U.S. patent application number 13/151207 was filed with the patent office on 2011-10-13 for synthetic immunoglobulin domains with binding properties engineered in regions of the molecule different from the complementarity determining regions.
This patent application is currently assigned to F-STAR BIOTECHNOLOGISCHE FORSCHUNGS-UND ENTWICKLUNGSGES.M.B.H.. Invention is credited to Gottfried HIMMLER, Florian RUKER, Gordana WOZNIAK-KNOPP.
Application Number | 20110251375 13/151207 |
Document ID | / |
Family ID | 36445146 |
Filed Date | 2011-10-13 |
United States Patent
Application |
20110251375 |
Kind Code |
A1 |
RUKER; Florian ; et
al. |
October 13, 2011 |
SYNTHETIC IMMUNOGLOBULIN DOMAINS WITH BINDING PROPERTIES ENGINEERED
IN REGIONS OF THE MOLECULE DIFFERENT FROM THE COMPLEMENTARITY
DETERMINING REGIONS
Abstract
Immunoglobulins which each have one or more amino acid
modifications in at least one structural loop region of such
immunoglobulins, where the modified loop region specifically binds
to an epitope of an antigen to which an unmodified immunoglobulin
does not significantly bind, obtained from display libraries.
Inventors: |
RUKER; Florian; (Vienna,
AT) ; HIMMLER; Gottfried; (Vienna, AT) ;
WOZNIAK-KNOPP; Gordana; (Vienna, AT) |
Assignee: |
F-STAR BIOTECHNOLOGISCHE
FORSCHUNGS-UND ENTWICKLUNGSGES.M.B.H.
Vienna
AT
|
Family ID: |
36445146 |
Appl. No.: |
13/151207 |
Filed: |
June 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13149871 |
May 31, 2011 |
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13151207 |
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11722517 |
Jun 28, 2007 |
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PCT/EP2006/050059 |
Jan 5, 2006 |
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13149871 |
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60641144 |
Jan 5, 2005 |
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Current U.S.
Class: |
530/387.3 ;
530/391.1; 530/391.3; 530/391.7 |
Current CPC
Class: |
C07K 16/00 20130101;
C07K 2317/55 20130101; A61P 37/08 20180101; C07K 2317/31 20130101;
C07K 2317/526 20130101; C07K 2318/20 20130101; A61P 33/02 20180101;
A61P 35/00 20180101; A61P 31/00 20180101; C07K 16/40 20130101; A61P
37/02 20180101; C07K 2319/30 20130101; C07K 19/00 20130101; C07K
2317/52 20130101; C40B 40/10 20130101; C07K 2317/522 20130101; C07K
2317/21 20130101; C07K 16/28 20130101 |
Class at
Publication: |
530/387.3 ;
530/391.3; 530/391.7; 530/391.1 |
International
Class: |
C07K 16/00 20060101
C07K016/00 |
Claims
1. A modified immunoglobulin comprising at least one structural
loop region of a CH3 constant domain, the structural loop region
having at least one amino acid modification with respect to a
structural loop region of a CH3 constant domain of an unmodified
immunoglobulin, thereby forming at least one modified loop region,
wherein the modified loop region specifically binds to an epitope
of an antigen, and wherein the structural loop region of the
unmodified immunoglobulin does not bind to the epitope, and wherein
the modified loop region is obtained from a display library
comprising at least 100 displayed immunoglobulins, each of the at
least 100 displayed immunoglobulins having a modified loop region
comprising an amino acid modification which is different than the
amino acid modifications of the modified loop regions of the
remaining displayed immunoglobulins.
2. The modified immunoglobulin of claim 1, wherein the modified
immunoglobulin is obtained from a library comprising
immunoglobulins displayed on the surface of a host.
3. The modified immunoglobulin of claim 2, wherein the host is
selected from the group consisting of mammalian cells, bacterial
cells, insect cells, and yeast cells.
4. The modified immunoglobulin of claim 1, wherein the modified
immunoglobulin is obtained from a library comprising
immunoglobulins displayed on the surface of phages, phagemids or
viruses.
5. The modified immunoglobulin of claim 1, wherein the modified
immunoglobulin is obtained from a library comprising
immunoglobulins displayed by an in vitro technology.
6. The modified immunoglobulin of claim 5, wherein the in vitro
technology is selected from the group consisting of polysome
display, mRNA display, and ribosome-inactivation display.
7. The modified immunoglobulin of claim 1, wherein the modified
immunoglobulin is obtained from a library comprising at least 1,000
displayed immunoglobulins.
8. The modified immunoglobulin of claim 1, wherein the modified
immunoglobulin is obtained from a library comprising at least
10,000 displayed immunoglobulins.
9. The modified immunoglobulin of claim 1, wherein the modified
immunoglobulin is selected from the group consisting of a full
length immunoglobulin, an Fc fragment, and a CH3 domain.
10. The modified immunoglobulin of claim 1, wherein the
immunoglobulin is of human origin.
11. The modified immunoglobulin of claim 1, wherein the modified
immunoglobulin is derived from IgG1.
12. The modified immunoglobulin of claim 1, wherein the amino acid
modification is selected from the group consisting of a deletion, a
substitution, an insertion, and a combination thereof.
13. The modified immunoglobulin of claim 1, wherein the modified
structural loop region comprises at least 6 amino acid
modifications.
14. The modified immunoglobulin of claim 1, wherein the modified
immunoglobulin comprises at least two modified loop regions.
15. The modified immunoglobulin of claim 1, wherein the modified
immunoglobulin is conjugated to a label or reporter molecule
selected from the group consisting of organic molecules, enzyme
labels, radioactive labels, colored labels, fluorescent labels,
chromogenic labels, luminescent labels, haptens, digoxigenin,
biotin, metal complexes, metals, colloidal gold and mixtures
thereof.
16. The modified immunoglobulin of claim 1, wherein the modified
loop region is within an amino acid sequence selected from the
group consisting of amino acids 7 to 21, amino acids 25 to 39,
amino acids 41 to 81, amino acids 83 to 85, amino acids 89 to 103,
and amino acids 106 to 117, where the numbering of the amino acid
position of the domains is according to the IMGT.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of and claims the benefit
of priority from U.S. patent application Ser. No. 13/149,871, filed
on May 31, 2011, which is a continuation of and claims the benefit
of priority from U.S. patent application Ser. No. 11/722,517, filed
on Jun. 21, 2007, which is the U.S. national phase under 35 U.S.C.
.sctn.371 of PCT International Application No. PCT/EP2006/050059,
filed on Jan. 5, 2006, which claims the benefit of priority under
35 U.S.C. 119(e) to U.S. Provisional Patent Application No.
60/641,144, filed on Jan. 5, 2005. The entire contents of the
foregoing patent applications are incorporated herein by reference
in their entireties.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] The entire content of a Sequence Listing titled
"SEQUENCE_LISTING.txt," created on Jun. 1, 2011 and having a size
of 62 kilobytes, which has been submitted in electronic form in
connection with the present application, is hereby incorporated by
reference herein in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to libraries of
immunoglobulins which each have one or more amino acid
modifications in at least one structural loop region of such
immunoglobulins, wherein the modified loop region specifically
binds to an epitope of an antigen to which an unmodified
immunoglobulin does not significantly bind.
BACKGROUND OF THE INVENTION
[0004] The general field is the engineering of proteins with the
aim to impart them with specific binding properties. More
specifically, the engineered proteins of relevance here are
immunoglobulins (antibodies), and even more specifically, single
domains or pairs or combinations of single domains of
immunoglobulins. The specific binding properties of immunoglobulins
are important features since they control the interaction with
other molecules such as antigens, and render immunoglobulins useful
for diagnostic and therapeutic applications.
[0005] The basic antibody structure will be explained here using as
example an intact IgG1 immunoglobulin.
[0006] Two identical heavy (H) and two identical light (L) chains
combine to form the Y-shaped antibody molecule. The heavy chains
each have four domains. The amino terminal variable domains (VH)
are at the tips of the Y. These are followed by three constant
domains: CH1, CH2, and the carboxy terminal CH3, at the base of the
Y's stem. A short stretch, the switch, connects the heavy chain
variable and constant regions. The hinge connects CH2 and CH3 (the
Fc fragment) to the remainder of the antibody (the Fab fragments).
One Fc and two identical Fab fragments can be produced by
proteolytic cleavage of the hinge in an intact antibody molecule.
The light chains are constructed of two domains, variable (VL) and
constant (CL), separated by a switch.
[0007] Disulfide bonds in the hinge region connect the two heavy
chains. The light chains are coupled to the heavy chains by
additional disulfide bonds. Asn-linked carbohydrate moieties are
attached at different positions in constant domains depending on
the class of immunoglobulin. For IgG1 two disulfide bonds in the
hinge region, between Cys235 and Cys238 pairs, unite the two heavy
chains. The light chains are coupled to the heavy chains by two
additional disulfide bonds, between Cys229s in the CH1 domains and
Cys214s in the CL domains. Carbohydrate moieties are attached to
Asn306 of each CH2, generating a pronounced bulge in the stem of
the Y.
[0008] These features have profound functional consequences. The
variable regions of both the heavy and light chains (VH) and (VL)
lie at the "tips" of the Y, where they are positioned to react with
antigen. This tip of the molecule is the side on which the
N-terminus of the amino acid sequence is located. The stem of the Y
projects in a way to efficiently mediate effector functions such as
the activation of complement and interaction with Fc receptors, or
ADCC and ADCP. Its CH2 and CH3 domains bulge to facilitate
interaction with effector proteins. The C-terminus of the amino
acid sequence is located on the opposite side of the tip, which can
be termed "bottom" of the Y. The structure of an intact IgG1 is
illustrated in FIG. 1a.
[0009] Two types of light chain, termed lambda (.lamda.) and kappa
(.kappa.), are found in antibodies. A given immunoglobulin either
has .kappa. chains or .lamda. chains, never one of each. No
functional difference has been found between antibodies having
.lamda. or .kappa. light chains.
[0010] The structural organization of the main human immunoglobulin
class monomers is shown in FIG. 1b. The classes differ in the
composition and sequence of their respective heavy chains. Both IgM
and IgE lack a hinge region but each contains an extra heavy-chain
domain (CH4). Numbers and locations of the disulfide bonds (lines)
linking the chains differ between the isotypes. They also differ in
the distribution of N-linked carbohydrate groups, symbolically
shown as circles.
[0011] Each domain in an antibody molecule has a similar structure
of two beta sheets packed tightly against each other in a
compressed antiparallel beta barrel. This conserved structure is
termed the immunoglobulin fold. The immunoglobulin fold of constant
domains contains a 3-stranded sheet packed against a 4-stranded
sheet. The fold is stabilized by hydrogen bonding between the beta
strands of each sheet, by hydrophobic bonding between residues of
opposite sheets in the interior, and by a disulfide bond between
the sheets. The 3-stranded sheet comprises strands C, F, and G, and
the 4-stranded sheet has strands A, B, E, and D. The letters A
through G denote the sequential positions of the beta strands along
the amino acid sequence of the immunoglobulin fold.
[0012] The fold of variable domains has 9 beta strands arranged in
two sheets of 4 and 5 strands. The 5-stranded sheet is structurally
homologous to the 3-stranded sheet of constant domains, but
contains the extra strands C' and C''. The remainder of the strands
(A, B, C, D, E, F, G) have the same topology and similar structure
as their counterparts in constant domain immunoglobulin folds. A
disulfide bond links strands B and F in opposite sheets, as in
constant domains. The immunoglobulin fold is illustrated in FIG. 2
for a constant and a variable domain of an immunoglobulin.
[0013] The variable domains of both light and heavy immunoglobulin
chains contain three hypervariable loops, or
complementarity-determining regions (CDRs). The three CDRs of a V
domain (CDR1, CDR2, CDR3) cluster at one end of the beta barrel.
The CDRs are loops that connect beta strands B-C, C'-C'', and F-G
of the immunoglobulin fold. The residues in the CDRs vary from one
immunoglobulin molecule to the next, imparting antigen specificity
to each antibody.
[0014] The VL and VH domains at the tips of antibody molecules are
closely packed such that the 6 CDRs (3 on each domain) cooperate in
constructing a surface (or cavity) for antigen-specific binding.
The natural antigen binding site of an antibody thus is composed of
the loops which connect strands B-C, C'-C'', and F-G of the light
chain variable domain and strands B-C, C'-C'', and F-G of the heavy
chain variable domain.
[0015] Using the 3D structure of a protein as an aid for design,
amino acid residues located on the surface of many proteins have
been randomized using the core structure of the protein as
scaffold. Examples for this strategy are described or reviewed in
the following references incorporated herein by reference: Nygren P
A, Uhlen M., Curr Opin Struct Biol. (1997) 7:463-9; Binz H K,
Amstutz P, Kohl A, Stumpp M T, Briand C, Forrer P, Grutter M G,
Pluckthun A. Nat. Biotechnol. (2004) 22:575-82; Vogt M, Skerra A.
Chembiochem. (2004) 5:191-9; U.S. Pat. No. 6,562,617.
[0016] The basic principle of this technique is based on the
obsernation that many proteins have a stable core, formed by
specific arrangements of secondary structure elements such as beta
sheets or alpha helices, which are interconnected by structures
such as loops, turns, or random coils. Typically, these latter
three structure elements are less crucial for the overall structure
of the protein, and amino acid residues in these structure elements
can be exchanged often without destroying the general fold of the
protein. A naturally occurring example for this design principle
are the CDRs of antibodies. Artificial examples include lipocalins,
ankyrins and other protein scaffolds.
[0017] The loops which are not CDR-loops in a native immunoglobulin
do not have antigen binding or epitope binding specificity but
contribute to the correct folding of the entire immunoglobulin
molecule and/or its effector or other functions and are therefore
called structural loops for the purpose of this invention.
[0018] In U.S. Pat. No. 6,294,654 it is shown that altered
antibodies can be made in which a peptide antigen can be
incorporated into a non-CDR loop of an antibody (Ab) in the CH1
region between the hinge region and the variable region, and the
resulting Ab can be taken up in an APC so that the peptide antigen
is presented on the surface of the APC in the context of MHC II,
and thereby produce an immune response. These inserted peptides are
epitopes and the overall structure of the carrier molecule is not
important. It was demonstrated that a ras peptide can be placed on
a (non-CDR) loop of an Immunoglobulin and the Immunoglobulin still
be secreted. There is stringent "quality control" in the cells
which prevent the Immunoglobulin from being secreted unless it is
properly folded, and altering the amino acid sequence of the loop
might cause the protein to fold into a structure which the cell
would detect as incorrect, and hence degrade it. Thus, besides the
examples shown it was considered to be difficult to further modify
the structural loops without changing the nature of the
Immunoglobulin.
[0019] US Pat Appl 2004/0101905 describes binding molecules
comprising a target binding site and a Fc effector peptide. The Fc
effector peptide is a peptide which interacts with effector
molecule. The insertion of an effector peptide into a non-CDR loop
of a CH1-domain of an immunoglobulin fragment has been shown.
[0020] Fc effector peptides are structures which are naturally
occurring in non-CDR loops of antibodies and are therefore expected
not to disturb the structure of the immunoglobulin if grafted to
onto different equivalent locations in an immunoglobulin.
[0021] Nevertheless every peptide grafted into a non-CDR loop
according to this disclosure has a high chance of being inactive by
the different structural environment it has been selected.
[0022] It is stated in both prior art documents mentioned above
that it is difficult to insert peptides into the loop that should
retain its structure and function, as it is critical not to disturb
the immunoglobulin folding structure as this is important for
function and secretion.
[0023] US Patent Applications 2004/0132101 and 2005/0244403
describe mutant immunoglobulins with altered binding affinity to an
effector ligand, which are natural ligands for structural loops of
antibodies. In this document a number of mutations in various
regions across the entire immunoglobulin molecule are described
which influence the effector function of the entire antibody.
[0024] Other prior art documents show that the immunoglobulin like
scaffold has been employed so far for the purpose of manipulating
the existing antigen binding site, thereby introducing novel
binding properties. So far however, only the CDR regions have been
engineered for antigen binding, in other words, in the case of the
immunoglobulin fold, only the natural antigen binding site has been
modified in order to change its binding affinity or specificity. A
vast body of literature exists which describes different formats of
such manipulated immunoglobulins, frequently expressed in the form
of single-chain Fv fragments (scFv) or Fab fragments, either
displayed on the surface of phage particles or solubly expressed in
various prokaryotic or eukaryotic expression systems. Among the
leading authors in the field are Greg Winter, Andreas Pluckthun and
Hennie Hoogenboom.
BRIEF SUMMARY OF THE INVENTION
[0025] It is an object of the present invention to provide
immunoglobulins with new antigen binding sites introduced, and
methods for engineering and manufacturing said immunoglobulins.
[0026] Therefore, the present invention relates to a method for
engineering an immunoglobulin comprising at least one modification
in a structural loop region of said immunoglobulin and determining
the binding of said immunoglobulin to an epitope of an antigen,
wherein the unmodified immunoglobulin does not significantly bind
to said epitope, comprising the steps of: [0027] providing a
nucleic acid encoding an immunoglobulin comprising at least one
structural loop region, [0028] modifying at least one nucleotide
residue of at least one of said structural loop regions, [0029]
transferring said modified nucleic acid in an expression system,
[0030] expressing said modified immunoglobulin, [0031] contacting
the expressed modified immunoglobulin with an epitope, and [0032]
determining whether said modified immunoglobulin binds to said
epitope.
[0033] In particular, the present invention relates to a method for
engineering an immunoglobulin binding specifically to an epitope of
an antigen selected from the group consisting of allergens, tumor
associated antigens, self antigens, enzymes, bacterial antigens,
fungal antigens, protozooal antigen and viral antigens. Through the
modification in the structural loop region the immunoglobulin may
be engineered bind to the epitope. In a preferred embodiment the
immunoglobulin is binding specifically to at least two such
epitopes, that differ from each other, either of the same antigen
or of different antigens.
[0034] For example, the method according to the invention refers to
engineering an immunoglobulin binding specifically to at least one
first epitope and comprising at least one modification in at least
one structural loop region of said immunoglobulin and determining
the specific binding of said at least one loop region to at least
one second epitope, the epitope being selected from the group of
antigens as mentioned above, wherein the unmodified structural loop
region (non-CDR region) does not specifically bind to said at least
one second epitope, comprising the steps of: [0035] providing a
nucleic acid encoding an immunoglobulin binding specifically to at
least one first epitope comprising at least one structural loop
region, [0036] modifying at least one nucleotide residue of at
least one of said loop regions encoded by said nucleic acid, [0037]
transferring said modified nucleic acid in an expression system,
[0038] expressing said modified immunoglobulin, [0039] contacting
the expressed modified immunoglobulin with said at least one second
epitope, and [0040] determining whether said modified
immunoglobulin binds specifically to the second epitope.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The present invention is further illustrated in the
following figures and examples without being restricted
thereto.
[0042] FIG. 1a shows the structure of an intact IgG1. Domains are
indicated with arrows.
[0043] FIG. 1b illustrates the structural organization of the main
human immunoglobulin isotype monomers. Disulfide bonds are shown as
lines, N-linked carbohydrate groups are shown as circles.
[0044] FIG. 2 shows the immunoglobulin fold for a constant (left)
and a variable (right) domain of an immunoglobulin. Beta strands
are indicated by arrows.
[0045] FIG. 3 shows a molecular model of the engineered CH3 domain
according to the present invention, with the randomized part
indicated by a solvent accessible surface. The surface is
circled.
[0046] FIG. 4 shows a schematic presentation of the PCRs used for
production of the fragments used for assembly of the mutated CH3
domain. PCR primers are indicated by arrows with their respective
5'-3' orientation, and vertical lines indicate the approximate
positions of the introduced restriction sites which were used for
assembly of the mutated gene. The following restriction sites are
contained on the primers for ligations of the PCR fragments:
CH3LNCO: NcoI; CH3LSAC and CH3CSAC: SacI; CH3CHIN and CH3RHIN:
HindIII; CH3RNOT: NotI.
[0047] FIG. 5 shows some examples of how the immunoglobulin domains
of the current application could be used. Randomized regions are
indicated by a star symbol. Specificities of the randomized regions
in one molecule can either be identical or different.
[0048] FIG. 6 shows a schematic presentation of the design of the
bispecific engineered CH3 domain. Names of primers are given in
boxes and arrows indicate the direction in which the primers are
elongated. Boxes with sloping lines indicate the relative positions
of regions that are randomized in this construct, boxes with
vertical lines indicate the relative positions of regions that were
introduced for generation of clone C24, and restriction sites used
for the cloning procedure are given.
[0049] FIG. 7 shows a schematic presentation of the design of the
bispecific engineered CH3 domain. The nucleotide sequence and its
translation is shown of the basic design of the bispecific
engineered CH3 domain. Red sequences indicate randomized regions in
order to generate the bispecific construct, while green boxes
indicate regions in which the sequence was randomized in order to
generate clone C24.
[0050] FIG. 8 shows the sequence listing of the sequences disclosed
herein.
DETAILED DESCRIPTION OF THE INVENTION
[0051] The method according to the invention preferably refers to
at least one modification in at least one structural loop region of
said immunoglobulin and determining the specific binding of said at
least one loop region to at least one antigen selected from the
group consisting of allergens, tumor associated antigens, self
antigens, enzymes, bacterial antigens, fungal antigens, viral
antigens and protozooal antigens, wherein the immunoglobulin
containing an unmodified structural loop region does not
specifically bind to said at least one antigen.
[0052] The term "immunoglobulins" according to the present
invention to be modified (as used herein the terms immunoglobulin
and antibody are interchangeable) may exhibit mono- or
multi-specific, or multivalent binding properties, at least two,
preferably at least three specific binding sites for epitopes of
e.g. antigens, effector molecules/proteins. Immunoglobulins
according to the invention are also functional fragments accepted
in the art, such as Fc, Fab, scFv, single chain dimers of CH/CL
domains, Fv, or other derivatives or combinations of the
immunoglobulins, domains of the heavy and light chains of the
variable region (such as Fd, V1, Vk, Vh) and the constant region of
an intact antibody such as CH1, CH2, CH3, CH4, Cl and Ck, as well
as mini-domains consisting of two beta-strands of an immunoglobulin
domain connected by a structural loop.
[0053] It is understood that the term "immunoglobulin", "modified
immunoglobulin" or "immunoglobulin according to the invention"
includes a derivative of immunoglobulins as well. A derivative is
any combination of one or more immunoglobulins of the invention and
or a fusion protein in which any domain or minidomain of the
immunoglobulin of the invention may be fused at any position of one
or more other proteins (such as other immunoglobulins, ligands,
scaffold proteins, enzymes toxins and the like). A derivative of
the immunoglobulin of the invention may also be obtained by binding
to other substances by various chemical techniques such as covalent
coupling, electrostatic interaction, disulphide bonding etc.
[0054] The other substances bound to the immunoglobulins may be
lipids, carbohydrates, nucleic acids, organic and anorganic
molecules or any combination thereof (e.g. PEG, prodrugs or drugs).
A derivative is also an immunoglobulin with the same amino acid
sequence but made completely or partly from non-natural or
chemically modified amino acids.
[0055] The engineered molecules according to the present invention
will be useful as stand-alone proteins as well as fusion proteins
or derivatives, most typically fused in such a way as to be part of
larger antibody structures or complete antibody molecules, or parts
thereof such as Fab fragments, Fc fragments, Fv fragments and
others. It will be possible to use the engineered proteins to
produce molecules which are monospecific, bispecific, trispecific,
and maybe even carry more specificities at the same time, and it
will be possible at the same time to control and preselect the
valency of binding at the same time according to the requirements
of the planned use of such molecules.
[0056] According to the present invention, binding regions to
antigens or antigen binding sites of all kinds of allergens, tumor
associated antigens, self antigens, enzymes, bacterial antigens,
fungal antigens, protozooal antigen and viral antigens, may be
introduced into a structural loop of a given antibody
structure.
[0057] The term "antigen" according to the present invention shall
mean molecules or structures known to interact or capable of
interacting with the CDR-loop region of immunoglobulins. Structural
loop regions of the prior art do not interact with antigens but
rather contribute to the overall structure and/or to the binding to
effector molecules.
[0058] The term "allergens, tumor associated antigens, self
antigens, enzymes, bacterial antigens, fungal antigens, protozooal
antigen and viral antigens" according to the present invention
shall include all allergens and antigens capable of being
recognised by an antibody structure, and fragments of such
molecules (especially substructures generally referred to as
"epitopes" (e.g. B-cell epitopes)), as long as they are
immunologically relevant, i.e. are also recognisable by natural or
monoclonal antibodies.
[0059] The term "epitope" according to the present invention shall
mean a molecular structure which may completely make up a specific
binding partner or be part of a specific binding partner to the
binding domain or the immunoglobulin of the present invention.
[0060] Chemically, an epitope may either be composes of a
carbohydrate, a peptide, a fatty acid, a anorganic substance or
derivatives thereof and any combinations thereof. If an epitope is
a polypeptide, it will usually include at least 3 amino acids,
preferably 8 to 50 amino acids, and more preferably between about
10-20 amino acids in the peptide. There is no critical upper limit
to the length of the peptide, which could comprise nearly the full
length of the polypeptide sequence. Epitopes can be either linear
or conformational epitopes. A linear epitope is comprised of a
single segment of a primary sequence of a polypeptide chain. Linear
epitopes can be contiguous or overlapping. Conformational epitopes
are comprised of amino acids brought together by folding of the
polypeptide to form a tertiary structure and the amino acids are
not necessarily adjacent to one another in the linear sequence.
[0061] Specifically, epitopes are at least part of diagnostically
relevant molecules, i.e. the absence or presence of an epitope in a
sample is qualitatively or quantitatively correlated to either a
disease or to the health status or to a process status in
manufacturing or to environmental and food status. Epitopes may
also be at least part of therapeutically relevant molecules, i.e.
molecules which can be targeted by the specific binding domain
which changes the course of the disease.
[0062] Preferred "allergens, tumor associated antigens, self
antigens, enzymes, bacterial antigens, fungal antigens, protozooal
antigen and viral antigens," are those allergens or antigens, which
have already been proven to be or are capable of being
immunologically or therapeutically relevant, especially those, for
which a clinical efficacy has been tested.
[0063] On the other hand, according to another aspect of the
present invention also other binding capacities may be introduced
in the structural loop regions, e.g. binding capacities for small
molecules, such as drugs or enzymes, catalytic sites of enzymes or
enzyme substrates or for a transition state analog of an enzyme
substrate.
[0064] Preferably the new antigen binding site in the structural
loops is foreign to the unmodified immunoglobulin. Thus targets
like effector molecules or Fc-receptors are preferably excluded
from the binding molecules and the specificity of the
immunoglobulins according to the invention.
[0065] Preferably, the new antigen binding sites in the structural
loops are introduced by substitution, deletion and/or insertion of
the immunoglobulin encoded by the selected nucleic acid.
[0066] According to another preferred embodiment of the present
invention the modification of at least one nucleotide results in a
substitution, deletion and/or insertion of the immunoglobulin
encoded by said nucleic acid.
[0067] The modification of the at least one loop region may result
in a substitution, deletion and/or insertion of 1 or more amino
acids, preferably a point mutation, exchange of whole loops, more
preferred the change of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 up to
30 amino acids.
[0068] Also preferred is the site directed random mutation. With
this method one or more specific amino acid residues of the loop
are exchanged or introduced using randomly generated inserts into
such structural loops. Alternatively preferred is the use of
combinatorial approaches.
[0069] The at least one loop region is preferably mutated or
modified by random, semi-random or, in particular, by site-directed
random mutagenesis methods. These methods may be used to make amino
acid modifications at desired positions of the immunoglobulin of
the present invention. In these cases positions are chosen
randomly, or amino acid changes are made using simplistic rules.
For example all residues may be mutated to alanine, referred to as
alanine scanning. Such methods may be coupled with more
sophisticated engineering approaches that employ selection methods
to screen higher levels of sequence diversity.
A preferred method according to the invention refers to the
randomly modified nucleic acid molecule which comprises at least
one nucleotide repeating unit having the sequence
5'-NNS-3',5'-NNN-3' or 5'-NNK-3'.
[0070] The randomly modified nucleic acid molecule may comprise the
above identified repeating units, which code for all known
naturally occurring amino acids.
[0071] As is well known in the art, there are a variety of
selection technologies that may be used for the identification and
isolation of proteins with certain binding characteristics and
affinities, including, for example, display technologies such as
phage display, ribosome display, cell surface display, and the
like, as described below. Methods for production and screening of
antibody variants are well known in the art. General methods for
antibody molecular biology, expression, purification, and screening
are described in Antibody Engineering, edited by Duebel &
Kontermann, Springer-Verlag, Heidelberg, 2001; and Hayhurst &
Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard &
Georgiou, 2000, Annu Rev Biomed Eng 2:339-76.
[0072] A "structural loop" or "non-CDR-loop" according to the
present invention is to be understood in the following manner:
immunoglobulins are made of domains with a so called immunoglobulin
fold. In essence, anti-parallel beta sheets are connected by loops
to form a compressed antiparallel beta barrel. In the variable
region, some of the loops of the domains contribute essentially to
the specificity of the antibody, i.e. the binding to an antigen.
These loops are called CDR-loops. All other loops of antibody
domains are rather contributing to the structure of the molecule
and/or the effector function. These loops are defined herein as
structural loops or non-CDR-loops.
[0073] The nucleic acid molecules encoding the modified
immunoglobulins (and always included throughout the whole
specification below: immunoglobulin fragments) may be cloned into
host cells, expressed and assayed for their binding specificities.
These practices are carried out using well-known procedures, and a
variety of methods that may find use in the present invention are
described in Molecular Cloning--A Laboratory Manual, 3.sup.rd Ed.
(Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001),
and Current Protocols in Molecular Biology (John Wiley & Sons).
The nucleic acids that encode the modified immunoglobulins of the
present invention may be incorporated into an expression vector in
order to express said immunoglobulins. Expression vectors typically
comprise an immunoglobulin operably linked, that is placed in a
functional relationship, with control or regulatory sequences,
selectable markers, any fusion partners, and/or additional
elements. The modified immunoglobulins of the present invention may
be produced by culturing a host cell transformed with nucleic acid,
preferably an expression vector, containing nucleic acid encoding
the modified immunoglobulins, under the appropriate conditions to
induce or cause expression of the modified immunoglobulins. The
methods of introducing exogenous nucleic acid molecules into a host
are well known in the art, and will vary with the host used. Of
course, also acellular or cell free expression systems for the
expression of modified immunoglobulins may be employed.
[0074] In a preferred embodiment of the present invention, the
modified immunoglobulins are purified or isolated after expression.
Modified immunoglobulins may be isolated or purified in a variety
of ways known to those skilled in the art. Standard purification
methods include chromatographic techniques, electrophoretic,
immunological, precipitation, dialysis, filtration, concentration,
and chromatofocusing techniques. Purification can often be enabled
by a particular fusion partner. For example, antibodys may be
purified using glutathione resin if a GST fusion is employed,
Ni.sup.+2 affinity chromatography if a His-tag is employed or
immobilized anti-flag antibody if a flag-tag is used. For general
guidance in suitable purification techniques, see Antibody
Purification: Principles and Practice, 3.sup.rd Ed., Scopes,
Springer-Verlag, NY, 1994. Of course, it is also possible to
express the modified immunoglobulins according to the present
invention on the surface of a host, in particular on the surface of
a bacterial, insect or yeast cell or on the surface of phages or
viruses.
[0075] Modified immunoglobulins may be screened using a variety of
methods, including but not limited to those that use in vitro
assays, in vivo and cell-based assays, and selection technologies.
Automation and high-throughput screening technologies may be
utilized in the screening procedures. Screening may employ the use
of a fusion partner or label, for example an enzyme, an immune
label, isotopic label, or small molecule label such as a
fluorescent or colorimetric dye or a luminogenic molecule.
[0076] In a preferred embodiment, the functional and/or biophysical
properties of the immunoglobulins are screened in an in vitro
assay. In a preferred embodiment, the antibody is screened for
functionality, for example its ability to catalyze a reaction or
its binding affinity to its target.
[0077] Assays may employ a variety of detection methods including
but not limited to chromogenic, fluorescent, luminescent, or
isotopic labels.
[0078] As is known in the art, a subset of screening methods are
those that select for favorable members of a library. The methods
are herein referred to as "selection methods", and these methods
find use in the present invention for screening modified
immunoglobulins. When immunoglobulins libraries are screened using
a selection method, only those members of a library that are
favorable, that is which meet some selection criteria, are
propagated, isolated, and/or observed. As will be appreciated,
because only the most fit variants are observed, such methods
enable the screening of libraries that are larger than those
screenable by methods that assay the fitness of library members
individually. Selection is enabled by any method, technique, or
fusion partner that links, covalently or noncovalently, the
phenotype of immunoglobulins with its genotype, that is the
function of a antibody with the nucleic acid that encodes it. For
example the use of phage display as a selection method is enabled
by the fusion of library members to the gene III protein. In this
way, selection or isolation of modified immunoglobulins that meet
some criteria, for example binding affinity to the immunoglobulin's
target, also selects for or isolates the nucleic acid that encodes
it. Once isolated, the gene or genes encoding modified
immunoglobulins may then be amplified. This process of isolation
and amplification, referred to as panning, may be repeated,
allowing favorable antibody variants in the library to be enriched.
Nucleic acid sequencing of the attached nucleic acid ultimately
allows for gene identification.
[0079] A variety of selection methods are known in the art that may
find use in the present invention for screening immunoglobulin
libraries. These include but are not limited to phage display
(Phage display of peptides and antibodies: a laboratory manual, Kay
et al., 1996, Academic Press, San Diego, Calif., 1996; Low-man et
al., 1991, Biochemistry 30:10832-10838; Smith, 1985, Science
228:1315-1317) and its derivatives such as selective phage
infection (Malmborg et al., 1997, J Mol Biol 273:544-551),
selectively infective phage (Krebber et al., 1997, J Mol Biol
268:619-630), and delayed infectivity panning (Benhar et al., 2000,
J Mol Biol 301:893-904), cell surface display (Witrrup, 2001, Curr
Opin Biotechnol, 12:395-399) such as display on bacteria (Georgiou
et al., 1997, Nat Biotechnol 15:29-34; Georgiou et al., 1993,
Trends Biotechnol 11:6-10; Lee et al., 2000, Nat Biotechnol
18:645-648; Jun et al., 1998, Nat Biotechnol 16:576-80), yeast
(Boder & Wittrup, 2000, Methods Enzymol 328:430-44; Boder &
Wittrup, 1997, Nat Biotechnol 15:553-557), and mammalian cells
(Whitehorn et al., 1995, Bio/technology 13:1215-1219), as well as
in vitro display technologies (Amstutz et al., 2001, Curr Opin
Biotechnol 12:400-405) such as polysome display (Mattheakis et al.,
1994, Proc Natl Acad Sci USA 91:9022-9026), ribosome display (Hanes
et al., 1997, Proc Natl Acad Sci USA 94:4937-4942), mRNA display
(Roberts & Szostak, 1997, Proc Natl Acad Sci USA
94:12297-12302; Nemoto et al., 1997, FEBS Lett 414:405-408), and
ribosome-inactivation display system (Zhou et al., 2002, J Am Chem
Soc 124, 538-543).
[0080] Other selection methods that may find use in the present
invention include methods that do not rely on display, such as in
vivo methods including but not limited to periplasmic expression
and cytometric screening (Chen et al., 2001, Nat Biotechnol
19:537-542), the antibody fragment complementation assay (Johnsson
& Varshaysky, 1994, Proc Natl Acad Sci USA 91:10340-10344;
Pelletier et al., 1998, Proc Natl Acad Sci USA 95:12141-12146), and
the yeast two hybrid screen (Fields & Song, 1989, Nature
340:245-246) used in selection mode (Visintin et al., 1999, Proc
Natl Acad Sci USA 96:11723-11728). In an alternate embodiment,
selection is enabled by a fusion partner that binds to a specific
sequence on the expression vector, thus linking covalently or
noncovalently the fusion partner and associated Fc variant library
member with the nucleic acid that encodes them. For example, PCT WO
00/22906; PCT WO 01/49058; PCT WO 02/04852; PCT WO 02/04853; PCT WO
02/08023; PCT WO 01/28702; and PCT WO 02/07466 describe such a
fusion partner and technique that may find use in the present
invention. In an alternative embodiment, in vivo selection can
occur if expression of the antibody imparts some growth,
reproduction, or survival advantage to the cell.
[0081] A subset of selection methods referred to as "directed
evolution" methods are those that include the mating or breeding of
favourable sequences during selection, sometimes with the
incorporation of new mutations. As will be appreciated by those
skilled in the art, directed evolution methods can facilitate
identification of the most favourable sequences in a library, and
can increase the diversity of sequences that are screened. A
variety of directed evolution methods are known in the art that may
find use in the present invention for screening antibody variants,
including but not limited to DNA shuffling (PCT WO 00/42561 A3; PCT
WO 01/70947 A3), exon shuffling (U.S. Pat. No. 6,365,377; Kolkman
& Stemmer, 2001, Nat Biotechnol 19:423-428), family shuffling
(Crameri et al., 1998, Nature 391:288-291; U.S. Pat. No.
6,376,246), RACHITT.TM. (Coco et al., 2001, Nat Biotechnol
19:354-359; PCT WO 02/06469), STEP and random priming of in vitro
recombination (Zhao et al., 1998, Nat Biotechnol 16:258-261; Shao
et al., 1998, Nucleic Acids Res 26:681-683), exonuclease mediated
gene assembly (U.S. Pat. No. 6,352,842; U.S. Pat. No. 6,361,974),
Gene Site Saturation Mutagenesis.TM. (U.S. Pat. No. 6,358,709),
Gene Reassembly.TM. (U.S. Pat. No. 6,358,709), SCRATCHY (Lutz et
al., 2001, Proc Natl Acad Sci USA 98:11248-11253), DNA
fragmentation methods (Kikuchi et al., Gene 236:159-167),
single-stranded DNA shuffling (Kikuchi et al., 2000, Gene
243:133-137), and AMEsystem.TM. directed evolution antibody
engineering technology (Applied Molecular Evolution) (U.S. Pat. No.
5,824,514; U.S. Pat. No. 5,817,483; U.S. Pat. No. 5,814,476; U.S.
Pat. No. 5,763,192; U.S. Pat. No. 5,723,323).
[0082] In a preferred embodiment, antibody variants are screened
using one or more cell-based or in vivo assays. For such assays,
purified or unpurified modified immunoglobulins are typically added
exogenously such that cells are exposed to individual
immunoglobulins or pools of immunoglobulins belonging to a library.
These assays are typically, but not always, based on the function
of the immunoglobulin; that is, the ability of the antibody to bind
to its target and mediate some biochemical event, for example
effector function, ligand/receptor binding inhibition, apoptosis,
and the like. Such assays often involve monitoring the response of
cells to the antibody, for example cell survival, cell death,
change in cellular morphology, or transcriptional activation such
as cellular expression of a natural gene or reporter gene. For
example, such assays may measure the ability of antibody variants
to elicit ADCC, ADCP, or CDC. For some assays additional cells or
components, that is in addition to the target cells, may need to be
added, for example example serum complement, or effector cells such
as peripheral blood monocytes (PBMCs), NK cells, macrophages, and
the like. Such additional cells may be from any organism,
preferably humans, mice, rat, rabbit, and monkey. Immunoglobulins
may cause apoptosis of certain cell lines expressing the target, or
they may mediate attack on target cells by immune cells which have
been added to the assay. Methods for monitoring cell death or
viability are known in the art, and include the use of dyes,
immunochemical, cytochemical, and radioactive reagents. For
example, caspase staining assays may enable apoptosis to be
measured, and uptake or release of radioactive substrates or
fluorescent dyes such as alamar blue may enable cell growth or
activation to be monitored. In a preferred embodiment, the
DELFIA.RTM. EuTDA-based cytotoxicity assay (Perkin Elmer, MA) may
be used. Alternatively, dead or damaged target cells may be
monitored by measuring the release of one or more natural
intracellular components, for example lactate dehydrogenase.
Transcriptional activation may also serve as a method for assaying
function in cell-based assays. In this case, response may be
monitored by assaying for natural genes or immunoglobulins which
may be upregulated, for example the release of certain interleukins
may be measured, or alternatively readout may be via a reporter
construct. Cell-based assays may also involve the measure of
morphological changes of cells as a response to the presence of
modified immunoglobulins. Cell types for such assays may be
prokaryotic or eukaryotic, and a variety of cell lines that are
known in the art may be employed. Alternatively, cell-based screens
are performed using cells that have been transformed or transfected
with nucleic acids encoding the variants. That is, antibody
variants are not added exogenously to the cells. For example, in
one embodiment, the cell-based screen utilizes cell surface
display. A fusion partner can be employed that enables display of
modified immunoglobulins on the surface of cells (Witrrup, 2001,
Curr Opin Biotechnol, 12:395-399).
[0083] In a preferred embodiment, the immunogenicity of the
modified immunoglobulins may be determined experimentally using one
or more cell-based assays. In a preferred embodiment, ex vivo Tcell
activation assays are used to experimentally quantitate
immunogenicity. In this method, antigen presenting cells and naive
T cells from matched donors are challenged with a peptide or whole
antibody of interest one or more times. Then, T cell activation can
be detected using a number of methods, for example by monitoring
production of cytokines or measuring uptake of tritiated thymidine.
In the most preferred embodiment, interferon gamma production is
monitored using Elispot assays (Schmittel et. al., 2000, J.
Immunol. Meth., 24: 17-24).
[0084] The biological properties of the modified immunoglobulins of
the present invention may be characterized in cell, tissue, and
whole organism experiments. As is known in the art, drugs are often
tested in animals, including but not limited to mice, rats,
rabbits, dogs, cats, pigs, and monkeys, in order to measure a
drug's efficacy for treatment against a disease or disease model,
or to measure a drug's pharmacokinetics, toxicity, and other
properties. The animals may be referred to as disease models.
Therapeutics are often tested in mice, including but not limited to
nude mice, SCID mice, xenograft mice, and transgenic mice
(including knockins and knockouts). Such experimentation may
provide meaningful data for determination of the potential of the
antibody to be used as a therapeutic. Any organism, preferably
mammals, may be used for testing. For example because of their
genetic similarity to humans, monkeys can be suitable therapeutic
models, and thus may be used to test the efficacy, toxicity,
pharmacokinetics, or other property of the modified immunoglobulins
of the present invention. Tests of the in humans are ultimately
required for approval as drugs, and thus of course these
experiments are contemplated. Thus the modified immunoglobulins of
the present invention may be tested in humans to determine their
therapeutic efficacy, toxicity, immunogenicity, pharmacokinetics,
and/or other clinical properties.
[0085] The modified immunoglobulins of the present invention may
find use in a wide range of antibody products. In one embodiment
the antibody variant of the present invention is used for therapy
or prophylaxis, for preparative or analytic use, as a diagnostic,
an industrial compound or a research reagent, preferably a
therapeutic. The antibody variant may find use in an antibody
composition that is monoclonal or polyclonal. In a preferred
embodiment, the modified immunoglobulins of the present invention
are used to kill target cells that bear the target antigen, for
example cancer cells. In an alternate embodiment, the modified
immunoglobulins of the present invention are used to block,
antagonize, or agonize the target antigen, for example by
antagonizing a cytokine or cytokine receptor. In an alternately
preferred embodiment, the modified immunoglobulins of the present
invention are used to block, antagonize, or agonize the target
antigen and kill the target cells that bear the target antigen.
[0086] In an alternately preferred embodiment, the modified
immunoglobulins of the present invention are used to block,
antagonize, or agonize growth factors or growth factor receptors
and kill the target cells that bear or need the target antigen. In
an alternately preferred embodiment, the modified immunoglobulins
of the present invention are used to block, antagonize, or agonize
enzymes and substrate of enzymes.
[0087] The modified immunoglobulins of the present invention may be
used for various therapeutic purposes. In a preferred embodiment,
an antibody comprising the modified immunoglobulins is administered
to a patient to treat a specific disorder. A "patient" for the
purposes of the present invention includes both humans and other
animals, preferably mammals and most preferably humans. By
"specific disorder" herein is meant a disorder that may be
ameliorated by the administration of a pharmaceutical composition
comprising a modified immunoglobulin of the present invention.
[0088] In one embodiment, a modified immunoglobulin according to
the present invention is the only therapeutically active agent
administered to a patient. Alternatively, the modified
immunoglobulin according the present invention are administered in
combination with one or more other therapeutic agents, including
but not limited to cytotoxic agents, chemotherapeutic agents,
cytokines, growth inhibitory agents, anti-hormonal agents, kinase
inhibitors, anti-angiogenic agents, cardioprotectants, or other
therapeutic agents. The modified immunoglobulins may be
administered concomitantly with one or more other therapeutic
regimens. For example, an antibody variant of the present invention
may be administered to the patient along with chemotherapy,
radiation therapy, or both chemotherapy and radiation therapy. In
one embodiment, the modified immunoglobulins of the present
invention may be administered in conjunction with one or more
antibodies, which may or may not comprise a antibody variant of the
present invention. In accordance with another embodiment of the
invention, the modified immunoglobulins of the present invention
and one or more other anti-cancer therapies are employed to treat
cancer cells ex vivo. It is contemplated that such ex vivo
treatment may be useful in bone marrow transplantation and
particularly, autologous bone marrow transplantation. It is of
course contemplated that the antibodies of the invention can be
employed in combination with still other therapeutic techniques
such as surgery.
[0089] A variety of other therapeutic agents may find use for
administration with the modified immunoglobulins of the present
invention. In one embodiment, the modified immunoglobulin is
administered with an anti-angiogenic agent, which is a compound
that blocks, or interferes to some degree, the development of blood
vessels. The anti-angiogenic factor may, for instance, be a small
molecule or a protein, for example an antibody, Fc fusion, or
cytokine, that binds to a growth factor or growth factor receptor
involved in promoting angiogenesis. The preferred anti-angiogenic
factor herein is an antibody that binds to Vascular Endothelial
Growth Factor (VEGF). In an alternate embodiment, the modified
immunoglobulin is administered with a therapeutic agent that
induces or enhances adaptive immune response, for example an
antibody that targets CTLA-4. In an alternate embodiment, the
modified immunoglobulin is administered with a tyrosine kinase
inhibitor, which is a molecule that inhibits to some extent
tyrosine kinase activity of a tyrosine kinase. In an alternate
embodiment, the modified immunoglobulins of the present invention
are administered with a cytokine. By "cytokine" as used herein is
meant a generic term for proteins released by one cell population
that act on another cell as intercellular mediators including
chemokines.
[0090] Pharmaceutical compositions are contemplated wherein
modified immunoglobulins of the present invention and one or more
therapeutically active agents are formulated. Formulations of the
antibody variants of the present invention are prepared for storage
by mixing said immunoglobulin having the desired degree of purity
with optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed., 1980), in the form of lyophilized formulations or
aqueous solutions. The formulations to be used for in vivo
administration are preferably sterile. This is readily accomplished
by filtration through sterile filtration membranes or other
methods. The modified immunoglobulins and other therapeutically
active agents disclosed herein may also be formulated as
immunoliposomes, and/or entrapped in microcapsules
[0091] Administration of the pharmaceutical composition comprising
a modified immunoglobulin of the present invention, preferably in
the form of a sterile aqueous solution, may be done in a variety of
ways, including, but not limited to, orally, subcutaneously,
intravenously, intranasally, intraotically, transdermally,
topically (e.g., gels, salves, lotions, creams, etc.),
intraperitoneally, intramuscularly, intrapulmonary (e.g., AERx.TM.
inhalable technology commercially available from Aradigm, or
Inhance.TM. pulmonary delivery system commercially available from
Inhale Therapeutics), vaginally, parenterally, rectally, or
intraocularly.
[0092] As used herein, the term "specifically binds" refers to a
binding reaction which is determinative of the cognate ligand of
interest in a heterogeneous population of molecules. Thus, under
designated conditions (e.g. immunoassay conditions in the case of
an immunoglobulin), the specified antibody binds to its particular
"target" and does not bind in a significant amount to other
molecules present in a sample. Comparable to CDRs of anti-bodies
the modified structural loop regions are antigen- or
molecule-binding protein moieties and not antigens as such.
[0093] The term "expression system" refers to nucleic acid
molecules containing a desired coding sequence and control
sequences in operable linkage, so that hosts transformed or
trans-fected with these sequences are capable of producing the
encoded proteins. In order to effect transformation, the expression
system may be included on a vector; however, the relevant DNA may
than also be integrated into the host chromosome.
[0094] According to a preferred embodiment of the present invention
the expression system comprises a vector. Any expression vector
known in the art may be used for this purpose as appropriate.
[0095] The modified immunoglobulin is preferably expressed in a
host, preferably in a bacterial, a yeast, a plant cell, in an
animal cell or in a plant or animal.
[0096] A wide variety of appropriate host cells may be used to
express the modified immunoglobulin, including but not limited to
mammalian cells (animal cells), plant cells, bacteria (e.g.
Bacillus subtilis, Escherichia coli), insect cells, and yeast (e.g.
Pichia pastoris, Saccharomyces cerevisiae). For example, a variety
of cell lines that may find use in the present invention are
described in the ATCC cell line catalog, available from the
American Type Culture Collection. Furthermore, also plants and
animals may be used as hosts for the expression of the
immunoglobulin according to the present invention. The expression
as well as the transfection vectors or cassettes may be selected
according to the host used.
[0097] Of course also acellular or cell free protein expression
systems may be used. In vitro transcription/translation protein
expression platforms, that produce sufficient amounts of protein
offer many advantages of a cell-free protein expression,
eliminating the need for laborious up- and down-stream steps (e.g.
host cell transformation, culturing, or lysis) typically associated
with cell-based expression systems.
[0098] Another aspect of the present invention relates to a method
for manufacturing an immunoglobulin or a pharmaceutical preparation
thereof comprising at least one modification in a structural loop
region of said immunoglobulin and determining the binding of said
immunoglobulin to an epitope of an antigen, wherein the unmodified
immunoglobulin does not significantly bind to said epitope,
comprising the steps of: [0099] providing a nucleic acid encoding
an immunoglobulin comprising at least one loop region, [0100]
modifying at least one nucleotide residue of at least one of said
loop regions, [0101] transferring said modified nucleic acid in an
expression system, [0102] expressing said modified immunoglobulin,
[0103] contacting the expressed modified immunoglobulin with an
epitope, [0104] determining whether said modified immunoglobulin
binds to said epitope, and [0105] providing the modified
immunoglobulin binding to said epitope and optionally finishing it
to a pharmaceutical preparation.
[0106] In particular the present invention relates to a method for
manufacturing a multi-specific immunoglobulin binding specifically
to at least one first molecule or a pharmaceutical preparation
thereof comprising at least one modification in at least one
structural loop region of said immunoglobulin and determining the
specific binding of said at least one loop region to at least one
second molecule selected from the group consisting of allergens,
tumor associated antigens, self antigens, enzymes, bacterial
antigens, fungal antigens, protozooal antigens and viral antigens,
wherein the immunoglobulin containing an unmodified structural loop
region does not specifically bind to said at least one second
molecule, comprising the steps of: [0107] providing a nucleic acid
encoding an immunoglobulin binding specifically to at least one
first molecule comprising at least one structural loop region,
[0108] modifying at least one nucleotide residue of at least one of
said loop regions encoded by said nucleic acid, [0109] transferring
said modified nucleic acid in an expression system, [0110]
expressing said modified immunoglobulin, [0111] contacting the
expressed modified immunoglobulin with said at least one second
molecule, and [0112] determining whether said modified
immunoglobulin binds specifically to the second molecule and [0113]
providing the modified immunoglobulin binding specifically to said
at least one second molecule and optionally finishing it to a
pharmaceutical preparation.
[0114] The engineering of more than one specificity into a member
of a specific binding pair is preferred (Kufer et al. (2004) Trends
in Biotechnology vol. 22 pages 238-244).
[0115] Numerous attempts have been made to produce multi-specific,
e.g. bispecific, monoclonal antibodies or antibody fragments. One
problem in the production of bispecific antibodies made of two
different polypeptide chains (heavy and light chain) is the
necessity to express four different chains (two heavy and two light
chains) in one cell resulting in a number of various combinations
of molecules which have to be separated from the desired bispecific
molecule in the mixture. Due to their similarity the separation of
these molecules is difficult and expensive. A number of techniques
have been employed to minimize the occurrence of such unwanted
pairings (Carter (2001) Journal of Immunological Methods, vol 248,
pages 7-15)
[0116] One solution to the problem is the production of one
polypeptide chain with two specificities, like e.g. two scFvs
linked to each other or the production of so-called diabodies. Such
molecules have been shown to be far away from the fold of a natural
molecule and are notoriously difficult to produce (LeGall et al.
(2004) Protein Engineering, Design & Selection vol 17 pages
357-366).
[0117] Another problem of the current design of bispecific
antibodies is the fact that even if the parent antibodies are
bivalently binding to their respective binding partner (e.g. IgG),
the resulting bispecific antibody is monovalent for each of the
respective binding partner.
[0118] The preferred multi-specific molecules of the present
invention solve these problems:
[0119] Expression of a bispecific molecule as one polypeptide chain
is possible (a modified Ig domain with two binding specificities,
see example section), which is easier to accomplish than the
expression of two antibody polypeptide chains (Cabilly et al. Proc.
Natl. Acad. Sci. USA 81:3273-3277 (1984)).
[0120] It can also be produced as an antibody like molecule (i.e.
made of 2 polypeptide chains), due to the fact that the second
specificity is located in the non-variable part of the molecule
there is no need for two different heavy chains or different light
chains. Thus, there is no possibility of wrong pairing of the two
chains.
[0121] An antibody of the present invention may consist of a heavy
chain and a light chain, which form together a variable region
binding to a specific binding partner the second specificity may be
formed by a modified loop of any of the structural loops of either
the heavy chain or the light chain. The binding site may also be
formed by more than one non-CDR loop which may be structurally
neighboured (either on the heavy chain or on the light chain or on
both chains).
[0122] The modified antibody or derivative may be a complete
antibody or an antibody fragment (e.g. Fab, CH1-CH2, CH2-CH3).
[0123] It may bind mono- or multi-valently to binding partners or
even with different valency for the different binding partners,
depending on the design.
[0124] As there are a number of various loops available for
selection and design of a specific binding site in the non-CDR
regions of heavy and light chains it is possible to design antibody
derivatives with even more than two specificities without the
problems mentioned above.
[0125] The specific binding domains within one polypeptide chain
may be connected with or without a peptide linker.
[0126] Some antibody classes can be regarded as multi-specific, in
particular bispecific, by nature: They bind to an antigen (which is
typically e.g. either a foreign structure or a cancer associated
structure) with the variable region and bind to Fc-effector
molecules with the Fc part (e.g. Fc receptors on various immune
cells or complement protein) thus enabling effects such as ADCC,
ADCP or CDC.
[0127] The Fc-effector molecules are bound by the Fc-part of an
immunoglobulin molecule (for IgG1 it consists of domains CH2 and
CH3) and a number of methods have been described to optimize
effector function by improvement of binding of the Fc-part of an
antibody molecule either by glycoengineering techniques (U.S. Pat.
No. 6,602,684) or by protein engineering either directly at the Fc
(US 2005/0054832) or indirectly by engineering outside the Fc (US
2005/02444403). Both, binding of the Fc region to Fc recept- or
and/or binding to complement proteins such Cq1 has been altered by
such techniques. Usually the binding affinity to such Fc-effector
molecules is seeked to improve as this correlates with improved
effector functions.
[0128] With the current invention it is possible to design antibody
binding to Fc-effector molecules outside the natural Fc binding
region. Modified loops in antibody domains other than the loops
involved in "natural" Fc-effector molecule binding can be selected
from a library or designed to bind to one or more Fc-effector
molecule. An antibody with such additional Fc-effector molecule
binding sites would either have stronger avidity to a certain
Fc-effector molecule or effector-cell displaying an Fc-effector
molecule and therefore may have an even stronger effect than
glycoengineered antibodies or otherwise improved Fc regions.
However, for certain embodiments of the present invention, the
effector characteristics of a given antibody to be modified should
not directly be changed but remain unaffected by the modification
in the structural loop according to the present invention.
[0129] Antibody fragments have certain advantages as compared to
whole antibodies. Fragments have usually good biodistribution
properties and can more easily be produced. However, most of the
antibody fragment designs lack effector functions and have short in
vivo half life (Holliger P, et al. Nat. Biotechnol. (2005)
23:1126-36.).
[0130] Neither CH1 nor C.kappa. or C.lamda. domains mediate
effector functions which is the reason why Fabs do not show ADCC,
ADCP or CDC. The WO 02/44215 describes binding molecules which
consists of the antigen binding site of an antibody and a peptide
binding Fc-effector molecules. In such a way an antibody fragment
displaying effector functions can be constructed. The peptide is
being incorporated into the binding molecule at a position that
does neither destroy the antigen binding nor the ability of the
peptide to bind to an Fc-effector molecule.
[0131] According to the present invention however, the binding to
Fc-effector molecules may be performed with modified immunoglobulin
domains which have been selected for Fc-effector molecule binding
from libraries of random loop sequences within a fixed scaffold of
an immunoglobulin domain. Therefore, it is possible to select for
specific loop sequences which would not bind to Fc-effector
molecules outside the Ig-domain scaffold. The polypeptides
resulting from the present invention may therefore preferably
consist of more than 100 amino acids.
[0132] In order to select for potential effector function of such
domains according to the present invention, libraries of mutant
CH1, C.kappa. or C.lamda. domains may be selected for binding to
Fc-receptors and/or complement factors such as C1q.
[0133] In order to increase in vivo half life of a molecule
consisting of or containing such a domain (e.g. CH1, CH2, CH3, CH4,
C.kappa. or C.lamda.), binding to FcRn may be selected for with
libraries of mutant e.g. CH1-, CH2-, CH3-, CH4-, C.kappa.- or
C.lamda.-domains according to the present invention.
[0134] FcRn-receptors for selection may be provided either on the
surface of cells expressing naturally the respective receptors or
by expression and purification of the extracellular part of the
respective receptor. For the purpose of this invention a first
screening on FcRn may select for mutant domains which can further
be tested in vitro and even further characterized in FACS
experiments by binding to cells expressing FcRn receptor. It can be
further characterized by affinity ranking of binding to various
recombinant FcRn, isoforms and allotypes e.g with surface plasmon
resonance techniques.
[0135] According to a preferred embodiment of the present invention
the immunoglobulin is of human or murine origin.
[0136] Since the modified immunoglobulin may be employed for
various purposes, in particular in pharmaceutical compositions, the
immunoglobulin is preferably of human or murine origin. Of course,
the modified immunoglobulin may also be a humanized or chimeric
immunoglobulin.
[0137] According to another preferred embodiment of the present
invention the human immunoglobulin is selected from the group
consisting of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and
IgM.
[0138] The murine immunoglobulin is preferably selected from the
group consisting of IgA, IgD, IgE, IgG1, IgG2A, IgG2B, IgG2C, IgG3
and IgM.
[0139] The modified immunoglobulin may be derived from one of the
above identified immunoglobulin classes.
[0140] The immunoglobulin comprises preferably a heavy and/or light
chain of the immunoglobulin or a part thereof.
[0141] The modified immunoglobulin may comprise a heavy and/or
light chain, at least one variable and/or constant domain.
[0142] The immunoglobulin according to the present invention
comprises preferably at least one constant and/or at least one
variable domain of the immunoglobulin or a part thereof including a
minidomain.
[0143] A constant domain is an immunoglobulin fold unit of the
constant part of an immunoglobulin molecule, also referred to as a
domain of the constant region (e.g. CH1, CH2, CH3, CH4, Ck,
Cl).
[0144] A variable domain is an immunoglobulin fold unit of the
variable part of an immunoglobulin, also referred to as a domain of
the variable region (e.g. Vh, Vk, V1, Vd)
[0145] A preferred immunoglobulin according to the invention
consists of a constant domain selected from the group consisting of
CH1, CH2, CH3, CH4, Igk-C, Igl-C, or a part thereof including a
minidomain, with at least one loop region, and is characterised in
that said at least one loop region comprises at least one amino
acid modification forming at least one modified loop region,
wherein said at least one modified loop region binds specifically
to at least one epitope of an antigen.
[0146] Another preferred immunoglobulin according to the invention
consists of a variable domain of a heavy or light chain, or a part
thereof including a minidomain, with at least one loop region, and
is characterised in that said at least one loop region comprises at
least one amino acid modification forming at least one modified
loop region, wherein said at least one modified loop region binds
specifically to at least one epitope of an antigen.
[0147] According to a preferred embodiment the constant domain is
selected from the group of CH1, CH2, CH3, CH4, Igk-C, Igl-C and
combinations thereof.
[0148] The modified immunoglobulin according to the present
invention may comprise one or more constant domains (e.g. at least
two, three, four, five, six, ten domains). If more than one domain
is present in the modified immunoglobulin these domains may be of
the same type or of varying types (e.g. CH1-CH1-CH2, CH3-CH3). Of
course also the order of the single domains may be of any kind
(e.g. CH1-CH3-CH2, CH4-CH1-CH3-CH2).
[0149] All numbering of the amino acid sequences of the
immunoglobulins is according to the IMGT numbering scheme (IMGT,
the international ImMunoGeneTics information; Lefranc et al., 1999,
Nucleic Acids Res. 27: 209-212; Ruiz et al., 2000 Nucleic Acids
Res. 28: 219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:
207-209; Lefranc et al., 2003, Nucleic Acids Res. 31: 307-310;
Lefranc et al., 2005, Dev Comp Immunol 29:185-203).
[0150] According to another preferred embodiment of the present
invention the modified loop regions of CH1, CH2, CH3 and CH4
comprise amino acids 7 to 21, amino acids 25 to 39, amino acids 41
to 81, amino acids 83 to 85, amino acids 89 to 103 and amino acids
106 to 117.
[0151] The loop regions of Igk-C and Igl-C of human origin comprise
preferably amino acids 8 to 18, amino acids 27 to 35, amino acids
42 to 78, amino acids 83 to 85, amino acids 92 to 100, amino acids
108 to 117 and amino acids 123 to 126.
[0152] The loop regions of Igk-C and Igl-C of murine origin
comprise preferably amino acids 8 to 20, amino acids 26 to 36,
amino acids 43 to 79, amino acids 83 to 85, amino acids 90 to 101,
amino acids 108 to 116 and amino acids 122 to 125.
[0153] The structural loop regions of the variable domain of the
immunoglobulin of human origin comprise preferably amino acids 8 to
20, amino acids 44 to 50, amino acids 67 to 76 and amino acids 89
to 101.
[0154] According to a preferred embodiment of the present invention
the structural loop regions of the variable domain of the
immunoglobulin of murine origin comprise amino acids 6 to 20, amino
acids 44 to 52, amino acids 67 to 76 and amino acids 92 to 101.
[0155] The above identified amino acid regions of the respective
immunoglobulins comprise loop regions to be modified.
[0156] The immunoglobulin according to the invention is preferably
of camel origin.
[0157] Camel antibodies comprise only one heavy chain and have the
same antigen affinity as normal antibodies consisting of light and
heavy chains. Consequently camel antibodies are much smaller than,
e.g., human antibodies, which allows them to penetrate dense
tissues to reach the antigen, where larger proteins cannot.
Moreover, the comparative simplicity, high affinity and specificity
and the potential to reach and interact with active sites, camel's
heavy chain antibodies present advantages over common antibodies in
the design, production and application of clinically valuable
compounds.
[0158] The immunoglobulin of camel origin comprises preferably at
least one constant domain selected from the group consisting of
CH1, CH2 and CH3.
[0159] According to a preferred embodiment of the present invention
the loop regions of CH1, CH2 and CH3 of the camel immunoglobulin
comprise amino acids 8 to 20, amino acids 24 to 39, amino acids 42
to 78, amino acids 82 to 85, amino acids 91 to 103 and amino acids
108 to 117.
[0160] According to a preferred embodiment of the present invention
the specific binding of the modified immunoglobulin to the molecule
is determined by a binding assay selected from the group consisting
of immunological assays, preferably enzyme linked immunosorbent
assays (ELISA), surface plasmon resonance assays, saturation
transfer difference nuclear magnetic resonance spectroscopy,
transfer NOE (trNOE) nuclear magnetic resonance spectroscopy,
competitive assays, tissue binding assays, live cell binding assays
and cellular extract assays.
[0161] Binding assays can be carried out using a variety of methods
known in the art, including but not limited to FRET (Fluorescence
Resonance Energy Transfer) and BRET (Bioluminescence Resonance
Energy Transfer)-based assays, AlphaScreen.TM. (Amplified
Luminescent Proximity Homogeneous Assay), Scintillation Proximity
Assay, ELISA (Enzyme-Linked Immunosorbent Assay), SPR (Surface
Plasmon Resonance, also known as BIACORE.RTM.), isothermal
titration calorimetry, differential scanning calorimetry, gel
electrophoresis, and chromatography including gel filtration. These
and other methods may take advantage of some fusion partner or
label.
[0162] The modified immunoglobulin is preferably conjugated to a
label selected from the group consisting of organic molecules,
enzyme labels, radioactive labels, colored labels, fluorescent
labels, chromogenic labels, luminescent labels, haptens,
digoxigenin, biotin, metal complexes, metals, colloidal gold and
mixtures thereof.
[0163] The modified immunoglobulin may be conjugated to other
molecules which allow the simple detection of said conjugate in,
for instance, binding assays (e.g. ELISA) and binding studies.
[0164] Another aspect of the present invention relates to a
immunoglobulin consisting of a constant domain selected from the
group consisting of CH1, CH2, CH3, CH4, Igk-C, Igl-C, or a part
thereof including minidomains, or combinations thereof, with at
least one loop region, characterised in that said at least one loop
region comprises at least one amino acid modification forming at
least one modified loop region, wherein said at least one modified
loop region binds specifically to at least one epitope of an
antigen
[0165] It is preferred to combine molecularly at least one modified
antibody domain (=binding to the specific partner via the
non-variable sequences or structural loop) with at least one other
binding molecule which can be an antibody, antibody fragment, a
soluble receptor, a ligand or another modified antibody domain.
[0166] The molecule is selected from the group consisting of
proteinaceous molecules, nucleic acids, and carbohydrates.
[0167] The loop regions of the modified immunoglobulins may
specifically bind to any kind of binding molecules, in particular
to proteinaceous molecules, proteins, peptides, polypeptides,
nucleic acids, glycans, carbohydrates, lipids, small organic
molecules, anorganic molecules. Of course, the modified
immunoglobulins may comprise at least two loop regions whereby each
of the loop regions may specifically bind to other molecules or
epitopes.
[0168] According to a preferred embodiment of the present invention
the molecule binding to the modified structural loop region is
selected from the group consisting of tumor associated antigens, in
particular EpCAM, tumor-associated glycoprotein-72 (TAG-72),
tumor-associated antigen CA 125, Prostate specific membrane antigen
(PSMA), High molecular weight melanoma-associated antigen
(HMW-MAA), tumor-associated antigen expressing Lewis Y related
carbohydrate, Carcinoembryonic antigen (CEA), CEACAM5, HMFG PEM,
mucin MUC1, MUC18 and cytokeratin tumor-associated antigen,
bacterial antigens, viral antigens, allergens, fluorescein,
lysozyme, toll-like receptor 9, erythropoietin, CD2, CD3, CD3E,
CD4, CD11, CD11a, CD14, CD18, CD19, CD20, CD22, CD23, CD25, CD28,
CD29, CD30, CD33 (p67 protein), CD38, CD40, CD40L, CD52, CD54,
CD56, CD80, CD147, GD3, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-6,
IL-6R, IL-8, IL-12, IL-15, IL-18, IL-23, interferon alpha,
interferon beta, interferon gamma; TNF-alpha, TNFbeta2, TNF.alpha.,
TNFalphabeta, TNF-R1, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL,
RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, TRAIL Receptor-1, A1
Adenosine Receptor, Lymphotoxin Beta Receptor, TACI, BAFF-R, EPO;
LFA-3, ICAM-1, ICAM-3, integrin beta1, integrin beta2, integrin
alpha4/beta7, integrin alpha2, integrin alpha3, integrin alpha4,
integrin alpha5, integrin alpha6, integrin alphav, alphaVbeta3
integrin, FGFR-3, Keratinocyte Growth Factor, VLA-1, VLA-4,
L-selectin, anti-Id, E-selectin, HLA, HLADR, CTLA-4, T cell
receptor, B7-1, B7-2, VNRintegrin, TGFbeta1, TGFbeta2, eotaxinl,
BLyS (B-lymphocyte Stimulator), complement C5, IgE, factor VII,
CD64, CBL, NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3),
Her4 (ErbB4), Tissue Factor, VEGF, VEGFR, endothelin receptor,
VLA-4, carbohydrates such as blood group antigens and related
carbohydrates, Galili-Glycosylation, Gastrin, Gastrin receptors,
tumor associated carbohydrates, Hapten NP-cap or NIP-cap, T cell
receptor alpha/beta, E-selectin, digoxin, placental alkaline
phosphatase (PLAP) and testicular PLAP-like alkaline phosphatase,
transferrin receptor, Heparanase I, human cardiac myosin,
Glycoprotein IIb/IIIa (GPIIb/IIIa), human cytomegalovirus (HCMV) gH
envelope glycoprotein, HIV gp120, HCMV, respiratory syncital virus
RSV F, RSVF Fgp, VNRintegrin, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB
gp120 V3 loop, respiratory syncytial virus (RSV) Fgp, Herpes
simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB
envelope glycoprotein, Clostridium perfringens toxin and fragments
thereof.
[0169] The modified immunoglobulin according to the present
invention may preferably bind to one of the molecules disclosed
above. These molecules comprise also allergens.
[0170] According to another preferred embodiment of the present
invention the amino acid residues of positions 15 to 17, 29 to 34,
85.4 to 85.3, 92 to 94, 97 to 98 and/or 108 to 110 of CH3 are
modified.
[0171] The modification of the immunoglobulin according to the
present invention is preferably a deletion, substitution or an
insertion.
[0172] According to the present invention at least 1, preferably at
least 2, 3, 4, 5, 6, 7, 8, 9, 10 and 15, amino acids are deleted,
substituted with other amino acids (also with modified amino acids)
or inserted into the loop region of the immunoglobulin. However,
the maximum number of amino acids inserted into a loop region of an
immunoglobulin may not exceed the number of 30, preferably 25, more
preferably 20, amino acids. The substitution and the insertion of
the amino acids occurs preferably randomly by methods known in the
art and as disclosed in the present patent application.
[0173] The immunoglobulin according to the invention is according
to a specific embodiment characterised in that the CH3 region
comprises SEQ ID No. 16 or SEQ ID No. 18, when EpCam binds to said
immunoglobulin, SEQ ID No. 20, when fluorescein binds to said
immunoglobulin, SEQ ID No. 22, 24, 26, 28, 30 or 32, when lysozyme
binds to said immunoglobulin, SEQ ID No. 34, 36, 38 or 40, when
TLR9 binds to said immunoglobulin, and SEQ ID No. 42, when lysozyme
and/or erythropoietin bind to said immunoglobulin.
[0174] According to a specific embodiment of the invention the
immunoglobulin is characterised in that it comprises SEQ ID No. 44
or SEQ ID No. 46, when lysozyme and gp41 bind to said
immunoglobulin.
[0175] The modified immunoglobulin is preferably conjugated to a
label or reporter molecule selected from the group consisting of
organic molecules, enzyme labels, radioactive labels, colored
labels, fluorescent labels, chromogenic labels, luminescent labels,
haptens, digoxigenin, biotin, metal complexes, metals, colloidal
gold and mixtures thereof.
[0176] Modified immunoglobulins conjugated to labels as specified
above may be used, for instance, in diagnostic methods.
[0177] Another aspect of the present invention relates to the use
of an immunoglobulin according to the present invention or
obtainable by a method according to the present invention for the
preparation of a vaccine for active immunization. Hereby the
immunoglobulin is either used as antigenic drug substance to
formulate a vaccine or used for fishing or capturing antigenic
structures for use in a vaccine formulation.
[0178] Another aspect of the present invention relates to the use
of an immunoglobulin according to the present invention or
obtainable by a method according to the present invention for the
preparation of a protein library of immunoglobulins.
[0179] Yet another aspect of the present invention relates to a
method for specifically binding and/or detecting a molecule
comprising the steps of:
[0180] (a) contacting a modified immunoglobulin according to the
present invention or a modified immunoglobulin obtainable by a
method according to the present invention with a test sample
suspected to contain said molecule, and
[0181] (b) detecting the potential formation of a specific
immunoglobulin/molecule complex.
[0182] Another aspect of the present invention relates to a method
for specifically isolating a molecule comprising the steps of:
[0183] (a) contacting a modified immunoglobulin according to the
present invention or a modified immunoglobulin obtainable by a
method according to the present invention with a sample containing
said molecule,
[0184] (b) separating the specific immunoglobulin/molecule complex
formed, and
[0185] (c) optionally isolating the molecule from said complex.
[0186] The immunoglobulins according to the present invention may
be used to isolate specifically molecules from a sample. If
multi-specific immunoglobulins are used more than one molecules may
be isolated from a sample. It is especially advantageous using
modified immunoglobulins in such methods because it allows, e.g.,
to generate a matrix having a homogeneous surface with defined
amounts of binding partners (i.e. Modified immunoglobulins)
immobilised thereon which able to bind to the molecules to be
isolated. In contrast thereto, if mono-specific binding partners
are used no homogeneous matrix can be generated because the single
binding partners do not bind with the same efficiency to the
matrix.
[0187] Another aspect of the present invention relates to a method
for targeting a compound to a target comprising the steps of:
[0188] (a) contacting a modified immunoglobulin according to the
present invention or a modified immunoglobulin obtainable by a
method according to the present invention capable to specifically
bind to said compound,
[0189] (b) delivering the immunoglobulin/compound complex to the
target.
[0190] Modified immunoglobulins according to the present invention
may be used to deliver at least one compound bound to the CDRs
and/or modified loop regions to a target. Such immunoglobulins may
be used to target therapeutic substances to a preferred site of
action in the course of the treatment of a disease.
[0191] Another aspect of the present invention relates to a protein
library comprising an immunoglobulin according to the present
invention or obtainable by the method according to the present
invention.
[0192] Preferred methods for constructing said library can be found
above and in the examples. The library according to the present
invention may be used to identify immunoglobulins binding to a
distinct molecule.
[0193] In particular the present invention relates to the use of a
protein library comprising an immunoglobulin according to the
present invention or obtainable by the method according to the
present invention for the design of immunoglobulin derivatives. An
existing immunoglobulin can be changed to introduce antigen binding
sites into any domain or minidomain by using a protein library of
the respective domain of at least 10, preferably 100, more
preferably 1000, more preferably 10000, more preferably 100000,
most preferably more than 1000000 variant domains with at least one
modified loop. The library is then screened for binding to the
specific antigen. After molecular characterization for the desired
properties the selected domain or minidomain is cloned into the
original immunoglobulin by genetic engineering techniques so that
it replaces the wild type region. Alternatively, only the DNA
coding for the loops or coding for the mutated amino acids may be
exchanged to obtain an immunoglobulin with the additional binding
site for the specific antigen.
[0194] The choice of the site for the mutated, antigen-specific
structural loop is dependent on the structure of the original
immunoglobulin and on the purpose of the additional binding site.
If, for example, the original molecule is a complete immunoglobulin
which needs to have inserted an additional antigen binding site
without disturbance of the effector function, the loops to be
modified would be selected from domains distant from CH2 and CH3
which are the natural binding partners to Fc-effect- or molecules.
If the original immunoglobulin is a Fab, modification of loops in
constant domains of the light chains or the heavy chains or the
respective variable domains is possible. To generate a library one
may prepare libraries of mutant original molecules which have
mutations in one or more structural loops of one or more domains.
The selection with complete mutated original molecules may have
some advantages as the selection for antigen binding with a
modified structural loop will deliver the sterically advantageous
modifications if tested also for the other properties the mutated
immunoglobulin should show.
[0195] The size requirement (i.e. the number of variant proteins)
of a protein library of a mutated domain or a minidomain or a
fusion molecule of a domain is dependent on the task. In general, a
library to generate an antigen binding site de novo needs to be
larger than a library used to further modify an already existing
engineered antigen binding site made of a modified structural loop
(e.g. for enhancing affinity or changing fine specificity to the
antigen).
[0196] The present invention also relates to an immunoglobulin
library or a nucleic acid library comprising a plurality of
immunoglobulins, e.g. a constant or variable domain, a minidomain
and/or at least one structural loop region contained in a
minidomain, or nucleic acid molecules encoding the same. The
library contains members with different modifications, wherein the
plurality is defined by the modifications in the at least one
structural loop region. The nucleic acid library preferably
includes at least 10 different members (resulting in one amino acid
exchange) and more preferably includes at least 100, more
preferably 1000 or 10000 different members (e.g. designed by
randomisation strategies or combinatory techniques). Even more
diversified individual member numbers, such as at least 1000000 or
at least 10000000 are also preferred.
[0197] A further aspect of the invention is the combination of two
different domains or minidomains selected from at least two
libraries according to the invention in order to generate
multispecific immunoglobulins. These selected specific
immunoglobulins may be combined with each other and with other
molecules, similar to building blocks, to design the optimal
arrangement of the domains or minidomains to get the desired
properties.
[0198] Furthermore, one or more modified immunoglobulins according
to to invention may be introduced at various or all the different
sites of a protein possible without destruction of the structure of
the protein. By such a "domain shuffling" technique new libraries
are created which can again be selected for the desired
properties.
[0199] The preferred library contains immunoglobulins according to
the invention, selected from the group consisting of domains of an
immunoglobulin, minidomains or derivatives thereof.
[0200] A preferred embodiment of the present invention is a binding
molecule for an antigen (antigen binding molecule) comprising at
least one immunoglobulin domain and a structural loop region being
modified according to the present invention to bind to the antigen,
wherein said binding molecule does not comprise variable domains of
an antibody. It may comprise other parts useable for antibody
activities (e.g. such as natural or modified effector regions
(sequences); however, it lacks the "natural" binding region of
antibodies, i.e. the variable domains in their naturally occurring
position. These antigen binding molecules according to the present
invention have the advantages described above for the present
molecules, yet without the specific binding activity of antibodies;
however with a newly introduced specific binding activity in the
structural loop region.
[0201] Preferably, these antigen binding molecules according to the
present invention comprise CH1, CH2, CH3, CH4, Igk-C, Igl-C and
combinations thereof; said combinations comprising at least two,
preferably at least four, especially at least six constant domains
and at least one structural loop region modified according to the
present invention. Preferably these structural loop regions are
either connected via structural loop region modified according to
the present invention or the structural loops being naturally
present between such two constant domains. An embodiment of these
antigen binding molecules according to the present invention
consists of the Fc region of an antibody with at least one
modification in a structural loop according to the present
invention. Also for the antigen binding molecules according to the
present invention it is preferred that the new antigen binding
sites in the structural loops are introduced by randomising
technologies, i.e. by exchanging one or more amino acid residues of
the loop by randomisation techniques or by introducing randomly
generated inserts into such structural loops. Alternatively
preferred is the use of combinatorial approaches.
[0202] According to another aspect, the present invention relates
to a modified immunoglobulin having an antigen binding site foreign
to the unmodified immunoglobulin and incorporated in one or more
structural loops. The term "foreign" means that the antigen binding
site is not naturally formed by the specific region of the
immunoglobulin, and a foreign binding partner, but not the natural
binding partner of an immunoglobulin, is bound by the antigen
binding site. This means that a binding partner, such as a
Fc-receptor or an effector of the immune system, is not considered
to be bound by the antigen binding site foreign to the unmodified
immunoglobulin.
[0203] Preferably, the antigen is selected from the group
consisting of pathogen antigen, tumour associated antigen, enzyme,
substrate, self antigen, organic molecule or allergen. More
preferred antigens are selected from the group consisting of viral
antigens, bacterial antigens or antigens from pathogens of
eukaryote or phages. Preferred viral antigens include HAV-, HBV-,
HCV-, HIV I-, HIV II-, Parvovirus-, Influenza-, HSV-, Hepatitis
Viruses, Flaviviruses, Westnile Virus, Ebola Virus, Pox-Virus,
Smallpox Virus, Measles Virus, Herpes Virus, Adenovirus, Papilloma
Virus, Polyoma Virus, Parvovirus, Rhinovirus, Coxsackie virus,
Polio Virus, Echovirus, Japanese Encephalitis virus, Dengue Virus,
Tick Borne Encephalitis Virus, Yellow Fever Virus, Coronavirus,
respiratory syncytial virus, parainfluenza virus, La Crosse Virus,
Lassa Virus, Rabies Viruse, Rotavirus antigens; preferred bacterial
antigens include Pseudomonas-, Mycobacterium-, Staphylococcus-,
Salmonella-, Meningococcal-, Borellia-, Listeria, Neisseria-,
Clostridium-, Escherichia-, Legionella-, Bacillus-, Lactobacillus-,
Streptococcus-, Enterococcus-, Corynebacterium-, Nocardia-,
Rhodococcus-, Moraxella-, Brucella, Campylobacter-,
Cardiobacterium-, Francisella-, Helicobacter-, Haemophilus-,
Klebsiella-, Shigella-, Yersinia-, Vibrio-, Chlamydia-,
Leptospira-, Rickettsia-, Mycobacterium-, Treponema-,
Bartonella-antigens. Preferred eukaryotic antigens of pathogenic
eukaryotes include antigens from Giardia, Toxoplasma, Cyclospora,
Cryptosporidium, Trichinella, Yeasts, Candida, Aspergillus,
Cryptococcus, Blastomyces, Histoplasma, Coccidioides.
[0204] Preferred immunoglobulins according to the present invention
comprise at least two antigen binding sites, the first site binding
to a first epitope, and the second site binding to a second
epitope.
[0205] According to a preferred embodiment, the present
immunoglobulin comprises at least two loop regions, the first loop
region binding to a first epitope, and the second loop region
binding to a second epitope. Either the at least first or at least
second loop region or both may be contain a structural loop. The
immunoglobulins according to the present inventions include the
fragments thereof known in the art to be functional which contain
the essential elements according to the present invention: the
structural loop region modified according to the present
invention.
[0206] Preferably, the immunoglobulin according to the present
invention is composed of at least two immunoglobulin domains, or a
part thereof including a minidomain, and each domain contains at
least one antigen binding site.
[0207] Also preferred is an immunoglobulin according to the
invention, which comprises at least one domain of the constant
region and/or at least one domain of the variable region of the
immunoglobulin, or a part thereof including a minidomain. Thus, a
variable domain, which is for example modified in the C-terminal
region, or the variable domain linked to a modified CH1 region, for
instance a modified CH1 minidomain, is one of the preferred
embodiments.
[0208] The preferred immunoglobulin according to the invention
comprises a domain that has at least 50% homology with the
unmodified domain.
[0209] The term "homology" indicates that polypeptides have the
same or conserved residues at a corresponding position in their
primary, secondary or tertiary structure. The term also extends to
two or more nucleotide sequences encoding the homologous
polypeptides.
[0210] "Homologous immunoglobulin domain" means an immunoglobulin
domain according to the invention having at least about 50% amino
acid sequence identity with regard to a full-length native sequence
immunoglobulin domain sequence or any other fragment of a
full-length immunoglobulin domain sequence as disclosed herein.
Preferably, a homologous immunoglobulin domain will have at least
about 50% amino acid sequence identity, preferably at least about
55% amino acid sequence identity, more preferably at least about
60% amino acid sequence identity, more preferably at least about
65% amino acid sequence identity, more preferably at least about
70% amino acid sequence identity, more preferably at least about
75% amino acid sequence identity, more preferably at least about
80% amino acid sequence identity, more preferably at least about
85% amino acid sequence identity, more preferably at least about
90% amino acid sequence identity, more preferably at least about
95% amino acid sequence identity to a native immunoglobulin domain
sequence, or any other specifically defined fragment of a
full-length immunoglobulin domain sequence as disclosed herein.
[0211] "Percent (%) amino acid sequence identity" with respect to
the immunoglobulin domain sequences identified herein is defined as
the percentage of amino acid residues in a candidate sequence that
are identical with the amino acid residues in the specific
immunoglobulin domain sequence, after aligning the sequence and
introducing gaps, if necessary, to achieve the maximum percent
sequence identity, and not considering any conservative
substitutions as part of the sequence identity. Alignment for
purposes of determining percent amino acid sequence identity can be
achieved in various ways that are within the skill in the art, for
instance, using publicly available computer software such as BLAST,
BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the
art can determine appropriate parameters for measuring alignment,
including any algorithms needed to achieve maximal alignment over
the full length of the sequences being compared.
[0212] % amino acid sequence identity values may be obtained as
described below by using the WU-BLAST-2 computer program (Altschul
et al., Methods in Enzymology 266:460-480 (1996)). Most of the
WU-BLAST-2 search parameters are set to the default values. Those
not set to default values, i.e., the adjustable parameters, are set
with the following values: overlap span=1, overlap fraction=0.125,
word threshold (T)=11, and scoring matrix=BLOSUM62. When WU-BLAST-2
is employed, a % amino acid sequence identity value is determined
by dividing (a) the number of matching identical amino acid
residues between the amino acid sequence of the immunoglobulin
domain of interest having a sequence derived from the native
immunoglobulin domain and the comparison amino acid sequence of
interest (i.e., the sequence against which the immunoglobulin
domain of interest is being compared which may be the unmodified
immunoglobulin domain) as determined by WU-BLAST-2 by (b) the total
number of amino acid residues of the non-randomized parts of the
immunoglobulin domain of interest. For example, in the statement "a
polypeptide comprising an amino acid sequence A which has or having
at least 80% amino acid sequence identity to the amino acid
sequence B", the amino acid sequence A is the comparison amino acid
sequence of interest and the amino acid sequence B is the amino
acid sequence of the immunoglobulin domain of interest.
[0213] In a preferred embodiment the immunoglobulin according to
the invention is a bispecific antibody or a bispecific single chain
antibody. Further preferred is that the immunoglobulin comprises a
bispecific domain or a part thereof including a minidomain.
[0214] The immunoglobulin according to the present invention may be
used for any purpose known in the art for immunoglobulins but also
enables applications which are depending on the combination of
specificities introduced by the present invention. Accordingly, the
immunoglobulins according to the present inventions are preferably
used for therapeutic and prophylactic use (e.g. as an active or
passive immunotherapy); for preparative and analytic use and for
diagnostic use.
[0215] Another aspect of the present invention relates to a kit of
binding partners containing
(a) a modified immunoglobulin having an antigen binding site
foreign to the immunoglobulin incorporated in one or more
structural loops, and (b) a binding molecule containing an epitope
of said antigen.
[0216] Such a binding molecule of this kit according to the present
invention may be used for identifying the binding specificity of
the modified immunoglobulin according to the present invention. By
using the binding molecule of this kit according to the present
invention, the potency of the modified immunoglobulins according to
the present invention may be determined.
[0217] Potency as defined here is the binding property of the
modified molecule to its antigen. The binding can be determined
quantitatively and/or qualitatively in terms of specificity and/or
affinity and/or avidity as used for quality control purposes.
[0218] Moreover, the binding molecule of a kit according to the
present invention may be used for selecting the modified
immunoglobulin according to the present invention from a library
consisting of at least 10, preferably at least 100, more preferably
at least 1000, more preferred at least 10000, especially at least
100000 immunoglobulins with different modifications in the
structural loops.
[0219] In accordance with the present invention, one of the key
features of the present invention is that the engineering of the
immunoglobulin domains takes place in regions which are not
normally involved in antigen binding, in other words, in regions
other than the CDRs of an antibody. It was observed that the
specific fold of immunoglobulin domains allows the introduction of
random mutations in regions which are structurally analogous to the
CDRs but different in position in sequence. The regions identified
by the present invention are, like CDRs, loop regions connecting
the beta strands of the immunoglobulin fold.
[0220] More specifically, it is described herein that by
introducing random mutations in the loops connecting beta strands
A-B and E-F of a human IgG1 CH3 domain, mutated CH3 domains were
selected that bind specifically to either Toll like receptor
9-peptide (TLR-9) or to hen egg lysozyme, which are a peptide and a
protein respectively that are not normally recognized and bound by
human CH3 domains of IgG1. The mutations introduced by us include
mutations in which selected amino acid residues in the wildtype
sequence were replaced by randomly chosen residues, and they also
include insertions of extra amino acid residues in the loops
mentioned above.
[0221] By analogy the immunoglobulin domains from any class of
immunoglobulins and from immunoglobulins from any species are
amenable to this type of engineering. Furthermore not only the
specific loops targeted in the present invention can be
manipulated, but any loop connecting beta strands in immunoglobulin
domains can be manipulated in the same way.
[0222] Engineered immunoglobulin domains from any organism and from
any class of immunoglobulin can be used according to the present
invention either as such (as single domains), or as part of a
larger molecule. For example, they can be part of an intact
immunoglobulin, which accordingly would have its "normal" antigen
binding region formed by the 6 CDRs and the new, engineered antigen
binding region. Like this, a multi-specific, e.g. bispecific,
immunoglobulin could be generated. The engineered immunoglobulin
domains can also be part of any fusion protein. The use of these
engineered immunoglobulin domains is in the general field of the
use of immunoglobulins.
[0223] The domains of the following immunoglobulins are understood
as immunoglobulin domains here:
[0224] for IgG, IgD and IgA: VL, CL, VH, CH1, CH2, CH3
[0225] for IgM and IgE: VL, CL, VH, CH1, CH2, CH3, CH4
[0226] 1. Single immunoglobulin domains randomized on one side,
i.e. either in loops connecting beta-strands B-C, D-E or F-G (the
"tip", with the exception of variable domains which are covered by
many patents) or beta-strands A-B, C-D, (C-C' and C''-D in the case
of variable domains) or E-F (the "bottom"). Single loops or any
combination of loops can be randomized. Residues can be changed,
deleted, or additional residues can be inserted.
[0227] 2. Single immunoglobulin domains randomized on both sides,
the tip and the bottom.
[0228] 3. any protein containing one of the single randomized
domains, such as: [0229] a) "single-chain CH3" dimers (scCH3),
scCH2, scCHl/CL, randomized on one or both sides [0230] b)
single-chain Fv randomized on the "bottom", i.e. on the side
opposite to the CDRs [0231] c) Fab fragments randomized at the
"bottom", i.e. on the C-terminal end of the CH1 and of the CL
domain [0232] d) Fc fragments (i.e. proteins consisting of CH2-CH3)
randomized on one or both sides [0233] e) complete immunoglobulins
randomized on the bottom of the Fc [0234] f) other suitable
domains
[0235] The primary advantages of the single domains: are very
similar to all the arguments that are used to promote camel VH
molecules ("nanobodies"). The randomized immunoglobulin domains are
very small proteins (molecular weight ca. 12-15 kDa, depending on
the number of inserted amino acid residues) and therefore will have
the following advantages as compared to conventional antibodies or
antibody fragments such as scFv and Fabs: recognizing uncommon or
hidden epitopes, binding into cavities or active sites of protein
targets, ease of manufacture, and many others. In the case of an
immunoglobulin domain that is randomized on both sides, a bivalent
or a bispecific molecule can be generated. The main advantages of
the single domains as part of fusion proteins is additional binding
properties can be engineered on any other protein.
[0236] It is contemplated that any expression system can be used to
make the proteins. An analogy to the single domains as described
here can be found in the antibodies from the camel, which only has
a VH but no VL In these proteins, only 3 CDRs (instead of 6 as in
"normal" antibodies are responsible for antigen binding).
[0237] The following patent references are incorporated herein by
reference as if set forth in their entirety herewith:
[0238] U.S. Pat. No. 6,294,654 Modified immunoglobulin molecule
incorporating an antigen in a non-CDR loop region
[0239] U.S. Pat. No. 5,844,094 Target binding polypeptide
[0240] U.S. Pat. No. 5,395,750 Methods for producing proteins which
bind to predetermined antigens
[0241] US 2004/0071690 High avidity polyvalent and polyspecific
reagents
[0242] US 2004/0018508 Surrogate antibodies and methods of
preparation and use thereof.
[0243] US 2003/0157091 Multi-functional proteins
[0244] US 2003/0148372 Method to screen phage display libraries
with different ligands
[0245] US 2002/0103345 Bispecific immunoglobulin-like antigen
binding proteins and method of production
[0246] US 2004/0097711 Immunoglobulin superfamily proteins
[0247] US 2004/0082508 Secreted proteins
[0248] US 2004/0063924 Secreted proteins
[0249] US 2004/0043424 Immunoglobulin superfamily proteins
[0250] U.S. Pat. No. 5,892,019 Production of a single-gene-encoded
immunoglobulin
[0251] U.S. Pat. No. 5,844,094 Target binding polypeptide
DESCRIPTION OF SPECIFIC EXAMPLES
Example 1
Construction of the CH3 Library and Phage Surface Display
[0252] The crystal structure of an IgG1 Fc fragment, which is
published in the Brookhaven Database as entry 1OQO.pdb was used to
aid in the design of the mutated CH3 domain.
[0253] The sequence which was used as the basis for construction of
the CH3 library is given in SEQ ID No. 1. In this sequence, the
first amino acid corresponds to Proline 343 of chain A of
Brookhaven database entry 1oqo.pdb. The last residue contained in
1oqo.pdb is Serine 102 of SEQ ID No. 1. After detailed analysis of
the structure of 1oqo.pdb and by visual inspection of the residues
forming the loops which connect the beta strands, it was decided to
randomize residues 17, 18 and 19, which are part of the loop
connecting beta strand A-B as well as 71, 72, 73, 76, and 77, which
are part of the loop connecting beta strand E-F of SEQ ID No. 1. A
molecular model of the engineered CH3 domain, with the randomized
part indicated by a solvent accessible surface is shown in FIG. 3.
The engineered gene was produced by a series of PCR reactions
followed by ligation of the resulting PCR products. To facilitate
ligation, some of the codons of the nucleotide sequence coding for
SEQ ID No. 1 were modified to produce restriction sites without
changing the amino acid sequences (silent mutations). For insertion
into the cloning vector pHEN1 (Nucleic Acids Res. 1991 Aug. 11;
19(15):4133-7. Multi-subunit proteins on the surface of filamentous
phage: methodologies for displaying antibody (Fab) heavy and light
chains. Hoogenboom H R, Griffiths A D, Johnson K S, Chiswell D J,
Hudson P, Winter G.) in frame with the pelB secretion signal, extra
nucleotide residues encoding Met-Ala were attached at the 5' end of
the sequence to create an NcoI restriction site. For the randomized
residues, the codon NNS (IUPAC code, where S means C or G) was
chosen which encodes all 20 naturally occurring amino acids, but
avoids 2 out of 3 stop codons. The engineered sequence is given as
a nucleotide sequence in SEQ ID No. 2 and as an amino acid sequence
in SEQ ID No. 3. The Letter X in SEQ ID No. 3 denotes randomized
amino acid residues. The sequences of the PCR primers used for
assembly of the mutated CH3 domain are given in SEQ ID No. 4
through 9. FIG. 4 shows a schematic presentation of the PCR
fragments generated for assembly of the mutated gene, and the
primers used therefor.
[0254] cDNA of the heavy chain of the human monoclonal antibody 3D6
(Felgenhauer M, Kohl J, Ruker F. Nucleotide sequences of the cDNAs
encoding the V-regions of H- and L-chains of a human monoclonal
antibody specific to HIV-1-gp41. Nucleic Acids Res. 1990 Aug. 25;
18(16):4927) was used as template for the PCR reactions. The 3 PCR
products were digested with Sad and/or HindIII respectively and
ligated together. The ligation product was further digested with
NcoI and NotI and ligated into the surface display phagemid vector
pHEN1, which had previously been digested with NcoI and NotI. A
number of selected clones were controlled by restriction analysis
and by DNA sequencing and were found to contain the insert as
planned, including the correctly inserted randomized sequences. For
the following steps of phage preparation, standard protocols were
followed. Briefly, the ligation mixture was transformed into E.
coli TG1 cells by electroporation. Subsequently, phage particles
were rescued from E. coli TG1 cells with helper phage M13-KO7.
Phage particles were then precipitated from culture supernatant
with PEG/NaCl in 2 steps, dissolved in water and used for selection
by panning or, alternatively, they were stored at minus 80.degree.
C.
Example 2
Construction of the CH3+3 Library
[0255] This library was constructed and cloned in the same way as
the CH3 library. The amino acid sequence of the construct is given
in SEQ ID No. 10, the corresponding nucleotide sequence in SEQ ID
No. 11, and the primers used for construction were SEQ ID No. 4-7,
SEQ ID No. 9 and SEQ ID No. 12.
Example 3
Construction of the CH3+5 Library
[0256] This library was constructed and cloned in the same way as
the CH3 library. The amino acid sequence of the construct is given
in SEQ ID No. 13, the corresponding nucleotide sequence in SEQ ID
No. 14, and the primers used for construction were SEQ ID No. 4-7,
SEQ ID No. 9 and SEQ ID No. 15.
Example 4
Panning of the Ch3-Phage Library on TLR-9 Peptide
[0257] 3 panning rounds were performed according to standard
protocols. Briefly, the following method was applied. Maxisorp
96-well plates (Nunc) were coated with a synthetic peptide
representing part of the sequence of Toll-like Receptor 9 (TLR-9).
200 .mu.l of the following solution were added per well: 0.1M
Na-carbonate buffer, pH 9.6, with the following concentrations of
dissolved peptide:
[0258] 1st panning round: 1 mg/ml TLR-9 peptide
[0259] 2nd panning round: 500 .mu.g/ml TLR-9 peptide
[0260] 3rd panning round: 100 .mu.g/ml TLR-9 peptide
[0261] Incubation was for 1 hour at 37.degree. C., followed by
blocking with 2% dry milk (M-PBS) with 200 .mu.l per well for 1
hour at room temperature.
[0262] The surface display phage library was then allowed to react
with the bound peptide by adding 100 .mu.l phage suspension and 100
.mu.l 4% dry milk (M-PBS), followed by incubation for 45 minutes
with shaking and for 90 minutes without shaking at room
temperature.
[0263] Unbound phage particles were washed away as follows. After
the 1st panning round: 10.times.300 .mu.l T-PBS, 5.times.300 .mu.l
PBS; after the 2nd panning round: 15.times.300 .mu.l T-PBS,
10.times.300 .mu.l PBS; after the 3rd panning round: 20.times.300
.mu.l T-PBS, 20.times.300 .mu.l PBS.
[0264] Elution of bound phage particles was performed by adding 200
.mu.l per well of 0.1 M glycine, pH 2.2, and incubation with
shaking for 30 minutes at room temperature. Subsequently, the phage
suspension was neutralized by addition of 60 .mu.l 2M Tris-Base,
followed by infection into E. coli TG1 cells by mixing 10 ml
exponentially growing culture with 0.5 ml eluted phage and
incubation for 30 minutes at 37.degree. C. Finally, infected
bacteria were plated on TYE medium with 1% glucose and 100 .mu.g/ml
ampicillin, and incubated at 30.degree. C. overnight.
TABLE-US-00001 TABLE 1 Results of the panning of the CH3-phage
library on TLR-9 peptide (Phage titers) Panning Concentration round
TLR-9 at panning Input (phage/ml) Output (phage/ml) 1st 1 mg/ml 6
.times. 10.sup.18 2 .times. 10.sup.10 2nd 0.5 mg/ml 4 .times.
10.sup.18 2 .times. 10.sup.10 3rd 0.1 mg/ml 4 .times. 10.sup.22 6
.times. 10.sup.10
Example 5
Cloning of Selected Clones of CH3 Mutants Selected Against TLR-9
for Soluble Expression
[0265] Phagemid DNA from the phage selected through the 3 panning
rounds was isolated with a midi-prep. DNA encoding mutated
CH3-regions was batch-amplified by PCR and cloned NcoI-NotI into
the vector pNOTBAD/Myc-His, which is the E. coli expression vector
pBAD/Myc-His (Invitrogen) with an inserted NotI restriction site to
facilitate cloning. Ligated constructs were transformed into E.
coli LMG194 cells (Invitrogen) with electroporation, and grown at
30.degree. C. on TYE medium with 1% glucose and ampicillin
overnight. Selected clones were inoculated into 200 .mu.l
2.times.YT medium with ampicillin, grown overnight at 30.degree.
C., and induced by adding L-arabinose to an end concentration of
0.1%. After expression at 16.degree. C. overnight, the cells were
harvested by centrifugation and treated with 100 .mu.l Na-borate
buffer, pH 8.0, at 4.degree. C. overnight for preparation of
periplasmic extracts. 50 .mu.l of the periplasmic extracts were
used in ELISA (see below).
Example 6
ELISA of Ch3 Mutants Selected Against TLR-9
[0266] Selected clones were assayed for specific binding to the
TLR-9 peptide by ELISA. [0267] Coating: Microtiter plate (NUNC,
Maxisorp), 100 .mu.l per well, 20 .mu.g TLR-9 peptide/ml 0.1 M
Na-carbonate buffer, pH 9.6, 1 h at 37.degree. C. [0268] Wash:
3.times.200 .mu.l PBS [0269] Blocking: 1% BSA-PBS, 1 h at RT [0270]
Wash: 3.times.200 .mu.l PBS [0271] Periplasmic extract binding: 50
.mu.l periplasmic extract 50 .mu.l 2% BSA-PBS, at room temperature
overnight [0272] Wash: 3.times.200 .mu.l PBS [0273] 1st antibody:
anti-His4 (Qiagen), 1:1000 in 1% BSA-PBS, 90 min at RT, 100 .mu.l
per well [0274] Wash: 3.times.200 .mu.l PBS [0275] 2nd antibody:
goat anti mouse*HRP(SIGMA), 1:1000 in 1% BSA-PBS, 90 min at RT, 100
.mu.l per well [0276] Wash: 3.times.200 .mu.l PBS [0277] Detection:
3 mg/ml OPD in Na-citrate/phosphate buffer, pH 4.5, 0.4 .mu.l 30%
H.sub.2O.sub.2 [0278] Stopping: 100 ml 3M H.sub.2SO.sub.4 [0279]
Absorbance read: 492/620 nm
[0280] Clones that gave a high signal in this first, preliminary
ELISA were cultured in a 20-ml volume at the same conditions as
described above. Their periplasmic extracts were isolated in 1/20
of the culture volume as described above and tested with ELISA (as
described above) for confirmation.
TABLE-US-00002 TABLE 2 Results of confirmation ELISA with antigen
without antigen A .sub.492/620 A .sub.492/620 clone 4 readings 1
reading A67 0.0435 0.019 B54 0.0937 0.051 C67 0.0295 0.013
Background (antigen alone) (12 parallel readings): 0.0115
Example 7
Panning of the CH3 and of the CH3+5-Phage Library on Hen Egg
Lysozyme
[0281] 3 panning rounds were performed. Maxisorp 96-well plates
(Nunc) were coated with hen egg lysozyme, by adding 200 .mu.l of
the following solution per well:
[0282] PBS, with the following concentrations of dissolved hen egg
lysozyme:
[0283] 1st panning round: 2 mg/ml HEL
[0284] 2nd panning round: 1 mg/ml HEL
[0285] 3rd panning round: 1 mg/ml HEL
[0286] Incubation was for 1 hour at 37.degree. C., followed by
blocking with 2% dry milk (M-PBS) with 200 .mu.l per well for 1
hour at room temperature.
[0287] The surface display phage library was then allowed to react
with the bound hen egg lysozyme by adding 100 .mu.l phage
suspension and 100 .mu.l 4% dry milk (M-PBS), followed by
incubation for 45 minutes with shaking and for 90 minutes without
shaking at room temperature.
[0288] Unbound phage particles were washed away as follows:
[0289] 1st panning round: 10.times.300 .mu.l T-PBS, 5.times.300
.mu.l PBS
[0290] 2nd panning round: 15.times.300 .mu.l T-PBS, 10.times.300
.mu.l PBS
[0291] 3rd panning round: 20.times.300 .mu.l T-PBS, 20.times.300
.mu.l PBS
[0292] Elution of bound phage particles was performed by adding 200
.mu.l per well of 0.1 M glycine, pH 2.2, and incubation with
shaking for 30 minutes at room temperature. Subsequently, the phage
suspension was neutralized by addition of 60 .mu.l 2M Tris-Base,
followed by infection into E. coli TG1 cells by mixture of 10 ml
exponentially growing culture with 0.5 ml eluted phage and
incubation for 30 minutes at 37.degree. C. Finally, infected
bacteria were plated on TYE medium with 1% glucose and 100 .mu.g/ml
ampicillin, and incubated at 30.degree. C. overnight.
TABLE-US-00003 TABLE 3 Results of the panning of phage library CH3
on hen egg lysozyme (Phage titers) Panning Concentration round HEL
at panning Input (phage/ml) Output (phage/ml) 1st 2 mg/ml 4.7
.times. 10.sup.10 2nd 1 mg/ml 1.29 .times. 10.sup.22 8.0 .times.
10.sup.9 3rd 1 mg/ml 5.71 .times. 10.sup.20 4.8 .times.
10.sup.10
TABLE-US-00004 TABLE 4 Results of the panning of the phage library
CH3+5 on hen egg lysozyme (HEL) (phage titers) Panning
Concentration round HEL at panning Input (phage/ml) Output
(phage/ml) 1st 2 mg/ml 8.3 .times. 10.sup.16 2.9 .times. 10.sup.9
2nd 1 mg/ml 2.1 .times. 10.sup.19 2.6 .times. 10.sup.9 3rd 1 mg/ml
5.4 .times. 10.sup.19 1.2 .times. 10.sup.10
Example 8
Cloning of Selected Clones of Example 7 for Soluble Expression
[0293] The cloning of selected clones for soluble expression was
performed as described above for the CH3 mutants selected against
TLR-9.
Example 9
Soluble Expression of Selected Clones of Example 7
[0294] The soluble expression of selected clones was performed as
described above for the CH3 mutants selected against TLR-9.
Periplasmic extracts were tested in a preliminary ELISA (protocol
see example 10)
[0295] Clones that gave a high signal in this first, preliminary
ELISA were cultured in a 20-ml volume at the same conditions as
described above. Their periplasmic extracts were isolated in 1/20
of the culture volume as described above and tested with ELISA (as
described in example 10) for confirmation.
Example 10
ELISA of CH3 Mutants Selected Against Hen Egg Lysozyme
[0296] Coating: Microtiter plate (NUNC, Maxisorp), 100 .mu.l per
well, 100 .mu.g hen egg lysozyme/ml in PBS, 1 h at 37.degree. C.
[0297] Wash: 3.times.200 .mu.l PBS [0298] Blocking: 1% BSA-PBS, 1 h
at RT [0299] Wash: 3.times.200 .mu.l PBS [0300] Periplasmic extract
binding: 50 .mu.l periplasmic extract 50 .mu.l 2% BSA-PBS, at room
temperature overnight [0301] Wash: 3.times.200 .mu.l PBS [0302] 1st
antibody: anti-His4 (Qiagen), 1:1000 in 1% BSA-PBS, 90 min at RT,
100 .mu.l per well [0303] Wash: 3.times.200 .mu.l PBS [0304] 2nd
antibody: goat anti mouse*HRP(SIGMA), 1:1000 in 1% BSA-PBS, 90 min
at RT (room temperature), 100 .mu.l per well [0305] Wash:
3.times.200 .mu.l PBS [0306] Detection: 3 mg/ml OPD in
Na-citrate/phosphate buffer, pH 4.5, 0.4 .mu.l 30% H.sub.2O.sub.2
[0307] Stopping: 100 ml 3M H.sub.2SO.sub.4 [0308] Absorbance read:
492/620 nm
TABLE-US-00005 [0308] TABLE 5 Results of confirmation ELISA of
C.sub.H3 mutants selected against hen egg lysozyme with antigen
without antigen A .sub.492/620 A .sub.492/620 clone 4 readings 1
reading B12 0.396 0.012 D10 0.415 0.026 D46 0.398 0.011 Background
(antigen alone) (12 parallel readings): 0.1763
TABLE-US-00006 TABLE 6 Results of confirmation ELISA with antigen
dilutions of C.sub.H3 mutants selected against hen egg lysozyme c
200 100 50 25 12.5 6.25 3.125 1.55 0.78 0.39 (.mu.g/ml) clone B12
0.707 0.532 0.432 0.297 0.192 0.150 0.148 0.049 0.034 0.015 D46
0.713 0.561 0.342 0.220 0.133 0.088 0.047 0.032 0.021 0.010 D10
0.715 0.685 0.571 0.368 0.231 0.175 0.171 0.068 0.047 0.026 - (nc)
0.449 0.360 0.165 0.072 0.038 0.023 0.017 0.013 0.009 0.007 nc: no
periplasmic extract added
[0309] It is noted that hen egg lysozyme reacts with anti-his.sub.4
antibody, therefore a relatively high background was observed.
TABLE-US-00007 TABLE 7 Results of confirmation ELISA of C.sub.H3+5
mutants selected against hen egg lysozyme with antigen without
antigen A .sub.492/620 A .sub.492/620 clone 4 readings 1 reading
A13 0.197 0.016 A66 0.461 0.019 B18 0.533 (5 readings) Not done B20
0.184 0.016 B68 0.535 0.019 B40 0.706 0.051 C24 0.352 0.072 D22
0.147 0.019 C22 0.439 0.017 D37 0.360 0.026 D40 0.559 0.034 D56
0.369 0.019 Background (antigen alone) (12 parallel readings):
0.1334 Note: hen egg lysozyme reacts with anti-his.sub.4 antibody,
therefore a relatively high background was observed.
Example 11
CL Library
[0310] Visual inspection of the crystal structure of an Fab
fragment (the structure of the Fab of the human monoclonal antibody
3D6 is used: RSCB Protein Data Bank Entry 1DFB.PDB (He X M, et al.
Proc Natl Acad Sci USA. 1992 Aug. 1; 89(15):7154-8) and
computer-aided analysis (e.g. Protein Explorer is used for this
purpose) of the secondary and tertiary structure of this protein)
allows to identify residues located in loop regions which connect
the beta-strands of the CL-domain scaffold. These residues comprise
amino acids 8 to 18, amino acids 27 to 35, amino acids 42 to 78,
amino acids 83 to 85, amino acids 92 to 100, amino acids 108 to 117
and amino acids 123 to 126 (numbering according to the IMGT
numbering system (Lefranc M P, et al. Nucleic Acids Res. 2005 Jan.
1; 33 (Database issue):D593-7; Lefranc M P, et al. Dev Comp
Immunol. 2005; 29(3):185-203)).
[0311] More specifically, residues 11, 12, 14-18, and 92-95 are
randomized within the human CL domain (SEQ ID No. 48).
Randomization is achieved by PCR amplification of the coding
sequences with PCR primers in which the positions of the relevant
codons are encoded by the nucleotide sequence 5'-NNS-3', which
potentially encodes for all 20 amino acids while avoiding 2 out of
3 stop codons. The library insert is amplified by two separate PCR
reactions, and the two PCR fragments are ligated together via a
HpyCH4IV restriction site which is introduced as a silent mutation
by the PCR primers. The primers further provide the restriction
endonuclease sites NcoI and NotI respectively for cloning into the
phage display vector pHEN (Hoogenboom H R, et al. Nucleic Acids
Res. 1991 Aug. 11; 19(15):4133-7). The C-terminal cystein of the CL
domain is not included for the phage display, but can be added
later on when a modified CL clone is used e.g. for the construction
of an Fab fragment.
[0312] As a template for PCR amplification, a plasmid such as
pRcCMV-3D6LC(Ruker F, et al. Ann N Y Acad. Sci. 1991 Dec. 27;
646:212-9), which contains as an insert the complete light chain of
the human monoclonal antibody, is used.
[0313] For the CL+3 (SEQ ID No. 50, 51) and the CL+5 (SEQ ID No.
52, 53) libraries, which contain additional residues inserted
between position 92 and 95 of the CL domain, primer CLRHPY3 and
CLRHPY5 are used respectively instead of primer CLRHPY.
[0314] The nucleotide and amino acid sequence of the final product
of the PCRs and ligations, cloned into the NcoI site of pHEN1,
which leads to the attachment of a pelB leader sequence to the
N-terminus of the construct is shown below (SEQ ID No. 48, 49):
TABLE-US-00008 +3 M K Y L L P T A A A G L L L L A A 1 ATGAAATACC
TATTGCCTAC GGCAGCCGCT GGATTGTTAT TACTCGCGGC NcoI +3 Q P A M A V A A
P S V F I F P P 51 CCAGCCGG CCGTGG CTGCACCATC TGTCTTCATC TTCCCGCCAT
+3 S Q A S V V C L L N 101 CTNNSNNSCA GNNSNNSNNS NNSNNSGCCT
CTGTTGTGTG CCTGCTGAAT +3 N F Y P R E A K V Q W K V D N A L 151
AACTTCTATC CCAGAGAGGC CAAAGTACAG TGGAAGGTGG ATAACGCCCT +3 Q S G N S
Q E S V T E Q D S K D 201 CCAATCGGGT AACTCCCAGG AGAGTGTCAC
AGAGCAGGAC AGCAAGGACA HpyCH4IV +3 S T Y S L S S T L T L Y E 251
GCACCTACAG CCTCAGCAGC ACCCTG TGNNSNNSNN SNNSTACGAG +3 K H K V Y A C
E V T H Q G L S S P 301 AAACACAAAG TCTACGCCTG CGAAGTCACC CATCAGGGCC
TGAGCTCGCC NotI +3 V T K S F N R G E A A A 351 CGTCACAAAG
AGCTTCAACA GGGGAGAG A Primer List for CL library: cllnco: (SEQ ID
No. 56) 5'-cttaccatgg ccgtggctgc accatctgtc ttcatcttcc cgc-catctnn
snnscagnns nnsnnsnnsn nsgcctctgt tgtgtgc-3' cllhpy: (SEQ ID No. 57)
5'-tgacaacgtc agggtgctgc tgaggc-3' clrhpy: (SEQ ID NO: 58)
5'-tcagaacgtt gnnsnnsnns nnstacgaga aacacaaagt c-3' clrhpy3: (SEQ
ID No. 59) 5'-tcagaacgtt gnnsnnsnns nnsnnsnnsn nstacgagaa
acacaaagtc-3' (SEQ ID No. 60) 5'- tcagaacgtt gnnsnnsnns nnsnnsnnsn
nsnnsnnsta cgagaaacac aaagtc-3' clrnot: (SEQ ID No. 61)
5'-catcgcggcc gcctctcccc tgttgaagct c-3'
[0315] A number of selected library clones (mutated CL domains
cloned in the phagmid vector pHEN1) are controlled by restriction
analysis and by DNA sequencing to contain the insert as planned,
including the correctly inserted randomized sequences. For the
following steps of phage preparation, standard protocols are
followed. Briefly, the ligation mixture is transformed into E. coli
TG1 cells by electroporation. Subsequently, phage particles are
rescued from E. coli TG1 cells with helper phage M13-KO7. Phage
particles are then precipitated from culture supernatant with
PEG/NaCl in 2 steps, dissolved in water and used for selection by
panning or, alternatively, they can be stored at minus 80.degree.
C.
Example 12
CH1 Library
[0316] Visual inspection of the crystal structure of an Fab
fragment (the structure of the Fab of the human monoclonal antibody
3D6 is used: RSCB Protein Data Bank Entry 1DFB.PDB) and
computer-aided analysis (Protein Explorer is used for this purpose)
of the secondary and tertiary structure of this protein allows to
identify residues located in loop regions which connect the
beta-strands of the CH1-domain scaffold. These residues comprise
amino acids 7 to 21, amino acids 25 to 39, amino acids 41 to 81,
amino acids 83 to 85, amino acids 89 to 103 and amino acids 106 to
117 (numbering according to the IMGT numbering system).
[0317] More specifically, residues 12-19 and 93-100 are randomized
within the human CH1 domain (SEQ ID No. 54, 55). Randomization is
achieved by PCR amplification of the coding sequences with PCR
primers in which the positions of the relevant codons are encoded
by the nucleotide sequence 5'-NNS-3', which potentially encodes for
all 20 amino acids while avoiding 2 out of 3 stop codons. The
library insert is amplified by two separate PCR reactions, and the
two PCR fragments are ligated together via a BstEII restriction
site which occurs naturally in the CH1 domain. The primers further
provide the restriction endonuclease sites NcoI and NotI
respectively for cloning into the phage display vector pHEN. The
C-terminal cystein of the CH1 domain is not included for the phage
display, but can be added later on when a modified CH1 clone is
used e.g. for the construction of an Fab fragment.
[0318] As a template for PCR amplification, a plasmid such as
pRcCMV-3D6HC, which contains as an insert the complete heavy chain
of the human monoclonal antibody, is used.
[0319] The nucleotide and amino acid sequence of the final product
of the PCRs and ligations, cloned into the NcoI site of pHEN.sup.1,
which leads to the attachment of a pelB leader sequence to the
N-terminus of the construct is shown below (SEQ ID No. 54, 55):
TABLE-US-00009 +3 M K Y L L P T A A A G L L L L A A 1 ATGAAATACC
TATTGCCTAC GGCAGCCGCT GGATTGTTAT TACTCGCGGC NcoI +3 Q P A M A A S T
K G P S V F P L 51 CCAGCCGG GCCGCCT CCACCAAGGG CCCATCGGTC
TTCCCCCTGG +3 A P S S A L G C L 101 CACCCTCCTC CNNSNNSNNS
NNSNNSNNSN NSNNSGCCCT GGGCTGCCTG +3 V K D Y F P E P V T V S W N S G
A 151 GTCAAGGACT ACTTCCCCGA ACCGGTGACG GTGTCGTGGA ACTCAGGCGC +3 L T
S G V H T F P A V L Q S S G 201 CCTGACCAGC GGCGTGCACA CCTTCCCGGC
TGTCCTACAG TCCTCAGGAC BstEII +3 L Y S L S S V V T V P 251
TCTACTCCCT CAGCAGCGT GTGC CCNNSNNSNN SNNSNNSNNS +3 T Y I C N V N H
K P S N T K V D 301 NNSACCTACA TCTGCAACGT GAATCACAAG CCCAGCAACA
CCAAGGTGGA NotI +3 K K V E P K S A A A 351 CAAGAAAGTT GAGCCCAAAT CT
A Primer List for CH1 library CH1LNCO: (SEQ ID No. 62)
5'-acgtccatgg ccgcctccac caagggccca tcggtcttcc ccctggcacc
ctcctccnns nnsnnsnnsn nsnnsnnsnn sgccctgggc tgcctg-gtc-3' CH1LBST:
(SEQ ID No. 63) 5'-ggcacggtca ccacgctgct gag-3' CH1RBST: (SEQ ID
No. 64) 5'-agcgtggtga ccgtgcccnn snnsnnsnns nnsnnsnnsa cctacatctg
caacgtgaat c-3' CH1RNOT: (SEQ ID No. 65) 5'-catagcggcc gcagatttgg
gctcaacttt cttgtc-3'
[0320] A number of selected library clones (mutated CH1 domains
cloned in the phagmid vector pHEN1) are controlled by restriction
analysis and by DNA sequencing to contain the insert as planned,
including the correctly inserted randomized sequences. For the
following steps of phage preparation, standard protocols are
followed. Briefly, the ligation mixture is transformed into E. coli
TG1 cells by electroporation. Subsequently, phage particles are
rescued from E. coli TG1 cells with helper phage M13-KO7. Phage
particles are then precipitated from culture supernatant with
PEG/NaCl in 2 steps, dissolved in water and used for selection by
panning or, alternatively, they can be stored at minus 80.degree.
C.
Example 13
Panning of the CH1-Phage Library on Hen Egg Lysozyme (HEL)
[0321] 3 panning rounds are performed with the CH1-phage library
(see example 12). Maxisorp 96-well plates (Nunc) are coated with
hen egg lysozyme, by adding 200 .mu.l of the following solution per
well: PBS, with the following concentrations of dissolved hen egg
lysozyme: [0322] 1.sup.st panning round: 2 mg/ml HEL [0323]
2.sup.nd panning round: 1 mg/ml HEL [0324] 3.sup.rd panning round:
1 mg/ml HEL
[0325] Incubation is for 1 hour at 37.degree. C., followed by
blocking with 2% dry milk (M-PBS) with 200 .mu.l per well for 1
hour at room temperature.
[0326] The surface display phage library is then allowed to react
with the bound hen egg lysozyme by adding 100 .mu.l phage
suspension and 100 .mu.l 4% dry milk (M-PBS), followed by
incubation for 45 minutes with shaking and for 90 minutes without
shaking at room temperature.
[0327] Unbound phage particles are washed away as follows: [0328]
1.sup.st panning round: 10.times.300 .mu.l T-PBS, 5.times.300 .mu.l
PBS [0329] 2.sup.nd panning round: 15.times.300 .mu.l T-PBS,
10.times.300 .mu.l PBS [0330] 3.sup.rd panning round: 20.times.300
.mu.l T-PBS, 20.times.300 .mu.l PBS
[0331] Elution of bound phage particles is performed by adding 200
.mu.l per well of 0.1 M glycine, pH 2.2, and incubation with
shaking for 30 minutes at room temperature. Subsequently, the phage
suspension is neutralized by addition of 60 .mu.l 2M Tris-Base,
followed by infection into E. coli TG1 cells by mixture of 10 ml
exponentially growing culture with 0.5 ml eluted phage and
incubation for 30 minutes at 37.degree. C. Finally, infected
bacteria are plated on TYE medium with 1% glucose and 100 .mu.g/ml
ampicillin, and incubated at 30.degree. C. overnight.
[0332] Cloning of Selected Clones of CH1 Mutants Selected Against
Lysozyme for Soluble Expression
[0333] Phagmid DNA from the phage selected through the 3 panning
rounds is isolated with a midi-prep. DNA encoding mutated
CH1-domains is batch-amplified by PCR and cloned NcoI-NotI into the
vector pNOTBAD/Myc-His, which is the E. coli expression vector
pBAD/Myc-His (Invitrogen) with an inserted NotI restriction site to
facilitate cloning. Ligated constructs are transformed into E. coli
LMG194 cells (Invitrogen) with electroporation, and grown at
30.degree. C. on TYE medium with 1% glucose and ampicillin
overnight. Selected clones are inoculated into 200 .mu.l 2.times.YT
medium with ampicillin, grown overnight at 30.degree. C., and
induced by adding L-arabinose to an end concentration of 0.1%.
After expression at 16.degree. C. overnight, the cells are
harvested by centrifugation and treated with 100 .mu.l Na-borate
buffer, pH 8.0, at 4.degree. C. overnight for preparation of
periplasmic extracts. 50 .mu.l of the periplasmic extracts are used
in ELISA.
[0334] Clones that give a high signal in this first, preliminary
ELISA are cultured in a 20-ml volume at the same conditions as
described above. Their periplasmic extracts are isolated in 1/20 of
the culture volume as described above and tested with ELISA (as
described below) for confirmation.
[0335] ELISA of CH1 Mutants Selected Against Hen Egg Lysozyme
[0336] Coating: Microtiter plate (NUNC, Maxisorp), 100 .mu.l per
well, 100 .mu.g hen egg lysozyme/ml in PBS, 1 h at 37.degree. C.
[0337] Wash: 3.times.200 .mu.l PBS [0338] Blocking: 1% BSA-PBS, 1 h
at RT [0339] Wash: 3.times.200 .mu.l PBS [0340] Periplasmic extract
binding: 50 .mu.l periplasmic extract 50 .mu.l 2% BSA-PBS, at room
temperature overnight [0341] Wash: 3.times.200 .mu.l PBS [0342]
1.sup.st antibody: anti-His.sub.4 (Qiagen), 1:1000 in 1% BSA-PBS,
90 min at RT, 100 .mu.l per well [0343] Wash: 3.times.200 .mu.l PBS
[0344] 2.sup.nd antibody: goat anti mouse*HRP(SIGMA), 1:1000 in 1%
BSA-PBS, 90 min at RT, 100 .mu.l per well [0345] Wash: 3.times.200
.mu.l PBS [0346] Detection: 3 mg/ml OPD in Na-citrate/phosphate
buffer, pH 4.5, 0.4 .mu.l 30% H.sub.2O.sub.2 [0347] Stopping: 100
ml 3M H.sub.2SO.sub.4 [0348] Absorbance read: 492/620 nm
[0349] Clones are interpreted as positive when their ELISA signal
is at least three times that of the background signal.
Example 14
Panning of the CL-Phage Library on Hen Egg Lysozyme (HEL)
[0350] 3 panning rounds are performed with the CL-phage library
(see e.lamda.ample 11). Maxisorp 96-well plates (Nunc) are coated
with hen egg lysozyme, by adding 200 .mu.l of the following
solution per well: PBS, with the following concentrations of
dissolved hen egg lysozyme: [0351] 1.sup.st panning round: 2 mg/ml
HEL [0352] 2.sup.nd panning round: 1 mg/ml HEL [0353] 3.sup.rd
panning round: 1 mg/ml HEL
[0354] Incubation is for 1 hour at 37.degree. C., followed by
blocking with 2% dry milk (M-PBS) with 200 .mu.l per well for 1
hour at room temperature.
[0355] The surface display phage library is then allowed to react
with the bound hen egg lysozyme by adding 100 .mu.l phage
suspension and 100 .mu.l 4% dry milk (M-PBS), followed by
incubation for 45 minutes with shaking and for 90 minutes without
shaking at room temperature.
Unbound phage particles are washed away as follows: [0356] 1.sup.st
panning round: 10.times.300 .mu.l T-PBS, 5.times.300 .mu.l PBS
[0357] 2.sup.nd panning round: 15.times.300 .mu.l T-PBS,
10.times.300 .mu.l PBS [0358] 3.sup.rd panning round: 20.times.300
.mu.l T-PBS, 20.times.300 .mu.l PBS
[0359] Elution of bound phage particles is performed by adding 200
.mu.l per well of 0.1 M glycine, pH 2.2, and incubation with
shaking for 30 minutes at room temperature. Subsequently, the phage
suspension is neutralized by addition of 60 .mu.l 2M Tris-Base,
followed by infection into E. coli TG1 cells by mixture of 10 ml
exponentially growing culture with 0.5 ml eluted phage and
incubation for 30 minutes at 37.degree. C. Finally, infected
bacteria are plated on TYE medium with 1% glucose and 100 .mu.g/ml
Ampicillin, and incubated at 30.degree. C. overnight.
[0360] Cloning of Selected Clones of CL Mutants Selected Against
Lysozyme for Soluble Expression
[0361] Phagmid DNA from the phage selected through the 3 panning
rounds is isolated with a midi-prep. DNA encoding mutated
CL-domains is batch-amplified by PCR and cloned NcoI-NotI into the
vector pNOTBAD/Myc-His, which is the E. coli expression vector
pBAD/Myc-His (Invitrogen) with an inserted NotI restriction site to
facilitate cloning. Ligated constructs are transformed into E. coli
LMG194 cells (Invitrogen) with electroporation, and grown at
30.degree. C. on TYE medium with 1% glucose and ampicillin
overnight. Selected clones are inoculated into 200 .mu.l 2xYT
medium with ampicillin, grown overnight at 30.degree. C., and
induced by adding L-arabinose to an end concentration of 0.1%.
After expression at 16.degree. C. overnight, the cells are
harvested by centrifugation and treated with 100 .mu.l Na-borate
buffer, pH 8.0, at 4.degree. C. overnight for preparation of
periplasmic extracts. 50 .mu.l of the periplasmic extracts are used
in ELISA.
[0362] Clones that give a high signal in this first, preliminary
ELISA are cultured in a 20-ml volume at the same conditions as
described above. Their periplasmic extracts are isolated in 1/20 of
the culture volume as described above and tested with ELISA (as
described below) for confirmation.
[0363] Elisa of CL Mutants Selected Against Hen Egg Lysozyme [0364]
Coating: Microtiter plate (NUNC, Maxisorp), 100 .mu.l per well, 100
.mu.g hen egg lysozyme/ml in PBS, 1 h at 37.degree. C. [0365] Wash:
3.times.200 .mu.l PBS [0366] Blocking: 1% BSA-PBS, 1 h at RT [0367]
Wash: 3.times.200 .mu.l PBS [0368] Periplasmic extract binding: 50
.mu.l periplasmic extract 50 .mu.l 2% BSA-PBS, at room temperature
overnight [0369] Wash: 3.times.200 .mu.l PBS [0370] 1.sup.st
antibody: anti-His.sub.4 (Qiagen), 1:1000 in 1% BSA-PBS, 90 min at
RT, 100 .mu.l per well [0371] Wash: 3.times.200 .mu.l PBS [0372]
2.sup.nd antibody: goat anti mouse*HRP(SIGMA), 1:1000 in 1%
BSA-PBS, 90 min at RT, 100 .mu.l per well [0373] Wash: 3.times.200
.mu.l PBS [0374] Detection: 3 mg/ml OPD in Na-citrate/phosphate
buffer, pH 4.5, 0.4 .mu.l 30% H.sub.2O.sub.2 [0375] Stopping: 100
ml 3M H.sub.2SO.sub.4 [0376] Absorbance read: 492/620 nm
[0377] Clones are interpreted as positive when their ELISA signal
is at least three times that of the background signal.
Example 15
Construction of an Immunoglobulin Domain which Is Randomized on
Both Sides (Bispecific Engineered C.sub.H3 Domain)
[0378] This example describes an engineered immunoglobulin domain
with two binding specificities.
[0379] The design of this engineered immunoglobulin domain
comprised the following strategy: [0380] an engineered C.sub.H3
domain, clone C24 (see example 10), derived from the C.sub.H3+5
library binding specifically to lysozyme was used as starting point
[0381] residues to be randomized were identified in this modified
CH3 domain which are connecting .beta.-strands of the
immunoglobulin fold, and which lie on the opposite side of the
domain compared to the residues that were mutated when generating
clone C24. [0382] PCR primers were designed that allowed
randomization of these residues and synthesis of this engineered
immunoglobulin domain in a procedure similar to the one described
above for the C.sub.H3, the C.sub.H3+3 and the C.sub.H3+5
libraries.
[0383] 4 PCR products containing randomised positions were ligated
and full-length inserts were amplified by PCR. Subsequently, they
were cloned in pHEN-1 via NcoI-NotI sites and transformed into E.
coli TG-1 cells to construct the library of about 10.sup.8
colonies. 20 randomly chosen colonies were sequenced and randomised
positions were found to be independently mutated. Also no "wild
type" (C24) sequence was observed. The phage library was generated
following standard protocols, and a phage titer of
6.32.times.10.sup.10 TU/ml was achieved.
[0384] In order to test bispecificity, recombinant human
Erythropoietin (rhEPO) was chosen as second antigen, while it was
expected that the construct retained its originally engineered
specificity for hen egg lysozyme. rhEPO-reactive phage was selected
in 4 panning rounds. In order to preserve the population of C24
clones that after mutagenesis still should bind hen egg lysozyme,
the first round of selection on rhEPO was followed by a round of
panning of the phage population on hen egg lysozyme (1 mg/ml in
PBS). 200 .mu.l of rhEPO was coated on the 5 wells of microtitre
plate (Maxisorp, Nunc) in 0.1 M Na-carbonate buffer, pH 9.6, in
decreasing concentrations in subsequent panning rounds (see Table
below). After blocking with 2% M-PBS, phage in the blocking agent
was allowed to bind at room temperature for 2 h. After 20 washes
with T-PBS and 20 with PBS, it was eluted with 0.1 M glycine, pH
2.2, and neutralised with 2M Tris. Eluted phage was used
immediately to infect exponentially growing TG-1. Infected cells
were selected on ampicilline-containing medium. Phage particles
were rescued from culture supernatants upon superinfection with
helper phage M13-KO7, concentrated with PEG and used in another
panning round. Input and output phage numbers were determined as
transforming units of E. coli after every panning round (Table
8).
TABLE-US-00010 TABLE 8 phage input phage output panning round
antigen (TU/ml) (TU/ml) 1 rhEPO, 500 .mu.g/ml 6.32 .times.
10.sup.10 1.9 .times. 10.sup.5 2 lysozyme, 1 mg/ml 6.16 .times.
10.sup.15 4.53 .times. 10.sup.10 3 rhEPO, 100 .mu.g/ml 6.07 .times.
10.sup.15 6.78 .times. 10.sup.10 4 rhEPO, 50 .mu.g/ml 8.42 .times.
10.sup.15 3.0 .times. 10.sup.11 5 rhEPO, 50 .mu.g/ml 5.12 .times.
10.sup.15 4.28 .times. 10.sup.10
[0385] Resulting colonies were scraped off the plates, cultured in
2.times.YT with ampicilline and their plasmid DNA was isolated with
a midi-prep. Inserts were amplified with a PCR, and then subcloned
into vector pNOTBAD and transformed into an E. coli strain E104.
4.times.72 colonies were cultured in 200 .mu.l 2.times.YT with
ampicilline and induced with 0.1% L-arabinose on the following day.
After 24 h expression at 16.degree. C., they were lysed with 200
.mu.l Na-borate buffer, pH 8.0 for 6 h at 4.degree. C. and
periplasmic extract was used in ELISA.
[0386] For ELISA, Maxisorp plates were coated with hen egg lysozyme
in PBS (20 .mu.g/ml) or rhEPO in 0.1 M Na-carbonate buffer, pH 9.6,
respectively, for 1 h at 37.degree. C. After blocking with 1%
BSA-PBS, periplasmic extract in the same blocking agent was allowed
to bind overnight. Binding was revealed with an anti-His-(4)
antibody and a goat anti-mouse IgG antibody, conjugated with HRP
(for hen egg lysozyme detection) or AP (for rhEPO detection).
Colour reaction of OPD conversion (HRP) was read at 492/620 nm
after being stopped with 1.25 M H.sub.2SO.sub.4, and pNPP
conversion (AP) was read at 405/620 nm. 14 clones with promising
absorbance values were selected for expression at 20-ml-scale.
After 24 h arabinose induction at 16.degree. C., the cells were
collected and lysed overnight in 1 ml Na-borate buffer at 4.degree.
C., and the lysate was used for ELISA. ELISA was performed as above
in 4 parallels, and wells without periplasmic extract and without
antigen were used as negative controls. Results (Table 9) were
achieved with clone according to SEQ ID No. 42, 43.
TABLE-US-00011 TABLE 9 absorbance on no periplasmic antigen binding
extract no antigen lysozyme A .sub.492/620 nm 0.299 0.110 0.018
rhEPO A .sub.405/620 nm 0.258 0.095 0.090
Example 16
Engineered C.sub.H3 Domains Provide Bispecificity in an Fab-like
format
[0387] In the construct used in this example, both the V, and the
V.sub.H chain of an antibody are fused to an engineered C.sub.H3
domain.
[0388] The VL and VH region of the human monoclonal antibody 3D6
(He X M, et al. Proc Natl Acad Sci USA. 1992 89:7154-8; Kohl J, et
al. Ann N Y Acad. Sci. 1991 646:106-14; Felgenhauer M, et al.
Nucleic Acids Res. 1990 18:4927), which recognizes an epitope on
gp41 of HIV-1 was used as fusion partner for the engineered
C.sub.H3 domain clone C24 which binds specifically to hen egg
lysozyme.
[0389] In order to promote the formation of the VL-CH3/VH-CH3 dimer
via a disulfide bond, the residues Ser-Cys were added to the
C-terminus of the C24 sequence.
[0390] The nucleotide- and amino acid sequences respectively of the
two chains, 3D6VL-C24 and 3D6VH-C24 are given in SEQ ID No. 47, 46
and SEQ ID No. 45, 44, respectively.
[0391] Primers were designed that allow the amplification of the
coding regions, introducing restriction sites at the same time
(silent mutations) which were used to ligate the coding regions
together. For expression of the genes, the Pichia pastoris
expression system was chosen. Constructs were cloned in suitable
Pichia pastoris expression vectors: 3D6VL-C24 was cloned in the
pPIC9K (final name: pPIC9K.sub.3LC) and 3D6VH-C24 (final name:
pPICZ3HC) was cloned in pPICZalphaA. Construct pPICZ3HC was
linearized with Bgl II, transformed into Pichia pastoris GS115 and
transformants were selected on zeocin-containing solid medium. One
of the transformants was subsequently used as a host cell for the
Sal I-linearized construct pPIC9K3LC. Double transformants were
then selected on RDB-medium.
[0392] Clones were inoculated into 30 ml YPG medium and grown until
OD.sub.600=10, and were then induced by the addition of 1% methanol
in BMMY medium. The induction was continued for 36 hours at
16.degree. C. Supernatants were removed by centrifugation and were
then concentrated about 10-times. Presence of the recombinant
protein was confirmed by a Western blot with an anti-His (4)
antibody, and was estimated to be at a concentration of
approximately 50-100 .mu.g/1 initial culture.
[0393] First functional tests wer performed with
10.times.-concentrated supernatant. Firstly, wells of Maxisorp
plates were coated with 20 .mu.g/ml hen egg lysozyme in PBS or 20
.mu.g/ml epitope of the antibody 3D6 in 0.1 M Na-carbonate buffer,
pH 9.6, respectively, for 1 h at 37.degree. C. The 3D6 epitope was
used in the form of a recombinantly produced GST-fusion protein.
After blocking with 1% BSA-PBS, concentrated supernatants were
allowed to bind overnight in the same blocking agent. Binding was
revealed with an anti-His (4) antibody and goat anti-mouse
antibody, conjugated to HRP, and visualised as colour reaction
resulting from OPD conversion at 492/620 nm (Table 10).
TABLE-US-00012 TABLE 10 ELISA signal Background Background antigen
(A .sub.492/620) (no antigen) (no supernatant) lysozyme 0.198 0.003
0.043 3D6 epitope 0.061 0.001 0.007
Sequence CWU 1
1
661108PRTHomo sapiens 1Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu1 5 10 15Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro 20 25 30Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn 35 40 45Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu 50 55 60Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val65 70 75 80Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 85 90 95Lys Ser Leu Ser
Leu Ser Pro Gly Lys Ala Ala Ala 100 1052332DNAArtificial
sequenceSynthetic oligonucleotide 2ccatggcccc ccgagaacca caggtgtaca
ccctgccccc atcccgggat gagctcnnsn 60nsnnscaggt cagcctgacc tgcctggtca
aaggcttcta tcccagcgac atcgccgtgg 120agtgggagag caatgggcag
ccggagaaca actacaagac cacgcctccc gtgctggact 180ccgacggctc
cttcttcctc tacagcaagc ttaccgtgnn snnsnnsagg tggnnsnnsg
240ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac
acacagaaga 300gcctctccct gtctccgggt aaagcggccg ca
3323110PRTArtificial sequenceSynthetic peptide 3Met Ala Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp1 5 10 15Glu Leu Xaa Xaa
Xaa Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60Phe
Leu Tyr Ser Lys Leu Thr Val Xaa Xaa Xaa Arg Trp Xaa Xaa Gly65 70 75
80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ala Ala Ala 100
105 110433DNAArtificial sequenceSynthetic oligonucleotide
4cttgccatgg ccccccgaga accacaggtg tac 33530DNAArtificial
sequenceSynthetic oligonucleotide 5agtcgagctc gtcacgggat gggggcaggg
30641DNAArtificial sequenceSynthetic oligonucleotide 6gtacgagctc
nnsnnsnnsc aagtcagcct gacctgcctg g 41732DNAArtificial
sequenceSynthetic oligonucleotide 7tgccaagctt gctgtagagg aagaaggagc
cg 32859DNAArtificial sequenceSynthetic oligonucleotide 8tgccaagctt
accgtgnnsn nsnnsaggtg gnnsnnsggg aacgtcttct catgctccg
59933DNAArtificial sequenceSynthetic oligonucleotide 9agttgcggcc
gctttacccg gagacaggga gag 3310113PRTArtificial sequenceSynthetic
peptide 10Met Ala Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp1 5 10 15Glu Leu Xaa Xaa Xaa Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu 35 40 45Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe 50 55 60Phe Leu Tyr Ser Lys Leu Thr Val Xaa Xaa
Xaa Xaa Xaa Xaa Arg Trp65 70 75 80Xaa Xaa Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His 85 90 95Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys Ala Ala 100 105
110Ala11341DNAArtificial sequenceSynthetic oligonucleotide
11ccatggcccc ccgagaacca caggtgtaca ccctgccccc atcccgtgac gagctcnnsn
60nsnnscaagt cagcctgacc tgcctggtca aaggcttcta tcccagcgac atcgccgtgg
120agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc
gtgctggact 180ccgacggctc cttcttcctc tacagcaagc ttaccgtgnn
snnsnnsnns nnsnnsaggt 240ggnnsnnsgg gaacgtcttc tcatgctccg
tgatgcatga ggctctgcac aaccactaca 300cacagaagag cctctccctg
tctccgggta aagcggccgc a 3411268DNAArtificial sequenceSynthetic
oligonucleotide 12tgccaagctt accgtgnnsn nsnnsnnsnn snnsaggtgg
nnsnnsggga acgtcttctc 60atgctccg 6813115PRTArtificial
sequenceSynthetic peptide 13Met Ala Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp1 5 10 15Glu Leu Xaa Xaa Xaa Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe 20 25 30Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60Phe Leu Tyr Ser Lys Leu
Thr Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65 70 75 80Arg Trp Xaa Xaa
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 85 90 95Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 110Ala
Ala Ala 11514347DNAArtificial sequenceSynthetic oligonucleotide
14ccatggcccc ccgagaacca caggtgtaca ccctgccccc atcccgtgac gagctcnnsn
60nsnnscaagt cagcctgacc tgcctggtca aaggcttcta tcccagcgac atcgccgtgg
120agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc
gtgctggact 180ccgacggctc cttcttcctc tacagcaagc ttaccgtgnn
snnsnnsnns nnsnnsnnsn 240nsaggtggnn snnsgggaac gtcttctcat
gctccgtgat gcatgaggct ctgcacaacc 300actacacaca gaagagcctc
tccctgtctc cgggtaaagc ggccgca 3471574DNAArtificial
sequenceSynthetic oligonucleotide 15tgccaagctt accgtgnnsn
nsnnsnnsnn snnsnnsnns aggtggnnsn nsgggaacgt 60cttctcatgc tccg
7416110PRTArtificial sequenceSynthetic peptide 16Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu1 5 10 15Gly Trp Pro
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 20 25 30Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 35 40 45Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 50 55
60Tyr Ser Lys Leu Thr Val Pro Lys Arg Trp Cys Val Ser Val Arg Trp65
70 75 80Pro Pro Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His 85 90 95Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
100 105 11017330DNAArtificial sequenceSynthetic oligonucleotide
17ccccgagaac cacaggtgta caccctgccc ccatcccgtg acgagctcgg ctggccgcaa
60gtcagcctaa cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag
120agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 180tccttcttcc tctacagcaa gcttaccgtg cccaagcggt
ggtgcgtgag cgtcaggtgg 240cccccgggga acgtcttctc atgctccgtg
atgcatgagg ctctgcacaa ccactacaca 300cagaagagcc tctccctgtc
tccgggtaaa 33018110PRTArtificial sequenceSynthetic peptide 18Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu1 5 10
15Ser Val Ser Gln Val Ser Pro Thr Cys Leu Val Lys Gly Phe Tyr Pro
20 25 30Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn 35 40 45Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu 50 55 60Tyr Ser Lys Leu Thr Val Ile Pro Phe Cys Arg Met Ser
Pro Arg Trp65 70 75 80Trp Ile Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His 85 90 95Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 100 105 11019330DNAArtificial sequenceSynthetic
oligonucleotide 19ccccgagaac cacaggtgta caccctgccc ccatcccgtg
acgagctctc ggtgtcgcaa 60gtcagcccga cctgcctggt caaaggcttc tatcccagcg
acatcgcagt ggagtgggag 120agcaatgggc agccggagaa caactacaag
accacgcctc ccgtgctgga ctccgacggc 180tccttcttcc tctacagcaa
gcttaccgtg atccccttct gcaggatgag ccccaggtgg 240tggatcggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca
300cagaagagcc tctccctgtc tccgggtaaa 33020105PRTArtificial
sequenceSynthetic peptide 20Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu1 5 10 15Glu Ala Leu Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 20 25 30Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn 35 40 45Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu 50 55 60Tyr Ser Lys Leu Thr Val
Arg Arg Asn Arg Trp Ser Trp Gly Asn Val65 70 75 80Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 85 90 95Lys Ser Leu
Ser Leu Ser Pro Gly Lys 100 10521315DNAArtificial sequenceSynthetic
oligonucleotide 21cctcgagaac cacaggtgta caccctgccc ccatcccgtg
acgagctcga ggcgctgcaa 60gtcagcctga cctgcctggt caaaggcttc tatcccagcg
acatcgccgt ggagtgggag 120agcaatgggc agccggagaa caactacaag
accacgcctc ccgtgctgga ctccgacggc 180tccttcttcc tctacagcaa
gcttaccgtg cggcgcaaca ggtggtcctg ggggaacgtc 240ttctcatgct
ccgtgatgca tgaggctctg cacaaccact acacacagaa gagcctctcc
300ctgtctccgg gtaaa 31522108PRTArtificial sequenceSynthetic peptide
22Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu1
5 10 15Gln Gly Ser Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro 20 25 30Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn 35 40 45Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu 50 55 60Tyr Ser Lys Leu Thr Val Lys Ser Arg Ala Thr Arg
Arg Trp Val Val65 70 75 80Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His 85 90 95Tyr Thr Gln Lys Asn Leu Ser Leu Ser
Pro Gly Lys 100 10523324DNAArtificial sequenceSynthetic
oligonucleotide 23ccccgagaac cacaggtgta caccctgccc ccatcccgtg
acgagctcca ggggagccaa 60gtcagcctga cctgcctggt caaaggcttc tatcccagcg
acatcgccgt ggagtgggag 120agcaatgggc agccggagaa caactacaag
accacgcctc ccgtgctgga ctccgacggc 180tccttcttcc tctacagcaa
gcttaccgtg aagtcgcgcg ccacccggag gtgggtggtg 240gggaacgtct
tttcttgctc cgtgatgcat gaggctctgc acaaccacta cacacagaag
300aacctctccc tgtctccggg taaa 32424107PRTArtificial
sequenceSynthetic peptide 24Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu1 5 10 15Ala Ile Gly Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 20 25 30Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn 35 40 45Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu 50 55 60Tyr Ser Lys Leu Thr Val
Arg Ser Thr Arg Asp Asn Arg Trp Leu Val65 70 75 80Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His 85 90 95Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 100 10525324DNAArtificial
sequenceSynthetic oligonucleotide 25ccccgagaac cacaggtgta
caccctgccc ccatcccgtg acgagctcgc gatcggccaa 60gtcagcctga cctgcctggt
caaaggcttc tatcccagcg acatcgccgt ggagtgggag 120agcaatgggc
agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc
180tccttcttcc tctacagcaa gcttaccgtg cgctcgacga gggacaacag
gtggctggtg 240gggaacgtct tctcatgctc cgtgatgcat gaggctctgc
acaaccacta cacacagaag 300agcctctccc tgtctccggg taaa
32426110PRTArtificial sequenceSynthetic peptide 26Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu1 5 10 15Ser Gly Ala
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 20 25 30Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 35 40 45Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 50 55
60Tyr Ser Lys Leu Thr Val Trp Phe Arg Gln Glu Gly Gly Met Arg Trp65
70 75 80Phe Ala Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His 85 90 95Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
100 105 11027330DNAArtificial sequenceSynthetic oligonucleotide
27ccccgagaac cacaggtgta caccctgccc ccatcccgtg acgagctcag cggggcgcaa
60gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag
120agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 180tccttcttcc tctacagcaa gcttaccgtg tggttcaggc
aggagggcgg catgaggtgg 240ttcgcgggga acgtcttctc atgctccgtg
atgcatgagg ctctgcacaa ccactacaca 300cagaagagcc tctccctgtc
tccgggtaaa 33028110PRTArtificial sequenceSynthetic peptide 28Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu1 5 10
15Val Leu Gly Gln Val Ser Pro Thr Cys Leu Val Lys Gly Phe Tyr Pro
20 25 30Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn 35 40 45Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu 50 55 60Tyr Gly Lys Leu Thr Val Pro Pro Arg Leu Lys Gly Trp
Pro Arg Trp65 70 75 80Gly Trp Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His 85 90 95Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 100 105 11029330DNAArtificial sequenceSynthetic
oligonucleotide 29ccccgagaac cacaggtgta caccctgccc ccatcccgtg
acgagctcgt cttggggcaa 60gtcagcccga cctgcctggt caaaggcttc tatcccagcg
acatcgccgt ggagtgggag 120agcaatgggc agccggagaa caactacaag
accacgcctc ccgtgctgga ctccgacggc 180tccttcttcc tctacggcaa
gcttaccgtg cccccgcggt tgaagggctg gccgaggtgg 240ggctggggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca
300cagaagagcc tctccctgtc tccgggtaaa 33030105PRTArtificial
sequenceSynthetic peptide 30Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu1 5 10 15Leu Ala Tyr Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 20 25 30Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn 35 40 45Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu 50 55 60Tyr Ser Lys Leu Thr Val
Val Ala Gly Arg Trp Thr Cys Gly Asn Val65 70 75 80Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 85 90 95Lys Ser Leu
Ser Leu Ser Pro Gly Lys 100 10531315DNAArtificial sequenceSynthetic
oligonucleotide 31ccccgagaac cacaggtgta caccctgccc ccatcccgtg
acgagctcct ggcgtaccaa 60gtcagcctga cctgcctggt caaaggcttc tatcccagcg
acatcgccgt ggagtgggag 120agcaatgggc agccggagaa caactacaag
accacgcctc ccgtgctgga ctccgacggc 180tccttcttcc tctacagcaa
gcttaccgtg gtggccggca ggtggacgtg cgggaacgtc 240ttctcatgct
ccgtgatgca tgaggctctg cacaaccact acacacagaa gagcctctcc
300ctgtctccgg gtaaa 31532110PRTArtificial sequenceSynthetic peptide
32Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu1
5 10 15Cys Val Pro Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro 20 25 30Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn 35 40 45Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu 50 55 60Tyr Ser Lys Leu Thr Val Val Leu Lys Val Val Gln
Ala Arg Arg Trp65 70 75 80Glu Val Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His 85 90 95Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys 100 105 11033330DNAArtificial sequenceSynthetic
oligonucleotide 33ccccgagaac cacaggtgta caccctgccc ccatcccgtg
acgagctctg cgtcccgcaa 60gtcagcctga cctgcctggt caaaggcttc tatcccagcg
acatcgccgt ggagtgggag 120agcaatgggc agccggagaa caactacaag
accacgcctc ccgtgctgga ctccgacggc 180tccttcttcc
tctacagcaa gcttaccgtg gtgctcaagg tcgtgcaggc gcgcaggtgg
240gaggtgggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa
ccactacaca 300cagaagagcc tctccctgtc tccgggtaaa
33034105PRTArtificial sequenceSynthetic peptide 34Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu1 5 10 15Gly Ile Ala
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 20 25 30Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 35 40 45Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 50 55
60Tyr Ser Lys Leu Thr Val Leu Gly Arg Arg Trp Thr Leu Gly Asn Val65
70 75 80Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln 85 90 95Lys Ser Leu Ser Leu Ser Pro Gly Lys 100
10535315DNAArtificial sequenceSynthetic oligonucleotide
35ccccgagaac cacaggtgta caccctgccc ccatcccggg acgagctcgg catcgcgcaa
60gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag
120agcaacgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 180tctttcttcc tctacagcaa gcttaccgtg ttgggccgca
ggtggaccct ggggaacgtc 240ttctcatgct ccgtgatgca tgaggctctg
cacaaccact acacacagaa gagcctctcc 300ctgtctccgg gtaaa
31536105PRTArtificial sequenceSynthetic peptide 36Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu1 5 10 15Gly Ile Ala
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 20 25 30Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 35 40 45Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 50 55
60Tyr Ser Lys Leu Thr Val Leu Gly Arg Arg Trp Thr Leu Gly Asn Val65
70 75 80Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln 85 90 95Lys Ser Leu Ser Leu Ser Pro Gly Lys 100
10537315DNAArtificial sequenceSynthetic oligonucleotide
37ccccgagaac cacaggtgta caccctgccc ccatcccgtg acgagctcgg catcgcgcaa
60gtcagcttga cctgcctggt caaaggcttt tatcccagcg acatcgccgt ggagtgggag
120agcaacgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 180tccttcttcc tctacagcaa gcttaccgtg ttgggccgca
ggtggaccct ggggaacgtc 240ttctcatgct ccgtgatgca tgaggctctg
cacaaccact acacacagaa gagcctctcc 300ctgtctccgg gtaaa
31538105PRTArtificial sequenceSynthetic peptide 38Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu1 5 10 15Leu Pro Cys
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 20 25 30Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 35 40 45Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 50 55
60Tyr Ser Lys Leu Thr Val Phe Cys Pro Arg Trp Leu Gly Gly Asn Val65
70 75 80Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln 85 90 95Lys Ser Leu Ser Leu Ser Pro Gly Lys 100
10539315DNAArtificial sequenceSynthetic oligonucleotide
39ccccgagaac cacaggtgta caccctgccc ccatcccgtg acgagctctt gccctgccaa
60gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag
120agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 180tctttcttcc tctacagcaa gcttaccgtg ttctgcccca
ggtggctggg ggggaacgtc 240ttctcatgct ccgtgatgca tgaggctctg
cacaaccact acacacagaa gagcctctcc 300ctgtctccgg gtaaa
31540105PRTArtificial sequenceSynthetic peptide 40Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu1 5 10 15Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 20 25 30Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 35 40 45Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 50 55
60Tyr Ser Lys Leu Thr Val Pro Cys Met Arg Trp Trp Gly Gly Asn Val65
70 75 80Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln 85 90 95Lys Ser Leu Ser Leu Ser Pro Gly Lys 100
10541315DNAArtificial sequenceSynthetic oligonucleotide
41ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag
60gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag
120agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga
ctccgacggc 180tccttcttcc tctacagcaa gcttaccgtg ccctgcatga
ggtggtgggg cgggaacgtc 240ttctcatgct ccgtgatgca tgaggctctg
cacaaccact acacacagaa gagcctctcc 300ctgtctccgg gtaaa
31542115PRTArtificial sequenceSynthetic peptide 42Arg Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu1 5 10 15Val Leu Gly
Gln Val Ser Leu Ala Cys Leu Val Lys Gly Phe Val Val 20 25 30Arg Leu
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 35 40 45Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Arg Gln Leu Ala 50 55
60Asp Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Pro Arg Leu Lys65
70 75 80Gly Trp Pro Arg Trp Gly Trp Gly Asn Val Phe Ser Cys Ser Val
Met 85 90 95Phe Leu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 100 105 110Pro Gly Lys 11543345DNAArtificial
sequenceSynthetic oligonucleotide 43cggcgagaac cacaggtgta
caccctgccc ccatcccgtg acgagctcgt cttggggcaa 60gtcagcctgg cctgcctcgt
gaaaggcttc gtggtccggt tgatcgccgt ggagtgggag 120agcaatgggc
agccggagaa caactacaag accacgcctc ccgttctaga ctccgacggc
180cggcagttgg cggactcctt cttcctctac agcaagctta ccgtgccccc
gcggttgaag 240ggctggccga ggtggggctg ggggaacgtc ttctcatgca
gtgtgatgtt cctggcgctg 300cacaaccact acacacagaa gagcctctcc
ctgtctccgg gtaaa 34544238PRTArtificial sequenceSynthetic peptide
44Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp
Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Gly Ile Ser Trp Asp Ser Ser Ser Ile Gly Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Met Ala Leu Tyr Tyr Cys 85 90 95Val Lys Gly Arg Asp Tyr Tyr Asp Ser
Gly Gly Tyr Phe Thr Val Ala 100 105 110Phe Asp Ile Trp Gly Gln Gly
Thr Met Val Thr Val Ser Ser Ala Ser 115 120 125Thr Lys Gly Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 130 135 140Val Leu Gly
Gln Val Ser Pro Thr Cys Leu Val Lys Gly Phe Tyr Pro145 150 155
160Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
165 170 175Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu 180 185 190Tyr Gly Lys Leu Thr Val Pro Pro Arg Leu Lys Gly
Trp Pro Arg Trp 195 200 205Gly Trp Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His 210 215 220Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys225 230 23545714DNAArtificial
sequenceSynthetic oligonucleotide 45gaagtgcagc tggtggagtc
tgggggaggc ttggtacagc ctggcaggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttaat gattatgcca tgcactgggt ccggcaagct 120ccagggaagg
gcctggagtg ggtctcaggt ataagttggg atagtagtag tataggctat
180gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa
ctccctgtat 240ctgcaaatga acagtctgag agctgaggac atggccttat
attactgtgt aaaaggcaga 300gattactatg atagtggtgg ttatttcacg
gttgcttttg atatctgggg ccaagggaca 360atggtcaccg tctcttcagc
ctccaccaag ggcccacagg tgtacaccct gcccccatcc 420cgtgacgagc
tcgtcttggg gcaagtcagc ccgacctgcc tggtcaaagg cttctatccc
480agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta
caagaccacg 540cctcccgtgc tggactccga cggctccttc ttcctctacg
gcaagcttac cgtgcccccg 600cggttgaagg gctggccgag gtggggctgg
gggaacgtct tctcatgctc cgtgatgcat 660gaggctctgc acaaccacta
cacacagaag agcctctccc tgtctccggg taaa 71446217PRTArtificial
sequenceSynthetic peptide 46Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Arg Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Val 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 Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Phe 85 90 95Gly Pro Gly
Thr Lys Val Asp Ile Lys Arg Thr Val Ala Glu Pro Gln 100 105 110Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Val Leu Gly Gln Val 115 120
125Ser Pro Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
130 135 140Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro145 150 155 160Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Gly Lys Leu Thr 165 170 175Val Pro Pro Arg Leu Lys Gly Trp Pro
Arg Trp Gly Trp Gly Asn Val 180 185 190Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205Lys Ser Leu Ser Leu
Ser Pro Gly Lys 210 21547651DNAArtificial sequenceSynthetic
oligonucleotide 47gacatccaga tgacccagtc tccttccacc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gagtattagt aggtggttgg
cctggtatca gcagaaacca 120gggaaagtcc ctaagctcct gatctataag
gcatctagtt tagaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagaa ttcactctca ccatcagcag cctgcagcct 240gatgattttg
caacttatta ctgccaacag tataatagtt attctttcgg ccctgggacc
300aaagtggata tcaaacgaac tgtggctgaa ccacaggtgt acaccctgcc
cccatcccgt 360gacgagctcg tcttggggca agtcagcccg acctgcctgg
tcaaaggctt ctatcccagc 420gacatcgccg tggagtggga gagcaatggg
cagccggaga acaactacaa gaccacgcct 480cccgtgctgg actccgacgg
ctccttcttc ctctacggca agcttaccgt gcccccgcgg 540ttgaagggct
ggccgaggtg gggctggggg aacgtcttct catgctccgt gatgcatgag
600gctctgcaca accactacac acagaagagc ctctccctgt ctccgggtaa a
65148129PRTArtificial sequenceSynthetic peptide 48Met Lys Tyr Leu
Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro
Ala Met Ala Val Ala Ala Pro Ser Val Phe Ile Phe Pro 20 25 30Pro Ser
Xaa Xaa Gln Xaa Xaa Xaa Xaa Xaa Ala Ser Val Val Cys Leu 35 40 45Leu
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 50 55
60Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp65
70 75 80Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Xaa
Xaa 85 90 95Xaa Xaa Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr
His Gln 100 105 110Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu Ala Ala 115 120 125Ala49387DNAArtificial sequenceSynthetic
oligonucleotide 49atgaaatacc tattgcctac ggcagccgct ggattgttat
tactcgcggc ccagccggcc 60atggccgtgg ctgcaccatc tgtcttcatc ttcccgccat
ctnnsnnsca gnnsnnsnns 120nnsnnsgcct ctgttgtgtg cctgctgaat
aacttctatc ccagagaggc caaagtacag 180tggaaggtgg ataacgccct
ccaatcgggt aactcccagg agagtgtcac agagcaggac 240agcaaggaca
gcacctacag cctcagcagc accctgacgt tgnnsnnsnn snnstacgag
300aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc
cgtcacaaag 360agcttcaaca ggggagaggc ggccgca 38750132PRTArtificial
sequenceSynthetic peptide 50Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala
Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala Val Ala Ala
Pro Ser Val Phe Ile Phe Pro 20 25 30Pro Ser Xaa Xaa Gln Xaa Xaa Xaa
Xaa Xaa Ala Ser Val Val Cys Leu 35 40 45Leu Asn Asn Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp 50 55 60Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp65 70 75 80Ser Lys Asp Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Xaa Xaa 85 90 95Xaa Xaa Xaa
Xaa Xaa Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val 100 105 110Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 115 120
125Glu Ala Ala Ala 13051396DNAArtificial sequenceSynthetic
oligonucleotide 51atgaaatacc tattgcctac ggcagccgct ggattgttat
tactcgcggc ccagccggcc 60atggccgtgg ctgcaccatc tgtcttcatc ttcccgccat
ctnnsnnsca gnnsnnsnns 120nnsnnsgcct ctgttgtgtg cctgctgaat
aacttctatc ccagagaggc caaagtacag 180tggaaggtgg ataacgccct
ccaatcgggt aactcccagg agagtgtcac agagcaggac 240agcaaggaca
gcacctacag cctcagcagc accctgacgt tgnnsnnsnn snnsnnsnns
300nnstacgaga aacacaaagt ctacgcctgc gaagtcaccc atcagggcct
gagctcgccc 360gtcacaaaga gcttcaacag gggagaggcg gccgca
39652134PRTArtificial sequenceSynthetic peptide 52Met Lys Tyr Leu
Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro
Ala Met Ala Val Ala Ala Pro Ser Val Phe Ile Phe Pro 20 25 30Pro Ser
Xaa Xaa Gln Xaa Xaa Xaa Xaa Xaa Ala Ser Val Val Cys Leu 35 40 45Leu
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 50 55
60Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp65
70 75 80Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Xaa
Xaa 85 90 95Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Glu Lys His Lys Val Tyr
Ala Cys 100 105 110Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser Phe Asn 115 120 125Arg Gly Glu Ala Ala Ala
13053402DNAArtificial sequenceSynthetic oligonucleotide
53atgaaatacc tattgcctac ggcagccgct ggattgttat tactcgcggc ccagccggcc
60atggccgtgg ctgcaccatc tgtcttcatc ttcccgccat ctnnsnnsca gnnsnnsnns
120nnsnnsgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc
caaagtacag 180tggaaggtgg ataacgccct ccaatcgggt aactcccagg
agagtgtcac agagcaggac 240agcaaggaca gcacctacag cctcagcagc
accctgacgt tgnnsnnsnn snnsnnsnns 300nnsnnsnnst acgagaaaca
caaagtctac gcctgcgaag tcacccatca gggcctgagc 360tcgcccgtca
caaagagctt caacagggga gaggcggccg ca 40254127PRTArtificial
sequenceSynthetic peptide 54Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala
Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 20 25 30Leu Ala Pro Ser Ser Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Ala Leu Gly 35 40 45Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn 50 55 60Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln65 70 75 80Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Xaa Xaa 85 90 95Xaa Xaa Xaa
Xaa Xaa Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 100 105 110Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Ala Ala Ala 115 120
12555381DNAArtificial sequenceSynthetic oligonucleotide
55atgaaatacc tattgcctac ggcagccgct ggattgttat tactcgcggc ccagccggcc
60atggccgcct ccaccaaggg cccatcggtc ttccccctgg caccctcctc cnnsnnsnns
120nnsnnsnnsn nsnnsgccct gggctgcctg gtcaaggact acttccccga
accggtgacg 180gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca
ccttcccggc tgtcctacag 240tcctcaggac tctactccct cagcagcgtg
gtgaccgtgc ccnnsnnsnn snnsnnsnns 300nnsacctaca tctgcaacgt
gaatcacaag
cccagcaaca ccaaggtgga caagaaagtt 360gagcccaaat ctgcggccgc a
3815687DNAArtificial sequenceSynthetic oligonucleotide 56cttaccatgg
ccgtggctgc accatctgtc ttcatcttcc cgccatctnn snnscagnns 60nnsnnsnnsn
nsgcctctgt tgtgtgc 875726DNAArtificial sequenceSynthetic
oligonucleotide 57tgacaacgtc agggtgctgc tgaggc 265841DNAArtificial
sequenceSynthetic oligonucleotide 58tcagaacgtt gnnsnnsnns
nnstacgaga aacacaaagt c 415950DNAArtificial sequenceSynthetic
oligonucleotide 59tcagaacgtt gnnsnnsnns nnsnnsnnsn nstacgagaa
acacaaagtc 506056DNAArtificial sequenceSynthetic oligonucleotide
60tcagaacgtt gnnsnnsnns nnsnnsnnsn nsnnsnnsta cgagaaacac aaagtc
566131DNAArtificial sequenceSynthetic oligonucleotide 61catcgcggcc
gcctctcccc tgttgaagct c 316299DNAArtificial sequenceSynthetic
oligonucleotide 62acgtccatgg ccgcctccac caagggccca tcggtcttcc
ccctggcacc ctcctccnns 60nnsnnsnnsn nsnnsnnsnn sgccctgggc tgcctggtc
996323DNAArtificial sequenceSynthetic oligonucleotide 63ggcacggtca
ccacgctgct gag 236461DNAArtificial sequenceSynthetic
oligonucleotide 64agcgtggtga ccgtgcccnn snnsnnsnns nnsnnsnnsa
cctacatctg caacgtgaat 60c 616536DNAArtificial sequenceSynthetic
oligonucleotide 65catagcggcc gcagatttgg gctcaacttt cttgtc
3666339DNAArtificial sequenceSynthetic oligonucleotide 66nnscgagaac
cacaggtgta caccctgccc ccatcccgtg acgagctcnn snnsnnscaa 60gtcagcctga
cctgcctcgt gaaaggcttc nnsnnsnnsn nsatcgccgt ggagtgggag
120agcaatgggc agccggagaa caactacaag accacgcctc ccgttctaga
ctccgacggc 180nnsnnsnnsn nsnnstcctt cttcctctac agcaagctta
ccgtgnnsnn snnsaggtgg 240nnsnnsggga acgtcttctc atgcagtgtg
atgnnsnnsn nsctgcacaa ccactacaca 300cagaagagcc tctccctgtc
tccgggtaaa gcggccgca 339
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