactcagcc acc 23 33 23 DNA Artificial Sequence Description of
Artificial Sequence Primer 33 tcttctgagc tgactcagga ccc 23 34 23
DNA Artificial Sequence Description of Artificial Sequence Primer
34 cacgttatac tgactcaacc gcc 23 35 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 35 caggctgtgc tgactcagcc
gtc 23 36 23 DNA Artificial Sequence Description of Artificial
Sequence Primer 36 aattttatgc tgactcagcc cca 23 37 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 37
cagrctgtgg tgacycagga gcc 23 38 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 38 cwgcctgtgc tgactcagcc
mcc 23 39 51 DNA Artificial Sequence Description of Artificial
Sequence Primer 39 accgcctcca ccgggcgcgc cttattaaca ctctcccctg
ttgaagctct t 51 40 50 DNA Artificial Sequence Description of
Artificial Sequence Primer 40 accgcctcca ccgggcgcgc cttattatga
acattctgta ggggccactg 50 41 50 DNA Artificial Sequence Description
of Artificial Sequence Primer 41 accgcctcca ccgggcgcgc cttattaaga
gcattctgca ggggccactg 50 42 56 DNA Artificial Sequence Description
of Artificial Sequence Primer 42 gtcctcgcaa ctgcggccca gccggccatg
gcccagrtgc agctggtgca rtctgg 56 43 56 DNA Artificial Sequence
Description of Artificial Sequence Primer 43 gtcctcgcaa ctgcggccca
gccggccatg gccsaggtcc agctggtrca gtctgg 56 44 56 DNA Artificial
Sequence Description of Artificial Sequence Primer 44 gtcctcgcaa
ctgcggccca gccggccatg gcccagrtca ccttgaagga gtctgg 56 45 56 DNA
Artificial Sequence Description of Artificial Sequence Primer 45
gtcctcgcaa ctgcggccca gccggccatg gccsaggtgc agctggtgga gtctgg 56 46
56 DNA Artificial Sequence Description of Artificial Sequence
Primer 46 gtcctcgcaa ctgcggccca gccggccatg gccgaggtgc agctggtgga
gwcygg 56 47 56 DNA Artificial Sequence Description of Artificial
Sequence Primer 47 gtcctcgcaa ctgcggccca gccggccatg gcccaggtgc
agctacagca gtgggg 56 48 56 DNA Artificial Sequence Description of
Artificial Sequence Primer 48 gtcctcgcaa ctgcggccca gccggccatg
gcccagstgc agctgcagga gtcsgg 56 49 56 DNA Artificial Sequence
Description of Artificial Sequence Primer 49 gtcctcgcaa ctgcggccca
gccggccatg gccgargtgc agctggtgca gtctgg 56 50 56 DNA Artificial
Sequence Description of Artificial Sequence Primer 50 gtcctcgcaa
ctgcggccca gccggccatg gcccaggtac agctgcagca gtcagg 56 51 24 DNA
Artificial Sequence Description of Artificial Sequence Primer 51
tgaggagacg gtgaccaggg tgcc 24 52 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 52 tgaagagacg gtgaccattg
tccc 24 53 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 53 tgaggagacg gtgaccaggg ttcc 24 54 24 DNA
Artificial Sequence Description of Artificial Sequence Primer 54
tgaggagacg gtgaccgtgg tccc 24 55 44 DNA Artificial Sequence
Description of Artificial Sequence Primer 55 accgcctcca ccagtgcact
tgacatccag wtgacccagt ctcc 44 56 44 DNA Artificial Sequence
Description of Artificial Sequence Primer 56 accgcctcca ccagtgcact
tgatgttgtg atgactcagt ctcc 44 57 44 DNA Artificial Sequence
Description of Artificial Sequence Primer 57 accgcctcca ccagtgcact
tgaaattgtg wtgacrcagt ctcc 44 58 44 DNA Artificial Sequence
Description of Artificial Sequence Primer 58 accgcctcca ccagtgcact
tgatattgtg atgacccaca ctcc 44 59 44 DNA Artificial Sequence
Description of Artificial Sequence Primer 59 accgcctcca ccagtgcact
tgaaacgaca ctcacgcagt ctcc 44 60 44 DNA Artificial Sequence
Description of Artificial Sequence Primer 60 accgcctcca ccagtgcact
tgaaattgtg ctgactcagt ctcc 44 61 41 DNA Artificial Sequence
Description of Artificial Sequence Primer 61 accgcctcca ccagtgcaca
gtctgtgctg actcagccac c 41 62 41 DNA Artificial Sequence
Description of Artificial Sequence Primer 62 accgcctcca ccagtgcaca
gtctgtgytg acgcagccgc c 41 63 41 DNA Artificial Sequence
Description of Artificial Sequence Primer 63 accgcctcca ccagtgcaca
gtctgtcgtg acgcagccgc c 41 64 39 DNA Artificial Sequence
Description of Artificial Sequence Primer 64 accgcctcca ccagtgcaca
rtctgccctg actcagcct 39 65 44 DNA Artificial Sequence Description
of Artificial Sequence Primer 65 accgcctcca ccagtgcact ttcctatgwg
ctgactcagc cacc 44 66 44 DNA Artificial Sequence Description of
Artificial Sequence Primer 66 accgcctcca ccagtgcact ttcttctgag
ctgactcagg accc 44 67 41 DNA Artificial Sequence Description of
Artificial Sequence Primer 67 accgcctcca ccagtgcaca cgttatactg
actcaaccgc c 41 68 41 DNA Artificial Sequence Description of
Artificial Sequence Primer 68 accgcctcca ccagtgcaca ggctgtgctg
actcagccgt c 41 69 44 DNA Artificial Sequence Description of
Artificial Sequence Primer 69 accgcctcca ccagtgcact taattttatg
ctgactcagc ccca 44 70 41 DNA Artificial Sequence Description of
Artificial Sequence Primer 70 accgcctcca ccagtgcaca grctgtggtg
acycaggagc c 41 71 41 DNA Artificial Sequence Description of
Artificial Sequence Primer 71 accgcctcca ccagtgcacw gcctgtgctg
actcagccmc c 41
* * * * *
References