U.S. patent application number 10/901011 was filed with the patent office on 2005-05-19 for binding polypeptides with restricted diversity sequences.
This patent application is currently assigned to GENENTECH, INC. Invention is credited to Fellouse, Frederic A., Sidhu, Sachdev.
Application Number | 20050106667 10/901011 |
Document ID | / |
Family ID | 34120173 |
Filed Date | 2005-05-19 |
United States Patent
Application |
20050106667 |
Kind Code |
A1 |
Fellouse, Frederic A. ; et
al. |
May 19, 2005 |
Binding polypeptides with restricted diversity sequences
Abstract
The invention provides variant CDRs comprising highly restricted
amino acid sequence diversity. These polypeptides provide a
flexible and simple source of sequence diversity that can be used
as a source for identifying novel antigen binding polypeptides. The
invention also provides these polypeptides as fusion polypeptides
to heterologous polypeptides such as at least a portion of phage or
viral coat proteins, tags and linkers. Libraries comprising a
plurality of these polypeptides are also provided. In addition,
methods of and compositions for generating and using these
polypeptides and libraries are provided.
Inventors: |
Fellouse, Frederic A.; (San
Francisco, CA) ; Sidhu, Sachdev; (San Francisco,
CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
GENENTECH, INC
|
Family ID: |
34120173 |
Appl. No.: |
10/901011 |
Filed: |
July 28, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60491877 |
Aug 1, 2003 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 530/388.22; 536/23.5 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 2317/565 20130101; C07K 16/22 20130101; C07K 2317/73 20130101;
A61P 37/02 20180101; C07K 2317/55 20130101; A61P 1/04 20180101;
A61P 17/00 20180101; A61P 31/04 20180101; A61P 9/10 20180101; A61P
43/00 20180101; C07K 2317/76 20130101; C07K 2317/92 20130101; A61P
1/16 20180101; A61P 11/00 20180101; A61P 17/02 20180101; C07K
2317/56 20130101; A61P 17/06 20180101; A61P 25/00 20180101; C07K
16/005 20130101; A61P 13/08 20180101; A61P 27/00 20180101; A61P
37/06 20180101; G01N 33/6845 20130101; A61P 15/00 20180101; A61P
35/02 20180101; C07K 16/32 20130101; A61P 19/02 20180101; C07K
2317/24 20130101; C07K 2317/31 20130101; A61P 27/02 20180101; A61P
1/18 20180101; A61P 35/00 20180101; A61P 29/00 20180101 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/325; 530/350; 536/023.5; 530/388.22 |
International
Class: |
C07H 021/04; C12P
021/04; C07K 014/705 |
Claims
1-25. (canceled)
26. a polypeptide comprising a variant CDRH1, H2, H3, L1, L2 and/or
L3, wherein the variant CDR has a variant amino acid in at least
one solvent accessible and highly diverse amino acid position,
wherein the variant amino acid is encoded by a restricted codon set
that encodes 10 or fewer amino acids:
27. The polypeptide of claim 26, wherein the polypeptide comprises
a variant CDRH3 comprising a variant amino acid in at least one of
positions 95, 96, 97, 98, 99, 100 and 100a, numbering of positions
according to the Kabat system.
28. The polypeptide of claim 26, wherein the polypeptide comprises
a variant CDRH3 comprising a variant amino acid in at least one of
positions 95, 96, 97, 98, 99, 100, and a position between 100 and
C-terminal sequence AMDY.
29. The polypeptide of claim 26, wherein the variant CDRH3
comprises an insertion of one or more positions, wherein said one
or more positions comprises an amino acid encoded by a restricted
codon set.
30. The polypeptide of claim 26, wherein the polypeptide comprises
a variant CDRH2 comprising a variant amino acid in at least one of
positions 50, 52, 53, 54, 56 and 58, numbering of positions
according to the Kabat system.
31. The polypeptide of claim 26, wherein the polypeptide comprises
a variant CDRH1 comprising a variant amino acid in at least one of
positions 28, 30, 31, 32 and 33, numbering of positions according
to the Kabat system.
32. The polypeptide of claim 26, wherein the polypeptide comprises
a variant CDRL3 comprising a variant amino acid in at least one of
positions 92, 93, 94, 95 and 97, numbering of positions according
to the Kabat system.
33. The polypeptide of claim 26, wherein the polypeptide comprises
a variant CDRL2 comprising a variant amino acid in at least one of
positions 51 and 54, numbering of positions according to the Kabat
system.
34. The polypeptide of claim 26, wherein the polypeptide comprises
a variant CDRL1 comprising a variant amino acid in at least one of
positions 29, 30, 31, 32 and 33, numbering of positions according
to the Kabat system.
35. The polypeptide of claim 26, wherein the restricted codon set
is TMT, WMT, RMC, RMG, RRC, RSA, MKC, YMT, RST, KMT, SRC, MRT, WMT,
or a combination thereof.
36. The polypeptide of claim 26, wherein the codon set is TMT
and/or KMT.
37-52. (canceled)
53. The polypeptide of claim 26, wherein the polypeptide is a heavy
chain antibody variable domain.
54. (canceled)
55. The polypeptide of claim 26, further comprising a dimerization
domain linked to C-terminal region of a heavy chain antibody
variable domain.
56. A polypeptide according to claim 55, wherein the dimerization
domain comprises a leucine zipper domain or a sequence comprising
at least one cysteine residue.
57. A polypeptide according to claim 56, wherein the dimerization
domain comprises a hinge region from an antibody and leucine
zipper.
58. A polypeptide according to claim 55, wherein the dimerization
domain is a single cysteine.
59-72. (canceled)
73. A library comprising a plurality of the polypeptide of claim
26, and wherein the library has at least 1.times.10.sup.4 distinct
antibody variable domain sequences.
74-90. (canceled)
91. A method comprising: a) constructing an expression vector
comprising a polynucleotide sequence which encodes a light chain
variable domain, a heavy chain variable domain or both of a source
antibody comprising at least one, two, three, four, five or all
CDRs of the source antibody selected from the group consisting of
CDR L1, L2, L3, H1, H2 and H3; and b) mutating at least one, two,
three, four, five or all CDRs of the source antibody at at least
one solvent accessible and highly diverse amino acid position using
a restricted codon set that encodes 10 or fewer amino acids.
92. The method of claim 91, wherein variant CDRH3 comprises an
amino acid sequence: (X1).sub.n-A-M wherein X.sub.1 is an amino
acid encoded by a restricted codon set that encodes 10 or fewer
amino acids, and n=3 to 20.
93. The method of claim 91, wherein variant CDRH2 comprises an
amino acid sequence: X1-I-X2-P-(X3)n-G-X4-T-X5-Y-A wherein X1, X2,
X3, X4 and/or X5 is an amino acid encoded by a restricted codon set
that encodes 10 or fewer amino acids, and n=1 to 2.
94. The method of claim 91, wherein variant CDRH1 comprises an
amino acid sequence: G-F-X1-I-(X2)n-I wherein X1 and/or X2 is an
amino acid encoded by a restricted codon set that encodes 10 or
fewer amino acids, and n=2 to 4.
95. The method of claim 91, wherein variant CDRL3 comprises an
amino acid sequence: Q-X1-(X2)n-P-X3-T-F wherein X1 is Q or
missing, and X2 and/or X3 is an amino acid encoded by a restricted
codon set that encodes 10 or fewer amino acids, and n=2 to 4.
96. The method of claim 91, wherein variant CDRL2 comprises an
amino acid sequence: Y-X1-A-S-X2-L wherein X1 and/or X2 is an amino
acid encoded by a restricted codon set that encodes 10 or fewer
amino acids.
97. The method of claim 91, wherein variant CDRL1 comprises an
amino acid sequence: S-Q-(X1)n-V wherein X1 is an amino acid
encoded by a restricted codon set that encodes 10 or fewer amino
acids, and n=3 to 5.
Description
RELATED APPLICATIONS
[0001] This application is a non-provisional application filed
under 37 CFR 1.53(b)(1), claiming priority benefit under 35 USC
119(e) to provisional application No. 60/491,877 filed Aug. 1,
2003, the contents of which are incorporated herein in its entirety
by reference.
FIELD OF THE INVENTION
[0002] The invention generally relates to variant CDRs diversified
using highly limited amino acid repertoires, and libraries
comprising a plurality of such sequences. The invention also
relates to fusion polypeptides comprising these variant CDRs. The
invention also relates to methods and compositions useful for
identifying novel binding polypeptides that can be used
therapeutically or as reagents.
BACKGROUND
[0003] Phage display technology has provided a powerful tool for
generating and selecting novel proteins that bind to a ligand, such
as an antigen. Using the techniques of phage display allows the
generation of large libraries of protein variants that can be
rapidly sorted for those sequences that bind to a target antigen
with high affinity. Nucleic acids encoding variant polypeptides are
fused to a nucleic acid sequence encoding a viral coat protein,
such as the gene III protein or the gene VIII protein. Monovalent
phage display systems where the nucleic acid sequence encoding the
protein or polypeptide is fused to a nucleic acid sequence encoding
a portion of the gene III protein have been developed. (Bass, S.,
Proteins, 8:309 (1990); Lowman and Wells, Methods: A Companion to
Methods in Enzymology, 3:205 (1991)). In a monovalent phage display
system, the gene fusion is expressed at low levels and wild type
gene III proteins are also expressed so that infectivity of the
particles is retained. Methods of generating peptide libraries and
screening those libraries have been disclosed in many patents (e.g.
U.S. Pat. No. 5,723,286, U.S. Pat. No. 5,432,018, U.S. Pat. No.
5,580,717, U.S. Pat. No. 5,427,908 and U.S. Pat. No.
5,498,530).
[0004] The demonstration of expression of peptides on the surface
of filamentous phage and the expression of functional antibody
fragments in the periplasm of E. coli was important in the
development of antibody phage display libraries. (Smith et al.,
Science (1985), 228:1315; Skerra and Pluckthun, Science (1988),
240:1038). Libraries of antibodies or antigen binding polypeptides
have been prepared in a number of ways including by altering a
single gene by inserting random DNA sequences or by cloning a
family of related genes. Methods for displaying antibodies or
antigen binding fragments using phage display have been described
in U.S. Pat. Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108,
6,172,197, 5,580,717, and 5,658,727. The library is then screened
for expression of antibodies or antigen binding proteins with
desired characteristics.
[0005] Phage display technology has several advantages over
conventional hybridoma and recombinant methods for preparing
antibodies with the desired characteristics. This technology allows
the development of large libraries of antibodies with diverse
sequences in less time and without the use of animals. Preparation
of hybridomas or preparation of humanized antibodies can easily
require several months of preparation. In addition, since no
immunization is required, phage antibody libraries can be generated
for antigens which are toxic or have low antigenicity (Hogenboom,
Immunotechniques (1988), 4:1-20). Phage antibody libraries can also
be used to generate and identify novel human antibodies.
[0006] Antibodies have become very useful as therapeutic agents for
a wide variety of conditions. For example, humanized antibodies to
HER-2, a tumor antigen, are useful in the diagnosis and treatment
of cancer. Other antibodies, such as anti-INF-.gamma. antibody, are
useful in treating inflammatory conditions such as Crohn's disease.
Phage display libraries have been used to generate human antibodies
from immunized, non-immunized humans, germ line sequences, or nave
B cell Ig repertories (Barbas & Burton, Trends Biotech (1996),
14:230; Griffiths et al., EMBO J. (1994), 13:3245; Vaughan et al.,
Nat. Biotech. (1996), 14:309; Winter EP 0368 684 B 1). Nave, or
nonimmune, antigen binding libraries have been generated using a
variety of lymphoidal tissues. Some of these libraries are
commercially available, such as those developed by Cambridge
Antibody Technology and Morphosys (Vaughan et al., Nature Biotech
14:309 (1996); Knappik et al., J. Mol. Biol. 296:57 (1999)).
However, many of these libraries have limited diversity.
[0007] The ability to identify and isolate high affinity antibodies
from a phage display library is important in isolating novel human
antibodies for therapeutic use. Isolation of high affinity
antibodies from a library is traditionally thought to be dependent,
at least in part, on the size of the library, the efficiency of
production in bacterial cells and the diversity of the library.
See, for e.g., Knappik et al., J. Mol. Biol. (1999), 296:57. The
size of the library is decreased by inefficiency of production due
to improper folding of the antibody or antigen binding protein and
the presence of stop codons. Expression in bacterial cells can be
inhibited if the antibody or antigen binding domain is not properly
folded. Expression can be improved by mutating residues in turns at
the surface of the variable/constant interface, or at selected CDR
residues. (Deng et al., J. Biol. Chem. (1994), 269:9533, Ulrich et
al., PNAS (1995), 92:11907-11911; Forsberg et al., J. Biol. Chem.
(1997), 272:12430). The sequence of the framework region is a
factor in providing for proper folding when antibody phage
libraries are produced in bacterial cells.
[0008] Generating a diverse library of antibodies or antigen
binding proteins is also important to isolation of high affinity
antibodies. Libraries with diversification in limited CDRs have
been generated using a variety of approaches. See, for e.g.,
Tomlinson, Nature Biotech. (2000), 18:989-994. CDR3 regions are of
interest in part because they often are found to participate in
antigen binding. CDR3 regions on the heavy chain vary greatly in
size, sequence and structural conformation.
[0009] Others have also generated diversity by randomizing CDR
regions of the variable heavy and light chains using all 20 amino
acids at each position. It was thought that using all 20 amino
acids would result in a large diversity of sequences of variant
antibodies and increase the chance of identifying novel antibodies.
(Barbas, PNAS 91:3809 (1994); Yelton, D E, J. Immunology, 155:1994
(1995); Jackson, J. R., J. Immunology, 154:3310 (1995) and Hawkins,
RE, J. Mol. Biology, 226:889 (1992)).
[0010] There have also been attempts to create diversity by
restricting the group of amino acid substitutions in some CDRs to
reflect the amino acid distribution in naturally occurring
antibodies. See, Garrard & Henner, Gene (1993), 128:103;
Knappik et al., J. Mol. Biol. (1999), 296:57. However, these
attempts have had varying success and have not been applied in a
systematic and quantitative manner. Creating diversity in the CDR
regions while minimizing the number of amino acid changes has been
a challenge. Furthermore, in some instances, once a first library
has been generated according to one set of criteria, it may be
desirable to further enhance the diversity of the first library.
However, this requires that the first library has sufficient
diversity and yet remain sufficiently small in size such that
further diversity can be introduced without substantially exceeding
practical limitations such as yield, etc.
[0011] Some groups have reported theoretical and experimental
analyses of the minimum number of amino acid repertoire that is
needed for generating proteins. However, these analyses have
generally been limited in scope and nature, and substantial
skepticism and questions remain regarding the feasibility of
generating polypeptides having complex functions using a restricted
set of amino acid types. See, for e.g., Riddle et al., Nat. Struct.
Biol. (1997), 4(10):805-809; Shang et al., Proc. Natl. Acad. Sci.
USA (1994), 91:8373-8377; Heinz et al., Proc. Natl. Acad. Sci. USA
(1992), 89:3751-3755; Regan & Degrado, Science (1988),
241:976-978; Kamteker et al., Science (1993), 262:1680-1685; Wang
& Wang, Nat. Struct. Biol. (1999), 6(11):1033-1038; Xiong et
al., Proc. Natl. Acad. Sci. USA (1995), 92:6349-6353; Heinz et al.,
Proc. Natl. Acad. Sci. USA (1992), 89:3751-3755; Cannata et al.,
Bioinformatics (2002), 18(8):1102-1108; Davidson et al., Nat.
Struct. Biol. (1995), 2(10):856-863; Murphy et al., Prot. Eng.
(2000), 13(3):149-152; Brown & Sauer, Proc. Natl. Acad. Sci.
USA (1999), 96:1983-1988; Akanuma et al., Proc. Natl. Acad. Sci.
(2002), 99(21):13549-13553; Chan, Nat. Struct. Biol. (1999),
6(11):994-996.
[0012] Thus, there remains a need to improve methods of generating
libraries that comprise functional polypeptides having a sufficient
degree of sequence diversity, yet are sufficiently amenable for
further manipulations directed at further diversification, high
yield expression, etc. The invention described herein meets this
need and provides other benefits.
DISCLOSURE OF THE INVENTION
[0013] The present invention provides simplified and flexible
methods of generating polypeptides comprising variant CDRs that
comprise sequences with restricted diversity yet retain target
antigen binding capability. Unlike conventional methods that are
based on the proposition that adequate diversity of target binders
can be generated only if a particular CDR(s), or all CDRs are
diversified, and unlike conventional notions that adequate
diversity is dependent upon the broadest range of amino acid
substitutions (generally by substitution using all or most of the
20 amino acids), the invention provides methods capable of
generating high quality target binders that are not necessarily
dependent upon diversifying a particular CDR(s) or a particular
number of CDRs of a reference polypeptide or source antibody. The
invention is based, at least in part, on the surprising and
unexpected finding that highly diverse libraries of high quality
comprising functional polypeptides capable of binding target
antigens can be generated by diversifying a minimal number of amino
acid positions with a highly restricted number of amino acid
residues. Methods of the invention are rapid, convenient and
flexible, based on using restricted codon sets that encode a low
number of amino acids. The restricted sequence diversity, and thus
generally smaller size of the populations (for e.g., libraries) of
polypeptides generated by methods of the invention allows for
further diversification of these populations, where necessary or
desired. This is an advantage generally not provided by
conventional methods. Candidate binder polypeptides generated by
the invention possess high-quality target binding characteristics
and have structural characteristics that provide for high yield of
production in cell culture. The invention provides methods for
generating these binder polypeptides, methods for using these
polypeptides, and compositions comprising the same.
[0014] In one aspect, the invention provides fusion polypeptides
comprising diversified CDR(s) and a heterologous polypeptide
sequence (preferably that of at least a portion of a viral
polypeptide), as single polypeptides and as a member of a plurality
of unique individual polypeptides that are candidate binders to
targets of interest. Compositions (such as libraries) comprising
such polypeptides find use in a variety of applications, for e.g.,
as pools of candidate immunoglobulin polypeptides (for e.g.,
antibodies and antibody fragments) that bind to targets of
interest. Such polypeptides may also be generated using
non-immunoglobulin scaffolds (for e.g., proteins, such as human
growth hormone, etc.). The invention encompasses various aspects,
including polynucleotides and polypeptides generated according to
methods of the invention, and systems, kits and articles of
manufacture for practicing methods of the invention, and/or using
polypeptides/polynucleotides and/or compositions of the
invention.
[0015] In one aspect, the invention provides a method of generating
a polypeptide comprising at least one, two, three, four, five or
all of variant CDRs selected from the group consisting of H1, H2,
H3, L1, L2 and L3, wherein said polypeptide is capable of binding a
target antigen of interest, said method comprising identifying at
least one (or any number up to all) solvent accessible and highly
diverse amino acid position in a reference CDR corresponding to the
variant CDR; and (ii) varying the amino acid at the solvent
accessible and high diverse position by generating variant copies
of the CDR using a restricted codon set (the definition of
"restricted codon set" as provided below).
[0016] Various aspects and embodiments of methods of the invention
are useful for generating and/or using a pool comprising a
plurality of polypeptides of the invention, in particular for
selecting and identifying candidate binders to target antigens of
interest. For example, the invention provides a method of
generating a composition comprising a plurality of polypeptides,
each polypeptide comprising at least one, two, three, four, five or
all of variant CDRs selected from the group consisting of H1, H2,
H3, L1, L2 and L3, wherein said polypeptide is capable of binding a
target antigen of interest, said method comprising identifying at
least one (or any number up to all) solvent accessible and highly
diverse amino acid position in a reference CDR corresponding to the
variant CDR; and (ii) varying the amino acid at the solvent
accessible and high diverse position by generating variant copies
of the CDR using a restricted codon set; wherein a plurality of
polypeptides are generated by amplifying a template polynucleotide
with a set of oligonucleotides comprising highly restricted
degeneracy in the sequence encoding a variant amino acid, wherein
said restricted degeneracy reflects the limited number of codon
sequences of the restricted codon set.
[0017] In another example, the invention provides a method
comprising: constructing an expression vector comprising a
polynucleotide sequence which encodes a light chain, a heavy chain,
or both the light chain and the heavy chain variable domains of a
source antibody comprising at least one, two, three, four, five or
all CDRs selected from the group consisting of CDR L1, L2, L3, H1,
H2 and H3; and mutating at least one, two, three, four, five or all
CDRs of the source antibody at at least one (or any number up to
all) solvent accessible and highly diverse amino acid position
using a restricted codon set.
[0018] In another example, the invention provides a method
comprising: constructing a library of phage or phagemid particles
displaying a plurality of polypeptides of the invention; contacting
the library of particles with a target antigen under conditions
suitable for binding of the particles to the target antigen; and
separating the particles that bind from those that do not bind to
the target antigen.
[0019] In any of the methods of the invention described herein, a
solvent accessible and/or highly diverse amino acid position can be
any that meet the criteria as described herein, in particular any
combination of the positions as described herein, for example any
combination of the positions described for the polypeptides of the
invention (as described in greater detail herein). Suitable variant
amino acids can be any that meet the criteria as described herein,
for example variant amino acids in polypeptides of the invention as
described in greater detail below.
[0020] Designing diversity in CDRs may involve designing diversity
in the length and/or in sequence of the CDR. For example, CDRH3 may
be diversified in length to be, for e.g., 7 to 19 amino acids in
length, and/or in its sequence, for e.g. by varying highly diverse
and/or solvent accessible positions with amino acids encoded by a
restricted codon set. In some embodiments, a portion of CDRH3 has a
length ranging from 5 to 22, 7 to 20, 9 to 15, or 11 to 13 amino
acids, and has a variant amino acid at one or more positions
encoded by a restricted codon set that encodes a limited number of
amino acids such as codon sets encoding no more than 10, 8, 6, 4 or
2 amino acids. In some embodiments, the C terminal end has an amino
acid sequence AM or AMDY.
[0021] In some embodiments, polypeptides of the invention can be in
a variety of forms as long as the target binding function of the
polypeptides is retained. In some embodiments, a polypeptide of the
invention is a fusion polypeptide (ie. a fusion of two or more
sequences from heterologous polypeptides). Polypeptides with
diversified CDRs according to the invention can be prepared as
fusion polypeptides to at least a portion of a viral coat protein,
for e.g., for use in phage display. Viral coat proteins that can be
used for display of the polypeptides of the invention comprise
protein p III, major coat protein pVIII, Soc (T4 phage), Hoc (T4
phage), gpD (lambda phage), pVI, or variants or fragments thereof.
In some embodiments, the fusion polypeptide is fused to at least a
portion of a viral coat protein, such as a viral coat protein
selected from the group consisting of pIII, pVIII, Soc, Hoc, gpD,
pVI, and variants or fragments thereof.
[0022] In some embodiments, in which the polypeptide with
diversified CDRs is one or more antibody variable domains, the
antibody variable domains can be displayed on the surface of the
virus in a variety of formats including ScFv, Fab, ScFv.sub.2,
F(ab').sub.2 and F(ab).sub.2. For display of the polypeptides in
bivalent manner, the fusion protein preferably includes a
dimerization domain. The dimerization domain can comprise a
dimerization sequence and/or a sequence comprising one or more
cysteine residues. The dimerization domain is preferably linked,
directly or indirectly, to the C-terminal end of a heavy chain
variable or constant domain (e.g., CH1). The structure of the
dimerization domain can be varied depending on whether the antibody
variable domain is produced as a fusion protein component with the
viral coat protein component (without an amber stop codon after
dimerization domain) or whether the antibody variable domain is
produced predominantly without viral coat protein component (eg.
with an amber stop codon after dimerization domain). When the
antibody variable domain is produced predominantly as a fusion
protein with viral coat protein component, one or more disulfide
bonds and/or a single dimerization sequence provides for bivalent
display. For antibody variable domains predominantly produced
without being fused to a viral coat protein component (eg. with
amber stop), it is preferable to have a dimerization domain
comprising both a cysteine residue and a dimerization sequence.
[0023] In addition, optionally, a fusion polypeptide can comprise a
tag that may be useful in purification, detection and/or screening
such as FLAG, poly-his, gD tag, c-myc, fluorescence protein or
B-galactosidase. In one embodiment, a fusion polypeptide comprises
a light chain variable or constant domain fused to a polypeptide
tag.
[0024] In another aspect of the invention, a polypeptide such as an
antibody variable domain is obtained from a single source or
template molecule. The source or template molecule is preferably
selected or designed for characteristics such as good yield and
stability when produced in prokaryotic or eukaryotic cell culture,
and/or to accommodate CDRH3 regions of varying lengths. The
sequence of the template molecule can be altered to improve folding
and/or display of the variable domain when presented as a fusion
protein with a phage coat protein component. For example, a source
antibody may comprise the amino acid sequence of the variable
domains of humanized antibody 4D5 (light chain variable domain
(FIG. 15; SEQ ID NO: 1)); (heavy chain variable domain (FIG. 15;
SEQ ID NO: 2)). For example, in an antibody variable domain of a
heavy or light chain, framework region residues can be modified or
altered from the source or template molecule to improve folding,
yield, display or affinity of the antibody variable domain. In some
embodiments, framework residues are selected to be modified from
the source or template molecule when the amino acid in the
framework position of the source molecule is different from the
amino acid or amino acids commonly found at that position in
naturally occurring antibodies or in a subgroup consensus sequence.
The amino acids at those positions can be changed to the amino
acids most commonly found in the naturally occurring antibodies or
in a subgroup consensus sequence at that position. In one
embodiment, framework residue 71 of the heavy chain may be R, V or
A. In another example, framework residue 93 of the heavy chain may
be S or A. In yet another example, framework residue 94 may be R, K
or T or encoded by MRT. In yet another example, framework residue
49 in the heavy chain may be alanine or glycine. Framework residues
in the light chain may also be changed. For e.g., the amino acid at
position 66 may be arginine or glycine.
[0025] Methods of the invention are capable of generating a large
variety of polypeptides comprising a diverse set of CDR sequences.
For e.g., in one embodiment, the invention provides a polypeptide
comprising a variant CDRH3 region that comprises an amino acid
sequence:
[0026] (X1).sub.n-A-M
[0027] wherein X.sub.1 is an amino acid encoded by a restricted
codon set, and n=a suitable number that would retain the functional
activity of the CDR. For e.g., n can be 3 to 20, 5-20, 7-20, 5-18
or 7-18. In one embodiment, n=7-20. In some embodiments, X1 is
encoded by codon set TMT, WMT, RMC, RMG, RRC, RSA, MKC, YMT, RST,
KMT, SRC, MRT, WMT, or a combination thereof. In one embodiment, X1
is encoded by codon set TMT and/or KMT. In one embodiment, the
amino acid sequence is (X1).sub.n-A-M-D-Y. In some embodiments, the
first X1 position corresponds to amino acid position 95 in CDRH3,
for e.g., position 95 of CDRH3 of antibody 4D5. In some
embodiments, the first X1 position corresponds to the position 33
residues after the end of CDRH2 and 2 residues after a cysteine. In
some embodiments, the first X1 position corresponds to the position
preceded by Cys-Xaa-Xaa, which in some embodiments is Cys-Ala-Arg
or Cys-Ser-Arg.
[0028] In one aspect, the invention provides a polypeptide
comprising a variant CDRH2 that comprises an amino acid
sequence:
[0029] X1-I-X2-P-(X3)n-G-X4-T-X5-Y-A
[0030] wherein X1, X2, X3, X4 and/or X5 is an amino acid encoded by
a restricted codon set, and n=a suitable number that would retain
the functional activity of the CDR. For e.g., n can be 1-5,1-3, or
1-2. In some embodiments, n=2. In some embodiments, the restricted
codon set is TMT, WMT, RMC, RMG, RRC, RSA, MKC, YMT, RST, KMT, SRC,
MRT, WMT, or a combination thereof. In some embodiments, the
restricted codon set is TMT and/or KMT.
[0031] In another aspect, the invention provides a polypeptide
comprising a variant CDRH1 that comprises an amino acid
sequence:
[0032] G-F-X1-I-(X2)n-I
[0033] wherein X1 and/or X2 is an amino acid encoded by a
restricted codon set, and n=a suitable number that would retain the
functional activity of the CDR. For e.g., n can be 14, 24 or 3-4.
In one embodiment, n=4. In some embodiments, the codon set is TMT,
WMT, RMC, RMG, RRC, RSA, MKC, YMT, RST, KMT, SRC, MRT, WMT, or a
combination thereof. In one embodiment, the codon set is TMT and/or
KMT.
[0034] In another aspect, the invention provides a polypeptide
comprising a variant CDRL3 that comprises an amino acid
sequence:
[0035] Q-X1-(X2)n-P-X3-T-F
[0036] wherein X1 is Q or missing, and
[0037] X2 and/or X3 is an amino acid encoded by a restricted codon
set, and n=a suitable number that would retain the functional
activity of the CDR. For e.g., n can be 1-4, 24 or 3-4. In one
embodiment, n=4. In some embodiments, the restricted codon set is
TMT, WMT, RMC, RMG, RRC, RSA, MKC, YMT, RST, KMT, SRC, MRT, WMT, or
a combination thereof. In one embodiment, the codon set is TMT
and/or KMT.
[0038] In another aspect, the invention provides a polypeptide
comprising a variant CDRL2 that comprises an amino acid
sequence:
[0039] Y-X1-A-S-X2-L
[0040] wherein X1 and/or X2 is an amino acid encoded by a
restricted codon set. In some embodiments, the restricted codon set
is TMT, WMT, RMC, RMG, RRC, RSA, MKC, YMT, RST, KMT, SRC, MRT, WMT,
or a combination thereof. In one embodiment, the codon set is TMT
and/or KMT.
[0041] In another aspect, the invention provides a polypeptide
comprising a variant CDRL1 that comprises an amino acid
sequence:
[0042] S-Q-(X1)n-V
[0043] wherein X1 is an amino acid encoded by a restricted codon
set, and n=a suitable number that would retain the functional
activity of the CDR. For e.g., n can be 1-5,2-5, 3-5 or 4-5. In one
embodiment, n=5. In some embodiments, the restricted codon set is
TMT, WMT, RMC, RMG, RRC, RSA, MKC, YMT, RST, KMT, SRC, MRT, WMT, or
a combination thereof. In one embodiment, the codon set is TMT
and/or KMT.
[0044] For clarity, where n is greater than 1 in CDR sequences
described herein, in a single variant CDR, amino acid X can be any
of the amino acids encoded by a particular restricted codon set.
For e.g., in a variant CDRH3 sequence wherein X1 is encoded by KMT
and n=4, the 4 X1 amino acids in the variant CDRH3 can be, for
e.g., AADY, AAAY, DSYA, SAYY, AAAA, SAAY, AAAY, AYDS, or any
combination of one or more of the four amino acids encoded by the
restricted codon set.
[0045] In one embodiment of the invention, a restricted codon set
encodes from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, or
only 2 amino acids. In some embodiments, a restricted codon set
encodes at least 2 but 10 or fewer, 8 or fewer, 6 or fewer, 4 or
fewer amino acids. In one embodiment, a restricted codon set is a
tetranomial codon set. In another embodiment, a restricted codon
set is a binomial codon set.
[0046] In yet another aspect, the invention provides a polypeptide
comprising a variant CDRH1, H2, H3, L1, L2 and/or L3, wherein the
variant CDR has a variant amino acid in at least one solvent
accessible and highly diverse amino acid position, wherein the
variant amino acid is encoded by a restricted codon set. In some
embodiments, the restricted codon set is TMT, WMT, RMC, RMG, RRC,
RSA, MKC, YMT, RST, KMT, SRC, MRT, WMT, or a combination thereof.
In one embodiment, the codon set is TMT and/or KMT. In some
embodiments, a variant CDR comprises an amino acid sequence as set
forth above.
[0047] In one aspect, the invention provides a polypeptide
comprising a variant CDRH3 comprising a variant amino acid in at
least one (or any number up to all) of positions 95, 96, 97, 98,
99, 100 and 100a, numbering of positions according to the Kabat
system. Typically, the C terminal residues of CDRH3 are kept
constant as AMDY (although some changes can be made as long as the
desired polypeptide characteristics (such as target antigen
binding) are substantially retained). In some embodiments, all
positions between 100 and A in the AMDY region comprise variant
amino acids. In some embodiments, at least one position between 100
and A in the AMDY region comprises a variant amino acid. In some
embodiments, a polypeptide comprises a variant CDRH3 comprising a
variant amino acid in at least one of positions 95, 96, 97, 98, 99,
100, and at least one position between 100 and C-terminal sequence
AMDY. In some embodiments of these polypeptides, the variant CDRH3
comprises an insertion of one or more residues/positions, wherein
said one or more positions comprises an amino acid encoded by a
restricted codon set. In some embodiments, said insertion comprises
1-15, 3-13, 5-11, or 7-9 residues/positions. In some embodiments,
said insertion comprises at least 1, at least 3, at least 5, at
least 7, at least 9, at least 11, at least 13 residues/positions.
In some embodiments, said insertion comprises 15 or fewer, 13 or
fewer, 111 or fewer, 9 or fewer, 7 or fewer, or 5 or fewer
residues/positions.
[0048] In one aspect, the invention provides a polypeptide
comprising a variant CDRH2 comprising a variant amino acid in at
least one (or any number up to all) of positions 50, 52, 53, 54, 56
and 58, numbering of positions according to the Kabat system.
[0049] In one aspect, the invention provides a polypeptide
comprising a variant CDRH1 comprising a variant amino acid in at
least one (or any number up to all) of positions 28, 30, 31, 32 and
33, numbering of positions according to the Kabat system.
[0050] In one aspect, the invention provides a polypeptide
comprising a variant CDRL3 comprising a variant amino acid in at
least one (or any number up to all) of positions 91, 92, 93, 94 and
96, numbering of positions according to the Kabat system.
[0051] In one aspect, the invention provides a polypeptide
comprising a variant CDRL2 comprising a variant amino acid in at
least one or both of positions 50 and 53, numbering of positions
according to the Kabat system.
[0052] In one aspect, the invention provides a polypeptide
comprising a variant CDRL1 comprising a variant amino acid in at
least one (or any number up to all) of positions 28, 29, 30, 31 and
32, numbering of positions according to the Kabat system.
[0053] In one aspect, the invention provides a polypeptide
comprising a variant CDR as described above, wherein the
polypeptide further comprises at least one, two, three, four or
five additional variant CDRs selected from the group consisting of
CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 or CDRL3, wherein a variant amino
acid is encoded by a restricted codon set. In some embodiments, the
restricted codon set is TMT, WMT, RMC, RMG, RRC, RSA, MKC, YMT,
RST, KMT, SRC, MRT, WMT, or a combination thereof. In one
embodiment, a restricted codon set encodes at least Y and/or S. In
one embodiment, a restricted codon set does not encode alanine. In
one embodiment, the restricted codon set encodes 4 or fewer amino
acids. In one embodiment, the restricted codon set encodes only 2
amino acids, which in one embodiment are Y and S. In one embodiment
of the invention, a restricted codon set encodes from 2 to 10, from
2 to 8, from 2 to 6, from 2 to 4, or only 2 amino acids. In some
embodiments, a restricted codon set encodes at least 2 but 10 or
fewer, 8 or fewer, 6 or fewer, 4 or fewer amino acids. In one
embodiment, a restricted codon set is a tetranomial codon set. In
another embodiment, a restricted codon set is a binomial codon set.
In one example, a polypeptide of the invention comprises a variant
CDRH3, and at least one additional variant CDR which is CDRH1
and/or CDRH2. In some embodiments, the polypeptide further
comprises at least one variant light chain CDR. In one embodiment,
a variant light chain CDR is CDRL3. In some embodiments, a
polypeptide of the invention further comprises a variant CDRL1
and/or CDRL2 (in some instances, in combination with a variant
CDRL3).
[0054] In one aspect, a polypeptide of the invention comprises at
least one, or both, of heavy chain and light chain antibody
variable domains, wherein the antibody variable domain comprises
one, two or three variant CDRs as described herein (for e.g., as
described in the foregoing).
[0055] In some embodiments, a polypeptide of the invention (in
particular those comprising an antibody variable domain) further
comprises an antibody framework sequence, for e.g., FR1, FR2, FR3
and/or FR4 for an antibody variable domain corresponding to the
variant CDR, the FR sequences obtained from a single antibody
template. In one embodiment, the FR sequences are obtained from a
human antibody. In one embodiment, the FR sequences are obtained
from a human consensus sequence (e.g., subgroup III consensus
sequence). In one embodiment, the framework sequences comprise a
modified consensus sequence as described herein (e.g., comprising
modifications at position 49, 71, 93 and/or 94 in the heavy chain,
and/or position 66 in the light chain). In one embodiment, each of
the FR has the sequence of antibody 4D5 (SEQ ID NO: 1).
[0056] In one aspect, the invention provides methods of generating
compositions comprising polypeptides and/or polynucleotides of the
invention. Accordingly, in one aspect, the invention provides a
method of generating a composition comprising a plurality of
polypeptides comprising:
[0057] a) generating a plurality of polypeptides comprising at
least one variant CDR of CDRH1 or CDRH2 or CDRH3 or mixtures
thereof wherein
[0058] i) polypeptides comprising variant CDRH3 comprise an amino
acid sequence:
[0059] (X1).sub.n-A-M
[0060] wherein X.sub.1 is an amino acid encoded by a restricted
codon set, and n=a suitable number that would retain the functional
activity of the CDR (for e.g., 3-20, 5-20, 7-20, 5-18, 7-18);
[0061] ii) polypeptides comprising variant CDRH2 comprise an amino
acid sequence:
[0062] X1-I-X2-P-(X3)n-G-X4-T-X5-Y-A
[0063] wherein X1, X2, X3, X4 and/or X5 is an amino acid encoded by
a restricted codon set, and n=a suitable number that would retain
the functional activity of the CDR (for e.g., 1-5,1-3, 1-2);
and
[0064] (iii) polypeptides comprising variant CDRH1 comprise an
amino acid sequence:
[0065] G-F-X1-I-(X2)n-I
[0066] wherein X1 and/or X2 is an amino acid encoded by a
restricted codon set, and n=a suitable number that would retain the
functional activity of the CDR (for e.g., 1-4,2-4, 3-4).
[0067] In some embodiments, a method of the invention also
comprises generating a plurality of polypeptides comprising a
variant CDRL1, CDRL2 or CDRL3 or mixtures thereof, wherein the
variant CDRs are formed with at least one variant amino acid in a
solvent accessible and highly diverse position; wherein the variant
amino acid is encoded by a restricted codon set. In one embodiment,
polypeptides comprising variant CDRL3 comprise an amino acid
sequence:
[0068] Q-X1-(X2)n-P-X3-T-F
[0069] wherein X1 is Q or missing, and
[0070] X2 and/or X3 is an amino acid encoded by a restricted codon
set, and n=a suitable number that would retain the functional
activity of the CDR (for e.g., 1-4, 2-4, 3-4). In one embodiment,
polypeptides comprising variant CDRL2 comprise an amino acid
sequence:
[0071] Y-X1-A-S-X2-L
[0072] wherein X1 and/or X2 is an amino acid encoded by a
restricted codon set. In one embodiment, polypeptides comprising
variant CDRL1 comprise an amino acid sequence:
[0073] S-Q-(X1)n-V
[0074] wherein X1 is an amino acid encoded by a restricted codon
set, and n=a suitable number that would retain the functional
activity of the CDR (for e.g., 1-5,2-5, 3-5,4-5).
[0075] In some aspects, the invention provides a polypeptide
comprising at least one, two, three, four, five or all of variant
CDRs selected from the group consisting of CDR L1, CDR L2, CDR L3,
CDR H1, CDR H2 and CDR H3, wherein the variant CDR is as described
above.
[0076] In some embodiments, a polypeptide of the invention
comprises a light chain and a heavy chain antibody variable domain,
wherein the light chain variable domain comprises at least 1, 2 or
3 variant CDRs selected from the group consisting of CDR L1, L2 and
L3, and the heavy chain variable domain comprises at least 1, 2 or
3 variant CDRs selected from the group consisting of CDR H1, H2 and
H3.
[0077] In some embodiments, a polypeptide of the invention is an
ScFv. In some embodiments, it is a Fab fragment. In some
embodiments, it is a F(ab).sub.2 or F(ab').sub.2 Accordingly, in
some embodiments, a polypeptide of the invention further comprises
a dimerization domain. In some embodiments, the dimerization domain
is located between an antibody heavy chain or light chain variable
domain and at least a portion of a viral coat protein. The
dimerization domain can comprise a dimerization sequence, and/or
sequence comprising one or more cysteine residues. The dimerization
domain is preferably linked, directly or indirectly, to the
C-terminal end of a heavy chain variable or constant domain. The
structure of the dimerization domain can be varied depending on
whether the antibody variable domain is produced as a fusion
protein component with the viral coat protein component (without an
amber stop codon after dimerization domain) or whether the antibody
variable domain is produced predominantly without viral coat
protein component (eg. with an amber stop codon after dimerization
domain). When the antibody variable domain is produced
predominantly as a fusion protein with viral coat protein
component, one or more disulfide bond and/or a single dimerization
sequence provides for bivalent display. For antibody variable
domains predominantly produced without being fused to a viral coat
protein component (eg. with amber stop), it is preferable, though
not required, to have a dimerization domain comprising both a
cysteine residue and a dimerization sequence. In some embodiments,
heavy chains of the F(ab).sub.2 dimerize at a dimerization domain
not including a hinge region. The dimerization domain may comprise
a leucine zipper sequence (for example, a GCN4 sequence such as
GRMKQLEDKVEELLSKNYHLENEVARLKKLVGERG (SEQ ID NO: 3)).
[0078] In some embodiments, a polypeptide of the invention further
comprises a light chain constant domain fused to a light chain
variable domain, which in some embodiments comprises at least one,
two or three variant CDRs. In some embodiments of polypeptides of
the invention, the polypeptide comprises a heavy chain constant
domain fused to a heavy chain variable domain, which in some
embodiments comprises at least one, two or three variant CDRs.
[0079] In some instances, it may be preferable to mutate a
framework residue such that it is variant with respect to a
reference polypeptide or source antibody. For example, framework
residue 71 of the heavy chain may be amino acid R, V or A. In
another example, framework residue 93 of the heavy chain may be
amino acid S or A. In yet another example, framework residue 94 of
the heavy chain may be amino acid R, K or T or encoded by MRT. In
yet another example, framework residue 49 of the heavy chain may be
amino acid A or G. Framework residues in the light chain may also
be mutated. For example, framework residue 66 in the light chain
may be amino acid R or G.
[0080] As described herein, a variant CDR refers to a CDR with a
sequence variance as compared to the corresponding CDR of a single
reference polypeptide/source antibody. Accordingly, the CDRs of a
single polypeptide of the invention preferably correspond to the
set of CDRs of a single reference polypeptide or source antibody.
Polypeptides of the invention may comprise any one or combinations
of variant CDRs. For example, a polypeptide of the invention may
comprise a variant CDRH1 and variant CDRH2. A polypeptide of the
invention may comprise a variant CDRH1, variant CDRH2 and a variant
CDRH3. In another example, a polypeptide of the invention may
comprise a variant CDRH1, variant CDRH2, variant CDRH3 and variant
CDRL3. In another example, a polypeptide of the invention comprises
a variant CDRL1, variant CDRL2 and variant CDRL3. Any polypeptide
of the invention may further comprise a variant CDRL3. Any
polypeptide of the invention may further comprise a variant
CDRH3.
[0081] In one embodiment, a polypeptide of the invention comprises
one or more variant CDR sequences as depicted in FIG. 5.
[0082] Polypeptides of the invention may be in a complex with one
another. For example, the invention provides a polypeptide complex
comprising two polypeptides, wherein each polypeptide is a
polypeptide of the invention, and wherein one of said polypeptides
comprises at least one, two or all of variant CDRs H1, H2 and H3,
and the other polypeptide comprises a variant light chain CDR
(e.g., CDR L3). A polypeptide complex may comprise a first and a
second polypeptide (wherein the first and second polypeptides are
polypeptides of the invention), wherein the first polypeptide
comprises at least one, two or three variant light chain CDRs, and
the second polypeptide comprises at least one, two or three variant
heavy chain CDRs. The invention also provides complexes of
polypeptides that comprise the same variant CDR sequences.
Complexing can be mediated by any suitable technique, including by
dimerization/multimerization at a dimerization/multimerization
domain such as those described herein or covalent interactions
(such as through a disulfide linkage) (which in some contexts is
part of a dimerization domain, for e.g. a dimerization domain may
contain a leucine zipper sequence and a cysteine).
[0083] In another aspect, the invention provides compositions
comprising polypeptides and/or polynucleotides of the invention.
For example, the invention provides a composition comprising a
plurality of any of the polypeptides of the invention described
herein. Said plurality may comprise polypeptides encoded by a
plurality of polynucleotides generated using a set of
oligonucleotides comprising degeneracy in the sequence encoding a
variant amino acid, wherein said degeneracy is that of the multiple
codon sequences of the restricted codon set encoding the variant
amino acid. A composition comprising a polynucleotide or
polypeptide or library of the invention may be in the form of a kit
or an article of manufacture (optionally packaged with
instructions, buffers, etc.).
[0084] In one aspect, the invention provides a polynucleotide
encoding a polypeptide of the invention as described herein. In
another aspect, the invention provides a vector comprising a
sequence encoding a polypeptide of the invention. The vector can
be, for e.g., a replicable expression vector (for e.g., the
replicable expression vector can be M13, f1, fd, Pf3 phage or a
derivative thereof, or a lambdoid phage, such as lambda, 21, phi80,
phi81, 82, 424, 434, etc., or a derivative thereof). The vector can
comprise a promoter region linked to the sequence encoding a
polypeptide of the invention. The promoter can be any suitable for
expression of the polypeptide, for e.g., the lac Z promoter system,
the alkaline phosphatase pho A promoter (Ap), the bacteriophage
l.sub.PL promoter (a temperature sensitive promoter), the tac
promoter, the tryptophan promoter, and the bacteriophage T7
promoter. Thus, the invention also provides a vector comprising a
promoter selected from the group consisting of the foregoing
promoter systems.
[0085] Polypeptides of the invention can be displayed in any
suitable form in accordance with the need and desire of the
practitioner. For e.g., a polypeptide of the invention can be
displayed on a viral surface, for e.g., a phage or phagemid viral
particle. Accordingly, the invention provides viral particles
comprising a polypeptide of the invention and/or polynucleotide
encoding a polypeptide of the invention.
[0086] In one aspect, the invention provides a population
comprising a plurality of polypeptide or polynucleotide of the
invention, wherein each type of polypeptide or polynucleotide is a
polypeptide or polynucleotide of the invention as described
herein.
[0087] In some embodiments, polypeptides and/or polynucleotides are
provided as a library, for e.g., a library comprising a plurality
of at least about 1.times.10.sup.4, 1.times.10.sup.5,
1.times.10.sup.6, 1.times.10.sup.7, 1.times.10.sup.8 distinct
polypeptide and/or polynucleotide sequences of the invention. In
another aspect, the invention also provides a library comprising a
plurality of the viruses or viral particles of the invention, each
virus or virus particle displaying a polypeptide of the invention.
A library of the invention may comprise viruses or viral particles
displaying any number of distinct polypeptides (sequences), for
e.g., at least about 1.times.10.sup.4, 1.times.10.sup.5,
1.times.10.sup.6, 1.times.10.sup.7, 1.times.10.sup.8 distinct
polypeptides.
[0088] In another aspect, the invention provides host cells
comprising a polynucleotide or vector comprising a sequence
encoding a polypeptide of the invention.
[0089] In another aspect, the invention provides methods for
selecting for high affinity binders to specific target antigens
such as growth hormone, bovine growth hormone, insulin like growth
factors, human growth hormone including n-methionyl human growth
hormone, parathyroid hormone, thyroxine, insulin, proinsulin,
amylin, an apoptosis protein, relaxin, prorelaxin, glycoprotein
hormones such as follicle stimulating hormone (FSH), leutinizing
hormone (LH), hemapoietic growth factor, fibroblast growth factor,
prolactin, placental lactogen, tumor necrosis factors, hepatocyte
growth factor, hepatocyte growth factor receptor (c-met), mullerian
inhibiting substance, mouse gonadotropin-associated polypeptide,
inhibin, activin, vascular endothelial growth factors, integrin,
nerve growth factors such as NGF-beta, insulin-like growth factor-I
and II, erythropoietin, osteoinductive factors, interferons, colony
stimulating factors, interleukins, bone morphogenetic proteins,
LIF,SCF, neutravidin, maltose binding protein, erbin GST, insulin,
IgG, FLT-3 ligand and kit-ligand.
[0090] The methods of the invention provide populations of
polypeptides (for e.g., libraries of polypeptides (eg. antibody
variable domains)) with one or more diversified CDR regions. These
libraries are sorted (selected) and/or screened to identify high
affinity binders to a target antigen. In one aspect, polypeptide
binders from the library are selected for binding to target
antigens, and for affinity. The polypeptide binders selected using
one or more of these selection strategies, may then be screened for
affinity and/or for specificity (binding only to target antigen and
not to non-target antigens).
[0091] In one aspect, a method of the invention comprises
generating a plurality of polypeptides with one or more diversified
CDR regions, sorting the plurality of polypeptides for binders to a
target antigen by contacting the plurality of polypeptides with a
target antigen under conditions suitable for binding; separating
the binders to the target antigen from those that do not bind;
isolating the binders; and identifying the high affinity binders
(or any binders having a desired binding affinity). The affinity of
the binders that bind to the target antigen can be determined using
a variety of techniques known in the art, for e.g., competition
ELISA such as described herein. Optionally, the polypeptides can be
fused to a polypeptide tag, such as gD, poly his or FLAG, which can
be used to sort binders in combination with sorting for the target
antigen.
[0092] Another embodiment provides a method of isolating or
selecting for an antibody variable domain that binds to a target
antigen from a library of antibody variable domains, said method
comprising: a) contacting a population comprising a plurality of
polypeptides of the invention with an immobilized target antigen
under conditions suitable for binding to isolate target antigen
polypeptide binders; b) separating the polypeptide binders from
nonbinders, and eluting the binders from the target antigen; c)
optionally, repeating steps a-b at least once (in some embodiments,
at least twice).
[0093] In some embodiments, a method may further comprise: d)
incubating the polypeptide binders with a concentration of labelled
target antigen in the range of 0.1 nM to 1000 nM under conditions
suitable for binding to form a mixture; e) contacting the mixture
with an immobilized agent that binds to the label on the target
antigen; f) eluting the polypeptide binders from the labelled
target antigen; g) optionally, repeating steps d) to f) at least
once (in some embodiments, at least twice), using a successively
lower concentration of labelled target antigen each time.
Optionally, the method may comprise adding an excess of unlabelled
target antigen to the mixture and incubating for a period of time
sufficient to elute low affinity binders from the labelled target
antigen.
[0094] Another aspect of the invention provides a method of
isolating or selecting for high affinity binders (or binders having
a desired binding affinity) to a target antigen. In one embodiment,
said method comprises: a) contacting a population comprising a
plurality of polypeptides of the invention with a target antigen,
wherein the antigen is provided at a concentration in the range of
about 0.1 nM to 1000 nM to isolate polypeptide binders to the
target antigen; b) separating the polypeptide binders from the
target antigen; c) optionally, repeating steps a-b at least once
(in some embodiments, at least twice), each time with a
successively lower concentration of target antigen to isolate
polypeptide binders that bind to lowest concentration of target
antigen; d) selecting the polypeptide binder that binds to the
lowest concentration of the target antigen for high affinity (or
any desired affinity) by incubating the polypeptide binders with
several different dilutions of the target antigen and determining
the IC50 of the polypeptide binder; and e) identifying a
polypeptide binder that has a desired affinity for the target
antigen. Said affinity can be, for e.g., about 0.1 nM to 200 nM,
0.5 nM to 150 nM, 1 nM to 100 nM, 25 nM to 75 nM.
[0095] Another embodiment provides an assay for isolating or
selecting polypeptide binders comprising (a) contacting a
population comprising a plurality of polypeptides of the invention
with a labelled target antigen, wherein the labeled target antigen
is provided at a concentration in a range of 0.1 nM to 1000 nM,
under conditions suitable for binding to form a complex of a
polypeptide binder and the labelled target antigen; b) isolating
the complexes and separating the polypeptide binder from the
labelled target antigen; c) optionally, repeating steps a-b at
least once, each time using a lower concentration of target
antigen. Optionally, the method may further comprise contacting the
complex of polypeptide binder and target antigen with an excess of
unlabelled target antigen. In one embodiment, the steps of the
method are repeated twice and the concentration of target in a
first round of selection is in the range of about 100 nM to 250 nM,
and, in a second round of selection (if performed) is in the range
of about 25 nM to 100 nM, and in the third round of selection (if
performed) is in the range of about 0.1 nM to 25 nM.
[0096] The invention also includes a method of screening a
population comprising a plurality of polypeptides of the invention,
said method comprising: a) incubating a first sample of the
population of polypeptides with a target antigen under conditions
suitable for binding of the polypeptides to the target antigen; b)
subjecting a second sample of the population of polypeptides to a
similar incubation but in the absence of the target antigen; (c)
contacting each of the first and second sample with immobilized
target antigen under conditions suitable for binding of the
polypeptides to the immobilized target antigen; d) detecting amount
of polypeptides bound to immobilized target antigen for each
sample; e) determining affinity of a particular polypeptide for the
target antigen by calculating the ratio of the amount of the
particular polypeptide that is bound in the first sample over the
amount of the particular polypeptide that is bound in the second
sample.
[0097] The libraries generated as described herein may also be
screened for binding to a specific target and for lack of binding
to nontarget antigens. In one aspect, the invention provides a
method of screening for a polypeptide, such as an antibody variable
domain of the invention, that binds to a specific target antigen
from a library of antibody variable domains, said method
comprising: a) generating a population comprising a plurality of
polypeptides of the invention; b) contacting the population of
polypeptides with a target antigen under conditions suitable for
binding; c) separating a binder polypeptide in the library from
nonbinder polypeptides; d) identifying a target antigen-specific
binder polypeptide by determining whether the binder polypeptide
binds to a non-target antigen; and e) isolating a target
antigen-specific binder polypeptide. In some embodiments, step (e)
comprises eluting the binder polypeptide from the target antigen,
and amplifying a replicable expression vector encoding said binder
polypeptide.
[0098] Combinations of any of the sorting/selection methods
described above may be combined with the screening methods. For
example, in one embodiment, polypeptide binders are first selected
for binding to an immobilized target antigen. Polypeptide binders
that bind to the immobilized target antigen can then be screened
for binding to the target antigen and for lack of binding to
nontarget antigens. Polypeptide binders that bind specifically to
the target antigen can be amplified as necessary. These polypeptide
binders can be selected for higher affinity by contact with a
concentration of a labelled target antigen to form a complex,
wherein the concentration range of labelled target antigen is from
about 0.1 nM to about 1000 nM, and the complexes are isolated by
contact with an agent that binds to the label on the target
antigen. A polypeptide binder can then be eluted from the labeled
target antigen and optionally, the rounds of selection are
repeated, each time a lower concentration of labelled target
antigen is used. The binder polypeptides that can be isolated using
this selection method can then be screened for high affinity using
for example, the solution phase ELISA assay as described in Example
8 or other conventional methods known in the art. Populations of
polypeptides of the invention used in methods of the invention can
be provided in any form suitable for the selection/screening steps.
For e.g., the polypeptides can be in free soluble form, attached to
a matrix, or present at the surface of a viral particle such as
phage or phagemid particle. In some embodiments of methods of the
invention, the plurality of polypeptides are encoded by a plurality
of replicable vectors provided in the form of a library. In
selection/screening methods described herein, vectors encoding a
binder polypeptide may be further amplified to provide sufficient
quantities of the polypeptide for use in repetitions of the
selection/screening steps (which, as indicated above, are optional
in methods of the invention).
[0099] In one embodiment, the invention provides a method of
selecting for a polypeptide that binds to a target antigen
comprising:
[0100] a) generating a composition comprising a plurality of
polypeptides of the invention as described herein;
[0101] b) selecting a polypeptide binder that binds to a target
antigen from the composition;
[0102] c) isolating the polypeptide binder from the nonbinders;
[0103] d) identifying binders of the desired affinity from the
isolated polypeptide binders.
[0104] In another embodiment, the invention provides a method of
selecting for an antigen binding variable domain that binds to a
target antigen from a library of antibody variable domains
comprising:
[0105] a) contacting the library of antibody variable domains of
the invention (as described herein) with a target antigen;
[0106] b) separating binders from nonbinders, and eluting the
binders from the target antigen and incubating the binders in a
solution with decreasing amounts of the target antigen in a
concentration from about 0.1 nM to 1000 nM;
[0107] c) selecting the binders that can bind to the lowest
concentration of the target antigen and that have an affinity of
about 0.1 nM to 200 nM.
[0108] In some embodiments, the concentration of target antigen is
about 100 to 250 nM, or about 25 to 100 nM.
[0109] In one embodiment, the invention provides a method of
selecting for a polypeptide that binds to a target antigen from a
library of polypeptides comprising:
[0110] a) isolating polypeptide binders to a target antigen by
contacting a library comprising a plurality of polypeptides of the
invention (as described herein) with an immobilized target antigen
under conditions suitable for binding;
[0111] b) separating the polypeptide binders in the library from
nonbinders and eluting the binders from the target antigen to
obtain a subpopulation enriched for the binders; and
[0112] c) optionally, repeating steps a-b at least once (in some
embodiments at least twice), each repetition using the
subpopulation of binders obtained from the previous round of
selection.
[0113] In some embodiments, methods of the invention further
comprise the steps of:
[0114] d) incubating the subpopulation of polypeptide binders with
a concentration of labelled target antigen in the range of 0.1 nM
to 1000 nM under conditions suitable for binding to form a
mixture;
[0115] e) contacting the mixture with an immobilized agent that
binds to the label on the target antigen;
[0116] f) detecting the polypeptide binders bound to labelled
target antigens and eluting the polypeptide binders from the
labelled target antigen;
[0117] g) optionally, repeating steps d) to f) at least once (in
some embodiments, at least twice), each repetition using the
subpopulation of binders obtained from the previous round of
selection and using a lower concentration of labelled target
antigen than the previous round.
[0118] In some embodiments, these methods further comprise adding
an excess of unlabelled target antigen to the mixture and
incubating for a period of time sufficient to elute low affinity
binders from the labelled target antigen.
[0119] In another embodiment, the invention provides a method of
isolating high affinity binders to a target antigen comprising:
[0120] a) contacting a library comprising a plurality of
polypeptides of the invention (as described herein) with a target
antigen in a concentration of at least about 0.1 nM to 1000 nM to
isolate polypeptide binders to the target antigen;
[0121] b) separating the polypeptide binders from the target
antigen to obtain a subpopulation enriched for the polypeptide
binders; and
[0122] c) optionally, repeating steps a) and b) at least once (in
some embodiments, at least twice), each repetition using the
subpopulation of binders obtained from the previous round of
selection and using a decreased concentration of target antigen
than the previous round to isolate polypeptide binders that bind to
lowest concentration of target antigen.
[0123] In one aspect, the invention provides an assay for selecting
polypeptide binders from a library comprising a plurality of
polypeptides of the invention (as described herein) comprising:
[0124] a) contacting the library with a concentration of labelled
target antigen in a concentration range of 0.1 nM to 1000 nM, under
conditions suitable for binding to form a complex of a polypeptide
binder and the labelled target antigen;
[0125] b) isolating the complexes and separating the polypeptide
binders from the labelled target antigen to obtain a subpopulation
enriched for the binders;
[0126] c) optionally, repeating steps a-b at least once (in some
embodiments, at least twice), each time using the subpopulation of
binders obtained from the previous round of selection and using a
lower concentration of target antigen than the previous round.
[0127] In some embodiments, the method further comprises adding an
excess of unlabelled target antigen to the complex of the
polypeptide binder and target antigen. In some embodiments, the
steps set forth above are repeated at least once (in some
embodiments, at least twice) and the concentration of target in the
first round of selection is about 100 nM to 250 nM, and in the
second round of selection is about 25 nM to 100 nM, and in the
third round of selection is about 0.1 nM to 25 nM.
[0128] In another aspect, the invention provides a method of
screening a library comprising a plurality of polypeptides of the
invention, said method comprising:
[0129] a) incubating a first sample of the library with a
concentration of a target antigen under conditions suitable for
binding of the polypeptides to the target antigen;
[0130] b) incubating a second sample of the library without a
target antigen;
[0131] c) contacting each of the first and second sample with
immobilized target antigen under conditions suitable for binding of
the polypeptide to the immobilized target antigen;
[0132] d) detecting the polypeptide bound to immobilized target
antigen for each sample;
[0133] e) determining affinity of the polypeptide for the target
antigen by calculating the ratio of the amounts of bound
polypeptide from the first sample over the amount of bound
polypeptide from the second sample.
[0134] In one embodiment, the invention provides a method
comprising:
[0135] (a) constructing an expression vector comprising a
polynucleotide sequence which encodes a light chain variable
domain, a heavy chain variable domain, or both, of a source
antibody comprising at least one, two, three, four, five or all
CDRs of the source antibody selected from the group consisting of
CDR L1, L2, L3, H1, H2 and H3; and
[0136] b) mutating at least one, two, three, four, five or all CDRs
of the source antibody at at least one solvent accessible and
highly diverse amino acid position using a restricted codon
set.
[0137] In one embodiment, a polypeptide in the population used in
methods of the invention comprises variant CDRH3 comprising an
amino acid sequence:
[0138] (X1).sub.n-A-M
[0139] wherein X.sub.1 is an amino acid encoded by a restricted
codon set, and n=a suitable number that would retain the functional
activity of the CDR.
[0140] In one embodiment, a polypeptide in the population used in
methods of the invention comprises variant CDRH2 comprising an
amino acid sequence:
[0141] X1-I-X2-P-(X3)n-G-X4-T-X5-Y-A
[0142] wherein X1, X2, X3, X4 and/or X5 is an amino acid encoded by
a restricted codon set, and n=a suitable number that would retain
the functional activity of the CDR.
[0143] In another embodiment, a polypeptide in the population used
in methods of the invention comprises variant CDRH1 comprising an
amino acid sequence:
[0144] G-F-X1-I-(X2)n-I
[0145] wherein X1 and/or X2 is an amino acid encoded by a
restricted codon set, and n=a suitable number that would retain the
functional activity of the CDR.
[0146] In one embodiment, a polypeptide in the population used in
methods of the invention comprises variant CDRL3 comprising an
amino acid sequence:
[0147] Q-X1-(X2)n-P-X3-T-F
[0148] wherein X1 is Q or missing, and
[0149] X2 and/or X3 is an amino acid encoded by a restricted codon
set, and n=a suitable number that would retain the functional
activity of the CDR.
[0150] In yet another embodiment, a polypeptide in the population
used in methods of the invention comprises variant CDRL2 comprising
an amino acid sequence:
[0151] Y-X1-A-S-X2-L
[0152] wherein X1 and/or X2 is an amino acid encoded by a
restricted codon set.
[0153] In still another embodiment, a polypeptide in the population
used in methods of the invention comprises variant CDRL1 comprising
an amino acid sequence:
[0154] S-Q-(X1)n-V
[0155] wherein X1 is an amino acid encoded by a restricted codon
set, and n=a suitable number that would retain the functional
activity of the CDR.
[0156] Diagnostic and therapeutic uses for binder polypeptides of
the invention are contemplated. In one diagnostic application, the
invention provides a method for determining the presence of a
protein of interest comprising exposing a sample suspected of
containing the protein to a binder polypeptide of the invention and
determining binding of the binder polypeptide to the sample. For
this use, the invention provides a kit comprising the binder
polypeptide and instructions for using the binder polypeptide to
detect the protein.
[0157] The invention further provides: isolated nucleic acid
encoding the binder polypeptide; a vector comprising the nucleic
acid, optionally, operably linked to control sequences recognized
by a host cell transformed with the vector; a host cell transformed
with the vector; a process for producing the binder polypeptide
comprising culturing this host cell so that the nucleic acid is
expressed and, optionally, recovering the binder polypeptide from
the host cell culture (e.g. from the host cell culture medium).
[0158] The invention also provides a composition comprising a
binder polypeptide of the invention and a carrier (e.g., a
pharmaceutically acceptable carrier) or diluent. This composition
for therapeutic use is sterile and may be lyophilized. Also
contemplated is the use of a binder polypeptide of this invention
in the manufacture of a medicament for treating an indication
described herein. The composition can further comprise a second
thereapeutic agent such as a chemotherapeutic agent, a cytotoxic
agent or an anti-angiogenic agent.
[0159] The invention further provides a method for treating a
mammal, comprising administering an effective amount of a binder
polypeptide of the invention to the mammal. The mammal to be
treated in the method may be a nonhuman mammal, e.g. a primate
suitable for gathering preclinical data or a rodent (e.g., mouse or
rat or rabbit). The nonhuman mammal may be healthy (e.g. in
toxicology studies) or may be suffering from a disorder to be
treated with the binder polypeptide of interest. In one embodiment,
the mammal is suffering from or is at risk of developing abnormal
angiogenesis (e.g., pathological angiogenesis). In one specific
embodiment, the disorder is a cancer selected from the group
consisting of colorectal cancer, renal cell carcinoma, ovarian
cancer, lung cancer, non-small-cell lung cancer (NSCLC),
bronchoalveolar carcinoma and pancreatic cancer. In another
embodiment, the disorder is a disease caused by ocular
neovascularisation, e.g., diabetic blindness, retinopathies,
primarily diabetic retinopathy, age-induced macular degeneration
and rubeosis. In another embodiment, the mammal to be treated is
suffering from or is at risk of developing an edema (e.g., an edema
associated with brain tumors, an edema associated with stroke, or a
cerebral edema). In another embodiment, the mammal is suffering
from or at risk of developing a disorder or illness selected from
the group consisting of rheumatoid arthritis, inflammatory bowel
disease, refractory ascites, psoriasis, sarcoidosis, arterial
arteriosclerosis, sepsis, burns and pancreatitis. According to
another embodiment, the mammal is suferring from or is at risk of
developing a genitourinary illness selected from the group
consisting of polycystic ovarian disease (POD), endometriosis and
uterine fibroids. In one embodiment, the disorder is a disease
caused by dysregulation of cell survival (e.g., abnormal amount of
cell death), including but not limited to cancer, disorders of the
immune system, disorders of the nervous system and disorders of the
vascular system. The amount of binder polypeptide of the invention
that is administered will be a therapeutically effective amount to
treat the disorder. In dose escalation studies, a variety of doses
of the binder polypeptide may be administered to the mammal. In
another embodiment, a therapeutically effective amount of the
binder polypeptide is administered to a human patient to treat a
disorder in that patient. In one embodiment, binder polypeptides of
this invention useful for treating inflammatory or immune diseases
described herein (e.g., rheumatoid arthritis) are Fab or scFv
antibodies. Accordingly, such binder polypeptides can be used in
the manufacture of a medicament for treating an inflammatory or
immune disease. A mammal that is suffering from or is at risk for
developing a disorder or illness described herein can be treated by
administering, a second therapeutic agent, simultaneously,
sequentially or in combination with, a polypeptide (e.g., an
antibody) of this invention. It should be understood that other
therapeutic agents, in addition to the second therapeutic agent,
can be administered to the mammal or used in the manufacture of a
medicament for the desired indications.
[0160] These polypeptides can be used to understand the role of
host stromal cell collaboration in the growth of implanted non-host
tumors, such as in mouse models wherein human tumors have been
implanted. These polypeptides can be used in methods of identifying
human tumors that can escape therapeutic treatment by observing or
monitoring the growth of the tumor implanted into a rodent or
rabbit after treatment with a polypeptide of this invention. The
polypeptides of this invention can also be used to study and
evaluate combination therapies with a polypeptide of this invention
and other therapeutic agents. The polypeptides of this invention
can be used to study the role of a target molecule of interest in
other diseases by administering the polypeptides to an animal
suffering from the disease or a similar disease and determining
whether one or more symptoms of the disease are alleviated.
[0161] For the sake of clarity, in the description herein, unless
specifically or contextually indicated otherwise, all amino acid
numberings are according to Kabat et al. (see further elaboration
in "Definitions" below).
BRIEF DESCRIPTION OF THE FIGURES
[0162] FIG. 1 illustrates CDR positions diversified in a library
based on a binomial codon set that encodes only Y and S. CDR
positions shown are numbered according to the Kabat
nomenclature.
[0163] FIG. 2 shows mutagenic oligonucleotides used in the
construction of two illustrative libraries that are based on a
binomial codon set that encodes only Y and S. These libraries are
referred to as YS-A and YS-B. Equimolar DNA degeneracies are
represented in the codon sets (M=A/C). Codon sets are represented
in the IUB code.
[0164] FIG. 3 shows enrichment ratios for libraries YADS-A and
YADS-B following 5 rounds of selection against various target
antigens.
[0165] FIG. 4 shows results of sorting of YS-A and YS-B libraries.
Number of specific binders obtained is shown. Numbers are shown as
X/Y, with X representing the number of specific clones (i.e., those
binding to the target antigen at least 10 times higher (based on
ELISA signal read at 450 nm) than the binding of bovine serum
albumin (BSA), and Y representing the number of clones screened for
a given library, round and target antigen.
[0166] FIG. 5 shows sequences of binders obtained from selection of
library YS-A and YS-B. Note: Asterisks correspond to absence of an
amino acid normally found in the corresponding position in a
template sequence.
[0167] FIG. 6 shows an illustrative set of restricted codon sets.
The codon sets shown are tetranomial, i.e., they each encode only 4
amino acids.
[0168] FIG. 7 shows the number of specific binders assessed by
phage ELISAs. Numbers are shown as X/Y, with X being the number of
specific binders, and Y being the number of clones screened.
[0169] FIG. 8 shows the number of unique clones obtained from
individual restricted diversity libraries for each target
antigen.
[0170] FIG. 9 shows mutagenic oligonucleotides used in the
construction of libraries YADS-A and YADS-B, which are based on
tetranomial codon sets that encode only 4 amino acids. Equimolar
DNA degeneracies are represented in the codon sets (W=T/G, K=T/A,
M=A/C). WMT encodes S, Y, T and N. KMT encodes Y, A, D and S. Codon
sets are represented in the IUB code.
[0171] FIG. 10 shows the number of specific binders assessed by
phage ELISAs for libraries YADS-A and YADS-B. Numbers are shown as
X[Y, with X being the number of specific binders, and Y being the
number of clones screened.
[0172] FIG. 11 shows values of IC50 of clones YS1-AP, YS2-AP and
YS3-AP with respect to its corresponding human target antigen and
cyno target antigen, measured by competitive phage ELISA
[0173] FIG. 12 shows light chain CDR positions that were
diversified in a library based on a tetranomial codon set (YADS).
The library is referred to as the YADS-II library. CDR positions
are numbered according to the Kabat nomenclature.
[0174] FIG. 13 shows mutagenic oligonucleotides used in the
construction of library YADS-II. Equimolar DNA degeneracies are
represented in the codon sets (K=T/G, M=A/C). KMT encodes Y, A, D
and S. Codon sets are represented in the IUB code.
[0175] FIG. 14 shows the results of screening YADS-II hVEGF
selectants. The figure shows clone number, BSA binding (measured by
phage ELISA--numbers lower than 0.200 were considered to be below
background and are indicated in bold character), and percent
inhibition of binding by 100 nM of human VEGF (numbers showing
inhibition greater than 75% are indicated in bold character).
[0176] FIG. 15 depicts the sequences of 4D5 light chain and heavy
chain variable domain (SED ID NO:1 & 2, respectively).
[0177] FIG. 16 graphically depicts results of phage ELISA of 3
binders obtained from a YADS library on plates coated with
different target antigens, shown for increasing amounts of
phage.
[0178] FIG. 17 shows values of association (k.sub.a), dissociation
rate (k.sub.d) and affinity (K.sub.d) of 3 binders for human VEGF
and murine VEGF.
[0179] FIG. 18 shows the DNA sequence of Ptac promoter driven
cassette for display of Fab-zip (SEQ ID NO: 4). Two open reading
frames are indicated. The first open reading frame encodes a malE
secretion signal, humanized 4D5 light chain variable and constant
domain. The second open reading frame encodes a stII secretion
signal, humanized 4D5 heavy chain variable domain, humanized 4D5
heavy chain first constant domain (CH1), zipper sequence, and
C-terminal of p3 (cP3).
[0180] FIG. 19 illustrates a bicistronic vector allowing expression
of separate transcripts for display of F(ab).sub.2. A suitable
promoter drives expression of the first and second cistron. The
first cistron encodes a secretion signal sequence (malE or stII), a
light chain variable and constant domain and a gD tag. The second
cistron encodes a secretion signal, a sequence encoding heavy chain
variable domain and constant domain 1 (CH1) and dimerization domain
and at least a portion of the viral coat protein.
[0181] FIG. 20 shows a 3-D modeled structure of humanized 4D5
showing CDR residues that form contiguous patches. Contiguous
patches are formed by amino acid residues 28, 29, 30, 31 and 32 in
CDRL1; amino acids residues 50 and 53 of CDRL2; amino acid residues
91, 92, 93, 94 and 96 of CDRL3; amino acid residues 28, 30, 31, 32,
33 in CDRH1; and amino acid residues 50, 52, 53, 54, 56, and 58 in
CDRH2.
[0182] FIG. 21 shows the frequency of amino acids (identified by
single letter code) in human antibody light chain CDR sequences
from the Kabat database. The frequency of each amino acid at a
particular amino acid position is shown starting with the most
frequent amino acid at that position at the left and continuing on
to the right to the least frequent amino acid. The number below the
amino acid represents the number of naturally occurring sequences
in the Kabat database that have that amino acid in that
position.
[0183] FIG. 22 shows the frequency of amino acids (identified by
single letter code) in human antibody heavy chain CDR sequences
from the Kabat database. The frequency of each amino acid at a
particular amino acid position is shown starting with the most
frequent amino acid at that position at the left and continuing on
to the right to the least frequent amino acid. The number below the
amino acid represents the number of naturally occurring sequences
in the Kabat database that have that amino acid in that position.
Framework amino acid positions 71, 93 and 94 are also shown.
[0184] FIG. 23 shows values of association (k.sub.a), dissociation
rate (k.sub.d) and affinity (K.sub.d) of two anti-VEGF binders
obtained from YS libraries (as described in Example 2) for human
VEGF and murine VEGF.
[0185] FIGS. 24A-F show the DNA (SEQ ID NO: 5) and amino acid (SEQ
ID NOs: 6 & 7, for light and heavy chain, respectively)
sequence of vector pV-0350-4, which is a vector that comprises a
dimerization domain between heavy chain constant CH1 domain and p3
sequences.
MODES FOR CARRYING OUT THE INVENTION
[0186] The invention provides novel, unconventional, greatly
simplified and flexible methods for diversifying CDR sequences
(including antibody variable domain sequences), and libraries
comprising a multiplicity, generally a great multiplicity of
diversified CDRs (including antibody variable domain sequences).
Such libraries provide combinatorial libraries useful for, for
example, selecting and/or screening for synthetic antibody clones
with desirable activities such as binding affinities and avidities.
These libraries are useful for identifying immunoglobulin
polypeptide sequences that are capable of interacting with any of a
wide variety of target antigens. For example, libraries comprising
diversified immunoglobulin polypeptides of the invention expressed
as phage displays are particularly useful for, and provide a high
throughput, efficient and automatable systems of, selecting and/or
screening for antigen binding molecules of interest. The methods of
the invention are designed to provide high affinity binders to
target antigens with minimal changes to a source or template
molecule and provide for good production yields when the antibody
or antigens binding fragments are produced in cell culture.
[0187] Methods and compositions of the invention provide numerous
additional advantages. For example, relatively simple variant CDR
sequences can be generated, using codon sets encoding a restricted
number of amino acids (as opposed to the conventional approach of
using codon sets encoding the maximal number of amino acids), while
retaining sufficient diversity of unique target binding sequences.
The simplified nature (and generally relatively smaller size) of
sequence populations generated according to the invention permits
further diversification once a population, or sub-population
thereof, has been identified to possess the desired
characteristics.
[0188] The simplified nature of sequences of target antigen binders
obtained by methods of the invention leaves significantly greater
room for individualized further sequence modifications to achieve
the desired results. For example, such sequence modifications are
routinely performed in affinity maturation, humanization, etc. By
basing diversification on restricted codon sets that encode only a
limited number of amino acids, it would be possible to target
different epitopes using different restricted codon sets, thus
providing the practitioner greater control of the diversification
approach as compared with randomization based on a maximal number
of amino acids. An added advantage of using restricted codon sets
is that undesirable amino acids can be eliminated from the process,
for e.g., methionine or stop codons, thus improving the overall
quality and productivity of a library. Furthermore, in some
instances, it may be desirable to limit the conformational
diversity of potential binders. Methods and compositions of the
invention provide the flexibility for achieving this objective. For
e.g., the presence of certain amino acids, such as tyrosine, in a
sequence results in fewer rotational conformations. As shown herein
in one embodiment of the invention, variant CDRs, and binders
comprising such variant CDRs, can be generated that contain
sequences that have a predominance of tyrosine residues.
[0189] Definitions
[0190] Amino acids are represented herein as either a single letter
code or as the three letter code or both.
[0191] The term "affinity purification" means the purification of a
molecule based on a specific attraction or binding of the molecule
to a chemical or binding partner to form a combination or complex
which allows the molecule to be separated from impurities while
remaining bound or attracted to the partner moiety.
[0192] The term "antibody" is used in the broadest sense and
specifically covers single monoclonal antibodies (including agonist
and antagonist antibodies), antibody compositions with polyepitopic
specificity, affinity matured antibodies, humanized antibodies,
chimeric antibodies, as well as antigen binding fragments (e.g.,
Fab, F(ab').sub.2, scFv and Fv), so long as they exhibit the
desired biological activity. In one embodiment, the term "antibody"
also includes human antibodies.
[0193] As used herein, "antibody variable domain" refers to the
portions of the light and heavy chains of antibody molecules that
include amino acid sequences of Complementarity Determining Regions
(CDRs; ie., CDR1, CDR2, and CDR3), and Framework Regions (FRs).
V.sub.H refers to the variable domain of the heavy chain. V.sub.L
refers to the variable domain of the light chain. According to the
compositions and methods used in this invention, the amino acid
positions assigned to CDRs and FRs may be defined according to
Kabat (Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md., 1987 and 1991)). Amino acid
numbering of antibodies or antigen binding fragments is also
according to that of Kabat.
[0194] As used herein, the term "Complementarity Determining
Regions (CDRs; ie., CDR1, CDR2, and CDR3) refers to the amino acid
residues of an antibody variable domain the presence of which are
necessary for antigen binding. Each variable domain typically has
three CDR regions identified as CDR1, CDR2 and CDR3. Each
complementarity determining region may comprise amino acid residues
from a "complementarity determining region" as defined by Kabat
(i.e. about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the
light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102
(H3) in the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (i.e. about residues 26-32
(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain
and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain
variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917
(1987)). In some instances, a complementarity determining region
can include amino acids from both a CDR region defined according to
Kabat and a hypervariable loop. For example, the CDRH1 of the heavy
chain of antibody 4D5 includes amino acids 26 to 35.
[0195] "Framework regions" (hereinafter FR) are those variable
domain residues other than the CDR residues. Each variable domain
typically has four FRs identified as FR1, FR2, FR3 and FR4. If the
CDRs are defined according to Kabat, the light chain FR residues
are positioned at about residues 1-23 (LCFR1), 3549 (LCFR2), 57-88
(LCFR3), and 98-107 (LCFR4) and the heavy chain FR residues are
positioned about at residues 1-30 (HCFR1), 3649 (HCFR2), 66-94
(HCFR3), and 103-113 (HCFR4) in the heavy chain residues. If the
CDRs comprise amino acid residues from hypervariable loops, the
light chain FR residues are positioned about at residues 1-25
(LCFR1), 3349 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the
light chain and the heavy chain FR residues are positioned about at
residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113
(HCFR4) in the heavy chain residues. In some instances, when the
CDR comprises amino acids from both a CDR as defined by Kabat and
those of a hypervariable loop, the FR residues can be adjusted
accordingly. For example, when CDRH1 includes amino acids H26-H35,
the heavy chain FR1 residues are at positions 1-25 and the FR2
residues are at positions 36-49.
[0196] As used herein, "codon set" refers to a set of different
nucleotide triplet sequences used to encode desired variant amino
acids. A set of oligonucleotides can be synthesized, for example,
by solid phase synthesis, including sequences that represent all
possible combinations of nucleotide triplets provided by the codon
set and that will encode the desired group of amino acids. A
standard form of codon designation is that of the IUB code, which
is known in the art and described herein. A codon set typically is
represented by 3 capital letters in italics, eg. NNK, NNS, XYZ, DVK
and the like. Synthesis of oligonucleotides with selected
nucleotide "degeneracy" at certain positions is well known in that
art, for example the TRIM approach (Knappek et al.; J. Mol. Biol.
(1999), 296:57-86); Garrard & Henner, Gene (1993), 128:103).
Such sets of oligonucleotides having certain codon sets can be
synthesized using commercial nucleic acid synthesizers (available
from, for example, Applied Biosystems, Foster City, Calif.), or can
be obtained commercially (for example, from Life Technologies,
Rockville, Md.). Therefore, a set of oligonucleotides synthesized
having a particular codon set will typically include a plurality of
oligonucleotides with different sequences, the differences
established by the codon set within the overall sequence.
Oligonucleotides, as used according to the invention, have
sequences that allow for hybridization to a variable domain nucleic
acid template and also can, but does not necessarily, include
restriction enzyme sites useful for, for example, cloning
purposes.
[0197] The term "restricted codon set", and variations thereof, as
used herein refers to a codon set that encodes a much more limited
number of amino acids than the codon sets typically utilized in art
methods of generating sequence diversity. In one aspect of the
invention, restricted codon sets used for sequence diversification
encode from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, or only
2 amino acids. In some embodiments, a restricted codon set used for
sequence diversification encodes at least 2 but 10 or fewer, 8 or
fewer, 6 or fewer, 4 or fewer amino acids. In a typical example, a
tetranomial codon set is used. Examples of tetranomial codon sets
include those listed in FIG. 6 (RMC, RMG, RRC, RSA, MKC, YMT, RST,
KMT, SRC, MRT and WMT). In another typical example, a binomial
codon set is used. Examples of binomial codon sets include TMT,
KAT, YAC, WAC, TWC, TYT, YTC, WTC, KTT, YCT, MCG, SCG, MGC, SGT,
GRT, GKT and GYT. Determination of suitable restricted codons, and
the identification of specific amino acids encoded by a particular
restricted codon, is well known and would be evident to one skilled
in the art. Determination of suitable amino acid sets to be used
for diversification of a CDR sequence can be empirical and/or
guided by criteria known in the art (for e.g., inclusion of a
combination of hydrophobic and hydrophilic amino acid types,
etc.)
[0198] An "Fv" fragment is an antibody fragment which contains a
complete antigen recognition and binding site. This region consists
of a dimer of one heavy and one light chain variable domain in
tight association, which can be covalent in nature, for example in
scFv. It is in this configuration that the three CDRs of each
variable domain interact to define an antigen binding site on the
surface of the V.sub.H-V.sub.L dimer. Collectively, the six CDRs or
a subset thereof confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv
comprising only three CDRs specific for an antigen) has the ability
to recognize and bind antigen, although usually at a lower affinity
than the entire binding site.
[0199] The "Fab" fragment contains a variable and constant domain
of the light chain and a variable domain and the first constant
domain (CHI) of the heavy chain. F(ab').sub.2 antibody fragments
comprise a pair of Fab fragments which are generally covalently
linked near their carboxy termini by hinge cysteines between them.
Other chemical couplings of antibody fragments are also known in
the art.
[0200] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally the Fv polypeptide
further comprises a polypeptide linker between the V.sub.H and
V.sub.L domains, which enables the scFv to form the desired
structure for antigen binding. For a review of scFv, see Pluckthun
in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg
and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
[0201] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H and
V.sub.L). By using a linker that is too short to allow pairing
between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in,
for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0202] The expression "linear antibodies" refers to the antibodies
described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995).
Briefly, these antibodies comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0203] "Cell", "cell line", and "cell culture" are used
interchangeably herein and such designations include all progeny of
a cell or cell line. Thus, for example, terms like "transformants"
and "transformed cells" include the primary subject cell and
cultures derived therefrom without regard for the number of
transfers. It is also understood that all progeny may not be
precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same function
or biological activity as screened for in the originally
transformed cell are included. Where distinct designations are
intended, it will be clear from the context.
[0204] "Control sequences" when referring to expression means DNA
sequences necessary for the expression of an operably linked coding
sequence in a particular host organism. The control sequences that
are suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, a ribosome binding site, and
possibly, other as yet poorly understood sequences. Eukaryotic
cells are known to utilize promoters, polyadenylation signals, and
enhancers.
[0205] The term "coat protein" means a protein, at least a portion
of which is present on the surface of the virus particle. From a
functional perspective, a coat protein is any protein which
associates with a virus particle during the viral assembly process
in a host cell, and remains associated with the assembled virus
until it infects another host cell. The coat protein may be the
major coat protein or may be a minor coat protein. A "major" coat
protein is generally a coat protein which is present in the viral
coat at preferably at least about 5, more preferably at least about
7, even more preferably at least about 10 copies of the protein or
more. A major coat protein may be present in tens, hundreds or even
thousands of copies per virion. An example of a major coat protein
is the p8 protein of filamentous phage.
[0206] The "detection limit" for a chemical entity in a particular
assay is the minimum concentration of that entity which can be
detected above the background level for that assay. For example, in
the phage ELISA, the "detection limit" for a particular phage
displaying a particular antigen binding fragment is the phage
concentration at which the particular phage produces an ELISA
signal above that produced by a control phage not displaying the
antigen binding fragment.
[0207] A "fusion protein" and a "fusion polypeptide" refers to a
polypeptide having two portions covalently linked together, where
each of the portions is a polypeptide having a different property.
The property may be a biological property, such as activity in
vitro or in vivo. The property may also be a simple chemical or
physical property, such as binding to a target antigen, catalysis
of a reaction, etc. The two portions may be linked directly by a
single peptide bond or through a peptide linker containing one or
more amino acid residues. Generally, the two portions and the
linker will be in reading frame with each other. Preferably, the
two portions of the polypeptide are obtained from heterologous or
different polypeptides.
[0208] "Heterologous DNA" is any DNA that is introduced into a host
cell. The DNA may be derived from a variety of sources including
genomic DNA, cDNA, synthetic DNA and fusions or combinations of
these. The DNA may include DNA from the same cell or cell type as
the host or recipient cell or DNA from a different cell type, for
example, from a mammal or plant. The DNA may, optionally, include
marker or selection genes, for example, antibiotic resistance
genes, temperature resistance genes, etc.
[0209] As used herein, "highly diverse position" refers to a
position of an amino acid located in the variable regions of the
light and heavy chains that have a number of different amino acid
represented at the position when the amino acid sequences of known
and/or naturally occurring antibodies or antigen binding fragments
are compared. The highly diverse positions are typically in the CDR
regions. In one aspect, the ability to determine highly diverse
positions in known and/or naturally occurring antibodies is
facilitated by the data provided by Kabat, Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md., 1987 and 1991). An internet-based database located at
http:/immuno/bme/nwu/edu provides an extensive collection and
alignment of human light and heavy chain sequences and facilitates
determination of highly diverse positions in these sequences.
According to the invention, an amino acid position is highly
diverse if it has preferably from about 2 to about 11, preferably
from about 4 to about 9, and preferably from about 5 to about 7
different possible amino acid residue variations at that position.
In some embodiments, an amino acid position is highly diverse if it
has preferably at least about 2, preferably at least about 4,
preferably at least about 6, and preferably at least about 8
different possible amino acid residue variations at that
position.
[0210] As used herein, "library" refers to a plurality of antibody
or antibody fragment sequences (for example, polypeptides of the
invention), or the nucleic acids that encode these sequences, the
sequences being different in the combination of variant amino acids
that are introduced into these sequences according to the methods
of the invention.
[0211] "Ligation" is the process of forming phosphodiester bonds
between two nucleic acid fragments. For ligation of the two
fragments, the ends of the fragments must be compatible with each
other. In some cases, the ends will be directly compatible after
endonuclease digestion. However, it may be necessary first to
convert the staggered ends commonly produced after endonuclease
digestion to blunt ends to make them compatible for ligation. For
blunting the ends, the DNA is treated in a suitable buffer for at
least 15 minutes at 15.degree. C. with about 10 units of the Klenow
fragment of DNA polymerase I or T4 DNA polymerase in the presence
of the four deoxyribonucleotide triphosphates. The DNA is then
purified by phenol-chloroform extraction and ethanol precipitation
or by silica purification. The DNA fragments that are to be ligated
together are put in solution in about equimolar amounts. The
solution will also contain ATP, ligase buffer, and a ligase such as
T4 DNA ligase at about 10 units per 0.5 .mu.g of DNA. If the DNA is
to be ligated into a vector, the vector is first linearized by
digestion with the appropriate restriction endonuclease(s). The
linearized fragment is then treated with bacterial alkaline
phosphatase or calf intestinal phosphatase to prevent self-ligation
during the ligation step.
[0212] A "mutation" is a deletion, insertion, or substitution of a
nucleotide(s) relative to a reference nucleotide sequence, such as
a wild type sequence.
[0213] As used herein, "natural" or "naturally occurring"
antibodies, refers to antibodies identified from a nonsynthetic
source, for example, from a differentiated antigen-specific B cell
obtained ex vivo, or its corresponding hybridoma cell line, or from
antibodies obtained from the serum of an animal. These antibodies
can include antibodies generated in any type of immune response,
either natural or otherwise induced. Natural antibodies include the
amino acid sequences, and the nucleotide sequences that constitute
or encode these antibodies, for example, as identified in the Kabat
database. As used herein, natural antibodies are different than
"synthetic antibodies", synthetic antibodies referring to antibody
sequences that have been changed from a source or template
sequence, for example, by the replacement, deletion, or addition,
of an amino acid, or more than one amino acid, at a certain
position with a different amino acid, the different amino acid
providing an antibody sequence different from the source antibody
sequence.
[0214] "Operably linked" when referring to nucleic acids means that
the nucleic acids are placed in a functional relationship with
another nucleic acid sequence. For example, DNA for a presequence
or secretory leader is operably linked to DNA for a polypeptide if
it is expressed as a preprotein that participates in the secretion
of the polypeptide; a promotor or enhancer is operably linked to a
coding sequence if it affects the transcription of the sequence; or
a ribosome binding site is operably linked to a coding sequence if
it is positioned so as to facilitate translation. Generally,
"operably linked" means that the DNA sequences being linked are
contiguous and, in the case of a secretory leader, contingent and
in reading frame. However, enhancers do not have to be contiguous.
Linking is accomplished by ligation at convenient restriction
sites. If such sites do not exist, the synthetic oligonucleotide
adapters or linkers are used in accord with conventional
practice.
[0215] "Phage display" is a technique by which variant polypeptides
are displayed as fusion proteins to at least a portion of coat
protein on the surface of phage, e.g., filamentous phage,
particles. A utility of phage display lies in the fact that large
libraries of randomized protein variants can be rapidly and
efficiently sorted for those sequences that bind to a target
antigen with high affinity. Display of peptide and protein
libraries on phage has been used for screening millions of
polypeptides for ones with specific binding properties. Polyvalent
phage display methods have been used for displaying small random
peptides and small proteins through fusions to either gene III or
gene VIII of filamentous phage. Wells and Lowman, Curr. Opin.
Struct. Biol., 3:355-362 (1992), and references cited therein. In
monovalent phage display, a protein or peptide library is fused to
a gene III or a portion thereof, and expressed at low levels in the
presence of wild type gene III protein so that phage particles
display one copy or none of the fusion proteins. Avidity effects
are reduced relative to polyvalent phage so that sorting is on the
basis of intrinsic ligand affinity, and phagemid vectors are used,
which simplify DNA manipulations. Lowman and Wells, Methods: A
companion to Methods in Enzymology, 3:205-0216 (1991).
[0216] A "phagemid" is a plasmid vector having a bacterial origin
of replication, e.g., Co1E1, and a copy of an intergenic region of
a bacteriophage. The phagemid may be used on any known
bacteriophage, including filamentous bacteriophage and lambdoid
bacteriophage. The plasmid will also generally contain a selectable
marker for antibiotic resistance. Segments of DNA cloned into these
vectors can be propagated as plasmids. When cells harboring these
vectors are provided with all genes necessary for the production of
phage particles, the mode of replication of the plasmid changes to
rolling circle replication to generate copies of one strand of the
plasmid DNA and package phage particles. The phagemid may form
infectious or non-infectious phage particles. This term includes
phagemids which contain a phage coat protein gene or fragment
thereof linked to a heterologous polypeptide gene as a gene fusion
such that the heterologous polypeptide is displayed on the surface
of the phage particle.
[0217] The term "phage vector" means a double stranded replicative
form of a bacteriophage containing a heterologous gene and capable
of replication. The phage vector has a phage origin of replication
allowing phage replication and phage particle formation. The phage
is preferably a filamentous bacteriophage, such as an M13, f1, fd,
Pf3 phage or a derivative thereof, or a lambdoid phage, such as
lambda, 21, phi80, phi81, 82, 424, 434, etc., or a derivative
thereof.
[0218] "Oligonucleotides" are short-length, single- or
double-stranded polydeoxynucleotides that are chemically
synthesized by known methods (such as phosphotriester, phosphite,
or phosphoramidite chemistry, using solid-phase techniques such as
described in EP 266,032 published 4 May 1988, or via deoxynucloside
H-phosphonate intermediates as described by Froeshler et al., Nucl.
Acids, Res., 14:5399-5407 (1986)). Further methods include the
polymerase chain reaction defined below and other autoprimer
methods and oligonucleotide syntheses on solid supports. All of
these methods are described in Engels et al., Agnew. Chem. Int. Ed.
Engl., 28:716-734 (1989). These methods are used if the entire
nucleic acid sequence of the gene is known, or the sequence of the
nucleic acid complementary to the coding strand is available.
Alternatively, if the target amino acid sequence is known, one may
infer potential nucleic acid sequences using known and preferred
coding residues for each amino acid residue. The oligonucleotides
can be purified on polyacrylamide gels or molecular sizing columns
or by precipitation.
[0219] DNA is "purified" when the DNA is separated from non-nucleic
acid impurities. The impurities may be polar, non-polar, ionic,
etc.
[0220] A "source antibody", as used herein, refers to an antibody
or antigen binding fragment whose antigen binding sequence serves
as the template sequence upon which diversification according to
the criteria described herein is performed. An antigen binding
sequence generally includes an antibody variable region, preferably
at least one CDR, preferably including framework regions.
[0221] As used herein, "solvent accessible position" refers to a
position of an amino acid residue in the variable regions of the
heavy and light chains of a source antibody or antigen binding
fragment that is determined, based on structure, ensemble of
structures and/or modeled structure of the antibody or antigen
binding fragment, as potentially available for solvent access
and/or contact with a molecule, such as an antibody-specific
antigen. These positions are typically found in the CDRs and on the
exterior of the protein. The solvent accessible positions of an
antibody or antigen binding fragment, as defined herein, can be
determined using any of a number of algorithms known in the art.
Preferably, solvent accessible positions are determined using
coordinates from a 3-dimensional model of an antibody (or portion
thereof, for e.g., an antibody variable domain, or CDR segment(s)),
preferably using a computer program such as the InsightII program
(Accelrys, San Diego, Calif.). Solvent accessible positions can
also be determined using algorithms known in the art (e.g., Lee and
Richards, J. Mol. Biol. 55, 379 (1971) and Connolly, J. Appl.
Cryst. 16, 548 (1983)). Determination of solvent accessible
positions can be performed using software suitable for protein
modeling and 3-dimensional structural information obtained from an
antibody (or portion thereof). Software that can be utilized for
these purposes includes SYBYL Biopolymer Module software (Tripos
Associates). Generally and preferably, where an algorithm (program)
requires a user input size parameter, the "size" of a probe which
is used in the calculation is set at about 1.4 Angstrom or smaller
in radius. In addition, determination of solvent accessible regions
and area methods using software for personal computers has been
described by Pacios ((1994) "ARVOMOL/CONTOUR: molecular surface
areas and volumes on Personal Computers." Comput. Chem. 18(4):
377-386; and (1995). "Variations of Surface Areas and Volumes in
Distinct Molecular Surfaces of Biomolecules." J. Mol. Model. 1:
46-53.)
[0222] A "transcription regulatory element" will contain one or
more of the following components: an enhancer element, a promoter,
an operator sequence, a repressor gene, and a transcription
termination sequence. These components are well known in the art.
U.S. Pat. No. 5,667,780.
[0223] A "transformant" is a cell which has taken up and maintained
DNA as evidenced by the expression of a phenotype associated with
the DNA (e.g., antibiotic resistance conferred by a protein encoded
by the DNA).
[0224] "Transformation" means a process whereby a cell takes up DNA
and becomes a "transformant". The DNA uptake may be permanent or
transient.
[0225] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues.
[0226] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody which does not possess those alteration(s).
Preferred affinity matured antibodies will have nanomolar or even
picomolar affinities for the target antigen. Affinity matured
antibodies are produced by procedures known in the art. Marks et
al. Bio/Technology 10:779-783 (1992) describes affinity maturation
by V.sub.H and V.sub.L domain shuffling. Random mutagenesis of CDR
and/or framework residues is described by: Barbas et al. Proc Nat.
Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155
(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et
al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol.
Biol. 226:889-896 (1992).
[0227] A "blocking" antibody or an "antagonist" antibody is one
which inhibits or reduces biological activity of the antigen it
bind. Preferred blocking antibodies or antagonist antibodies
substantially or completely inhibit the biological activity of the
antigen.
[0228] An "agonist antibody", as used herein, is an antibody which
mimics at least one of the functional activities of a polypeptide
of interest.
[0229] To increase the half-life of the antibodies or polypeptide
containing the amino acid sequences of this invention, one can
attach a salvage receptor binding epitope to the antibody
(especially an antibody fragment), as described, e.g., in U.S. Pat.
No. 5,739,277. For example, a nucleic acid molecule encoding the
salvage receptor binding epitope can be linked in frame to a
nucleic acid encoding a polypeptide sequence of this invention so
that the fusion protein expressed by the engineered nucleic acid
molecule comprises the salvage receptor binding epitope and a
polypeptide sequence of this invention. As used herein, the term
"salvage receptor binding epitope" refers to an epitope of the Fc
region of an IgG molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
or IgG.sub.4) that is responsible for increasing the in vivo serum
half-life of the IgG molecule (e.g., Ghetie, V et al., (2000) Ann.
Rev. Immunol. 18:739-766, Table 1). Antibodies with substitutions
in an Fc region thereof and increased serum half-lives are also
described in WO00/42072 (Presta, L.), WO 02/060919; Shields, R. L.,
et al., (2001) JBC 276(9):6591-6604; Hinton, P. R., (2004) JBC
279(8):6213-6216). In another embodiment, the serum half-life can
also be increased, for example, by attaching other polypeptide
sequences. For example, antibodies of this invention or other
polypeptide containing the amino acid sequences of this invention
can be attached to serum albumin or a portion of serum albumin that
binds to the FcRn receptor or a serum albumin binding peptide so
that serum albumin binds to the antibody or polypeptide, e.g., such
polypeptide sequences are disclosed in WO01/45746. In one preferred
embodiment, the serum albumin peptide to be attached comprises an
amino acid sequence of DICLPRWGCLW. In another embodiment, the
half-life of a Fab according to this invention is increased by
these methods. See also, Dennis, M. S., et al., (2002) JBC
277(38):35035-35043 for serum albumin binding peptide
sequences.
[0230] A "disorder" is any condition that would benefit from
treatment with a substance/molecule or method of the invention.
This includes chronic and acute disorders or diseases including
those pathological conditions which predispose the mammal to the
disorder in question. Non-limiting examples of disorders to be
treated herein include malignant and benign tumors; non-leukemias
and lymphoid malignancies; neuronal, glial, astrocytal,
hypothalamic and other glandular, macrophagal, epithelial, stromal
and blastocoelic disorders; and inflammatory, immunologic and other
angiogenesis-related disorders.
[0231] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0232] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer",
"cancerous", "cell proliferative disorder", "proliferative
disorder" and "tumor" are not mutually exclusive as referred to
herein.
[0233] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth/proliferation. Examples of cancer
include but are not limited to, carcinoma, lymphoma, blastoma,
sarcoma, and leukemia. More particular examples of such cancers
include squamous cell cancer, small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of
the lung, cancer of the peritoneum, hepatocellular cancer,
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical
cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,
breast cancer, colon cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney cancer, liver
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma and various types of head and neck cancer.
[0234] Dysregulation of angiogenesis can lead to many disorders
that can be treated by compositions and methods of the invention.
These disorders include both non-neoplastic and neoplastic
conditions. Neoplastics include but are not limited those described
above. Non-neoplastic disorders include but are not limited to
undesired or aberrant hypertrophy, arthritis, rheumatoid arthritis
(RA), psoriasis, psoriatic plaques, sarcoidosis, atherosclerosis,
atherosclerotic plaques, diabetic and other proliferative
retinopathies including retinopathy of prematurity, retrolental
fibroplasia, neovascular glaucoma, age-related macular
degeneration, diabetic macular edema, corneal neovascularization,
corneal graft neovascularization, corneal graft rejection,
retinal/choroidal neovascularization, neovascularization of the
angle (rubeosis), ocular neovascular disease, vascular restenosis,
arteriovenous malformations (AVM), meningioma, hemangioma,
angiofibroma, thyroid hyperplasias (including Grave's disease),
corneal and other tissue transplantation, chronic inflammation,
lung inflammation, acute lung injury/ARDS, sepsis, primary
pulmonary hypertension, malignant pulmonary effusions, cerebral
edema (e.g., associated with acute stroke/closed head
injury/trauma), synovial inflammation, pannus formation in RA,
myositis ossificans, hypertropic bone formation, osteoarthritis
(OA), refractory ascites, polycystic ovarian disease,
endometriosis, 3rd spacing of fluid diseases (pancreatitis,
compartment syndrome, burns, bowel disease), uterine fibroids,
premature labor, chronic inflammation such as IBD (Crohn's disease
and ulcerative colitis), renal allograft rejection, inflammatory
bowel disease, nephrotic syndrome, undesired or aberrant tissue
mass growth (non-cancer), hemophilic joints, hypertrophic scars,
inhibition of hair growth, Osler-Weber syndrome, pyogenic granuloma
retrolental fibroplasias, scleroderma, trachoma, vascular
adhesions, synovitis, dermatitis, preeclampsia, ascites,
pericardial effusion (such as that associated with pericarditis),
and pleural effusion.
[0235] As used herein, "treatment" refers to clinical intervention
in an attempt to alter the natural course of the individual or cell
being treated, and can be performed either for prophylaxis or
during the course of clinical pathology. Desirable effects of
treatment include preventing occurrence or recurrence of disease,
alleviation of symptoms, diminishment of any direct or indirect
pathological consequences of the disease, preventing metastasis,
decreasing the rate of disease progression, amelioration or
palliation of the disease state, and remission or improved
prognosis. In some embodiments, antibodies of the invention are
used to delay development of a disease or disorder.
[0236] An "effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result.
[0237] A "therapeutically effective amount" of a substance/molecule
of the invention, agonist or antagonist may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of the substance/molecule, agonist or
antagonist to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the substance/molecule, agonist or
antagonist are outweighed by the therapeutically beneficial
effects. A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically but not necessarily,
since a prophylactic dose is used in subjects prior to or at an
earlier stage of disease, the prophylactically effective amount
will be less than the therapeutically effective amount.
[0238] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents e.g.
methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described below.
A tumoricidal agent causes destruction of tumor cells.
[0239] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem Intl. Ed.
Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antiobiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, carminomycin, carzinophilin, chromomycinis,
dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM. doxorubicin (including
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2"-trichlorotriethylam- ine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., TAXOL.RTM. paclitaxel
(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.TM.
Cremophor-free, albumin-engineered nanoparticle formulation of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. doxetaxel (Rhne-Poulenc Rorer, Antony, France);
chloranbucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.);
oxaliplatin; leucovovin; vinorelbine (NAVELBINE.RTM.); novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such
as retinoic acid; capecitabine (XELODA.RTM.); pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone, and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
[0240] Also included in this definition are anti-hormonal agents
that act to regulate, reduce, block, or inhibit the effects of
hormones that can promote the growth of cancer, and are often in
the form of systemic, or whole-body treatment. They may be hormones
themselves. Examples include anti-estrogens and selective estrogen
receptor modulators (SERMs), including, for example, tamoxifen
(including NOLVADEX.RTM. tamoxifen), EVISTA.RTM. raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. toremifene; anti-progesterones;
estrogen receptor down-regulators (ERDs); agents that function to
suppress or shut down the ovaries, for example, leutinizing
hormone-releasing hormone (LHRH) agonists such as LUPRON.RTM. and
ELIGARD.RTM. leuprolide acetate, goserelin acetate, buserelin
acetate and tripterelin; other anti-androgens such as flutamide,
nilutamide and bicalutamide; and aromatase inhibitors that inhibit
the enzyme aromatase, which regulates estrogen production in the
adrenal glands, such as, for example, 4(5)-imidazoles,
aminoglutethimide, MEGASE.RTM. megestrol acetate, AROMASIN.RTM.
exemestane, formestanie, fadrozole, RIVISOR.RTM. vorozole,
FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole. In addition,
such definition of chemotherapeutic agents includes bisphosphonates
such as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.),
DIDROCAL.RTM. etidronate, NE-58095, ZOMETA.RTM. zoledronic
acid/zoledronate, FOSAMAX.RTM. alendronate, AREDIA.RTM.
pamidronate, SKELID.RTM. tiludronate, or ACTONEL.RTM. risedronate;
as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine
analog); antisense oligonucleotides, particularly those that
inhibit expression of genes in signaling pathways implicated in
abherant cell proliferation, such as, for example, PKC-alpha, Raf,
H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such
as THERATOPE.RTM. vaccine and gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase
small-molecule inhibitor also known as GW572016); and
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
[0241] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell whose
growth is dependent upon activity of a target molecule of interest
either in vitro or in vivo. Thus, the growth inhibitory agent may
be one which significantly reduces the percentage of target
molecule-dependent cells in S phase. Examples of growth inhibitory
agents include agents that block cell cycle progression (at a place
other than S phase), such as agents that induce G1 arrest and
M-phase arrest. Classical M-phase blockers include the vincas
(vincristine and vinblastine), taxanes, and topoisomerase II
inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. (W B Saunders:
Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and
docetaxel) are anticancer drugs both derived from the yew tree.
Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer), derived from the
European yew, is a semisynthetic analogue of paclitaxel
(TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and docetaxel
promote the assembly of microtubules from tubulin dimers and
stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0242] "Doxorubicin" is an anthracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyx-
o-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,1-trihydroxy-8-(hydroxyacetyl-
)-1-methoxy-5,12-naphthacenedione.
[0243] A "variant" or "mutant" of a starting or reference
polypeptide (for e.g., a source antibody or its variable
domain(s)/CDR(s)), such as a fusion protein (polypeptide) or a
heterologous polypeptide (heterologous to a phage), is a
polypeptide that 1) has an amino acid sequence different from that
of the starting or reference polypeptide and 2) was derived from
the starting or reference polypeptide through either natural or
artificial (manmade) mutagenesis. Such variants include, for
example, deletions from, and/or insertions into and/or
substitutions of, residues within the amino acid sequence of the
polypeptide of interest. For example, a fusion polypeptide of the
invention generated using an oligonucleotide comprising a
restricted codon set that encodes a sequence with a variant amino
acid (with respect to the amino acid found at the corresponding
position in a source antibody/antigen binding fragment) would be a
variant polypeptide with respect to a source antibody and/or
antigen binding fragment and/or CDR. Thus, a variant CDR refers to
a CDR comprising a variant sequence with respect to a starting or
reference polypeptide sequence (such as that of a source antibody
and/or antigen binding fragment and/or CDR). A variant amino acid,
in this context, refers to an amino acid different from the amino
acid at the corresponding position in a starting or reference
polypeptide sequence (such as that of a source antibody and/or
antigen binding fragment and/or CDR). Any combination of deletion,
insertion, and substitution may be made to arrive at the final
variant or mutant construct, provided that the final construct
possesses the desired functional characteristics. In some of the
examples described herein, binder sequences contain point mutations
such as deletions or additions. For example, a VEGF clone from the
YADS library exhibits a missing Q in CDRL3 which was not the result
of vector construction. In another example, the Q in position 89 of
the 4D5 CDRL3 was intentionally deleted in the construction of the
vector. The amino acid changes also may alter post-translational
processes of the polypeptide, such as changing the number or
position of glycosylation sites. Methods for generating amino acid
sequence variants of polypeptides are described in U.S. Pat. No.
5,534,615, expressly incorporated herein by reference.
[0244] A "wild type" or "reference" sequence or the sequence of a
"wild type" or "reference" protein/polypeptide, such as a coat
protein, or a CDR or variable domain of a source antibody, maybe
the reference sequence from which variant polypeptides are derived
through the introduction of mutations. In general, the "wild type"
sequence for a given protein is the sequence that is most common in
nature. Similarly, a "wild type" gene sequence is the sequence for
that gene which is most commonly found in nature. Mutations may be
introduced into a "wild type" gene (and thus the protein it
encodes) either through natural processes or through man induced
means. The products of such processes are "variant" or "mutant"
forms of the original "wild type" protein or gene.
[0245] A "plurality" of a substance, such as a polypeptide or
polynucleotide of the invention, as used herein, generally refers
to a collection of two or more types or kinds of the substance.
There are two or more types or kinds of a substance if two or more
of the substances differ from each other with respect to a
particular characteristic, such as the variant amino acid found at
a particular amino acid position. For example, there is a plurality
of polypeptides of the invention if there are two or more
polypeptides of the invention that are substantially the same,
preferably identical, in sequence except for the sequence of a
variant CDR or except for the variant amino acid at a particular
solvent accessible and highly diverse amino acid position. In
another example, there is a plurality of polynucleotides of the
invention if there are two or more polynucleotides of the invention
that are substantially the same, preferably identical, in sequence
except for the sequence that encodes a variant CDR or except for
the sequence that encodes a variant amino acid for a particular
solvent accessible and highly diverse amino acid position.
[0246] The invention provides methods for generating and isolating
novel target antigen binding polypeptides, such as antibodies or
antigen binding fragments, that can have a high affinity for a
selected antigen. A plurality of different binder polypeptides are
prepared by mutating (diversifying) one or more selected amino acid
positions in a source antibody light chain variable domain and/or
heavy chain variable domain with restricted codon sets to generate
a library of with variant amino acids in at least one CDR sequence,
wherein the number of types of variant amino acids is kept to a
minimum (i.e., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, or
only 2, but generally at least 2). The amino acid positions include
those that are solvent accessible, for example as determined by
analyzing the structure of a source antibody, and/or that are
highly diverse among known and/or natural occurring immunoglobulin
polypeptides. A further advantage afforded by the limited nature of
diversification of the invention is that additional amino acid
positions other than those that are highly diverse and/or solvent
accessible can also be diversified in accordance with the need or
desire of the practitioner; examples of these embodiments are
described herein.
[0247] The amino acid positions that are solvent accessible and
highly diverse are preferably those in the CDR regions of the
antibody variable domains selected from the group consisting of
CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, CDRH3, and mixtures thereof.
Amino acid positions are each mutated using a restricted codon set
encoding a limited number of amino acids, the choice of amino acids
generally being independent of the commonly occurring amino acids
at each position. In some embodiments, when a solvent accessible
and highly diverse position in a CDR region is to be mutated, a
codon set is selected that encodes preferably from 2 to 10,
preferably from 2 to 8, preferably from 2 to 6, preferably from 2
to 4, preferably only 2 amino acids. In some embodiments, when a
solvent accessible and highly diverse position in a CDR region is
to be mutated, a codon set is selected that encodes preferably from
2 to 10, from 3 to 9, from 4 to 8, from 5 to 7 amino acids. In some
embodiments, a codon set encodes at least 2, but 10 or fewer, 8 or
fewer, 6 or fewer, 4 or fewer amino acids. CDR sequences can also
be diversified by varying the length, for e.g., for CDRH3, variant
CDRH3 regions can be generated that have different lengths and/or
are randomized at selected positions using restricted codon
sets.
[0248] The diversity of the library of the polypeptides comprising
variant CDRs is designed using codon sets that encode only a
limited number of amino acids, such that a minimum but sufficient
amount of sequence diversity is introduced into a CDR. The number
of positions mutated in the CDR is minimized and the variant amino
acids at each position are designed to include a limited number of
amino acids, independent of the amino acids that deemed to be
commonly occurring at that position in known and/or naturally
occurring CDRs. Preferably, a single antibody, including at least
one CDR, is used as the source antibody. It is surprising that a
library of antibody variable domains having diversity in sequences
and size can be generated using a single source antibody as a
template and targeting diversity to particular positions using an
unconventionally limited number of amino acid substitutions.
[0249] Design of Diversity of Antibody Variable Domains
[0250] In one aspect of the invention, high quality libraries of
antibody variable domains are generated. The libraries have
restricted diversity of different sequences of CDR sequences, for
e.g., diversity of the antibody variable domains. The libraries
include high affinity binding antibody variable domains for one or
more antigens, including, for example, neutravidin, an apoptosis
protein (AP), maltose binding protein 2 (MBP2), erbin-GST, insulin,
murine and human VEGF. The diversity in the library is designed by
selecting amino acid positions that are solvent accessible and
highly diverse in a single source antibody and mutating those
positions in at least one CDR using restricted codon sets. The
restricted codon set preferably encodes preferably fewer 10, 8, 6,
4 amino acids, or encodes only 2 amino acids.
[0251] One source antibody is humanized antibody 4D5, but the
methods for diversification can be applied to other source
antibodies whose sequence is known. A source antibody can be a
naturally occurring antibody, synthetic antibody, recombinant
antibody, humanized antibody, germ line derived antibody, chimeric
antibody, affinity matured antibody, or antigen binding fragment
thereof. The antibodies can be obtained from a variety of mammalian
species including humans, mice and rats. In some embodiments, a
source antibody is an antibody that is obtained after one or more
initial affinity screening rounds, but prior to an affinity
maturation step(s). A source antibody may be selected or modified
to provide for high yield and stability when produced in cell
culture.
[0252] Antibody 4D5 is a humanized antibody specific for a
cancer-associated antigen known as Her-2 (erbB2). The antibody
includes variable domains having consensus framework regions; a few
positions were reverted to mouse sequence during the process of
increasing affinity of the humanized antibody. The sequence and
crystal structure of humanized antibody 4D5 have been described in
U.S. Pat. No. 6,054,297, Carter et al, PNAS 89:4285 (1992), the
crystal structure is shown in J. Mol. Biol. 229:969 (1993) and
online at www/ncbi/nih/gov/structure/mmdb(MMDB#s-990-9- 92).
[0253] A criterion for generating diversity in antibody variable
domains is to mutate residues at positions that are solvent
accessible (as defined above). These positions are typically found
in the CDRs, and are typically on the exterior of the protein.
Preferably, solvent accessible positions are determined using
coordinates from a 3-dimensional model of an antibody, using a
computer program such as the InsightII program (Accelrys, San
Diego, Calif.). Solvent accessible positions can also be determined
using algorithms known in the art (e.g., Lee and Richards, J. Mol.
Biol. 55, 379 (1971) and Connolly, J. Appl. Cryst. 16, 548 (1983)).
Determination of solvent accessible positions can be performed
using software suitable for protein modeling and 3-dimensional
structural information obtained from an antibody. Software that can
be utilized for these purposes includes SYBYL Biopolymer Module
software (Tripos Associates). Generally and preferably, where an
algorithm (program) requires a user input size parameter, the
"size" of a probe which is used in the calculation is set at about
1.4 Angstrom or smaller in radius. In addition, determination of
solvent accessible regions and area methods using software for
personal computers has been described by Pacios ((1994)
"ARVOMOL/CONTOUR: molecular surface areas and volumes on Personal
Computers", Comput. Chem. 18(4): 377-386; and "Variations of
Surface Areas and Volumes in Distinct Molecular Surfaces of
Biomolecules." J. Mol. Model. (1995), 1: 46-53).
[0254] In some instances, selection of solvent accessible residues
is further refined by choosing solvent accessible residues that
collectively form a minimum contiguous patch, for example when the
reference polypeptide or source antibody is in its 3-D folded
structure. For example, as shown in FIG. 21, a compact (minimum)
contiguous patch is formed by residues selected for
CDRH1/H2/H3/L1/L2/L3 of humanized 4D5. A compact (minimum)
contiguous patch may comprise only a subset (for example, 2-5 CDRs)
of the full range of CDRs, for example, CDRH1/H2/H3/L3. Solvent
accessible residues that do not contribute to formation of such a
patch may optionally be excluded from diversification. Refinement
of selection by this criterion permits the practitioner to
minimize, as desired, the number of residues to be diversified. For
example, residue 28 in H1 can optionally be excluded in
diversification since it is on the edge of the patch. However, this
selection criterion can also be used, where desired, to choose
residues to be diversified that may not necessarily be deemed
solvent accessible. For example, a residue that is not deemed
solvent accessible, but forms a contiguous patch in the 3-D folded
structure with other residues that are deemed solvent accessible
may be selected for diversification. An example of this is
CDRL1-29. Selection of such residues would be evident to one
skilled in the art, and its appropriateness can also be determined
empirically and according to the needs and desires of the skilled
practitioner.
[0255] The solvent accessible positions identified from the crystal
structure of humanized antibody 4D5 for each CDR are as follows
(residue position according to Kabat):
[0256] CDRL1: 28, 30, 31, 32
[0257] CDRL2: 50, 53
[0258] CDRL3: 91, 92, 93, 94, 96
[0259] CDRH1: 28, 30, 31, 32, 33
[0260] CDRH2: 50, 52, 52A, 53, 54, 55, 56, 57, 58.
[0261] In addition, in some embodiments, residue 29 of CDRL1 may
also be selected based on its inclusion in a contiguous patch
comprising other solvent accessible residues. All or a subset of
the solvent accessible positions as set forth above may be
diversified in methods and compositions of the invention. For e.g.,
in some embodiments, in CDRH2, only positions 50, 52, 53, 54, 56
and 58 are diversified.
[0262] Another criterion for selecting positions to be mutated are
those positions which show variability in amino acid sequence when
the sequences of known and/or natural-antibodies are compared. A
highly diverse position refers to a position of an amino acid
located in the variable regions of the light or heavy chains that
have a number of different amino acids represented at the position
when the amino acid sequences of known and/or natural
antibodies/antigen binding fragments are compared. The highly
diverse positions are preferably in the CDR regions. The positions
of CDRH3 are all considered highly diverse. According to the
invention, amino acid residues are highly diverse if they have
preferably from about 2 to about 11 (although the numbers can range
as described herein) different possible amino acid residue
variations at that position.
[0263] In one aspect, identification of highly diverse positions in
known and/or naturally occurring antibodies is facilitated by the
data provided by Kabat, Sequences of Proteins of Immunological
Interest (National Institutes of Health, Bethesda, Md., 1987 and
1991). An internet-based database located at
http/immuno/bme/nwu/edu provides an extensive collection and
alignment of human light and heavy chain sequences and facilitates
determination of highly diverse positions in these sequences. The
diversity at the solvent accessible positions of humanized antibody
4D5 in known and/or naturally occurring light and heavy chains is
shown in FIGS. 22 and 23.
[0264] In one aspect of the invention, the highly diverse and
solvent accessible residues in at least one, two, three, four, five
or all of CDRs selected from the group consisting of CDRL1, CDRL2,
CDRL3, CDRH1, CDRH2, CDRH3, and mixtures thereof are mutated (i.e.,
randomized using restricted codon sets as described herein). For
example, a population of polypeptides may be generated by
diversifying at least one solvent accessible and/or highly diverse
residue in CDRL3 and CDRH3 using restricted codons. Accordingly,
the invention provides for a large number of novel antibody
sequences formed by replacing at least one solvent accessible and
highly diverse position of at least one CDR of the source antibody
variable domain with variant amino acids encoded by a restricted
codon. For example, a variant CDR or antibody variable domain can
comprise a variant amino acid in one or more amino acid positions
28, 30, 31, 32 and/or 33 of CDRH1; and/or in one or more amino acid
positions 50, 52, 53, 54, 56 and/or 58 of CDRH2; and/or in one or
more amino acid positions 28, 29, 30 and/or 31 of CDRL1; and/or in
one or more amino acid positions 50 and/or 53 in CDRL2; and/or in
one or more amino acid positions 91, 92, 93, 94 and/or 96 in CDRL3.
The variant amino acids at these positions are encoded by
restricted codon sets, as described herein.
[0265] As discussed above, the variant amino acids are encoded by
restricted codon sets. A codon set is a set of different nucleotide
triplet sequences which can be used to form a set of
oligonucleotides used to encode the desired group of amino acids. A
set of oligonucleotides can be synthesized, for example, by solid
phase synthesis, containing sequences that represent all possible
combinations of nucleotide triplets provided by the codon set and
that will encode the desired group of amino acids. Synthesis of
oligonucleotides with selected nucleotide "degeneracy" at certain
positions is well known in that art. Such sets of nucleotides
having certain codon sets can be synthesized using commercial
nucleic acid synthesizers (available from, for example, Applied
Biosystems, Foster City, Calif.), or can be obtained commercially
(for example, from Life Technologies, Rockville, Md.). Therefore, a
set of oligonucleotides synthesized having a particular codon set
will typically include a plurality of oligonucleotides with
different sequences, the differences established by the codon set
within the overall sequence. Oligonucleotides, as used according to
the invention, have sequences that allow for hybridization to a
variable domain nucleic acid template and also can include
restriction enzyme sites for cloning purposes.
[0266] In one aspect, the restricted repertoire of amino acids
intended to occupy one or more of the solvent accessible and highly
diverse positions in CDRs of humanized antibody 4D5 are determined
(based on the desire of the practitioner, which can be based on any
of a number of criteria, including specific amino acids desired for
particular positions, specific amino acid(s) desired to be absent
from a particular position, size of library desired, characteristic
of antigen binders sought, etc.).
[0267] Heavy chain CDR3s (CDRH3s) in known antibodies have diverse
sequences, structural conformations, and lengths. CDRH3s are often
found in the middle of the antigen binding pocket and often
participate in antigen contact. The design of CDRH3 is thus
preferably developed separately from that of the other CDRs because
it can be difficult to predict the structural conformation of CDRH3
and the amino acid diversity in this region is especially diverse
in known antibodies. In accordance with the present invention,
CDRH3 is designed to generate diversity at specific positions
within CDRH3, for e.g., positions 95, 96, 97, 98, 99, 100 and 100a
(for e.g., according to Kabat numbering in 4D5). In some
embodiments, diversity is also generated by varying CDRH3 length
using restricted codon sets. Length diversity can be of any range
determined empirically to be suitable for generating a population
of polypeptides containing substantial proportions of antigen
binding proteins. For example, polypeptides comprising variant
CDRH3 can be generated having the sequence (X1).sub.n-A-M, wherein
X1 is an amino acid encoded by a restricted codon set, and n is of
various lengths, for example, n=3-20, 5-20, 7-20, 5-18 or 7-18.
Other examples of possible n values are 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 and 20. Illustrative embodiments of
oligonucleotides that can be utilized to provide for variety in
CDRH3 sequence length include those shown in FIG. 2 and FIG. 9.
[0268] It is contemplated that the sequence diversity of libraries
created by introduction of variant amino acids in a particular CDR,
for e.g., CDRH3, can be increased by combining the variant CDR with
other CDRs comprising variations in other regions of the antibody,
specifically in other CDRs of either the light or heavy chain
variable sequences. It is contemplated that the nucleic acid
sequences that encode members of this set can be further
diversified by introduction of other variant amino acids in the
CDRs of either the light or heavy chain sequences, via codon sets.
Thus, for example, in one embodiment, CDRH3 sequences from fusion
polypeptides that bind a target antigen can be combined with
diversified CDRL3, CDRH1, or CDRH2 sequences, or any combination of
diversified CDRs.
[0269] It should be noted that in some instances framework residues
may be varied relative to the sequence of a source antibody or
antigen binding fragment, for example, to reflect a consensus
sequence or to improve stability or display. For example, framework
residues 49, 93, 94 or 71 in the heavy chain may be varied. Heavy
chain framework residue 93 may be serine or alanine (which is the
human consensus sequence amino acid at that position.) Heavy chain
framework residue 94 may be changed to reflect framework consensus
sequence from threonine to arginine or lysine. Another example of a
framework residue that may be altered is heavy chain framework
residue 71, which is R in about 1970 polypeptides, V in about 627
polypeptides and A in about 527 polypeptides, as found in the Kabat
database. Heavy chain framework residue 49 may be alanine or
glycine. In addition, optionally, the 3 N-terminal amino acids of
the heavy chain variable domain can be removed. In the light chain,
optionally, the arginine at amino acid position 66 can be changed
to glycine.
[0270] In one aspect, the invention provides vector constructs for
generating fusion polypeptides that bind with significant affinity
to potential ligands. These constructs comprise a dimerizable
domain that when present in a fusion polypeptide provides for
increased tendency for heavy chains to dimerize to form dimers of
Fab or Fab' antibody fragments/portions. These dimerization domains
may include, eg. a heavy chain hinge sequence (for e.g., a sequence
comprising TCPPCPAPELLG (SEQ ID NO: 120) that may be present in the
fusion polypeptide. Dimerization domains in fusion phage
polypeptides bring two sets of fusion polypeptides (LC/HC-phage
protein/fragment (such as pIII)) together, thus allowing formation
of suitable linkages (such as interheavy chain disulfide bridges)
between the two sets of fusion polypeptide. Vector constructs
containing such dimerization domains can be used to achieve
divalent display of antibody variable domains, for example the
diversified fusion proteins described herein, on phage. Preferably,
the intrinsic affinity of each monomeric antibody fragment (fusion
polypeptide) is not significantly altered by fusion to the
dimerization domain. Preferably, dimerization results in divalent
phage display which provides increased avidity of phage binding,
with significant decrease in off-rate, which can be determined by
methods known in the art and as described herein. Dimerization
domain-containing vectors of the invention may or may not also
include an amber stop codon after the dimerization domain.
[0271] Dimerization can be varied to achieve different display
characteristics. Dimerization domains can comprise a sequence
comprising a cysteine residue, a hinge region from a full-length
antibody, a dimerization sequence such as leucine zipper sequence
or GCN4 zipper sequence or mixtures thereof. Dimerization sequences
are known in the art, and include, for example, the GCN4 zipper
sequence (GRMKQLEDKVEELLSKNYHLENEVARLKKLVGERG) (SEQ ID NO: 3). The
dimerization domain is preferably located at the C-terminal end of
the heavy chain variable or constant domain sequence and/or between
the heavy chain variable or constant domain sequence and any viral
coat protein component sequence. An amber stop codon may also be
present at or after the C-terminal end of the dimerization domain.
In one embodiment, wherein an amber stop codon is present, the
dimerization domain encodes at least one cysteine and a dimerizing
sequence such as leucine zipper. In another embodiment, wherein no
amber stop codon is present, the dimerization domain may comprise a
single cysteine residue.
[0272] The polypeptides of the invention can also be fused to other
types of polypeptides in order to provide for display of the
variant polypeptides or to provide for purification, screening or
sorting, and detection of the polypeptide. For embodiment involving
phage display, the polypeptides of the invention are fused to all
or a portion of a viral coat protein. Examples of viral coat
protein include protein PIII, major coat protein, pVIII, Soc, Hoc,
gpD, pVI and variants thereof. In addition, the variant
polypeptides generated according to the methods of the invention
can optionally be fused to a polypeptide marker or tag such as
FLAG, polyhistidine, gD, c-myc, B-galactosidase and the like.
[0273] Methods of Generating Libraries of Randomized Variable
Domains
[0274] Methods of substituting an amino acid of choice into a
template nucleic acid are well established in the art, some of
which are described herein. For example, libraries can be created
by targeting solvent accessible and/or highly diverse positions in
at least one CDR region for amino acid substitution with variant
amino acids using the Kunkel method. See, for e.g., Kunkel et al.,
Methods Enzymol. (1987), 154:367-382. Generation of randomized
sequences is also described below in the Examples.
[0275] The sequence of oligonucleotides includes one or more of the
designed restricted codon sets for different lengths of CDRH3 or
for the solvent accessible and highly diverse positions in a CDR. A
codon set is a set of different nucleotide triplet sequences used
to encode desired variant amino acids. Codon sets can be
represented using symbols to designate particular nucleotides or
equimolar mixtures of nucleotides as shown below according to the
IUB code. Typically, a codon set is represented by three capital
letters eg. KMT, TMT and the like.
[0276] IUB Codes
[0277] G Guanine
[0278] A Adenine
[0279] T Thymine
[0280] C Cytosine
[0281] R (A or G)
[0282] Y (C or T)
[0283] M (A or C)
[0284] K (G or T)
[0285] S (C or G)
[0286] W (A or T)
[0287] H (A or C or T)
[0288] B (C or G or T)
[0289] V (A or C or G)
[0290] D (A or G or T)
[0291] N (A or C or G or T)
[0292] For example, in the codon set TMT, T is the nucleotide
thymine; and M can be A or C. This codon set can present multiple
codons and can encode only a limited number of amino acids, namely
tyrosine and serine.
[0293] Oligonucleotide or primer sets can be synthesized using
standard methods. A set of oligonucleotides can be synthesized, for
example, by solid phase synthesis, containing sequences that
represent all possible combinations of nucleotide triplets provided
by the restricted codon set and that will encode the desired
restricted group of amino acids. Synthesis of oligonucleotides with
selected nucleotide "degeneracy" at certain positions is well known
in that art. Such sets of oligonucleotides having certain codon
sets can be synthesized using commercial nucleic acid synthesizers
(available from, for example, Applied Biosystems, Foster City,
Calif.), or can be obtained commercially (for example, from Life
Technologies, Rockville, Md.). Therefore, a set of oligonucleotides
synthesized having a particular codon set will typically include a
plurality of oligonucleotides with different sequences, the
differences established by the codon set within the overall
sequence. Oligonucleotides, as used according to the invention,
have sequences that allow for hybridization to a CDR (for e.g., as
contained within a variable domain) nucleic acid template and also
can include restriction enzyme sites for cloning purposes.
[0294] In one method, nucleic acid sequences encoding variant amino
acids can be created by oligonucleotide-mediated mutagenesis of a
nucleic acid sequence encoding a source or template polypeptide
such as the antibody variable domain of 4D5. This technique is well
known in the art as described by Zoller et al. Nucleic Acids Res.
10:6487-6504(1987). Briefly, nucleic acid sequences encoding
variant amino acids are created by hybridizing an oligonucleotide
set encoding the desired restricted codon sets to a DNA template,
where the template is the single-stranded form of the plasmid
containing a variable region nucleic acid template sequence. After
hybridization, DNA polymerase is used to synthesize an entire
second complementary strand of the template that will thus
incorporate the oligonucleotide primer, and will contain the
restricted codon sets as provided by the oligonucleotide set.
Nucleic acids encoding other source or template molecules are known
or can be readily determined.
[0295] Generally, oligonucleotides of at least 25 nucleotides in
length are used. An optimal oligonucleotide will have at least 12
to 15 nucleotides that are completely complementary to the template
on either side of the nucleotide(s) coding for the mutation(s).
This ensures that the oligonucleotide will hybridize properly to
the single-stranded DNA template molecule. The oligonucleotides are
readily synthesized using techniques known in the art such as that
described by Crea et al., Proc. Natl. Acad. Sci. USA, 75:5765
(1978).
[0296] The DNA template is generated by those vectors that are
either derived from bacteriophage M13 vectors (the commercially
available M13mp18 and M13mp19 vectors are suitable), or those
vectors that contain a single-stranded phage origin of replication
as described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus,
the DNA that is to be mutated can be inserted into one of these
vectors in order to generate single-stranded template. Production
of the single-stranded template is described in sections 4.21-4.41
of Sambrook et al., above.
[0297] To alter the native DNA sequence, the oligonucleotide is
hybridized to the single stranded template under suitable
hybridization conditions. A DNA polymerizing enzyme, usually T7 DNA
polymerase or the Klenow fragment of DNA polymerase I, is then
added to synthesize the complementary strand of the template using
the oligonucleotide as a primer for synthesis. A heteroduplex
molecule is thus formed such that one strand of DNA encodes the
mutated form of gene 1, and the other strand (the original
template) encodes the native, unaltered sequence of gene 1. This
heteroduplex molecule is then transformed into a suitable host
cell, usually a prokaryote such as E. coli JM101. After growing the
cells, they are plated onto agarose plates and screened using the
oligonucleotide primer radiolabelled with a 32-Phosphate to
identify the bacterial colonies that contain the mutated DNA.
[0298] The method described immediately above may be modified such
that a homoduplex molecule is created wherein both strands of the
plasmid contain the mutation(s). The modifications are as follows:
The single stranded oligonucleotide is annealed to the
single-stranded template as described above. A mixture of three
deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine
(dGTP), and deoxyribothymidine (dTT), is combined with a modified
thiodeoxyribocytosine called dCTP-(aS) (which can be obtained from
Amersham). This mixture is added to the template-oligonucleotide
complex. Upon addition of DNA polymerase to this mixture, a strand
of DNA identical to the template except for the mutated bases is
generated. In addition, this new strand of DNA will contain
dCTP-(aS) instead of dCTP, which serves to protect it from
restriction endonuclease digestion. After the template strand of
the double-stranded heteroduplex is nicked with an appropriate
restriction enzyme, the template strand can be digested with ExoIII
nuclease or another appropriate nuclease past the region that
contains the site(s) to be mutagenized. The reaction is then
stopped to leave a molecule that is only partially single-stranded.
A complete double-stranded DNA homoduplex is then formed using DNA
polymerase in the presence of all four deoxyribonucleotide
triphosphates, ATP, and DNA ligase. This homoduplex molecule can
then be transformed into a suitable host cell.
[0299] As indicated previously the sequence of the oligonucleotide
set is of sufficient length to hybridize to the template nucleic
acid and may also, but does not necessarily, contain restriction
sites. The DNA template can be generated by those vectors that are
either derived from bacteriophage M13 vectors or vectors that
contain a single-stranded phage origin of replication as described
by Viera et al. ((1987) Meth. Enzymol., 153:3). Thus, the DNA that
is to be mutated must be inserted into one of these vectors in
order to generate single-stranded template. Production of the
single-stranded template is described in sections 4.21-4.41 of
Sambrook et al., supra.
[0300] According to another method, a library can be generated by
providing upstream and downstream oligonucleotide sets, each set
having a plurality of oligonucleotides with different sequences,
the different sequences established by the codon sets provided
within the sequence of the oligonucleotides. The upstream and
downstream oligonucleotide sets, along with a variable domain
template nucleic acid sequence, can be used in a polymerase chain
reaction to generate a "library" of PCR products. The PCR products
can be referred to as "nucleic acid cassettes", as they can be
fused with other related or unrelated nucleic acid sequences, for
example, viral coat protein components and dimerization domains,
using established molecular biology techniques.
[0301] The sequence of the PCR primers includes one or more of the
designed codon sets for the solvent accessible and highly diverse
positions in a CDR region. As described above, a codon set is a set
of different nucleotide triplet sequences used to encode desired
variant amino acids.
[0302] Oligonucleotide sets can be used in a polymerase chain
reaction using a variable region nucleic acid template sequence as
the template to create nucleic acid cassettes. The variable region
nucleic acid template sequence can be any portion of the light or
heavy immunoglobulin chains containing the target nucleic acid
sequences (ie., nucleic acid sequences encoding amino acids
targeted for substitution). The variable region nucleic acid
template sequence is a portion of a double stranded DNA molecule
having a first nucleic acid strand and complementary second nucleic
acid strand. The variable region nucleic acid template sequence
contains at least a portion of a variable domain and has at least
one CDR. In some cases, the variable region nucleic acid template
sequence contains more than one CDR. An upstream portion and a
downstream portion of the variable region nucleic acid template
sequence can be targeted for hybridization with members of an
upstream oligonucleotide set and a downstream oligonucleotide
set.
[0303] A first oligonucleotide of the upstream primer set can
hybridize to the first nucleic acid strand and a second
oligonucleotide of the downstream primer set can hybridize to the
second nucleic acid strand. The oligonucleotide primers can include
one or more codon sets and be designed to hybridize to a portion of
the variable region nucleic acid template sequence. Use of these
oligonucleotides can introduce two or more codon sets into the PCR
product (ie., the nucleic acid cassette) following PCR. The
oligonucleotide primer that hybridizes to regions of the nucleic
acid sequence encoding the antibody variable domain includes
portions that encode CDR residues that are targeted for amino acid
substitution.
[0304] The upstream and downstream oligonucleotide sets can also be
synthesized to include restriction sites within the oligonucleotide
sequence. These restriction sites can facilitate the insertion of
the nucleic acid cassettes [ie., PCR reaction products] into an
expression vector having additional antibody sequences. Preferably,
the restriction sites are designed to facilitate the cloning of the
nucleic acid cassettes without introducing extraneous nucleic acid
sequences or removing original CDR or framework nucleic acid
sequences.
[0305] Nucleic acid cassettes can be cloned into any suitable
vector for expression of a portion or the entire light or heavy
chain sequence containing the targeted amino acid substitutions
generated. According to methods detailed in the invention, the
nucleic acid cassette is cloned into a vector allowing production
of a portion or the entire light or heavy chain sequence fused to
all or a portion of a viral coat protein (ie., creating a fusion
protein) and displayed on the surface of a particle or cell. While
several types of vectors are available and may be used to practice
this invention, phagemid vectors are the preferred vectors for use
herein, as they may be constructed with relative ease, and can be
readily amplified. Phagemid vectors generally contain a variety of
components including promoters, signal sequences, phenotypic
selection genes, origin of replication sites, and other necessary
components as are known to those of ordinary skill in the art.
[0306] In another embodiment, wherein a particular variant amino
acid combination is to be expressed, the nucleic acid cassette
contains a sequence that is able to encode all or a portion of the
heavy or light chain variable domain, and is able to encode the
variant amino acid combinations. For production of antibodies
containing these variant amino acids or combinations of variant
amino acids, as in a library, the nucleic acid cassettes can be
inserted into an expression vector containing additional antibody
sequence, for example all or portions of the variable or constant
domains of the light and heavy chain variable regions. These
additional antibody sequences can also be fused to other nucleic
acid sequences, such as sequences which encode viral coat protein
components and therefore allow production of a fusion protein.
[0307] Vectors
[0308] One aspect of the invention includes a replicable expression
vector comprising a nucleic acid sequence encoding a gene fusion,
wherein the gene fusion encodes a fusion protein comprising a
CDR-containing polypeptide (such as an antibody variable domain),
or an antibody variable domain and a constant domain, fused to all
or a portion of a viral coat protein. Also included is a library of
diverse replicable expression vectors comprising a plurality of
gene fusions encoding a plurality of different fusion proteins
including a plurality of the fusion polypeptides generated with
diverse sequences as described above. The vectors can include a
variety of components and may be constructed to allow for movement
of antibody variable domain between different vectors and/or to
provide for display of the fusion proteins in different
formats.
[0309] Examples of vectors include phage vectors and phagemid
vectors (which is illustrated extensively herein, and described in
greater detail above). A phage vector generally has a phage origin
of replication allowing phage replication and phage particle
formation. The phage is generally a filamentous bacteriophage, such
as an M13, f1, fd, Pf3 phage or a derivative thereof, or a lambdoid
phage, such as lambda, 21, phi80, phi81, 82, 424, 434, etc., or a
derivative thereof.
[0310] Examples of viral coat proteins include infectivity protein
PIII (sometimes also designated p3), major coat protein PVIII, Soc
(T4), Hoc (T4), gpD (of bacteriophage lambda), minor bacteriophage
coat protein 6 (pVI) (filamentous phage; J Immunol Methods. 1999
Dec. 10;231 (1-2):39-51), variants of the M13 bacteriophage major
coat protein (P8) (Protein Sci 2000 April; 9(4):647-54). The fusion
protein can be displayed on the surface of a phage and suitable
phage systems include M13KO7 helper phage, M13R408, M13-VCS, and
Phi X 174, pJuFo phage system (J. Virol. 2001 August;
75(15):7107-13.v), hyperphage (Nat Biotechnol. 2001 January;
19(1):75-8). The preferred helper phage is M13KO7, and the
preferred coat protein is the M 13 Phage gene III coat protein. The
preferred host is E. coli, and protease deficient strains of E.
coli. Vectors, such as the fth 1 vector (Nucleic Acids Res. 2001
May 15;29(10):E50-0) can be useful for the expression of the fusion
protein.
[0311] The expression vector also can have a secretory signal
sequence fused to the DNA encoding a CDR-containing fusion
polypeptide (for e.g., each subunit of an antibody, or fragment
thereof). This sequence is typically located immediately 5' to the
gene encoding the fusion protein, and will thus be transcribed at
the amino terminus of the fusion protein. However, in certain
cases, the signal sequence has been demonstrated to be located at
positions other than 5' to the gene encoding the protein to be
secreted. This sequence targets the protein to which it is attached
across the inner membrane of the bacterial cell. The DNA encoding
the signal sequence may be obtained as a restriction endonuclease
fragment from any gene encoding a protein that has a signal
sequence. Suitable prokaryotic signal sequences may be obtained
from genes encoding, for example, LamB or OmpF (Wong et al., Gene,
68:1931 (1983), MalE, PhoA and other genes. In one embodiment, a
prokaryotic signal sequence for practicing this invention is the E.
coli heat-stable enterotoxin II (STII) signal sequence as described
by Chang et al., Gene 55:189 (1987), and/or malE.
[0312] As indicated above, a vector also typically includes a
promoter to drive expression of the fusion polypeptide. Promoters
most commonly used in prokaryotic vectors include the lac Z
promoter system, the alkaline phosphatase pho A promoter (Ap), the
bacteriophage l.sub.PL promoter (a temperature sensitive promoter),
the tac promoter (a hybrid trp-lac promoter that is regulated by
the lac repressor), the tryptophan promoter, and the bacteriophage
T7 promoter. For general descriptions of promoters, see section 17
of Sambrook et al. supra. While these are the most commonly used
promoters, other suitable microbial promoters may be used as
well.
[0313] The vector can also include other nucleic acid sequences,
for example, sequences encoding gD tags, c-Myc epitopes,
poly-histidine tags, fluorescence proteins (eg., GFP), or
beta-galactosidase protein which can be useful for detection or
purification of the fusion protein expressed on the surface of the
phage or cell. Nucleic acid sequences encoding, for example, a gD
tag, also provide for positive or negative selection of cells or
virus expressing the fusion protein. In some embodiments, the gD
tag is preferably fused to an antibody variable domain which is not
fused to the viral coat protein component. Nucleic acid sequences
encoding, for example, a polyhistidine tag, are useful for
identifying fusion proteins including antibody variable domains
that bind to a specific antigen using immunohistochemistry. Tags
useful for detection of antigen binding can be fused to either an
antibody variable domain not fused to a viral coat protein
component or an antibody variable domain fused to a viral coat
protein component.
[0314] Another useful component of the vectors used to practice
this invention is phenotypic selection genes. Typical phenotypic
selection genes are those encoding proteins that confer antibiotic
resistance upon the host cell. By way of illustration, the
ampicillin resistance gene (ampr), and the tetracycline resistance
gene (tetr) are readily employed for this purpose.
[0315] The vector can also include nucleic acid sequences
containing unique restriction sites and suppressible stop codons.
The unique restriction sites are useful for moving antibody
variable domains between different vectors and expression systems,
especially useful for production of full-length antibodies or
antigen binding fragments in cell cultures. The suppressible stop
codons are useful to control the level of expression of the fusion
protein and to facilitate purification of soluble antibody
fragments. For example, an amber stop codon can be read as Gln in a
supE host to enable phage display, while in a non-supE host it is
read as a stop codon to produce soluble antibody fragments without
fusion to phage coat proteins. These synthetic sequences can be
fused to one or more antibody variable domains in the vector.
[0316] It is sometimes beneficial to use vector systems that allow
the nucleic acid encoding an antibody sequence of interest, for
example a CDR having variant amino acids, to be easily removed from
the vector system and placed into another vector system. For
example, appropriate restriction sites can be engineered in a
vector system to facilitate the removal of the nucleic acid
sequence encoding an antibody or antibody variable domain having
variant amino acids. The restriction sequences are usually chosen
to be unique in the vectors to facilitate efficient excision and
ligation into new vectors. Antibodies or antibody variable domains
can then be expressed from vectors without extraneous fusion
sequences, such as viral coat proteins or other sequence tags.
[0317] Between nucleic acid encoding antibody variable or constant
domain (gene 1) and the viral coat protein component (gene 2), DNA
encoding a termination or stop codon may be inserted, such
termination codons including UAG (amber), UAA (ocher) and UGA
(opel). (Microbiology, Davis et al., Harper & Row, New York,
1980, pp. 237, 24547 and 374). The termination or stop codon
expressed in a wild type host cell results in the synthesis of the
gene 1 protein product without the gene 2 protein attached.
However, growth in a suppressor host cell results in the synthesis
of detectable quantities of fused protein. Such suppressor host
cells are well known and described, such as E. coli suppressor
strain (Bullock et al., BioTechniques 5:376-379 (1987)). Any
acceptable method may be used to place such a termination codon
into the mRNA encoding the fusion polypeptide.
[0318] The suppressible codon may be inserted between the first
gene encoding an antibody variable or constant domain, and a second
gene encoding at least a portion of a phage coat protein.
Alternatively, the suppressible termination codon may be inserted
adjacent to the fusion site by replacing the last amino acid
triplet in the antibody variable domain or the first amino acid in
the phage coat protein. The suppressible termination codon may be
located at or after the C-terminal end of a dimerization domain.
When the plasmid containing the suppressible codon is grown in a
suppressor host cell, it results in the detectable production of a
fusion polypeptide containing the polypeptide and the coat protein.
When the plasmid is grown in a non-suppressor host cell, the
antibody variable domain is synthesized substantially without
fusion to the phage coat protein due to termination at the inserted
suppressible triplet UAG, UAA, or UGA. In the non-suppressor cell
the antibody variable domain is synthesized and secreted from the
host cell due to the absence of the fused phage coat protein which
otherwise anchored it to the host membrane.
[0319] In some embodiments, the CDR being diversified (randomized)
may have a stop codon engineered in the template sequence (referred
to herein as a "stop template"). This feature provides for
detection and selection of successfully diversified sequences based
on successful repair of the stop codon(s) in the template sequence
due to incorporation of the oligonucleotide(s) comprising the
sequence(s) for the variant amino acids of interest. This feature
is further illustrated in the Examples below.
[0320] The light and/or heavy chain antibody variable or constant
domains can also be fused to an additional peptide sequence, the
additional peptide sequence providing for the interaction of one or
more fusion polypeptides on the surface of the viral particle or
cell. These peptide sequences are herein referred to as
"dimerization domains". Dimerization domains may comprise at least
one or more of a dimerization sequence, or at least one sequence
comprising a cysteine residue or both. Suitable dimerization
sequences include those of proteins having amphipathic alpha
helices in which hydrophobic residues are regularly spaced and
allow the formation of a dimer by interaction of the hydrophobic
residues of each protein; such proteins and portions of proteins
include, for example, leucine zipper regions. Dimerization domains
can also comprise one or more cysteine residues (e.g. as provided
by inclusion of an antibody hinge sequence within the dimerization
domain). The cysteine residues can provide for dimerization by
formation of one or more disulfide bonds. In one embodiment,
wherein a stop codon is present after the dimerization domain, the
dimerization domain comprises at least one cysteine residue. The
dimerization domains are preferably located between the antibody
variable or constant domain and the viral coat protein
component.
[0321] In some cases the vector encodes a single antibody-phage
polypeptide in a single chain form containing, for example, both
the heavy and light chain variable regions fused to a coat protein.
In these cases the vector is considered to be "monocistronic",
expressing one transcript under the control of a certain promoter.
For example, a vector may utilize a promoter (such as the alkaline
phosphatase (AP) or Tac promoter) to drive expression of a
monocistronic sequence encoding VL and VH domains, with a linker
peptide between the VL and VH domains. This cistronic sequence may
be connected at the 5' end to a signal sequence (such as an E. coli
malE or heat-stable enterotoxin II (STII) signal sequence) and at
its 3' end to all or a portion of a viral coat protein (such as the
bacteriophage pIII protein). The fusion polypeptide encoded by a
vector of this embodiment is referred to herein as "ScFv-pIII". In
some embodiments, a vector may further comprise a sequence encoding
a dimerization domain (such as a leucine zipper) at its 3' end,
between the second variable domain sequence (for e.g., V.sub.H) and
the viral coat protein sequence. Fusion polypeptides comprising the
dimerization domain are capable of dimerizing to form a complex of
two scFv polypeptides (referred to herein as "(ScFv)2-pIII)").
[0322] In other cases, the variable regions of the heavy and light
chains can be expressed as separate polypeptides, the vector thus
being "bicistronic", allowing the expression of separate
transcripts. In these vectors, a suitable promoter, such as the
Ptac or PhoA promoter, is used to drive expression of a bicistronic
message. A first cistron encoding, for example, a light chain
variable and constant domain, may be connected at the 5' end to a
signal sequence, such as E. coli malE or heat-stable enterotoxin II
(STII) signal sequence, and at the 3' end to a nucleic acid
sequence encoding a tag sequence, such as gD tag. A second cistron,
encoding, for example, a heavy chain variable domain and constant
domain CH1, is connected at its 5' end to a signal sequence, such
as E. coli malE or heat-stable enterotoxin II (STII) signal
sequence, and at the 3' end to all or a portion of a viral coat
protein.
[0323] In one embodiment of a vector which provides a bicistronic
message and for display of F(ab').sub.2-pIII, a suitable promoter,
such as Ptac or PhoA (AP) promoter, drives expression of a first
cistron encoding a light chain variable and constant domain
operably linked at 5' end to a signal sequence such as the E. coli
malE or heat stable enteroxtoxin II (STII) signal sequence, and at
the 3' end to a nucleic acid sequence encoding a tag sequence such
as gD tag. The second cistron encodes, for example, a heavy chain
variable and constant domain operatively linked at 5' end to a
signal sequence such as E. coli malE or heat stable enterotoxin II
(STI) signal sequence, and at 3' end has a dimerization domain
comprising IgG hinge sequence and a leucine zipper sequence
followed by at least a portion of viral coat protein.
[0324] Display of Fusion Polypeptides
[0325] Fusion polypeptides of a CDR-containing polypeptide (for
e.g., an antibody variable domain) can be displayed on the surface
of a cell, virus, or phagemid particle in a variety of formats.
These formats include single chain Fv fragment (scFv), F(ab)
fragment and multivalent forms of these fragments. For example,
multivalent forms include a dimer of ScFv, Fab, or F(ab'), herein
referred to as (ScFv).sub.2, F(ab).sub.2 and F(ab').sub.2,
respectively. The multivalent forms of display are advantageous in
some contexts in part because they have more than one antigen
binding site which generally results in the identification of lower
affinity clones and also allows for more efficient sorting of rare
clones during the selection process.
[0326] Methods for displaying fusion polypeptides comprising
antibody fragments, on the surface of bacteriophage, are well known
in the art, for example as described in patent publication number
WO 92/01047 and herein. Other patent publications WO 92/20791; WO
93/06213; WO 93/11236 and WO 93/19172, describe related methods and
are all herein incorporated by reference. Other publications have
shown the identification of antibodies with artificially rearranged
V gene repertoires against a variety of antigens displayed on the
surface of phage (for example, H. R. Hoogenboom & G. Winter J.
Mol. Biol. 227 381-388 1992; and as disclosed in WO 93/06213 and WO
93/11236).
[0327] When a vector is constructed for display in a scFv format,
it includes nucleic acid sequences encoding an antibody variable
light chain domain and an antibody variable heavy chain variable
domain. Typically, the nucleic acid sequence encoding an antibody
variable heavy chain domain is fused to a viral coat protein
component. One or both of the antibody variable domains can have
variant amino acids in at least one CDR region. The nucleic acid
sequence encoding the antibody variable light chain is connected to
the antibody variable heavy chain domain by a nucleic acid sequence
encoding a peptide linker. The peptide linker typically contains
about 5 to 15 amino acids. Optionally, other sequences encoding,
for example, tags useful for purification or detection can be fused
at the 3' end of either the nucleic acid sequence encoding the
antibody variable light chain or antibody variable heavy chain
domain or both.
[0328] When a vector is constructed for F(ab) display, it includes
nucleic acid sequences encoding antibody variable domains and
antibody constant domains. A nucleic acid encoding a variable light
chain domain is fused to a nucleic acid sequence encoding a light
chain constant domain. A nucleic acid sequence encoding an antibody
heavy chain variable domain is fused to a nucleic acid sequence
encoding a heavy chain constant CH1 domain. Typically, the nucleic
acid sequence encoding the heavy chain variable and constant
domains are fused to a nucleic acid sequence encoding all or part
of a viral coat protein. One or both of the antibody variable light
or heavy chain domains can have variant amino acids in at least one
CDR. In some embodiments, the heavy chain variable and constant
domains are expressed as a fusion with at least a portion of a
viral coat protein, and the light chain variable and constant
domains are expressed separately from the heavy chain viral coat
fusion protein. The heavy and light chains associate with one
another, which may be by covalent or non-covalent bonds.
Optionally, other sequences encoding, for example, polypeptide tags
useful for purification or detection, can be fused at the 3' end of
either the nucleic acid sequence encoding the antibody light chain
constant domain or antibody heavy chain constant domain or
both.
[0329] In some embodiments, a bivalent moiety, for example, a
F(ab).sub.2 dimer or F(ab').sub.2 dimer, is used for displaying
antibody fragments with the variant amino acid substitutions on the
surface of a particle. It has been found that F(ab').sub.2 dimers
generally have the same affinity as F(ab) dimers in a solution
phase antigen binding assay but the off rate for F(ab').sub.2 are
reduced because of a higher avidity. Therefore, the bivalent format
(for example, F(ab').sub.2) is a particularly useful format since
it can allow for the identification of lower affinity clones and
also allows more efficient sorting of rare clones during the
selection process.
[0330] Introduction of Vectors into Host Cells
[0331] Vectors constructed as described in accordance with the
invention are introduced into a host cell for amplification and/or
expression. Vectors can be introduced into host cells using
standard transformation methods including electroporation, calcium
phosphate precipitation and the like. If the vector is an
infectious particle such as a virus, the vector itself provides for
entry into the host cell. Transfection of host cells containing a
replicable expression vector which encodes the gene fusion and
production of phage particles according to standard procedures
provides phage particles in which the fusion protein is displayed
on the surface of the phage particle.
[0332] Replicable expression vectors are introduced into host cells
using a variety of methods. In one embodiment, vectors can be
introduced into cells using electroporation as described in
WO/00106717. Cells are grown in culture in standard culture broth,
optionally for about 6-48 hours (or to OD.sub.600=0.6-0.8) at about
37.degree. C., and then the broth is centrifuged and the
supernatant removed (e.g. decanted). Initial purification is
preferably by resuspending the cell pellet in a buffer solution
(e.g. 1.0 mM HEPES pH 7.4) followed by recentriguation and removal
of supernatant. The resulting cell pellet is resuspended in dilute
glycerol (e.g. 5-20% v/v) and again recentrifuged to form a cell
pellet and the supernatant removed. The final cell concentration is
obtained by resuspending the cell pellet in water or dilute
glycerol to the desired concentration.
[0333] A particularly preferred recipient cell is the
electroporation competent E. coli strain of the present invention,
which is E. coli strain SS320 (Sidhu et al., Methods Enzymol.
(2000), 328:333-363). Strain SS320 was prepared by mating MC1061
cells with XL1-BLUE cells under conditions sufficient to transfer
the fertility episome (F' plasmid) or XL1-BLUE into the MC1061
cells. Strain SS320 has been deposited with the American Type
Culture Collection (ATCC), 10801 University Boulevard, Manassas,
Va. USA, on Jun. 18, 1998 and assigned Deposit Accession No. 98795.
Any F' episome which enables phage replication in the strain may be
used in the invention. Suitable episomes are available from strains
deposited with ATCC or are commercially available (CJ236, CSH18,
DHF', JM101, JM103, JM105, JM107, JM109, JM110), KS1000, XL1-BLUE,
71-18 and others).
[0334] The use of higher DNA concentrations during electroporation
(about 10.times.) increases the transformation efficiency and
increases the amount of DNA transformed into the host cells. The
use of high cell concentrations also increases the efficiency
(about 10.times.). The larger amount of transferred DNA produces
larger libraries having greater diversity and representing a
greater number of unique members of a combinatorial library.
Transformed cells are generally selected by growth on antibiotic
containing medium.
[0335] Selection (Sorting) and Screening for Binders to Targets of
Choice
[0336] Use of phage display for identifying target antigen binders,
with its various permutations and variations in methodology, are
well established in the art. One approach involves constructing a
family of variant replicable vectors containing a transcription
regulatory element operably linked to a gene fusion encoding a
fusion polypeptide, transforming suitable host cells, culturing the
transformed cells to form phage particles which display the fusion
polypeptide on the surface of the phage particle, followed by a
process that entails selection or sorting by contacting the
recombinant phage particles with a target antigen so that at least
a portion of the population of particles bind to the target with
the objective to increase and enrich the subsets of the particles
which bind from particles relative to particles that do not bind in
the process of selection. The selected pool can be amplified by
infecting host cells, such as fresh XL1-Blue cells, for another
round of sorting on the same target with different or same
stringency. The resulting pool of variants are then screened
against the target antigens to identify novel high affinity binding
proteins. These novel high affinity binding proteins can be useful
as therapeutic agents as antagonists or agonists, and/or as as
diagonostic and research reagents.
[0337] Fusion polypeptides such as antibody variable domains
comprising the variant amino acids can be expressed on the surface
of a phage, phagemid particle or a cell and then selected and/or
screened for the ability of members of the group of fusion
polypeptides to bind a target antigen which is typically an antigen
of interest. The processes of selection for binders to target can
also be include sorting on a generic protein having affinity for
antibody variable domains such as protein L or a tag specific
antibody which binds to antibody or antibody fragments displayed on
phage, which can be used to enrich for library members that display
correctly folded antibody fragments (fusion polypeptides).
[0338] Target proteins, such as receptors, may be isolated from
natural sources or prepared by recombinant methods by procedures
known in the art. Target antigens can include a number of molecules
of therapeutic interest.
[0339] A variety of strategies of selection (sorting) for affinity
can be used. One example is a solid-support method or plate sorting
or immobilized target sorting. Another example is a
solution-binding method.
[0340] For the solid support method, the target protein may be
attached to a suitable solid or semi solid matrix which are known
in the art such as agarose beads, acrylamide beads, glass beads,
cellulose, various acrylic copolymers, hydroxyalkyl methacrylate
gels, polyacrylic and polymethacrylic copolymers, nylon, neutral
and ionic carriers, and the like. Attachment of the target protein
to the matrix may be accomplished by methods described in Methods
in Enzymology, 44 (1976), or by other means known in the art.
[0341] After attachment of the target antigen to the matrix, the
immobilized target is contacted with the library expressing the
fusion polypeptides under conditions suitable for binding of at
least a subset of the phage particle population with the
immobilized target antigen. Normally, the conditions, including pH,
ionic strength, temperature and the like will mimic physiological
conditions. Bound particles ("binders") to the immobilized target
are separated from those particles that do not bind to the target
by washing. Wash conditions can be adjusted to result in removal of
all but the high affinity binders. Binders may be dissociated from
the immobilized target by a variety of methods. These methods
include competitive dissociation using the wild-type ligand (e.g.
excess target antigen), altering pH and/or ionic strength, and
methods known in the art. Selection of binders typically involves
elution from an affinity matrix with a suitable elution material
such as acid like 0.1M HCl or ligand. Elution with increasing
concentrations of ligand could elute displayed binding molecules of
increasing affinity.
[0342] The binders can be isolated and then re-amplified in
suitable host cells by infecting the cells with the viral particles
that are binders (and helper phage if necessary, e.g. when viral
particle is a phagemid particle) and the host cells are cultured
under conditions suitable for amplification of the particles that
display the desired fusion polypeptide. The phage particles are
then collected and the selection process is repeated one or more
times until binders of the target antigen are enriched in a way.
any number of rounds of selection or sorting can be utilized. One
of the selection or sorting procedures can involve isolating
binders that bind to a generic affinity protein such as protein L
or an antibody to a polypeptide tag present in a displayed
polypeptide such as antibody to the gD protein or polyhistidine
tag.
[0343] One aspect of the invention involves selection against
libraries of the invention using a novel selection method which is
termed "solution-binding method". The invention allows solution
phase sorting with much improved efficiency over conventional
solution sorting methods. The solution binding method may be used
for finding original binders from a random library or finding
improved binders from a library that was designated to improve
affinity of a particular binding clone or group of clones. The
method comprises contacting a plurality of polypeptides, such as
those displayed on phage or phagemid particles (library), with a
target antigen labelled or fused with a tag molecule. The tag could
be biotin or other moieties for which specific binders are
available. The stringency of the solution phase can be varied by
using decreasing concentrations of labelled target antigen in the
first solution binding phase. To further increase the stringency,
the first solution binding phase can be followed by a second
solution phase having high concentration of unlabelled target
antigen after the initial binding with the labelled target in the
first solution phase. Usually, 100 to 1000 fold of unlabelled
target over labelled target is used in the second phase (if
included). The length of time of incubation of the first solution
phase can vary from a few minutes to one to two hours or longer to
reach equilibrium. Using a shorter time for binding in this first
phase may bias or select for binders that have fast on-rate. The
length of time and temperature of incubation in second phase can be
varied to increase the stringency. This provides for a selection
bias for binders that have slow rate of coming off the target
(off-rate). After contacting the plurality of polypeptides
(displayed on the phage/phagemid particles) with a target antigen,
the phage or phagemid particles that are bound to labelled targets
are separated from phage that do not bind. The particle-target
mixture from solution phase of binding is isolated by contacting it
with the labelled target moiety and allowing for its binding to, a
molecule that binds the labelled target moiety for a short period
of time (eg. 2-5 minutes). The initial concentration of the
labelled target antigen can range from about 0.1 nM to about 1000
nM. The bound particles are eluted and can be propagated for next
round of sorting. Multiple rounds of sorting are preferred using a
lower concentration of labelled target antigen with each round of
sorting.
[0344] For example, an initial sort or selection using about 100 to
250 nM labelled target antigen should be sufficient to capture a
wide range of affinities, although this factor can be determined
empirically and/or to suit the desire of the practitioner. In the
second round of selection, about 25 to 100 nM of labelled target
antigen may be used. In the third round of selection, about 0.1 to
25 nM of labled target antigen may be used. For example, to improve
the affinity of a 100 nM binder, it may be desirable to start with
20 nM and then progress to 5 and 1 nM labelled target, then,
followed by even lower concentrations such as about 0.1 nM labelled
target antigen.
[0345] The conventional solution sorting involves use of beads like
strepavidin-coated beads, which is very cumbersome to use and often
results in very low efficiency of phage binders recovery. The
conventional solution sorting with beads takes much longer than 2-5
minutes and is less feasible to adapt to high throughput automation
than the invention described above.
[0346] As described herein, combinations of solid support and
solution sorting methods can be advantageously used to isolate
binders having desired characteristics. After selection/sorting on
target antigen for a few rounds, screening of individual clones
from the selected pool generally is performed to identify specific
binders with the desired properties/characteristics. Preferably,
the process of screening is carried out by automated systems to
allow for high-throughput screening of library candidates.
[0347] Two major screening methods are described below. However,
other methods known in the art may also be used in the methods of
the invention. The first screening method comprises a phage ELISA
assay with immobilized target antigen, which provides for
identification of a specific binding clone from a non-binding
clone. Specificity can be determined by simultaneous assay of the
clone on target coated well and BSA or other non-target protein
coated wells. This assay is automatable for high throughput
screening.
[0348] One embodiment provides a method of selecting for an
antibody variable domain that binds to a specific target antigen
from a library of antibody variable domain by generating a library
of replicable expression vectors comprising a plurality of
polypeptides; contacting the library with a target antigen and at
least one nontarget antigen under conditions suitable for binding;
separating the polypeptide binders in the library from the
nonbinders; identifying the binders that bind to the target antigen
and do not bind to the nontarget antigen; eluting the binders from
the target antigen; and amplifying the replicable expression
vectors comprising the polypeptide binder that bind to a specific
antigen.
[0349] The second screening assay is an affinity screening assay
that provides for screening for clones that have high affinity from
clones that have low affinity in a high throughput manner. In the
assay, each clone is assayed with and without first incubating with
target antigen of certain concentration for a period of time (for
e.g 30-60 minutes) before application to target coated wells
briefly (e.g. 5-15 minutes). Then bound phage is measured by usual
phage ELISA method, eg. using anti-M 13 HRP conjugates. The ratio
of binding signal of the two wells, one well having been
preincubated with target and the other well not preincubated with
target antigen is an indication of affinity. The selection of the
concentraion of target for first incubation depends on the affinity
range of interest. For example, if binders with affinity higher
than 10 nM are desired, 100 nM of target in the first incubation is
often used. Once binders are found from a particular round of
sorting (selection), these clones can be screened with affinity
screening assay to identify binders with higher affinity.
[0350] Combinations of any of the sorting/selection methods
described above may be combined with the screening methods. For
example, in one embodiment, polypeptide binders are first selected
for binding to immobilized target antigen. Polypeptide binders that
bind to the immobilized target antigen can then be amplified and
screened for binding to the target antigen and for lack of binding
to nontarget antigens. Polypeptide binders that bind specifically
to the target antigen are amplified. These polypeptide binders can
then selected for higher affinity by contact with a concentration
of a labelled target antigen to form a complex, wherein the
concentration ranges of labelled target antigen from about 0.1 nM
to about 1000 nM, the complexes are isolated by contact with an
agent that binds to the label on the target antigen. The
polypeptide binders are then eluted from the labled target antigen
and optionally, the rounds of selection are repeated, each time a
lower concentration of labelled target antigen is used. The high
affinity polypeptide binders isolated using this selection method
can then be screened for high affinity using a variety of methods
known in the art, some of which are described herein.
[0351] These methods can provide for finding clones with high
affinity without having to perform long and complex competition
affinity assays on a large number of clones. The intensive aspect
of doing complex assays of many clones often is a significant
obstacle to finding best clones from a selection. This method is
especially useful in affinity improvement efforts where multiple
binders with similar affinity can be recovered from the selection
process. Different clones may have very different efficiency of
expression/display on phage or phagemid particles. Those clones
more highly expressed have better chances being recovered. That is,
the selection can be biased by the display or expression level of
the variants. The solution-binding sorting method of the invention
can improve the selection process for finding binders with high
affinity. This method is an affinity screening assay that provides
a significant advantage in screening for the best binders quickly
and easily.
[0352] After binders are identified by binding to the target
antigen, the nucleic acid can be extracted. Extracted DNA can then
be used directly to transform E. coli host cells or alternatively,
the encoding sequences can be amplified, for example using PCR with
suitable primers, and sequenced by typical sequencing method.
Variable domain DNA of the binders can be restriction enzyme
digested and then inserted into a vector for protein
expression.
[0353] Populations comprising polypeptides having CDR(s) with
restricted sequence diversity generated according to methods of the
invention can be used to isolate binders against a variety of
targets, including those listed in FIGS. 3, 4, 5, 8. These binders
may comprise one or more variant CDRs comprising diverse sequences
generated using restricted codons. In some embodiments, a variant
CDR is CDRH3 comprising sequence diversity generated by amino acid
substitution with restricted codon sets and/or amino acid
insertions resulting from varying CDRH3 lengths. Illustrative
oligonucleotides useful for generating fusion polypeptides of the
invention include those listed in FIGS. 2, 9, 14. One or more
variant CDRs may be combined. In some embodiments, only CDRH3 is
diversified. In other embodiments, two or more heavy chain CDRs,
including CDRH3, are variant. In other embodiments, one or more
heavy chain CDRs, excluding CDRH3, are variant. In some
embodiments, at least one heavy chain and at least one light chain
CDR are variant. In some embodiments, at least one, two, three,
four, five or all of CDRs H1, H2, H3, L1, L2 and L3 are
variant.
[0354] In some cases, it can be beneficial to combine one or more
diversified light chain CDRs with novel binders isolated from a
population of polypeptides comprising one or more diversified heavy
chain CDRs. This process may be referred to as a 2-step process. An
example of a 2-step process comprises first determining binders
(generally lower affinity binders) within one or more libraries
generated by randomizing one or more CDRs, wherein the CDRs
randomized in each library are different or, where the same CDR is
randomized, it is randomized to generate different sequences.
Binders from a heavy chain library can then be randomized with CDR
diversity in a light chain CDRs by, for e.g. a mutagenesis
technique such as that of Kunkel, or by cloning (cut-and-paste (eg.
by ligating different CDR sequences together)) the new light chain
library into the existing heavy chain binders that has only a fixed
light chain. The pool can then be further sorted against target to
identify binders possessing increased affinity. For example,
binders (for example, low affinity binders) obtained from sorting
an H1/H2/H3 may be fused with library of an L1/L2/L3 diversity to
replace its original fixed L1/L2/L3, wherein the new libraries are
then further sorted against a target of interest to obtain another
set of binders (for example, high affinity binders). Novel antibody
sequences can be identified that display higher binding affinity to
any of a variety of target antigens.
[0355] In some embodiments, libraries comprising polypeptides of
the invention are subjected to a plurality of sorting rounds,
wherein each sorting round comprises contacting the binders
obtained from the previous round with a target antigen distinct
from the target antigen(s) of the previous round(s). Preferably,
but not necessarily, the target antigens are homologous in
sequence, for example members of a family of related but distinct
polypeptides, such as, but not limited to, cytokines (for example,
alpha interferon subtypes).
[0356] Generation of Libraries Comprising Variant CDR-Containing
Polypeptides
[0357] Libraries of variant CDR polypeptides can be generated by
mutating the solvent accessible and/or highly diverse positions in
at least one CDR of an antibody variable domain. Some or all of the
CDRs can be mutated using the methods of the invention. In some
embodiments, it may be preferable to generate diverse antibody
libraries by mutating positions in CDRH1, CDRH2 and CDRH3 to form a
single library or by mutating positions in CDRL3 and CDRH3 to form
a single library or by mutating positions in CDRL3 and CDRH1, CDRH2
and CDRH3 to form a single library.
[0358] A library of antibody variable domains can be generated, for
example, having mutations in the solvent accessible and/or highly
diverse positions of CDRH1, CDRH2 and CDRH3. Another library can be
generated having mutations in CDRL1, CDRL2 and CDRL3. These
libraries can also be used in conjunction with each other to
generate binders of desired affinities. For example, after one or
more rounds of selection of heavy chain libraries for binding to a
target antigen, a light chain library can be replaced into the
population of heavy chain binders for further rounds of selection
to increase the affinity of the binders.
[0359] In one embodiment, a library is created by substitution of
original amino acids with a limited set of variant amino acids in
the CDRH3 region of the variable region of the heavy chain
sequence. According to the invention, this library can contain a
plurality of antibody sequences, wherein the sequence diversity is
primarily in the CDRH3 region of the heavy chain sequence.
[0360] In one aspect, the library is created in the context of the
humanized antibody 4D5 sequence, or the sequence of the framework
amino acids of the humanized antibody 4D5 sequence. Preferably, the
library is created by substitution of at least residues 95-100a of
the heavy chain with amino acids encoded by the TMT, KMT or WMT
codon set, wherein the TMT, KMT or WMT codon set is used to encode
a limited set of variant amino acids for every one of these
positions. Examples of suitable oligonucleotide sequences include,
but are not limited to, those listed in FIG. 2 and FIG. 9 and can
be determined by one skilled in the art according to the criteria
described herein.
[0361] In another embodiment, different CDRH3 designs are utilized
to isolate high affinity binders and to isolate binders for a
variety of epitopes. For diversity in CDRH3, multiple libraries can
be constructed separately with different lengths of H3 and then
combined to select for binders to target antigens. The range of
lengths of CDRH3 generated in this library can be 3-20; 5-20, 7-20,
5-18 or 7-18 amino acids, although lengths different from this can
also be generated. Diversity can also be generated in CDRH1 and
CDRH2, as indicated above. In one embodiment of a library,
diversity in H1 and H2 is generated utilizing the oligonucleotides
illustrated in FIGS. 2 and 9. Other oligonucleotides with varying
sequences can also be used. Oligonucleotides can be used singly or
pooled in any of a variety of combinations depending on practical
needs and desires of the practitioner. In some embodiments,
randomized positions in heavy chain CDRs include those listed in
FIG. 1.
[0362] Multiple libraries can be pooled and sorted using solid
support selection and solution sorting methods as described herein.
Multiple sorting strategies may be employed. For example, one
variation involves sorting on target bound to a solid, followed by
sorting for a tag that may be present on the fusion polypeptide
(eg. anti-gD tag) and followed by another sort on target bound to
solid. Alternatively, the libraries can be sorted first on target
bound to a solid surface, the eluted binders are then sorted using
solution phase binding with decreasing concentrations of target
antigen. Utilizing combinations of different sorting methods
provides for minimization of selection of only highly expressed
sequences and provides for selection of a number of different high
affinity clones.
[0363] Of the binders isolated from the pooled libraries as
described above, it has been discovered that in some instances
affinity may be further improved by providing limited diversity in
the light chain. Light chain diversity may be, but is not
necessarily, generated in this embodiment as follows: in CDRL1,
positions to be diversified include amino acid positions 28, 29,
30, 31, 32; in CDRL2, positions to be diversified include amino
acid positions 50, 51, 53, 54, 55; in CDRL3, positions to be
diversified include amino acid positions 91, 92, 93, 94, 95, 97. In
one embodiment, the randomized positions are those listed in FIG.
13.
[0364] High affinity binders isolated from the libraries of these
embodiments are readily produced in bacterial and eukaryotic cell
culture in high yield. The vectors can be designed to readily
remove sequences such as gD tags, viral coat protein component
sequence, and/or to add in constant region sequences to provide for
production of full length antibodies or antigen binding fragments
in high yield.
[0365] Any combination of codon sets and CDRs can be diversified
according to methods of the invention. Examples of suitable codons
in various combinations of CDRs are illustrated in FIGS. 2, 6, 9,
13.
[0366] Vectors, Host Cells and Recombinant Methods
[0367] For recombinant production of an antibody polypeptide of the
invention, the nucleic acid encoding it is isolated and inserted
into a replicable vector for further cloning (amplification of the
DNA) or for expression. DNA encoding the antibody is readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody). Many vectors are available. The choice of vector depends
in part on the host cell to be used. Generally, preferred host
cells are of either prokaryotic or eukaryotic (generally mammalian)
origin.
[0368] Generating Antibodies Using Prokaryotic Host Cells:
[0369] Vector Construction
[0370] Polynucleotide sequences encoding polypeptide components of
the antibody of the invention can be obtained using standard
recombinant techniques. Desired polynucleotide sequences may be
isolated and sequenced from antibody producing cells such as
hybridoma cells. Alternatively, polynucleotides can be synthesized
using nucleotide synthesizer or PCR techniques. Once obtained,
sequences encoding the polypeptides are inserted into a recombinant
vector capable of replicating and expressing heterologous
polynucleotides in prokaryotic hosts. Many vectors that are
available and known in the art can be used for the purpose of the
present invention. Selection of an appropriate vector will depend
mainly on the size of the nucleic acids to be inserted into the
vector and the particular host cell to be transformed with the
vector. Each vector contains various components, depending on its
function (amplification or expression of heterologous
polynucleotide, or both) and its compatibility with the particular
host cell in which it resides. The vector components generally
include, but are not limited to: an origin of replication, a
selection marker gene, a promoter, a ribosome binding site (RBS), a
signal sequence, the heterologous nucleic acid insert and a
transcription termination sequence.
[0371] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR322, a plasmid
derived from an E. coli species. pBR322 contains genes encoding
ampicillin (Amp) and tetracycline (Tet) resistance and thus
provides easy means for identifying transformed cells. pBR322, its
derivatives, or other microbial plasmids or bacteriophage may also
contain, or be modified to contain, promoters which can be used by
the microbial organism for expression of endogenous proteins.
Examples of pBR322 derivatives used for expression of particular
antibodies are described in detail in Carter et al., U.S. Pat. No.
5,648,237.
[0372] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, bacteriophage such as .lambda.GEM.TM.-11 may be utilized
in making a recombinant vector which can be used to transform
susceptible host cells such as E. coli LE392.
[0373] The expression vector of the invention may comprise two or
more promoter-cistron pairs, encoding each of the polypeptide
components. A promoter is an untranslated regulatory sequence
located upstream (5') to a cistron that modulates its expression.
Prokaryotic promoters typically fall into two classes, inducible
and constitutive. Inducible promoter is a promoter that initiates
increased levels of transcription of the cistron under its control
in response to changes in the culture condition, e.g. the presence
or absence of a nutrient or a change in temperature.
[0374] A large number of promoters recognized by a variety of
potential host cells are well known. The selected promoter can be
operably linked to cistron DNA encoding the light or heavy chain by
removing the promoter from the source DNA via restriction enzyme
digestion and inserting the isolated promoter sequence into the
vector of the invention. Both the native promoter sequence and many
heterologous promoters may be used to direct amplification and/or
expression of the target genes. In some embodiments, heterologous
promoters are utilized, as they generally permit greater
transcription and higher yields of expressed target gene as
compared to the native target polypeptide promoter.
[0375] Promoters suitable for use with prokaryotic hosts include
the PhoA promoter, the .beta.-galactamase and lactose promoter
systems, a tryptophan (trp) promoter system and hybrid promoters
such as the tac or the trc promoter. However, other promoters that
are functional in bacteria (such as other known bacterial or phage
promoters) are suitable as well. Their nucleotide sequences have
been published, thereby enabling a skilled worker operably to
ligate them to cistrons encoding the target light and heavy chains
(Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors
to supply any required restriction sites.
[0376] In one aspect of the invention, each cistron within the
recombinant vector comprises a secretion signal sequence component
that directs translocation of the expressed polypeptides across a
membrane. In general, the signal sequence may be a component of the
vector, or it may be a part of the target polypeptide DNA that is
inserted into the vector. The signal sequence selected for the
purpose of this invention should be one that is recognized and
processed (i.e. cleaved by a signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process the
signal sequences native to the heterologous polypeptides, the
signal sequence is substituted by a prokaryotic signal sequence
selected, for example, from the group consisting of the alkaline
phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II
(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment
of the invention, the signal sequences used in both cistrons of the
expression system are STII signal sequences or variants
thereof.
[0377] In another aspect, the production of the immunoglobulins
according to the invention can occur in the cytoplasm of the host
cell, and therefore does not require the presence of secretion
signal sequences within each cistron. In that regard,
immunoglobulin light and heavy chains are expressed, folded and
assembled to form functional immunoglobulins within the cytoplasm.
Certain host strains (e.g., the E. coli trxB.sup.- strains) provide
cytoplasm conditions that are favorable for disulfide bond
formation, thereby permitting proper folding and assembly of
expressed protein subunits. Proba and Pluckthun Gene, 159:203
(1995).
[0378] The present invention provides an expression system in which
the quantitative ratio of expressed polypeptide components can be
modulated in order to maximize the yield of secreted and properly
assembled antibodies of the invention. Such modulation is
accomplished at least in part by simultaneously modulating
translational strengths for the polypeptide components.
[0379] One technique for modulating translational strength is
disclosed in Simmons et al., U.S. Pat. No. 5,840,523. It utilizes
variants of the translational initiation region (TIR) within a
cistron. For a given TIR, a series of amino acid or nucleic acid
sequence variants can be created with a range of translational
strengths, thereby providing a convenient means by which to adjust
this factor for the desired expression level of the specific chain.
TIR variants can be generated by conventional mutagenesis
techniques that result in codon changes which can alter the amino
acid sequence, although silent changes in the nucleotide sequence
are preferred. Alterations in the TIR can include, for example,
alterations in the number or spacing of Shine-Dalgarno sequences,
along with alterations in the signal sequence. One method for
generating mutant signal sequences is the generation of a "codon
bank" at the beginning of a coding sequence that does not change
the amino acid sequence of the signal sequence (i.e., the changes
are silent). This can be accomplished by changing the third
nucleotide position of each codon; additionally, some amino acids,
such as leucine, serine, and arginine, have multiple first and
second positions that can add complexity in making the bank. This
method of mutagenesis is described in detail in Yansura et al.
(1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.
[0380] Preferably, a set of vectors is generated with a range of
TIR strengths for each cistron therein. This limited set provides a
comparison of expression levels of each chain as well as the yield
of the desired antibody products under various TIR strength
combinations. TIR strengths can be determined by quantifying the
expression level of a reporter gene as described in detail in
Simmons et al. U.S. Pat. No. 5,840,523. Based on the translational
strength comparison, the desired individual TIRs are selected to be
combined in the expression vector constructs of the invention.
[0381] Prokaryotic host cells suitable for expressing antibodies of
the invention include Archaebacteria and Eubacteria, such as
Gram-negative or Gram-positive organisms. Examples of useful
bacteria include Escherichia (e.g., E. coli), Bacilli (e.g., B.
subtilis), Enterobacteria, Pseudomonas species (e.g., P.
aeruginosa), Salmonella typhimurium, Serratia marcescans,
Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or
Paracoccus. In one embodiment, gram-negative cells are used. In one
embodiment, E. coli cells are used as hosts for the invention.
Examples of E. coli strains include strain W3110 (Bachmann,
Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American
Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No.
27,325) and derivatives thereof, including strain 33D3 having
genotype W3110 .DELTA.fhuA (.DELTA.tonA) ptr3 lac Iq lacL8
.DELTA.ompT.DELTA.(nmpc-fepE) degP4I kan.sup.R (U.S. Pat. No.
5,639,635). Other strains and derivatives thereof, such as E. coli
294 (ATCC 31,446), E. coli B, E. coli.sub..lambda. 1776 (ATCC
31,537) and E. coli RV308(ATCC 31,608) are also suitable. These
examples are illustrative rather than limiting. Methods for
constructing derivatives of any of the above-mentioned bacteria
having defined genotypes are known in the art and described in, for
example, Bass et al., Proteins, 8:309-314 (1990). It is generally
necessary to select the appropriate bacteria taking into
consideration replicability of the replicon in the cells of a
bacterium. For example, E. coli, Serratia, or Salmonella species
can be suitably used as the host when well known plasmids such as
pBR322, pBR325, pACYC177, or pKN4]0 are used to supply the
replicon. Typically the host cell should secrete minimal amounts of
proteolytic enzymes, and additional protease inhibitors may
desirably be incorporated in the cell culture.
[0382] Antibody Production
[0383] Host cells are transformed with the above-described
expression vectors and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0384] Transformation means introducing DNA into the prokaryotic
host so that the DNA is replicable, either as an extrachromosomal
element or by chromosomal integrant. Depending on the host cell
used, transformation is done using standard techniques appropriate
to such cells. The calcium treatment employing calcium chloride is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO. Yet another technique used is
electroporation.
[0385] Prokaryotic cells used to produce the polypeptides of the
invention are grown in media known in the art and suitable for
culture of the selected host cells. Examples of suitable media
include luria broth (LB) plus necessary nutrient supplements. In
some embodiments, the media also contains a selection agent, chosen
based on the construction of the expression vector, to selectively
permit growth of prokaryotic cells containing the expression
vector. For example, ampicillin is added to media for growth of
cells expressing ampicillin resistant gene.
[0386] Any necessary supplements besides carbon, nitrogen, and
inorganic phosphate sources may also be included at appropriate
concentrations introduced alone or as a mixture with another
supplement or medium such as a complex nitrogen source. Optionally
the culture medium may contain one or more reducing agents selected
from the group consisting of glutathione, cysteine, cystamine,
thioglycollate, dithioerythritol and dithiothreitol.
[0387] The prokaryotic host cells are cultured at suitable
temperatures. For E. coli growth, for example, the preferred
temperature ranges from about 20.degree. C. to about 39.degree. C.,
more preferably from about 25.degree. C. to about 37.degree. C.,
even more preferably at about 30.degree. C. The pH of the medium
may be any pH ranging from about 5 to about 9, depending mainly on
the host organism. For E. coli, the pH is preferably from about 6.8
to about 7.4, and more preferably about 7.0.
[0388] If an inducible promoter is used in the expression vector of
the invention, protein expression is induced under conditions
suitable for the activation of the promoter. In one aspect of the
invention, PhoA promoters are used for controlling transcription of
the polypeptides. Accordingly, the transformed host cells are
cultured in a phosphate-limiting medium for induction. Preferably,
the phosphate-limiting medium is the C.R.A.P medium (see, for e.g.,
Simmons et al., J. Immunol. Methods (2002), 263:133-147). A variety
of other inducers may be used, according to the vector construct
employed, as is known in the art.
[0389] In one embodiment, the expressed polypeptides of the present
invention are secreted into and recovered from the periplasm of the
host cells. Protein recovery typically involves disrupting the
microorganism, generally by such means as osmotic shock, sonication
or lysis. Once cells are disrupted, cell debris or whole cells may
be removed by centrifugation or filtration. The proteins may be
further purified, for example, by affinity resin chromatography.
Alternatively, proteins can be transported into the culture media
and isolated therein. Cells may be removed from the culture and the
culture supernatant being filtered and concentrated for further
purification of the proteins produced. The expressed polypeptides
can be further isolated and identified using commonly known methods
such as polyacrylamide gel electrophoresis (PAGE) and Western blot
assay.
[0390] In one aspect of the invention, antibody production is
conducted in large quantity by a fermentation process. Various
large-scale fed-batch fermentation procedures are available for
production of recombinant proteins. Large-scale fermentations have
at least 1000 liters of capacity, preferably about 1,000 to 100,000
liters of capacity. These fermentors use agitator impellers to
distribute oxygen and nutrients, especially glucose (the preferred
carbon/energy source). Small scale fermentation refers generally to
fermentation in a fermentor that is no more than approximately 100
liters in volumetric capacity, and can range from about 1 liter to
about 100 liters.
[0391] In a fermentation process, induction of protein expression
is typically initiated after the cells have been grown under
suitable conditions to a desired density, e.g., an OD.sub.550 of
about 180-220, at which stage the cells are in the early stationary
phase. A variety of inducers may be used, according to the vector
construct employed, as is known in the art and described above.
Cells may be grown for shorter periods prior to induction. Cells
are usually induced for about 12-50 hours, although longer or
shorter induction time may be used.
[0392] To improve the production yield and quality of the
polypeptides of the invention, various fermentation conditions can
be modified. For example, to improve the proper assembly and
folding of the secreted antibody polypeptides, additional vectors
overexpressing chaperone proteins, such as Dsb proteins (DsbA,
DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl
cis,trans-isomerase with chaperone activity) can be used to
co-transform the host prokaryotic cells. The chaperone proteins
have been demonstrated to facilitate the proper folding and
solubility of heterologous proteins produced in bacterial host
cells. Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou et
al., U.S. Pat. No. 6,083,715; Georgiou et al., U.S. Pat. No.
6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem.
275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem.
275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210.
[0393] To minimize proteolysis of expressed heterologous proteins
(especially those that are proteolytically sensitive), certain host
strains deficient for proteolytic enzymes can be used for the
present invention. For example, host cell strains may be modified
to effect genetic mutation(s) in the genes encoding known bacterial
proteases such as Protease III, OmpT, DegP, Tsp, Protease I,
Protease Mi, Protease V, Protease VI and combinations thereof. Some
E. coli protease-deficient strains are available and described in,
for example, Joly et al. (1998), supra; Georgiou et al., U.S. Pat.
No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et
al., Microbial Drug Resistance, 2:63-72 (1996).
[0394] In one embodiment, E. coli strains deficient for proteolytic
enzymes and transformed with plasmids overexpressing one or more
chaperone proteins are used as host cells in the expression system
of the invention.
[0395] Antibody Purification
[0396] In one embodiment, the antibody protein produced herein is
further purified to obtain preparations that are substantially
homogeneous for further assays and uses. Standard protein
purification methods known in the art can be employed. The
following procedures are exemplary of suitable purification
procedures: fractionation on immunoaffinity or ion-exchange
columns, ethanol precipitation, reverse phase HPLC, chromatography
on silica or on a cation-exchange resin such as DEAE,
chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel
filtration using, for example, Sephadex G-75.
[0397] In one aspect, Protein A immobilized on a solid phase is
used for immunoaffinity purification of the antibody products of
the invention. Protein A is a 41 kD cell wall protein from
Staphylococcus aureas which binds with a high affinity to the Fc
region of antibodies. Lindmark et al (1983) J. Immunol. Meth.
62:1-13. The solid phase to which Protein A is immobilized is
preferably a column comprising a glass or silica surface, more
preferably a controlled pore glass column or a silicic acid column.
In some applications, the column has been coated with a reagent,
such as glycerol, in an attempt to prevent nonspecific adherence of
contaminants.
[0398] As the first step of purification, the preparation derived
from the cell culture as described above is applied onto the
Protein A immobilized solid phase to allow specific binding of the
antibody of interest to Protein A. The solid phase is then washed
to remove contaminants non-specifically bound to the solid phase.
Finally the antibody of interest is recovered from the solid phase
by elution.
[0399] Generating Antibodies Using Eukaryotic Host Cells:
[0400] The vector components generally include, but are not limited
to, one or more of the following: a signal sequence, an origin of
replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence.
[0401] (i) Signal Sequence Component
[0402] A vector for use in a eukaryotic host cell may also contain
a signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide of
interest. The heterologous signal sequence selected preferably is
one that is recognized and processed (i.e., cleaved by a signal
peptidase) by the host cell. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for
example, the herpes simplex gD signal, are available.
[0403] The DNA for such precursor region is ligated in reading
frame to DNA encoding the antibody.
[0404] (ii) Origin of Replication
[0405] Generally, an origin of replication component is not needed
for mammalian expression vectors. For example, the SV40 origin may
typically be used only because it contains the early promoter.
[0406] (iii) Selection Gene Component
[0407] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, where relevant, or (c) supply
critical nutrients not available from complex media.
[0408] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0409] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the antibody nucleic acid, such as DHFR, thymidine
kinase, metallothionein-I and -II, preferably primate
metallothionein genes, adenosine deaminase, ornithine
decarboxylase, etc.
[0410] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium that contains methotrexate (Mtx), a competitive
antagonist of DHFR. An appropriate host cell when wild-type DHFR is
employed is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity (e.g., ATCC CRL-9096).
[0411] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding an antibody, wild-type DHFR protein, and another
selectable marker such as aminoglycoside 3'-phosphotransferase
(APH) can be selected by cell growth in medium containing a
selection agent for the selectable marker such as an
aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See
U.S. Pat. No. 4,965,199.
[0412] (iv) Promoter Component
[0413] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the antibody polypeptide nucleic acid. Promoter sequences are known
for eukaryotes. Virtually alleukaryotic genes have an AT-rich
region located approximately 25 to 30 bases upstream from the site
where transcription is initiated. Another sequence found 70 to 80
bases upstream from the start of transcription of many genes is a
CNCAAT region where N may be any nucleotide. At the 3' end of most
eukaryotic genes is an AATAAA sequence that may be the signal for
addition of the poly A tail to the 3' end of the coding sequence.
All of these sequences are suitably inserted into eukaryotic
expression vectors.
[0414] Antibody polypeptide transcription from vectors in mammalian
host cells is controlled, for example, by promoters obtained from
the genomes of viruses such as polyoma virus, fowlpox virus,
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, from
heat-shock promoters, provided such promoters are compatible with
the host cell systems.
[0415] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the Rous Sarcoma
Virus long terminal repeat can be used as the promoter.
[0416] (v) Enhancer Element Component
[0417] Transcription of DNA encoding the antibody polypeptide of
this invention by higher eukaryotes is often increased by inserting
an enhancer sequence into the vector. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the antibody
polypeptide-encoding sequence, but is preferably located at a site
5' from the promoter.
[0418] (vi) Transcription Termination Component
[0419] Expression vectors used in eukaryotic host cells will
typically also contain sequences necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are
commonly available from the 5' and, occasionally 3', untranslated
regions of eukaryotic or viral DNAs or cDNAs. These regions contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA encoding an antibody. One useful
transcription termination component is the bovine growth hormone
polyadenylation region. See WO94/11026 and the expression vector
disclosed therein.
[0420] (vii) Selection and Transformation of Host Cells
[0421] Suitable host cells for cloning or expressing the DNA in the
vectors herein include higher eukaryote cells described herein,
including vertebrate host cells. Propagation of vertebrate cells in
culture (tissue culture) has become a routine procedure. Examples
of useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney
cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR(CHO,
Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse
sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells;
and a human hepatoma line (Hep G2).
[0422] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0423] (viii) Culturing the Host Cells
[0424] The host cells used to produce an antibody of this invention
may be cultured in a variety of media. Commercially available media
such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),
(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium
((DMEM), Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0425] (ix) Purification of Antibody
[0426] When using recombinant techniques, the antibody can be
produced intracellularly, or directly secreted into the medium. If
the antibody is produced intracellularly, as a first step, the
particulate debris, either host cells or lysed fragments, are
removed, for example, by centrifugation or ultrafiltration. Where
the antibody is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0427] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a C.sub.H3 domain, the Bakerbond
ABX.TM.resin (J. T. Baker, Phillipsburg, N.J.) is useful for
purification. Other techniques for protein purification such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin SEPHAROSE.TM. chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation are also available
depending on the antibody to be recovered.
[0428] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
[0429] Activity Assays
[0430] The antibodies of the present invention can be characterized
for their physical/chemical properties and biological functions by
various assays known in the art.
[0431] The purified immunoglobulins can be further characterized by
a series of assays including, but not limited to, N-terminal
sequencing, amino acid analysis, non-denaturing size exclusion high
pressure liquid chromatography (HPLC), mass spectrometry, ion
exchange chromatography and papain digestion.
[0432] In certain embodiments of the invention, the immunoglobulins
produced herein are analyzed for their biological activity. In some
embodiments, the immunoglobulins of the present invention are
tested for their antigen binding activity. The antigen binding
assays that are known in the art and can be used herein include
without limitation any direct or competitive binding assays using
techniques such as western blots, radioimmunoassays, ELISA (enzyme
linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation assays, fluorescent immunoassays, and protein A
immunoassays.
[0433] In one embodiment, the present invention contemplates an
altered antibody that possesses some but not all effector
functions, which make it a desired candidate for many applications
in which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In certain embodiments, the Fc
activities of the produced immunoglobulin are measured to ensure
that only the desired properties are maintained. In vitro and/or in
vivo cytotoxicity assays can be conducted to confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc
receptor (FcR) binding assays can be conducted to ensure that the
antibody lacks Fc.gamma.R binding (hence likely lacking ADCC
activity), but retains FcRn binding ability. The primary cells for
mediating ADCC, NK cells, express Fc.gamma.RIII only, whereas
monocytes express Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR
expression on hematopoietic cells is summarized in Table 3 on page
464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). An
example of an in vitro assay to assess ADCC activity of a molecule
of interest is described in U.S. Pat. No. 5,500,362 or 5,821,337.
Useful effector cells for such assays include peripheral blood
mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). C1q
binding assays may also be carried out to confirm that the antibody
is unable to bind C1q and hence lacks CDC activity. To assess
complement activation, a CDC assay, for e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed. FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the
art, for e.g. those desribed in the Examples section.
[0434] Humanized Antibodies
[0435] The present invention encompasses humanized antibodies.
Various methods for humanizing non-human antibodies are known in
the art. For example, a humanized antibody can have one or more
amino acid residues introduced into it from a source which is
non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al. (1986)
Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;
Verhoeyen et al. (1988) Science 239:1534-1536), by substituting
hypervariable region sequences for the corresponding sequences of a
human antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially
less than an intact human variable domain has been substituted by
the corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
hypervariable region residues and possibly some FR residues are
substituted by residues from analogous sites in rodent
antibodies.
[0436] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework for the humanized
antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al.
(1987) J. Mol. Biol. 196:901. Another method uses a particular
framework derived from the consensus sequence of all human
antibodies of a particular subgroup of light or heavy chains. The
same framework may be used for several different humanized
antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA,
89:4285; Presta et al. (1993) J. Immunol., 151:2623.
[0437] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to one
method, humanized antibodies are prepared by a process of analysis
of the parental sequences and various conceptual humanized products
using three-dimensional models of the parental and humanized
sequences. Three-dimensional immunoglobulin models are commonly
available and are familiar to those skilled in the art. Computer
programs are available which illustrate and display probable
three-dimensional conformational structures of selected candidate
immunoglobulin sequences. Inspection of these displays permits
analysis of the likely role of the residues in the functioning of
the candidate immunoglobulin sequence, i.e., the analysis of
residues that influence the ability of the candidate immunoglobulin
to bind its antigen. In this way, FR residues can be selected and
combined from the recipient and import sequences so that the
desired antibody characteristic, such as increased affinity for the
target antigen(s), is achieved. In general, the hypervariable
region residues are directly and most substantially involved in
influencing antigen binding.
[0438] Antibody Variants
[0439] In one aspect, the invention provides antibody fragment
comprising modifications in the interface of Fc polypeptides
comprising the Fc region, wherein the modifications facilitate
and/or promote heterodimerization. These modifications comprise
introduction of a protuberance into a first Fc polypeptide and a
cavity into a second Fc polypeptide, wherein the protuberance is
positionable in the cavity so as to promote complexing of the first
and second Fc polypeptides. Methods of generating antibodies with
these modifications are known in the art, for e.g., as described in
U.S. Pat. No. 5,731,168.
[0440] In some embodiments, amino acid sequence modification(s) of
the antibodies described herein are contemplated. For example, it
may be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the antibody are prepared by introducing appropriate nucleotide
changes into the antibody nucleic acid, or by peptide synthesis.
Such modifications include, for example, deletions from, and/or
insertions into and/or substitutions of, residues within the amino
acid sequences of the antibody. Any combination of deletion,
insertion, and substitution is made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics. The amino acid alterations may be introduced in
the subject antibody amino acid sequence at the time that sequence
is made.
[0441] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells (1989) Science, 244:1081-1085. Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed
immunoglobulins are screened for the desired activity.
[0442] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue or the antibody fused to a cytotoxic
polypeptide. Other insertional variants of the antibody molecule
include the fusion to the N- or C-terminus of the antibody to an
enzyme (e.g. for ADEPT) or a polypeptide which increases the serum
half-life of the antibody.
[0443] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule replaced by a different residue. The sites of
greatest interest for substitutional mutagenesis include the
hypervariable regions, but FR alterations are also contemplated.
Conservative substitutions are shown in Table 2 under the heading
of "preferred substitutions". If such substitutions result in a
change in biological activity, then more substantial changes,
denominated "exemplary substitutions" in the table below, or as
further described below in reference to amino acid classes, may be
introduced and the products screened.
1 Original Exemplary Preferred Residue Substitutions Substitutions
Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln;
His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser
Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H)
Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Leu Phe;
Norleucine Leu (L) Norleucine; Ile; Val; Ile Met; Ala; Phe Lys (K)
Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val;
Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser
Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V)
Ile; Leu; Met; Phe; Leu Ala; Norleucine
[0444] Substantial modifications in the biological properties of
the antibody are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Amino acids may be grouped
according to similarities in the properties of their side chains
(in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth
Publishers, New York (1975)):
[0445] (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P),
Phe (F), Trp (W), Met (M)
[0446] (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr
(Y), Asn (N), Gln (O)
[0447] (3) acidic: Asp (D), Glu (E)
[0448] (4) basic: Lys (K), Arg (R), His(H)
[0449] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties:
[0450] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0451] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0452] (3) acidic: Asp, Glu;
[0453] (4) basic: His, Lys, Arg;
[0454] (5) residues that influence chain orientation: Gly, Pro;
[0455] (6) aromatic: Trp, Tyr, Phe.
[0456] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, more preferably, into the remaining (non-conserved)
sites.
[0457] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further development will have improved biological
properties relative to the parent antibody from which they are
generated. A convenient way for generating such substitutional
variants involves affinity maturation using phage display. Briefly,
several hypervariable region sites (e.g. 6-7 sites) are mutated to
generate all possible amino acid substitutions at each site. The
antibodies thus generated are displayed from filamentous phage
particles as fusions to the gene III product of M13 packaged within
each particle. The phage-displayed variants are then screened for
their biological activity (e.g. binding affinity) as herein
disclosed. In order to identify candidate hypervariable region
sites for modification, alanine scanning mutagenesis can be
performed to identify hypervariable region residues contributing
significantly to antigen binding. Alternatively, or additionally,
it may be beneficial to analyze a crystal structure of the
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues are candidates for substitution according to the
techniques elaborated herein. Once such variants are generated, the
panel of variants is subjected to screening as described herein and
antibodies with superior properties in one or more relevant assays
may be selected for further development.
[0458] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
[0459] It may be desirable to introduce one or more amino acid
modifications in an Fc region of the immunoglobulin polypeptides of
the invention, thereby generating a Fc region variant. The Fc
region variant may comprise a human Fc region sequence (e.g., a
human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions including that of a hinge cysteine.
[0460] In accordance with this description and the teachings of the
art, it is contemplated that in some embodiments, an antibody used
in methods of the invention may comprise one or more alterations as
compared to the wild type counterpart antibody, for e.g. in the Fc
region. These antibodies would nonetheless retain substantially the
same characteristics required for therapeutic utility as compared
to their wild type counterpart. For e.g., it is thought that
certain alterations can be made in the Fc region that would result
in altered (i.e., either improved or diminished) C1q binding and/or
Complement Dependent Cytotoxicity (CDC), for e.g., as described in
WO99/51642. See also Duncan & Winter Nature 322:738-40 (1988);
U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO94/29351
concerning other examples of Fc region variants.
[0461] Immunoconjugates
[0462] The invention also pertains to immunoconjugates, or
antibody-drug conjugates (ADC), comprising an antibody conjugated
to a cytotoxic agent such as a chemotherapeutic agent, a drug, a
growth inhibitory agent, a toxin (e.g., an enzymatically active
toxin of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0463] The use of antibody-drug conjugates for the local delivery
of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit
tumor cells in the treatment of cancer (Syrigos and Epenetos (1999)
Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997)
Adv. Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278)
theoretically allows targeted delivery of the drug moiety to
tumors, and intracellular accumulation therein, where systemic
administration of these unconjugated drug agents may result in
unacceptable levels of toxicity to normal cells as well as the
tumor cells sought to be eliminated (Baldwin et al., (1986) Lancet
pp. (Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal
Antibodies '84: Biological And Clinical Applications, A. Pinchera
et al. (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity
is sought thereby. Both polyclonal antibodies and monoclonal
antibodies have been reported as useful in these strategies
(Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87).
Drugs used in these methods include daunomycin, doxorubicin,
methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins
used in antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer
Inst. 92(19): 1573-1581; Mandler et al (2000) Bioorganic & Med.
Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.
13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc.
Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al
(1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res.
53:3336-3342). The toxins may effect their cytotoxic and cytostatic
effects by mechanisms including tubulin binding, DNA binding, or
topoisomerase inhibition. Some cytotoxic drugs tend to be inactive
or less active when conjugated to large antibodies or protein
receptor ligands.
[0464] ZEVALIN.RTM. (ibritumomab tiuxetan, Biogen/Idec) is an
antibody-radioisotope conjugate composed of a murine IgG1 kappa
monoclonal antibody directed against the CD20 antigen found on the
surface of normal and malignant B lymphocytes and .sup.111In or
.sup.90Y radioisotope bound by a thiourea linker-chelator (Wiseman
et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al
(2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol.
20(10):2453-63; Witzig et al (2002) J. Clin. Oncol.
20(15):3262-69). Although ZEVALIN has activity against B-cell
non-Hodgkin's Lymphoma (NHL), administration results in severe and
prolonged cytopenias in most patients. MYLOTARG.TM. (gemtuzumab
ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate
composed of a hu CD33 antibody linked to calicheamicin, was
approved in 2000 for the treatment of acute myeloid leukemia by
injection (Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos.
4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116;
5,767,285; 5,773,001). Cantuzumab mertansine (Immunogen, Inc.), an
antibody drug conjugate composed of the huC242 antibody linked via
the disulfide linker SPP to the maytansinoid drug moiety, DM1, is
advancing into Phase II trials for the treatment of cancers that
express CanAg, such as colon, pancreatic, gastric, and others.
MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an
antibody drug conjugate composed of the anti-prostate specific
membrane antigen (PSMA) monoclonal antibody linked to the
maytansinoid drug moiety, DM1, is under development for the
potential treatment of prostate tumors. The auristatin peptides,
auristatin E (AE) and monomethylauristatin (MMAE), synthetic
analogs of dolastatin, were conjugated to chimeric monoclonal
antibodies cBR96 (specific to Lewis Y on carcinomas) and cAC10
(specific to CD30 on hematological malignancies) (Doronina et al
(2003) Nature Biotechnology 21(7):778-784) and are under
therapeutic development.
[0465] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re. Conjugates of the antibody and cytotoxic
agent are made using a variety of bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate
(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al.,
Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0466] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothecene,
and CC 1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
[0467] Maytansine and Maytansinoids
[0468] In one embodiment, an antibody (full length or fragments) of
the invention is conjugated to one or more maytansinoid
molecules.
[0469] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the
disclosures of which are hereby expressly incorporated by
reference.
[0470] Maytansinoid-Antibody Conjugates
[0471] In an attempt to improve their therapeutic index, maytansine
and maytansinoids have been conjugated to antibodies specifically
binding to tumor cell antigens. Immunoconjugates containing
maytansinoids and their therapeutic use are disclosed, for example,
in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425
235 B1, the disclosures of which are hereby expressly incorporated
by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds the
HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoid
conjugate was tested in vitro on the human breast cancer cell line
SK-BR-3, which expresses 3.times.10.sup.5 HER-2 surface antigens
per cell. The drug conjugate achieved a degree of cytotoxicity
similar to the free maytansinoid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
[0472] Antibody-Maytansinoid Conjugates (Immunoconjugates)
[0473] Antibody-maytansinoid conjugates are prepared by chemically
linking an antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. An average of 3-4
maytansinoid molecules conjugated per antibody molecule has shown
efficacy in enhancing cytotoxicity of target cells without
negatively affecting the function or solubility of the antibody,
although even one molecule of toxin/antibody would be expected to
enhance cytotoxicity over the use of naked antibody. Maytansinoids
are well known in the art and can be synthesized by known
techniques or isolated from natural sources. Suitable maytansinoids
are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the
other patents and nonpatent publications referred to hereinabove.
Preferred maytansinoids are maytansinol and maytansinol analogues
modified in the aromatic ring or at other positions of the
maytansinol molecule, such as various maytansinol esters.
[0474] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and
Chari et al., Cancer Research 52:127-131 (1992). The linking groups
include disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile
groups, as disclosed in the above-identified patents, disulfide and
thioether groups being preferred.
[0475] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 [1978]) and
N-succinimidyl-4-(2-pyridylthio)- pentanoate (SPP) to provide for a
disulfide linkage.
[0476] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
[0477] Calicheamicin
[0478] Another immunoconjugate of interest comprises an antibody
conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma..sub.1.sup.1, .alpha..sub.2.sup.1,
.alpha..sub.3.sup.1, N-acetyl-.gamma..sub.1.sup.1, PSAG and
.theta..sup.1.sub.1 (Hinman et al., Cancer Research 53:3336-3342
(1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the
aforementioned U.S. patents to American Cyanamid). Another
anti-tumor drug that the antibody can be conjugated is QFA which is
an antifolate. Both calicheamicin and QFA have intracellular sites
of action and do not readily cross the plasma membrane. Therefore,
cellular uptake of these agents through antibody mediated
internalization greatly enhances their cytotoxic effects.
[0479] Other Cytotoxic Agents
[0480] Other antitumor agents that can be conjugated to the
antibodies of the invention include BCNU, streptozoicin,
vincristine and 5-fluorouracil, the family of agents known
collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[0481] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0482] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0483] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
antibodies. Examples include At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32,
Pb.sup.212 and radioactive isotopes of Lu. When the conjugate is
used for detection, it may comprise a radioactive atom for
scintigraphic studies, for example tc.sup.99m or I.sup.123, or a
spin label for nuclear magnetic resonance (NMR) imaging (also known
as magnetic resonance imaging, mri), such as iodine-123 again,
iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.
[0484] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal,CRC Press 1989) describes other methods
in detail.
[0485] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat.
No. 5,208,020) may be used.
[0486] The compounds of the invention expressly contemplate, but
are not limited to, ADC prepared with cross-linker reagents: BMPS,
EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB,
SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate) which are commercially
available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,
U.S.A). See pages 467-498, 2003-2004 Applications Handbook and
Catalog.
[0487] Preparation of Antibody Drug Conjugates
[0488] In the antibody drug conjugates (ADC) of the invention, an
antibody (Ab) is conjugated to one or more drug moieties (D), e.g.
about 1 to about 20 drug moieties per antibody, through a linker
(L). The ADC of Formula I may be prepared by several routes,
employing organic chemistry reactions, conditions, and reagents
known to those skilled in the art, including: (1) reaction of a
nucleophilic group of an antibody with a bivalent linker reagent,
to form Ab-L, via a covalent bond, followed by reaction with a drug
moiety D; and (2) reaction of a nucleophilic group of a drug moiety
with a bivalent linker reagent, to form D-L, via a covalent bond,
followed by reaction with the nucleophilic group of an
antibody.
Ab-(L-D).sub.p I
[0489] Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine,
and (iv) sugar hydroxyl or amino groups where the antibody is
glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic
and capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and linker reagents including: (i) active
esters such as NHS esters, HOBt esters, haloformates, and acid
halides; (ii) alkyl and benzyl halides such as haloacetamides;
(iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain
antibodies have reducible interchain disulfides, i.e. cysteine
bridges. Antibodies may be made reactive for conjugation with
linker reagents by treatment with a reducing agent such as DTT
(dithiothreitol). Each cysteine bridge will thus form,
theoretically, two reactive thiol nucleophiles. Additional
nucleophilic groups can be introduced into antibodies through the
reaction of lysines with 2-iminothiolane (Traut's reagent)
resulting in conversion of an amine into a thiol.
[0490] Antibody drug conjugates of the invention may also be
produced by modification of the antibody to introduce electrophilic
moieties, which can react with nucleophilic subsituents on the
linker reagent or drug. The sugars of glycosylated antibodies may
be oxidized, e.g. with periodate oxidizing reagents, to form
aldehyde or ketone groups which may react with the amine group of
linker reagents or drug moieties. The resulting imine Schiff base
groups may form a stable linkage, or may be reduced, e.g. by
borohydride reagents to form stable amine linkages. In one
embodiment, reaction of the carbohydrate portion of a glycosylated
antibody with either glactose oxidase or sodium meta-periodate may
yield carbonyl (aldehyde and ketone) groups in the protein that can
react with appropriate groups on the drug (Hermanson, Bioconjugate
Techniques). In another embodiment, proteins containing N-terminal
serine or threonine residues can react with sodium meta-periodate,
resulting in production of an aldehyde in place of the first amino
acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146;
U.S. Pat. No. 5,362,852). Such aldehyde can be reacted with a drug
moiety or linker nucleophile.
[0491] Likewise, nucleophilic groups on a drug moiety include, but
are not limited to: amine, thiol, hydroxyl, hydrazide, oxime,
hydrazine, thiosemicarbazone, hydrazine carboxylate, and
arylhydrazide groups capable of reacting to form covalent bonds
with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; (iii) aldehydes, ketones, carboxyl, and
maleimide groups.
[0492] Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g., by recombinant techniques or
peptide synthesis. The length of DNA may comprise respective
regions encoding the two portions of the conjugate either adjacent
one another or separated by a region encoding a linker peptide
which does not destroy the desired properties of the conjugate.
[0493] In yet another embodiment, the antibody may be conjugated to
a "receptor" (such streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
[0494] Antibody Derivatives
[0495] The antibodies of the present invention can be further
modified to contain additional nonproteinaceous moieties that are
known in the art and readily available. Preferably, the moieties
suitable for derivatization of the antibody are water soluble
polymers. Non-limiting examples of water soluble polymers include,
but are not limited to, polyethylene glycol (PEG), copolymers of
ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,
polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and
dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
propropylene glycol homopolymers, prolypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols (e.g., glycerol),
polyvinyl alcohol, and mixtures thereof. Polyethylene glycol
propionaldehyde may have advantages in manufacturing due to its
stability in water. The polymer may be of any molecular weight, and
may be branched or unbranched. The number of polymers attached to
the antibody may vary, and if more than one polymers are attached,
they can be the same or different molecules. In general, the number
and/or type of polymers used for derivatization can be determined
based on considerations including, but not limited to, the
particular properties or functions of the antibody to be improved,
whether the antibody derivative will be used in a therapy under
defined conditions, etc.
[0496] Pharmaceutical Formulations
[0497] Therapeutic formulations comprising an antibody of the
invention are prepared for storage by mixing the antibody having
the desired degree of purity with optional physiologically
acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the
form of aqueous solutions, lyophilized or other dried formulations.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, histidine and other organic
acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0498] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0499] The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0500] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0501] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the
immunoglobulin of the invention, which matrices are in the form of
shaped articles, e.g., films, or microcapsule. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated immunoglobulins remain
in the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0502] Uses
[0503] An antibody of the present invention may be used in, for
example, in vitro, ex vivo and in vivo therapeutic methods.
Antibodies of the invention can be used as an antagonist to
partially or fully block the specific antigen activity in vitro, ex
vivo and/or in vivo. Moreover, at least some of the antibodies of
the invention can neutralize antigen activity from other species.
Accordingly, the antibodies of the invention can be used to inhibit
a specific antigen activity, e.g., in a cell culture containing the
antigen, in human subjects or in other mammalian subjects having
the antigen with which an antibody of the invention cross-reacts
(e.g. chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or
mouse). In one embodiment, the antibody of the invention can be
used for inhibiting antigen activities by contacting the antibody
with the antigen such that antigen activity is inhibited.
Preferably, the antigen is a human protein molecule.
[0504] In one embodiment, an antibody of the invention can be used
in a method for inhibiting an antigen in a subject suffering from a
disorder in which the antigen activity is detrimental, comprising
administering to the subject an antibody of the invention such that
the antigen activity in the subject is inhibited. Preferably, the
antigen is a human protein molecule and the subject is a human
subject. Alternatively, the subject can be a mammal expressing the
antigen with which an antibody of the invention binds. Still
further the subject can be a mammal into which the antigen has been
introduced (e.g., by administration of the antigen or by expression
of an antigen transgene). An antibody of the invention can be
administered to a human subject for therapeutic purposes. Moreover,
an antibody of the invention can be administered to a non-human
mammal expressing an antigen with which the immunoglobulin
cross-reacts (e.g., a primate, pig or mouse) for veterinary
purposes or as an animal model of human disease. Regarding the
latter, such animal models may be useful for evaluating the
therapeutic efficacy of antibodies of the invention (e.g., testing
of dosages and time courses of administration). Blocking antibodies
of the invention that are therapeutically useful include, for
example but are not limited to, anti-HER2, anti-VEGF, anti-IgE,
anti-CD11, anti-interferon, anti-interferon receptor,
anti-hepatocyte growth factor (HGF), anti-c-met, and anti-tissue
factor antibodies. The antibodies of the invention can be used to
treat, inhibit, delay progression of, prevent/delay recurrence of,
ameliorate, or prevent diseases, disorders or conditions associated
with abnormal expression and/or activity of one or more antigen
molecules, including but not limited to malignant and benign
tumors; non-leukemias and lymphoid malignancies; neuronal, glial,
astrocytal, hypothalamic and other glandular, macrophagal,
epithelial, stromal and blastocoelic disorders; and inflammatory,
angiogenic and immunologic disorders.
[0505] In one aspect, a blocking antibody of the invention is
specific to a ligand antigen, and inhibits the antigen activity by
blocking or interfering with the ligand-receptor interaction
involving the ligand antigen, thereby inhibiting the corresponding
signal pathway and other molecular or cellular events. The
invention also features receptor-specific antibodies which do not
necessarily prevent ligand binding but interfere with receptor
activation, thereby inhibiting any responses that would normally be
initiated by the ligand binding. The invention also encompasses
antibodies that either preferably or exclusively bind to
ligand-receptor complexes. An antibody of the invention can also
act as an agonist of a particular antigen receptor, thereby
potentiating, enhancing or activating either all or partial
activities of the ligand-mediated receptor activation.
[0506] In certain embodiments, an immunoconjugate comprising an
antibody conjugated with a cytotoxic agent is administered to the
patient. In some embodiments, the immunoconjugate and/or antigen to
which it is bound is/are internalized by the cell, resulting in
increased therapeutic efficacy of the immunoconjugate in killing
the target cell to which it binds. In one embodiment, the cytotoxic
agent targets or interferes with nucleic acid in the target cell.
Examples of such cytotoxic agents include any of the
chemotherapeutic agents noted herein (such as a maytansinoid or a
calicheamicin), a radioactive isotope, or a ribonuclease or a DNA
endonuclease.
[0507] Antibodies of the invention can be used either alone or in
combination with other compositions in a therapy. For instance, an
antibody of the invention may be co-administered with another
antibody, chemotherapeutic agent(s) (including cocktails of
chemotherapeutic agents), other cytotoxic agent(s), anti-angiogenic
agent(s), cytokines, and/or growth inhibitory agent(s). Where an
antibody of the invention inhibits tumor growth, it may be
particularly desirable to combine it with one or more other
therapeutic agent(s) which also inhibits tumor growth. For
instance, an antibody of the invention may be combined with an
anti-VEGF antibody (e.g., AVASTIN) and/or anti-ErbB antibodies
(e.g. HERCEPTIN.RTM. anti-HER2 antibody) in a treatment scheme,
e.g. in treating any of the diseases described herein, including
colorectal cancer, metastatic breast cancer and kidney cancer.
Alternatively, or additionally, the patient may receive combined
radiation therapy (e.g. external beam irradiation or therapy with a
radioactive labeled agent, such as an antibody). Such combined
therapies noted above include combined administration (where the
two or more agents are included in the same or separate
formulations), and separate administration, in which case,
administration of the antibody of the invention can occur prior to,
and/or following, administration of the adjunct therapy or
therapies.
[0508] The antibody of the invention (and adjunct therapeutic
agent) is/are administered by any suitable means, including
parenteral, subcutaneous, intraperitoneal, intrapulmonary, and
intranasal, and, if desired for local treatment, intralesional
administration. Parenteral infusions include intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous
administration. In addition, the antibody is suitably administered
by pulse infusion, particularly with declining doses of the
antibody. Dosing can be by any suitable route, for e.g. by
injections, such as intravenous or subcutaneous injections,
depending in part on whether the administration is brief or
chronic.
[0509] The antibody composition of the invention will be
formulated, dosed, and administered in a fashion consistent with
good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular
mammal being treated, the clinical condition of the individual
patient, the cause of the disorder, the site of delivery of the
agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners.
The antibody need not be, but is optionally formulated with one or
more agents currently used to prevent or treat the disorder in
question. The effective amount of such other agents depends on the
amount of antibodies of the invention present in the formulation,
the type of disorder or treatment, and other factors discussed
above. These are generally used in the same dosages and with
administration routes as used hereinbefore or about from 1 to 99%
of the heretofore employed dosages.
[0510] For the prevention or treatment of disease, the appropriate
dosage of an antibody of the invention (when used alone or in
combination with other agents such as chemotherapeutic agents) will
depend on the type of disease to be treated, the type of antibody,
the severity and course of the disease, whether the antibody is
administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the
antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg)
of antibody is an initial candidate dosage for administration to
the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment is sustained until a desired suppression
of disease symptoms occurs. One exemplary dosage of the antibody
would be in the range from about 0.05 mg/kg to about 10 mg/kg.
Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or
10 mg/kg (or any combination thereof) may be administered to the
patient. Such doses may be administered intermittently, e.g. every
week or every three weeks (e.g. such that the patient receives from
about two to about twenty, e.g. about six doses of the antibody).
An initial higher loading dose, followed by one or more lower doses
may be administered. An exemplary dosing regimen comprises
administering an initial loading dose of about 4 mg/kg, followed by
a weekly maintenance dose of about 2 mg/kg of the antibody.
However, other dosage regimens may be useful. The progress of this
therapy is easily monitored by conventional techniques and
assays.
[0511] Articles of Manufacture
[0512] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
etc. The containers may be formed from a variety of materials such
as glass or plastic. The container holds a composition which is by
itself or when combined with another composition effective for
treating, preventing and/or diagnosing the condition and may have a
sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). At least one active agent in the
composition is an antibody of the invention. The label or package
insert indicates that the composition is used for treating the
condition of choice, such as cancer. Moreover, the article of
manufacture may comprise (a) a first container with a composition
contained therein, wherein the composition comprises an antibody of
the invention; and (b) a second container with a composition
contained therein, wherein the composition comprises a further
cytotoxic agent. The article of manufacture in this embodiment of
the invention may further comprise a package insert indicating that
the first and second antibody compositions can be used to treat a
particular condition, for e.g. cancer. Alternatively, or
additionally, the article of manufacture may further comprise a
second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0513] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLE 1
Construction of Phage-Displayed Fab Libraries with CDR Residues
Randomized as Only Tyr or Ser
[0514] Phage-displayed Fab libraries were constructed using a
phagemid vector that resulted in the display of bivalent Fab
moieties dimerized by a leucine zipper domain inserted between the
Fab heavy chain and the C-terminal domain of the gene-3 minor coat
protein (P3C). This vector comprises the sequence shown in FIG. 18
(SEQ ID NO:4). The vector (schematically illustrated in FIG. 19)
comprises the humanized antibody 4D5 variable domains under the
control of the IPTG-inducible Ptac promoter. The humanized antibody
4D5 is an antibody which has mostly human consensus sequence
framework regions in the heavy and light chains, and CDR regions
from a mouse monoclonal antibody specific for Her-2. The method of
making the anti-Her-2 antibody and the identity of the variable
domain sequences are provided in U.S. Pat. Nos. 5,821,337 and
6,054,297.
[0515] Two libraries were constructed. Library YS-A was constructed
with randomized residues in all three heavy chain CDRs, while
Library YS-B was constructed with randomized residues in all three
heavy chain CDRs and light chain CDR3. The specific residues that
were randomized are shown in the FIG. 1.
[0516] At each of the randomized positions, the wild-type codon was
replaced by a degenerate TMT codon (M=A/C in an equimolar ratio)
that encoded for Tyr and Ser in an equimolar ratio. In addition,
the length of CDRH3 was varied by using oligonucleotides that
replaced the 7 wild-type codons between positions 101 to 107 with
varying numbers of TMT codons (7 to 20 for Library YS-A and 7 to 15
for Library YS-B). In addition, the CDRL3 of Library YS-B was
randomized so that 50% of the library members contained a deletion
at position number 91 while the other 50% contained the wildtype
Gln residue at this position.
[0517] Libraries were constructed using the method of Kunkel
(Kunkel, T. A., Roberts, J. D. & Zakour, R. A., Methods
Enzymol. (1987), 154, 367-382) with previously described methods
(Sidhu, S. S., Lowman, H. B., Cunningham, B. C. & Wells, J. A.,
Methods Enzymol. (2000), 328, 333-363). A unique "stop template"
version of the Fab display vector was used to generate both
libraries YS-A and YS-B. We used a template phagemid designated
pV0350-4 (the phagemid vector comprises the sequence shown in FIG.
24; SEQ ID NO: 5) with TAA stop codons inserted at positions 30,
33, 52, 54, 56, 57, 60, 102, 103, 104, 107, 108 of the heavy chain.
No stops were introduced in the light chain CDR3. Mutagenic
oligonucleotides with degenerate TMT codons at the positions to be
diversified were used to simultaneously introduce CDR diversity and
repair the stop codons. The oligonucleotide sequences are shown in
FIG. 2. For both libraries, diversity was introduced into CDR-H1
and CDR-H2 with oligonucleotides H1 and H2, respectively. For
Library YS-A, diversity was introduced into CDR-H3 with an
equimolar mixture of oligonucleotides H3-7, H3-8, H3-9, H3-10,
H3-11, H3-12, H3-13, H3-14, H3-15, H3-16, H3-17, H3-18, H3-19, and
H3-20. For library YS-B, diversity was introduced into CDR-H3 with
an equimolar mixture of oligonucleotides H3-7, H3-8, H3-9, H3-10,
H3-11, H3-12, H3-13, H3-14, and H3-15. For library YS-B, diversity
was introduced into CDR-L3 with an equimolar mixture of
oligonucleotides L3a and L3b. The mutagenic oligonucleotides for
all CDRs to be randomized were incorporated simultaneously in a
single mutagenesis reaction, so that simultaneous incorporation of
all the mutagenic oligonucleotides resulted in the introduction of
the designed diversity at each position and simultaneously repaired
all the TAA stop codons, thus generating an open reading frame that
encoded a Fab library member fused to a homodimerizing leucine
zipper and P3C.
[0518] The mutagenesis reactions were electroporated into E. coli
SS320 (Sidhu et al., supra), and the transformed cells were grown
overnight in the presence of M13-KO7 helper phage (New England
Biolabs, Beverly, Mass.) to produce phage particles that
encapsulated the phagemid DNA and displayed Fab fragments on their
surfaces. Each library contained greater than 5.times.10.sup.9
unique members.
EXAMPLE 2
Selection of Specific Antibodies from the Nave Libraries YS-A and
YS-B
[0519] Phage from library YS-A or YS-B (Example I) were cycled
through rounds of binding selection to enrich for clones binding to
targets of interest. Eight target proteins were analyzed separately
with each library: human VEGF, murine VEGF, neutravidin, an
apoptosis protein (AP), maltose binding protein, erbin-GST fusion,
and Insulin. The binding selections were conducted using previously
described methods (Sidhu et al., supra).
[0520] NUNC 96-well Maxisorp immunoplates were coated overnight at
4.degree. C. with capture target (5 .mu.g/mL) and blocked for 2 h
with Superblock TBS (tris-buffered saline) (Pierce). After
overnight growth at 37.degree. C., phage were concentrated by
precipitation with PEG/NaCl and resuspended in Superblock TBS,
0.05% Tween 20 (Sigma), as described previously (Sidhu et al.,
supra). Phage solutions (.about.10.sup.12 phage/mL) were added to
the coated immunoplates. Following a 2 h incubation to allow for
phage binding, the plates were washed 10 times with PBS, 0.05%
Tween 20. Bound phage were eluted with 0.1 M HCl for 10 min and the
eluant was neutralized with 1.0 M Tris base. Eluted phage were
amplified in E. coli XL1-blue and used for further rounds of
selection.
[0521] The libraries were subjected to 5 rounds of selection
against each target protein, and at each round, titers were
obtained for phage binding to either the target protein or blank
wells coated with Superblock TBS. The titer of phage bound to
target-coated wells divided by the titer of phage bound to the
blank wells was defined as an enrichment ratio used to quantify
specific binding of phage pools to the target protein; larger
enrichment ratios indicate higher specific binding. The enrichment
ratios observed after 3, 4, or 5 rounds of selection are shown in
FIG. 3.
[0522] Individual clones from each round of selection were grown in
a 96-well format in 500 RL of 2YT broth supplemented with
carbenicillin and M13-VCS, and the culture supernatants were used
directly in phage ELISAs (Sidhu et al., supra) to detect
phage-displayed Fabs that bound to plates coated with target
protein but not to plates coated with BSA. Specific binders were
defined as those phage clones that exhibited an ELISA signal at
least 15-fold greater on target-coated plates in comparison with
BSA-coated plates. Individual clones were screened after 2 rounds
of selection for binding to human VEGF or after 5 rounds of
selection for the other target proteins. These data were used to
calculate the percentage of specific binders, and the results for
each library against each target protein are shown in FIG. 4; it
can be seen that each library produced binders against each target
protein, with the exception of the YS-A library with respect to
MBP2.
[0523] Individual clones representing specific binders were
subjected to DNA sequence analysis, and the sequences of the
randomized CDR positions for some of the targets are shown in FIG.
5. It can be seen that, for each target protein, it was possible to
select specific binders that contained only Tyr or Ser at the
randomized positions (although some non-designed mutations were
observed, which were likely created during library construction
probably due to impurities in the oligonucleotides). Furthermore,
the sequences of specific binders were unique to the target protein
against which they were selected.
[0524] Two anti-VEGF binders were tested for their affinity with
respect to hVEGF and MVEGF. BIAcore data was obtained according to
Chen et al., J Mol Biol. (1999), 293(4):865-81. Briefly, binding
affinities of hVEGF binders for hVEGF and mVEGF were calculated
from association and dissociation rate constants measured using a
BIAcore.TM.-2000 surface plasmon resonance system (BIAcore, Inc.,
Piscataway, N.J.). A biosensor chip was activated for covalent
coupling of VEGF using
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
(BIAcore, Inc., Piscataway, N.J.) instructions. hVEGF or mVEGF was
buffer-exchanged into 10 mM sodium acetate, pH 4.8 and diluted to
approximately 30 .mu.g/ml. Aliquots of VEGF were injected at a flow
rate of 2 microL/minute to achieve approximately 200-300 response
units (RU) of coupled protein. A solution of 1 M ethanolamine was
injected as a blocking agent. For kinetics measurements, twofold
serial dilutions of Fab were injected in PBS/Tween buffer (0.05%
Tween20 in phosphate-buffered saline) at 25.degree. C. at a flow
rate of 10 microL/minute. Equilibrium dissociation constants, Kd
values from surface plasmon resonance measurements were calculated
as k.sub.off/k.sub.on. The BIAcore.TM. data is summarized in FIG.
23.
[0525] The IC50 values for selected anti-AP clones were determined
by phage ELISA, as described previously (Sidhu et al., supra). The
values are shown in FIG. 11.
EXAMPLE 3
Construction of a Phage-Displayed Fab Library (F0505) with CDR
Residues Randomized with Tetranomial Codons Encoding Four Amino
Acids
[0526] Phage displayed libraries were constructed, as described in
Example 1, with a previously described phagemid designed to display
bivalent Fab moieties dimerized by a leucine zipper domain inserted
between the Fab heavy chain and the C-terminal domain of the gene-3
minor coat protein (P3C) (as described in Example 1). CDR positions
in the heavy chain were randomized, positions as shown in FIG. 1.
Eleven separate mutagenesis reactions were performed with each
mutagenesis reaction designed to randomize the CDR positions with a
tetranomial codon that encoded for only four amino acids. In each
mutagenesis reaction, the CDR positions were simultaneously
replaced with only one type of tetranomial codon. The eleven
tetranomial codons used for the eleven mutagenesis reactions and
the amino acids they encode are shown in FIG. 6. For each
mutagenesis, three mutagenic oligonucleotides were used, with each
designed to introduce diversity into one of the three heavy chain
CDRs. The sequences of the oligonucleotides were as follows:
2 (SEQ ID NO: 8) CDR-H1: GCA GCT TCT GGC TTC XXX ATT XXX XXX XXX
XXX ATA CAC TGG GTG CGT (SEQ ID NO: 9) CDR-H2: CTG GAA TGG GTT GCA
XXX ATT XXX CCA XXX XXX GGT XXX ACT XXX TAT GCC GAT AGC GTC (SEQ ID
NO: 10) CDR-H3: GTC TAT TAT TGT AGC CGC XXX XXX XXX XXX XXX XXX XXX
ATG GAC TAC TGG
[0527] In each oligonucleotide "XXX" denotes a degenerate codon at
which the wild-type codon was replaced with one of the tetranomial
codons shown in FIG. 6.
[0528] The eleven mutagenesis reactions were pooled and
electroporated into E. coli SS320 (Sidhu et al., supra), and the
transformed cells were grown overnight in the presence of M13-KO7
helper phage (New England Biolabs, Beverly, Mass.) to produce phage
particles that encapsulated the phagemid DNA and displayed Fab
fragments on their surfaces. The library contained
2.6.times.10.sup.10 unique members, and it was named library
F0505.
EXAMPLE 4
Selection of Specific Antibodies from the Tetranomial Nave Library
F0505
[0529] Phage from library F0505 (Example 3) were cycled through
rounds of binding selection to enrich for clones binding four
different targets: IGF, h-VEGF, anti-hGH, hGH binding protein. The
binding selections were conducted using previously described
methods (Sidhu et al., supra).
[0530] NUNC 96-well Maxisorp immunoplates were coated overnight at
4.degree. C. with capture target (5 .mu.g/mL) and blocked for 2 h
with BSA (Sigma). After overnight growth at 37.degree. C., phage
were concentrated by precipitation with PEG/NaCl and resuspended in
PBS, 0.5% BSA, 0.05% Tween 20 (Sigma), as described previously
(Sidhu et al., supra). Phage solutions (.about.10.sup.12 phage/mL)
were added to the coated immunoplates. Following a 2 h incubation
to allow for phage binding, the plates were washed 10 times with
PBS, 0.05% Tween20. Bound phages were eluted with 0.1 M HCl for 10
min and the eluant was neutralized with 1.0 M Tris base. Eluted
phage were amplified in E. coli XL1-blue and used for further
rounds of selection.
[0531] The libraries were subjected to 4 rounds of selection
against each target protein. After rounds 2 and 3, individual
clones from each round and each target selection were grown in a
96-well format in 500 .mu.L of 2YT broth supplemented with
carbenicillin and M13-VCS, and the culture supernatants were used
directly in phage ELISAs (Sidhu et al., supra) to detect
phage-displayed Fabs that bound to plates coated with target
protein but not to plates coated with BSA. A clone is considered to
be a specific binder if the ELISA signal on plates coated with
target protein was at least 10 times greater than the signal on BSA
coated plates. The number of specific binders for each round and
each target is tabulated in FIG. 7.
[0532] The specific clones were subjected to DNA sequence analysis.
The library of origin for each of the unique sequence were
determined and summarized in FIG. 8.
EXAMPLE 5
Construction of Phage-Displayed Fab Libraries YADS-A and YADS-B
[0533] Two phage displayed libraries (YADS-A and YADS-B) were
constructed, as described in Example 1, with a previously described
phagemid designed to display bivalent Fab moieties dimerized by a
leucine zipper domain inserted between the Fab heavy chain and the
C-terminal domain of the gene-3 minor coat protein (P3C) (as
described in Example 1). CDR positions in the heavy chain were
randomized, positions as shown in FIG. 1. The oligonucleotide
sequences are shown in FIG. 9.
[0534] For library YADS-A, two separate mutagenesis reactions were
performed. In the first reaction, diversity was introduced into
CDR-H1, CDR H2 and CDR-H3 with oligonucleotides YADS-H1, YADS-H2
and YADS-H3-7, respectively. This resulted in the introduction of
degenerate codons that encoded for the four amino acids tyrosine,
alanine, aspartate, and serine. In the second reaction, diversity
was introduced into CDR-H1, CDR H2 and CDR-H3 with oligonucleotides
YTNS-H1, YTNS-H2 and YTNS-H3-7, respectively. This resulted in the
introduction of degenerate codons that encoded for the four amino
acids tyrosine, threonine, asparagine, and serine. The two
reactions were pooled.
[0535] For library YADS-B, 13 separate mutagenesis reactions were
peformed. The reactions resulted in the introduction of degenerate
codons that encoded for the four amino acids tyrosine, alanine,
aspartate, and serine. In each reaction, diversity was introduced
into CDR-H1 and CDR-H2 with oligonucleotides YADS-H1 and YADS-H2.
For each reaction, one of the following oligonucleotides was used
to introduce diversity into CDR-H3: YADS-H3-3, YADS-H3-4,
YADS-H3-5, YADS-H3-6, YADS-H3-7, YADS-H3-8, YADS-H3-9, YADS-H3-10,
YADS-H3-11, YADS-H3-12, YADS-H3-13, YADS-H3-14, or YADS-H3-15. The
13 reactions were pooled.
[0536] For both libraries, the pooled mutagenesis reactions were
electroporated in E. coli SS320 (Sidhu et al., supra). The
transformed cells were grown overnight in the presence of M13-KO7
helper phage (New England Biolabs, Beverly, Mass.) to produce phage
particles that encapsulated the phagemid DNA and displayed Fab
fragments on their surfaces. The size of library YADS-A and YADS-B
were both 7.times.10.sup.9.
EXAMPLE 6
Selection of Anti-hVEGF Specific Antibodies from YADS-A and YADS-B
Nave Libraries
[0537] Phage from library YADS-A and YADS-B (Example 5) were cycled
seperately through rounds of binding selection to enrich for clones
binding to h-VEGF. The binding selections were conducted using
previously described methods (Sidhu et al., supra).
[0538] NUNC 96-well Maxisorp immunoplates were coated overnight at
4.degree. C. with capture target (5 .mu.g/mL) and blocked for 2 h
with BSA (Sigma). After overnight growth at 37.degree. C., phage
were concentrated by precipitation with PEG/NaCl and resuspended in
PBS, 0.5% BSA, 0.05% Tween 20 (Sigma), as described previously
(Sidhu et al., supra). Phage solutions (.about.10.sup.12 phage/mL)
were added to the coated immunoplates. Following a 2 h incubation
to allow for phage binding, the plates were washed 10 times with
PBS, 0.05% Tween20. Bound phages were eluted with 0.1 M HCl for 10
min and the eluant was neutralized with 1.0 M Tris base. Eluted
phage were amplified in E. coli XL1-blue and used for further
rounds of selection.
[0539] The libraries were subjected to 4 rounds of selection
against each target protein. Individual clones from each round were
grown in a 96-well format in 500 .mu.L of 2YT broth supplemented
with carbenicillin and M13-VCS, and the culture supernatants were
used directly in phage ELISAs (Sidhu et al., supra) to detect
phage-displayed Fabs that bound to plates coated with target
protein but not to plates coated with BSA. A clone was considered
to be a specific binder if the ELISA signal on target coated plates
was at least 20 times greater than that on BSA coated plates. The
results are tabulated in FIG. 10. Multiple unique sequences of
specific binders were obtained (data not shown).
EXAMPLE 7
Construction of Library YADS-II for Affinity Maturation of
VEGF-Binding Clones Isolated from Libraries YADS-A and YADS-B
[0540] The sequencing of VEGF-binding clones selected from
libraries YADS-A and YADS-B (Examples 5 and 6) revealed 24 unique
clones in which the randomized heavy chain CDR positions contained
only tyrosine, alanine, asparte, or serine. We wanted to improve
the affinity of 16 of these clones by introducing diversity into
the light chain CDRs with degenerate codons that encoded for only
tyrosine, alanine, aspartate, or serine.
[0541] The Kunkel method of site-directed mutagenesis (Kunkel et
al., supra) was used to construct 16 "stop template" versions of
phagemids used in this Example. Codons in the light chain CDRs
(positions 29, 32, 51, 54, 55, 93, 94 and 97) were replaced with
TAA stop codons. Sixteen separate mutagenesis reactions (one with
each template) were performed with three oligonucleotides designed
to simultaneously repair the stop codons and introduce degenerate
codons encoding for tyrosine, alanine, aspartate, and serine. The
mutagenic oligonucleotides YADS-L1, YADS-L2, and YADS-L3 were used
to introduce diversity into CDR-L 1, CDR-L2, and CDR-L3,
respectively. The oligonucleotide sequences are shown in FIG. 13
and the light chain CDR sites that were randomized are shown in
FIG. 12.
[0542] The 16 mutagenesis reactions were pooled and electroporated
into E. coli SS320 (Sidhu et al., supra). The transformed cells
were grown overnight in the presence of M13-KO7 helper phage (New
England Biolabs, Beverly, Mass.) to produce phage particles that
encapsulated the phagemid DNA and displayed Fab fragments on their
surfaces. The library contained 6.5.times.10.sup.9 unique members,
and it was named library YADS-II.
EXAMPLE 8
Selection of Anti-hVEGF Specific Antibodies from YADS-II
Library
[0543] Phage from library YADS-II (Example 7) were cycled through
rounds of binding selection to enrich for clones binding h-VEGF.
The binding selections were conducted as follows.
[0544] Library YADS-II was selected on solid support followed by
two rounds of selection in solution. For the first round of
selection, NUNC 96-well Maxisorp immunoplates were coated overnight
at 4.degree. C. with capture h-VEGF (5 .mu.g/mL) and blocked for 2
h with BSA (Sigma). After overnight growth at 37.degree. C., phage
were concentrated by precipitation with PEG/NaCl and resuspended in
PBS, 0.5% BSA, 0.05% Tween 20 (Sigma), as described previously
(Sidhu et al., supra). Phage solutions (.about.10.sup.12 phage/mL)
were added to the coated immunoplates. Following a 2 h incubation
to allow for phage binding, the plates were washed 10 times with
PBS, 0.05% Tween20. Bound phages were eluted with 0.1 M HCl for 10
min and the eluant was neutralized with 1.0 M Tris base. Eluted
phage were amplified in E. coli XL1-blue and used for further
rounds of selection.
[0545] For both following rounds of selection, the selection was
done in solution. After overnight growth at 37.degree. C., phage
were concentrated by precipitation with PEG/NaCl and resuspended in
Superblock 1% TBS (Pierce), 0.05% Tween 20 (Sigma), as described
above. Phage solutions (200 .mu.L at a concentration close to
10.sup.12 phage/mL) were incubated with biotinylated h-VEGF at a
concentration of 25 nM. After 2 hours of incubation at room
temperature with gentle shaking, 800 uL of Superblock plus 0.05%
Tween 20 was added. 800 uL of this dilution was incubated on 8
wells coated with neutravidin (Pierce) at 5 ng/uL and saturated
with Superblock solution. After an incubation of 5 minutes at room
temperature with gentle shaking, the plates were washed 10 times
with PBS 0.05% Tween20. The phage was eluted with 100 uL of HCl 100
mM per well and neutralized with IM TRIS base. Eluted phage were
amplified in E. coli XL I-blue.
[0546] Two hundred individual clones from each round were grown in
a 96-well format in 500 .mu.L of 2YT broth supplemented with
carbenicillin and M13-VCS, and the culture supernatants were used
directly in phage ELISAs (Sidhu et al., supra) to detect
phage-displayed Fabs that bound to plates coated with target
protein but not to plates coated with BSA. A clone was considered
to be a specific binder if the ELISA signal on target coated plates
was at least 20 times greater than that on BSA coated plates. The
results are tabulated in FIG. 14.
[0547] Based on the amount of inhibition of binding by 100 nM of
hVEGF, three binders were further analyzed. The measurement of
binding on other proteins (FIG. 16) was determined for these three
binders. These binders were expressed as Fab proteins in E. coli,
and their binding affinities to hVEGF and mVEGF measured by Biacore
as described in Example 2. Data is summarized in FIG. 17.
[0548] All publications (including patents and patent applications)
cited herein are hereby incorporated in their entirety by
reference.
Sequence CWU 1
1
130 1 109 PRT Artificial sequence Humanized antibody light chain 1
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10
15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn 20
25 30 Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45 Leu Leu Ile Tyr Ser Ala Ser Phe Leu Glu Ser Gly Val Pro
Ser 50 55 60 Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu
Thr Ile 65 70 75 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln 80 85 90 His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly
Thr Lys Val Glu 95 100 105 Ile Lys Arg Thr 2 120 PRT Artificial
sequence Humanized antibody heavy chain 2 Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys 20 25 30 Asp Thr Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45 Glu Trp Val
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr 50 55 60 Ala Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser 65 70 75 Lys
Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90
Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr 95 100
105 Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110
115 120 3 35 PRT Artificial sequence GCN4 sequence 3 Gly Arg Met
Lys Gln Leu Glu Asp Lys Val Glu Glu Leu Leu Ser 1 5 10 15 Lys Asn
Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu 20 25 30 Val
Gly Glu Arg Gly 35 4 2383 DNA Artificial sequence Phage display
vector 4 gaaatgagct gttgacaatt aatcatcggc tcgtataatg tgtggaattg 50
tgagcggata acaatttcac acaggaaaca gccagtccgt ttaggtgttt 100
tcacgagcac ttcaccaaca aggaccatag attatgaaaa taaaaacagg 150
tgcacgcatc ctcgcattat ccgcattaac gacgatgatg ttttccgcct 200
cggcttatgc atccgatatc cagatgaccc agtccccgag ctccctgtcc 250
gcctctgtgg gcgatagggt caccatcacc tgccgtgcca gtcaggatgt 300
gaatactgct gtagcctggt atcaacagaa accaggaaaa gctccgaagc 350
ttctgattta ctcggcatcc ttcctctact ctggagtccc ttctcgcttc 400
tctggtagcc gttccgggac ggatttcact ctgaccatca gcagtctgca 450
gccggaagac ttcgcaactt attactgtca gcaacattat actactcctc 500
ccacgttcgg acagggtacc aaggtggaga tcaaacgaac tgtggctgca 550
ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac 600
tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga gaggccaaag 650
tacagtggaa ggtggataac gccctccaat cgggtaactc ccaggagagt 700
gtcacagagc aggacagcaa ggacagcacc tacagcctca gcagcaccct 750
gacgctgagc aaagcagact acgagaaaca caaagtctac gcctgcgaag 800
tcacccatca gggcctgagc tcgcccgtca caaagagctt caacagggga 850
gagtgtggtg ccagctccgg tatggctgat ccgaaccgtt tccgcggtaa 900
ggacctggca taactcgagg ctgatcctct acgccggacg catcgtggcc 950
ctagtacgca agttcacgta aaaagggtaa ctagaggttg aggtgatttt 1000
atgaaaaaga atatcgcatt tcttcttgca tctatgttcg ttttttctat 1050
tgctacaaac gcgtacgctg agatctccga ggttcagctg gtggagtctg 1100
gcggtggcct ggtgcagcca gggggctcac tccgtttgtc ctgtgcagct 1150
tctggcttca acattaaaga cacctatata cactgggtgc gtcaggcccc 1200
gggtaagggc ctggaatggg ttgcaaggat ttatcctacg aatggttata 1250
ctagatatgc cgatagcgtc aagggccgtt tcactataag cgcagacaca 1300
tccaaaaaca cagcctacct acaaatgaac agcttaagag ctgaggacac 1350
tgccgtctat tattgtagcc gctggggagg ggacggcttc tatgctatgg 1400
actactgggg tcaaggaacc ctggtcaccg tctcctcggc ctccaccaag 1450
ggcccatcgg tcttccccct ggcaccctcc tccaagagca cctctggggg 1500
cacagcggcc ctgggctgcc tggtcaagga ctacttcccc gaaccggtga 1550
cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 1600
gctgtcctac agtcctcagg actctactcc ctcagcagcg tggtgaccgt 1650
gccctccagc agcttgggca cccagaccta catctgcaac gtgaatcaca 1700
agcccagcaa caccaaggtc gacaagaaag ttgagcccaa atcttgtgac 1750
aaaactcaca catgcccgcc gtgcccagca ccagaactgc tgggcggccg 1800
catgaaacag ctagaggaca aggtcgaaga gctactctcc aagaactacc 1850
acctagagaa tgaagtggca agactcaaaa aacttgtcgg ggagcgcgga 1900
aagcttagtg gcggtggctc tggttccggt gattttgatt atgaaaagat 1950
ggcaaacgct aataaggggg ctatgaccga aaatgccgat gaaaacgcgc 2000
tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt 2050
gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa 2100
tggtgctact ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg 2150
gtgacggtga taattcacct ttaatgaata atttccgtca atatttacct 2200
tccctccctc aatcggttga atgtcgccct tttgtcttta gcgctggtaa 2250
accatatgaa ttttctattg attgtgacaa aataaactta ttccgtggtg 2300
tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttctacg 2350
tttgctaaca tactgcgtaa taaggagtct taa 2383 5 7171 DNA Artificial
sequence Phage display vector 5 gaattcaact tctccatact ttggataagg
aaatacagac atgaaaaatc 50 tcattgctga gttgttattt aagcttgccc
aaaaagaaga agagtcgaat 100 gaactgtgtg cgcaggtaga agctttggag
attatcgtca ctgcaatgct 150 tcgcaatatg gcgcaaaatg accaacagcg
gttgattgat caggtagagg 200 gggcgctgta cgaggtaaag cccgatgcca
gcattcctga cgacgatacg 250 gagctgctgc gcgattacgt aaagaagtta
ttgaagcatc ctcgtcagta 300 aaaagttaat cttttcaaca gctgtcataa
agttgtcacg gccgagactt 350 atagtcgctt tgtttttatt ttttaatgta
tttgtaacta gtacgcaagt 400 tcacgtaaaa agggtatgta gaggttgagg
tgattttatg aaaaagaata 450 tcgcatttct tcttgcatct atgttcgttt
tttctattgc tacaaatgcc 500 tatgcatccg atatccagat gacccagtcc
ccgagctccc tgtccgcctc 550 tgtgggcgat agggtcacca tcacctgccg
tgccagtcag gatgtgtcca 600 ctgctgtagc ctggtatcaa cagaaaccag
gaaaagctcc gaagcttctg 650 atttactcgg catccttcct ctactctgga
gtcccttctc gcttctctgg 700 tagcggttcc gggacggatt tcactctgac
catcagcagt ctgcagccgg 750 aagacttcgc aacttattac tgtcagcaat
cttatactac tcctcccacg 800 ttcggacagg gtaccaaggt ggagatcaaa
cgaactgtgg ctgcaccatc 850 tgtcttcatc ttcccgccat ctgatgagca
gttgaaatct ggaactgcct 900 ctgttgtgtg cctgctgaat aacttctatc
ccagagaggc caaagtacag 950 tggaaggtgg ataacgccct ccaatcgggt
aactcccagg agagtgtcac 1000 agagcaggac agcaaggaca gcacctacag
cctcagcagc accctgacgc 1050 tgagcaaagc agactacgag aaacacaaag
tctacgcctg cgaagtcacc 1100 catcagggcc tgagctcgcc cgtcacaaag
agcttcaaca ggggagagtg 1150 tggtgccagc tccggtatgg ctgatccgaa
ccgtttccgc ggtaaggacc 1200 tggcataact cgaggctgat cctctacgcc
ggacgcatcg tggccctagt 1250 acgcaagttc acgtaaaaag ggtaactaga
ggttgaggtg attttatgaa 1300 aaagaatatc gcatttcttc ttgcatctat
gttcgttttt tctattgcta 1350 caaacgcgta cgctgaggtt cagctggtgg
agtctggcgg tggcctggtg 1400 cagccagggg gctcactccg tttgtcctgt
gcagcttctg gcttcaacat 1450 taaagacacc tatatacact gggtgcgtca
ggccccgggt aagggcctgg 1500 aatgggttgc aaggatttat cctacgaatg
gttatactag atatgccgat 1550 agcgtcaagg gccgtttcac tataagcgca
gacacatcca aaaacacagc 1600 ctacctacaa atgaacagct taagagctga
ggacactgcc gtctattatt 1650 gtagccgctg gggaggggac ggcttctatg
ctatggacta ctggggtcaa 1700 ggaacactag tcaccgtctc ctcggcctcc
accaagggcc catcggtctt 1750 ccccctggca ccctcctcca agagcacctc
tgggggcaca gcggccctgg 1800 gctgcctggt caaggactac ttccccgaac
cggtgacggt gtcgtggaac 1850 tcaggcgccc tgaccagcgg cgtgcacacc
ttcccggctg tcctacagtc 1900 ctcaggactc tactccctca gcagcgtggt
gaccgtgccc tccagcagct 1950 tgggcaccca gacctacatc tgcaacgtga
atcacaagcc cagcaacacc 2000 aaggtcgaca agaaagttga gcccaaatct
tgtgacaaaa ctcacggccg 2050 catgaaacag ctagaggaca aggtcgaaga
gctactctcc aagaactacc 2100 acctagagaa tgaagtggca agactcaaaa
aacttgtcgg ggagcgcgga 2150 aagcttagtg gcggtggctc tggttccggt
gattttgatt atgaaaagat 2200 ggcaaacgct aataaggggg ctatgaccga
aaatgccgat gaaaacgcgc 2250 tacagtctga cgctaaaggc aaacttgatt
ctgtcgctac tgattacggt 2300 gctgctatcg atggtttcat tggtgacgtt
tccggccttg ctaatggtaa 2350 tggtgctact ggtgattttg ctggctctaa
ttcccaaatg gctcaagtcg 2400 gtgacggtga taattcacct ttaatgaata
atttccgtca atatttacct 2450 tccctccctc aatcggttga atgtcgccct
tttgtcttta gcgctggtaa 2500 accatatgaa ttttctattg attgtgacaa
aataaactta ttccgtggtg 2550 tctttgcgtt tcttttatat gttgccacct
ttatgtatgt attttctacg 2600 tttgctaaca tactgcgtaa taaggagtct
taatcatgcc agttcttttg 2650 gctagcgccg ccctatacct tgtctgcctc
cccgcgttgc gtcgcggtgc 2700 atggagccgg gccacctcga cctgaatgga
agccggcggc acctcgctaa 2750 cggattcacc actccaagaa ttggagccaa
tcaattcttg cggagaactg 2800 tgaatgcgca aaccaaccct tggcagaaca
tatccatcgc gtccgccatc 2850 tccagcagcc gcacgcggcg catctcgggc
agcgttgggt cctggccacg 2900 ggtgcgcatg atcgtgctcc tgtcgttgag
gacccggcta ggctggcggg 2950 gttgccttac tggttagcag aatgaatcac
cgatacgcga gcgaacgtga 3000 agcgactgct gctgcaaaac gtctgcgacc
tgagcaacaa catgaatggt 3050 cttcggtttc cgtgtttcgt aaagtctgga
aacgcggaag tcagcgccct 3100 gcaccattat gttccggatc tgcatcgcag
gatgctgctg gctaccctgt 3150 ggaacaccta catctgtatt aacgaagcgc
tggcattgac cctgagtgat 3200 ttttctctgg tcccgccgca tccataccgc
cagttgttta ccctcacaac 3250 gttccagtaa ccgggcatgt tcatcatcag
taacccgtat cgtgagcatc 3300 ctctctcgtt tcatcggtat cattaccccc
atgaacagaa attccccctt 3350 acacggaggc atcaagtgac caaacaggaa
aaaaccgccc ttaacatggc 3400 ccgctttatc agaagccaga cattaacgct
tctggagaaa ctcaacgagc 3450 tggacgcgga tgaacaggca gacatctgtg
aatcgcttca cgaccacgct 3500 gatgagcttt accgcaggat ccggaaattg
taaacgttaa tattttgtta 3550 aaattcgcgt taaatttttg ttaaatcagc
tcatttttta accaataggc 3600 cgaaatcggc aaaatccctt ataaatcaaa
agaatagacc gagatagggt 3650 tgagtgttgt tccagtttgg aacaagagtc
cactattaaa gaacgtggac 3700 tccaacgtca aagggcgaaa aaccgtctat
cagggctatg gcccactacg 3750 tgaaccatca ccctaatcaa gttttttggg
gtcgaggtgc cgtaaagcac 3800 taaatcggaa ccctaaaggg agcccccgat
ttagagcttg acggggaaag 3850 ccggcgaacg tggcgagaaa ggaagggaag
aaagcgaaag gagcgggcgc 3900 tagggcgctg gcaagtgtag cggtcacgct
gcgcgtaacc accacacccg 3950 ccgcgcttaa tgcgccgcta cagggcgcgt
ccggatcctg cctcgcgcgt 4000 ttcggtgatg acggtgaaaa cctctgacac
atgcagctcc cggagacggt 4050 cacagcttgt ctgtaagcgg atgccgggag
cagacaagcc cgtcagggcg 4100 cgtcagcggg tgttggcggg tgtcggggcg
cagccatgac ccagtcacgt 4150 agcgatagcg gagtgtatac tggcttaact
atgcggcatc agagcagatt 4200 gtactgagag tgcaccatat gcggtgtgaa
ataccgcaca gatgcgtaag 4250 gagaaaatac cgcatcaggc gctcttccgc
ttcctcgctc actgactcgc 4300 tgcgctcggt cgttcggctg cggcgagcgg
tatcagctca ctcaaaggcg 4350 gtaatacggt tatccacaga atcaggggat
aacgcaggaa agaacatgtg 4400 agcaaaaggc cagcaaaagg ccaggaaccg
taaaaaggcc gcgttgctgg 4450 cgtttttcca taggctccgc ccccctgacg
agcatcacaa aaatcgacgc 4500 tcaagtcaga ggtggcgaaa cccgacagga
ctataaagat accaggcgtt 4550 tccccctgga agctccctcg tgcgctctcc
tgttccgacc ctgccgctta 4600 ccggatacct gtccgccttt ctcccttcgg
gaagcgtggc gctttctcat 4650 agctcacgct gtaggtatct cagttcggtg
taggtcgttc gctccaagct 4700 gggctgtgtg cacgaacccc ccgttcagcc
cgaccgctgc gccttatccg 4750 gtaactatcg tcttgagtcc aacccggtaa
gacacgactt atcgccactg 4800 gcagcagcca ctggtaacag gattagcaga
gcgaggtatg taggcggtgc 4850 tacagagttc ttgaagtggt ggcctaacta
cggctacact agaaggacag 4900 tatttggtat ctgcgctctg ctgaagccag
ttaccttcgg aaaaagagtt 4950 ggtagctctt gatccggcaa acaaaccacc
gctggtagcg gtggtttttt 5000 tgtttgcaag cagcagatta cgcgcagaaa
aaaaggatct caagaagatc 5050 ctttgatctt ttctacgggg tctgacgctc
agtggaacga aaactcacgt 5100 taagggattt tggtcatgag attatcaaaa
aggatcttca cctagatcct 5150 tttaaattaa aaatgaagtt ttaaatcaat
ctaaagtata tatgagtaaa 5200 cttggtctga cagttaccaa tgcttaatca
gtgaggcacc tatctcagcg 5250 atctgtctat ttcgttcatc catagttgcc
tgactccccg tcgtgtagat 5300 aactacgata cgggagggct taccatctgg
ccccagtgct gcaatgatac 5350 cgcgagaccc acgctcaccg gctccagatt
tatcagcaat aaaccagcca 5400 gccggaaggg ccgagcgcag aagtggtcct
gcaactttat ccgcctccat 5450 ccagtctatt aattgttgcc gggaagctag
agtaagtagt tcgccagtta 5500 atagtttgcg caacgttgtt gccattgctg
caggcatcgt ggtgtcacgc 5550 tcgtcgtttg gtatggcttc attcagctcc
ggttcccaac gatcaaggcg 5600 agttacatga tcccccatgt tgtgcaaaaa
agcggttagc tccttcggtc 5650 ctccgatcgt tgtcagaagt aagttggccg
cagtgttatc actcatggtt 5700 atggcagcac tgcataattc tcttactgtc
atgccatccg taagatgctt 5750 ttctgtgact ggtgagtact caaccaagtc
attctgagaa tagtgtatgc 5800 ggcgaccgag ttgctcttgc ccggcgtcaa
cacgggataa taccgcgcca 5850 catagcagaa ctttaaaagt gctcatcatt
ggaaaacgtt cttcggggcg 5900 aaaactctca aggatcttac cgctgttgag
atccagttcg atgtaaccca 5950 ctcgtgcacc caactgatct tcagcatctt
ttactttcac cagcgtttct 6000 gggtgagcaa aaacaggaag gcaaaatgcc
gcaaaaaagg gaataagggc 6050 gacacggaaa tgttgaatac tcatactctt
cctttttcaa tattattgaa 6100 gcatttatca gggttattgt ctcatgagcg
gatacatatt tgaatgtatt 6150 tagaaaaata aacaaatagg ggttccgcgc
acatttcccc gaaaagtgcc 6200 acctgacgtc taagaaacca ttattatcat
gacattaacc tataaaaata 6250 ggcgtatcac gaggcccttt cgtcttcaat
acaggtagac ctttcgtaga 6300 gatgtacagt gaaatccccg aaattataca
catgactgaa ggaagggagc 6350 tcgtcattcc ctgccgggtt acgtcaccta
acatcactgt tactttaaaa 6400 aagtttccac ttgacacttt gatccctgat
ggaaaacgca taatctggga 6450 cagtagaaag ggcttcatca tatcaaatgc
aacgtacaaa gaaatagggc 6500 ttctgacctg tgaagcaaca gtcaatgggc
atttgtataa gacaaactat 6550 ctcacacatc gacaaaccaa tacaatacag
gtagaccttt cgtagagatg 6600 tacagtgaaa tccccgaaat tatacacatg
actgaaggaa gggagctcgt 6650 cattccctgc cgggttacgt cacctaacat
cactgttact ttaaaaaagt 6700 ttccacttga cactttgatc cctgatggaa
aacgcataat ctgggacagt 6750 agaaagggct tcatcatatc aaatgcaacg
tacaaagaaa tagggcttct 6800 gacctgtgaa gcaacagtca atgggcattt
gtataagaca aactatctca 6850 cacatcgaca aaccaataca atctacaggt
agacctttcg tagagatgta 6900 cagtgaaatc cccgaaatta tacacatgac
tgaaggaagg gagctcgtca 6950 ttccctgccg ggttacgtca cctaacatca
ctgttacttt aaaaaagttt 7000 ccacttgaca ctttgatccc tgatggaaaa
cgcataatct gggacagtag 7050 aaagggcttc atcatatcaa atgcaacgta
caaagaaata gggcttctga 7100 cctgtgaagc aacagtcaat gggcatttgt
ataagacaaa ctatctcaca 7150 catcgacaaa ccaatacaat c 7171 6 256 PRT
Artificial sequence Light chain variable domain 6 Met Lys Lys Asn
Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe 1 5 10 15 Ser Ile Ala
Thr Asn Ala Tyr Ala Ser Asp Ile Gln Met Thr Gln 20 25 30 Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 35 40 45 Thr
Cys Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala Trp Tyr 50 55 60
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala 65 70
75 Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 80
85 90 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
95 100 105 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro
Pro 110 115 120 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
Val Ala 125 130 135 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys 140 145 150 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro 155 160 165 Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser 170 175 180 Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser 185 190 195 Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr 200 205 210 Glu Lys Glu Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu 215 220 225 Ser Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys Gly Ala
230 235 240 Ser Ser Gly Met Ala Asp Pro Asn Arg Phe Arg Gly Lys Asp
Leu 245 250 255 Ala 7 445 PRT Artificial sequence Heavy chain
variable domain 7 Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met
Phe Val Phe 1 5 10 15 Ser Ile Ala Thr Asn Ala Tyr Ala Glu Val Gln
Leu Val Glu Ser 20 25 30 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser Cys 35 40 45 Ala Ala Ser Gly Phe Asn Ile Lys Asp
Thr Tyr Ile His Trp Val 50 55 60 Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Ala Arg Ile Tyr 65 70 75 Pro Thr Asn Gly Tyr Thr Arg
Tyr Ala Asp Ser Val Lys Gly Arg 80 85 90 Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr Leu Gln 95 100 105 Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser 110 115 120 Arg Trp Gly Gly
Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 125 130 135 Gly Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 140 145 150 Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 155 160 165 Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 170 175 180
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 185 190
195 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 200
205 210 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
215 220 225 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys 230 235 240 Val Glu Pro Lys Ser Cys Asp Lys Thr His Gly Arg Met
Lys Gln 245 250 255 Leu Glu Asp Lys Val Glu Glu Leu Leu Ser Lys Asn
Tyr His Leu 260 265 270 Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val
Gly Glu Arg Gly 275 280 285 Lys Leu Ser Gly Gly Gly Ser Gly Ser Gly
Asp Phe Asp Tyr Glu 290 295 300 Lys Met Ala Asn Ala Asn Lys Gly Ala
Met Thr Glu Asn Ala Asp 305 310 315 Glu Asn Ala Leu Gln Ser Asp Ala
Lys Gly Lys Leu Asp Ser Val 320 325 330 Ala Thr Asp Tyr Gly Ala Ala
Ile Asp Gly Phe Ile Gly Asp Val 335 340 345 Ser Gly Leu Ala Asn Gly
Asn Gly Ala Thr Gly Asp Phe Ala Gly 350 355 360 Ser Asn Ser Gln Met
Ala Gln Val Gly Asp Gly Asp Asn Ser Pro 365 370 375 Leu Met Asn Asn
Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser 380 385 390 Val Glu Cys
Arg Pro Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu 395 400 405 Phe Ser
Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val Phe 410 415 420 Ala
Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser Thr 425 430 435
Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser 440 445 8 48 DNA Artificial
sequence Oligonucleotide 8 gcagcttctg gcttcnnnat tnnnnnnnnn
nnnatacact gggtgcgt 48 9 60 DNA Artificial sequence Oligonucleotide
9 ctggaatggg ttgcannnat tnnnccannn nnnggtnnna ctnnntatgc 50
cgatagcgtc 60 10 51 DNA Artificial sequence Oligonucleotide 10
gtctattatt gtagccgcnn nnnnnnnnnn nnnnnnnnna tggactactg 50 g 51 11
48 DNA Artificial sequence Oligonucleotide 11 gcagcttctg gcttctmtat
ttmttmttmt tmtatacact gggtgcgt 48 12 60 DNA Artificial sequence
Oligonucleotide 12 ctggaatggg ttgcatmtat ttmtccatmt tmtggttmta
cttmttatgc 50 cgatagcgtc 60 13 54 DNA Artificial sequence
Oligonucleotide 13 gtctattatt gtagccgctm ttmttmttmt tmttmttmtg
ctatggacta 50 ctgg 54 14 57 DNA Artificial sequence Oligonucleotide
14 gtctattatt gtagccgctm ttmttmttmt tmttmttmtt mtgctatgga 50
ctactgg 57 15 60 DNA Artificial sequence Oligonucleotide 15
gtctattatt gtagccgctm ttmttmttmt tmttmttmtt mttmtgctat 50
ggactactgg 60 16 63 DNA Artificial sequence Oligonucleotide 16
gtctattatt gtagccgctm ttmttmttmt tmttmttmtt mttmttmtgc 50
tatggactac tgg 63 17 66 DNA Artificial sequence Oligonucleotide 17
gtctattatt gtagccgctm ttmttmttmt tmttmttmtt mttmttmttm 50
tgctatggac tactgg 66 18 69 DNA Artificial sequence Oligonucleotide
18 gtctattatt gtagccgctm ttmttmttmt tmttmttmtt mttmttmttm 50
ttmtgctatg gactactgg 69 19 48 DNA Artificial sequence
Oligonucleotide 19 gcagcttctg gcttcwmtat twmtwmtwmt wmtatacact
gggtgcgt 48 20 60 DNA Artificial sequence Oligonucleotide 20
ctggaatggg ttgcawmtat twmtccawmt wmtggtwmta ctwmttatgc 50
cgatagcgtc 60 21 42 DNA Artificial sequence Oligonucleotide 21
gtctattatt gtagccgcwm twmtwmtgct atggactact gg 42 22 45 DNA
Artificial sequence Oligonucleotide 22 gtctattatt gtagccgcwm
twmtwmtwmt gctatggact actgg 45 23 48 DNA Artificial sequence
Oligonucleotide 23 gtctattatt gtagccgcwm twmtwmtwmt wmtgctatgg
actactgg 48 24 51 DNA Artificial sequence Oligonucleotide 24
gtctattatt gtagccgcwm twmtwmtwmt wmtwmtgcta tggactactg 50 g 51 25
54 DNA Artificial sequence Oligonucleotide 25 gtctattatt gtagccgcwm
twmtwmtwmt wmtwmtwmtg ctatggacta 50 ctgg 54 26 57 DNA Artificial
sequence Oligonucleotide 26 gtctattatt gtagccgcwm twmtwmtwmt
wmtwmtwmtw mtgctatgga 50 ctactgg 57 27 60 DNA Artificial sequence
Oligonucleotide 27 gtctattatt gtagccgcwm twmtwmtwmt wmtwmtwmtw
mtwmtgctat 50 ggactactgg 60 28 63 DNA Artificial sequence
Oligonucleotide 28 gtctattatt gtagccgcwm twmtwmtwmt wmtwmtwmtw
mtwmtwmtgc 50 tatggactac tgg 63 29 66 DNA Artificial sequence
Oligonucleotide 29 gtctattatt gtagccgcwm twmtwmtwmt wmtwmtwmtw
mtwmtwmtwm 50 tgctatggac tactgg 66 30 69 DNA Artificial sequence
Oligonucleotide 30 gtctattatt gtagccgcwm twmtwmtwmt wmtwmtwmtw
mtwmtwmtwm 50 twmtgctatg gactactgg 69 31 72 DNA Artificial sequence
Oligonucleotide 31 gtctattatt gtagccgcwm twmtwmtwmt wmtwmtwmtw
mtwmtwmtwm 50 twmtwmtgct atggactact gg 72 32 75 DNA Artificial
sequence Oligonucleotide 32 gtctattatt gtagccgcwm twmtwmtwmt
wmtwmtwmtw mtwmtwmtwm 50 twmtwmtwmt gctatggact actgg 75 33 78 DNA
Artificial sequence Oligonucleotide 33 gtctattatt gtagccgcwm
twmtwmtwmt wmtwmtwmtw mtwmtwmtwm 50 twmtwmtwmt wmtgctatgg actactgg
78 34 48 DNA Artificial sequence Oligonucleotide 34 gcagcttctg
gcttckmtat tkmtkmtkmt kmtatacact gggtgcgt 48 35 60 DNA Artificial
sequence Oligonucleotide 35 ctggaatggg ttgcakmtat tkmtccakmt
kmtggtkmta ctkmttatgc 50 cgatagcgtc 60 36 54 DNA Artificial
sequence Oligonucleotide 36 gtctattatt gtagccgckm tkmtkmtkmt
kmtkmtkmtg ctatggacta 50 ctgg 54 37 51 DNA Artificial sequence
Oligonucleotide 37 acctgccgtg ccagtcagkm tkmtkmtkmt kmtgtagcct
ggtatcaaca 50 g 51 38 48 DNA Artificial sequence Oligonucleotide 38
ccgaagcttc tgatttackm tgcatcckmt ctctactctg gagtccct 48 39 54 DNA
Artificial sequence Oligonucleotide 39 acttattact gtcagcaakm
tkmtkmtkmt ccakmtacgt tcggacaggg 50 tacc 54 40 9 PRT Artificial
sequence Synthetic CDR sequence 40 Gly Phe Ser Ile Tyr Ser Tyr Ser
Ile 1 5 41 12 PRT Artificial sequence Synthetic CDR sequence 41 Ala
Ser Ile Ser Pro Tyr Ser Gly Tyr Thr Ser Tyr 1 5 10 42 18 PRT
Artificial sequence Synthetic CDR sequence 42 Ser Arg Tyr Ser Ser
Tyr Tyr Ser Tyr Tyr Tyr Ser Ser Ser Ser 1 5 10 15 Tyr Ser Tyr 43 6
PRT Artificial sequence Synthetic CDR sequence 43 Ser Ser Ser Ser
Pro Tyr 1 5 44 9 PRT Artificial sequence Synthetic CDR sequence 44
Gly Phe Ser Ile Tyr Ser Tyr Ser Ile 1 5 45 12 PRT Artificial
sequence Synthetic CDR sequence 45 Ala Ser Ile Ser Pro Tyr Tyr Gly
Tyr Thr Ser Tyr 1 5 10 46 14 PRT Artificial sequence Synthetic CDR
sequence 46 Ser Arg Ser Ser Tyr Ser Tyr Tyr Ser Ser Ser Ser Ser Tyr
1 5 10 47 6 PRT Artificial sequence Synthetic CDR sequence 47 Tyr
Tyr Tyr Tyr Pro Ser 1 5 48 9 PRT Artificial sequence Synthetic CDR
sequence 48 Gly Phe Ser Ile Tyr Ser Ser Ser Ile 1 5 49 12 PRT
Artificial sequence Synthetic CDR sequence 49 Ala Ser Ile Tyr Pro
Tyr Tyr Gly Tyr Thr Ser Tyr 1 5 10 50 12 PRT Artificial sequence
Synthetic CDR sequence 50 Ser Arg Ser Tyr Tyr Ser Ser Tyr Tyr Tyr
Tyr Ser 1 5 10 51 9 PRT Artificial sequence Synthetic CDR sequence
51 Gly Phe Ser Ile Ser Ser Ser Ser Ile 1 5 52 12 PRT Artificial
sequence Synthetic CDR squence 52 Ala Ser Ile Ser Pro Tyr Ser Gly
Tyr Thr Ser Tyr 1 5 10 53 12 PRT Artificial sequence Synthetic CDR
sequence 53 Ser Arg Ser Ser Tyr Ser Tyr Tyr Ser Ser Tyr Tyr 1 5 10
54 9 PRT Artificial sequence Synthetic CDR sequence 54 Gly Phe Ser
Ile Ser Ser Ser Ser Ile 1 5 55 12 PRT Artificial sequence Synthetic
CDR sequence 55 Ala Ser Ile Ser Pro Tyr Ser Gly Tyr Thr Ser Tyr 1 5
10 56 12 PRT Artificial sequence Synthetic CDR sequence 56 Ser Arg
Tyr Ser Tyr Ser Tyr Tyr Ser Ser Tyr Tyr 1 5 10 57 9 PRT Artificial
sequence Synthetic CDR sequence 57 Gly Phe Tyr Ile Ser Tyr Ser Ser
Ile 1 5 58 12 PRT Artificial sequence Synthetic CDR sequence 58 Ala
Ser Ile Ser Pro Ser Ser Gly Tyr Thr Ser Tyr 1 5 10 59 12 PRT
Artificial sequence Synthetic CDR sequence 59 Ser Arg Ser Ser Tyr
Tyr Ser Tyr Ser Ser Tyr Tyr 1 5 10 60 9 PRT Artificial sequence
Synthetic CDR sequence 60 Val Phe Ser Ile Asp Tyr Tyr Tyr Ile 1 5
61 12 PRT Artificial sequence Synthetic CDR sequence 61 Ala Ser Ile
Ser Pro Tyr Ser Gly Ser Thr Ser Tyr 1 5 10 62 16 PRT Artificial
sequence Synthetic CDR sequence 62 Ser Arg Ser Tyr Ser Tyr Ser Ser
Ser Tyr Tyr Tyr Tyr Ser Tyr 1 5 10 15 Ser 63 9 PRT Artificial
sequence Synthetic CDR sequence 63 Gly Phe Ser Ile Ser Tyr Ser Ser
Ile 1 5 64 12 PRT Artificial sequence Synthetic CDR sequence 64 Ala
Ser Ile Ser Pro Tyr Ser Gly Tyr Thr Ser Tyr 1 5 10 65 21 PRT
Artificial sequence Synthetic CDR sequence 65 Ser Arg Ser Ser Tyr
Tyr Tyr Ser Ser Ser Tyr Tyr Tyr Tyr Tyr 1 5 10 15 Ser Ser Tyr Ser
Ser Ser 20 66 9 PRT Artificial sequence Synthetic CDR sequence 66
Gly Phe Ser Ile Tyr Ser Ser Ser Ile 1 5 67 12 PRT Artificial
sequence Synthetic CDR sequence 67 Ala Ser Ile Tyr Pro Ser Tyr Gly
Tyr Thr Ser Tyr 1 5 10 68 22 PRT Artificial sequence Synthetic CDR
sequence 68 Ser Arg Ser Ser Ser Tyr Tyr Ser Ser Tyr Tyr Ser Tyr Tyr
Tyr 1 5 10 15 Ser Ser Tyr Ser Tyr Ser Ser 20 69 9 PRT Artificial
sequence Synthetic CDR sequence 69 Gly Phe Ser Ile Tyr Tyr Ser Tyr
Ile 1 5 70 12 PRT Artificial sequence Synthetic CDR sequence 70 Ala
Ser Ile Ser Pro Tyr Tyr Gly Tyr Thr Ser Tyr 1 5 10 71 22 PRT
Artificial sequence Synthetic CDR sequence 71 Ser Arg Ser Ser Tyr
Ser Tyr Ser Tyr Ser Tyr Ser Ser Ser Ser 1 5 10 15 Tyr Ser Tyr Tyr
Ser Ser Ser 20 72 9 PRT Artificial sequence Synthetic CDR sequence
72 Gly Phe Ser Ile Tyr Tyr Ser Tyr Ile 1 5 73 12 PRT Artificial
sequence Synthetic CDR sequence 73 Ala Ser Ile Ser Pro Ser Ser Gly
Tyr Thr Ser Tyr 1 5 10 74 21 PRT Artificial sequence Synthetic CDR
sequence 74 Ser Arg Tyr Tyr Tyr Ser Tyr Ser Tyr Ser Tyr Ser Tyr Tyr
Ser 1 5 10 15 Ser Ser Ser Tyr Ser Ser 20 75 9 PRT Artificial
sequence Synthetic CDR sequence 75 Gly Phe Ser Ile Tyr Tyr Ser Ser
Ile 1 5 76 12 PRT Artificial sequence Synthetic CDR sequence 76 Ala
Ser Ile Tyr Pro Tyr Ser Gly Ser Thr Ser Tyr 1 5 10 77 22 PRT
Artificial sequence Synthetic CDR sequence 77 Ser Arg Tyr Tyr Ser
Tyr Tyr Ser Ser Tyr Tyr Tyr Ser Ser Ser 1 5 10 15 Ser Ser Ser Ser
Tyr Ser Ser 20 78 9 PRT Artificial sequence Synthetic CDR sequence
78 Gly Phe Ser Ile Tyr Ser Tyr Ser Ile 1 5 79 12 PRT Artificial
sequence Synthetic CDR sequence 79 Ala Ser Ile Ser Pro Tyr Ser Gly
Ser Thr Ser Tyr 1 5 10 80 22 PRT Artificial sequence Synthetic CDR
sequence 80 Ser Arg Ser Ser Tyr Ser Tyr Ser Tyr Tyr Tyr Ser Tyr Tyr
Ser 1 5 10 15 Tyr Ser Tyr Ser Tyr Ser Ser 20 81 9 PRT Artificial
sequence Synthetic CDR sequence 81 Gly Phe Tyr Ile Ser Tyr Ser Ser
Ile 1 5 82 12 PRT Artificial sequence Synthetic CDR sequence 82 Ala
Ser Ile Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr 1 5 10 83 15 PRT
Artificial sequence Synthetic CDR sequence 83 Ser Arg Ser Ser Tyr
Ser Ser Ser Ser Tyr Ser Ser Tyr Tyr Ser 1 5 10 15 84 9 PRT
Artificial sequence Synthetic CDR sequence 84 Gly Phe Ser Ile Ser
Ser Tyr Ser Ile 1 5 85 12 PRT Artificial sequence Synthetic CDR
sequence 85 Ala Ser Ile Ser Pro Tyr Tyr Gly Ser Thr Ser Tyr 1 5 10
86 12 PRT Artificial sequence Synthetic CDR sequence 86 Ser Arg Ser
Ser Ser Tyr Ser Ser Tyr Tyr Ser Ser 1 5 10 87 9 PRT Artificial
sequence Synthetic CDR sequence 87 Gly Phe Ser Ile Tyr Ser Tyr Tyr
Ile 1 5 88 12 PRT Artificial sequence Synthetic CDR sequence 88 Ala
Ser Ile Ser Pro Tyr Ser Gly Tyr Thr Tyr Tyr 1 5 10 89 21 PRT
Artificial sequence Synthetic CDR sequence 89 Ser Arg Ser Ser Tyr
Tyr Tyr Tyr Tyr Ser Tyr Ser Ser Ser Ser 1 5 10 15 Ser Ser Tyr Tyr
Tyr Ser 20 90 9 PRT Artificial sequence Synthetic CDR sequence 90
Gly Phe Ser Ile Ser Ser Ser Ser Ile 1 5 91 12 PRT Artificial
sequence Synthetic CDR sequence 91 Ala Ser Ile Ser Pro Tyr Tyr Gly
Tyr Thr Tyr Tyr 1 5 10 92 20 PRT Artificial sequence Synthetic CDR
sequence 92 Ser Arg Ser Tyr Tyr Ser Tyr Ser Ser Ser Ser Tyr Ser Tyr
Tyr 1 5 10 15 Tyr Tyr Tyr Tyr Tyr 20 93 9 PRT Artificial sequence
Synthetic CDR sequence 93 Gly Phe Ser Ile Tyr Tyr Ser Ser Ile 1 5
94 12 PRT Artificial sequence Synthetic CDR sequence 94 Ala Tyr Ile
Ser Pro Ser Ser Gly Ser Thr Tyr Tyr 1 5 10 95 19 PRT Artificial
sequence Synthetic CDR sequence 95 Ser Arg Ser Tyr Ser Phe Leu Leu
Ser Tyr Ser Ser Tyr Ser Ser 1 5 10 15 Tyr Tyr Ser Ser 96 9 PRT
Artificial sequence Synthetic CDR sequence 96 Gly Phe Ser Ile Tyr
Ser Tyr Ser Ile 1 5 97 11 PRT Artificial sequence Synthetic CDR
sequence 97 Ala Ser Ile Ser Pro Tyr Tyr Gly Thr Ser Tyr 1 5 10 98
18 PRT Artificial sequence Synthetic CDR sequence 98 Ser Arg Tyr
Ser Tyr Ser Ser Ser Tyr Ser Ser Ser Tyr Tyr Ser 1 5 10 15 Tyr Ser
Ser 99
9 PRT Artificial sequence Synthetic CDR sequence 99 Ala Phe Ser Ile
Ser Tyr Ser Tyr Ile 1 5 100 12 PRT Artificial sequence Synthetic
CDR sequence 100 Ala Ser Ile Tyr Pro Ser Ser Gly Ser Thr Ser Tyr 1
5 10 101 17 PRT Artificial sequence Synthetic CDR sequence 101 Ser
Arg Ser Tyr Ser Phe Tyr Ser Ser Tyr Tyr Ser Tyr Tyr Tyr 1 5 10 15
Ser Ser 102 9 PRT Artificial sequence Synthetic CDR sequence 102
Gly Phe Ser Ile Tyr Ser Tyr Asn Ile 1 5 103 12 PRT Artificial
sequence Synthetic CDR sequence 103 Ala Ser Ile Ser Pro Tyr Ser Gly
Tyr Thr Tyr Tyr 1 5 10 104 21 PRT Artificial sequence Synthetic CDR
sequence 104 Ser Arg Ser Ser Tyr Tyr Tyr Tyr Tyr Ser Tyr Ser Ser
Ser Ser 1 5 10 15 Ser Ser Tyr Tyr Tyr Ser 20 105 9 PRT Artificial
sequence Synthetic CDR sequence 105 Gly Phe Tyr Ile Tyr Ser Ser Ser
Ile 1 5 106 11 PRT Artificial sequence Synthetic CDR sequence 106
Ala Ser Ile Ser Pro Tyr Ser Gly Thr Ser Tyr 1 5 10 107 14 PRT
Artificial sequence Synthetic CDR sequence 107 Ser Arg Ser Tyr Ser
Ser Ser Ser Tyr Tyr Ser Ser Tyr Tyr 1 5 10 108 9 PRT Artificial
sequence Synthetic CDR sequence 108 Gly Phe Tyr Ile Tyr Ser Ser Ser
Ile 1 5 109 12 PRT Artificial sequence Synthetic CDR sequence 109
Ala Ser Ile Tyr Pro Tyr Ser Gly Tyr Thr Ser Tyr 1 5 10 110 14 PRT
Artificial sequence Synthetic CDR sequence 110 Ser Arg Tyr Ser Tyr
Tyr Ser Tyr Ser Ser Tyr Ser Tyr Ser 1 5 10 111 9 PRT Artificial
sequence Synthetic CDR sequence 111 Gly Phe Tyr Ile Tyr Ser Ser Ser
Ile 1 5 112 12 PRT Artificial sequence Synthetic CDR sequence 112
Ala Ser Ile Ser Pro Ser Ser Gly Tyr Thr Ser Tyr 1 5 10 113 14 PRT
Artificial sequence Synthetic CDR sequence 113 Ser Arg Tyr Ser Ser
Tyr Ser Tyr Ser Ser Tyr Ser Tyr Ser 1 5 10 114 9 PRT Artificial
sequence Synthetic CDR sequence 114 Gly Phe Tyr Ile Tyr Ser Ser Ser
Ile 1 5 115 12 PRT Artificial sequence Synthetic CDR sequence 115
Ala Ser Ile Tyr Pro Ser Ser Gly Tyr Thr Ser Tyr 1 5 10 116 14 PRT
Artificial sequence Synthetic CDR sequence 116 Ser Arg Tyr Ser Ser
Tyr Ser Tyr Ser Ser Tyr Ser Tyr Ser 1 5 10 117 9 PRT Artificial
sequence Synthetic CDR sequence 117 Gly Phe Ser Ile Ser Ser Ser Ser
Ile 1 5 118 12 PRT Artificial sequence Synthetic CDR sequence 118
Ala Ser Ile Tyr Pro Ser Ser Gly Ser Thr Ser Tyr 1 5 10 119 14 PRT
Artificial sequence Synthetic CDR sequence 119 Ser Arg Ser Ser Ser
Tyr Ser Tyr Ser Ser Tyr Ser Tyr Ser 1 5 10 120 12 PRT Artificial
sequence Dimerization domain sequence 120 Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly 1 5 10 121 72 DNA Artificial sequence
Oligonucleotide 121 gtctattatt gtagccgctm ttmttmttmt tmttmttmtt
mttmttmttm 50 ttmttmtgct atggactact gg 72 122 75 DNA Artificial
sequence Oligonucleotide 122 gtctattatt gtagccgctm ttmttmttmt
tmttmttmtt mttmttmttm 50 ttmttmttmt gctatggact actgg 75 123 78 DNA
Artificial sequence Oligonucleotide 123 gtctattatt gtagccgctm
ttmttmttmt tmttmttmtt mttmttmttm 50 ttmttmttmt tmtgctatgg actactgg
78 124 81 DNA Artificial sequence Oligonucleotide 124 gtctattatt
gtagccgctm ttmttmttmt tmttmttmtt mttmttmttm 50 ttmttmttmt
tmttmtgcta tggactactg g 81 125 84 DNA Artificial sequence
Oligonucleotide 125 gtctattatt gtagccgctm ttmttmttmt tmttmttmtt
mttmttmttm 50 ttmttmttmt tmttmttmtg ctatggacta ctgg 84 126 87 DNA
Artificial sequence Oligonucleotide 126 gtctattatt gtagccgctm
ttmttmttmt tmttmttmtt mttmttmttm 50 ttmttmttmt tmttmttmtt
mtgctatgga ctactgg 87 127 90 DNA Artificial sequence
Oligonucleotide 127 gtctattatt gtagccgctm ttmttmttmt tmttmttmtt
mttmttmttm 50 ttmttmttmt tmttmttmtt mttmtgctat ggactactgg 90 128 93
DNA Artificial sequence Oligonucleotide 128 gtctattatt gtagccgctm
ttmttmttmt tmttmttmtt mttmttmttm 50 ttmttmttmt tmttmttmtt
mttmttmtgc tatggactac tgg 93 129 54 DNA Artificial sequence
Oligonucleotide 129 gcaacttatt actgtcagtm ttmttmttmt ccatmtacgt
tcggacaggg 50 tacc 54 130 54 DNA Artificial sequence
Oligonucleotide 130 acttattact gtcagcaatm ttmttmttmt ccatmtacgt
tcggacaggg 50 tacc 54
* * * * *
References