U.S. patent application number 16/716137 was filed with the patent office on 2020-05-28 for immunoglobulin frameworks which demonstrate enhanced stability in the intracellular environment and methods of identifying same.
The applicant listed for this patent is Novartis AG. Invention is credited to Alcide Barberis, Dominik Escher, Stefan Ewert, Adrian Auf Der Maur, Kathrin Tissot.
Application Number | 20200165322 16/716137 |
Document ID | / |
Family ID | 29553590 |
Filed Date | 2020-05-28 |
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United States Patent
Application |
20200165322 |
Kind Code |
A1 |
Tissot; Kathrin ; et
al. |
May 28, 2020 |
IMMUNOGLOBULIN FRAMEWORKS WHICH DEMONSTRATE ENHANCED STABILITY IN
THE INTRACELLULAR ENVIRONMENT AND METHODS OF IDENTIFYING SAME
Abstract
Compositions are provided, which can be used as frameworks for
the creation of very stable and soluble single-chain Fv antibody
fragments. These frameworks have been selected for intracellular
performance and are thus ideally suited for the creation of scFv
antibody fragments or scFv antibody libraries for applications
where stability and solubility are limiting factors for the
performance of antibody fragments, such as in the reducing
environment of a cell. Such frameworks can also be used to identify
highly conserved residues and consensus sequences which demonstrate
enhanced solubility and stability.
Inventors: |
Tissot; Kathrin;
(Deutschland, DE) ; Ewert; Stefan; (Zurich,
CH) ; Maur; Adrian Auf Der; (Zurich, CH) ;
Barberis; Alcide; (Zurich, CH) ; Escher; Dominik;
(Huenenberg, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis AG |
Basel |
|
CH |
|
|
Family ID: |
29553590 |
Appl. No.: |
16/716137 |
Filed: |
December 16, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16175002 |
Oct 30, 2018 |
10570190 |
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16716137 |
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15340195 |
Nov 1, 2016 |
10125186 |
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16175002 |
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14469276 |
Aug 26, 2014 |
9518108 |
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15340195 |
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10515241 |
Jul 18, 2005 |
8853362 |
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PCT/EP03/05324 |
May 21, 2003 |
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14469276 |
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60382649 |
May 22, 2002 |
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60438256 |
Jan 3, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/21 20130101;
C07K 2317/622 20130101; G01N 33/6857 20130101; A61K 48/00 20130101;
C07K 2317/82 20130101; C07K 2317/80 20130101; C07K 16/00 20130101;
A61P 43/00 20180101; C07K 2317/94 20130101; C07K 2317/55 20130101;
C07K 2317/567 20130101 |
International
Class: |
C07K 16/00 20060101
C07K016/00; G01N 33/68 20060101 G01N033/68 |
Claims
1. A single-chain antibody comprising a human variable light chain
framework (VL) and a human variable heavy chain framework (VH)
having the general structure: NH.sub.2-VL-linker-VH-COOH; or
NH.sub.2-VH-linker-VL-COOH wherein the single-chain antibody has
the VH framework and the VL framework of AH, BH, CH, DH, EH, FH,
GH, AI, BI, CI, DI, EI, FI, GI, AJ, BJ, CJ, DJ, EJ, FJ, GJ, AK, BK,
CK, DK, EK, FK, or GK wherein A is the amino acid sequence (Seq.
Id. No. 1) TABLE-US-00001
EIVMTQSPSTLSASVGDRVIITCRASQSISSWLAWYQQKPGKAPKLLIY
KASSLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQYKSYWTFG QGTKLTVLG;
B is the amino acid sequence (Seq. Id. No. 2) TABLE-US-00002
EIVLTQSPSSLSASVGDRVILTCRASQGIRNELAWYQQRPGKAPKRLIY
AGSILQSGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQYYSLPYMF GQGTKVDIKR;
C is the amino acid sequence (Seq. Id. No. 3) TABLE-US-00003
EIVMTQSPATLSVSPGESAALSCRASQGVSTNVAWYQQKPGQAPR
LLIYGATTRASGVPARFSGSGSGTEFTLTINSLQSEDFAAYYCQQYKHW
PPWTFGQGTKVEIKR;
D is the amino acid sequence (Seq. Id. No. 4) TABLE-US-00004
QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLI
YDNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSG
VVFGGGTKLTVLG;
E is the amino acid sequence (Seq. Id. No. 5) TABLE-US-00005
EIVLTQSPATLSLSPGERATLSCRASQTLTHYLAWYQQKPGQAPR
LLIYDTSKRATGVPARFSGSGSGTDFILTISSLEPEDSALYYCQQRNSW
PHTFGGGTKLEIKR;
F is the amino acid sequence (Seq. Id. No. 6) TABLE-US-00006
SYVLTQPPSVSVAPGQTATVTCGGNNIGSKSVHWYQQKPGQAPVL
VVYDDSDRPSGIPERFSGSNSGNTATLTIRRVEAGDEADYYCQVWDSSS
DHNVFGSGTKVEIKR;
G is the amino acid sequence (Seq. Id. No. 7) TABLE-US-00007
LPVLTQPPSVSVAPGQTARISCGGNNIETISVHWYQQKPGQAPVL
VVSDDSVRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSS
DYVVFGGGTKLTVLG;
H is the amino acid sequence (Seq. Id. No. 8) TABLE-US-00008
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAHV
LRFLEWLPDAFDIWGQGTLVTVSS;
I is the amino acid sequence (Seq. Id. No. 9) TABLE-US-00009
EIVLTQSPSSLSASLGDRVTITCRASQSISSYLNWYQQKPGKAPK
LLIYAASSSQSGVPSRFRGSESGTDFILTISNLQPEDFATYYCQQSYRTP
FTFGPGTKVEIKR;
J is the amino acid sequence (Seq. Id. No. 10) TABLE-US-00010
VQLVQSGAEVKKPGASVKVSCTASGYSFTGYFLHWVRQAPGQGLEWMGRI
NPDSGDTIYAQKFQDRVILTRDTSIGTVYMELTSLTSDDTAVYYCARVPR
GTYLDPWDYFDYWGQGTLVTVSS;
and K is the amino acid sequence (Seq. Id. No. 11) TABLE-US-00011
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL
EWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY
CAKDAGIAVAGTGFDYWGQGTLVTVSSS.
2. A single-chain antibody of claim 1 fused to a second protein
moiety to yield a fusion construct of the general structure:
NH.sub.2-VL-linker-VH-second protein-COOH; or NH.sub.2-second
protein-VL-linker-VH-COOH.
3. The single chain antibody of claim 1, wherein orientation of the
VH and VL regions is reversed.
4. The single chain antibody according to claim 1 wherein the VL
framework is of the kappa1, lambda 1 or 3 type.
5. The single chain antibody according to claim 2, wherein the
second protein provides a read-out for intracellular assays.
6. A single chain antibody framework comprising a human variable
light chain framework (VL) and a human variable heavy chain
framework (VH), the single chain antibody framework being selected
from the group consisting of: AH, BH, CH, DH, EH, FH, GH, AI, BI,
CI, DI, EI, FI, GI, AJ, BJ, CJ, DJ, EJ, FJ, GJ, AK, BK, CK, DK, EK,
FK, and GK wherein A is the amino acid sequence (Seq. Id. No. 1)
TABLE-US-00012 EIVMTQSPSTLSASVGDRVIITCRASQSISSWLAWYQQKPGKAPKLLIYK
ASSLESGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQYKSYWTFGQG TKLTVLG;
B is the amino acid sequence (Seq. Id. No. 2) TABLE-US-00013
EIVLTQSPSSLSASVGDRVTLTCRASQGIRNELAWYQQRPGKAPKRLIYA
GSILQSGVPSRFSGSGSGTEFTLTISSLQPEDVAVYYCQQYYSLPYMFGQ GTKVDIKR;
C is the amino acid sequence (Seq. Id. No. 3) TABLE-US-00014
EIVMTQSPATLSVSPGESAALSCRASQGVSTNVAWYQQKPGQAPR
LLIYGATTRASGVPARFSGSGSGTEFTLTINSLQSEDFAAYYCQQYKHWP
PWTFGQGTKVEIKR;
D is the amino acid sequence (Seq. Id. No. 4) TABLE-US-00015
QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQLPGTAPQLLIY
DNTKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSGVV FGGGTKLTVLG;
E is the amino acid sequence (Seq. Id. No. 5) TABLE-US-00016
EIVLTQSPATLSLSPGERATLSCRASQTLTHYLAWYQQKPGQAPR
LLIYDTSKRATGVPARFSGSGSGTDFILTISSLEPEDSALYYCQQRNSWP
HTFGGGTKLEIKR;
F is the amino acid sequence (Seq. Id. No. 6) TABLE-US-00017
SYVLTQPPSVSVAPGQTATVTCGGNNIGSKSVHWYQQKPGQAPVL
VVYDDSDRPSGIPERFSGSNSGNTATLTIRRVEAGDEADYYCQVWDSSSD
HNVFGSGTKVEIKR;
G is the amino acid sequence (Seq. Id. No. 7) TABLE-US-00018
LPVLTQPPSVSVAPGQTARISCGGNNIETISVHWYQQKPGQAPVL
VVSDDSVRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSD
YVVFGGGTKLTVLG;
H is the amino acid sequence (Seq. Id. No. 8) TABLE-US-00019
QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA
ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAHV
LRFLEWLPDAFDIWGQGTLVTVSS;
I is the amino acid sequence (Seq. Id. No. 9) TABLE-US-00020
EIVLTQSPSSLSASLGDRVTITCRASQSISSYLNWYQQKPGKAPK
LLIYAASSSQSGVPSRFRGSESGTDFILTISNLQPEDFATYYCQQSYRTP
FTFGPGTKVEIKR;
J is the amino acid sequence (Seq. Id. No. 10) TABLE-US-00021
VQLVQSGAEVKKPGASVKVSCTASGYSFTGYFLHWVRQAPGQGLEWMGRI
NPDSGDTIYAQKFQDRVILTRDTSIGTVYMELTSLTSDDTAVYYCARVPR
GTYLDPWDYFDYWGQGTLVTVSS;
and K is the amino acid sequence (Seq. Id. No. 11) TABLE-US-00022
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGL
EWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY
CAKDAGIAVAGTGFDYWGQGTLVTVSSS.
7. A single chain antibody selected from the group consisting of
variants of the single chain antibody according to claim 6.
8. A single chain antibody selected from the group consisting of
derivatives of the single chain antibody according to claim 6.
9. A method of using the single chain antibody according to claim 6
in target validation, diagnostic applications, library construction
or therapeutic applications.
10. A method of using at least two framework sequences of claim 6
in the identification of a conserved framework residue class.
11. The method according to claim 10, wherein the conserved
framework residue class is selected from the group consisting of:
polar but uncharged R groups; positively charged R groups;
negatively charged R groups; hydrophobic R groups; and special
amino acids.
12. A method of using at least two framework sequences of claim 6
in the identification of at least one conserved framework
sequence.
13. The method according to claim 12, wherein the conserved
framework sequence is 2-5 residues.
14. The method according to claim 12, wherein the conserved
framework sequence is 5-10 residues.
15. The method according to claim 12, wherein the conserved
framework sequence is 10-25 residues.
16. The method according to claim 12, wherein the conserved
framework sequence has gaps.
17. An antibody comprising the VL or the VH or both from the single
chain framework according to claim 6.
18. An antibody fragment comprising the VL or the VH or both from
the single chain framework according to claim 6.
19. A nucleic acid capable of encoding the single chain antibody
according to claim 1.
20. A vector comprising the nucleic acid according to claim 19.
21. A host cell comprising the nucleic acid according to claim 19.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 16/175,002 filed Oct. 30, 2018, which is a divisional of
U.S. patent application Ser. No. 15/340,195 filed Nov. 1, 2016 that
issued as U.S. Pat. No. 10,125,186 on Nov. 13, 2018, that is a
divisional of U.S. patent application Ser. No. 14/469,276 filed
Aug. 26, 2014 that issued as U.S. Pat. No. 9,518,108 on Dec. 13,
2016, that is a divisional of U.S. patent application Ser. No.
10/515,241, filed Jul. 18, 2005 that issued as U.S. Pat. No.
8,853,362 on Oct. 7, 2014, which is a 371 National Stage Entry of
International Application Serial No. PCT/EP03/05324, filed May 21,
2003 (now pending) which claims priority to U.S. Provisional Patent
Application Ser. No. 60/438,256 filed Jan. 3, 2003 (expired), and
U.S. Provisional Patent Application Ser. No. 60/382,649, filed May
22, 2002 (expired), the entire contents of which are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to protein chemistry, molecular
biology, and immunology.
BACKGROUND OF THE RELATED ART
[0003] Antibodies can recognize and target almost any molecule with
high specificity and affinity. This characteristic has been
exploited to turn these natural proteins into powerful tools for
diagnostic and therapeutic applications. Advances in recombinant
DNA technology have facilitated the manipulation, cloning, and
expression of antibody genes in a wide variety of non-lymphoid
cells (Skerra, 1988; Martineau, 1998; Verma, 1998). A number of
different antibody fragments have been constructed to best suit the
various applications. The smallest entity that retains the full
antigen-binding capacity of the whole parental immunoglobulin is
the single-chain Fv fragment (scFv) (Bird, 1988). This antibody
fragment comprises the variable regions of the heavy and the light
chains linked by a flexible peptide-linker, which allows the
expression of the protein from a single gene.
[0004] Antibody fragments have several important advantages in
comparison to the entire immunoglobulin molecule. Due to their
smaller size, the expression is facilitated and the yield is
enhanced in a variety of expression host cells, such as E. coli
cells (Pluckthun, 1996). Moreover, antibody fragments allow
improved tumour penetration in in vivo applications (Yokota, 1992)
and they can be linked covalently to various effector molecules for
therapeutic approaches.
[0005] Naturally occurring antibodies, which are secreted by plasma
cells, have evolved to function in an extracellular, oxidizing
environment. To obtain their functional, folded structure, they
generally require the formation of disulfide-bridges within the
separate domains, which are crucial for the stability of the
immunoglobulin fold. In contrast to full-length antibodies, scFv or
Fab antibody fragments can, in principle, be functionally expressed
in a reducing environment inside any cell and directed to any
compartment to target intracellular proteins and thus evoke
specific biological effects (Biocca, 1991). Indeed, some
intracellular single chain antibody fragments, which are called
intrabodies, have been applied successfully to modulate the
function of intracellular target proteins in different biological
systems. Thus, resistance against viral infections has been
demonstrated in plant biotechnology (Tavladoraki, 1993; Benvenuto,
1995), binding of intrabodies to HIV proteins has been
shown(Rondon, 1997), and binding to oncogene products (Biocca,
1993; Cochet, 1998; Lener, 2000) has been described. Moreover,
intracellular antibodies promise to be a valuable tool in
characterizing the function of a vast number of genes now
identified through the sequencing of the human genome (Richardson,
1995; Marasco, 1997). For example, they can be used in a functional
genomics approach to block or modulate the activity of newly
identified proteins, thereby contributing to the understanding of
their functions. Finally, intrabodies have potential diagnostic and
therapeutic applications, for example in gene therapy settings.
[0006] Despite these great prospects, the generation of functional
intrabodies is still limited by their instability and insolubility
or propensity to aggregate. The reducing environment of the
cytoplasm prevents the formation of the conserved intrachain
disulfide bridges, thus rendering a high percentage of antibody
fragments unstable and, as a consequence, non-functional inside the
cell (Biocca, 1995; Proba, 1997). Stability and solubility of
antibody fragments therefore represents a major obstacle for the
application of intrabodies as potential modulators of protein
function in vivo. So far, no predictions can be made about the
sequence requirements that render an antibody fragment functional
in an intracellular environment.
[0007] There is, therefore, a need for antibody fragments which
perform well in a broad range of different cell types and can thus
be used as frameworks for diverse binding specificities. Such
frameworks can be used to construct libraries for intracellular
screening or can serve as an acceptor for the binding portions of
an existing antibody.
[0008] Besides being uniquely suited for intracellular
applications, such antibody fragments or whole antibodies based on
very stable variable domain frameworks also have a distinct
advantage over other antibodies in numerous extracellular and in
vitro applications. When such frameworks are produced in an
oxidizing environment, their disulfide-bridges can be formed,
further enhancing their stability and making them highly resistant
towards aggregation and protease degradation. The in vivo half-life
(and thus the resistance towards aggregation and degradation by
serum proteases) is, besides affinity and specificity, the
single-most important factor for the success of antibodies in
therapeutic or diagnostic applications (Willuda, 1999). The
half-life of antibody fragments can further be increased through
the covalent attachment of polymer molecules such as poly-ethylene
glycol (PEG) (Weir, 2002). Stable molecules of this type represent
a significant advance in the use of antibodies, especially, but not
exclusively, when the Fc functionality is not desired.
[0009] The great practical importance of antibody-fragment
libraries has motivated research in this area. Winter (EP 0368684)
has provided the initial cloning and expression of antibody
variable region genes. Starting from these genes he has created
large antibody libraries having high diversity in both the
complementary determining regions (CDRs) as well as in the
framework regions. Winter does not disclose, however, the
usefulness of different frameworks for library construction.
[0010] The teaching of Pluckthun (EP 0859841), on the other hand,
has tried to improve the library design by limiting the frameworks
to a defined number of synthetic consensus sequences. Protein
engineering efforts involving introduction of a large amount of
rationally designed mutations have previously suggested mutations
towards the respective consensus sequence as a suitable means for
the improvement of the stability of isolated variable
immunoglobulin domains (Ohage 1999; Ohage 1999 and U.S. Pat. No.
5,854,027, hereby incorporated by reference).
[0011] Pluckthun (EP 0859841) discloses methods for the further
optimization of binding affinities based on these consensus
sequences. The Pluckthun patent also acknowledges the ongoing
increase in knowledge concerning antibodies and accordingly aims at
including such future findings in the library design. However, no
possible further improvements of the synthetic consensus frameworks
are suggested.
[0012] The teachings of Winter, Pluckthun and others (e.g.
Soderlind, WO 0175091) have thus tried to create large antibody
libraries with a focus on high diversity in the CDRs for selection
and application of the selected scFvs under oxidizing conditions.
All of these libraries are, however, not optimized for
intracellular applications and thus not useful for selection and
applications in a reducing environment, or other conditions which
set special requirements on stability and solubility of the
expressed antibody fragment.
[0013] The qualities required for antibody fragments to perform
well in a reducing environment, e.g. the cytoplasm of prokaryotic
and eukaryotic cells, are not clear. The application of
intracellular antibodies or "intrabodies" is therefore currently
limited by their unpredictable behavior under reducing conditions,
which can affect their stability and solubility properties (Biocca,
1995; Worn, 2000). Present patent applications (EP1040201,
EP1166121 and WO0200729) and publications (Visintin, 1999)
concerning intracellular screening for intrabodies focus on the
screening technology but do not disclose specific antibody
sequences which are functional in eukaryotic cells, in particular
in yeast, and, thus, useful for library construction in this
context.
[0014] Visintin and Tse have independently described the isolation
of a so-called intracellular consensus sequence (ICS) (Visintin,
2002; Tse, 2002). This sequence was derived from a number of
sequences that had been isolated from an
antigen-antibody-interaction screen in yeast. The input into the
intracellular screen was, however, heavily biased due to prior
phage-display selection. Thus, all but one of the input-sequences
belonged to the VH 3 subgroup in the case of Visintin et al. The
published consensus sequence ICS is fully identical to the
consensus sequence for the human VH 3 subgroup described by Knappik
(2000) and EP0859841. 60 of the 62 amino acids of the ICS are also
identical to the general human VH-domain consensus sequence which
was proposed by Steipe as a basis for the construction of variable
domains with enhanced stability (U.S. Pat. No. 6,262,238, hereby
incorporated by reference). These works were, in turn, based on
earlier sequence collections (i.e., Kabat, 1991 and definitions of
variable domain subgroups and structural determinants (Tomlinson,
1992; Williams, 1996; Chothia, 1989 and Chothia, 1987). However,
because the input to the intrabody selection was so heavily biased
(i.e., in the case of Visintin et al. all but one of the VH domains
was VH3), the isolation of VH3 sequences from intracellular
screening is not particularly surprising. Due to the heavy bias of
their input library, the work of Tse et al. and Visintin et al.
does not provide a thorough evaluation of the human variable domain
repertoire as would be provided by an unbiased inquiry and as is
required to identify the useful intrabody frameworks present in the
human repertoire.
[0015] We have previously described a system, which allows for the
selection of stable and soluble intrabodies in yeast, independent
of their antigen-binding specificity (Auf der Maur (2001),
WO0148017). This approach allows efficient screening of scFv
libraries and the isolation of specific frameworks, which are
stable and soluble in the reducing environment of the yeast cell.
The objective remains to actually isolate framework sequences and
use the patterns in a first step to predict what sequence types
would be most stable in the reducing environment and in a second
step identify by analysis, recombination and further in vivo and in
vitro experiments the optimal sequence.
BRIEF SUMMARY OF THE INVENTION
[0016] The present invention fills a missing link in the field of
antibody generation. It provides antibody variable domain framework
sequences with superior characteristics regarding stability and
solubility. These are crucial features for many relevant
applications, such as in diagnostics, therapy or research. These
frameworks can be used for grafting of existing
binding-specificities or for the generation of antibody libraries
with high stability and solubility.
[0017] ScFv libraries were used for the isolation of frameworks
which are stable and soluble in the reducing environment of the
yeast cell. The performance of the isolated frameworks has
subsequently been characterized in human cell lines and in in vitro
experiments. The described frameworks can directly serve as
acceptor backbones for existing binding specificities or to
construct CDR libraries by randomization of one or more of the
hypervariable loops for use in reducing or otherwise challenging
environments. The isolated variable domain sequences have further
been analyzed by alignment to identify preferred sequence families.
From those preferred variable domain sequence families, optimal
sequences were chosen based on a structural analysis which excludes
sequences containing framework residues which disturb the
immunoglobulin fold. The identified variable domain sequence
candidates were subsequently recombined in all possible variations
and the optimal combinations of variable domains of the light and
heavy chain were selected by analysis of their performance in
yeast, mammalian cells and in vitro.
[0018] These optimized scFvs and their constituting variable domain
frameworks, as well as other antibody fragments or whole antibodies
derived thereof, are ideal as, for example, acceptor backbones for
existing binding specificities or for the construction of CDR
libraries by randomization of one or more of the hypervariable
loops for use in reducing or otherwise challenging environments.
Antibodies suitable for intracellular applications are by
definition more stable and soluble. Accordingly, their use will
also be advantageous in applications outside the intracellular
environment.
[0019] The invention provides compositions comprising frameworks of
antibody variable domains and single-chain Fv antibody (ScFv)
fragments which can be incorporated into various antibody fragments
or whole antibodies. Classes of antibody variable domains fragments
are provided which are the most stable and soluble and thus best
suited for intracellular applications. Specific framework sequences
of antibody variable domains and scFv antibody fragments which show
the highest performance in intracellular assays are also provided.
The invention also provides specific framework sequences of
antibody variable domains and synthetic combinations of variable
domains of the light and heavy chain in scFv fragments which are,
for example, optimal for intracellular applications and show an
optimal performance in vitro regarding stability and
solubility.
[0020] The invention provides single-chain framework reagents that
have the general structures:
[0021] NH.sub.2-VL-linker-VH-COOH or
[0022] NH.sub.2-VH-linker-VL-COOH.
[0023] In another embodiment of the invention the single-chain
framework may be fused to a second protein moiety to yield a fusion
construct of the general structure:
[0024] NH.sub.2-VL-linker-VH-second protein-COOH
[0025] NH.sub.2-second protein-VL-linker-VH-COOH.
[0026] The orientation of the VH and VL regions in these fusion
constructs may be reversed.
[0027] In another embodiment of the invention the variable domains
may be incorporated into a Fab fragment, which may additionally be
fused to a second protein moiety to yield fusion constructs of the
general structure:
[0028] NH.sub.2-VH-CH-second protein-COOH and
NH.sub.2-VL-CL-COOH
[0029] The second protein may be fused to either Nor C-terminus of
either the heavy or the light chain.
[0030] In a preferred embodiment, the second protein of the
single-chain or Fab framework fusion construct is a protein which
provides a read-out for intracellular assays, either directly or
via transcriptional activation.
[0031] Another object of the invention is to provide framework
classes of antibody variable domains and sequences of variable
domains and scFvs which are suitable for grafting the hypervariable
loops from existing antibodies, for example, in order to obtain
antibodies which are functional in a reducing or otherwise
challenging environment.
[0032] Another object of the invention is to provide framework
classes of antibody variable domains and sequences of variable
domains and scFvs which, for example, through randomization of one
or more of the hypervariable loops of such frameworks, are suitable
for the creation of libraries for use in a reducing or otherwise
challenging environment.
[0033] Another object of the invention is the use of the disclosed
sequences in the identification of conserved residues and consensus
sequences.
[0034] The antibodies or antibody fragments resulting from the use
of the disclosed frameworks can be used as reagents in target
validation and in therapy, prevention and diagnosis of human,
animal and plant diseases. The antibodies can be used in the form
of protein or DNA encoding such a protein and are not limited to
intracellular applications.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 shows the result of a typical "quality control"
screen in yeast assayed by activation of lacZ expression (see, for
example, Example 1). The selected, positive clones (black) were
identified in several different screens and the corresponding
sequences of the positive clones can be found in FIGS. 12 and 13.
The selected sequences are compared to the positive control, the
very stable lambda-graft (dark grey).
[0036] FIG. 2 shows the performance of the frameworks isolated from
a typical "quality control" screen in yeast (black) in the human
cell line Hela, assayed by the activation of luciferase expression
in comparison to the very stable lambda-graft (dark grey). The
positive control Gal4-VP16 (white) gives the maximally possible
level of transcriptional activation in the system. Luciferase
activity has been corrected for transfection efficiency.
[0037] FIG. 3 shows the in vivo performance of the superior
framework combinations assayed in yeast by the activation of lacZ
expression. The framework sequences (black) are compared to the
positive control (the very stable lambda-graft (dark grey)). The
numbering of the frameworks is as described in FIG. 16.
[0038] FIG. 4 shows the in vivo performance of the superior
framework combinations assayed in the human cell line Hela by the
activation of luciferase expression and illustrated in comparison
to the very stable lambda-graft (dark grey). The positive control,
Gal4-VP16 (white) gives the maximal possible level of
transcriptional activation in the system. Luciferase activity has
been corrected for transfection efficiency.
[0039] FIG. 5 shows the in vivo performance of the superior
framework combinations assayed by the amount of soluble protein
produced in the cytoplasm of yeast strain S. cerevisiae JPY9.
[0040] FIG. 6A shows the expression behavior of selected framework
combinations (2.1, 3.1, 4.1, 5.1, 5.2, and 5.3) in the periplasm of
E. coli. The arrow indicates the location of the band corresponding
to the scFv frameworks. FIG. 6B shows the expression behavior of
selected framework combinations (7.3, 2.4, 3.4, 4.4, 5.4, and 6.4)
in the periplasm of E. coli. The arrow indicates the location of
the band corresponding to the scFv frameworks.
[0041] FIG. 7 shows the in vivo performance of selected superior
framework combinations assayed in three human cell lines (Hela,
(black), Saos-2 (dark grey) and HEK 293 (white)), by the activation
of luciferase expression and illustrated in comparison to the very
stable lambda-graft. The positive control Gal4-VP16 gives the
maximal possible level of transcriptional activation in the system.
Luciferase activity has been corrected for transfection
efficiency.
[0042] FIG. 8A represents the resistance towards aggregation at
37.degree. C. of selected framework combinations (for frameworks
2.4 and 5.2) as quantified by the amount of monomeric protein
present before and after incubation as indicated in PBS-buffer.
FIG. 8B represents the resistance towards aggregation at 37.degree.
C. of selected framework combinations (for frameworks 4.4, 6.4 and
7.3) as quantified by the amount of monomeric protein present
before and after incubation as indicated in PBS-buffer.
[0043] FIG. 9 represents the resistance towards protease
degradation aggregation in human serum at 37.degree. C. of selected
framework combinations, quantified by the amount of soluble
full-length protein present before and after prolonged
incubation.
[0044] FIG. 10 shows the in vivo performance of two selected
binders on the novel framework 7.3 in the Fab-context, assayed in
yeast interaction assay by the activation of lacZ expression.
Expression of the Fab-chains is from a bi-directional
galactose-inducible promoter, on either an ars/cen or a 2 micron
vectors. Expression from the Fab vector yields the antibody light
chain and a VH-CH1-Gal4-AD fusion protein. Binders are directed
against human Polo-like kinase1 (hPLK1). Binding to the target is
compared with the unspecific binding to an unrelated antigen and
the binding of the un-randomized framework 7.3. Note that the
corresponding scFv that have been included for reference are
expressed from an actin promoter (2 micron).
[0045] FIG. 11 shows the in vivo performance of the scFv frameworks
in the Fab-context assayed by the amount of soluble protein
produced in the cytoplasm of the yeast strain JPY9. Expression of
the Gal4-AD-scFv fusion (actin/2 micron) is compared with the
expression of the corresponding Fab-construct, and with the parent
framework 7.3 as a Fab, both from two different vectors
(Gal-inducible, ars/cen and 2 micron). Expression from the Fab
vector yields the antibody light chain and a VH-CH1-Gal4-AD fusion
protein, which is detected in this blot.
[0046] FIG. 12 shows an alignment of all VH-domain framework
sequences selected from various "quality control" screens in
yeast.
[0047] FIG. 13 shows an alignment of all VL-domain framework
sequences selected from various "quality control" screens in
yeast.
[0048] FIG. 14 shows an alignment of randomly picked sequences from
the library.
[0049] FIG. 15 shows a statistical analysis of the sub-class
frequency for VH- and VL-domains in the sequences isolated with the
"quality control" system. Only those sequences were considered
which were subsequently found to be positive in the quantitative
yeast assay. The selected sequences are compared with the
unselected library as determined from a limited number of random
sequences (FIG. 14).
[0050] FIG. 16 shows the sequences used for further recombination
and evaluation of the best combinations in scFvs and their
respective abbreviations (abb.), sources and sub-family.
DETAILED DESCRIPTION OF THE INVENTION
[0051] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention,
suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0052] As used herein, "identity" refers to the sequence similarity
between two polypeptides, molecules or between two nucleic acids.
When a position in both of the two compared sequences is occupied
by the same base or amino acid monomer subunit (for instance, if a
position in each of the two DNA molecules is occupied by adenine,
or a position in each of two polypeptides is occupied by a lysine),
then the respective molecules are homologous at that position. The
"percentage identity" between two sequences is a function of the
number of matching positions shared by the two sequences divided by
the number of positions compared.times.100. For instance, if 6 of
10 of the positions in two sequences are matched, then the two
sequences have 60% identity. By way of example, the DNA sequences
CTGACT and CAGGTT share 50% homology (3 of the 6 total positions
are matched). Generally, a comparison is made when two sequences
are aligned to give maximum homology. Such alignment can be
provided using, for instance, the method of Needleman et al., J.
Mol Biol. 48: 443-453 (1970), implemented conveniently by computer
programs such as the Align program (DNAstar, Inc.).
[0053] "Similar" sequences are those which, when aligned, share
identical and similar amino acid residues, where similar residues
are conservative substitutions for, or "allowed point mutations"
of, corresponding amino acid residues in an aligned reference
sequence. In this regard, a "conservative substitution" of a
residue in a reference sequence is a substitution by a residue that
is physically or functionally similar to the corresponding
reference residue, e.g., that has a similar size, shape, electric
charge, chemical properties, including the ability to form covalent
or hydrogen bonds, or the like. Thus, a "conservative substitution
modified" sequence is one that differs from a reference sequence or
a wild-type sequence in that one or more conservative substitutions
or allowed point mutations are present. The "percentage positive"
between two sequences is a function of the number of positions that
contain matching residues or conservative substitutions shared by
the two sequences divided by the number of positions
compared.times.100. For instance, if 6 of 10 of the positions in
two sequences are matched and 2 of 10 positions contain
conservative substitutions, then the two sequences have 80%
positive homology.
[0054] "VH domain" refers to the variable part of the heavy chain
of an immunoglobulin molecule.
[0055] "VL domain" refers to the variable part of the light chain
of an immunoglobulin molecule.
[0056] VH or VL "subtype" refers to the subtype defined by the
respective consensus sequence as defined in Knappik (2000). The
term "subfamily" or "subclass" is used as synonym for "subtype".
The term "subtype" as used herein refers to sequences sharing a
high degree of identity and similarity with the respective
consensus sequence representing their subtype. Whether a certain
variable domain sequence belongs to a "subtype" is determined by
alignment of the sequence with either all known human germline
segments of the respective domain, or the defined consensus
sequences and subsequent identification of the greatest homology.
Methods for determining homologies and grouping of sequences by
using search matrices, such as BLOSUM (Henikoff 1992) are well
known to the person skilled in the art.
[0057] "Amino acid consensus sequence" as used herein refers to an
amino acid sequence, which can be generated using a matrix of at
least two or preferably more aligned amino acid sequences, and
allowing for gaps in the alignment, it is possible to determine the
most frequent amino acid residue at each position. The consensus
sequence is that sequence which comprises the amino acids which are
most frequently represented at each position. In the event that two
or more amino acids are equally represented at a single position,
the consensus sequence includes both or all of those amino
acids.
[0058] The amino acid sequence of a protein can be analyzed at
various levels. For example, conservation or variability could be
exhibited at the single residue level, multiple residue level,
multiple residue with gaps etc. Residues could exhibit conservation
of the identical residue or could be conserved at the class level.
Examples of amino acid classes include polar but uncharged R groups
(Serine, Threonine, Asparagine and Glutamine); positively charged R
groups (Lysine, Arginine, and Histidine); negatively charged R
groups (Glutamic acid and Aspartic acid); hydrophobic R groups
(Alanine, Isoleucine, Leucine, Methionine, Phenylalanine,
Tryptophan, Valine and Tyrosine); and special amino acids
(Cysteine, Glycine and Proline). Other classes are known to one of
skill in the art and may be defined using structural determinations
or other data to assess substitutability. In that sense a
substitutable amino acid could refer to any amino acid which could
be substituted and maintain functional conservation at that
position.
[0059] "Polynucleotide consensus sequence" as used herein refers to
a nucleotide sequence, which can be generated using a matrix of at
least two or preferably more aligned nucleic acid sequences, and
allowing for gaps in the alignment, it is possible to determine the
most frequent nucleotide at each position. The consensus sequence
is that sequence which comprises the nucleotides which are most
frequently represented at each position. In the event that two or
more nucleotides are equally represented at a single position, the
consensus sequence includes both or all of those nucleotides.
[0060] "Structural sub-element" as used herein refers to stretches
of amino acid residues within a protein or polypeptide that
correspond to a defined structural or functional part of the
molecule. These can be loops (i.e. CDR loops of an antibody) or any
other secondary or functional structure within the protein or
polypeptide (i.e., domains, .alpha.-helices, .beta.-sheets,
framework regions of antibodies, etc.). A structural sub-element
can be identified using known structures of similar or homologous
polypeptides, or by using the above mentioned matrices of aligned
amino acid sequences. Here the variability at each position is the
basis for determining stretches of amino acid residues which belong
to a structural sub-element (e.g. hypervariable regions of an
antibody).
[0061] "Sub-sequence" as used herein refers to a genetic module
which encodes at least one structural sub-element. It is not
necessarily identical to a structural sub-element.
[0062] "Antibody CDR" as used herein refers to the complementarity
determining regions of the antibody which consist of the antigen
binding loops as defined by Kabat et al. (1991). Each of the two
variable domains of an antibody Fv fragment contain, for example,
three CDRs.
[0063] "Antibody" as used herein is a synonym for "immunoglobulin".
Antibodies according to the present invention may be whole
immunoglobulins or fragments thereof, comprising at least one
variable domain of an immunoglobulin, such as single variable
domains, Fv (Skerra, 1988), scFv (Bird, 1988; Huston, 1988), Fab,
(Fab')2 or other fragments well known to a person skilled in the
art.
[0064] "Antibody framework" as used herein refers to the part of
the variable domain, either VL or VH, which serves as a scaffold
for the antigen binding loops of this variable domain (Kabat et
al., 1991).
[0065] Rationally engineered scFv fragments have demonstrated a
clear correlation between the thermodynamic stability of a scFv
fragment and its in vivo performance (Worn, 2000; Auf der Maur,
2001). Using a recently developed system named "Quality Control"
(Auf der Maur, 2001), specific antibody variable domain framework
sequences which are suitable for intracellular applications have
been isolated (FIGS. 12 and 13), characterized (FIGS. 1 and 2) and
further improved (FIGS. 3 to 9 and FIG. 14). As observed in our
previous experiments, well performing frameworks selected in the
intracellular assay show a high in vitro stability as demonstrated
by their resistance to aggregation and protease degradation at
37.degree. C. (FIGS. 8 and 9). Moreover, a pattern emerged which
allows a selection of frameworks for intracellular applications on
a more general basis, depending on their framework subfamily (FIG.
15). Specific antibody variable domain sequences useful for
intracellular applications are disclosed here, as well as the
general pattern. This allows, on the one hand, the use of these
sequences as framework donors in grafting experiments to obtain
functional intrabodies which retain the binding specificity of the
loop donor. Additionally, antibody libraries can be constructed
using the disclosed sequences as frameworks. Such libraries are
suitable for intracellular selection systems under reducing
conditions, such as those in prokaryotic and eukaryotic cells.
Additionally, the disclosed sequences may be used to identify, for
example, conserved sequences or residues or motifs. The grafting of
structural sub-elements, for example, those of the binding loops of
an antibody (e.g. Jung, 1997), as well as the making of libraries
of antibodies or fragments thereof (e.g. Vaughan, 1996; Knappik,
2000) has been described in detail and is well known to a person
skilled in the art.
[0066] Because intracellular applications expose the antibody
fragments to very unfavorable conditions (i.e. increased
temperatures, reducing environment), the sequences disclosed in the
present invention have acquired features that make them resistant
to the most adverse conditions. Therefore, when compared to
"average" sequences, the disclosed sequences are of outstanding
stability and solubility as is demonstrated by their resistance
towards aggregation and protease degradation (FIGS. 8 and 9). These
features, together with their excellent expression yield make the
disclosed antibody framework sequences uniquely suitable not only
for intracellular use, but especially for all therapeutic and
diagnostic applications where long half-life, robustness, and ease
of production are of great concern.
[0067] The present invention enables the design of polypeptide
sequences comprising at least the variable part of an antibody that
are useful for applications in a reducing or otherwise challenging
environment. In a first embodiment, the invention provides a
collection of antibody framework sequences useful for intracellular
applications (FIGS. 12 and 13). In a first step, a library of
diverse sequences is screened independent of binding affinity using
the Quality control system in yeast. The isolated sequences can be
evaluated for their intracellular performance in yeast and in
mammalian cells (FIGS. 1 and 2).
[0068] In one embodiment of the invention, the collection of
isolated sequences is analyzed by alignment to identify the
antibody variable domain sub-classes and consensus sequences that
are suitable for intracellular applications.
[0069] In a further preferred embodiment of the invention, the
collection of antibody framework sequences described above is
further analyzed by alignment to each other and grouping into
sub-families. All frameworks belonging to one sub-type are compared
regarding their intracellular performance in yeast and in mammalian
cells (FIGS. 1 and 2, as an example) and regarding the occurrence
of negative, neutral or positive exchanges in their amino-acid
sequence relative to the respective sub-type consensus. A person
skilled in the art can distinguish between positive, neutral and
negative changes based on the structural environment of the
particular exchanged residue in the immunoglobulin domain.
Subsequently, framework sequences of variable antibody domains are
chosen which show the best intracellular performance and which are
devoid of negative exchanges compared to their respective sub-type
consensus. Preferably, sequences are selected which further contain
amino-acid exchanges which are considered positive.
[0070] In a further preferred embodiment, the selected antibody
variable domains of the heavy and the light chain are subsequently
recombined in all possible combinations into scFv fragments, in
order to identify the combinations with the highest stability and
solubility. To this end the novel, recombined scFv fragments are
evaluated for their performance under reducing conditions in
intracellular interaction assays in yeast (FIG. 3) and in mammalian
cell lines (FIGS. 4 and 7) and for soluble intracellular expression
in yeast (FIG. 5). Promising combinations are further evaluated for
their behavior under oxidizing conditions by analyzing the
periplasmic expression yield in E. coli (FIG. 6), the resistance to
aggregation at elevated temperatures (FIG. 8) and the resistance to
aggregation and protease degradation upon prolonged incubation in
human serum at 37.degree. C. (FIG. 9). These data are used to
identify the scFv framework best suitable for any specific
application, either intracellular, or under oxidizing
conditions.
[0071] The selected and optimized framework sequences disclosed
herein have a significant advantage not only in intracellular
applications, but in all applications which can profit from
increased stability and/or solubility of the scFv. Examples are the
long-term storage at high concentrations required for diagnostic
applications, and prolonged functional half-life in serum at
37.degree. C. (as required, for example, in therapeutic
applications). According to one aspect of the present invention,
there is provided an intrabody framework comprising a single-chain
framework having the general structure:
[0072] NH.sub.2-VL-linker-VH-COOH; or [0073]
NH.sub.2-VH-linker-VL-COOH
[0074] wherein the VH framework is of subtype 1a, 1b or 3.
[0075] In another embodiment, the orientation of the VH and VL
regions is reversed in the single chain framework described
above.
[0076] According to one aspect of the present invention, there is
provided an intrabody framework comprising a single-chain framework
having the general structure:
[0077] NH.sub.2-VL-linker-VH-COOH; or
[0078] NH.sub.2-VH-linker-VL-COOH
[0079] wherein the VH framework is of subtype 1a, 1b or 3 and the
VL framework is of subtype .lamda.1, .lamda.3 or .kappa.1.
[0080] In another embodiment, the invention provides a single-chain
framework fused to a second protein moiety to yield a fusion
construct of the general structure:
[0081] NH.sub.2-VL-linker-VH-second protein-COOH; or
[0082] NH.sub.2-second protein-VL-linker-VH-COOH wherein the VH
framework is of subtype 1a, 1b or 3 and the VL framework is of
subtype .lamda.1, .lamda.3 or .kappa.1.
[0083] In another embodiment, the orientation of the VH and VL
regions in these fusion constructs may be reversed.
[0084] In another embodiment, the variable domains may be
incorporated into a Fab fragment which may additionally be fused to
a second protein moiety to yield fusion constructs of the general
structure:
[0085] NH.sub.2-VH-CH-second protein-COOH and
NH.sub.2-VL-CL-COOH
[0086] The second protein may be fused to either Nor C-terminus of
either the heavy or the light chain.
[0087] As disclosed herein, there is a very strong preference in
intracellular applications for VH framework of the subtype 3, but
also for 1a and 1b. Regarding the light chain variable domain (VL),
there is a clear preference by numbers for frameworks of the kappa
1 type, but lambda 1 and 3 are also enriched. These framework
sub-types, i.e. VH 1a, 1b and 3 combined with a kappa 1, lambda for
3 VL domain are therefore best suited for intracellular use and
other applications which require he folding properties of the scFv.
Therefore, in order to reduce the amount of molecules which are not
functional in the reducing environment, libraries for intracellular
screening systems should preferentially be constructed from a
mixture of these framework sub-types.
[0088] In a preferred embodiment, the VH domain of the antibody
fragments of the invention is of the subgroup 1a, 1b or 3.
[0089] In a preferred embodiment, the VL domain of the antibody
fragments of the invention is of the subgroup kappa1, lambda 1 or
3.
[0090] In a preferred embodiment, antibody fragments used as
frameworks are selected from the group consisting of: 1.1, 2.1,
3.1, 4.1, 5.1, 1.2, 2.2, 3.2, 4.2, 5.2, 1.3, 2.3, 3.3, 4.3, 5.3,
7.3, 1.4, 2.4, 3.4, 4.4, 5.4, and 6.4 as described in FIG. 16.
[0091] In one embodiment of the invention, at least two and
preferably more frameworks are identified and then analyzed. A
database of the protein sequences may be established where the
protein sequences are aligned with each other. The alignment can
then be used to define, for example, residues, sub-elements,
sub-sequence or subgroups of framework sequences which show a high
degree of similarity in both the sequence and, if that information
is available, in the structural arrangement.
[0092] The length of the sub-elements is preferably, but not
exclusively ranging between 1 amino acid (such as one residue in
the active site of an enzyme or a structure-determining residue)
and 150 amino acids (for example, whole protein domains). Most
preferably, the length ranges between 3 and 25 amino acids, such as
most commonly found in CDR loops of antibodies.
[0093] In another embodiment, consensus nucleic acid sequences,
which are predicted from the analysis are synthesized. This can be
achieved by any one of several methods well known to the
practitioner skilled in the art, for example, by total gene
synthesis or by PCR-based approaches.
[0094] In another embodiment, the nucleic acid sequences are cloned
into a vector. The vector could be a sequencing vector, an
expression vector or a display (e.g. phage display) vector, all
which are well known to those of skill in the art. A vector could
comprise one nucleic acid sequence, or two or more nucleic
sequences, either in different or the same operon. In the last
case, they could either be cloned separately or as contiguous
sequences.
[0095] In one embodiment, the polypeptides have an amino acid
pattern characteristic of a particular species. This can for
example be achieved by deducing the consensus sequences from a
collection of homologous proteins of just one species, most
preferably from a collection of human proteins.
[0096] A further embodiment of the present invention relates to
fusion proteins by providing for a DNA sequence which encodes both
the polypeptide, as described above, as well as an additional
moiety.
[0097] In further embodiments, the invention provides for nucleic
acid sequences, vectors containing the nucleic acid sequences, host
cells containing the vectors, and polypeptides obtainable according
to the methods described herein.
[0098] In a further embodiment, the invention provides for
synthesizing or otherwise placing restriction sites at the end of
the nucleic acid sequences of the invention allowing them to be
cloned into suitable vectors.
[0099] In a further preferred embodiment, the invention provides
for vector systems being compatible with the nucleic acid sequences
encoding the polypeptides. The vectors comprise restriction sites,
which would be, for example, unique within the vector system and
essentially unique with respect to the restriction sites
incorporated into the nucleic acid sequences encoding the
polypeptides, except for example the restriction sites necessary
for cloning the nucleic acid sequences into the vector.
[0100] In another embodiment, the invention provides for a kit,
comprising one or more of the list of nucleic acid sequences,
recombinant vectors, polypeptides, and vectors according to the
methods described above, and, for example, suitable host cells for
producing the polypeptides.
[0101] All of the above embodiments of the present invention can be
effected using standard techniques of molecular biology known to
one skilled in the art.
[0102] In another embodiment, the nucleic acid sequence is any
sequence capable of encoding the polypeptides of the invention.
[0103] In another embodiment, the inventive nucleic acids are used
in gene therapy.
[0104] In another embodiment, the single chain framework is a
variant of any one of sequences 1.1, 2.1, 3.1, 4.1, 5.1, 1.2, 2.2,
3.2, 4.2, 5.2, 1.3, 2.3, 3.3, 4.3, 5.3, 7.3, 1.4, 2.4, 3.4, 4.4,
5.4, 6.4 (FIG. 16), where "variant" as used herein refers to a
sequence that exhibits 90% or greater identity, while maintaining
enhanced stability.
[0105] In another embodiment, the single chain framework is a
derivative of any one of sequences 1.1, 2.1, 3.1, 4.1, 5.1, 1.2,
2.2, 3.2, 4.2, 5.2, 1.3, 2.3, 3.3, 4.3, 5.3, 7.3, 1.4, 2.4, 3.4,
4.4, 5.4, 6.4 (FIG. 16) where "derivative" as used herein refers to
a sequence that maintains only those amino acids that are critical
to the function and stability of the molecule. Isolated neutral or
positive exchanges in the framework as described in example 3, are
not considered to be relevant change to the antibody frameworks of
the present invention.
[0106] In a preferred embodiment of the invention, the single chain
framework is fused to a second protein, wherein that protein
provides a read-out for intracellular assays. The read-out can be
either direct, for example in the form of a fusion to a detectable
protein, e.g. GFP (green fluorescent protein), enhanced blue
fluorescent protein, enhanced yellow fluorescent, protein enhanced
cyan fluorescent protein which can be observed by fluorescence, or
other fusion partners with different detection methods.
Alternatively, a read-out can be achieved through transcriptional
activation of a reporter gene, where the fusion partner in the
scFv-fusion protein is either a transcriptional activator, such as
the Gal4 activation domain, or a DNA-binding protein, such as the
LexA- or Gal4 DNA-binding domain, which activates the transcription
of a reporter gene of an enzyme, such as .beta.-galctosidase,
luciferase, .alpha.-galactosidase, .beta.-glucuronidase,
chloramphenicol acetyl transferase and others, which in turn
provide a read-out. Fusion proteins, which provide a read out are
well known to one of skill in the art.
[0107] Another embodiment of the invention is an antibody
comprising a framework described herein.
[0108] Another embodiment of the invention is the use of the
antibody of the instant invention.
[0109] A further preferred embodiment of the invention is the use
of the described framework classes of antibody variable domains and
sequences of variable domains and scFvs for grafting of
hypervariable loops from existing antibodies, in order to obtain
antibodies which are functional in a reducing or otherwise
challenging environment.
[0110] Another further preferred embodiment of the invention is the
use of the described framework classes of antibody variable domains
and sequences of variable domains and scFvs, for example through
randomization of one or more of the hypervariable loops of such
frameworks, for the creation of libraries for applications in a
reducing or otherwise challenging environment.
[0111] As would be apparent to one of ordinary skill in the art,
the inventive molecules described herein may be used in diagnostic
and therapeutic applications, target validation and gene
therapy.
[0112] The invention may be illustrated by the following examples,
which are not intended to limit the scope of the invention in any
way.
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[0157] The invention is further illustrated in the following
non-limiting examples.
Example 1
[0158] Selection of intrabody frameworks through screening of a
human library in the "quality control" system in yeast Screening
with the "quality control" system for stable frameworks was
essentially performed as described in detail by Auf der Maur
(WO0148017, Auf der Maur 2001, each hereby incorporated by
reference).
[0159] The plasmids for expression of the scFv-fusion constructs
for screening in yeast were derived from pESBA-Act (Worn, 2000). It
contains the yeast TRP1 gene for transformation selection in S.
cerevisiae and the 2 micron origin of replication to ensure high
copy numbers. Moreover it has a constitutive actin promoter for
strong expression and the GAL11 transcriptional termination
sequence, separated by a multiple cloning site. For handling in
bacterial systems, it also has a bacterial origin of replication
and the amp resistance gene.
[0160] The Gal4 activation domain (AD amino acids 768-881) was
originally amplified by PCR using pGAD424 (Clontech) as template
with primers including the SV40 T-antigen nuclear localization
signal N-terminal to the Gal4-AD. The DNA-fragments encoding amino
acids 263-352 of Gal11P were amplified by PCR and cloned in frame,
N-terminal to the SV40-NLS-Gal4-AD-construct. The human scFv
library, amplified from human spleen-cell cDNA as described
elsewhere (Welschhof, 1995; Krebber, 1997; de Haard, 1999), was
cloned in frame, N-terminal to this fusion construct via
SfiI-sites, and in the orientation V.sub.L-linker-V.sub.H where the
linker has the sequence (GGGS).sub.4. Expression thus yields a
fusion protein of the general structure scFv-Gal11p-SV40
NLS-Gal4AD.
[0161] Screening was carried out in the yeast strain S. cerevisiae
YDE172 (MAT.alpha. ura3-52 leu2.DELTA.1 trp1d63 his3.DELTA.200
lys2.DELTA. 385 gal4.DELTA. 11) (Auf der Maur, 2001), which was
derived from the strain JPY9 (Escher, 2000) by integrating the
divergently oriented LacZ and HIS3 reporter genes under the control
of the natural UAS.sub.G from Gal1GAL10 regulatory sequences into
the his3.DELTA.200 locus. Transcriptional activation of the
reporter system is mediated by the Gal4-AD moiety of the
scFv-fusion construct, following the specific interaction of its
Gal11P moiety with the Gal4-DNA-binding-domain (DBD, amino acids
1-100). The Gal4-DBD is provided by expression from a second
plasmid, pMP83. It contains the yeast LEU2 gene for transformation
selection in S. cerevisiae and the ARS CEN origin of replication.
Moreover, it has a constitutive actin promoter for strong
expression and the GAL11 transcriptional termination sequence. For
handling in bacterial systems, it also has a bacterial origin of
replication and the amp resistance gene.
[0162] For screening, the yeast strain S. cerevisiae YDE172 was
co-transformed with a scFv-library as fusion construct on the
pESBA-Act2 vector while the pMP83-vector provided the Gal4-DBD. A
standard lithium acetate transformation protocol was used (Agatep,
1998). Following transformation, the cells were plated on drop-out
plates (-Trp/-Leu/-His) containing 80 mM 3-aminotriazole. Colonies
were picked after 3 days incubation at 30.degree. C. and
re-streaked on drop-out plates (-Trp/-Leu/-His) containing 80 mM
3-aminotriazole. Those that re-grew were tested for LacZ-expression
by development of blue color in a filter assay on plates containing
the substrate X-Gal. Positive clones were taken for further
analysis involving isolation of the scFv-carrying plasmid from
yeast, transformation into E. coli DH5a, isolation of plasmid from
single colonies of E. coli and re-transformation into freshly
prepared yeast strain S. cerevisiae YDE172 for the assay as
described below. All methods were performed according to standard
procedures, well known to a person of ordinary skill in the
art.
[0163] In addition, a modified screening procedure was used were
the scFv was directly fused to both a DNA-binding domain (LexA
amino acids 1-202) and an activation domain (Gal4, amino acids
768-881) to yield a fusion construct of the following structure:
scFv-LexA-NLS-Gal4AD. The plasmids for expression of the
scFv-fusion constructs for screening in yeast were derived from
pESBA-Act2. It contains the yeast TRP1 gene for transformation
selection in S. cerevisiae and the 2 micron origin of replication
to ensure high copy numbers. Moreover, it has a constitutive actin
promoter (for strong expression) and the GAL11 transcriptional
termination sequence separated by a multiple cloning site. For
handling in bacterial systems, it also has a bacterial origin of
replication and the amp resistance gene.
[0164] Screening was carried out in the yeast strain S. cerevisiae
ImmunaLHB (MAT.alpha. ura3-52 leu2.DELTA.1 trp1d63 his3.DELTA.200
lys2.DELTA. 385) which was derived from the strain JPY5 by
integrating the divergently oriented LacZ and HIS3 reporter genes
under the control of a bi-directional promoter with six
LexA-binding sites (integrating reporter plasmid pDE200, Escher
2000) into the his3.DELTA.200 locus and by integrating the LEU2
reporter gene under the control of a promoter with eight
LexA-binding sites (derived from EGY48) into the leu2.DELTA.1
locus. Transcriptional activation of the reporter system is
mediated by the Gal4-AD moiety of the scFv-fusion construct.
Screening was carried out essentially as described above using
drop-out medium (-Trp/-Leu/-His) and 3-aminotriazole concentrations
up to 40 mM.
Example 2
[0165] Evaluation of In Vivo Performance
[0166] a) In Yeast
[0167] For quantitative analysis of the performance of the selected
frameworks in yeast (FIGS. 1 and 3), S. cerevisiae-strain Immuna
LHB was transformed with the isolated scFvs as LexA-Gal4-AD-fusion
constructs on the pESBA-Act2 vector by following a standard lithium
acetate transformation protocol (Agatep, 1998). Following
transformation, the cells were plated on drop-out plates (Trp). 2
ml overnight-cultures in drop-out medium (-Trp) were inoculated in
duplicates from streaks containing several colonies and grown at
30.degree. C. Cultures were diluted in 1 ml drop-out medium (-Trp)
to an optical density at 600 nm (OD600) of 0.7. They were then
grown at 30.degree. C. for 2 h. For the assay, 100 .mu.l cell
culture were taken, mixed with 900 .mu.l buffer, 45 .mu.l
Chloroform and 30 .mu.l 0.1% SDS, vortexed and incubated at room
temperature for 5 minutes. The color development was initiated by
the addition of 0.2 ml ONPG (4 mg/ml) and stopped with 0.5 ml
Na.sub.2CO.sub.3 (1 M). The activity was calculated by taking into
account the OD600 of the assay culture, as well as the incubation
time of the color development and the culture volume used
[0168] Clones that were at least equal to or better than the
positive control (the very stable lambda-graft described before
(Worn, 2000; Auf der Maur, 2001)) were sequenced to identify the
framework subtype (framework subtype definitions according to
Tomlinson, (1992), Cox, (1994) and Williams, (1996)). Sequencing
revealed a striking preference for certain framework subtypes. For
the heavy chain variable domain (VH), framework subtypes 2 and 6
were never found and 4 was markedly reduced among the positive
clones. Corrected for the performance of the isolated sequences in
the yeast intracellular assay, there is a very strong preference
for VH framework of the subtype 3, but also for 1a and 1b in
intracellular applications. Regarding the light chain variable
domain (VL), there is a clear preference for frameworks of the
kappa 1, lambda 1 and lambda 3 sub-types (FIG. 15).
[0169] These framework subtypes, i.e. VH 1a, 1b and 3 combined with
a kappa 1, lambda 1 and lambda 3 VL domain are therefore best
suited for intracellular use and other applications with stringent
requirements concerning the folding properties of the scFv.
Libraries for intracellular screening systems should, for example,
preferentially be constructed from a mixture of these framework
subtypes only, to reduce the amount molecules which are not
functional in the reducing environment.
[0170] b) In Mammalian Cells
[0171] Hela cell line was used for quantitative analysis of the
performance of the selected frameworks in human cells (FIGS. 2, 4
and 7). The luciferase reporter gene was provided from a
co-transfected pGL3 (Promega) reporter plasmid containing the
luciferase under the control of the natural Gal4 UAS. The mammalian
expression vectors used for transient transfection contains the
Gal4 (1-147) fused on the C-terminus to the VP16-AD under the
control of a CMV promoter. The isolated scFvs were cloned in frame,
C-terminal to a Gal4(1-147)-VP16-fusion to yield a
Gal4(1-147)-VP16-scFv-fusion protein upon expression. Cells were
cultured in DMEM supplemented with 2.5% FCS and 2 mM 1-glutamine.
Transient transfections were carried out according to the
Polyfect-protocol (Qiagen) in 60 mm tissue culture plates using
0.01-0.1 .mu.g of the vector containing the scFv-construct, 0.5
.mu.g of a CMV promoter-driven Gal4(1-147)-VP16-scFv expression
plasmid and 0.5 .mu.g of a LacZ expression vector as reference for
transfection efficiency. Cells were harvested 24-48 hours after
transfection, resupended in 1000 .mu.l buffer and lysed by three
freeze-thaw-cycles. The cell lysate was centrifuged and the
supernatant assayed for luciferase activity using luciferase assay
solution (Promega) and for LacZ activity according to the standard
protocol. The obtained luciferase activity was corrected with the
LacZ activity to account for the variation in transfection
efficiency.
Example 3
[0172] Multiple Alignment and Analysis of the Sequence
Comparison
[0173] To elucidate the general pattern of framework sequences
suitable for intracellular applications, all positive clones (i.e.
those that grow under selective conditions in the quality control
system) were isolated and the part coding for the scFvs was
sequenced. Subsequently, the scFv sequences were divided in their
light and heavy-chain component to allow alignment of the
respective domains (FIGS. 12 and 13) according to the structural
adjusted numbering scheme of immunoglobulin domains by Honegger
(2001).
[0174] To allow evaluation of the obtained data, an alignment
representing the unselected library was generated (FIG. 14). In
order to obtain unselected sequences, the library was transformed
in E. coli cells which do not express the scFv-genes and clones
were picked at random for plasmid isolation and sequencing of the
scFv-sequence. The library covers the human antibody repertoire as
expected and thus has no bias towards specific subgroups, other
than expected by the expression pattern generally found in
humans.
[0175] The VH and VL sequences were grouped according to their
subgroup. Changes to the subgroup-specific consensus sequence were
highlighted. A person skilled in the art can distinguish between
positive, neutral and negative changes based on the structural
environment of the particular exchanged residue (e.g. Honegger,
2001). An exchange of a residue belonging to a particular group of
amino acids to a residue of the same group is in general validated
as a neutral exchange. An exchange of a residue belonging to the
group of hydrophobic amino acid pointing into the hydrophobic core
of the protein to one amino acid of the group of polar but
uncharged or positively or negatively charged amino acids would be
highly unfavorable because unsatisfied hydrogen donor/acceptor
sites disturb tight packing of the hydrophobic core. Such a change
is therefore considered negative. An exchange of a residue
belonging to the group of polar but uncharged residues at the
surface of the immunoglobulin domain to an amino acid of the group
of positively or negatively charged residues is highly favorable as
the solubility of the protein is increased. Such a change is
therefore validated positively, whereas the exchange from a polar
to a hydrophobic residue is highly unfavorable as the solubility of
the protein is decreased and is therefore validated negatively. At
positions with a conserved positive phi-angle, an exchange of any
amino acid to glycine is validated positively whereas an exchange
of gylcine to any amino acid is validated negatively because
glycine is the only amino acid which is able to form a positive
phi-angle. The loss of a conserved salt bridge between positions
45-53, 45-100, 77-100 and 108-137 because of an exchange from an
amino acid of the group of positively or negatively charged
residues to an uncharged amino acid results in a decreased
thermodynamic stability and is therefore considered negative.
[0176] Finally, we chose 7 VL domains and 4 VH domains that were
preferentially selected during the quality control (i.e. showing
the least negative and most positive exchanges from the consensus
sequence and cover the subgroups) and that each show high in vivo
performance in yeast. The sequences are summarized in FIG. 16 and
include two V.kappa.1 (k I 27 (1.x) and k III 25(2.x)), two
V.kappa.3 (k IV 103 (3.x) and k IV135 (5.x)), one V.lamda.1 (k IV
107 (4.x)), two V.lamda.3 (a33 (7.x) and a43 (6.x)), one VH1b (a33
(x.3)) and three VH3 (a fw10 (x.2), a43 (x.4) and a44 (x.1)). These
VL and VH domains were shuffled giving 22 novel combinations in the
scFv format (1.1, 2.1, 3.1, 4.1, 5.1, 1.2, 2.2, 3.2, 4.2, 5.2, 1.3,
2.3, 3.3, 4.3, 5.3, 7.3, 1.4, 2.4, 3.4, 4.4, 5.4, 6.4).
Example 4
[0177] Evaluation of In Vivo Performance of Shuffled Domains
[0178] a) Performance in an Intracellular Assay in Yeast and
Mammalian Cells
[0179] The 22 combinations were tested for their in vivo
performance in yeast and mammalian cells as described in example 2
(FIGS. 3 and 4).
[0180] b) Expression of Soluble Protein Under Reducing Conditions
in Yeast
[0181] To compare the yields of soluble protein upon expression
under reducing conditions, the selected frameworks were expressed
as a fusion to Gal4 AD in the cytoplasm of yeast S. cerevisiae. The
fusion constructs on the pESBA-Act2 vector had the general
structure Gal4 AD-scFv. They were transformed as described above
into the yeast S. cerevisiae strain JPY9 and plated on -Trp,
drop-out plates.
[0182] 5 ml overnight-cultures in drop-out medium (Trp) were
inoculated from streaks containing several colonies and grown at
30.degree. C. Cultures were diluted in 50 ml drop-out medium (-Trp)
to an optical density at 600 nm (OD600) of 0.5. They were grown at
30.degree. C. for 5 h. For the native cell extract, 2.5 ml cell
culture normalized to an OD600 of 3 were harvested by
centrifugation, frozen in liquid nitrogen and subsequently
resuspended in 75 .mu.l Y-PER (Pierce) containing protease
inhibitor (PMSF). The resuspended cell pellet was vortexed shortly
and incubated (slightly shaking) at 20.degree. C. for 20 min.
Insoluble and aggregated material was pelleted at maximal speed in
an eppendorf centrifuge at 4.degree. C. for 10 min. The supernatant
was mixed with loading dye, heated to 100.degree. C. for 5 min. and
separated on a 12% SDS-PAGE. The soluble Gal4 AD-scFv fusion
constructs were visualized by western blotting via detection of the
Gal4-moiety with an anti-Gal4AD monoclonal mouse antibody (Santa
Cruz Biotechnology) as a primary antibody and an
anti-mouse-peroxidase conjugate (Sigma) as secondary antibody and
using a chemoluminescent substrate (Pierce) (FIG. 5). SDS-PAGE and
western blotting procedures are well known to a person of ordinary
skill in the art.
[0183] c) Expression Behavior in the Periplasm of E. coli
[0184] For evaluation of periplasmic expression behavior in E. coli
(FIG. 6), isolated scFvs-frameworks were cloned in a bacterial
vector harbouring the cam resistance gene (catR) and the lacI
repressor gene (Krebber, 1997), with a N-terminal pelB-leader
sequence and a C-terminal his-tag under the control of the lac
promoter/operator. Competent E. coli JM83 were transformed with
these plasmids. 50 ml dYT-medium containing 35 mg/l chloramphenicol
in shaking flasks was inoculated 1:40 with an over-night culture
and incubated at 30.degree. C. Cells were induced at an OD600 of
0.8 with 1 mM IPTG and harvested after 3 hours of induction by
centrifugation. The pellet was resuspended in 50 mM Tris, pH 7.5,
500 mM NaCl and normalized to an OD600 of 10. Samples of each scFv
fragments were analyzed either directly (total extract) or after
sonification followed by centrifugation (soluble fraction) by
SDS-PAGE. The amount of soluble protein was estimated from the
Coomassie-stained gel.
Example 5
[0185] Detailed Evaluation of 5 Combinations with Superior
Properties for Extracellular Use
[0186] Five combinations were chosen as examples which show good
performance both in yeast and mammalian intracellular assays, yield
soluble protein during expression in yeast and E. coli, and cover
the subgroups which were preferentially selected during the quality
control (2.4, 4.4, 5.2, 6.4 and 7.3, see FIG. 16 for details). We
analysed these combinations in greater detail to further evaluate
their use under reducing, as well as oxidizing conditions.
[0187] a) Performance in an Intracellular Assay in Different
Mammalian Cells
[0188] The quantitative analysis of the performance of the five
combinations in human cells was carried out using Hela cells and in
addition using the human osteosarcoma cell line Saos-2 and the
human embryonal kidney cell line HEK293 as performed in Example 2
(FIG. 7).
[0189] b) Performance In Vitro
[0190] Expression and Purification
[0191] For evaluation of the in vitro performance, the five
superior combinations were expressed in the periplasm of E. coli
(FIG. 6). The amount of 0.1 l dYT-medium containing 35 mg/l
chloramphenicol in shaking flasks was inoculated 1:40 with an
over-night culture and incubated at 30.degree. C. Cells were
induced at an OD550 of 1.5 with 1 mM IPTG and harvested after 2
hours of induction by centrifugation. For purification of the
scFvs, the cell pellet was resuspended and lysed by sonication.
Following centrifugation in SS34 at 20 krpm, 4.degree. C. for 30
minutes, the supernatant was applied to a Ni-MC-affinity column
(Hilrap.TM. Chelating HP, 1 ml, Amersham Pharmacia) at pH 7.5 and
eluted with 200 mM imidazol using an Akta Basic system from
Amersham Pharmacia. The purity of the scFv fragments was greater
than 98% as determined by SDS-PAGE (data not shown). The
concentration of the purified protein was determined using the
calculated extinction coefficient at 280 nm. The yield of soluble
purified protein was normalized to a culture volume of 1 l with an
OD600 of 10 and varied from 8 to over 55 mg.
[0192] Resistance to Aggregation
[0193] Resistance towards aggregation has been shown to correlate
with thermodynamic stability (Worn, 1999) in vitro and the
efficiency of tumor localization in a xenografted tumor model in
mice (Willuda, 1999). In order to test for the stability,
resistance to aggregation and reversibility of unfolding, 200 .mu.l
samples of the purified proteins at concentrations of 6 .mu.M in 50
mM Tris, pH 7.5, 100 mM NaCl were either kept 4 days at 4.degree.
C. or 4 days at 37.degree. C. or 3 days at 4.degree. C. followed by
an incubation of 15 or 60 minutes at 100.degree. C., slow cooling
down to room temperature and an overnight incubation at 4.degree.
C. The oligomeric state of each sample was subsequently analyzed on
a gel filtration column equilibrated with 50 mM Tris, pH 7.5, 100
mM NaCl to estimate the amount of aggregated versus monomeric
material (FIG. 8). The proteins were injected on a Superdex-75
column (Amersham Pharmacia) in a volume of 100 .mu.l and a
flow-rate of 1 ml/min on a Akta Basic system (Amersham
Pharmacia).
[0194] Resistance to Protease Degradation
[0195] To determine the stability of the isolated frameworks
towards protease degradation, a parameter that is important for
therapeutic applications, we incubated the purified frameworks in
human serum at 37.degree. C. (FIG. 9).
[0196] Purified, his-tagged scFv-protein (see above) at a
concentration of 50 .mu.M was diluted tenfold into human serum to a
final concentration of 5 .mu.M in 90% serum. The samples were then
either incubated at 37.degree. C. for either 3 days or 1 day, or
taken directly for loading. Before loading insoluble and aggregated
material was pelleted at maximal speed in an eppendorf centrifuge
at 4.degree. C. for 10 min. The supernatant was diluted six-fold
with a loading dye to reduce the amount of serum loaded on the gel,
heated to 100.degree. C. for 5 min. and separated on a 12%
SDS-PAGE. The soluble his-tagged scFv fragments were visualized by
western blotting via detection of the his-tag with an anti-his
monoclonal mouse antibody (Qiagen) as primary and an
anti-mouse-peroxidase conjugate (Sigma) as secondary antibody and
using a chemoluminescent substrate (Pierce). SDS-PAGE and western
blotting procedures are well known to a person of ordinary skill in
the art.
Example 6
[0197] Selection of Antigen Binders Through Screening of a
Randomized CDR-Library on the Framework 7.3 in the Interaction
Screening System in Yeast
[0198] Screening with the interaction system for antigen binders
was essentially performed as described in detail before (Auf der
Maur, 2002).
[0199] The plasmids for expression of the scFv-fusion constructs
for screening in yeast were derived from pESBA-Act2. It contains
the yeast TRP1 nutritional marker and the 2 micron origin of
replication. Moreover it has a constitutive actin promoter for
strong expression and the GAL11 transcriptional termination
sequence, separated by a multiple cloning site. For handling in
bacterial systems, it also has a bacterial origin of replication
and the amp resistance gene.
[0200] The Gal4 activation domain (AD amino acids 768-881) was
originally amplified by PCR using pGAD424 (Clontech) as template
with primers including the SV40 T-antigen nuclear localization
signal N-terminal to the Gal4-AD. The scFv library was obtained by
PCR-amplification of the scFv-framework 7.3 using primers
randomizing 7 amino acids within the CDR3 of VH. The resulting
PCR-product was cloned in the framework 7.3, present in the vector
in the orientation V.sub.L-linker-V.sub.H, as a C-terminal fusion
to Gal4-AD. Expression thus yields a fusion protein of the general
structure Gal4-AD-scFv.
[0201] Screening was carried out in the yeast strain S. cerevisiae
Immuna LHB (MAT.alpha. ura3-52 leu2.DELTA.1 trp1d63 his3.DELTA.200
lys2.DELTA. 385). It was derived from the strain JPY5 by
integrating the divergently oriented LacZ and HIS3 reporter genes
under the control of a bi-directional promoter with six
LexA-binding sites (integrating reporter plasmid pDE200, Escher
2000) into the his3.DELTA.200 locus and by integrating the LEU2
reporter gene under the control of a promoter with eight
LexA-binding sites (derived from EGY48) into the leu2.DELTA.1
locus.
[0202] Transcriptional activation of the reporter system is
mediated by the Gal4-AD moiety of the scFv-fusion construct,
following the specific interaction of its scFv moiety with the
antigen-moiety of the bait-fusion protein. The bait-fusion protein
consists of the kinase domain of the human polo-like kinase 1
(hPlk1-KD) fused C-terminal to the DNA-binding LexA protein. The
kinase domain (amino acid 2-332) was PCR amplified from a hPlk1
cDNA using the upstream primer 5'-tgctctagaagt gctgcagtgactgcag-3'
(Seq. Id.No. 12) and downstream primer
5'-ggttgtcgacttacaggctgctgggagcaatcg-3' (Seq. Id. No. 13). The
resulting PCR product was cloned C-terminal of LexA via XbaI and
SalI into the bait vector. The bait vector contains the URA3
nutritional marker and an Ars Cen origin of replication. Expression
of the bait-fusion protein is driven by a constitutively active
actin promoter. Transcription is terminated by the GAL11
termination sequence. The bait vector also carries a bacterial
origin of replication and the amp resistance gene for propagation
in bacterial systems.
[0203] For screening the yeast strain S. cerevisiae Immuna LHB was
co-transformed with a scFv-library as fusion to Gal4-AD on the
pESBA-Act2 vector and the bait-vector providing the LexA-hPLK1-KD
fusion by following a standard lithium acetate transformation
protocol (Agatep, 1998). Following transformation, the cells were
plated on drop-out plates (-Trp/-Leu/-Ura). Colonies were picked
after 3 to 5 days incubation at 30.degree. C. and re-streaked on
drop-out plates (-Trp/-Leu/-Ura). Those that re-grew were tested
for LacZ expression by development of blue color in a filter assay
on plates containing the substrate X-Gal. Positive clones were
taken for further analysis involving isolation of the scFv-carrying
plasmid from yeast, transformation into E. coli DH5a, isolation of
plasmid from single colonies of E. coli, sequencing and
re-transformation into freshly prepared yeast strain S. cerevisiae
Immuna LHB for the assay as described below. All methods were
performed according to standard procedures, well known to a person
of ordinary skill in the art.
Example 7
[0204] Evaluation of In Vivo Performance of Fab-constructs Derived
from Novel scFv Frameworks
[0205] To evaluate the beneficial effect of using stable variable
domain frameworks on different antibody formats, Fab expression
vector were constructed for use in the yeast interaction
screen.
[0206] a) Fab Constructs for Intracellular Screening in Yeast
[0207] Two different expression vectors were constructed to allow
different expression levels. The vectors are based on either yEplac
112 (2 micron) or yCplac22 (ars/cen) backbones (Gietz, 1988). Both
contain the yeast TRP1 nutritional marker, an inducible,
bi-directional Gal1/Gal10 promoter, a bacterial origin of
replication and the amp resistance gene for handling in bacterial
systems. In one direction, the VH domain of the framework 7.3 was
cloned N-terminal to the CH1-domain of IgG1 including the
C-terminal cysteine, followed by a linker and the Gal4 activation
domain (AD amino acids 768-881) including the SV40 T-antigen. On
the other side, the VL domain of the framework 7.3 was cloned
N-terminal to the CL (lambda)-domain including the C-terminal
cysteine. The terminators are Gal11 terminator on the side of the
heavy chain and Cyclin 1 terminator on the side of the light
chain.
[0208] b) Performance in an Intracellular Assay in Yeast
[0209] For quantitative analysis of the performance of the antigen
binders in scFv and Fab format in yeast (FIGS. 1 and 3), S.
cerevisiae strain Immuna LHB was co-transformed with the isolated
scFvs as Gal4-AD-fusion constructs on the pESBA-Act2 vector and the
bait vector containing the LexA-hPLK1-KD fusion by following a
standard lithium acetate transformation protocol (Agatep, 1998).
Following transformation, the cells were plated on drop-out plates
(-Trp, -Ura, Glc). 2 ml overnight-cultures in drop-out medium
(-Trp, -Ura, Glc) were inoculated in duplicates from streaks
containing several colonies and grown at 30.degree. C. Cultures
were diluted in 1 ml drop-out medium (-Trp, -Ura, Gal) to an
optical density at 600 nm (OD600) of 0.7. They were grown at
30.degree. C. for 5 h. The assay was carried out as described
above.
[0210] c) Expression of Soluble Protein Under Reducing Conditions
in Yeast
[0211] To compare the yields of soluble protein upon expression
under reducing conditions, the scFv and Fab constructs, together
with the hPLK1-KD-bait vector, as described above were expressed in
the cytoplasm of yeast S. cerevisiae. They were transformed as
described above into the yeast strain YDE173 and plated on -Trp,
-Ura, drop-out plates containing glucose.
[0212] 5 ml overnight-cultures in drop-out medium (-Trp, -Ura, Glc)
were inoculated from streaks containing several colonies and grown
at 30.degree. C. Cultures were diluted in YPAG to an optical
density at 600 nm (OD600) of 0.5. They were grown at 30.degree. C.
for 7.5 h. For the native cell extract, 2.5 ml cell culture
normalized to an OD600 of 3 were harvested by centrifugation,
frozen in liquid nitrogen and subsequently resuspended in 75 .mu.l
Y-PER (Pierce). The resuspended cell pellet was vortexed shortly
and incubated slightly shaking at 20.degree. C. for 20 min.
Subsequently insoluble and aggregated material were pelleted at
maximal speed in an eppendorf centrifuge at 4.degree. C. for 10
min. The supernatant was mixed with loading dye, heated to
100.degree. C. for 5 min and separated on a 12% SDS-PAGE. The
soluble Gal4-AD-scFv fusion and the heavy chain part of the Fab
fused to the Gal4-AD were visualized by western blotting via
detection of the Gal4-moiety with an anti-Gal4-AD monoclonal mouse
antibody (Santa Cruz Biotechnology) as primary and an
anti-mouse-peroxidase conjugate (Sigma) as secondary antibody and
using a chemoluminescent substrate (Pierce) (FIG. 11). SDS-PAGE and
western blotting procedures are well known to a person of ordinary
skill in the art.
Sequence CWU 1
1
151107PRTArtificial sequenceantibody framework 1Glu Ile Val Met Thr
Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Ile
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys
Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser
Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Lys Ser Tyr Trp Thr
85 90 95Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly 100
1052108PRTArtificial sequenceantibody framework 2Glu Ile Val Leu
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Leu Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Glu 20 25 30Leu Ala
Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr
Ala Gly Ser Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Leu Pro
Tyr 85 90 95Met Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg 100
1053109PRTArtificial sequenceantibody framework 3Glu Ile Val Met
Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Ser Ala
Ala Leu Ser Cys Arg Ala Ser Gln Gly Val Ser Thr Asn 20 25 30Val Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr
Gly Ala Thr Thr Arg Ala Ser Gly Val Pro Ala Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Ser Leu Gln Ser65
70 75 80Glu Asp Phe Ala Ala Tyr Tyr Cys Gln Gln Tyr Lys His Trp Pro
Pro 85 90 95Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100
1054111PRTArtificial sequenceantibody framework 4Gln Ser Val Leu
Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln1 5 10 15Lys Val Thr
Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Asp Asn 20 25 30Tyr Val
Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Gln Leu Leu 35 40 45Ile
Tyr Asp Asn Thr Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55
60Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln65
70 75 80Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser
Leu 85 90 95Ser Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly 100 105 1105108PRTArtificial sequenceantibody framework 5Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Leu Thr His Tyr
20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile 35 40 45Tyr Asp Thr Ser Lys Arg Ala Thr Gly Val Pro Ala Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Glu Pro65 70 75 80Glu Asp Ser Ala Leu Tyr Tyr Cys Gln Gln Arg
Asn Ser Trp Pro His 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg 100 1056109PRTArtificial sequenceAntibody framework 6Ser
Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln1 5 10
15Thr Ala Thr Val Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val
20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val
Tyr 35 40 45Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser
Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Arg Arg Val
Glu Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
Ser Ser Ser Asp His 85 90 95Asn Val Phe Gly Ser Gly Thr Lys Val Glu
Ile Lys Arg 100 1057109PRTArtificial sequenceantibody framework
7Leu Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln1 5
10 15Thr Ala Arg Ile Ser Cys Gly Gly Asn Asn Ile Glu Thr Ile Ser
Val 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val
Val Ser 35 40 45Asp Asp Ser Val Arg Pro Ser Gly Ile Pro Glu Arg Phe
Ser Gly Ser 50 55 60Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg
Val Glu Ala Gly65 70 75 80Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp
Asp Ser Ser Ser Asp Tyr 85 90 95Val Val Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly 100 1058124PRTArtificial sequenceantibody framework
8Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala His Val Leu Arg Phe Leu Glu
Trp Leu Pro Asp Ala Phe Asp 100 105 110Ile Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 1209108PRTArtificial sequenceantibody
framework 9Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Leu Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser Ser Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45Tyr Ala Ala Ser Ser Ser Gln Ser Gly Val Pro
Ser Arg Phe Arg Gly 50 55 60Ser Glu Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Asn Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ser Tyr Arg Thr Pro Phe 85 90 95Thr Phe Gly Pro Gly Thr Lys
Val Glu Ile Lys Arg 100 10510123PRTArtificial sequenceantibody
framework 10Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala Ser1 5 10 15Val Lys Val Ser Cys Thr Ala Ser Gly Tyr Ser Phe Thr
Gly Tyr Phe 20 25 30Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met Gly 35 40 45Arg Ile Asn Pro Asp Ser Gly Asp Thr Ile Tyr
Ala Gln Lys Phe Gln 50 55 60Asp Arg Val Thr Leu Thr Arg Asp Thr Ser
Ile Gly Thr Val Tyr Met65 70 75 80Glu Leu Thr Ser Leu Thr Ser Asp
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg Val Pro Arg Gly Thr Tyr
Leu Asp Pro Trp Asp Tyr Phe Asp Tyr 100 105 110Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 12011122PRTArtificial sequenceantibody
framework 11Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asp Ala Gly Ile Ala
Val Ala Gly Thr Gly Phe Asp Tyr Trp 100 105 110Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 1201228DNAArtificial sequencePCR Primer
12tgctctagaa gtgctgcagt gactgcag 281333DNAArtificial sequencePCR
Primer 13ggttgtcgac ttacaggctg ctgggagcaa tcg 3314122PRTArtificial
SequenceConsensus sequence 14Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile
Phe Gly Thr Ala Ala Asn Tyr Ala Gln Lys 50 55 60Phe Gln Gly Arg Val
Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala65 70 75 80Tyr Met Glu
Leu Ser Ser Leu Arg Ser Glu Ala Asp Thr Ala Val Tyr 85 90 95Tyr Cys
Ala Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
12015122PRTArtificial SequenceConsensus sequence 15Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Tyr Met
His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asn Pro Asn Ser Gly Gly Thr Asx Asn Tyr Ala Gln Lys 50 55
60Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala65
70 75 80Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asx Asp Thr Ala Val
Tyr 85 90 95Tyr Cys Ala Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp
Tyr Trp 100 105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120
* * * * *
References